Fish Physiology: Molecular Endocrinology of Fish

FISH PHYSIOLOGY VOLUME XZZZ Molecular Endocrinology of Fish CONTRIBUTORS BENOIT AUPERIN K.-M. C H A N SHU JIN CHAN T ...

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FISH PHYSIOLOGY VOLUME XZZZ Molecular Endocrinology of Fish

CONTRIBUTORS BENOIT AUPERIN K.-M. C H A N SHU JIN CHAN T H O M A S T. C H E N CLARA M. C H E N G S T E P H E N J . DUGUAY HARRY P. E L S H O L T Z J. N. FRYER C H O Y L. H E W S H U I C H I HIRAOKA H I R O S H I KAWAUCHI KAORU KUBOKAWA YVES LE D R E A N K. L E D E R I S D A V I D W. L E S C H E I D S O N A L I MAJUMDAR ADAM MARSH J O H N E. McRORY T H O M A S P. M O M M S E N Y 0SH ITAKA NAGAH AM A

Y. OKAWARA MASAO O N 0 FARZAD P A K D E L D A V I D B. PARKER PATRICK P R U N E T D. R I C H T E R CHR. SCHONROCK MIKE SHAMBLOTT NANCY M. S H E R W O O D D O N A L D F. S T E I N E R KUNIMASA S U Z U K I M I N O R U TANAKA Y.-L. T A N G AKIHISA URANO YVES VALOTAIRE GRAHAM F. W A G N E R FEI X I O N G MASAKANE YAMASHITA B.-Y. YANG MICHIYASU YOSHIKUNI

FISH PHYSIOLOGY Edited by N A N C Y M. S H E R W O O D DEPARTMENT OF BIOLOGY UNIVERSITY OF VICTORIA VICTORIA, BRITISH COLUMBIA, CANADA

C H O Y L. H E W DEPARTMENT OF BIOCHEMISTRY RESEARCH INSTITUTE, HOSPITAL FOR SICK CHILDREN, TORONTO, AND DEPARTMENTS OF CLINICAL BIOCHEMISTRY AND BIOCHEMISTRY UNIVERSITY OF TORONTO TORONTO, ONTARIO, CANADA

Series Editors ANTHONY P. FARRELL DEPARTMENT OF BIOLOGICAL SCIENCES SIMON FRASER UNIVERSITY BURNABY, BRITISH COLUMBIA, CANADA

DAVID J. RANDALL DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOUVER, BRITISH COLUMBIA, CANADA

VOLUME XIII Molecular Endocrinology of Fish

ACADEMIC PRESS San Diego New York Boston

London

Sydney Tokyo Toronto

This book is printed on acid-free paper.

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Copyright 0 1994 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX Library of Congress Cataloging-in-Publication Data (Revised for vol. 13) Hoar, William Stewart, date. Fish physiology. (v. 13: Fish physiology series) Beginning with v. 8 editors vary. Includes bibliographies and indexes. Contents: v. 1. Excretion, ionic regulation, and metabolism.--[etc.]--v. 12. The cardiovascular system (2 v.)--v. 13. Molecular endocrinology of fish. 1. Fishes--Physiology--Collected works. I. Randall, David J., date. 11. Conte, Frank P., date. 111. Title. IV. Series. 597'.01 76-84233 QL 639. I .H6 ISBN 0-12-350405-8 (v. 5) ISBN 0-12-350437-6

PRINTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 Q W 9 8 7 6

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CONTENTS CONTRIBUTORS

xi

PREFACE

xv xvii

OF OTHERVOLUMES CONTENTS

I. Brain Hormones 1.

Molecular Evolution of Growth Hormone-Releasing Hormone and Gonadotropin-Releasing Hormone Nancy M . Sherwood, David B . Parker, John E . McRory, and David W. Lescheid I. Introduction

11. GHRH-PACAP

111. Gonadotropin-Releasing Hormone IV. Intertwining of Function in the GnRH and GHRH Families References

2.

3 4 29 50 51

Corticotropin-Releasing Factors Acting on the Fish Pituitary: Experimental and Molecular Analysis K . Lederis,]. N . Fryer, Y. Okawara, Chr. Schonrock, and D . Richter I. Introduction

11. ACTH-Releasing Peptides and Their Receptors

111. CRF, Its Protein Precursors, cDNAs, and Genes IV. Evolutionary Considerations for CRF-UI References V

68 69 78 90 94

CONTENTS

vi

Expression of the Vasotocin and Isotocin Gene Family in Fish Akihisa Urano, Kaoru Kubokawa, and Shuichi Hiraoka

3.

I . Introduction Genes, cDNAs, and Precursors Divergence of V T and IT Gene Expression VT and IT Gene Expression in Osmotic Adaptation Conclusion References

11. 111. IV. V.

102 108 117 122 127 128

11. Pituitary Hormones 4. Control of Teleost Gonadotropin Gene Expression Fei Xiong, Kunimasa Suzuki, and C h o y L. H e w I. Introduction 11. Duality of Teleost Gonadotropins 111. Genomic Organization of Teleost Gonadotropins IV. Control of Gonadotropin Gene Expression V. Conclusion References

5.

The Somatolactin Gene Masao Ono and Hiroshi Kawauchi

I. Somatolactin 11. Somatolactin Gene 111. Regulation of Somatolactin Gene Expression IV. Conclusion References

6.

135 136 140 142 153 1S4

159 164

168 173 174

Structure and Evolution of Fish Growth Hormone and Insulinlike Growth Factor Genes T h o m a s T . C h e n , A d a m Marsh, Mike Shamblott, K . - M . C h a n , Y.-L. Tang, Clara M . Cheng, and B.-Y. Yang I. Introduction

11. Conserved Domains of Fish Growth Hormones

179 181

CONTENTS

111. IV. V. VI. VII. VIII. IX.

Conserved Domains of Fish Prolactins and Somatolactins Genomic Organization of Fish GH, PRL, and SL Genes Ancestral Gene of the Fish Growth Hormone Gene Family A Functional Model of Fish Growth Hormone Gene Family Fish IGF I and IGF I1 mRNAs Age- and Tissue-Specific Levels of Five IGF mRNAs Concluding Remarks References

vii 185 189 191 194 197 200 202 203

111. Other Hormones Structure and Expression of Insulinlike Growth Factor Genes in Fish Shu ] i n Chan and Donald F . Steiner

7.

I. Introduction 11. IGF Activity in Fish 111. Cloning of Fish IGF cDNAs and Genes

IV. Expression and Regulation of IGF V. Summary and Perspective References

213 214 215 220 22 1 222

8. Molecular Aspects of Pancreatic Peptides Stephen J. Duguay and Thomas P . Mommsen I. Introduction 11. Insulin 111. Glucagon and Glucagonlike Peptide

IV. Somatostatin V. Pancreatic Polypeptide and Related Peptides References

9.

226 226 231 250 258 262

The Molecular Biology of the Corpuscles of Stannius and Regulation of Stanniocalcin Gene Expression Graham F . Wagner I. Introduction

11. A Brief History of Discovery

111. Molecular Cloning of Eel and Salmon Stanniocalcin IV. Structural Comparisons of Eel and Salmon Stanniocalcin

273 275 276 278

CONTENTS

viii V. Studies on Tissue-Specific Expression of the Stanniocalcin Gene VI. Localization of Stanniocalcin mRNA in CS Cells by in Situ Hybridization VII. Calcium Regulation of Stanniocalcin Cell Activity VIII. Conclusions References

IV. 10.

289 30 1 302

Comparative Aspects of Pituitary Development and Pit-l Function Sonali Majumdar and Harry P . Elsholtz I. Introduction

111. Differentiation of Adenohypophysial Cell Types IV. Transcription Factor Pit-1 V. Comparison of Pit-1 in Mammals and Teleost Fish: Studies on the PRL Target Gene VI. Conclusion References

309 310 311 313 320 324 325

Structure and Regulation of Genes for Estrogen Receptors Yves Le Drkan, Farzad Pakdel, and Yves Valotaire I. Introduction

11. The Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor

111. The Rainbow Trout Estogen Receptor Gene 1V. Conclusion References

12.

285

Hormone Regulation

11. Comparative Organization of the Pituitary Gland

11.

283

331 337 349 357 357

Prolactin Receptors Patrick Prunet and Benoit Auperin

I. Introduction 11. Prolactin Receptors in Mammalian Tissues 111. Prolactin Receptors in Fish References

367 369 372 385

CONTENTS

ix

Regulation of Oocyte Maturation in Fish Yoshitaka Nagahama, Michiyasu Yoshikuni, Masakane Yamashita, and Minoru Tanaka

13.

I. Introduction 11. 111. IV. V.

Phenomenology Structure of Follicles Gonadotropin: Primary Mediator of Oocyte Maturation Maturation-Inducing Hormone (MIH): Secondary Mediator of Oocyte Maturation VI. Maturation-Promoting Factor (MPF):Tertiary Mediator of Oocyte Maturation VII. Conclusions References

393 394 395 398 400 4 19 428 430

AUTHORINDEX

44 1

SYSTEMATIC INDEX

473

SUBJECTINDEX

479

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CONTRIBUTORS Numbers in purentheses indicute the puges on which the authors' contributions begin.

Benoit Auperin (367), Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cbdex, France K.-M. Chan' ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202

Shu Jin Chan (213), Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Thomas T. Chen ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Clara M. Cheng ( 1 79), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Stephen J. Duguay (225), Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Harry P. Elsholtz (309), Department of Clinical Biochemistry and Banting G Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada M5G 1L5

'

Present address: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong.

xi

xii

CONTRIBUTORS

J. N. Fryer (67),Department ofAnatomy and Neurobiology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 Choy L. Hew (135), Department of Biochemistry, Research Institute, Hospital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada MSG 1 L5 Shuichi Hiraoka (1O l ) , Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060, Japan, and Laboratory of Molecular Biology, Ocean Research l n stitute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan Hiroshi Kawauchi (159), Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, lwate 022-01, Japan Kaoru Kubokawa (1 O l ) , Laboratory of Molecular Biology, Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan Yves Le Drean (331), Laboratoire de Biologie Molkculaire, U R A , CNRS 256, Universitk de Rennes I , 35042 Rennes Cddex, France

K. Lederis (67),Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Alberta, Canada T2N 1N4 David W. Lescheid (3),Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2 Sonali Majumdar (309), Department of Clinical Biochemistry and Banting G Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada MSG 1L5 Adam Marsh (179), Center of Marine Biotechnology, University of Maryland Biotechnology lnstitute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 John E. McRory (3), Department of Biology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2 Thomas P. Mommsen (225),Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 3P6

CONTRIBUTORS

xiii

Yoshitaka Nagahama (393), Laboratory of Reproductive Biology, De-

partment of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444, Japan Y. Okawara (67),Department of Anatomy and Neurobiology, University of Ottawa, Ottawa, Ontario, Canada KIN 6N5 Masao Ono (159), Department of Molecular Biology, School of Medi-

cine, Kitasato University, Sagamihara, Kanagawa 228, Japan Farzad Pakdel(331), Laboratoire de Biologie Molkculaire, URA, CNRS 256, Universitk de Rennes I , 35042 Rennes Ckdex, France David B. Parker2 (3),Department of Biology, University of Victoria,

Victoria, British Columbia, Canada V8W 2Y2 Patrick Prunet (367), Laboratoire de Ph ysiologie des Poissons, INRA,

Campus de Beaulieu, 35042 Rennes Ckdex, France D. Richter (67), lnstitut f u r Zellbiochemie und Klinische Neurobiologie, Universitats-Krankenhaus Eppendorf, Universitat Hamburg, W-20246 Hamburg, Federal Republic of Germany Chr. Schonrock (67), Institut f u r Zellbiochemie und Klinische Neuro-

biologie, Universitats-Krankenhaus Eppendorf, Universitat Hamburg, W-20246 Hamburg, Federal Republic of Germany Mike Shamblott (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21 202 Nancy M . Sherwood (3),Department of Biology, University of Victo-

ria, Victoria, British Columbia, Canada V8W 2Y2 Donald F. Steiner (213), Howard Hughes Medical Institute and De-

partment of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637 Kunimasa Suzuki (135),Department of Biochemistry, Research Insti-

tute, Hospital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L5

’

Present address: The Clayton Foundation, Laboratory for Peptide Biology, The Salk Institute, La Jolla, California 92037.

xiv

CONTRIBUTORS

Minoru Tanaka (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444,Japan Y.-L. Tang3 (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Akihisa Urano (1Ol), Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060, Japan Yves Valotaire (331),Laboratoire de Biologie Moleculaire, U R A , CNRS 256, Universitk de Rennes I , 35042 Rennes Ckdex, France Graham F. Wagner (273),Department of Physiology, Faculty of Medicine, University of Western Ontario, London, Ontario, Canada N6A 5C1 Fei Xiong (135), Department of Biochemistry, Research Institute, Hos-

pital f o r Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada M5G 1L5 Masakane Yamashita (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okaxaki 444,Japan

B.-Y. Yang (179), Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland 21202 Michiyasu Yoshikuni (393), Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute f o r Basic Biology, Okazaki 444, Japan

Present address: American Red Cross, 15601Crabs Branch Way, Rockville, Maryland 20855.

PREFACE

In this volume our aim is to highlight some of the exciting research that has emerged on molecular biology of fish hormones, their receptors, and regulation. Like studies in biomedical sciences, comparative vertebrate studies have found that molecular biological techniques are a powerful and indispensable tool for advancing our knowledge of gene structure, evolution, and regulation of fish hormones. Comparative studies of the structure of these hormones, at both the protein and the DNA level, provide important clues about the structurefunction relationship of the hormones, as well as their evolutionary history and mechanisms of action. Similarly, elucidation of regulatory DNA sequences is a prerequisite for studying tissue and celltype specificity, temporal expression ofthese hormones, and regulation by various factors. Largest in number and most diverse of the vertebrates, fish have an immense variety of life cycles, developmental stages, body structures, and physiological mechanisms. Clearly, fish offer a natural laboratory for elucidating the role of hormones in adaptation to a variety of environments. As shown in this book, knowledge of the structural basis of fish hormones has made possible major advances in the understanding of fish neuropeptides (Chapters 1-3); pituitary hormones, including the novel somatolactin (Chapters 4-6); and hormones related to growth, metabolism, and ion regulation (Chapters 7-9). Pioneering work on regulation by hormones and of hormones is presented in Chapters 4 and 10-13. Important advances are expected in this area in the next five to ten years. Finally, recent data on the estrogen and prolactin receptors are presented (Chapters 11 and 12).Here we see the intricate balance that exists between hormones and receptors, and the physiological implications of their relationship. xv

xvi

PREFACE

Finally, we thank Dave Randall and Tony Farrell for the invitation to prepare this volume and for their kindly guidance. We also acknowledge with gratitude the help, suggestions, and patience of Dr. Charles Crumly and Heidi Inman of Academic Press. NANCY M . SHERWOOD CHOY L. HEW

CONTENTS OF OTHER VOLUMES Volume I The Body Compartments and the Distribution of Electrolytes W. N . Holmes and Edward M . Donaldson The Kidney Cleveland P . Hickman, Jr., and Benjamin F . Trump Salt Secretion Frank P . Conte The Effects of Salinity on the Eggs and Larvae of Teleosts F . G. T . Holliday Formation of Excretory Products Roy P . Forster and Leon Goldstein Intermediary Metabolism in Fishes P . W. Hochachka Nutrition, Digestion, and Energy Utilization Arthur M . Phillips, Jr. AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECT

INDEX

Volume I1

The Pituitary Gland: Anatomy and Histophysiology J . N . Ball and Bridget 1. Baker The Neurohypophysis A . M . Perks Prolactin (Fish Prolactin or Paralactin) and Growth Hormone J . N . Ball Thyroid Function and Its Control in Fishes Aubrey Gorbman xvii

xviii

CONTENTS OF OTHER VOLUMES

The Endocrine Pancreas August Epple The Adrenocortical Steroids, Adrenocorticotropin and the Corpuscles of Stannius I. ChesterJones, D. K . 0. Chan, I. W. Henderson, a n d ] . N . Ball The Ultimobranchial Glands and Calcium Regulation D. Harold C o p p Urophysis and Caudal Neurosecretory System Howard A. Bern AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume I11

Reproduction William S. Hoar Hormones and Reproductive Behavior in Fishes N . R . Liley Sex Differentiation

Toki-o Yamamoto Development: Eggs and Larvae 1.H . S. Blaxter Fish Cell and Tissue Culture Ken Wolfand M . C. Quimby Chromatophores and Pigments Ryozo Fujii Bioluminescence J . A. C . Nicol Poisons and Venoms Findlay E . Russell AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume IV Anatomy and Physiology of the Central Nervous System Jerald J. Berstein

CONTENTS OF OTHER VOLUMES

T h e Pineal Organ James Clarke Fenwick Autonomic Nervous System Graeme Campbell T h e Circulatory System D . J . Randall Acid-Base Balance C . Albers Properties of Fish Hemoglobins Austen Riggs

Gas Exchange in Fish D . J . Randall T h e Regulation of Breathing G . Shelton Air Breathing in Fishes Kjell Johansen The Swim Bladder as a Hydrostatic Organ Johan B . Steen Hydrostatic Pressure Malcolm S. Gordon Immunology of Fish John E. Cushing AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume V

Vision: Visual Pigments F . W. Munz Vision: Electrophysiology of the Retina T . Tomita Vision: The Experimental Analysis of Visual Behavior David lngle Chemoreception Toshiaki J . Hara

xix

xx

CONTENTS OF OTHER VOLUMES

Temperature Receptors R . W. Murray Sound Production and Detection William N . Tavolga The Labyrinth 0. Lowenstein The Lateral Line Organ Mechanoreceptors Ake Flock The Mauthner Cell I . Diamond Electric Organs M . V . L. Bennett Electroreception M . V. L. Bennett AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume VI The Effect of Environmental Factors on the Physiology of Fish F. E . J . Fry Biochemical Adaptation to the Environment P . W. Hochachka and G . N . Somero Freezing Resistance in Fishes Arthur L. DeVries Learning and Memory Henry Gleitman and Paul Rozin The Ethological Analysis of Fish Behavior Gerard P . Baerends Biological Rhythms Horst 0. Schwassmann Orientation and Fish Migration Arthur D . Hasler Special Techniques D . J . Randall and W. S. Hoar AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

CONTENTS OF OTHER VOLUMES

Volume VII Form, Function, and Locomotory Habits in Fish C. C. Lindsey Swimming Capacity F . W . H . Beamish Hydrodynamics: Nonscombroid Fish Paul W . Webb Locomotion by Scombrid Fishes: Hydromechanics, Morphology, and Behavior John J . Magnuson Body Temperature Relations of Tunas, Especially Skipjack E . Don Stevens and William H . Neil1 Locomotor Muscle Quentin Bone The Respiratory and Circulatory Systems during Exercise David R . Jones and David J. Randall Metabolism in Fish during Exercise William R . Driedzic and P . W. Hochachka

AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume VIII Nutrition C. B . Cowey and J . R . Sargent Feeding Strategy Kim D . Hyatt The Brain and Feeding Behavior Richard E . Peter Digestion Ragner Fange and David Grove Metabolism and Energy Conversion during Early Development Charles Terner Physiological Energetics J . R . Brett and T . D . D. Groves

xxi

xxii

CONTENTS OF OTHER VOLUMES

Cytogenetics J . R. Gold Population Genetics Fred W. Allendorf and Fred M . Utter Hormonal Enhancement of Growth Edward M . Donaldson, U Y H . M . Fagerlund, David A. Higgs, and J . R. McBride Environment Factors and Growth 3. R. Brett Growth Rates and Models W. E . Ricker AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume IXA Reproduction in Cyclostome Fishes and Its Regulation Aubrey Gorbman Reproduction in Cartilaginous Fishes (Chondrichthyes) J . M . Dodd The Brain and Neurohormones in Teleost Reproduction Richard E . Peter The Cellular Origin of Pituitary Gonadotropins in Teleosts P. G. W. J . v a n Oordt and J. Peute Teleost Gonadotropins: Isolation, Biochemistry, and Function David R. l d l e r and T . B u n Ng The Functional Morphology of Teleost Gonads Yoshitaka Nagahnma The Gonadal Steroids A. Fostier, B.Jalabert, R. Billard, B. Breton, and Y . Zohar

Yolk Formation and Differentiation in Teleost Fishes T . B u n Ng and David R . Idler An Introduction to Gonadotropin Receptor Studies in Fish G l e n V a n D e r Kraak AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX

CONTENTS OF OTHER VOLUMES

xxiii

Volume IXB

Hormones, Pheromones, and Reproductive Behavior in Fish N . R . Liley and N. E . Stacey Environmental Influences on Gonadal Activity in Fish T. J . L a m Hormonal Control of Oocyte Final Maturation and Ovulation in Fishes Fredrick W. Goetz Sex Control and Sex Reversal in Fish under Natural Conditions S. T . H . Chan and W. S . B. Yeung Hormonal Sex Control and Its Application to Fish Culture George A. Hunter and Edward M . Donaldson Fish Gamete Preservation and Spermatozoan Physiology Joachim Stoss Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish Edward M . Donaldson and George A . Hunter Chromosome Set Manipulation and Sex Control in Fish Gary H. Thorgaard AUTHOR

INDEX-SYSTEMATIC INDEX-SUBJECT INDEX

Volume XA

General Anatomy of the Gills George Hughes Gill Internal Morphology Pierre Laurent Innervation and Pharmacology of the Gills Stefan Nilsson Model Analysis of Gas Transfer in Fish Gills Johannes Piiper and Peter Scheid Oxygen and Carbon Dioxide Transfer across Fish Gills David Randall and Charles Daxboeck Acid-Base Regulation in Fishes Norbert Heisler

xxiv

CONTENTS OF OTHER VOLUMES

Physicochemical Parameters for Use in Fish Respiratory Physiology Robert 6 . Boutilier, Thomas A. Heming, and George K . Iwama AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECTINDEX

Volume XB Water and Nonelectrolyte Permeation Jacques Isaia Branchial Ion Movements in Teleosts: The Role of Respiratory and Chloride Cells P. Payan, J. P. Girard, and N . Mayer-Gostan Ion Transport and Gill ATPases Guy de Renzis and Michel Bornancin Transepithelial Potentials in Fish Gills W. T . W. Potts The Chloride Cell: The Active Transport of Chloride and the Paracellular Pathways J . A. Zadunaisky Hormonal Control of Water Movement across the Gills J. C. Rankin and Liana Bolis Metabolism of the Fish Gill Thomas P. Momnzsen The Roles of Gill Permeability and Transport Mechanisms in Euryhalinity David H . Evans The Pseudobranch: Morphology and Function Pierre Laurent and Suzanne Dunel-Erh Perfusion Methods for the Study of Gill Physiology S . F . Perry, P. S.Davie, C. Daxboeck, A . G . Ellis, and D. G . Smith AUTHOR

INDEX-SYSTEMATIC

INDEX-SUBJECT

INDEX

Volume XIA Pattern and Variety in Development J . H . S . Blaxter Respiratory Gas Exchange, Aerobic Metabolism, and Effects of Hypoxia during Early Life Peter J . Rombough

CONTENTS OF OTHER VOLUMES

xxv

Osmotic and Ionic Regulation in Teleost Eggs and Larvae D. F . Alderdice Sublethal Effects of Pollutants on Fish Eggs and Larvae H. von Westernhagen Vitellogenesis and Oocyte Assembly Thomas P. Mommsen and Patrick J . Walsh Yolk Absorption in Embryonic and Larval Fishes Thomas A . Heming and Randal K . Buddington Mechanisms of Hatching in Fish Kenjiro Yamagami AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX Volume XIB The Maternal-Embryonic Relationship in Viviparous Fishes John P. Worums, Bryon D. Grove, and Julian Lombardi First Metamorphosis John H . Youson Factors Controlling Meristic Variation C . C . Lindsey The Physiology of Smolting Salmonids W . S . Hoar Ontogeny of Behavior and Concurrent Developmental Changes in Sensory Systems in Teleost Fishes David L. G . Noakes and Jean-Guy J . Godin AUTHORINDEX-SYSTEMATIC INDEX-SUBJECTINDEX Volume XIIA

The Heart Anthony P. Farrell and David R. Jones The Arterial System P. G . Bushnell, David R. Jones, and Anthony P. Farrell The Venous System Geoffrey H . Satchel1 The Secondary Vascular System J . F. Steffensen and J . P. Lomholt

xxvi

CONTENTS OF OTHER VOLUMES

Cardiac Energy Metabolism William R. Driedzic Excitation-Contraction Coupling in the Teleost Heart Glen F . Tibbits, Christopher D . Moyes, and Leif Hove-Madsen AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume XIIB

Fish Blood Cells Ragnar Funge Chemical Properties of the Blood D . G. McDonald and C. L. Milligan Blood and Extracellular Fluid Volume Regulation: Role of the Renin-Angiotensin System, Kallikrein-Kinin System, and Atrial Natriuretic Peptides Kenneth R. Olson Catecholamines D . J. Randall and S. F. Perry Cardiovascular Control by Purines, 5-Hydroxytryptamine, and Neuropeptides Stefan Nilsson and Susanne Holmgren Nervous Control of the Heart and Cardiorespiratory Interactions E . W. Taylor Afferent Inputs Associated with Cardioventilatory Control in Fish Mark L. Burleson, Neal J . Smatresk, and William K . Milsom AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX

BRAIN HORMONES

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1 MOLECULAR EVOLUTION OF GROWTH HORMONE-RELEASING HORMONE AND GONADOTROPIN-RELEASING HORMONE Nancy M . Sherwood, David B . Parker,]ohn E . McRory, and David W. Lescheid Department of Biology, University of Victoria Victoria, British Columbia, Canada

I. Introduction 11. GHRH-PACAP

A. Identification B. Phylogenetic Studies C. Structural Analysis D. Functional Roles of GHRH and PACAP E. Evolution of GHRH and PACAP 111. Gonadotropin-Releasing Hormone A. Identification B. Phylogenetic Studies C. Structural Analysis D. Questions Regarding Localization and Function of GnRH E. Evolution of GnRH and GHRH Families IV. Intertwining of Function in the GnRH and GHRH Families References

I. INTRODUCTION This chapter is concerneG with two of the most funamental processes in life: growth and reproduction. Single cells grow and divide whether they are isolated or part of a multicellular organism, but the emergence of the nervous system in multicellular animals provided a new and overriding control on growth and reproduction. The mechanism by which the nervous system coordinated these slow processes of growth and reproduction was by the secretion of neuropeptides, and a number of such neuropeptides have been identified in inverte3 FISH PHYSIOLOGY, VOL. XI11

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction Ln any form reserved.

4

NANCY M. SHERWOOD ET AL.

brates from coelenterates to protochordates. In vertebrates the orderly sequence of maturational changes that lead to growth and reproduction is also influenced by neuropeptides that act as releasing factors, neuromodulators, and even local hormones in nonneural tissue. Hence, vertebrates and invertebrates share the use of neuropeptides whether the action is direct or indirect for altering growth and reproduction. Fish are a pivotal group in which to consider neuropeptides related to growth and reproduction. Two distinct families of peptides in fish have been associated with the neural control of reproduction or growth, gonadotropin-releasing hormone (GnRH) and growth hormonereleasing hormone (GHRH). These hormones have specific actions in releasing gonadotropins or growth hormone from the pituitary, although some crossover of function has been reported. The primary structures of fish GnRHs and GHRHs have been only recently identified, but they have clear structural similarities to those of other vertebrates so that shared ancestral hormones can be postulated. Fish are also intermediates in the deuterostome line of evolution that is thought to have led to mammals. The relationship of fish GnRH and GHRH to peptides in invertebrates is not yet clear, but it is assumed that both have links to invertebrate peptides. In any event, fish provide a varied group for consideration of the origin, function, and evolution of the neuropeptides of GnRH, GHRH, and a newly discovered peptide, pituitary adenylate cyclase activating polypeptide (PACAP), which is related to GHRH in fish and found in the same precursor. Mammals have two separate genes, one of which encodes only the classic GHRH peptide, whereas the other gene encodes a precursor with both PACAP and a PACAP-related peptide (PRP), the latter having sequence similarity to GHRH. In contrast, fish (salmon, catfish, and sturgeon) have a precursor that contains a GHRH-like peptide in addition to the PACAP hormone, but to date a second gene that encodes only GHRH has not been found. The question is whether the niammalian GHRH gene arose from a gene duplication after the bony fish separated from the tetrapod line.

11. GHRH-PACAP A. Identification 1. GHRH IN TETRAPODS In 1959 Seymour Reichlin provided one of the first indications that growth hormone is under the control of the brain. He showed that

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

5

rats with hypothalamic lesions grew less well than control animals (Reichlin, 1960a,b). By 1964 it was known that rat hypothalamic extracts contained a substance that specifically caused the release of growth hormone from rat pituitary cells in vitro (Deuben and Meites, 1964). Several different growth hormone-releasing substances were isolated and partially purified (Dhariwal et al., 1965; Frohman et al., 1971; Schally et al., 1971; Stachura et al., 1972), but none proved to be authentic GHRH. The isolation and characterization of a growth hormone-releasing factor proved elusive until 1982. At that time two independent groups, working with separate pancreatic islet cell tumors, isolated and characterized GH-releasing peptides. Vale and co-workers found a 40-aminoacid peptide with a free carboxy terminus within their tumor extract (Rivier et al., 1982) (Table I). Guillemin and colleagues also found a CHRH,-4,OH form in the same pancreatic tumor (Esch et al., 1982). In addition, the Guillemin group isolated a 44-amino-acid, amidated GHRH peptide, as well as 40-amino-acid and 37-amino-acid peptides with free carboxy terminals in a different tumor (Guillemin et al., 1982). By 1984 the hypothalamic form of GHRH was shown to b e identical to the pancreatic hormone (Ling et al., 1 9 8 4 ~ )To . date, seven mammalian GHRH peptides and one nonmammalian GHRH-like peptide have been isolated and sequenced (Fig. 1A). Further information on GHRH has been obtained from molecular studies, but is mainly limited to mammals. The sequence of GHRH cDNA is known for human pancreatic tumor (Gubler et al., 1983; Mayo et al., 1983),rat hypothalamus (Mayo et al., 1985b), rat placenta (Gonzhles-Crespo and Boronat, 1991), mouse hypothalamus (M. A. Frohman et al., 1989), and mouse placenta (Suhr et al., 1989). Only the human (Mayo et al., 1985a)and rat (Mayo et al., 198513) genes are known (Table I). For birds, reptiles, and amphibians, indirect evidence suggests that a GHRH-like molecule also exists and has similar functions compared with mammalian GHRH peptides. For example, synthetic hGHRH,-,,NH2 stimulated the release of GH either in vivo or in vitro from the pituitary of the chicken (Perez et al., 1987), dwarf chicken (Harvey et al., 1984), turtle (Denver and Licht, 1991), and frog (Malagon et al., 1991). 2. GHRH IN FISH Although the structure of GHRH for a nonmammalian species was not published until 1992, there was substantial indirect evidence that fish contained a GHRH-like substance with similar function, immuno-

Table I Identification of PRP/GHRH/PACAP Sequcences Source of sequence

Q,

Peptide

Species

Tissue

Form

PRP

Human

Testes

1-29, 1-48

Peptide

cDNA

X X

1-29, 1-48 Sheep Rat

Brain Brain

Human

Tumor"

GHRH

Tumor" Tumora Brain Tumor" Tumor" Rat

Brain Brain

1-29, 1-48 1-29, 1-48 1-44 NH2 1-40 OH 1-37 OH 1-40 OH 1-40 OH 1-44 NH2 1-44 NH2 1-44 NH2 1-44 NH2 1-43 OH 1-43 OH 1-43 OH

Gene

X X

References Ohkubo et ul. (1992) Hosoya et al. (1992) Ohkubo et u1. (1992) Ohkubo et al. (1992) Ogi et al. (1990)

X

Guillemin et al. (1982)

X X X

Rivier et al. (1982) Esch et ul. (1982) Ling et al. ( 1 9 8 4 ~ ) Gubler et al. (1983) Mayo et al. (1983) Mayo et nl. (1985a) Spiess et al. (1983) Mayo et al. (198513) Mayo et al. (1985b)

X X X

X X X

Mouse

PACAP

cow Pig Goat Sheep Carp Salmon Human

Sheep

Rat Salmon Frog ~

Pancreatic tumor.

Placenta

1-43 OH

X

Brain Placenta Brain Brain Brain Brain Brain Brain

1-42 OH 1-42 OH 1-44 NHZ 1-44 NH, 1-44 NHZ 1-44 NH, 1-45 OH 1-45 OH

X X

Testes Brain Brain Brain Brain Brain Brain Brain Brain Brain

1-38 1-38 1-38 1-38 1-38 1-27 1-38 1-38 1-38 1-38

Gonzales-Crespo and Boronat (1991) M. A. Frohman et d . (1989) Suhr et u1. (1989) Esch e t a / . (1983) Bohlen et a / . (1983) Brazeau et al. (1984) Brazeau et d . (1984) Vaughan et u/. (1992) Parker et a / . (1993)

X X X X X X X

X X X X

Kimura et a / . (1990) Kimura et u/. (1990) Ohkubo et al. (1992) Hosoya et al. (1992) Miyata et d . (1989) Miyata et a / . (1990) Kimura et d . (1990) Ogi et ul. (1990) Parker et a / . (1993) Chartrel et (I/. (1991)

8

NANCY hl. SHERWOOD ET A L .

A SALMON

25 30 35 40 45 I I I I I EADGMFNKAYRKALGQLSARKYLHSLMAKRVGGGSTMEDDTEPLS-OH

CARP

H

STURGEON H

5

10

15

20

I

I

I

I

MI

N

-OH

S

-OH

EEEEN ENS

-OH

T V

I

V

V S

CATFISH

H

MOUSE

HV A 1 TTN

RAT

H

A 1 TSS

RI

SHEEP

Y

A 1 TNS

I

L QDI NRQQ ERNQEQGAKVR --NHz

GOAT

Y

A 1 TNS

V

L QDI NRQQ ERNQEQGAKVR --N&

cow

Y

A 1 TNS

V

L QDI NRQQ ERNQEQGAKVR --N&

PIG

Y

A 1 TNS

V

L QDI SRQQ ERNQEQGARVR --NH2

HUMAN

Y

A 1 TNS

V

L QDI SRQQ ESNQERGARAR --N&

LLDR L D I V

B SALMON

S

L S

Y

IQDI NKQ- ERIQEQ--RAR

Y

L

5

10

15

20

25

30

I

I

I

I

I

I

D

G

I

F

T

-OH

E I NRQQ ERNQEQ--RSRFN-OH

D

S

Y

S

35 I

R

Y

FROG

K

IK

HUMAN

K V K

CATFISH

H

T V

R

R

~

F

Fig. 1. Comparison of vertebrate GHRH and PACAP peptides. (A) Salmon GHRH amino acids are compared with those oftwo other fish and seven mammals. (B) Comparison of amino acids for the known forms of PACAP. Residues are coded by a single letter and only residues that differ from the salmon sequence are shown. For maximal alignment, a dash is inserted to shift the sequence.

reactivity, and chromatographic behavior compared with mammalian GHRH peptides. For example, in 1966 Olivereau and Ball showed that severing the connections between the hypothalamus and pituitary resulted in poor growth rates and a significant decrease in the number of growth hormone cells in the molly Poecilia formosa. This suggested

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

9

that the hypothalamus in this species may exert a dominant stimulatory influence over growth hormone secretion (Donaldson et aZ., 1979). In 1984 Peter and associates demonstrated that intraperitoneal injections of hGHRH,_,,NH, stimulated GH release from sexually regressed goldfish, Carussius auratus (Peter et al., 1984), although hGHRH,-,,NH, did not release GH fi-om goldfish pituitaries in vitro (Marchant and Peter, 1989). Immunoreactive GHRH neurons were detected in a number of fish species and irGHRH-like molecules were partially characterized with HPLC (Table I). By 1990 a carp GHRH-like molecule was available (even though its sequence had not yet been published) and it was shown that the carp GHRH,-,,OH and carp GHRH1-2yNH2forms not only caused GH release from cultured rainbow trout (Oncorhyncus mykiss) pituitary cells (Luo et al., 1990; Luo and McKeown, l99la), but the 45-aminoacid form stimulated GH release from goldfish (C. auratus) pituitaries both in citro and in cico (Vaughan et al., 1992). However, a number of hormones, such as thyroid hormones, glucocorticoids (Donaldson et al., 1979; Nishioka et al., 1985; Luo and McKeown, 1991b), and insulinlike growth factor I (McCormick et aZ., 1992), can influence the release of GH in some teleosts under certain conditions, making it difficult to determine the true GH releaser in fish. cDNAs have been ioslated for a GHRH-like peptide in three fish: sockeye salmon (Oncorhynchus nerka) (Parker et al., 1993),Thai catfish (Clarias macrocephaZus) ( J . E. McRory personal communication), and white sturgeon (Ascipenser transmontanus) (D. W. Lescheid personal communication). The physiological studies of these molecules are in progress.

3. PACAP An unexpected discovery in mammals was the presence of an mRNA coding for a precursor with two peptides: one peptide had sequence similarity with GHRH and the other was pituitary adenylate cyclase activating polypeptide (PACAP). The mammalian GHHH-like peptide (named PACAP-related peptide, PRP) has not been shown to release GH, whereas PACAP released GH and three other pituitary hormones as well (Hart et aZ., 1992). PACAP was originally isolated based on its ability to increase cyclic AMP (CAMP)accumulation in cultured rat pituitary cells. This approach was unique because the other hypothalamic neurohormones had been isolated using assays for specific physiological functions, like the release of growth hormone or gonadotropins. In 1989 Miyata and co-workers isolated and characterized the 38-amino-acid form of PACAP from sheep hypothalami (Miyata et al., 1989)(Table I and Fig. 1B). A 27-amino-acid form, identical with the N-terminal region of

10

NANCY M. SHEHWOOD E?' AL.

PACAP1-38, was isolated from the same ovine hypothalamic extracts the following year (Miyata et al., 1990). Sheep (Kimura et al., 1990), rat (Ogi et al., 1990), and human (Kimura et al., 1990; Ohkubo et al., 1992) PACAP cDNAs have been characterized. By 1992 the human PACAP gene had been isolated (Hosoya et al., 1992), but the essential biological function of PACAP is still unknown. In other tetrapods, a PACAP1-38peptide has been isolated from the European green frog (Chartrel et al., 1991). In fish, PACAP has been isolated from three species. Our laboratory has cloned PACAP cDNAs for sockeye salmon (0.nerka) (Parker et al., 1993), Thai catfish (C. macrocephalus) ( J . E. McRory, personal communication), and white sturgeon (A. transmontanus) (I>. W. Lescheid, personal communication). We have recently isolated the GHKH/ PACAP gene from sockeye salmon (0.nerka) (D. B. Parker and N. M. Sherwood, personal communication). B. Phylogenetic Studies

1.

IMMUNOCYTOCHEMISTKY OF

GHRH

IN

TETRAPODS

Immunoreactive GHRH perikarya have been detected in the arcuate and ventromedial nuclei in humans, monkeys (Bloch et al., 1983, 1984;Bresson et al., 1984),and rats (Ishikawaetal., 1986).The paraventricular nucleus in guinea pigs also contains GHRH (Beauvillain et al., 1987).The GHRH nerve fibers that originate in these nuclei project to the median eminence and terminate on the portal vascular system. In addition, immunoreactive GHRH is present in the duodenum (Bruhn et n l . , 1985), testis (Berry and Pescovitz, 1988; Moretti et al., lYgOb), ovary (Moretti et al., l99Ob), and placenta (Baird et al., 1985; Meigan et al., 1988), suggesting alternative functions for the peptide. In the only amphibian species (Rana temporaria) examined for immunoreactive GHRH neurons, cells were detected in the magnocellular portion of the preoptic nuclei and gave rise to nerve fibers running in both the external and internal layers of the median eminence (Marivoet et ul., 1988).

2. IMMUNOCYTOCHEMISTRY OF GHRH IN FISH In fish, immunocytochemistry was used to detect a GHRH-like niolecule in the brain of the cod (Gadus morhua) (Pan et al., 1985a,b), sea bass (Dicentrarchus Zabrax) (Marivoet et al., 1988),rainbow trout (0.mykiss) (Luo and McKeown, 1989; Olivereau et al., 1990), and eight other species of teleost fish, including eels (Anguilla anguilla, A. rostrata), goldfish (C. auratus), carp (Cyprinus curpio), chinook

1.

MOLECULAR EVOLUTION OF

GHKH

AND

GnRH

11

salmon (0.tshawytscha),trout ( S a l m o f a r i o ) ,mullet (Mugil rumada), and sculpin (Myoxocephalus octodecimspinosus) (Olivereau et al., 1990). In cod there was cross-reactivity in neurons of the preoptic area and lateral tuberal nucleus and in fibers of the pars nervosa of the pituitary with an antiserum made against the GHRH,-,,OH molecule, but only cells in the rostral pars distalis stained with a GHRH,-,,NH, antiserum (Pan et al., 1985a). In most teleost studies, however, human GHRH1-,,NH, antiserum stains immunoreactive perikarya in the preoptic nuclei and to a lesser extent in the lateral tuberal nucleus. In the eel, carp, goldfish, and salmon, the irGHRH fibers did not enter the rostral pars distalis and only a few fibers were seen in close association with the somatotrophs (Olivereau e t al., 1990).Instead, the immunoreactive fibers from these species and a variable number of fibers from mullet and sculpin terminated in the intermediate or neurointermediate lobe of the pituitary.

3. IMMUNOCYTOCHEMISTRY OF PACAP Immunocytochemical methods were used originally to show the presence of immunoreactive PACAP in the hypothalamus and septum of sheep (Koves et al., 1990). A dense network of PACAP fibers was seen in both external and internal zones of the median eminence, pituitary stalk, and in close contact with the hypophysial capillaries. PACAP immunoreactivity was not limited to the hypothalamus, but was seen also in the posterior pituitary. Within spider monkey and human brains, a similar distribution of PACAP-immunoreactive elements existed in the supraoptic and paraventricular nuclei (Vigh e t ul., 1991). In rats, PACAP perikarya in the hypothalamus were located in the supraoptic, paraventricular, anterior commissural, periventricular, and perifornical nuclei (Koves et al., 1991). Extrahypothalamic regions that have immunoreactivity to PACAP include the central thalamic nuclei, amygdaloid complex, bed nucleus of stria terminalis, septum, hippocampus, cingulate cortex, and entorhinal cortex (Koves et al., 1991). PACAP-immunoreactive fibers outside of the brain were detected in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep, and squirrel monkeys (Uddman et al., 1991). Also, immunoreactive PACAP was found in rats in the following tissues, which are listed from highest to lowest concentration: testis, posterior pituitary, adrenal gland, duodenum, stomach, jejunum, ileum, anterior pituitary, colon, ovary, epididymis, and lung. Other organs also had immunoreactive PACAP, but the concentration was less than 1ng/g wet tissue (Arimura et al., 1991). PACAP localization in fish has not been reported.

12

NAKCY M. SIIEKWOOD ET AL.

4. CHKOMATOGKAPHY OF GHRH

IN

FISH

Liquid chromatography has been utilized to isolate and characterize irGHRH-like molecules from a few fish species. Pan et uZ. (1985b) used exclusion chromatography of extracted codfish G. nzorhuu brains to isolate three fractions that reacted with a hGHRH antiserum. Codfish brains were also analyzed with high performance liquid chromatography (HPLC) by Ackland et al. (1989) to partially purify an irGHRH molecule that had a similar HPLC retention time in comparison to hGHRH,_,,NH,. Chum salmon (Oncorhynchus ketu) and coho salmon (0.kisutch) were shown to have an irGHRH-like molecule that could be detected with a hGHRH,-,,NH, antiserum (Parker and Sherwood, 1990). It was not until Vaughan et ul. (1992) extracted 16,000 carp (C. curpio) hypothalami and used HPLC methods that a nonmammalian GHRH-like peptide was identified and sequenced for the first time. PACAP from fish has not been studied using chromatography.

C . Structural Analysis 1. P w r I m

SEQUENCES

GHRH peptides have been characterized in human, rat, mouse, cow, sheep, pig, goat, and carp (C. carpio) (Fig. 1A). Most of the peptides are 44 amino acids long, with tyrosine as the initial aniinoterminal residue and an aniidated carboxy terminus. The exceptions to this rule are rat, mouse, and carp GHRH, which are 43, 42, and 45 amino acids long, respectively, with histidine at the N terminus and free acid at the C terminus. Of the mammalian GHRHs, pig GHRH is closest to human GHRH with only three amino acid differences, whereas mouse GHRH is the most distinct with 18 amino acid changes. The carp GHRH peptide is only 41% (18/44) identical to human GHRH,-,,NH,. PACAP peptides have been iodated only from ovine hypothalamus and frog brain. In the ovine hypothalamus, the two forms of PACAP are identical in the first 27 amino acids, but one form is extended to 38 aniino acids. Both PACAP1-38and PACAP,-27 are encoded by the same exon in humans. It is not yet known whether PACAP,_2-;is a posttranslational derivative of PACAP,-38 or whether it is directly cleaved from a common precursor (Hosoya et al., 1992; Okazaki et uZ., 1992) (Fig. 2). Frog PACAP is 38 amino acids long and has only 1 amino acid substitution compared to ovine PACAP.

Salmon GHRH/PACAP Precursor 22

1

173 R

R K R K K R

KR

RRKKKGKR

GRR

1 1 1 1 I 111 l l l l l

Sign1

Human GHRH Precursor 108

20

1

RR Signal

RK

RK

R RRRGR

I I I I IIII

Fig. 2. Comparison of the salmon and human proteins for GHRH, PRP, and PACAP precursors. The number ofamino acids for each precursor is at the upper right. Possible cleavage sites are indicated by single lines for one amino acid or black bands for two amino acids. The single-letter code is used for amino acids: R, arginine, K, lysine, and G for glycine, the amino acid that donates the final amide group. Possible mature peptides are shown below each precursor. For the peptides shown, the only ones that have been isolated from normal tissue are PACAP,* and PACAP,; from sheep; GHRH,, from rat; GHRH, from human, cow, pig, goat, and sheep; and GHRH45from carp.

14

NANCY M. SHERWOOD ET AL.

2. DNA SEQUENCES

The cDNAs for GHRH and PRP/PACAP have been isolated from a number of mammals (Table I). Although the mature forms of the PRP peptide have not been isolated, the peptide structure can be deduced from the cDNA sequence. In the cDNA, PRP is just upstream of' the region that encodes PACAPl-38 in the human, rat, and sheep (Figs. 2 and 3). Human PRP is only 48% similar to human GHRH (Ohkubo et al., 1992). Like the mammalian PRP/PACAP cDNAs, the fish GHRH-like/ PACAP cDNAs for sockeye salmon (0.nerka), Thai catfish (C. macrocephalus),and white sturgeon (A. transmontanus)contain four distinct consecutive regions: a signal peptide, a cryptic peptide, a GHRH-like region, and a PACAP region (Fig. 3). These precursors are similar to the glucagon (Heinrich et al., 1984) and the VIP (Bodner et al., 1985; Itoh et al., 1983) precursors that also contain consecutive coding regions for two different mature neuropeptides. Of all the fish GHRH-like regions sequenced to date, sturgeon (A. transmontanus) GHRH is closest (45%) to human GHRH1-,,NH2; sockeye salmon (0.nerka) and carp (C. carpio) GHRH are a close second (41%); and Thai catfish ( C . macrocephalus) GHKH has the least (32%) sequence identity. A comparison between human PRP and fish GHRH-like peptides shows that the similarity ranges from 62% for sturgeon to 55% for carp. 3. GENESTRUCTURE AND COPYNUMBER Several genes have been isolated and sequenced (Table I ) , including a human and rat GHRH gene, a human PRPIPACAP gene, and a salmon (0.nerka) GHRH-like/PACAP gene. In humans, GHRH is a single gene, 10 kilobases long, and separated into five exons. Splicing of the human GHRH transcript can occur to yield either a 107- or 108amino-acid preproGHRH (Gubler et al., 1983; Mayo et al., 1985a).The rat GHRH transcript encodes a 104-amino-acid preproGHRH (Mayo et al., 1985b). PRP/PACAP in humans is encoded on a single gene, contains five exons (Fig. 4), and is transcribed into a preprohormone of 176 amino acids. The salmon GHRH-like/PACAP gene also has five exons and has at least two copies or, alternatively, allelic polymorphism (Parker et al., 1993). The salmon gene can be transcribed into an mRNA that encodes a 173-amino-acid preprohormone. The human and rat GHRH genes have conventional TATA and CCAAT boxes, required for the accurate initiation of transcription in most eukaryotic promoters, whereas the human PRP/PACAP and the salmon GHRH-like PACAP genes do not contain a TATA or CCAAT

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

15

HUMAN PRP/PACAP

OVlNE PRP/PACAP

RAT PRP/PACAP

SALMON GHRH-ILe/PACAP

STURGEON GHRH-like/PACAP

CATFISH GHRH-k?/PACAP

Fig. 3. Comparison of mammalian PHP/PACAP precursors and fish GI
sequence. However, the 5’ flanking region of the human PRP/PACAP gene does have several conventional consensus sequences for binding transcription factors; these include CRE (CAMP response element), TRE (thyroid hormone response element) or AP-1 (TPA response element), and GHF-1 or Pit-1 (GH factor response element) regulatory sites (Hosoya et al., 1992). Other genes like the one encoding IGF1 also lack TATA and CCAAT regions with the result that several transcription start sites are used (LeRoith and Roberts, 1993).

16

NANCY Sl. SHEKWOOD E?' AL.

A

I

N

Ill

U

V

.GwMkU PACAP 3.45 kb

I

II

N

II

V

hFW/PACAP 6.487 kb

N

I

V

exm

htrm

B

I

Y

250

2000

V

hFW/PACAP

I

II

Ill

IV

v

VI

VY

hVlP

250

hum

2000

Fig. 4. (A) A schematic diagram of the salnlon (s) GHHH-IikeiPACAP, llulnan (11) PHP/PACAP, and hCHHH genes. ( B ) A schematic diagram of' the hPHPiPACAP and hY1P genes. Boxes represent exons and lines denote introns.

4. TISSUE AND DEVELOPMENTAL EXPRESSION Within the human hypothalanius the GHRH gene is transcribed into a 750-base-pair (bp) mRNA species, including the poly A signal. Alternate RNA splicing, particularly between exoiis 4 and 5, results in much of the species variation in the 3' end of the niature peptide (L. A. Frohman et al., 1989a).

1. MOLECULAR

EVOLUTION OF

GHRH

AND

GnRH

17

The GHRH gene is actively transcribed in several nonneural tissues, including rat and mouse testes (Berry and Pescovitz, 1990),ovaries (Bagnato et al., 1992), and placenta (Suhr et al., 1989; GonzfilezCrespo and Boronat, 1991; Mizobuchi et al., 1991).GHRH gene expression in the rat and mouse testes results in a stable 1750-bp mRNA species and a 3350-bp species that is expressed at a specific time during development (Berry and Pescovitz, 1990; Suhr et al., 1989). It is interesting to note that administration of exogenous G H had no effect on testicular GHRH mRNA levels (Berry and Pescovitz, 1990). This is in contrast to the negative feedback effect of GH on hypothalamic GHRH gene expression and may reflect tissue-specific differences in GHRH gene regulation. The rat ovary contains a GHRH-like peptide that is immunologically indistinguishable from hypothalamic GHRH. Also within rat ovarian tissue there are 1.75-kb fragments, as well as 3.2-kb and 3.6-kb fragments, that hybridize with an oligonucleotide probe of rat GHRH in a Northern blot (Bagnato et al., 1992). These larger mRNA species in rat ovary and testes may be unprocessed precursors of the 750-bp precursor present in the brain or alternatively physiologically significant gene transcripts that arose because of tissue-specific initiation, termination, or differential splicing (Bagnato et al., 1992). The more abundant form of rat placental GHRH mRNA is the same size as rat hypothalamic GHRH and encodes an identical preproGHRH protein, suggesting that one of the mature peptide forms in placenta is identical with hypothalamic GHRH. The less abundant transcript of GHRH in the placenta is larger than the hypothalamic form (Suhr et nl., 1989). In developmental studies, Northern blots or dot blot analysis did not detect GHRH gene transcripts in testis until 2 days after birth, but indicated a rise in expression levels from Day 21 to a maximal level at Day 30. This developmental expression of the GHRH gene in this tissue was not affected by the expression of IGF-I and IGF-I1 gene expression (Berry and Pescovitz, 1990). PACAP cDNAs have been isolated from neural and nonneural tissues in the human, sheep, rat, and salmon (0.nerka).Northern analysis shows that the mRNA species in the human brain were significantly larger, that is 2.4, 2.6, and 3.0 kh compared to positively hybridizing bands of only 1.2 kb in the testes (Hosoya et al., 1992). Ovine and rat hypothalanius contain only one detectable PACAP mRNA, which is 3 kb. It is not yet known whether these smaller mRNA species in the testes and cortex encode the same precursor. In catfish ( C . macrocepha h ) , analysis of tissue expression for the GHRH-like/PACAP gene indicates expression in the brain, stomach, testes, and ovary, but not

18

NAIVCY M. SHEKWOOD ET AL.

in the pancreas, pituitary, muscle, and liver ( J . E. McHory, personal communication). PCR studies suggest that both long and short precursors are expressed in salmon (0.nerka) (D. B. Parker and N. M. Sherwood, unpublished).

D. Functional Roles of GHRH and PACAP It is well known that GHRH directly stimulates the release of growth hormone from the pituitaries of human, cow, pig, and rat (see Campbell and Scanes, 1992; Frohman et al., 1992). Within the lower vertebrates, human GHRH has been shown to initiate the release of GH from chicken adenohypophyseal cells in uitro (Perez et al., 1987), dwarf chicken pituitaries in vivo (Harvey et al., 1984), immature and adult turtle pituitaries in vitro (Denver and Licht, 1989, l W l ) , frog pituitaries in uiuo (Malagon et al., 1991), and goldfish (C. auratus) pituitaries in vivo (Peter et al., 1984).In contrast, hGHRH adminstered by injections, pellets, or pumps resulted in neither growth in salmon (E. M. Donaldson and N. M. Sherwood, personal observation) nor release of GH from pituitary cells in rainbow trout (0.mykiss) (Luo et al., 1990) or from goldfish pituitaries in u i t m (Marchant and Peter, 1989).The difference between the studies may lie in the requirement for species-specific GHRH. Indeed, carp GHRH stimulates the release of GH from pituitary cells cultured from rainbow trout (0.mykiss) (Luo et aI., 1990) and from perfused goldfish (C. auratus) pituitaries (Vaughan et al., 1992) in a dose-dependent manner. There is indirect evidence that GHRH is involved not only with growth, but also with reproductive function. GHRH immunoreactivity has been reported in the gonads and placenta for several mammals (see Sections II,B and IV). GHRH may also affect follicular maturation based on evidence that GHRH binds to rat granulosa cells and stimulates cell proliferation and FSH-induced CAMP production (Moretti et nl., 1990a). As its name implies, PACAP stimulates adenylate cyclase in pituitary cells from rat (Miyata et al., 1989) and frog (Chartrel et al., 1991). Miyata et al. (1989) found that PACAP did not affect the release of pituitary hormones in a static cell culture; it did stimulate the release of GH, prolactin, ACTH, and LH, but not TSH or FSH in superfused rat pituitary cells. In a dispersed pituitary cell culture, PACAP released G H , ACTH, and LH but not prolactin (Hart et d., 1992). In pituitary clonal cell lines, PACAP stimulated the release of GH, prolactin, and peptide 7B2 from GH3 cells and released ACTH and peptide 7B2 1992). from AtT-20 cells (Propato-Mussafiri et d.,

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

19

Synergistically, PACAP enhanced GnRH’s effect of releasing LH and FSH in static cell cultures. Synergistic effects were not observed between PACAP and GHRH, CRF, or TRH on the release of GH, ACTH, or TSH (Culler and Paschall, 1991). Numerous other functions in tissues external to the pituitary have been reported. PACAP,-,8 was shown to reduce food intake in mice (Morley et al., 1992), act as a vasodilator on femoral blood flow in dogs (Naruse et al., 1993), be a potent relaxant of the rat ileum (Katsoulis et al., 1993), increase heart rate in sheep (Sawangiaroen et al., 1992), stimulate amylase and protein secretion from the pancreas (Mungan et al., 1991),and stimulate ion secretion in the rat jejunum (Cox, 1992). E. Evolution of GHRH and PACAP 1. RELATIONSHIP OF FISHAND MAMMALIAN FORMS The sturgeon (A. transmontanus), catfish ( C . macrocephalus), and salmon (0.nerka)PACAP peptides clearly belong to the PACAP family and glucagon superfamily of proteins. The membership is based not only on protein sequence identity and preprohormone structure but, in the case of the salmon gene, on the exodintron organization. The sockeye salmon (0.nerka) GHRH/PACAP gene is the first nonmammalian gene to be identified within the glucagon superfamily (D. B. Parker and N. M. Sherwood, unpublished). Structural organization of the salmon gene with five exons is the same as the human PRP/ PACAP gene. The major structural difference between human PRP/ PACAP and salmon GHRH/PACAP genes is the small size of the salmon introns. Each exon of the salmon gene encodes a distinct domain of the GHRH/PACAP preprohormone mRNA, which is characteristic of all members of the glucagon superfamily (Bell, 1986). Identification of the salmon GHRH/PACAP gene places its emergence before the time that fish and tetrapods diverged. The size and sequence similarity of the exons encoding peptide domains is well conserved between the salmon and human PACAP genes. However, PACAP encoded on exon 5 has even higher sequence similarity than the peptide encoded on exon 4. This implies that a greater functional constraint exists on PACAP than the GHRH-like peptide. It is also possible that the tetraploid nature of salmon (Allendorf and Thorgaard, 1984) has allowed more sequence drift in this GHRH-like region. The theory is that one of the duplicated genes remains conserved, whereas the other sequence is free to change. Hence, gene duplication followed by point mutations can result in the evolution of a gene with a novel product and function (Niall, 1982).

20

NANCY 11. SIIERWOOD ET AL.

Conservation of exon length may also be linked to structural and/ or functional significance of the encoded domain. Exon 4 (GHRH1-:?J of the salmon GHRH/PACAP gene is equal in length (105 bp) to the exon encoding the same region for both human GHRH and human PHM (peptide histidine methionine). On the other hand, exon 4 encoding the PRP region ofthe human PACAP gene is six nucleotides shorter than the related exons of the genes for salmon GHRH/PACAP, human PHM/VIP, and human GHRH. We do not know whether differences in exon size affect the function of the protein products. The salmonid GHRH/PACAP and mammalian PRP/PACAP precursors have dibasic enzyme processing sites within the GHRH(PRP) and PACAP peptide coding regions that could result in the production of shorter peptides (Fig. 2). PACAP,-,,NH, has been identified and found to be bioactive in mammals (see earlier discussion). Salmon (0. nerka), sturgeon (A. trunsmontunus), and catfish ( C . macrocephalus) also have dibasic residue sites for cleavage to process a PACAP,-,,NH,, although this peptide has not yet been isolated from fish. Two other cleavage products, a 19- and 13-residue peptide, could be processed from PACAP in fish and mammals. Salmon GHRH-like peptide and mammalian PRP and GHRH also have enzyme processing sites resulting in shorter peptide products (Fig. 2). Like human GHRH, salmon GHRH-like peptide could be cleaved to produce 19- and 10-residue molecules. A single Arg processing site in human GHRH could result in a 28-residue peptide similar to the salmon GHRH-like peptide of 28 amino acids. The conservation of these enzyme processing sites may be critical for the inactivation of the longer biological forms, or peptide functions that are presently unknown. 2. HIGHCONSERVATION OF PACAP

The high conservation ( 8 9 4 2 % )of PACAP1-38NH2between such divergent groups as fish (salmon, catfish, sturgeon) and mammals suggests that structural and/or functional constraints existed for this molecule during its evolution (Fig. 1B). Like the GHRH family, amino acid substitutions in the PACAP family are found in the C terminus, except in sturgeon, which has a Glu at position 15 in place of Lys. PACAP,-,,NH, has 100% sequence identity in all known PACAPs except for one amino acid substitution in sturgeon. The tight conservation of the amino-terminal region may explain why PACAP,_,,NH, has similar potency as PACAP,_,,NH, in stimulating adenylate cyclase activity (see Arimura, 1992a,b).

1.

MOLECULAR EVOLUTION OF

21

GHRH .4ND GnRH POTENCY

GHRH

1NH2

Tyr-AIa-AspAla

1.o 0.0002

Ib P

NH2

0.00002 0.000003

I OH

ITW

0.70

7"

IOH

I Tvr

0.34 0.27 0.25 0.0002

1

40

His

OH

0.35

Ala

1OH

0.01

I TrP

OH

0.003

I Ala-Tvr

IOH

0.007

Arg-Tyr

OH

0.002

Fig. 5. Comparison of' human GHRH1_,, or GHHHl_," with truncated, substituted, or extended forms of GHHH. Potency was measured by the release of GH from rat anterior pituitary cell cultures. A considerable reduction of potency is observed if the N-terminal residues are deleted or ifthe N-terminal Tyr or His is substituted or extended. Data from Guillemin et al. (1982),Ling et (11. (1984a,h) and Guillemin (1986).

3.

CONSERVATION OF CRITICAL

IN

AMINOACIDS

GHRH

Evidence that salmon and sturgeon GHRH-like peptides may be functionally related to the mammalian GHKHs can also be based on the conservation of specific amino acids required for biological activity. A comparison of' the salmon and sturgeon GHRH-like peptides to

22

NANCY M. SHERWOOD ET AL.

the mammalian GHRHs shows the highest conservation in the amino terminus (1-29) of the molecules (Fig. IA). Except for Leu,, at the carboxy-terminal end, G1y32is the last conserved residue. This position also correlates to the exodintron splice site in both the PACAP and GHRH genes. The importance of the high conservation of the N terminus of GHRH has been shown by deletion studies (Fig. 5). GHRHl-zlNH2 is the shortest C-terminally deleted synthetic fragment to have biological activity, but the potency is reduced to only 0.0001% of the activity of hGHRHl-,,NHz (Fig. 5) (Ling et al., l984b). Biological activity was not observed with a GHRHl-,,NH2 fragment. GHRH1-29NH2is the shortest fragment that exhibits full intrinsic activity; the potency is about 50% of that of hGHRH,-,,NH, (Ling et al., 1984b). All the mammalian GHRH peptides begin with a tyrosine or, in the case of rat (Bohlen et al., 1984) and mouse (Suhr et al., 1989; M. Frohman et al., 1989), a histidine (Fig. 6), which is required for receptor binding (Ling et al., 1984b; Coy et al., 1987). Like rat and mouse GHRH, salmon and sturgeon GHRH-like peptides have a histidine at position 1. Substitution of position 1 in mammalian GHRH, with other amino acids or the N-terminal addition of an amino acid, reduces biological potency (Fig. 5). The importance of the N-terminal amino acids (positions 1 and 2) is also illustrated by inactivation of GHRH through enzymatic cleavage of the first two amino acids by dipeptidylpeptidase type IV in plasma (L. A. Frohman et al., 1989a,b). Furthermore, GHRH3-,,NH, is biologically inactive (L. A. Frohman et al., 198%; Campbell et al., 1991). Salmon and sturgeon GHRH-like peptides have a phenylalanine at position 6, which is conserved in all known GHRHs except catfish, and is important for their biological activity (Coy et al., 1987). Methionine at position 27 is also conserved in all mammalian GHRHs and in fish GHRH-like peptides, except for catfish. Although methionine is not required for bioactivity and can be replaced b y norvaline or norleucine (Guillemin, 1986; Coy et al., 1987), oxidation of methionine reduces the biological activity of GHRH (Rivier et al., 1982; Campbell et al., 1991). Catfish GHRH-like peptide may be a 45- or 48-amino-acid protein. Ifthe precursor is cleaved at Arg-Arg, the Ala would be the first amino acid, but if cleaved after Arg-Arg-X-X-Thr, then His is in position 1 as shown in Fig. 1A. If the former cleavage site is used, only catfish GHRH-like peptide differs in having Ala in position 1 in contrast to the aromatic residue that is conserved in all other GHRHs. If the latter site is cleaved, then His in position 1 is similar to the fish, mouse, and rat GHRH peptides.

1.

23

MOLECULAR EVOLUTION OF GHRH AND GnRH 1

44

OH m i e m In vltro

GHRH44 (Human, pig, cow, goat. sheep)

GHRH 43 (rat) GHRH 42 (mouse) GHRH-llke 45 (Salmon.

sturgeon)

PACAP 38

(Human, sheep, rat)

PACAP 38

Salmon

GIP 42 (Human) GLP 31 (Salmon) Glucagon 29

OH

(Human)

Glucagon 29 (Salmon)

VIP28 (Human) VIP 28 (Dogfish) Secretin 27(Human) PHM 27 (Human) PHI 27 (Rat) PACAP-related peptlde 48

lIsa

1-OH

(Human, sheep, rat)

Fig. 6. Comparison of the glucagon superfamily peptides regarding length, Nterminal residues, and C-terminal modifications. All members have His or Tyr at the N terminus regardless oftheir ability to release GH, with the exception ofthe mammalian PACAP-related peptide (PRP).

4. FUNCTIONAL QUESTIONS REGARDING PRP

The function of the mammalian PRP is not known. In contrast to PACAP, not a single function for PRP has been reported to date. Unlike other members ofthis superfamily, PRP does not begin with a histidine or tyrosine, which are required for their biological activity (Fig. 6). Therefore, in the case of PRP, it may not have a biological function. Alternatively, it may assist in folding of the precursor, act as a secreted

24

NANCY kl. SHEHWOOD ET AL.

peptide with a function unrelated to GH release, or act as a releaser of GH. The existence of GHRH encoded on a separate gene implies that PRP is not essential for GH release.

5. PURPOSE OF Two PEPTIDES IN ONEPRECURSOR The physiological significance of two peptides processed from one precursor must be considered for GHRH(PRP)/PACAP, PHMIVIP, and glucagon/GLP genes. An increasing number of studies show colocalization of a variety of peptides such as GHRH and neuropeptide Y (NPY) (Ciofi et al., 1987), or GHRH and vasotocin (Marivoet et ul., 1987). Processing of more than one peptide from a single prohormone rather than from two precursors might have an advantage if both peptides regulate the same physiological response. It remains to be shown whether the GHRH-like (PRP) and PACAP molecules are (1)cosecreted and have coordinated actions, (2) cosecreted, but have unrelated functions, (3) cosecreted, but only one peptide is bioactive (e.g., hPACAP but not hPRP), or (4) released singly from a shortened precursor containing only one of the encoded peptides such as salmon PACAP. For coordinated actions, the two peptides niay act on different or overlapping sites. One cosecreted peptide may act to release a hormone from the target cells, whereas the other peptide may play a role in gene expression or cell growth. It is also possible that one of the peptides acts on a subset of the same cells. For example, GHRH and PACAP both stimulate GH release, but PACAP acts only on a subpopulation of the somatotropic cells (Yada et nl., 1993). In an example of overlapping function, PACAP stimulates the release of interleukin-6 from folliculostellate cells in the rat pituitary (Tatsuno et ul., 1991), which in turn may promote GH release from somatotropic cells (Spangelo et al., 1989). Hiikfelt et ul. (1983) have proposed a model in which three neuropeptides [peptide histidine isoleucine (PHI), corticotropin releasing factor (CRF), and enkephalin] that are synthesized and secreted from the same neuron are responsible for the corrdinated release of GH, ACTH, and prolactin. Mechanisms by which both peptides, PACAP and GHRH-like peptide (or PRP), are cosecreted but only one is bioactive include alternate splicing of the RNA or enzymatic cleavage of the precursor. In salmon, alternate slicing produces a short precursor which contains only PACAP (Fig. 7). The region deleted in the short cDNA precursor corresponds to the excision of exon 4 containing the GHRH domain o f t h e gene. This exon encodes the last 3 amino acids of the cryptic peptide and the first 32 amino acids of the GHRH-like molecule. The

1.

25

MOLECULAR EVOLUTION OF GHRH AND GnRH SP

GHRH-LIKE

PACAP

GHRH-like Tyr Pro Pro Glu Lys TACCCACCG GAG AAA

II

~

&- - -

I-

-

Gly Gly Ser Thr Met Glu Ay,

- T GGA GGG AGC ACC ATG GAA GAC

-

Tyr Pro Pro Glu Lys Ser Gly Gly Ser Thr Met Glu Asp TACCCACCGGAGAAA AGT GGAGGGAGCACTATGGAAGAC

Fig. 7. A line diagram of the proposed exodintron organization of the salmon (s) GHRH/PACAP gene and processing of the RNA transcript to obtain the short preprohornione. Alternate splicing of the RNA transcript results in the loss of exon IV, which contains the first 32 amino acids of the sGHRH sequence. Excision of exon IV results in a new codon (AGT) that creates a serine in the precursor and allows for inframe reading of the remaining sequence. This short precursor would encode only PACAP.

gene structure shows exon/intron splice sites at nucleotide positions

233-234 and 338-339 in the full-length cDNA (Fig. 7). The other mechanism for creating only one bioactive peptide from a two-peptide precursor depends on the use of alternate cleavage sites in the protein. For the fish GHRH/PACAP precursors, the presence or absence of an endoprotease for processing at a single Arg cleavage site may determine if one or both peptides are secreted (Fig. 2). THE GHRH CRYPTIC PEPTIDES

6. ROLEOF

AND

PACAP

The fish and human GHRH(PRP)/PACAP precursors, like those of other gene families, have greater sequence divergence in exons that do not code for functional peptides (Fig. 3) (Heilig et ul., 1980; Li et al., 1985).An example is the cryptic area, which may be important for the folding of'the precursor prior to cleavage, but is not thought to be

26

NANCY hl. SHERWOOD ET AL.

secreted as a functional peptide. The cryptic areas in the fish and mammalian precursors have little sequence similarity, but show conservation of the secondary structure based on hydrophobicity. On the other hand, the 3’ untranslated region, which is expected to be like the cryptic peptide in having little conservation of nucleotide bases, shows surprising similarity between the human and salmon. The conserved 3’ untranslated region may be related to control of mRNA translation.

7.EVOLUTION

OF THE

PACAP GENE

The PRP/PACAP and the PHM/VIP genes are the most closely related ofthe superfamily members. One hypothesis about this relationship is that an exon duplication gave rise to an ancestral gene that coded for two peptides: a PHM/PRP consensus region and a VIP/ PACAP consensus region. Later, a gene duplication event is thought to have resulted in two genes: PHM/VIP and PRP/PACAP (Ohkubo et al., 1992). After duplication of the ancestral gene, either an exon was lost in the PACAP gene or gained in the VIP gene (Figs. 8 and 9). Both the salmon GHRH/PACAP and mammalian PRP/PACAP genes are unique in comparison to the other glucagon superfamily members in that they have neither an intron located in the carboxyterminal peptide nor an intron in the 3’ untranslated region (Figs. 4A and 4B).

8. OKICINOF

THE

MAMMALIAN GHRH GENE

One of the most interesting aspects in the evolution of the glucagon superfamily is the point at which the GHRH gene arose. GHRH may have arisen after duplication of one exon in the ancestral gene (Fig. 9). Thereafter, two pathways for the evolution of GHRH are likely. In the first scheme, GHRH may have arisen from a gene duplication prior to the evolution of the PACAP and VIP genes (Fig. 9) (Ohkubo et al., 1992).This assumption is based on the low sequence identity between GHRH, PHM( I), and PRP (Ohkubo et al., 1992).In the second scheme, it is possible that GHRH arose from either the VIP or the PACAP gene as shown in Figs. 8A, SB, and 9. In both cases exon 111, encoding only the cryptic peptide, would be lost from the gene. Alternatively, if the new GHRH gene arose from PACAP, it would gain an intron within the 3’ untranslated region, but if it arose from the VIP gene it would lose an exon near the 3’ end (see Fig. 8). Campbell and Scanes (1992) consider the GHRH gene to have arisen prior to the emergence of the vertebrates, approximately 750 million years ago. However, a cDNA or gene encoding only a GHRH peptide has not been identified in the nonmammalian vertebrates. If

1.

MOLECULAR EVOLUTION OF

27

GHRH AND GnRH

A I

II

I

I1

IY

IV

V

111

YTRON QAINED IV v

GHRKPACAPgene MON n.h-1.wapod split

GENE DUPLICATION In lokapods

EXON LOST

MW

GHRH gone

)-(

I

II

111

V

IV

PRP-PACAP gem

ACA

3'U

B PHWPHCVIP gene hypoheticel -1rd

gene

U O NLo81

U O N LOST

new GHRH gone

II

ni

N

V

VI

PHMPHCVIP gene (mammals)

Fig. 8. Two possible schemes for the derivation of the C H R H gene in mammals are shown. (A) A scheme in which the C H R H gene evolved from the ancestral GHRHPACAP gene by loss o f exon 111 and addition of an intron in exon V. (B) A scheme in which the GHRH gene in mammals evolved from the ancestral PHMIPHI-VIP gene by loss of exons I11 and VI.

an additional GHRH peptide (gene) is not found in fish, the emergence of the GHRH gene would be placed some time after the divergence of the tetrapods from fish, approximately 400 million years ago. This means GHRH has undergone rapid changes in its sequence as well as acquiring the function of GH release.

28

NANCY bl. SHEKW’OOD ET AL.

Exon duplication

Gene duplications

n

--

Gene duplication

\

Exon

\

duplication

\

\

-cH_I

\

A

Gene duplication

I I

Gene duplication

\

\

{GHTF

I I

t

_ _ _ _ _ _ _ _ _ _ _ _ _ -J

Fig. 9. A schematic diagram ofthe hypothetical evolution ofthe glucagon superfanlily. An ancestal gene may have contained a single exon which duplicated to give two cxons encoding two peptides. One branch may have given rise to precursors such as gastric inhibitory peptide (GIP), secretin (SECR), and glucagon (GLUC), whereas the other branch gave rise to precursors containing GHRH, PACAP, and VIP. The most plausible explanation for the origin of GHKH is that a duplication ofthe ancestrd gene gave rise to the PHIiVIP and PRPiPACAP genes from which a second duplication of one of these two genes gave rise to the GHRH gene.

9.

EVOLUTION OF THE

GLUCAGON SUPERFAMILY

By comparing the structural organization of the precursors in the glucagon superfamily it is suggested that two lines within the superfamily evolved, one containing PACAP, VIP, and glucagon, and the other consisting of GHRH, GIP, and secretin. In the glucagon-VIP-PACAP group, each precursor contains tandem sequences coding for two or

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

29

more functional peptides, whereas in the other group, each precursor has a single functional peptide plus a cryptic sequence without known function (Fig. 9). The pathway to this diversification may have initially involved exori amplification with duplication of the full genes later (Heinrich et al., 1984; Hosoya et al., 1992). The progenitor gene may have had an exon containing at least 100 nucleotides which encoded the first 27-30 amino acids of the bioactive peptide. This assumption is based on the fact that the first 27 amino acids are the most highly conserved between the superfamily members and that this region is encoded on a separate exon. Thereafter, the family genes would have evolved through point mutations and nucleotide additions and deletions. Gene duplication and divergent evolution from an ancestral gene has occurred in many protein superfamilies and families, including immunoglobulins (Rogers et al., 1980),globins (Breathnach and Chambon, 1981), growth hormone and prolactin (see Kawauchi et al., 1990), and vasopressin and oxytocin (Acher, 1981). The key to understanding the evolution ofthe glucagon superfamily depends on identification of the genes in lower vertebrates and perhaps invertebrates. At present only the GHRH-like/PACAP gene has been isolated for fish. Missing are the genes for VIP, GIP, secretin, and glucagon.

111. GONADOTROPIN-RELEASING HORMONE A. Identification

The first fish GnRH to be sequenced was isolated from chum salmon (0.keta) brains (Table 11).The primary structure did not leave any doubt that salmon GnRH (sGnRH) was closely related to mammalian GnRH (mGnRH). Both peptides have 10 amino acids, 80% sequence similarity, and the same N-terminal (pGlu) and C-terminal (NH,) modifications. In the 10 years since the structure of sGnRH was published, seven distinct GnRH peptides have been isolated and sequenced from fish: lamprey GnRH-I and GnRH-111, salmon GnRH, dogfish GnRH, catfish GnRH, chicken GnRH-11, and mammalian GnRH (Fig. 10 and Table 11). The vertebrate GnRH family to date has eight distinct forms of GnRH with a sequence similarity among the forms of 50% or greater (Table 111).In tetrapods, only three forms ofGnRH are known: mammalian GnRH, chicken GnRH-I, and chicken GnRH-I1 (Table 11). The

Table I1 Identification of GnEH Sequences Source of sequence Peptide

Form

Species

M

Hunran

Tissrlc

Peptide

cDNA

Gene

References

GnRH

M h4 hl bI XI

Placenta Placenta Placenta Brain Brain

X X X X X X X

Pig Sheep Rat

Brain Brain Brain Ovary Lynrphocyte

X X X X X X

M

X

M

Mouse Frog Sturgeon

Brain Brain

X X

C-I1 c-I1

Chicken Alligator

Brain Brain

X

XI

X

Tan and Rousseau (1982) Seeburg and Adelman (1984) Radovick et al. (1990) Adelman et al. (1986) Radovick et 01. (1990) Adelman et al. (1986) Hayflick et a1. (1989) Radovick et al. (1990) Matsuo et a1. (1971) Burgris et al. (1972) Adelman et al. (1986) Oikawa et a1. (1990) Azad et al. (1991) Adelman et ul. (1986) Bond et al. (1989) Kepa et al. (1992) Mason et a1. (1986) Conlon et (11. (1993) D. W. Lescheid et ul., personal conmrunication Miyamoto et a1. (1984) Lovejoy et a1. (1991b)

C-I1 C-I1 C-I1 C-I1 c-I1 C-I1

Frog Catfish

Ratfish Dogfish

Brain Brain Brain Brain Brain Brain

C-I c-I

Chicken Alligator

Brain Brain

S

Salmon

Brain Brain Brain Brain Brain

S S SI s2

X

Miyamoto et (11. (1983) Lovejoy et al. (1991b)

X

Sherwood et al. (1983) Klungland et al. (19924 Suzuki et al. (1992) Ashihara et al. (1993) Ashihara et a1. (1993) Klungland et al. (1992a) Coe et al. (19924 Klungland et d . (1992b) Klungland et a1. (1992b) Bond et ul. (1991) Gothilf et al. (1993)

X

Ngmvongchon et al. (1992a) Bogerd et al. (1992) Bogerd et al. (1994)

X X X

X X X X X

S S

S S

S S CF CF CF DF L-I L-111

Trout Cichlid Str. bass

Conlon et a1. (1993) Ngamvongchon et al. (l992a) Bogerd et al. (1992) Bogerd et d . (1994) Lovejoy et al. (l99la) Lovejoy et a1. (1992,)

X X X

X

Brain Brain

Catfish

Brain Brain Brain

X X

Dogfish

Brain

Lamprey

Brain

Lamprey

Brain

X X X

Lovejoy et a1. (1992a) Sherwood et al. (1986) Sower et al. (1993)

32

NANCY kl. SHEHWOOD EZ A L .

1 SALMON

2

3

4

5

6

7

8

9

10

pGLU-HIS-TRO-SER-TYR-GLY-TRP-LEU-PRO-GLY-NE2

DOGFISH CATFISH

mm5AX.l CHICKEN I

LAMPREY I

pGLU-HIS

SER

Fig. 10. Comparison of eight GnRH peptides. Boxed amino acids indicate residue changes with respect co the salmon form.

GnRH names reflect the animal from which the forms were first isolated. For example, chicken GnRH-I1 was identified in chicken brain extracts, but has since been identified by primary structure in ratfish (Hydrolagus colliei), dogfish (Squalus acanthias), catfish ( C . macrocephalus), frog, and alligator. Mammalian GnRH was isolated originally from pig, sheep, and other mammals, but has recently been sequenced from brain extracts of sturgeon (A. transmontunus) and frog. Table IV shows that the family of GnRH peptides is not complete. GnRH has been studied in 47 species offish by one or more methods: high-performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA); amino acid composition; amino acid sequencing; and cDNA or genomic analysis. The GnRH forms tentatively identified by the HPLC-RIA method may include more novel forms than listed because detection depends on specificity of the antisera. In fish, all forms of GnRH have been identifed initially by protein chemistry, although once identified, several of the known forms of GnRH have been shown to exist in additional species by the use of molecular biological techniques. Table I1 shows that the primary structure of salmon GnRH has been confirmed in several other fish species by using molecular biological methods (Bond et al., 1991; Klungland et al., 1992a,b; Suzuki et al., 1992; Ashihara et al., 1993; Gothilf et al., 1993; Bogerd and Goos, 1993). The structure for the GnRH gene has been presented for only two fish: Atlantic salmon (Salrno s,zlar) (Klungland et al., 1992b) and sockeye salmon (0.nerka) (Coe et al., 1992b) (Fig. 11).

1.

MOLECULAR EVOLUTION OF

GHRH

AND

33

GnRH

Table I11 Amino Acid Sequence Similarity (70)Among Vertebrate GnRHs ~~

L-I Lamprey GnRH-I Lamprey GnRH-111 Chicken GnRH-I1 Dogfish GnRH

Salmon GnRH Catfish GnRH Mammal GnRH Chicken GnRH-I

L-I L-111 c-I1 DF S CF

M c-I

L-111

C-I1

DF

S

CF

M

100 80 80

100

C-I

100 70

100

60

80

100

60

80

90

100

60

70

80

YO

100

50 50 50

70 60 60

80 70 70

80 70 70

70 80 80

YO

100

B. Phylogenetic Studies 1. IMMUNOCYTOCHEMISTRY

The location of GnRH neurons in the fish brain shows several evolutionary trends. All three classes of fish have GnRH cell bodies in the more ventral aspects of the brain, but the location of the cell bodies and axon terminals has changed among the classes. The GnRH cell bodies are found in or near at least one of the following structures: olfactory nerves or bulbs, ventral telencephalon, septum, preoptic region, hypothalamus, or midbrain. The location is thought to reflect the migration pathway of GnRH neurons, all of which appear to develop in the olfactory placode, but enter the brain from the olfactory and rostra1 telencephalic region. The studies providing evidence of this path are limited to a few species of fish, but are numerous in tetrapods. From the GnRH cell bodies, axons extend to a number of brain areas outside of the hypothalamus in addition to termination points in or near the pituitary. The three classes of fish have distinct locations for the GnRH terminals that are thought to cause gonadotropin release from the pituitary. a. Agnatha. In the class Agnatha, containing the most phylogenetically ancient fish, several species of lamprey have been shown to have GnRH neurons that are located in a very restricted region ofthe brain. The cell bodies are in the caudal part ofthe telencephalon (the preoptic region) and their axons terminate in the neurohypophysis, the third ventricle, or in extrahypothalamic regions in the brain (Crim et aZ., 1979a,b; Nozaki and Kobayashi, 1979; Crim, 1981; Nozaki et al., 1984a; J. C. King et aZ., 1988). The absence of GnRH neurons associated with

. U

L

0

o

0 . 0 0 0

1

Moray eel (Gymnothorax fimbriatus) Herring (Clupea harengus pullasi) M ilkfish (Chunos chanos) Goldfish (Carassius auratus) Catfish (Clarius macrocephalus) Catfish (Cluricis gariepinus) Catfish (Clarins batrachus) Chum salmon (Oncorhynchus keta) Sockeye salmon (Oncorhynchus nerka) Masu salmon (Oncorhynchus masou) Chinook salmon (Oncorhynchus tshawytschu) Rainbow trout (Ortcorhynchus m y kiss) Atlantic salmon (Salnio sular) Brown trout (Salmo trutta) Brook Trout (Saloelinus fontinalis) Hake (Merluccius caperisis)

3

0

0 0

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Shih et al. (1988) Sherwood (1986)

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Sherwood et al. (1984) Sherwood and Harve: (1986)

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0

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0

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0

Coe et a1. (1992b)

0

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0

Klungland e t a / . (l992a)

0

Klungland et a / . (1992a)

0

Klungland et ul. (19921,)

0

Klungland et

0

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0

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(11.

(1992a)

36

J

u a,

-

F

IP

s

Bluefin tuna (Thunnus thynnus) Black sea bream (Acunthopagrus schlegeli) Red sea bream (Pagrus major) Red spotted grouper (Epinephelus akaara) Japanese flounder (Paralichthys olioaceus) Wrasse (Corisjulis) Winter flounder (Pseudopleuronectesamericanus)

0

0

0

Okuzawa et a / . (1993)

0

0

0

Okuzawa et u1. (1993)

0

0

0

Okuzawa et ul. (1993)

0

0

0

Okuzawa et al. (1993)

0

0

0

Okuzawa et d.(1993)

H. Powell et u1. (1986)

0 0

0

0

Idler and Everdrd (1987) ~

~~

c4

0, The primary structure or amino acid composition of GnRH has been determined for the species listed. 0, The GnRH form is identified indirectly by a combination of HPLC and HIA.

38

NANCY 31. SIIERWOOD ET AL.

Atlantic Salmon I

II

111

IV

Pacific Salmon

Rat

1

I

1

Human

Fig. 11. Comparison of the GnHH genes of Atlantic salmon, Pacific salmon, rat, and human. Exons I, 11,111, and IV are labeled. Introns are shown as lines. The coding for the signal peptide is stippled, the hormone is black, the processing site and GnRHassociated peptide (GAP) are white, and the 5’ and 3’ untranslated regions are shown b y diagonal lines.

the agnathan terminal nerve in the olfactory-anterior telencephalicsepta1 region is a noticeable difference in these jawless fish compared with other classes of fish (Demski, 1984; Meyer et al., 1987; J. C. King et nl., 1988).Also, the termination ofall the axons entering the pituitary in the neurohypophysis differs from that of the cartilaginous and bony fish. The current hypothesis is that GnRH reaches the anterior pituitary from the blood vessels in the neurohypophysis or from the cerebral spinal fluid in the third ventricle ( J . C. King et nl., 1988). 12. Chondrichthyes. In the class Chondrichthyes, the location of immunoreactive GnRH neurons has been studied only in elasmobranchs, which includes sharks, skates, and rays. In contrast to agnathans, cartilaginous fish have GnRH neurons that are outside of the hrain and arranged in ganglia that lie along the terminal nerve near the olfactory bulbs (Stell, 1984; Demski and Fields, 1988). The GnRH axons from these ganglia pass into the brain to be distributed primarily to the telencephalon. Only a few GnRH fibers go to the hypothalamus and median eminence region (Nozaki et al., 198413; Demski, 1987; Demski et al., 1987; Wright and Demski, 1993). Unlike other vertebrates, elasmobranchs rarely have GnRH cell bodies in the hypothalamus and median eminence (Nozaki et al., 1984b, Lovejoy et al., 1992a;

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

39

Wright and Demski, 1993). The delivery of GnRH to the pituitary is thought to be by the systemic circulation, which receives GnRH from axon terminals at the base of the telencephalon (Sherwood and Lovejoy, 1993; Wright and Demski, 1993). Another novel aspect of the location of GnRH neurons in cartilaginous fish is the presence of a large, well-defined nucleus of GnRH cells in the midbrain with at least some axons projecting into the spinal cord. The function and origin of the midbrain GnRH cells are not clear, although some of the fibers may secrete GnRH into the cerebral spinal fluid (Wright and Demski, 1991, 1993). The presence of midbrain GnRH neurons in cartilaginous fish is in contrast to their absence in jawless fish and their scarcity in bony fish. c. Osteichthyes. Like the cartilaginous fish, bony fish have GnRH neurons that are associated with the terminal nerve (Miinz et al., 1982; Nozaki et al., 1984b; Stell et al., 1984; Demski, 1984; Grober et al., 1987). Unlike the cartilaginous fish, most bony fish, but not all, have GnRH neurons that are inside the brain (Nozaki et al., 198413). The location of GnRH neurons inside the forebrain supports the idea that there is a migratory GnRH pathway similar to that in tetrapods. In this pathway GnRH neurons begin outside of the brain in the olfactory placode and then move from the nasal cavity, across the cribiform plate, and into the forebrain (see Section III,C,4). Examples of GnRH neurons at all levels of the path have been reported in adult fish. In the catfish Clarias batrachus, some GnRH neurons remain in the olfactory epithelium (Subhedar and Rama Krishna, 1988).In other teleosts such as goldfish (C. auratus),brown trout (Salmo trutta, L.), and masu salmon (Oncorhynchus masou), GnRH neurons are found only as far forward as the olfactory nerves (Stell et al., 1984; Kah et al., 1986; Breton et al., 1986; Suzuki et al., 1992), but in many other teleosts, the rostra1 position of GnRH neurons is slightly more posterior, that is, in the junction between the olfactory nerve and bulb or in the anterior olfactory bulb (Nozaki et al., 198413; Schreibman et al., 1984; Amano et al., 1991). In addition there is a second clump of GnRH cell bodies just below the posterior olfactory bulb in the Japanese eel (Anguilla japonica) and American eel (Anguilla rostrata) (Nozaki et al., 1984b; Grober et al., 1987). Just caudal at the junction of the olfactory bulbs and the ventral telencephalon is a cluster of GnRH cell bodies, the nucleus olfactoretinalis, in a variety of fish, including the platyfish (Xiphophorus maculatus), blue gill (Lepomis macrochirus), sea bass (Dicentrarchus labrax), sole (Solea solea L.), catfish (Clarias batrachus, Linn.), molly (Poecilia latipinna),and guppy (Poe-

40

NANCY M . SHERWOOD ET AL.

cilia reticulata Peters) (Schreibman et al., 1979; Miinz et al., 1981, 1982; Halpern-Sebold and Schreibman, 1983; Nunez Rodriguez et al., 1985; Zentel et al., 1987; Subhedar and Rama Krishna, 1988; Batten et al., 1990; Oka and Ichikawa, 1990; Amano et al., 1991; Kah et al., 1991). The GnRH neurons in the forebrain have been considered to be part of the terminal nerve. Although not yet studied in fish, the GnRH neurons in the mouse fetus follow by about one day the migratory path of the terminal and vomeronasal nerves (Wray et a1.,1989a,b). This suggests that the GnRH and terminal nerve systems are separate, but intermingle in their final locations. In teleosts, GnRH neurons almost always occur in the telencephalon (ventral and/or preoptic areas) and sometimes in the diencephalon (usually in the lateral tuberal nucleus); axons extend from the preoptic or hypothalamic neurons into the anterior pituitary and sometimes into the neurohypophysis, the third ventricle, and to brain areas outside of the hypothalamus (Goos and Murathanoglu, 1977; Schreibman et al., 1979; Nozaki and Kobayashi, 1979; Nozaki et al., 1984a,b; Miinz et al., 1981,1982; Borg et al., 1982; Halpern-Sebold and Schreibman, 1983; Goos et al., 1985; Nunez Rodriguez et aZ., 1985; Breton et al., 1986; Kah et al., 1986; Subhedar and Rama Krishna, 1988; Garcia Ayala et al., 1989; Schafer et al., 1989; Batten et al., 1990; Oka and Ichikawa, 1990; Coe et al., 1992a; Amano et al., 1991; Khan and Thomas, 1993).Finally, in some teleosts the midbrain contains a group of GnRH neurons with at least some axons extending down the spinal cord (Munz et a,?., 1981; Borg et al., 1982; Kah et al., 1986,1991; Miller and Kreibel, 1986; Subhedar and Rama Krishna, 1988; Batten et al., 1990; Amano et al., 1991; Khan and Thomas, 1993). There is a reduction in the number of GnRH neurons in the midbrain in teleosts compared with cartilaginous fish. This decrease in GnRH neurons may be an evoluntionary trend. Alternatively, the form(s) of GnRH in the teleost midbrain neurons may not be detected b y antisera used in studies to date. The latter explanation seems less likely as Amano et al. (1991,1992) present evidence that the midbrain neurons in masu salmon (0.masou) contain cGnRH-11. A reduction in midbrain GnRH neurons in teleosts may not be critical because teleosts without midbrain GnRH cells have an extensive distribution of GnRH fibers in the brain from the olfactory bulb to the spinal cord (Oka and Ichikawa, 1990). Most bony fish, like cartilaginous and jawless fish, lack a portal blood system for transport of GnRH from the brain to the gonadotrophs in the anterior pituitary. Unlike the other two classes of fish, GnRH axons in many bony fish, especially teleosts,

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

41

terminate in the anterior pituitary near the gonadotrophs, although in some teleosts, GnRH fibers also end in the neurohypophysis close to the anterior pituitary (see Nozaki et al., 1984a,b; Goos et al., 1985). Some of the more primitive bony fish and lobe-fin fish have the beginnings of a portal blood system. In conclusion, variations in the location of GnRH cell bodies and their axons may be a reflection of different developmental patterns or alternate routes for delivery of GnRH to the pituitary. 2. CHROMATOGRAPHY An important key to understanding the evolution of GnRH within the varied species of fish has been to screen for GnRH in brain extracts using high-performance liquid chromatography combined with radioimmunoassay. These methods are used to tentatively identify novel and known forms of GnRH in brain extracts. It is also the method used to show that multiple forms of GnRH are present in all vertebrates studied, except for single forms in ratfish ( H . colliei) and eutherian mammals. The resulting phylogenetic map for distribution of GnRH forms is shown in Table IV and Fig. 12. The most important part of the distribution chart is the GnRH forms that have been identified from individual species by primary structure. The other forms of GnRH are tentative until they have been isolated and sequenced. The evidence that multiple forms of GnRH exist within a species has been based on experiments with pools of brains. Hence, multiple forms in a species could be derived from a polymorphism or a gene duplication with subsequent changes. An evolutionary scheme for GnRH based on this chromatographic data is discussed in Section II1,E. C. Structural Analysis 1. PEPTIDESEQUENCES The structure of the eight known peptide sequences is shown in Table 11. The seven primary structures identified in fish were originally determined using protein chemistry. Critical amino acids in GnRH include those at both termini because they are essential for recognition of the GnRH receptor, whereas release of LH and FSH from the pituitary depends on residues 1-3 (see Sherwood et al., 1993b).Also, tryptophan in position 7 appears to be important in the binding of GnRH to its receptor, at least in teleosts. For example, Trp7 is found in the goldfish endogenous forms of GnRH (sGnRH and

42

NANCY M . SHERWOOD ET A L .

PRRTERTEBRATES EARLY VERTEBRATES JAWLESS FISH

CARTILAGINOUS FISH 1-

Elumobranchr

-

BONY FISH EARLY

-

BONY FISH EARLY TELEOSTS BONY FISH -TELEOSTS CaRIh

salmon

t.0

-

BONY FISH LATE TELEOSTS seabream AMPHIBIANS

IUI

REPTILES BIRDS EARLY U W L S LATE PLACENTAL MAMMALS

Fig. 12. Hypothetical scheme for the evolution of known forms of GnRH in vertebrates from an ancestral GnRH (A). Numbers indicate the amino acid differences between the two GnRH forms compared. T h e scheme is based on the known distribution of the nine forms of GnRH for which primary structure is known. T h e forms of GnRH are shown as: A, ancestral; C-I, chicken I; C-11, chicken 11; CF, catfish; DF, dogfish; L - I , lamprey I; L-111,lamprey 111; M, mammal; S, salmon; SB, sea bream.

cGnRH-11), and these two forms of GnRH are approximately two- to three-fold more effective than mGnRH in releasing gonadotropin I1 in goldfish. The substitution ofTrp7into mCnRH improved its potency to the level of the endogenous forms (Millar et al., 1989; Habibi et al., 1992).

1.

MOLECULAR EVOLUTION OF

GHRH

AND

GnRH

43

2. D N A SEQUENCES

The cDNAs isolated from fish brains have been sequenced for three types of GnRH: salmon, chicken-11, and catfish (Table 11). Several insights were immediately obvious from the cDNA sequences. First, each form of GnRH is encoded on a separate gene. Second, each cDNA includes coding not only for the GnRH peptide but also for a GnRHassociated peptide (GAP). Third, each cDNA encodes a signal peptide as expected for the preprohormone of a secreted peptide. Fourth, the overall organization of the cDNAs isolated from fish is similar to that of the cDNA for mammalian GnRH isolated from mammalian brain or placenta in that the cDNA encodes a signal peptide, GnRH hormone, and GAP. Fifth, the deduced amino acid sequence of each hormone matches the primary structure determined by amino acid sequencing. The most striking difference in the cDNAs is the coding region for the GnRH-associated peptide. The length of GAP in the precursor molecule is 56 amino acids for mGnRH (in human, rat, and mouse), 54 amino acids for sGnRH (in cichlids) or 46 amino acids in sGnRH (in 7 salmonid species), 46 residues for catfish GnRH (in catfish), and 49 residues for chicken-11 GAP (in catfish). The length of the GAP peptides that are released into the blood may be shorter than the ones deduced from the cDNA because the GAPs identified to date have potential dibasic enzyme cleavage sites (Lys-Lys, Lys-Lys-Arg, or ArgLys-Lys-Arg) near the C terminus. The sequence similarity among these GAP peptides is very low even if the best alignment is used. Although there is an 80% conservation of amino acids in GAP among 7 salmonid species (Atlantic salmon, S. salar; sockeye salmon, 0. nerka; chinook salmon, 0. tshawytscha; m a w salmon, 0. masou; rainbow trout, 0. mykiss; brook trout, Salvelinus fontinalis; and brown trout, S. trutta), there is a modest 60% conservation between the African cichlid Haplochromis burtoni and masu salmon GAPs, only 15% sequence similarity between masu salmon and rat, and 8% between masu salmon and human. In contrast, the GnRH peptides (mammalian and salmon) are 80% conserved and the amidation-cleavage site (GlyLys-Arg) is fully conserved in the mammals and fish listed here. Analysis of the GAP sequences suggests that the function of GAP may be restricted to folding of the prohormone for processing inside of the secretory vesicle or an unknown function after secretion. The latter seems unlikely because 8% sequence similarity between fish and human GAPs is of questionable significance and also GAP receptors have not been reported. This evolutionary approach questions the function

44

NANCY M. SHERWOOD ET AL.

of GAP as an inhibitor of prolactin release, unless the function is newly acquired in mammals. 3. GENESTRUCTURE AND COPYNUMBER

Two genes for GnRH have been isolated and sequenced from fish: Pacific sockeye salmon (Oncorhynchus nerka) and Atlantic salmon (Salmo salar) (see Table 11). Both genes encode the preprohormone for the salmon form of GnRH. A comparison of these two salmon GnRH genes with the two mammalian GnRH genes from human and rat shows that all of these genes have four exons with the 5' untranslated region on the first exon (Fig. 11). Exon 2 encodes the signal peptide, decapeptide, Gly-Lys-Arg amidation-cleavage site, and part of the associated peptide (GAP,-l1). The middle part of GAP is on the third exon and the final part of GAP along with the 3' untranslated region is on the fourth exon. The lack of tandem sequences encoding different forms of GnRH supports the hypothesis that multiple forms of GnRH arose by complete duplication of the gene and subsequent nucleotide changes in one or both genes rather than duplication of exon 2 only. An obvious difference in the GnRH gene organization is the shortness of introns 2 and 3 in the salmon genes compared with the mammalian genes (Fig. 11). Also, a comparison of the sockeye salmon (0. nerka) and Atlantic salmon ( S . salar) GnRH genes shows a short deletion in the second intron in 0. nerka. The functional importance of these GnRH intron changes is not known. There are also nucleotide base substitutions throughout the sockeye compared with the Atlantic salmon gene, but none is in the coding region for the GnRH decapeptide. A substantial deletion ofover 1000 bases is present in the proximal 5' flanking region of the Pacific compared with the Atlantic salmon GnRH gene (von Schalburg and Sherwood, personal communication). The 5' flanking region in the Atlantic salmon GnRH gene contains two estrogen response element (ERE)-like motifs that bind the human estrogen receptor (Klungland et al., 1993).This salmon ERE-like motif is unusual because of the long interspace (8or 9 nucleotides) between the palindromic half-sites compared with the tetrapod EREs ( 3 nucleotides), but does not appear to hinder binding. Only a single GnRH gene is reported to be present in mouse, rat, and human genomic DNA based on Southern blots (see Bond and Adelman, 1993). However, the presence of GnRH-I1 in a placental mammal (musk shrew, Dellovade et al., 1993) marsupials, and monotremes suggests that a second GnRH gene will be found for more mammalian species. In fish, two distinct GnRH cDNAs have been isolated from one species, African catfish ( C . gariepinus); the cDNAs

1.

MOLECULAR EVOLUTION OF

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45

were catfish GnRH and cGnRH-I1 (Bogerd et al., 1994). Also, salmon, a tetraploid species, is reported to have two separate cDNAs, and presumably genes, for sGnRH (Ashihara e t al., 1993). These fish are known to have cGnRH-11, but neither the cDNA nor gene has been reported. The difficulty in detecting multiple GnRH genes on Southern blots even with low stringency conditions may reflect entensive changes in the nucleotide bases that have occurred over hundreds of millions of years in all regions of the GnRH cDNAs except the 39 bases coding for the hormone and processing site. Nonetheless, it should be possible eventually to construct a phylogenetic map for GnRH based on the distribution and gene structure of the eight or more forms of GnRH. 4. TISSUE AND DEVELOPMENTAL EXPRESSION Northern blot analysis of poly (A)+ mRNA for sGnRH from African cichlid (Bond et al., 1991)and masu salmon (Suzuki et al., 1992)brains showed the presence of a single RNA species of approximately 600 nucleotides. The GnRH mRNA was detected in tissue from whole brains (Bond et al., 1991) or from two brain regions: the diencephalon and the telencephalon with attached olfactory bulbs (Suzuki et al., 1992). Also, expressed mRNA for sGnRH from the hypothalamus of Atlantic salmon has been detected using PCR (Klungland et al., 1992b). Likewise, PCR was used to show that mRNA for catfish GnRH and chicken GnRH-I1 from the African catfish Clarias gariepinus brains was present (Bogerd et al., 1994). In addition to the brain, mRNA for mammalian GnRH has been detected in the placenta, ovary, and spleen lymphocytes of mammals (see Table 11). There are reports that immunoreactive GnRH can also be detected in pancreas (Seppala and Wahlstrom, 1980a), mammary gland tumors (Seppala and Wahlstrom, 198Ob), and breast carcinoma cell lines (Harris et al., 1991). In fish, the expression of GnRH outside of the brain has been demonstrated only for the retina using immunocytochemistry (Stell et al., 1984; Subhedar and Rama Krishna, 1988). Developmental studies in fish have provided only indirect evidence that GnRH neurons migrate from the olfactory placode toward the brain. During development in a teleost such as platyfish, the GnRH neurons are detected first in an anterior brain region, the nucleus olfactoretinalis, later in the nucleus praeopticus periventricularis, and finally in the more posterior region of the nucleus lateralis tuberis near the pituitary stalk (Halpern-Sebold and Schreibman, 1983). One interpretation of these data is that the GnRH neurons are moving from an anterior to posterior direction during development of the brain.

46

NANCY M. SHERWOOD ET AL.

Likewise, in immature salmon (30-70 g) compared to l-year-old maturing salmon (240-400 g), sGnRH mRNA is expressed only in the anterior regions, whereas in the older salmon there are intense in situ hybridization signals for sGnRH mRNA in brain regions that are both anterior (olfactory nerve and ventral olfactory bulb) and posterior (ventral telencephalon and preoptic areas) (Suzuki et al., 1992). It is not clear ifthis distribution in immature fish results from incomplete migration of GnRH neurons from the anterior to the posterior areas or whether the GnRH neurons are in place in the telencephalon and preoptic areas but do not fully express GnRH. Direct evidence for migration of GnRH neurons is needed for fish. The idea that GnRH migration occurs in fish is based on direct evidence from developmental studies in tetrapods showing that GnRH neurons originate in the olfactory placode (Schwanzel-Fukuda and Silverman, 1980; Schwanzel-Fukuda et al., 1985; Wirsig and Leonard, 1986; Muske and Moore, 1988; Wray et al., 1989a,b; SchwanzelFukuda and Pfaff, 1989; Daikoku-Ishido et al., 1990; Norgren and Lehman, 1991; Murakami et al., 1992). Additional proof for the migration theory has been obtained from humans, in which the genetic disease known as Kallmann’s syndrome results in the failure of the reproductive system to mature because the GnRH neurons do not migrate from the olfactory placode into the brain (Schwanzel-Fukuda et al., 1989). The deleted gene (KALIG-1) has been shown to normally produce a protein product that is closely related in structure to a neural cell adhesion molecule, which is thought to be essential for neuron migration (Ballabio et al., 1989; Franco et al., 1991).

D. Questions Regarding Localization and Function of GnRH Evidence that two or more forms of GnRH are present in most species of fish (see Sherwood et al., 1993b) suggests that distinct forms of GnRH may differ in function. Future studies will require not only determination of the number of GnRH forms in a species, but also use of appropriate antisera or probes to distinguish the location of the GnRH types within the brain of a single species. Early studies using extracts from different regions of the brain showed that there is a differential but overlapping location of sGnRH and cGnRH-I1 in the goldfish brain, but the technique could not determine if cell bodies or fibers were the source of GnRH (Yu et ul., 1988). A study with specific antisera to these two forms of GnRH suggests that the masu salmon (0.masou) has sGnRH neurons in the anterior brain with

1.

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47

axons extending to the pituitary and many brain areas, but cGnRH-I1 neurons in the midbrain with fibers also extending to many brain regions, but not to the pituitary (Amano et al., 1991). However, it appears the expression GnRH varies with the species. Goldfish (C. aurata) have both sGnRH and cGnRH-I1 fibers in the pituitary (Yu et al., 1988; Kobayashi et al., 1992) and catfish (C. gariepinus) have cfGnRH and cGnRH-I1 fibers in the pituitary (Zandbergen et al., 1992); but masu salmon (0.rnasou) appear to have only sGnRH in axons that end in the pituitary (Amano et al., 1991). These findings share a dilemma with all immunological studies in that the antisera showed some cross-reactivity with other forms of GnRH. In the study b y Amano and associates, the antiserum against cGnRH-I1 has 16% cross-reactivity with salmon GnRH. A more acceptable method for specificity in GnRH studies is to localize GnRH neurons by in situ hybridization. A probe designed to match a unique part of the GAP sequence would clearly distinguish different forms of GnRH. One such study used a probe specific for sGnRH mRNA to confirm that sGnRH is localized as described in the foregoing and in the olfactory nerve (Suzuki et al., 1992). Another study in salmon is needed to localize cGnRH-I1 by in situ hybridization. Localization of GnRH cell bodies and their axon terminals containing different forms of GnRH in specific brain regions is assumed to be the key for deducing hypotheses about novel GnRH functions that can be tested experimentally. The critical question is whether multiple GnRH forms have discrete functions and are differentially regulated. Although two forms of GnRH are present in the catfish pituitary, the role of these GnRHs is not known (Zandbergen et al., 1992; Schulz et al., 1993; Bogerd et ul., 1994).

E. Evolution of GnRH and GHRH Families 1. EVOLUTION OF THE GNRH FAMILY The studies in Table IV show that chicken GnRH-I1 is distributed throughout cartilaginous and bony fish, but has not been proven to exist in jawless fish. Salmon GnRH is found only in teleosts. The lamprey GnRH forms are confirmed for lamprey, but only tentatively in teleosts. Dogfish and catfish GnRH are confirmed only for their respective species to date. Mammalian GnRH has been isolated and sequenced from sturgeon (D. W. Lescheid, J . F. F. Powell, 0. Bukovskaya, I. Barannikova, W. H. Fischer, and N. M. Sherwood, personal

48

NANCY M. SHERWOOD ET AL.

communications) and has also been detected by cross-reactivity in other primitive bony fish (Shenvood et al., 1991) and eel (Anguilla anguilla) ( J . A. King et al., 1990). Chicken GnRH-I has not been isolated and sequenced from any fish. A phylogenetic map has been deduced from these studies (Fig. 12) using both the known distribution of GnRH forms among species and the principle of parsimony. Earlier reports suggested that the ancestral GnRH molecule may have been closer to lamprey GnRH-I, which is the outlier compared to other known forms of GnRH (Sower et al., 1993), or that the ancestral GnRH was closer to cGnRH-I1 based on a greater reliance on parsimony (Grober, 1993).These schemes, however, must be rooted in the observed distribution of GnRH and not exclusively in parsimony. Salmon and dogfish GnRH may differ by only one amino acid, which is important for parsimony considerations, but distribution studies to date suggest that neither peptide resulted from a single amino substitution in the other peptide (Fig. 12). Rather, dogfish GnRH has been observed only in the elasmobranch subdivision of cartilaginous fish and salmon GnRH appears only in the teleostean subdivision of bony fish and apparently only when mammalian GnRH disappears. Hence parsimony has to be overridden in favor of the suggestion that dogfish GnRH was derived from chicken GnRHI1 and salmon GnRH is derived from mammalian GnRH. Both parsimony and distribution arguments support the idea that chicken GnRHI has evolved from mammalian GnRH; there is only one amino acid difference between them and, also, mammalian GnRH is present in primitive bony fish, amphibians, and mammals, but disappears in all reptiles and birds when chicken GnRH-I appears (Fig. 12). Catfish GnRH could have evolved from chicken GnRH-I1 (two amino acids different), from mammalian (two amino acids different), or from salmon (three amino acids different) based on the distribution of the peptides and known phylogenetic history of the fish. In Fig. 12, catfish GnRH is shown as evolving from mammalian GnRH, but more proof is needed. To further elucidate the evolution of GnRH, it remains to be determined whether (1)GnRH can be isolated and sequenced from invertebrates, (2) the structure of invertebrate GnRH is closer to cGnRH-I1 or lamprey GnRH, ( 3 ) known forms are present in other species of fish, (4)mGnRH arose in primitive bony fish from an unknown ancestral gene or from cGnRH-I1 because of a gene or exon duplication of the cGnRH-I1 gene followed b y point mutations, (5) catfish GnRH arose from m, c-11, or sGnRH, and (6)other novel forms of GnRH exist in fish.

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M O L E C U L A R EVOLUTION OF

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2. COMPARATIVE EVOLUTION OF GNRH AND GHRH The neuropeptide GnRH is part of a family, whereas GHRH is part of an expanded superfamily. The basis of this statement is that proteins within a family usually differ at fewer than half of their amino acid positions (Dayhoff, 1976). The eight known forms of GnRH do not differ from one another by more than 50% of their amino acids, but the GnRHs are not related to other peptides beyond the decapeptide family. This classification comes primarily from vertebrate studies, although immunoreactive GnRH has been reported for a protochordate (Georges and Dubois, 1980; Dufour et al., 1988; Kelsall et al., 1990). Also, there is some sequence similarity between GnRH and yeast a mating factor (Loumaye et al., 1982), but GnRH-like molecules in animals that evolved after yeast and before vertebrates have not been directly identified. In contrast, GHRH as part ofthe glucagon superfamily is related to a number of families including PACAP, glucagon, VIP, and PHM/PHI that have evolved from an ancestral gene through successive gene duplication. The structures of the GnRH peptides and genes compared to those of GHRH are more highly conserved in vertebrate evolution. The length of GnRH apparently does not change between lamprey (Petromyzon marinus) and humans and 50% or more of the amino acids are conserved. In contrast, the GHRH peptides vary from 42 residues in mice to 45 in salmon and possibly 48 in catfish. The question is whether fish GHRH-like molecules belong to the mammalian GHRH family if a family is defined as 50% amino acid conservation. The answer depends on whether the full-length molecule of 45 amino acids or the biologically active core of 29 amino acids is compared to the mammalian GHRHs. It is true that salmon GHRH,-,, is only 41% conserved compared with human GHRH,-,,, but salmon GHRH,-,, is 55% similar compared with human GHRH. The comparison of these truncated molecules may be more meaningful because hGHRH,-,,NH, contains full intrinsic activity and potency in vitro (Rivier et al., 1982) and because the salmon and human truncated molecules are each encoded on one exon. The variable part of the GHRH peptides is in the Cterminal region encoded on a separate exon. It is difficult to determine whether salmon GHRH is closer to the GHRH family or the PHI and PHM family. The N-terminal amino acids of the salmon GHRH-like molecule share 59% similarity (16 of 27 amino acids) with PHI (rat and pig), 67% (18 of 27 amino acids) with PHM (human), and 55% (16 of 29 amino acids) with GHRH (human). Therefore, the dividing line between the families is not yet

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clear if based only on a structural comparison. The function and receptor binding of the peptides are certainly important in determining family membership. The ability of GnRH and GHRH to release pituitary hormones in mammals is well established. Whether fish with multiple forms of GnRH in the brain use one or more forms of GnRH to release gonadotropins is not clear and may vary with the species. Proof that the GHRHlike molecule releases GH in fish is still limited (Vaughan et al., 1992; Luo et al., 1990), and further studies are essential to prove that a second GHRH is not needed to explain GH release. A comparison of expression of GnRH and GHRH/PACAP in tissues outside of the brain in fish is important as a first step in the study of' functions other than the release of pituitary hormones.

IV. INTERTWINING OF FUNCTION IN THE GnRH AND GHRH FAMILIES There appears to be some crossover in the functions of the GnRH and GHRH/PACAP molecules. In fish, but not mammals, GnRH is reported to release GH from the pituitary. Several forms of GnRH have been tested in vitro and found to effectively release GH; the forms were sGnRH, mGnRH, cfGnRH, and dfGnRH (Marchant et al., 1989; Ngamvongchon et al., 1992a; Lovejoy et al., 199213). In turn, GHRH and PACAP may play a role in reproduction. Several studies have examined the effect of PACAP on the anterior pituitary, but the results depend on the experimental parameters. In some experiments PACAP stimulated the release of LH (Miyata et al., 1989; Hart et al., 1992; Osuga et al., 1992), but not in others (Miyata et al., 1989; see Arimura, 1992a,b).Also, PACAP was shown to enhance the release of LH and FSH if given with mGnRH in static cell cultures (Culler and Paschall, 1991). GHRH and PACAP may act as local hormones in the gonads. In catfish ( C . macrocephalus) testes, an mRNA transcript for GHRH/ PACAP has been detected ( J . E. McRory, D. B. Parker, S. Ngamvongchon, and N. M. Sherwood, personal communication). In mammals, both GHRH and PACAP are present in the ovary and testes. GHRH mRNA is expressed in the rat testis (Berry and Pescovitz, 1990) and ovary (Bagnato et al., 1992).Within the testes, immunoreactive GHRH was localized in both early and mature sperm cells (Pescovitz et ul., 1990; Srivastava et al., 1993). Receptors for GHRH are also present in gonads. For example, in granulosa cells GHRH binds to a GHRH/

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VIP receptor and enhances FSH-induced CAMPformation (Moretti et al., 1990a). The presence oflarge amounts of PACAP in the testes (the total content of one testis is equal to that of the brain; Arimura et al., 1991) and the presence of highly specific PACAP receptors in spermatogonia and mature spermatozoa (see Arimura, 1992a,b) suggest a role in reproduction. Immunoreactive PACAP is also present in rat ovaries, although the concentration is considerably lower than in the brain or testis (Arimura et al., 1991); ovarian receptors for PACAP are type I1 in which both PACAP and VIP bind (see Arimura, 1992b). Thus, it is possible that GHRH, GHRH-like, and PACAP molecules have an important paracrine or autocrine role in the gonads during development and reproduction.

ACKNOWLEDGMENTS The research described in this chapter was supported by the Medical Research Council of Canada (NSERC and Department of Fisheries and Oceans Grant). We also gratefully acknowledge the financial assistance of a British Columbia Science Council G.R.E.A.T. Fellowship (to D.B.P.) and King-Platt Fellowships (to D.B.P. and D.W.L.).

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Schwanzel-Fukuda, M., Morrell, J. I., and Pfaff, D. W. (1985).Ontogenesis of neurons producing luteinizing hormone-releasing hormone (LHRH) in the nervus terminalis of the rat. J. Cornp. Neurol. 238, 348-364. Schwanzel-Fukuda, M., Bick, D., and Pfaff, D. W. (1989). Luteinizing hormonereleasing hormone (LHRH)-expressingcells do not migrate normally in an inherited hypogonadal (Kallman) syndrome. M o l . Bruin Res. 6, 311-326. Seeburg, P. H., and Adelman, J. P. (1984).Characterization of cDNA for precursor of human luteinizing hormone releasing hormone. Nature (London) 311, 666-668. Seppala, M., and Wahlstrom, T. (1980a).Identification of luteinizing hormone-releasing factor and alpha-subunit of glycoprotein hormones in human pancreatic islets. Life sci. 27, 395-397. Seppala, M., and Wahlstrom, T. (l980b).Identification of luteinizing hormone-releasing factor and alpha subunit of glycoprotein hormones in ductal carcinoma of the mammary gland. l n t . J . Cancer 26,267-268. Shenvood, N. M . (1986). Evolution of a neuropeptide family: Gonadotropin-releasing hormone. Am. 2001.26, 1041-1054. Sherwood, N. M., and Harvey, B. (1986).Topical absorption of gonadotropin-releasing hormone (GnRH) in goldfish. Gen. Comp. Endocrinol. 61, 13-19. Shenvood, N. M., and Lovejoy, D. A. (1993).Gonadotropin-releasing hormone in cartilaginous fishes: Structure, location, and transport. Enciron. B i d . Fishes 38, 197-208. Sherwood, N. M . , and Sower, S. A. (1985). A new family member for gonadotropinreleasing hormone. Neuropeptides 6, 205-214. Sherwood, N., Eiden, L., Brownstein, M., Spiess, J., Rivier, J., and Vale, W. (1983). Characterization of a teleost gonadotropin-releasing hormone. Proc. Natl. Acad. Sci. U.S.A.80, 2794-2798. Sherwood, N. M., Harvey, B., Brownstein, M. J., and Eiden, L. E. (1984).Conadotropinreleasing hormone (Gn-RH) in striped mullet (Mugil cephalus), milkfish (Chanos chanos), and rainbow trout (Salrno gairdneri): Comparison with salmon Gn-RH. Gen. Comp. Endocrinol. 55, 174-181. Sherwood, N. M., Sower, S. A., Marshak, D. H., Fraser, B. A,, and Brownstein, M. J. (1986). Primary structure of gonadotropin-releasing hormone from lamprey brain. I . Biol. Chem. 261,4812-4819. Sherwood, N. M., Doroshov, S., and Lance, V. (1991).Gonadotropin-releasing hormone (GnRH) in bony fish that are phylogenetically ancient: Reedfish (Calumoichthys calaburicus), sturgeon (Acipenser transrnontanus). and alligator gar (Lepisosteus spatula). Gen. Comp. Endocrinol. 84,44-57. Sherwood, N. M., Grier, H. J . , Warby, C., Peute, J., and Taylor, R. G . (1993a). Gonadotropin-releasing hormones, including a novel form, in snook Centropornus undecirnalis, in comparison with forms in black sea bass Centropristis striutu. Regul. Pept. 46,523-534. Sherwood, N. M., Lovejoy, D. A., and Coe, I. R. (1993b). Origin of mammalian gonadotropin-releasing hormones. Endocr. Reu. 14, 241-254. Shih, S. H., Yu, K. L., and Peter, R. E. (1988).Molecular forms of gonadotropin-releasing hormone in the brain areas of Gyrnnothorux fimbriatus (Bennett). Program, 1st International Symposium Fish Endocrinology. Edmonton, Alberta, Canada, June 12-17, p. 44. (abstract). Sower, S. A., Chiang, Y.-C., Lovas, S . , and Conlon, J . M. (1993).Primary structure and biological activity of a third gonadotropin-releasing hormone from lainprey brain. Endocrinology (Baltimore)132, 1125-1131.

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Spangelo, B. L., Judo, A. M., Isakson, P. C., and MacLeod, R. M . (1989).Interleukin6 stimulates anterior pituitary hormone release in uitro. Endocrinology (Baltimore) 125,575-577. Spiess, J., Rivier, J., and Vale, W. (1983).Characterization of rat hypothalamic growth hormone releasing factor. Nature (London)303, 532-534. Srivastava, C. H., Collard, M. W., Rothrock, J. K., Peredo, M. J., Berry, S. A., and Pescovitz, 0. H . (1993). Germ cell localization of a testicular growth hormonereleasing hormone-like factor. Endocrinology (Baltimore) 133, 83-89. Stachura, M. E., Dhariwal, A. P. S., and Frohman, L. A. (1972).Growth hormone synthesis and release in uitro: Effects of partially purified ovine hypothalamic extract. Endocrinology (Baltimore)91, 1071-1078. Stell, W. K. (1984). Luteinizing hormone-releasing hormone (LHRH)- and pancreatic polypeptide (PP)-immunoreactive neurons in the terminal nerve of the spiny dogfish, Squalus ucanthias. Anat. Rec. 208, 173A-174A (abstract). Stell, W. K., Walker, S. E., Chohan, K. S., and Ball, A. K. (1984). The goldfish nervus terminalis: A luteinizing hormone-releasing hormone and molluscan cardioexcitatory peptide immunoreactive olfactoretinal pathway. Proc. Natl. Acnd. Sci. U.S.A. 81,940-944. Subhedar, N., and Rama Krishna, N. S. (1988).Immunocytochemical localization of LHRH in the brain and pituitary of the catfish, Clurius bntruchus (Linn). Gen. Comp. Endocrinol. 72,431-442. Suhr, S. T., Rahal, J. O., and Mayo, K. E. (1989). Mouse growth hormone releasing hormone: Precursor structure and expression in brain and placenta. Mol. Endocrinol. 3,1693-1700. Suzuki, M., Hyodo, S., Kobayashi, M., Aida, K., and Urano, A. (1992). Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the m a w salmon. J . Mol. Endocrinol. 9, 73-82. Tan, L., and Rousseau, P. (1982). The chemical identity of the immunoreactive LHRHlike peptide biosynthesized in the human placenta. Biochem. Biophys. Res. Commun. 109, 1061-1071. Tatsuno, I., Somogyvsri-Vigh, A,, Mizuno, K., Gottschall, P. E., Hidaka, H., and Arimura, A. (1991). Neuropeptide regulation of interleukin-6 production from the pituitary: Stimulation by pituitary adenylate cyclase activating polypeptide and calcitonin gene-related peptide. Endocrinology (Baltimore) 129, 179771804, Uddman, R., Luts, A,, Arimura, A., and Sundler, F. (1991). Pituitary adenylate cyclaseactivating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract. Cell Tissue Res. 265, 197-201. Vaughan, 1. M., Rivier, J . E., Spiess, J., Peng, C . , Chang, J. P., Peter, R. E., and Vale, W. W. (1992). Isolation and characterization of hypothalamic growth hormone-releasing hormone from common carp, C yprinus carpio. Neuroendocrinology 56, 539-549. Vigh, S., Arimura, A., Koves, K., Somogyvari-Vigh, A,, Sitton, J., and Fermin, C. D. (1991). Immunohistochemical localization of the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP),in human and primate hypothalamus. Peptides 12,313-318. Wirsig, C . R., and Leonard, C . M. (1986).The terminal nerve projects centrally in the hamster. Neuroscience (Oxford) 19, 709-717. Wray, S., Grant, P., and Gainer, H. (1989a). Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc. Natl. Acud. Sci. U.S.A. 86, 8132-8136.

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311-321. Yada, T., Vigh, S., and Arimura, A. (1993).Pituitary adenylate cyclase activating polypeptide (PACAP) increases cytosolic-free calcium concentration in folliculostellate cells and somatotropes of rat pituitary. Peptides 14,235-239. Y u , K. L., Sherwood, N. M., and Peter, R. E. (1988). Differential distribution of two molecular forms of gonadotropin-releasing hormone in discrete brain areas of goldfish (Carassius auratus). Peptides 9, 625-630. Zandbergen, M . A., Peute, J., Verkley, A. J., and Goos, H. J. Th. (1992). Application of cryosubstitution in neurohormone- and neurotransmitter-immunocytochemistry. Histochemistry 97, 133-139. Zentel, H. J., Jennes, L., Heinboth, R., and Stumpf, W. E. (1987).Ontogeny of gonadotropin releasing hormone and gonadotropin immunoreactivity in brain and pituitary of normal and estrogen-treated guppies, Poecilia reticulata Peters. Cell Tissue Res.

249,227-234.

2 C O R T I C O T R O P I N - R E L E A S I N G FACTORS A C T I N G O N THE F I S H PITUITARY: E X P E R I M E N T A L A N D M O L E C U L A R ANALYSIS K . LEDERIS Department of Phar~nacologyand Therapeutics University of Calgary, Calgary, Alberta, Canada

J. N . FRYER AND Y. OKAWARA Department of Anatomy and Neurobiology University of Ottawa, Ottawa, Ontario, Canada

CHR. SCHONROCK A N D D. RICHTER Institut fiir Zellbiochemie und klinische Neurobiologie Universitats-Krankenhaus Eppendorf, Universitit Hamburg 20246 Hamburg, Federal Republic of Germany

I. Introduction 11. ACTH-Releasing Peptides and Their Receptors A. The Peptides B. CRF Receptors C . CRF Receptor Localization and Distribution 111. CRF, Its Protein Precursors, cDNAs, and Genes A. CRF Precursors and Genes B. Localization of CRF-UI-AVT in Sucker and Goldfish Central Nervous System C. CRF mRNA in Sucker and Goldfish Brain D. CRF-UI Genes IV. Evolutionary Considerations for CRF-UI References 67 FISH PHYSIOLOGY, VOI,. XI11

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I. INTRODUCTION An understanding of the interplay between components of the hypothalamic-pituitary-adrenal (interrenal) [HPA(I)] system is not as well defined for nonmammalian vertebrates as for mammals. Numerous publications have appeared on the mammalian HPA( I ) system, especially after the discovery of a 41-residue corticotropin-releasing factor (CRF) from sheep hypothalamic extracts (Vale et al., 1981).Vale and colleagues were modest in drawing readers’ attention to the notion that this CRF may or may not be the definitive hypothalamic hormone that regulates corticotropin (ACTH) secretion, in view of a 30-year search for CRF. Quite fittingly, since the recommendation by Schally and colleagues (Patthy et al., 1985), there is now a general consensus that this 41-residue CRF peptide is the corticotropin-releasing hormone (CRH). One of the main reasons for the long-lasting search for the CRH was the widely held expectation that this putative hypothalamic hormone would be another small peptide like all the other hypothalamic hormones previously identified. The intensive search for CRH started on the strength of classical endocrinological experiments. These experiments, including ablation of the “gland” and interruption of its blood supply from the pituitary portal vessels, were carried out by the English physiologist Geoffrey Harris (1948). During the 1950s and 1960s, many leading laboratories reported the isolation of ACTH-releasing “factors” of 1-2 kDa (for the historical background, see Saffran and Schally, 1977). The term CRF was first used by Saffran et al. (1955) to designate the active ACTH-releasing substance in their extracts. Apart from two early reports that CRF may be a larger peptide (Slusher and Roberts, 1954; Porter and Rumsfeld, 1959),other reports did not appear until a recent one showing that CRF may be a peptide of about 4 kDa. Interestingly, among these reports was one from Vale and Rivier (1977) on the purification of a large 4-kDa peptide from an extract of approximately a half million sheep hypothalami. The 4-kDa substance was subsequently purified, sequenced, and determined to be a 41-residue ovine CRF by Vale and colleagues (Vale et al., 1981). George Fink (1981),commenting in Nuture immediately after the publication of this report on CRF (Vale et al., 1981), suggested that the finding was likely to constitute a quantum leap in our understanding of the endocrine response to stress. The term stress had been coined earlier by Selye (1936) and was based on a “triad” of phenomena, that is, adrenocortical enlargement, involution of the thymolympathic

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complex, and bleeding gut ulcers in the rat. Fink concluded his appraisal of the newly reported CRF by stating that “only after the dust has settled will we know whether Vale and colleagues have indeed found the holy grail of neuroendocrinology.” Judging from the continuous stream of published work centered around CRH, Fink may not have exaggerated the significance of this discovery. In fishes, the identity of a physiological ACTH-releasing hormone has not been finalized, even though the structures of several CRF or CRF-like substances (peptides) have been identified, their actions determined, and their gene expression elucidated (for a review, see Lederis et at!., 1993).Therefore, the term CRF will be used throughout this review when describing piscine neuropeptides with corticotropinreleasing activities. The presence of corticotropin-releasing activity in extracts of the fish caudal neurosecretory system was initially observed by Roy (1962). Evidence for ACTH-releasing activity in extracts of goldfish hypothalami was obtained with goldfish pituitary cells in culture (Sage and Purrott, 1969) and in dexamethasone-blocked goldfish in t h o (Fryer and Peter, 1977a). The localization of CRF production, as well as the center for the control of ACTH secretion, was ascribed to the nucleus preopticus (NPO) and nucleus lateral tuberalis (NLT) of the goldfish hypothalamus (Fryer and Peter, 1977b). After the discovery of ovine hypothalamic CRF, urotensin I (UI), a homologlie ofCRF, was isolated from the urophysis of the sucker Catostomus commersoni (Lederis et ul., 1982) and the carp Cyprinus carpi0 (Ichikawa et al., 1982). Investigations of CRF physiological mechanisms in teleosts were carried out with synthetic CRF or CRF-like peptides (Fryer, 1989). In addition, the components controlling interrenal function were elucidated by the identification of teleostean CRF deduced from the threemembered fish CRF gene family, that is, CRF1, CRF2, and UI (Okawara et al., 1988; Morley et ul., 1991).

11. ACTH-RELEASING PEPTIDES AND THEIR RECEPTORS A. The Peptides A list of known CRF and CRF-like peptides has been presented by Lederis et al. (1993). In the interim, at least three more CRF/UIlike insect peptides have been identified, complementing the tobacco hornworm moth Manduca serta diuretic peptide (DPI). These include

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l Goat CRF

L Bovine CRF Human CRF Rat CRF

l-h

Pig CRF Minor Whine Sucker CRF 1 White Sucker CRF 2 Pig CRF Major

Xenopus laevis CRF 1 / 2 Carp Urotensin I

White Sucker Urotensin 1

Flounder Urotensin I Sole Urotensin I Frog Sauvagine

1

Tobacco Hornworm Diuretic Hormone II

Fig. 1. Dendrogram of CRF and CRF-related peptide sequences. Alignment of thc following sequences was performed with the aid of the “pileup” program (gapweight 3.0; gaplengthweight 0.1) from the Genetics Computer Group software package: goat CRF (Ling et al., 1984), sheep CRF (Vale et a/., 1981), bovine CRF (Esch et u / . , 19841, human CRF (Shibahara et a / . , 1983), rat CRF (Rivier et ( I / . , 1983),pig minor and major

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(1)a diuretic peptide (DPII) isolated and characterized from Mundzcca sexta (Blackburn et al., 1991), (2) a peptide closely homologous with DPI and DPII from the locust Locusta rnigratoria (Lehmberg et nl., 1991), and ( 3 )a 46-residue DP peptide from the cockroach Periplaneta urnericana (Kay et al., 1991). Whatever phylogenetic or functional relationships exist between the vertebrate peptides of the CRF/UI family and the insect DP peptides, innumerable other bioactive peptides homologous with the CRF/UI family are likely to be found in the not too distant future, given the huge number of extant invertebrates. It seems likely that further homologues and analogues will be determined based on the wide distribution of other peptide hormones from prokaryotes to complex forms of animal life (LeRoith et al., 1980). Among the CRF peptides and their genes in vertebrates, one of the “missing links” has now been filled. The peptide or gene structure of CRF was only known for several mammalian and one teleostean fish species. The structures of two highly homologous CRF genes have now been characterized in an amphibian (Xenopus laevis). The predicted amino acid sequences in the two gene products for the mature 41-residue X . Zaevis CRF peptides is the same. The peptides differ from both the human CRH and the major form of the teleost (sucker). CRF in three positions: a substitution ofAla for Ser in position 1, Ile for Leu in position 27, and Asp for Glu in position 39 (StenzelPoore et al., 1992). The structure of the X . laevis CRF expands the short list of authentic CRF peptides in vertebrates and confirms the conservation of structure in fish, amphibians, and mammals, including humans (Fig. 1). Despite the temptation to further speculate on the evolution of the hormonal cascade controlling the steroidogenic activity of the adrenals in general, and of fish interrenal cells in particular, it is advisable to be cautious. Given our present ability to make significant progress rapidly, it is quite likely that advances in CRF gene structure and function will be available shortly. This is likely to be matched by new information, in parallel, on CRF receptor structure, gene expression, CRFs (Patthy et al., 1985),sucker CRFs (Morley et al., 1991),carp urotensin I (Ichikawa et al., 1982), sucker urotensin I (Lederis et a/., 1982),flounder urotensin 1 (Conlon et d., 1990),sole urotensin I (McMaster et al., 1988), frog SVG (Montecucchi et ul., 1979), and tobacco hornworm diuretic hormone I (Kataoka et a/., 1989), Loczlsta migratoria diuretic hormone (Lehmberg et aZ., 1991), and tobacco hornworm diuretic hormone I1 (Blackburn et al., 1991). On the dendrogram, distance along the horizontal axis is proportional to the differences between sequences, whereas the distance along the vertical axis has n o significance.

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and function. It follows that elucidation of changes during evolution as well as of mechanisms controlling the HPA(1) axis in vertebrates and other forms of animal life will alleviate the need for guesswork. Whereas the expression of at least two CRF genes in the sucker and the toad is explainable based on genomic tetraploidy in both (Ferris and Whitt, 1978; Kobe1 and Pasquier, 1986), a number of biological and physiological questions cannot be answered without additional experimental evidence. Although the function of the two CRF peptides in the sucker has not been tested, the a priori assumption is that one amino acid substitution (Ala/Val in position 28) in these sucker CRF peptides may not affect receptor binding or potency to a significant degree. Only if there were an important difference in the activity would there be a need to investigate the degree of expression of the two genes. The precise function of the definitive CRH will require identification ofthe “effector (hormone),receptor and feedback components linked to mechanisms for processing, transport, storage, secretion and metabolism of all the components” (Niall, 1982).

B. CRF Receptors Evidence for the existence and localization of CRF receptors was first indicated when extracts of the fish caudal neurosecretory system were shown to increase the concentration of fish corticosteroid in the plasma, presumably by stimulating pituitary ACTH secretion (Roy, 1962). The presence of ACTH in the fish pituitary had been indirectly proven by the ascorbic acid depletion test (Rinfret and Hane, 1955). In retrospect, the findings by Roy (1962) had already shown, unknowingly, the occurrence of a substance in the caudal neurosecretory system with homology to CRF. Only when synthetic U I became available and its CRF-like activity was compared with that of the ovine CRF did its close structural homology with mammalian CRF become apparent (Lederis et al., 1982; Vale et al., 1982). Soon thereafter, the potency of UI was compared with ovine CRF and the amphibian skin CRF-like peptide, sauvagine (SVG), in dispersed and superfused anterior pituitary cells of the goldfish (Fryer et al., 1983). Neurosecretory cells of the teleost neurohypophyseal system produce arginine vasotocin (AVT) and isotocin (IST), which also affect ACTH secretion. AVT and IST are nonapeptides structurally homologous to arginine vasopressin (AVP) and oxytocin of the mammalian neurohypophyseal system. In mammals, AVP stimulates the release of ACTH (see Rivier and Vale, 1983). In dexamethasone-blocked goldfish, AVT, IST, and AVP induce cortisol secretion (Fryer and Leung,

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1982). I n vitro AVT and IST are virtually equipotent, but about three times more potent than AVP in stimulating the release of ACTH from superfused goldfish anterior pituitary cells (Fryer and Lederis, 1986). However, compared to ovine CRF or UI, the AVT, IST, and AVP peptides demonstrate a lower intrinsic activity for stimulation of ACTH release (Fig. 2). The maximum ACTH-releasing activity of ovine CRF or UI is about twice that of the neurohypophyseal nonapeptides. Mammalian studies investigating the role of AVP in the regulation of ACTH release have shown that the ACTH-releasing activity of ovine CRF is potentiated severalfold by AVP (Rivier and Vale, 1983). In contrast, investigations of goldfish anterior pituitary cells undertaken to determine possible interactions of AVP, AVT, and IST on ovine CRF- and UI-induced ACTH release in citro revealed no such potentiation (Fryer et aZ., 1985).These observations suggest different mechanisms for the actions of the neurohypophyseal nonapeptides in stimulating ACTH release in teleosts and mammals. In goldfish, it appears that the neurohypophyseal peptides stimulate the release of ACTH from the corticotropes directly, whereas in mammals, such as the rat, the ACTH-releasing activity of the neurohypophyseal peptides is mediated indirectly through a potentiation of the ACTH-releasing activity of CRF (Rivier and Vale, 1983). Figure 2 summarizes the ACTH-releasing activities of UI, some CRFs, and some neurohypophyseal peptides on superfused goldfish pituitary cells. Interestingly, the CRF-like peptides, UI, ovine CRF, and SVG, have been shown to be effective agents in stimulating the concomitant release of ACTH and a-MSH from superfused goldfish melanotropes (Tran et al., 1989, 1990). The relative potency of the neuropeptides in stimulating the release of ACTH and a-MSH from goldfish melanotropes was the same as that observed for the ACTH-releasing activity in corticotropes (i.e., UI was two to three times as potent as ovine CRF or SVG). A secretory response of the melanotropes upon stimulation with CRF-like peptides is a common feature of the pituitary in at least three vertebrate classes-Teleostei, Amphibia, and Mammalia. Ovine CRF was effective in stimulating the release of a-MSH from the rat pars intermedia (Meunier et al., 1982; Kraicer et al., 1986). However, it is not known if UI or SVG also possesses a-MSH-releasing activity in mammalian melanotropes. In amphibian X. Zaevis melanotropes, ovine CRF, UI, and SVG stimulate the release of a-MSH and endorphin (Verburg-van Kemende et al., 1987). In contrast, these peptides failed to evoke the release of hormone from the melanotropes of Rana ridibunda (Tonon et al., 1986). Investigations in salmonid species have revealed stress-induced elevations of a-MSH and ACTH

K. LEDEKIS ET A L .

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LOG PEPTIDE nM Fig. 2. ACTH-releasing activities of C R F s a n d urotensins. AVP, arginine vasopressin; AVT, arginine vasotocin; IST, isotocin; oCKF, ovine C R F ; r/h C R F , ratihumatr CRF; sCRF, sucker (Cutostornus) CRF; U1, sucker (Cntostomtts) UI.

in rainbow trout (Oncorhynchus mykiss) (Sumpter et ul., 1986) and of a-MSH, ACTH, and endorphin in brown trout (Snlmo truttn)(Sumpter et nl., 1985).The response is conceivably mediated by stress-induced CRF release. It appears that stimulation ofthe release of proopiomelanocortin-derived peptides from both teleost corticotropes and nielanotropes is mediated, at least in part, hy CRF-UI peptides. Mammalian studies have shown that human angiotensin I1 (hAII) stimulates the release of ACTH from the anterior pituitary (Sobel, 1983). Also, investigations in the goldfish have demonstrated that A11 has ACTH-releasing activity (Weld and Fryer, 1987). In goldfish anterior pituitary cells in zjitro, human A11 was a potent ACTH secretag o g u e and proved to be about 10 times more potent than sallnon A11

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or human angiotensin I (AI). Although A1 must be converted to A11 to effect ACTH release from mammalian corticotropes, the angiotensinconverting enzyme inhibitor captopril, which inhibits the conversioll of A1 to AII, was unable to block salmon AI-stimulated ACTH release from goldfish corticotropes. This finding suggests that the angiotensin receptor mediating ACTH release in the goldfish corticotrope is less discriminating than the mammalian receptor and binds both A1 and AII. Support for this concept resides in the observation that three sarcosine analogues of AII, which are antagonists of A11 in mammals, are ineffective in blocking human AII-stimulated ACTH release in the goldfish. Several studies in mammals have indicated that A11 may potentiate the ACTH-releasing activity of CRF. Concentrations of A11 that elicited modest increases in ACTH release from goldfish pituitary cells failed to potentiate the ACTH-releasing activity of ovine CRF or sucker UI (Weld et al., 1987; Weld and Fryer, 1988). These findings demonstrate that angiotensins have ACTH-releasing activity in goldfish corticotropes and suggest that the ACTH-releasing activity of angiotensinlike molecules was established early in the evolution of the vertebrate HPA(1) axis. Analysis of the CRF-like activity of UI in fishes and mammals and comparison of this activity of UI with its unique vasoactive properties in mammalian arterial systems suggests that there may be two or more distinct subtypes of the CRF receptor. T h e first proof is the hemodynamic effects of UI and of ovine CRF in mammals, where UI has been shown to have a uniquely specific vasodilatory activity exclusively in the superior (anterior) mesenteric artery region of the dog (MacCannell et al., 1982) with a potency higher than ovine CRF by a factor of about 20: 1. The second proof is that UI is equipotent with ovine CRF in ACTH release from cultured anterior pituitary cells of the rat, in 1300 and in vitru (Rivier et al., 1983), but there is significantly higher potency of UI than that of ovine CRF in the release of ACTH from superfused pituitary cells of the goldfish (Fryer et al., 1983). The third proof is that the UI-fragment, UI,-28 has no measurable ACTH releasing activity in the rat or the dog (C. Rivier and J. Cowan, respectively, personal communications), but has significant and specific mammalian mesenteric vasodilatory activity, comparable to that of ovine CRF (i,e.,about 5% of UI; Lederis et ul., 1985a), and is an interesting antagonist/partial agonist of pituitary ACTH secretion in the goldfish (Lederis et al., 1985b). Finally, the potency of the major synthetic fish CRF (sCRF1) on ACTH release from superfused goldfish anterior pituitary cells does not differ significantly from that ofovine CRF, but is only one-third to one-half that of UI (Fig. 2).

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The tentative conclusion, based on the preceding summary, is that at least two types of CRF receptors are present in mammals, but there is 110 evidence at present for two receptor types or classes in fish. Type one CRF receptor (CRF-RI) would be that in goldfish pituitary corticotropes and in mammalian mesenteric artery membranes for reasons of (a) high potency of UI as compared with ovine CRF and (b) similar responses in both systems to UI,-,8. Type two (CRF-RII) would be in mammalian corticotropes, because of (a) similar receptor recognition and potency for UI, ovine CRF (and sucker CRF), and SVG and (b) no receptor recognition for UI,_28. It seems at this time that a third receptor subtype (CRF-RIII) would be that of amphibian corticotropes. The amphibian CRF receptor does not recognize SVG or UI, but it binds and responds to ovine CRF and amphibian hypothalamic extracts, that is, presumably to amphibian CRF (Tonon et al., 1986). This tentative classification of CRF receptor subtypes cannot be analyzed further without experimental testing in appropriate vertebrate classes. The predicted conformation of CRF and some of the related peptides (e.g., UI, SVG) at their receptors would give virtually identical a-helical conformations and the equally close distribution of their hydrophobic and hydrophilic domains would be compatible with their overlapping biological activity (Snell, 1984). This would support a one receptor type for these various peptides but indicates several mechanisms for signal transduction and/or different mechanisms controlling postreceptor events. Indeed, biochemical characterization, which initially indicated that CRF receptors of markedly different properties were present in the mammalian pituitary and brain, is now consistent with the view that the ligand-binding part of the CRF receptors in both anterior pituitary and brain is a polypeptide of40-50 kDa (Grigoriadis and deSouza, 1989). Until information on the biochemical characterization ofCRF receptors in different tissues of various vertebrates can be related to the evidence for different receptor types summarized here, the existence of CRF receptor subtypes corresponding to the differential ligand binding properties cannot be excluded. At the same time, allowances have to be made that different receptor subtypes may utilize various postreceptor signaling pathways such as G-protein coupling, CAMP, protein kinase C, and also a host of many other “second messenger” systems. After all, the involvement of multifactorial input predicated by multiple homeostatic requirements of steroidogenesis would clearly require the requisite variables at all levels of the fish HPA(1) axis. The fish axis may not have any less complex physiological require-

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ments than the mammalian system (Aguilera et al., 1983, 1987; Owens and Nemeroff, 1991). C. CRF Receptor Localization and Distribution Direct experimental evidence on the characterization, localization, and distribution of CRF receptors in fish is still wanting. An extrapolation based on available information of localization of CRF/UI in the brain, pituitary, and peripheral tissues of fishes will at least provide some indication of the questions that need answers. The physiological roles and mechanisms of action are not yet known either for CRF or for UI, the latter originating from the neurons of the caudal and neurosecretory system and from the hypothalamuslmidbrain (Morley et al., 1991). Therefore, information available on either CRF or UI has to be treated as applying to CRF. Only chemical and biological characterization of CRF receptors in fish will make definitive studies on the receptor identity, distribution, and function possible. UI-specific binding sites in goldfish kept in fresh water or dilute seawater have been characterized with regard to ligand binding, to variations in binding in the osmotically challenged fish, and also to receptor specificity by means of competitive displacement of 1251labeled UI (Woo et al., 1985a,b; Lederis et al., 198513). Competitive displacement relationships between '"1-labeled UI and CRF do not exclude the possibility that these binding studies may have measured CRF-binding sites and thus indicated a possible role for fish CRF (Okawara et al., 1988; Morley et al., 1991) in osmoregulation as has been suggested previously (Bern et al., 1985). The tissue distribution of UI-binding sites observed in these experiments is not too dissimilar from the distribution and localization of CRF receptors in mammals (Dave et al., 1985; DeSouza, 1987). The involvement of CRF in the functioning of the immune system has been studied extensively in mammals (for review, see Owens and Nemeroff, 1991). Similar involvement of the HPA( I ) system has also been shown in the coho salmon (Tripp et al., 1987; Schreck and Bradford, 1990). As in much of the foregoing review of the CRF receptor properties and distribution, it remains to be seen if HPA( 1)-immune system interactions in teleost fishes will also show a localization of CRF receptors on different cellular elements of the immune system as is the case in mammals (for review, see Owens and Nemeroff, 1991). One of the intriguing questions in teleosts is the type of interaction between CRF nerve terminals and corticotrope cells in the pituitary. What kind of an effector-(CRF)-target cell (corticotrope) mechanism

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regulates the innervation of these and other adenohypophysial cells in teleosts: synaptoid? receptor-binding? postreceptor events? These are some of the basic avenues of fish neuroendocrinology that remain to be explored.

111. CRF, ITS PROTEIN PRECURSORS, cDNAs, AND GENES A. CRF Precursors and Genes Advances in molecular biology have led to the isolation of cDNAs and genes that encode CRF precursors, thus revealing the sequence and structural features of this family of proteins and the organization of' the corresponding genes. The precursors from ovine (Furutani et ul., 1983), human (Shibahara et al., 1983), rat (Jingami et al., 1985a), and sucker CRF (Okawara et al., 1988; Morley et al., 1991) and carp UI (Ishida et d., 1986)share a common structure consisting of a signal peptide, a cryptic peptide of variable length and unknown function, arid the hormone moiety (Fig. 3). The processing of these precursors is initiated by removal of the signal sequence. Cleavage sites for signal peptides are usually characterized by polar amino acids, especially small, neutral residues at positions 1 and 3 relative to the cleavage position (von Heijne, 1983, 1986). The amino acid residues surrounding the proposed signal cleavage sites for CRF and UI precursors indicated in Fig. 3 are consistent with the predictions of von Heijne. The processing signal, which is found between the cryptic peptide and the peptide hormone, and could be recognized by specific proteases in secretory vesicles, consists of two to four basic amino acids. All CKF and CRF-related peptides analyzed thus far are amidated C-terminally and, interestingly, the carboxy-terminal Gly and Lys residues that are found in all known precursor proteins form a typical processing signal, with the Lys residue as a proteolytic cleavage site and the Gly residue as an amidating signal. Alignment of human, rat, ovine, and sucker CRF precursor proteins exhibits varying degrees of identity to each other. Typical values are shown in Table I. Further analysis shows that the highest area of identity between the precursors is found in the hormone moiety (100% identity between rat and human sequences) and in the conserved motif in the cryptic region (93% identity between rat and human sequences). This well-conserved 10- to 13-amino-acid stretch (Fig. 3) is surrounded b y basic amino acids, which suggests that this short peptide could be

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White S u c k e r CRF 2 White Sucker CRF 1 Human CRF R a t CRF

...MRLnFLV t T m a L L V A F p ...MkLnFLV t T v a L L V A F p ...MRLpLLV SaGVLLVALl ...MRLrLLV SaGmLLVALs

.......... ...... D p d e .......... ..... .Dpdg pQHPQPMFF QPpPqsEQPQ ... P Q P L n F L Q P . . ..EQPQ sQHPQPLSFF Q P l P . . .QPQ i P G a 1 P t L & .T . P r d L S L M n s q l d d v l l n q a g d g a m s y l -QHPQPLSFF

QP -P ---QPQ

EPQ--PVL-R

120 ........RAL.. QLQLtQRvLE ........RAL.. QLQLtQRlLE

eaSqySk etsqypk sGSRpSpEqA rGSRpShDqA ssSRiSpDkv ..rnlgaqkA -GSR-S-D-A

tANFFRvLLQ AANFFRvLLQ AANFFRALLQ qqvLhlphFp AANFFRALLQ

QLlLPrRsLD QLQMPQRPLD .PrRPLD taQLhsphqD QLQLPQRPLD

...

SPrSpPdTYp SlrSSPdTYp SPaSSlLagg SPnStPLTag rAAPL S P a a S P L a s r

ibet .......... ..........

VGEEYFLRLG N

VGEklLqyLq MGEEYFLRLG N - N K - P W L

SP-SSPLTY-

gkvGNvgrWd gkvGNvgrWd Spaa.LAERG Sste.LAERG SpaG.pAkRG nsleeLtEFs S--GNLAERG

AP-ERERRSE

gnyALralds gnyALralds arnALGghQe aedALGghQg tenALGsr& APaaRk

................

---ALG--Q-

E P P I S L D L T F HLLREVLEMA RAEQLAQQAH SNRKMMEI E P P I S L D L T F HLLREVLEMA RAEQLAQQAH SNRKLMEI E P P I S L D L T F HLLREVLEMA RAEQLAQQAH SNRKLMEI E P P I S L D L T F HLLREVLEMA RAEQLAQQAH SNRKLMEIIG K

Fig. 3. Alignment of CRF and urotensin I precursor sequences. Alignment of sucker (Morley et al., 1991), human (Shibahara et al., 1983), rat (Jingami et uZ., 1985a), sheep CRF (Furutani et al., 1983), and carp urotensin I protein precursors (Ishida et d., 1986) was performed with the aid of the “pileup” program (gapweight 3.0; gaplengthweight 0.1) from the Genetics Computer Group software package (Devereux, 1989). Amino acids in capital letters are conserved in all precursor sequences (see consensus). The signal sequence cleavage site is indicated by a vertical arrow and amino acids typically surrounding the cleavage site are boxed (I). The conserved motif present in all CHF precursor structures is shown in box 11. The third and largest box denotes the mature CRF peptide.

released by monobasic cleavage from the precursor. A search of the Swiss-Prot data base (release 25.0,4/93)with the amino acid sequences ofthe conserved motifs of sucker, rat, sheep, and human CRFs revealed that by far the best homologies of these sequences were found to each other. Several groups of other proteins, such as DNA- and RNAprocessing enzymes, the regulatory chain of CAMP protein kinase type 11, the p-chain precursor of the acetylcholine receptor, and potassium channel proteins, showed a relationship to the conserved motif se-

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Table I Pairwise Alignment of C R F Protein Precursor Sequences“

White sucker CRFl Sheep C R F Rat CRF Human CRF

White sucker CRF

Sheep CRF

Rat CHF

100 46.3 54.0 51.2

100 68.3 77.2

100 81.8

Human CRF

100 ~~

~

The figures indicate percentage identities of the CRF precursors of various species as sucker (Morley et al., 1991),sheep (Furutani et ul., 1983),rat (Jingami et ul., l985a), and human (Shibahara et ul., 1983) compared with each other. T h e alignments were performed using the “gap” program (gapweight 3.0; lengthweight 0.1) of the Genetics Computer group software package (Devereux, 1989). “

quences. All the amino acid sequences corresponding to residues within the conserved motifs were clustered around a tyrosine residue in the first third of the conserved sequences. However, it is difficult to draw any conclusions from these sequence relationships, because these proteins have very different functions and locations within a cell. Because the conserved motifs do not have any relationship to known peptides or peptide precursors, no function can yet be assigned to these “potential peptides.” Two different cDNA molecules that encode the CRF-like precursors (sCRF1 and sCRF2) were cloned from a cDNA library constructed from the anterior hypothalamus (NP0)-midbrain region of the sucker. Both CRF precursors are very similar (93% at the DNA and 91% at the amino acid level). In the hormone moiety only one amino acid is substituted (N-terminal Val for Ala) between these sequences. The cDNA sequences for both sucker C R F precursors possess multiple polyadenylation signals, which is a characteristic feature for human (Shibahara et al., 1983),rat (Thompson e t ul., 1987),and sheep (Roche et d . ,1988)CRF genes. In sucker at least two of these signals appear to be used during RNA processing. Northern blot analysis using hypothalamic polyA+ RNA shows two mRNA transcripts that differ in length by -500 b p (1300 and 1800 bp) when probed with a sCRF 1cDNA fragment under conditions allowing cross-hybridization of s C R F l and sCRF2; the shorter transcript is more abundant than the longer one. It is noteworthy that this 500-bp difference in the length of these transcripts is reflected by the spacing of two polyadenylation sites of the sCRFl cDNA sequence (Morley et d . , 1991).

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B. Localization of CRF-UI-AVT in Sucker and Goldfish Central Nervous System Inimunocytochemistry has demonstrated the presence of CRF and UI immunoreactivities in the brain and caudal spinal cord of the sucker and goldfish. In the sucker, U I immunoreactivity was observed in all identifiable secretory cells of the caudal neurosecretory system, in their processes that projected to the urophysis, and in thin beaded fibers coursing along the spinal cord, brain stem, hypothalamus, and telencephalon (Yulis et ul., 1986). Perikarya reacting with an ovine CRF antiserum, but not with a UI antiserum, were visualized in the parvocellular and magnocellular regions of the NPO, and immunoreactive ovine CRF fibers were observed in the preoptic-neurohypophyseal tract and in the rostral pars distalis and neurointermediate lobe of the pituitary in close proximity to the corticotropes and melanotropes, respectively. Perikarya reacting with a UI antiserum, but not with an ovine CRF antiserum, were visualized in the NLT but not in the NPO. Thus the sucker hypothalamus contains two distinct yet structurally similar peptides, iminunoreactive to UI and ovine CRF, residing in neurons in separate regions, the NLT and NPO, respectively (Yulis and Lederis, 1986; Yulis et ul., 1986). Immunocytochemical studies in the goldfish hypothalamus have shown a similar localization o fCRFUI peptides in the NPO and NLT and have revealed the presence of additional populations of neurons elaborating CRF-UI-like peptides (Fryer and Lederis, 1988; Fryer, 1989). Ovine CRF immunoreactivity was found in the magnocellular and parvocellular perikarya of the nucleus preopticus (NPO), in the preoptic-neurohypophyseal tract, and in fibers of the rostral pars distalis and neurointermediate lobe. CRF-immunoreactive (ir) cell bodies were also found in the nucleus lateralis tuberis (NLT),nucleus recessus lateralis pars lateralis (NRLI), and nucleus recessus lateralis pars medialis (NRLm) of the hypothalamus, and in the ventral telencephalon. UI-ir perikarya were observed in the same regions and appeared to be the same perikarya that crossreacted with the CRF antiserum. However, when the UI antiserum was passed through an ovine CRF affinity column, to remove antibodies cross-reacting with ovine CRF, immunoreactivity was again observed in perikarya of the NLT, NRL1, NRlm, and ventral telencephalon, but not in the NPO. These immunocytochemical observations indicate the presence of two struturally similar, yet distinct, CRFlike peptides in the goldfish hypothalamus. T h e CRF-like peptide is elaborated by perikarya of the NPO, and the UI-like peptide by peri-

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karya ofthe NLT, NRL1, NRLm and ventral telencephalon. Immunocytochemical studies using a sucker CRF antiserum provided identical results obtained with the ovine CRF antiserum (Figs. 4 and 5). A diagram illustrating the distribution of sCRF immunoreactivity in the goldfish brain and pituitary gland is presented in Fig. 6. Studies in the goldfish have explored further the relationship between the pituitary-adrenocortical axis and the caudal neurosecretory system (Arnt et al, 1990). Immunocytochemistry with a sucker UI or a sucker urotensin I1 (UII) antiserum combined with morphonietric analysis was used to investigate the effects of corticoid status on the secretory activity of UI-ir or UII-ir neurons in the caudal neurosecretory system. Iinmunocytochemistry on adjacent sections treated with UI or UII antiserum revealed a colocalization of UI and UII immunoreactivities in many, but not all, of the neurosecretory perikarya of the caudal neurosecretory system. Metopirone administration resulted in significant decreases for both cellular and nuclear areas of the UI-ir and UII-ir neurons, with an increase in the intensity of the UI but not the UII immunostaining reaction. Dexamethasone administration resulted in significant increases in both cellular and nuclear areas of the UI-ir but not for the UII-ir perikarya, with a decrease in the intensity of the UI but not the UII immunostaining reaction. These results suggest that adrenocorticosteroid hormones promote the selective secretion of UI, but not of UII, by the goldfish caudal neurosecretory 5 ystem. Immunocytochemical studies in the sucker (Yulis et d.,1986), goldfish (Fryer and Lederis, 1988),and eel (Anguilla anguilla) (01'ivereau et al., 1988)have revealed a colocalization of CRF and AVT inimunoreactivities in magnocellular and parvocelluiar perikarya of the NPO. These findings are similar to the results from the mammalian hypothalamus, where immunocytochemical studies have revealed a colocalization of AVP with CRF in perikarya of the paraventricular nucleus (Tramu et al., 1983). The apparent redundancy in both the niammalian and teleostean neurohypophyseal systems of two neuropeptides with ACTH-releasing activity being released from the same neuron is indeed intriguing. Evidence that corticosteroids inhibit the release and/or biosynthesis of AVP, as well as of CRF, from neurons of the NPO has come from studies in the goldfish. Cortisol pellet implants in the NPO have been shown to reduce stress-induced elevations of circulating cortisol (Fryer and Peter, 1 9 7 7 ~ )Further . evidence that the biosynthesis and release of AVT in the goldfish is negatively regulated by cortisol is indicated from alterations in corticoid status by pharmacological adrenalectomy. Following metopyrone administra-

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Q

Fig. 4. Immunoreactive perikarya in the goldfish brain (a) "PO, nucleus preopticus; (b) V1, area telencephali ventralis pars lateralis; (c) NKL1, nucleus recessus lateralis; (d) NLT, nucleus lateralis tuberis; and (e) NRLni, nucleus recessus lateralis pars medialis. Bar denotes 20 p m .

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Fig. 5. sCRF-immunoreactive fibers in the goldfish pituitary gland. RPD, rOStrdl pars distalis; NIL, neurointermediate lobe.

Fig. 6. Distribution ofimrnunoreactive sucker CRF in the goldfish brain and pituitary gland. MES, mesencephalon; NIL, neurointermediate lobe; OT, optic tectum; NLT, nucleus lateralis tuberis; NRL1, nucleus recessus alteralis pars lateralis; NRLm, nucleus recessus lateralis pars medialis; TEL, telencephalon; V1, area telencephali ventralis pars lateralis; RPD, rostra1 pars distalis; NPO, nucleus preopticus.

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tion, which reduces circulating levels of cortisol by inhibiting interrenal 11-p hydroxylase activity, there was a decrease in AVT immunostaining of perikarya of the NPO with an increase in the cross-sectional area of the nucleus for both parvocellular and magnocellular neurons (Fryer and Lederis, 1988), suggesting that AVT synthesis and release were enhanced. Conversely, dexamethasone administration resulted in an increase in AVT immunostaining of perikarya of the NPO and a reduction in the cross-sectional area of the nucleus for both parvocellular and magnocellular neurons, suggesting an inhibition of AVT synthesis and release. The involvement of the NPO and NLT in the control of ACTH secretion from the goldfish pituitary (Fryer and Peter, 1977a,b; Fryer, 1989) suggested that the two nuclei may subserve different functional roles in the regulation of ACTH release. As a means of addressing this possibility, a study was undertaken using goldfish to examine the effects of destruction of the NPO, the NLT, or the NPO and NLT combined, on pituitary ACTH content and plasma cortisol (Lederis et al., 198513).Lesions of the NPO did not affect pituitary ACTH content, but reduced plasma cortisol to virtually undetectable levels. Lesions of the NLT reduced pituitary ACTH content by about 50% and reduced plasma cortisol significantly. Combined lesions of both the NPO and NLT diminished pituitary ACTH content and reduced plasma cortisol to undetectable levels. These observations suggest that the function of the NLT, which elaborates the more UI-like CRF peptide, may be to control, preferentially, ACTH biosynthesis, whereas the function of the NPO, which also elaborates the more CRF-like peptide and the neurohypophyseal peptides AVT and IST, may be to stimulate preferentially ACTH release. This type of dual control has not been identified in mammals. C. CRF mRNA in Sucker and Goldfish Brain Information on the location of neuronal CRF synthesis was provided by a combination of Northern blot experiments and in situ hybridization. CRF transcripts can be detected in two nuclei of the sucker hypothalamus, namely, the NPO and NLT by Northern blot hybridization (Morley et al., 1991). Expression of the CRF genes (experiments done under conditions allowing the cross-hybridization of sCRFl and sCRF2 species) was much more pronounced in the NPO than in NTL, even though significant levels of CRF mRNA synthesis in the latter nucleus were shown. Consistent with the data described here, CRF mRNA was detected in the sucker NPO in both magnocellular and

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parvocellular neurons by in situ hybridization (Okawara et al., 1992). Immunolabeled CRF peptide could also be detected in these neurons when in situ hybridization and immunohistochemistry were compared on adjacent sections of the sucker hypothalamus. Cell bodies were labeled using both methods described on parallel sections, showing that these cells produce both CRF mRNA and peptide. CRF immunoreactivity was detected in the perikarya of the parvo- and magnocellular neurons, suggesting that these cells store this peptide. Although this area has cells that can be immunolabeled, the cells do not react with CRF mRNA-specific probes. Either these cells were not synthesizing mRNA at the time of analysis or the mRNA had a short half-life. Hence not all brain cells capable of CRF synthesis are actively synthesizing peptide precursors. Certain stimuli may be necessary to initiate mRNA and peptide production in these CRF cells. In situ hybridization studies in the goldfish have demonstrated that CRF, AVT, and IST mRNAs are localized in magnocellular and parvocellular perikarya of the NPO scattered throughout the nucleus with a similar distribution pattern (Figs. 7 and 8) (Okawara and Fryer, 1991). A co-expression of CRF mRNA with AVT mRNA was observed for both magnocellular and parvocellular perikarya, whereas co-expression of CRF mRNA with IST mRNA was observed only in parvocellular perikarya. Co-expression of AVT mRNA with IST mRNA in either magnocellular or parvocellular perikarya was not observed. Furthermore, perikarya expressing CRF mRNA were also observed in the NLT, NRL1, NRLm, and ventral telencephalon in complete agreement with the distribution of CRF-immunoreactive perikarya observed using the sucker CRF antiserum (Fig. 8). Similar studies in mammals did not reveal any co-expression of AVP mRNA and oxytocin (OXT) mRNA in perikarya of the paraventricular nucleus or supraoptic nucleus (Ichimiya et al., 1989). However, a transient co-expression of' AVP mRNA with OXT mRNA was reported in parturient rats (Mezey and Kiss, 1991).The absence ofco-expression of AVT mRNA with IST mRNA in magnocellular perikarya of the NPO has been reported in the rainbow trout (Hyodo and Urano, 1991).The observation that CRF mRNA is co-expressed in the same perikaryon of the teleost NPO with AVT mRNA or IST mRNA indicates that the elaboration of neurohypophyseal nonapeptide hormones with a CRF peptide by the same neuron is a principle that was established early in the evolution o f the neurohypophyseal system. Because CRF, AVT, and IST stimulate the release of ACTH from the goldfish pituitary, studies of the coexpression of CRF mRNA with AVT mRNA and of CRF mRNA with IST mRNA under various experimental conditions such as stress or

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Fig. 7. In situ hybridization for CRF mRNA in the goldfish NPO with a 35S-lalxled oligonucleotide probe under (a) bright field illumination and (b)dark field illumination.

alterations ofcorticoid status will be ofconsiderable interest in elucidating the roles played by these peptides in the regulation of the teleost HPA(1) axis.

D. CRF-UI Genes Not only the sites of CKF synthesis but also the factors that influence the rate of C R F mRNA synthesis are of interest. Both metopyrone treatment and urophysectomy are known to change the rate of CRF and UI synthesis. After 5 days of metopyrone treatment, CRF mRNA levels were found to be significantly increased, as detected by in situ hybridization, in the sucker hypothalamus ( Y . Okawara and K. Lederis, 1994, unpublished). This method was utilized to detect sCRF1- or sCRF2-specific mRNA transcripts with oligonucleotide probes in the sucker (Morley et al., 1991).This result suggests that changes in corticosteroid concentrations may have a regulatory impact on CRF synthesis

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Fig. 8. In situ hybridization on adjacent sections ofthe NPO ofthe goldfish hypothalanitis using digoxigenin-labeled oligonucleotide probes with a digoxigenin antibody conjugated with alkaline phosphatase a s reporter showing CRF mHNA (a, c), AVT

mHNA (b), or IST niKNA (d). Perikarya co-expressing CRF mRNA with AVT mHNA or IST mRNA are indicated by arrows. Bar denotes 20 pin.

b y influencing CRF gene transcription. In addition to higher levels of

CRF-specific transcripts, the hybridization pattern of mRNA prepared from metopyrone-treated fish differed from that of untreated (control) fish. In treated fish the expression of the longer transcript (1800 hp) was significantly increased and, in addition, a third 4.0-kb transcript was detected, which might reflect the presence of an incompletely spliced CRF transcript. Urophysectomy was known to selectively increase the production of UI in brains of the sucker and goldfish (Woo et ul., 1985a; Yulis and Lederis, 1986). In Northern blot hybridization experiments, no difference in CRF mRNA levels was seen between urophysectomized and control fish. However, UI transcripts were elevated in the NLT after urophysectomy, whereas no effect was seen in the NPO (Morley et al., 1991). Thus urophysectomy appears to stirnulate only the transcription of the UI gene in the NLT, which implies

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that tissue-specific elements must be involved in the induction of UI gene transcription. Although no direct information is currently available on the structure of sucker or goldfish UI genes, a carp UI cDNA has been isolated (Ishida et al., 1986). The deduced sequence of the precursor is composed of 145 amino acid residues with the same organization of signal peptide, cryptic peptide, and hormone moiety, as commonly found in CRF peptides. The overall sequence identity to sucker C R F l precursors is low (34%), but in the hormone moiety the similarity is much higher at 75.6%. A dendrogram of all known CRF and UI peptide sequences (Fig. 1)reveals that UI peptides form a distinct group and are more closely related to frog SVG than to CRF peptides. Fish CRF genes have not been isolated to date. However, Southern blot analysis of sucker genomic D N A was performed using a sCRFl c D N A fragment. The hybridization pattern, together with the information of two distinct cDNA sequences, provided evidence that two distinct genes must exist in the sucker genome (Morley et al., 1991). This phenomenon might be explained by the fact that catostomids are tetraploid, with a complement of 100 chromosomes per genome (Uyeno and Smith, 1972). It is noteworthy that two genes are also present for sucker isotocin and vasotocin peptides (Figueroa et al., 1989; Morley et al., 1990). CRF genes have been isolated and analyzed for the human (Shibahara et al., 1983), rat (Thompson et al., 1987), and sheep (Roche et al., 1988) and exhibit a highly conserved structural organization. Only a single intron (700-800 bp) is found in the 5' untranslated region. Hence, the first exon contains 160-1700 bp of the 5' untranslated region. The second exon consists of the reminder of the 5' untranslated region, the complete coding sequence, and the 3' untranslated region. Comparison of the 5' flanking regions of CRF genes shows a high degree of sequence conservation in the region of -350 to 1, which is even higher than in the protein coding region. These sequences may be important in the regulation of gene transcription or tissue-specific expression and are conserved in all CRF genes analyzed to date. When the 5' flanking regions of ovine, human, and rat genes (Roche et al., 1988) are aligned, several TATA boxes and CAAT boxes are highly conserved in position and sequence. The human CRH gene has at least two sites of transcription initiation (Robinson et al., 1989). A CAMP responsive element is also present in the 5' flanking region of rat, human and sheep CRF genes (Roche et al., 1988). This was shown to be functional in a transfection assay using rat PC 12 cells and a construct containing the rat CAMP responsive element and a chloramphenicol acetyltransferase reporter gene (Seasholtz et al., 1988).

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T h e ovine CKF gene contains five glucocorticoid responsive elements that are not conserved in the human CRF gene. However, the influence of glucocorticoids on CRF gene transcription has been shown in human, rat, and ovine species. In humans, opposite effects of glucocorticoids on mRNA transcription could be demonstrated; a stimulation of CRF mRNA synthesis was documented in the placenta, in contrast to a suppression in the hypothalamus (Robinson et al., 1989).Experiments in sheep and rats also confirmed the glucocorticoid sensitivity of CRF gene transcription as adrenalectomy, which significantly decreases the glucocorticoid level, increased hypothalamic CRF mRNA in both species. In sheep, dexamethasone treatment elevating the glucocorticoid level reduced CRF mRNA level by -50% (Roche et al., 1988; Jingami et al., 1985b). As metopyrone treatment in the sucker led to a significant increase in CRF mRNA level, speculations are very tentative about the glucocorticoid responsive elements in fish CRF genes or the glucocorticoid induction of protein factors that regulate CRF gene transcription. This regulation of CRF synthesis appears to operate in both lower and higher vertebrates. Although fish genes for C R F and UI have not yet been isolated, cDNA sequences have been cloned from the sucker (Okawara et al., 1988).Analysis of sequence motifs characterizing splice junctions [donor sequences: (C/A)AG 1GT(A/G)AGTand acceptor sequences (T/ C),,N(C/T)AG.1G, where arrow denotes a splice site] (Mount, 1982) in the sucker CRF cDNA sequences revealed that an AAG//G motif is present 46 b p upstream of the start codon, AAG representing part o f a splice donor site whereas the G residue could be part o f a splice acceptor site. In the 5' untranslated region of the sucker CRF2 cDNA sequence only AG J, G motifs were found. The position of one of these sequences corresponds to the one in the sucker C R F l sequence. If these motifs indicate the presence of introns in the 5' untranslated regions of the CRF gene, they would be similarly positioned as their mammalian counterparts. How the expression of two distinct CRF encoding genes is regulated and how the tissue-specific expression of CKF and U I genes is achieved requires further investigations.

IV. EVOLUTIONARY CONSIDERATIONS FOR CRF-UI Determination of the precursor structures and gene organizations of CKF and CRF-related peptides may contribute toward the elucidation of the evolution of these molecules. UI and CRF peptides have

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been isolated from fish (see Lederis et al., 1993) and their homology (e.g., a 53% identity for sucker peptides) as well as the analysis of their biological properties (Lederis et al., 1985a,b) have widened the scope of this family of peptides. Analysis of the precursor sequences ofthese peptides (Furutani et al., 1983; Shibahara et al., 1983; Jingami et al., 1985a; Ishida et ul., 1986; et uZ.,Okawara et al., 1988) revealed a conserved organization, including a signal peptide, a cryptic peptide, and the hormone moiety. The origin of teleost fish (300 million years ago) and hominids (70million years ago) indicates a remarkable conservation of the CRF structure and sequence over time. Assuming that a common ancestral gene existed for these peptides, the question arises whether this ancestral gene encoded a product more like UI or CRF. In the sucker, two CRF genes are expressed; two corresponding mRNA transcripts have been detected (Morley et al., 1991), and two distinct peptides have been identified by HPLC (McMaster et al., 1990). These CRF peptides differ by only one amino acid at position 28 (alanine in sCRFl and valine in sCRF2). Two different CRF peptides (CRF major and CRF minor) have also been isolated from porcine species (Patthy et al., 1985). The amount ofCRF major is twice that of CRF minor. In this case, the peptides differ by a single amino acid substitution at position 40 (asparagine in CRF major and isoleucine in CRF minor). Why are two nearly identical CRF peptides synthesized in the fish and porcine species? An evolutionary advantage might exist in possessing two genes in order to synthesize more RNA and consequently more protein at a given time. Alternatively, the two genes might be differentially expressed, and the one variation in the amino acid sequence might be important for a specific function. The general strategy of evolution seems to be an increase of DNA by a succession of mutational events, this creates modified and new proteins providing organisms with better chances for survival or reproduction. The increase of genomic DNA is achieved by gene duplication, which may occur as a result of a variety of alternative steps: unequal exchange between sister chromatids of one chromosome, unequal crossing-over between two homologous chromosomes during meiosis, regional gene duplication, and/or polyploidization. The first three of these mechanisms lead to a tandem organization of genes on the same chromosome. However, genes that are amplified by polyploidization are located on distinct chromosomes. All four mechanisms described here are assumed to have contributed to vertebrate evolution during the aquatic stage. Analysis of the isoenzyme patterns of LDH, the chromosome numbers, and DNA content of several members of fish families and

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higher vertebrates contributed to the development ofa model of evolutionary events leading from primitive chordate forms to mammals (Ohno et al., 1968).The first step, the evolution of ancestral vertebrates with about 48 chromosomes, may have been initiated by tetraploidization, which involves a duplication of the chromosomal complement of primitive chordate forms, with a chromosome complement of around 28 chromosomes. As chromosome numbers can only be analyzed in extant species, these numbers have obviously changed since the early genome duplication event. Thus only rough estimations are possible. But this early stage in vertebrate evolution may still be represented by many fish that normally have a diploid chromosomal complement of46-50 chromosomes. In the second step, another duplication of the genome, followed by regional gene duplications, is thought to have occurred before the descendants of the crossopterygian fish left the aquatic environment, the event that led to the evolution of higher vertebrates including mammals. This view may help to understand the evolution of CRF and UI in fish, but it does not explain why in most higher vertebrates only one CRF and no urotensin exist. If an increase in DNA content can be equated with evolutionary progression it may be argued that the U I gene represents the more ancient form, because the protein precursors demonstrate a progressive increase in length: carp UI contains 145 amino acid residues, sCRF1 and 2 contain 162 amino acid residues, and human CRF has 196 residues. This gain in length of the protein precursor may be argued to have accompanied the ascension on the evolutionary tree. Alternatively, it cannot be excluded that in primitive ancestral chordates a combined CRF/UI gene might have existed, which was duplicated during the tetraploidization event that led to the evolution of' vertebrates. After several mutational events, these genes might have given rise to CRF-like and UI-like genes, which differ from each other in terms of tissue specificity and biological properties. In catostomid fish a special situation is found: these fish are tetraploid and can have a genomic complement of up to 100 chromosomes. Family members of the Cyprinidae, for example, barbs, may possess about 50 diploid chromosomes and have a DNA content that represents -20% of that found in mammals. Other members of this family, such as carp and goldfish, have twice as many chromosomes (about 100) and a DNA content that amounts to -50% of that of mammals. From these examples it may be argued that the simplest way to achieve such a gain of DNA and chromosomes is a tetraploidization step (Ohno et uZ., 1968).

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The CRF gene of catostomids was duplicated along with the tetraploidization event in other subfamilies of Cyprinidae. Both genes differ from each other by about 10% at the nucleotide and amino acid level. Assuming a rate of amino acid divergence of 1% substitutions per 10 million years (Wilson et al., 1977), duplication of the CRF gene by tetraploidization could have occurred approximately 100 million years ago. A similar gene duplication event is assumed for sucker vasotocin (VT) and isotocin peptide precursors (Mohr et al., 1990). Two isotocin precursors that differed by 10%in amino acid sequence were isolated (Figueroa et al., 1989; Morley et al., 1990). In contrast, three vasotocin precursors were identified; two of them (VT-l and VT-1’) were 90% identical at the amino acid level, whereas the third one (VT-2) was only 55% identical to VT-1 and VT-1’ sequences. These values suggest that the vasotocin precursors, VT-1 and VT-2, were the result ofa gene duplication at least 450 million years ago (Heierhorst et al., 1989; Mohr et al., 1990). In addition, this duplication may have been a regional one, restricted to the vasotocin gene and further restricted to certain fish families. A second duplication event about 100 million yers ago would then have led to he VT-1 and VT-1‘ genes. As the identity values of sCRFl and sCRF2 (90%) as well as isotocin 1 and 2 precursors (90%)are similar, all of these genes would seem to have duplicated at the same time, by the mechanism of polyploidization, which characterizes the evolution ofcatostomids in the family of Cyprinidae. To date, only one UI gene has been cloned from the carp. U I sequences for all other fish species have been obtained by peptide sequencing. The isolation of a second UI gene might well be anticipated from tetraploid fish species, which would be expected to be -90% identical at the level of nucleotide sequence. However, it is also possible that the second gene is no longer transcribed, resulting in a silent gene or pseudogene. The sequences of sucker UI and C R F l precursors are only 34% identical. Assuming a duplication of an ancestral gene that combined both CRF-like and UI-like functions, this event would have to be placed approximately 660 million years ago. There is little evidence to support the hypothesis that the evolution of vertebrates was accompanied by a polyploidization event because it has not been possible to deduce vertebrate origins from the fossil record. Nevertheless, the fossil record does show that around 620 million years ago a large array of morphological forms had evolved, which does not rule out such a polyploidization event.

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In mammals, usually only one type of CRF precursor has been found. To date, UI-like peptides have not been detected. Although UI as well as the corresponding mRNA can be detected in fish brain, this peptide is usually associated with the urophysis, a fish-specific organ that is not found in mammals. During the transition from aquatic life to terrestrial life, the function of UI may have become less important, leading to a silencing or loss of the UI gene during higher vertebrate evolution. A similar mechanism may have led to the loss of the second CRF gene, which might have existed in the early stages of terrestrial life.

ACKNOWLEDGMENTS The authors would like to thank €3. Goodwin for typing the nianuscript.

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37,467-469. Tran, T. N., Fryer, J. N., Bennett, H. P. J., and Vaudry, H. (1989). TRH stimulates the release of POMC-derived peptides from goldfish melanotropes. Peptides 10, 835-84 1. Tran, T. N., Fryer, J. N., Lederis, K., and Vaudry, H . (1990). CRF, urotensin I, and sauvagine stimulate the release of POMC-derived peptides from goldfish neurointermediate lobe cells. Gen. Comp. Endocrinol. 78, 351-360. Tripp, R. A,, Maule, A. G., Schreck, C. B., and Kaattari, S. L. (1987). Cortisol mediated suppression of salmonid lymphocyte responses in uitro. Deo. Comp. Zrnrnunol. 11,

565-576. Uyeno, T., and Smith, G. R. (1972).Tetraploid origin of the karyotype of catostomid fishes. Science 125,644-646. Vale, W., and Rivier, C. (1977). Effects of a putative hypothalamic CRF and known substances on the secretion of radioimmunoassayable ACTH by cultured anterior pituitary cells. Proc. Endocrinol. Soc. 59, 217 (abstract). Vale, W., Spiess, J., Rivier, C.,and Rivier, J. (1981).Characterization ofa41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and p-endorphin. Science 213, 1394-1397. Verburg-van Kemande, B. hl. L., Jenks, B. G., Cruijserr, P. M. J. M.. Dings, A., Tonon, M. C., and Vaudry, H. (1987).Regulation of MSH release from the neurointermediate lobe of Xenopus luevis by CRF-like peptides. Peptides 8, 1093-1100. von Heijne, G . (1983). Patterns of amino acids near signal-sequence cleavage sites. Eur.

J. Biochem. 133, 17-21. von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. NucIeic Acids Res. 14, 4683-4690. Weld, M. M., and Fryer, J. N. (1987). Stimulation by angiotensins I and I1 of ACTH release from goldfish anterior pituitary cells columns. Gen. Comp. Endocrinol. 68,

19-27. Weld, M . M., and Fryer, J. N. (1988).Angiotensin I1 stimulation ofteleost adrenocorticotropic hormone release: Interactions with urotensin I and corticotropin-releasing factor. Gen. Comp. Endocrinol. 69, 335-340.

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Weld, M . M., Fryer, J . N., Rivier, J., and Lederis, K. (1987). Inhibition of CRF and urotensin I-stimulated ACTH release from goldfish pituitary cell columns by the CRF analogue alpha-helical CRF (9-41).Regul. P e p t . 19, 273-280. Wilson, A. C., Carlson, S. S., and White, T. J. (1977). Biochemical evolution. Annu. Rev. Biochem. 46,573-639. Woo, N. Y. S., Hontela, A,, Fryer, J . N., Kobayashi, Y., and Lederis, K. (1985a).Activation of hypothalamo-hypophyseal-interrenal system by urophysectomy in goldfish. Am. J. Physiol. 248, R197-R201. Woo, N. Y. S., Wong, K. L., Hontela, A., Fryer, J. N., Kobayashi, Y., and Lederis, K. (1985b). In uitro urotensin binding to tissues and in ~ i u ostimulation of hypothalamo-hypophysio-interrenal function by urophysectomy in the goldfish. In “Neurosecretion and the Biology of Neuropeptides” (H. Kobayashi, H. A. Bern, and A. Urano, eds.), pp. 471-478. Japan Science Society Press, Tokyo/SpringerVerlag, Berlin. Yulis, C. R., and Lederis, K. (1986). The distribution of “extraurophyseal” urotensin 1immunoreactivity in the central nervous system of Catostomus cornmersoni after urophysectomy. Neurosci. Lett. 70, 75-80. Yulis, C. R., and Lederis, K. (1987). Co-localization of the immunoreactivities ofcorticotropin releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni. Cell Tissue Res. 247, 267-273. Yulis, C. R., Lederis, K., Wong, K.-L., and Fisher, A. W. F. (1986). Localization of urotensin I- and corticotropin-releasing factor-like immunoreactivity in the central nervous system of Catostomus commersoni. Peptides 7 , 79-86.

3 EXPRESSION OF THE VASOTOCIN AND ISOTOCIN G E N E FAMILY IN F I S H AKIHISA U R A N O Division of Biological Sciences, Graduate School of Science Hokkaido University, Sapporo, Hokkaido 060, Japan

KAORU KUBOKAWA Laboratory of Molecular Biology, Ocean Research Institute University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan

S H U I C H l HIRAOKA Division of Biological Sciences, Graduate School of Science Hokkaido University, Sapporo, Hokkaido 060, and Laboratory of Molecular Biology, Ocean Research Institute University of Tokyo, Minamidai, Nakano-ku, Tokyo 164, Japan

I. Introduction A. Neurohypophysial Hormones in Fish B. Magnocellular Neurosecretory Cells C. Biological Roles of Vasotocin and Isoticin D. Responsiveness of NSC to Physiological Stimuli E. Why Molecular Study? 11. Genes, cDNAs, and Precursors A. Structure of VT and IT cDNAs and Precursors B. Structure of VT and IT Genes C. Evolutionary Pathway of VT and IT Genes 111. Divergence of VT and IT Gene Expression A. Hybridization Probes B. Northern Blot Analyses IV. VT and IT Gene Expression in Osmotic Adaptation A. Physiological and Histological Backgrounds B. Effects of Transfer Experiments on VT and IT mRNAs V. Conclusion References 101 FISH PHYSIOLOGY. VOL. XI11

Copyright 0 1994 by Academic Press, Inc.

All rights of reproduction in any form reserved.

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I. INTRODUCTION Keurosecretory systems are involved in the regulation of various aspects of physiological events in both vertebrates and invertebrates. Important roles for peptidergic neurohormones in intercellular communication have been revealed by approaches using morphological, physiological, pharmacological, immunological, biochemical, and, now, molecular methods. In vertebrates the classical hypothalamic neurosecretory system, which secretes neurohypophysial hormones, has served as a good model system in these approaches, although many other peptidergic neurohormones are now known. In this chapter we first review the current status of studies on neurohypophysial hormones in fish because such information is basic for understanding the biological meaning of gene expression.

A. Neurohypophysial Hormones in Fish Since the first isolation and characterization of mammalian neurohypophysial hormones by Du Vigneaud and associates (1953a,b), more than ten distinct nonapeptide principles have been characterized in a wide variety of vertebrates (Acher, 1985, Acher et uZ., 1991). They can be classified into two groups: the vasopressin (VP) or basic peptide family and the oxytocin (OT) or neutral peptide family (Table I ) . Vertebrate species, except for the cyclostomes, generally have one VP-like peptide and one OT-like peptide. Only vasotocin has been characterized as a neurohypophysial hormone in cyclostomes (Lane et al., 1988). The teleostean neurohypophyses examined to date contain vasotociri ( V T ) as a VP-family member and isotocin (IT) as an OT-fkmily member. It is, however, still possible that we have overlooked important neurohypophysial principles (Maetz and Lahlou, 1974) because of a lack of appropriate bioassays using fish target organs. Among 15,000teleostean species, neurohypophysial hormones have been purified and characterized from only a very limited number of species. In this sense, it is interesting that sturgeons, unlike other bony fish, may have a peptide with oxytocic activity; this peptide, however, is distinct from the known OT-like peptides and is present in only small amounts (Rouille et al., 1991). According to Acher and associates (1985, 1991), the situation in cartilaginous fishes is rather more complicated than that in teleosts. Dogfishes have vasotocin only as a VP-like hormone, whereas two OT-

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Table I Vasopressinlike and Oxytocinlike Peptides in Vertebrates Peptide

Amino acid sequence

Vasopressin family Arg-vasopressin Lys-vasopressin Phenypressin Vasotocin

1 2 3 4 5 6 7 8 9" <:ys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-a Cys-Tyr-Phe-Gin-Asn-Cys-Pro-Lys-Gly-a Cys-Phe-Phe-Gln-Asn-Cys-Pro-Arg-Gly-a Cys-Tyr- Ile -Gln-Asn-Cys- Pro- Arg-Gly-a

Oxytocin family Oxytocin Mesotocin Isotocin Glurnitocin Aspargtocin Valitocin Asvatocin Phasvatocin

Cys-Tyr- Ile Cys-Tyr- lle Cys-Tyr- Ile Cys- Tyr - Ile Cys-Tyr- Ile Cys-Tyr- Ile Cys-Tyr- Ile

' C terminus

1

2

3

Phvletic distribution

Mammals Mammals Metatherians Nonmammals

4 5 6 7 8 9 -Gln-Asn-Cys-Pro-Leu-Gly-a Mammals

-Gin-Asn-Cys-Pro- lle -Gly-a -Ser-Asn-Cys-Pro- Ile -Gly-a - Ser - Asn -Cys- Pro - Glu - Gly -a -Asn-Asn-Cys-Pro-Leu-Gly-a -Gln-Asn-Cys-Pro- Val -Gly-a -Am-Asn-Cys-Pro- Val -Gly-a Cys-Tyr-Phe-Asn-Asn-Cys-ProVal -6 l y - a

Metatherians, nonmarnmals Bony fish Ray

Shark Shark Shark Shark

is amidated.

like hormones have been identified in each of Squalus acanthias and Scyliorhinus caniculus; aspargtocin and valitocin are present in the former, and asvatocin and phasvatocin in the latter. Whereas VP- and OT-like peptides are considered to be derived from a common ancestral molecule by gene duplication, two OT-like peptides in cartilaginous fish might be produced by a second gene duplication. A previous claim that holocephalian fish have VT and OT has been confirmed by a chemical identification study in ratfish (Michel et al.,

1993b). The presence in lungfish of tetrapod neurohypophysial hormones, for example, vasotocin and mesotocin (MT) (Acher et al., 1970; Michel et al., 1993a), as well as the amphibianlike structure of the hypothalamo-hypophysial region, had raised an important question about the evolutionary pathway from fish to amphibia. To solve this question, it will be interesting to analyze the precursor structures ofthe neurohypophysial hormones in Crossopterygii and Dipnoi.

B. Magnocellular Neurosecretory Cells Neurohypophysial hormones are produced mainly by hypothalamic neurosecretory cells (NSCs) in the magnocellular part of preoptic nucleus (PONnig) and are released from neurosecretory axon terminals

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in the neurohypophysial neurohemal region. The concept of neurosecretion first arose from a histological study of the brain of the fish Phoxinus laevis (Scharrer, 1928) and was rapidly developed by many researchers (Scharrer and Scharrer, 1954). Immunohistochemistry accelerated our understanding of the morphological characteristics of VT and IT neurons. The localization of VT and IT immunoreactivity (ir) in separate PONmg neurons was shown in goldfish, plaice, and rainbow trout (Goossens et al., 1977), and, for goldfish, in electrophysiologically characterized preoptic neurons, which were marked with a fluorescent dye (Reaves and Hayward, 1980).However, there were no clear subgroups of VT-ir and IT-ir neurons within the PONmg. The localization of VT and IT mRNAs by an in situ hybridization technique, in which unique synthetic oligonucleotides were used as molecular probes, further confirmed that VT and IT genes are expressed in separate PONmg NSCs in the hypothalamus of rainbow trout (Hyodo and Urano, l99la). Although not extensively examined in fish, several authors have reported colocalizaion in neurons of VT-ir with immunoreactivity o f other neuropeptides such as angiotensin I1 (Yamada et al., 1985), corticotropin-releasing factor (Yulis and Lederis, 1987; Olivereau et al., 1988; Batten et al., 1990), and growth hormone-releasing factor (Batten et al., 1990). IT-ir occasionally coexists with enkephalin-ir in a few PONmg neurons (Batten et al., 1990). These neurons that simultaneously express two or more neuropeptide genes are useful for analyzing integrative regulatory mechanisms. C. Biological Roles of Vasotocin and Isotocin

Maets and Lahlou (1974) wrote “Knowledge of the chemistry and phylogenetic distribution ofthe neurohypophysial hormones has made enormous progress. . . . In contrast, our knowledge of the biological role of these hormones in the lower vertebrates has been lagging.” The situation seems to be quite similar at present. We can add only a small amount of knowledge to their excellent review that was written almost 20 years ago.

1. V T RECEPTORSIN

THE

BRAIN

Specific 3H-labeled Arg-VP (AVP) binding sites, which probably present VT receptors, were localized in the brain and pituitary of sea bass b y light microscopic autoradiography. The PONmg was included

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among several 3H-labeled AVP binding sites in the brain (Moons et al., 1989). Neurohypophysial hormones may be neurotransmitters or neuromodulators in the teleost brain because V T and IT directly applied to eel PONmg neurons by microiontophoresis excited electrical activity of a considerable number of PONmg units (Sugita and Urano, 1986). VT RECEPTOR 2. BKANCHIAL T h e presence of a single type of VT receptor with high affinity but low capacity was shown by a binding study in gill cells isolated from eels that were fresh water (FW) adapted or seawater (SW) adapted ( K d : 3.21 nM in FW and 1.05 nM in SW). The VT binding suggests a direct action of VT on the gill epithelium of teleosts (Guibbolini et al., 1988).The action ofVT and IT on adenylate cyclase was inhibitory, suggesting that these hormones probably act through the G(i)-protein in the membrane fractions from the gill epithelium of rainbow trout (Guibbolini and Lahlou, 1992). 3. VT

AND

IT

IN

REPRODUCTION

The involvement of neurohypophysial hormones in spawning and parturition in some teleostean species seems to be established by many studies (see Maetz and Lahlou, 1974).In these studies, spawning in oviparous fish and parturition in viviparous fish was induced by the application of VT and/or IT; contraction of the genital tracts was observed with a physiological dose of VT and IT. However, the role ofneurohypophysial hormones in fish reproduction is not clearly understood compared to in other vertebrate classes (Urano, 1988; Moore, 1992). D. Responsiveness of NSCs to Physiological Stimuli

The biological role of the hypothalamo-neurohypophysial system in fish is rather ambiguous. We presume that fish are like mammals

(Hyodo and Urano, 199lb)in that the NSCs are responsive to physiological stimuli. These signals can facilitate or inhibit regulation of homeo-

stasis and reproduction. The response of NSCs to such stimuli is modulation of secretory activity, that is, release of stored neurohypophysial hormone as an acute phasic event, and/or synthesis of hormone that may be accompanied later by morphological changes (Fig. 1).

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TRANSPORT

RELEASE

BLOOD C A P I L L A R Y

Fig. 1. Schematic diagram showing characteristic features o f a hypothalamic nciirosecretory cell that sends axon collaterals to a neurohemal region and also to other neurons. Various transmitter candidates, such as acetylcholine, monoaniines, amino acids, and neiiropeptides, control not only electrical activity but secretory activity, including expression of neurohormone gene and release of mature hormone. Lipophilic Iioriiioiies, for example, steroid hormones and tlryroicl hormones, niay regulate expression of various genes by both cis and trans mechanisms. Upon integration of' such signals, a neurohormone can b e released either from varicosities as a local hormone 01ti-om ordinary presynaptic terminals as a transmitter or a modulator and affects second tiiesseiiger systems within target cells.

1. ELECTRICAL ACTIVITYAND

HORMONE SECRETION

T h e amount of' neurohypophysial hormone released by a NSC is correlated with its electrical activity, both the discharge rate and the duration of burst (Dyball et al., 1985).Recent evidence indicates that excitation of neurons, including hypothalamic NSCs, further activates a second messenger system, which in turn modulates expression of certain genes through immediate-early genes by a mechanism of' stimulus-transcription coupling (Morgan and Curran, 1991). Knowledge about the responsiveness of NSCs to physiological stimuli is thus crucial in studies of neurohormonal gene expression.

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Histochemical and cytochemical studies suggest that teleost PONmg neurons can be affected by various neurotransmitters, for example, acetylcholine (Uemura, 1965), monoamines (Urano, 1971; Parent, 1983), and neuropeptides (Cumming et al., 1982). Although the available electrophysiological data are quite limited concerning chemical transmitters that act on the NSCs in fish, Sugita and Urano (1986)used in witro preparations ofthe eel brain to show that a considerable portion of PONmg units were excited by either glutamic acid, acetylcholine (probably through muscarinic receptors), dopamine, noradrenaline, VT, IT, and angiotensin 11. These chemicals can facilitate release as well as expression of VT and IT precursor genes through second messenger systems. 2. MORPHOLOGICAL CHANGES WITH REPRODUCTIVE ACTIVITY In some teleost species, seasonal changes in the content of neurosecretory material in the neurohypophysis appeared to be correlated with reproductive behavior (see Maetz and Lahlou, 1974). Garlov and Khutinaev (1991) precisely followed histological changes in all parts of the preoptico-neurohypophysial neurosecretory system of the pink salmon (Oncorhynchus gorhuscha) during spawning using quantitative methods. They found activation of every phase of secretory activity, including synthesis, transport, and release, at the beginning of spawning. Such changes can be elicited by an elevation of the intraovarian pressure, which was reported to induce hypertrophy of PONmg neurons (Subhedar et al., 1987). The question here is whether these morphological changes of teleost NSCs during reproduction are coupled with gene expression of neurohypophysial hormone precursors, and if so, how gonadal hormones are involved in regulation of gene expression.

E. Why Molecular Study? It is well established in mammals (see review by Hyodo and Urano, 1991b) that biologically meaningful stimuli can modulate expression of genes encoding precursors of peptidergic neurohormones, as well as electrical activity of neurosecretory neurons. The molecular events are rapidly initiated, probably through second messenger systems and immediate-early genes, prior to the morphological changes. In this way, various aspects of the hypothalamo-neurohypophysial system have been clarified by use of molecular techniques. It is now indispensable to incorporate recombinant DNA techniques into physio-

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logical studies for understanding the biological roles of the hypothalamic neurosecretory system in fish.

11. GENES, cDNAS, AND PRECURSORS Recent progress in cloning and sequence analyses of cDNAs and genes encoding VP and O T families means that molecular tools can now be used to study the expression of genes for neurohypophysial hormones. To date, more than 20 nucleotide sequences have been determined in various vertebrate species. In fish, the first cDNAs and genes encoding VT and IT precursors were cloned from white sucker (Catostomus comrnersoni) (Figueroa et al., 1989; Heierhorst et al., 1989; Morley et al., 1990).Thereafter, cDNAs for V T and IT precursors were reported in three salmonid fishes, chum salmon (Oncorhynchus keta) (Heierhorst et al., 1990; Hyodo et al., 1991), masu salmon (0.masou) (Suzuki et al., 1992),and sockeye salmon (0.nerka) (Hiraoka et al., 1993). Molecular information on both hagfishes and lamprey VT precursors has been added to these teleostean sequences (Heierhorst et al., 1992; Suzuki et al., 1993). At present, 14 cDNA nucleotide sequences and in turn 14 deduced amino acid sequences of VT and IT precursors are available for studies on the expression of the VT and IT gene families in fish. Furthermore, sequence data for fish in combination with amphibian (Nojiri et al., 1987) and mammalian sequences (Land et al., 1982, 1983; Sausville et al., 1985) enable us to speculate about the evolutionary pathway of neurohypophysial hormones in gnathostomes.

A. Structure of VT and IT cDNAs and Precursors

As discussed in the next section, catostomids and salmonids are tetraploid and tend to have a pair of genes for each hormone. Cloning studies ofcDNAs for VT and IT precursors showed this is true for V T and IT genes. VT and IT precursors in salmonids form two distinct gene groups, each composed of a pair of V T and IT genes. They are referred to as VT-I, VT-11, IT-I, and IT-11. The cDNAs for VT-I1 and IT-I1 are longer than those for VT-I and IT-I, respectively, in both white sucker and chum salmon. The existence of two distinct genes for each of the VT and IT precursors in salmonid fish is probably due to the tetraploidization that is considered to have taken place in a cyprinidlike ancestral fish (Ohno et al., 1968).

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EXPRESSION OF THE V A S O T O C I N A N D I S O T O C I N GENE FAMILY

109

1. STRUCTURE OF VT AND IT PRECURSORS The V T and I T precursors consist of three parts, a signal peptide, a hormone moiety, and a cysteine-rich protein called neurophysin (Fig. 2). The hormone moiety is connected to neurophysin by the residues Gly-Lys-Arg, known to serve as potential signals for hormone cleavage at the Lys-Arg site and for amidation of the C terminus at Gly. As shown in Fig. 2, these structures are common in the precursors of toad VT and MT, mammalian VP and OT, and cyclostome VT. The position of each cysteine residues in the hormone and neurophysin domains is highly conserved in both VT and IT precursors among the fish species examined. The central region of neurophysin, which consists of approximately 67 amino acid residues, is rich in cysteine and highly homologous to the corresponding region of mammalian and amphibian neurophysins. Interestingly, the codon for the fourth cysteine residue in the neurophysin is changed to a stop codon by a point mutation from C to G in

VT and VP precursors

I T , WIT and OT precursors

N P

I

N P

I1

Fig. 2. Structural organization of neurohypophysial hormone precursors in hagfish (Eptatretus stouti) (Heierhorst et al., l992), chum salmon (Oncorhynchus keta) (Hyodo et al., 1991), toad (Bufofaponicus) (Nojiri et aZ., 1987), and human (Sausville et a / . , 1985).Abbreviations indicate moieties of precursors: SP, signal peptide; VT, vasotocin; VP, vasopressin; IT, isotocin; MT, mesotocin; OT, oxytocin; NP, neurophysin; CP, copeptin. GKR implies Gly-Lys-Arg residues connecting hormone moiety to neurophysin.

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the VT-I1 cDNA of chum salmon (Hyodo et al., 1991). However, in the study b y Heierhorst et al. (l990), this change was not found in the same hormone cDNA from the same species. A reason for this microheterogeneity might be a geographical variation in the fish between Japan and Canada where the samples were obtained.

2. NEUROPHYSIN AND COPEPTIN The size of the VT and IT neurophysins in salmon is larger than that of toad MT and mammalian OT. The V T and IT neurophysins of fish have an extended C-terminal region with approximately 30 amino acids including a leucine-rich segment. This additional portion may correspond to copeptin, which is a glycopeptide moiety in mammalian VP but is lost in mammalian OT and amphibian MT. This portion in fish lacks the glycosylation site, but Heierhorst et al. (1990) indicated that the glycosylation site could be produced by a single mutation in the neurophysins of VT-I and IT-I. The amino acid sequences of copeptin among mammals and the corresponding part in teleosts and hagfishes are more variable than other parts of the precursor molecules. Nonetheless, they are characterized as leucine-rich segments. Based on the presence of copeptin or its comparable moiety in the C-terminal region of the precursor, neurohypophysial hormones can be divided into two groups; one includes mammalian VP, amphibian VT, fish VT, and fish IT, and the other includes mammalian OT and amphibian MT. Although neurohypophysial hormones are classified into VP and OT families according to the amino acid sequence of the hormone moiety, the structures of cDNA and deduced precursors suggest the possibility that IT belongs in the VP rather than the OT family. Unlike their sucker counterparts, salmon V T precursors possess a putative processing signal that may result in the posttranslational cleavage of a copeptinlike moiety from the C-terminal end of salmon neurophysins. However, it has been shown that conformational constraints preclude such a second cleavage in an amphibian vasotocin precursor. This has led to the suggestion that the use of such putative cleavage sites occurs only after the transition from VT in nonmammalian vertebrates to VP in mammals (Urano et al., 1992).

3 . SEQUENCE HOMOLOGY Nucleotide deletions are found in VT-I and IT-I1 when compared to their matching genes. VT-I precursor lacks one amino acid in its signal peptide and two amino acids in neurophysin. The latter are

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located in the variant and constant domains, respectively. IT-I1 lacks four continuing codons in its neurophysin region. The presence of such deletions indicates that the divergence of VT genes is greater than that of the IT genes. Nucleotide sequences of cDNAs encoding salmon VT and IT precursors were compared to calculate homology scores from the HOMOGAPN program of the GENETYX sequence analysis software package (Software Development Co. Ltd., Tokyo, Japan). In chum salmon, homology scores between I and I1 of VT or IT are greater than 60%, although homology scores between IT and VT are 56 to 59%, except for a 62.3% homology score between IT-I and VT-I. Divergence of the VT precursors is considered to be greater than that of the IT precursors.

B. Structure of VT and IT Genes

The nucleotide sequences ofhormonal genes are essential for analysis of the physiological signals that regulate gene expression. It is therefore quite important to analyze the regulatory elements located in the 5' region upstream to the coding region, although little is known for the fish VT and IT genes. 1. CODING REGIONOF VT

AND

IT GENES

Among various species ofteleosts, both the cDNAs and genes encoding VT and IT precursors have been sequenced from white sucker (Figueroa et al., 1989; Morley et al., 1990). Two types of cDNAs were obtained for each of the VT and IT precursors. The two genes corresponding to VT-I and I1 are composed of three exons and two introns, the positions of which are identical to those in VP genes, their mammalian counterparts. The first exon (exon A in Fig. 3 ) encodes a signal peptide, the hormone VT, and the N-terminal portion of neurophysin. The second exon (exon B) encodes the central conserved portion of neurophysin, and the third exon (exon C) encodes the Cterminal portion of neurophysin including the leucine-rich segment, which corresponds to mammalian copeptin. In contrast to the mammalian OT gene consisting of three exons and two introns, neither type of IT gene in white sucker has introns (Fig. 3 ) .This gene organization is supporting evidence for an independent evolutionary pathway of teleost isotocin and tetrapod oxytocin after bifurcation from a common ancestral molecule (see Fig. 5).

AKIHISA URANO E T AL.

112 Piecurear

HN ,

vT-l

SP

IVr-11

NP

ECOOH

Gene

EXON A

5

EXON 0

EXONC

3

Gene

5

VT-I1

Precursor

-COOH

IT-I SINGLE EXON

5'

3

Gene

Fig. 3. Structural organization of white sucker VT-I, L'I-11, and IT-I precursors and their genes (Heierhorst et ul., 1989; Morlev et ul., 1990). Protein-coding regions in the genes are shown as boxes, and introns as disconnected lines. SP, signal peptide; VT, vasotocin; NP, neurophysin; IT, isotocin.

2. REGULATORY ELEMENTS IN THE UPSTREAM REGION The presence of at least two genes each for V T and IT precursors is clearly shown for four salmonid species by Southern blot analysis (Fig. 4),although the patterns of expression differ among species and will be described in the next section. Differences in the expression of the VT and I T gene family may be due in part to alterations in the nucleotide sequence in the 5' upstream region, which is responsible for the regulation of gene expression (Hyodo and Urano, 199lb). Elements involved in such regulation have been examined in mammals, but regrettably not in fish. It is clear in mammalian gene expression that the 5' upstream region of hormonal genes integrates various intracellular signals by interacting with DNA binding proteins (Adan et al., 1993).

C. Evolutionary Pathway of VT and IT Genes T h e molecular evolution of neurohypophysial nonapeptides has attracted the attention of many comparative endocrinologists. On the basis of the sequences of nine amino acids and phyletic distributions,

3.

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EXPRESSION OF THE VASOTOCIN A N D ISOTOCIN GENE FAMILY

Rainbow trout

Masu salmon

Sockeye salmon

Chum salmon

Vasotocin I II -

1 2 3 1 2 3

I II -

1 2 3 1 2 3

I II 1 2 3 1 2 3

1)

1:EcoRI 2: HlrKl 111 3:PStl

I -

I 2 3 1 2 3

lsotocin

I I1 I II I II I II -

I 2 3 1 2 3

I 2 3 1 2 3

I 2 3 1 2 3

1 2 3 1 2 3

-20 -5

-2 kbP

Fig. 4. Southern blot analyses o f V T and IT genes in salmonids showing the presence of at least two genes ( I and 11) for each of the VT and I T precursors. Cenoinic DNA was prepared from the liver of single fishes, species of which were rainbow trout (Oncorhynchus mykiss), masu salmon (0.musou), sockeye salmon (0.nerka), and chum salmon (0.ketu). DNA samples were digested separately with Eco HI (lane l), Hind 111 (lane 2), and Pst I (lane 3 ) . Molecular probes used for hybridization were chum salmon VT-I, VT-11, IT-I, and IT-I1cDNAs that were labeled with "P-labeled CTP b y use of a Multiprime DNA labeling system (Aniersham). T h e type of probe is shown above the lane numbers. T h e distributional patterns of positive bands differ between I and I1 for both VT and IT.

they have proposed various schemes for an evolutionary pathway of VP and OT families. The accumulation of molecular information mentioned in the foregoing now enable us to perform a statistical estimation of the evolutionary pathway.

1. SYNONYMOUS SIJBSTITUTION RATE Hyodo et al. (1991)analyzed the evolutionary relationships among the neurohypophysial hormone precursors by using the method of Miyata et al. (1985),in which nucleotide sites and substitutions are

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classified as synonymous and nonsynonymous. The structures ofmarnnialian (Ruppert et al., 1984) and white sucker (Morley et al., 1990) genes, toad cDNAs (Nojiri et al., 1987), and the presence of three exons were considered in the estimation. The nucleotide sequences of precursors were thus divided into three regions: region A, the moiety which encodes the signal peptide, the hormone, and the N-terminal portion of neurophysin; region B, the segment that encodes the central conserved portion of neurophysin; and region C, the segment that encodes the C-terminal portion of neurophysin and copeptin. In each region, the evolutionary distances in the phylogenetic tree among neurohypophysial hormone precursors were estimated by the formula T = c K s / 2 v , where T = evolutionary distance between two sequences; c K s = a mean value of corrected synonymous nucleotide substitution rates; and 0 = 3.1 x lO-')/locus/year, the mutation rate calculated from mammalian genomic data (Miyata et ul., 1985). The values of c K s are almost the same for vasotocin and isotocin precursor genes, and the mean mutation rate (c) ofgenes encoding the neurohypophysial hormone precursors was estimated as 8.4 x lO-'/Iocus/year in t el eost s . The rate o f nucleotide substitution for region B was considerably lower than for the other two regions in terms of both synonymous and nonsynonymous substitutions. This result coincides with the fact that region B includes the part of neurophysin that is highly conserved among species used for the estimation of the substitution rates. Furthermore, the occurrence of gene conversion in the central portions of neurophysins was suggested for salmonid fishes, similar to that found i n the human equivalents (Sausville e t al., 1985), whereas the rate of nucleotide substitution for the conserved regions of V T and MT neurophysins in the toad differed less than those corresponding to the other two segments. On the basis of the estimated evolutionary distances and the structural organization of the precursors, Hyodo et al. (1991) have proposed a model describing the evolutionary pathway of neurohypophysial hormone precursors. Teleost VT and IT precursors may be derived from an ancestral fish VT precursor by gene duplication about 230-270 million years ago. A second gene duplication led to different genes for salmonid V T and IT precursors about 100-125 million years ago. Tetraploidization of salmonid fish (Ohno et al., 1968), which might be the basis of the second gene duplication, would have preceded the gene conversion event approximately 45 million years ago. A diver-

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gence time of 370 million years was estimated between toad V T and MT precursors. It would be reasonable to consider that teleost I T and amphibian MT genes were derived directly from the corresponding ancestral VT genes by gene duplication. 2. MAXIMUM LIKELIHOOD AND BOOT STHAP Miyata’s method employed by Hyodo et al. (1991)for the analysis of the evolution of salmonid neurohypophysial hormones is effective for the calculation of relatively close phylogenetic distances. However, for analysis of a distant phylogenetic relationship, comparison of nucleotide sequences is not valid because too many substitutions could occur, especially in the third position of the codon, and nucleotide frequencies are different among lineages. The method called PROTML, which is based on the maximum likelihood (Adachi and Hasegawa, 1992a,b),uses the amino acid sequence instead ofthe nucleotide sequence. In this method, a novel algorithm for searching tree topologies, called “star decomposition,” is employed. This algorithm seems to be effective in finding the best tree from a large number of possible tree topologies. The algorithm “star decomposition” is similar to the procedure employed in the neighbor-joining method that uses the distance matrix (Saitou and Nei, 1987).The PROTML can be used for both rooted and unrooted trees. For the 17 known neurohypophysial hormone precursors, aligned sequences of 119 amino acids were used for the calculation of the phylogenetic distances among species. The sequences included the signal peptide, mature hormone, and neurophysin, but not copeptin. Gap positions that were observed in some species were not included in the calculation. Characteristics of the phylogenetic tree obtained by PROTML were as follows. A large divergence occurred in the common ancestral neurohypophysial hormone of vertebrates. It bifurcated into the tetrapod and teleost trees. The tetrapod tree further separated into two groups, the toad VT group and the group ofremainders that includes toad MT, mammalian VP, and OT. Not only OT but also VP had a common ancestral molecule with toad M T (Fig. 5). Along the tree, copeptin disappeared in two lineages, one in the toad M T lineage and the other in the mammalian OT lineage. Although the sequences used for calculation did not include the copeptin region, the resultant phylogenetic tree fits fairly well with the phylogenetic relationships based on the presence or absence of copeptin in tetrapods. Furthermore, the tree clearly explains the presence of two groups of conserved common sequences in neurophysins; one group is neuro-

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I Teleost I Tetrapod 1-

I IT 1 - V

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Fig. 5. Phylogenetic tree ofneurohypophysial hormone precursors in gnathostomes. Amino acid sequence data from teleostean, amphibian, and mammalian species were used for estimation of relationships by maximum likelihood analysis.

physins of toad VT and teleost VT and IT, and another group is in neurophysins of toad M T and mammalian OT and VP. In the teleost tree, two branches appeared; one is the VT branch including both type I and 11, and another is the IT branch, which also includes the two types. To analyze the teleost tree more precisely, the Boot Strap probability was calculated for 11 likely trees in addition to the best one mentioned in the preceding. The probability of the best tree shown in Fig. 5 is 0.2784, and those for the next three trees are 0.2534, 0.2411, and 0.2066, respectively. The other trees show a probability of less than 0.006. The various trees differed in the pattern ofthe modes ofdiversification, so that a judgment ofa correct tree might be difficult. A serious problem here is that there is little information on the characteristics of gene conversion and duplication events in the genes encoding precursors of neurohypophysial hormones. Such information is crucial to determine the mosaic evolution of VP and OT gene families. Further studies of the molecular structures of hormone genes are required to estimate a more detailed pathway for the evolution of hormonal molecules.

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111. DIVERGENCE OF VT AND

IT GENE EXPRESSION

The hypothalamic NSCs are considered to be responsive to stimuli that are involved in the regulation of various physiological phenomena. The responses of the NSCs to such stimuli are changes in secretory activity, including expression of VT and IT genes. We can now study the effects of physiological stimuli on VT and IT gene expression because the nucleotide sequences ofcDNAs encoding teleost neurohypophysial hormones have been elucidated in salmonid and catostomid fishes. Such information is important for clarification of the mechanisms that control the secretory activity of NSCs, and further for understanding of the biological roles of the hypothalamic neurosecretory system in fish. One problem is that salmonid and catostomid fishes are tetraploid. Cell nuclei in salmon actually contain about double the amount of' DNA and number of chromosomes when compared to other species in the kin order, Clupeiformes (Ohno et al., 1968).Probably because of this, pairs of genes for various hypothalamic and pituitary hormones were found in salmonid fishes and white sucker. For example, two different cDNAs were cloned and sequence-analyzed for each of the VT and IT precursors in chum salmon (Heierhorst et al., 1990; Hyodo et al., 1991) and white sucker (Figueroa et al., 1989; Heierhorst et al., 1989). Southern blot analyses ofgenomic DNA from individual salmon have shown the presence of at least two types of V T and IT genes in masu salmon, sockeye salmon, and rainbow trout in addition to chum salmon (Fig. 4). Furthermore, two different cDNAs encoding precursors of salmon-type gonadotropin-releasing hormone in socke y e salmon (Ashihara et al., 1992) and two genes encoding melaninconcentrating hormone in chum salmon (Ono et al., 1988) were cloned and sequence-analyzed. Considerable information has accumulated on nucleotide sequences of genes and mRNAs encoding peptide hormones in salmonid fishes and white sucker, making it possible to use these species in studies of hormonal gene expression. Hence, we need to confirm whether two genes for the same hormone are expressed equally or differently. Clarification of this problem is requisite for a quantitative study of gene expression, and also for analyses of regulatory mechanisms of gene expression, when a tetraploid fish is used as an experimental animal. We therefore will discuss in this section the expression patterns of VT and IT genes in salmon.

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A. Hybridization Probes There are several molecular techniques by which expression of particular genes can be examined. These techniques, including Northern blotting, dot/slot blot analysis, and in situ hybridization histochemistry, utilize unique base pair matching between tissue mRNA and specific molecular probes. However, the probes required for the analysis of gene expression are usually highly specific, often making crossspecies hybridization impossible (Urano et al., 1988). We were concerned this would be true also among salmonids. If so, it would be difficult to obtain nucleotide sequences of VT and IT cDNAs for each of the various salmonid species used as experimental animals. Our experience described in the following may be informative in this respect.

1. SYNTHETIC OLIGONUCLEOTIDE PROBES For in situ hybridization histochemistry of hypothalamic sections from several vertebrate species, we applied a 22mer synthetic oligonucleotide probe in which the nucleotide sequence corresponded to the sequence for the cyclic region (GGACGATGTAGGTCTTGACGGG) oftoad VT and mammalian OT. This VT/OT probe gave intense hybridization signals in the hypothalamic magnocellular nuclei of the Japanese toad, the bullfrog, the mouse, and the rat. On the other hand, only weak signals were localized in the preoptic nucleus of Xenopus laevis, and no signals above background were seen in the hypothalamic magnocellular neurons of the eel, Japanese quail, or zebra finch. A comparison of some sequences known in these species indicates that oligonucleotide probes of about 20mer can recognize a sequence with one mismatch, that is, a point mutation, if' the design of the probe is appropriate.

2. RELIABILITY OF c D N A PROBES Depending on the stringency of the hybridization conditions, cDNA probes labeled with 32P or 35S by a random priming method can be sufficiently specific to discriminate four genes and four mRNAs for VT-I, VT-11, IT-I, and IT-I1 precursors in individual chum salmon (see Figs. 4 and 6). The conditions of hybridization with a labeled cDNA probe that we adopted in our Southern and Northern blot analyses (see Hyodo et al., 1991)theoretically cannot discriminate nucleotide sequences that are less than 80% identical. This value is much higher than the identities of about 60 to 70% among the nucleotide sequences of four precursor cDNAs in chum salmon.

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Masu salmon Sockeye salmon Chum salmon

Vasotocin

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lsotocin

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Fig. 6. Northern blot analysis of mRNA from hypothalami of several salmonid species, showing the expression pattern of genes encoding neurohypophysial hormone precursors.

3 . CROSS-SPECIES HYBRIDIZATION IN SALMONIDS The specificity of base pairing in hybridization techniques mentioned earlier is often inconvenient for studies of gene expression in fish because the cDNA or the sequence for a certain neurohormone obtained from one species cannot be used in other species that are phylogenetically distant. cDNA probes encoding VT and IT precursors of chum salmon did not yield positive hybridization signals in a Northern blot analysis of goldfish hypothalamic poly(A+)KNA ( S . Hyodo, unpublished data, 1990).The evolutionary distances (cKs, the number of synonymous substitutions per nucleotide site) of VT and IT precursor genes between chum salmon and goldfish could not be estimated because of the lack of nucleotide sequences of goldfish VT and IT genes. Nonetheless, considering the distance obtained from fossil records, we presume that the evolutionary distance between salmon and goldfish might be similar to those between salmonids and catostomids. The use of heterologous molecular probes in distant species should be avoided in a quantitative study. Studies of gene expression require the use of specific molecular probes, the nucleotide sequences of which have been obtained usually b y cloning and sequence analysis. However, cloning and sequence

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analysis of a hormonal cDNA is time- and money-consuming. Therefore, we have tested by Southern blot analyses the usefulness ofcDNAs encoding chum salmon VT and IT precursors in other salmonid species, including masu salmon, rainbow trout, and sockeye salmon (Hiraoka et al., 1993). The identity of the nucleotide sequence for the IT-I cDNA of sockeye salmon was estimated in comparison with those for other peptides of the OT family. The sockeye salmon IT-I cDNA is highly similar to those of chum salmon (99.0%) and masu salmon (96.8%), whereas it is considerably different from chum salmon IT-I1 cDNA (62.7% identity). The magnitude of this difference is comparable to the distances from white sucker IT-I, toad MT, and human OT cDNAs. These values for sequence identity mean that chum salmon cDNAs encoding VT-I, VT-11, IT-I, and IT-I1 can be used as unique molecular probes to analyze the expression of the VT and IT gene family in the brains of various salmonid species. As shown in Fig. 4,chum salmon cDNA probes can clearly recognize corresponding sequences in the Southern blot analyses of genomic DNAs in the salmonid species examined. Nucleotide sequences for hormone precursors other than VT and IT precursors are also highly similar in the genus Oncorhynchus, as shown by growth hormone cDNAs in the chum salmon, rainbow trout, and coho salmon. Sequence data in one salmonid species therefore can be applicable to molecular studies to analyze the expression of hormonal genes in other salmonid species.

B. Northern Blot Analyses Expression of neurohypophysial hormone genes in hypothalamic NSCs may be influenced by various environmental and physiological conditions. It is also subject to diurnal/circadian, lunar, seasonal, or circannual control, although almost nothing is known of these effects in fish. Moreover, all salmonid species that we examined have a set of duplicated genes for VT and also for IT precursors, suggesting that the magnitudes of transcription may differ between two genes encoding the same hormone. The result of Northern blot analysis clearly showed that this is true, and also the patterns of expression are different among the following four species: masu salmon, rainbow trout, sockeye salmon, and chum salmon (Fig. 6). Phylogenetically, masu salmon and rainbow trout are considered to be more primitive than sockeye and chum salmon (Numachi 1984). The location and the intensity of hybridization positive bands of mRNA suggest that the VT-I, VT-11, IT-I, and IT-I1 genes are highly

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expressed in sockeye salmon and chum salmon, whereas the expression of VT-I1 gene is scarce in m a w salmon and rainbow trout. Thus, strikingly more intense hybridization signals for VT-I mRNA than for VT-I1 mRNA were detected in the latter species. In contrast to the VT genes, the hybridization band pattern for IT mRNAs among different species showed a similar tendency, that is, the signals indicating the amounts of IT-I mRNA are more intense or at least similar when compared to those of IT-I1 mRNA. In spite of the difference in the intensity of hybridization signals, the lengths of mRNAs encoding identical neurohypophysial hormones remained nearly the same among all species examined. Interpretation of these results is somewhat problematic. The amount of cellular or tissue mRNA shown by the hybridization techniques is a result ofboth transcription and degradation activities, which determine the turnover rate. Thus, the amount of mRNA does not reflect the magnitude of mRNA synthesis, that is, the rate of gene expression, unless the turnover rate ofthe mRNA is determined. Nevertheless, this concern can be eliminated in the present case for the reason stated in the following. In the rat, the sizes of VP and OT mRNAs, as well as their amounts, were increased following sodium loading and water deprivation, which are well-known physiological stimuli that facilitate secretory activity of hypothalamic NSCs (for review see Hyodo and Urano, 1991b). The increase in the size of mature mRNA, which was detected within 2 hr after the commencement of stimulation, is probably due to an increase in the poly(A) tail size because removal of the poly(A) tail reduced the lengths of the VP mRNA in control and experimental groups to exactly the same size. A similar increase in the length of VT mRNA has been observed in anuran amphibians (S. Hyodo, personal communication, 1993).The poly(A) tail of eukaryotic mRNA is considered to be involved in the regulation of mRNA stability, that is, the turnover rate of mRNA, and translation efficiency. The lengths of mRNAs for identical neurohypophysial hormone precursors were nearly the same among examined salmonids, suggesting that the difference in the intensity of hybridization signals may not reflect the turnover rates, but the transcriptional activities of VT and IT genes. VT and IT cDNAs have similar 3’-noncoding regions that affect turnover ofmRNA; this also supports the idea that transcription and not turnover is affected. We therefore consider that the varied patterns of hybridization positive bands in Fig. 6 show the divergence in expression of neurohypophysial hormone precursor genes among salmonids. Phylogenetically similar species, such as masu salmon and rainbow trout, or sock-

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e y e salmon and chum salmon, showed similar tendencies in VT gene expression, suggesting that the divergent patterns of gene expression, particularly of VT genes, reflect molecular heterogeneity in the loci involved in regulation oftranscription of VT and IT genes. Thus, the analysis of the structure of genomic DNAs encoding VT and IT in tetraploid fish is important for understanding the evolution of the regulation of gene expression.

IV. VT AND IT GENE EXPRESSION IN OSMOTIC ADAPTATION One important benefit of recombinant DNA techniques is that we can now measure or estimate the changes in the amount of specific mRNAs in tissues, or even in individual cells that accompany a physiological event. However, there are many methodological problems that might be solved in the near future. Expression of hormonal genes includes various aspects of hormone synthesis, such as transcription and translation of genetic information, and further processing of the hormone precursor. However, we need to have information not only on synthetic activity, but also on releasing activity to understand the physiological significance of gene expression in neuroendocrine cells. The use ofradioimmunoassay (RIA) and/or immunohistochemistry in combination with molecular hybridization techniques would be the minimum requirement in this respect, but published data that fulfill this requirement are rare for fish studies at present.

A. Physiological and Histological Backgrounds The biological role of neurohypophysial hormones in water and salt metabolism of fish does not seem to be uniform among species, in contrast to an antidiuretic and sodium-retaining function in tetrapods.

1. CLASSICAL HISTOCHEMICAL STUDIES Responses to the modification of environmental salinity in the fish neurosecretory system were examined by nse of classical histochemical techniques. The latter can uniquely stain neurosecretory materials in hypothalamic NSCs, although the results showed some contradictions among species concerning the effects oftransfer from fresh water ( F W ) to seawater (SW) and vice versa. The important results in such papers were cited in a review by Maetz and Lahlou (1974), and also in reports concerning secretory activity of the vasotocinergic neurose1991; Perrott et d., 1991; Hyodo and cretory system (Haruta et d., Urano, l99la); these results will not be repeated in this paper.

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2. ELECTRICAL ACTIvrrY Also, electrical responses of single PONmg units to Na+ appeared not to be uniform among fishes. In the goldfish, application of diluted seawater to the olfactory chemosensory system resulted in suppression of electrical activity in PONmg neurons, that is, hyperpolarization and a decrease in spontaneous firing rates (Kandel, 1964). In contrast, Na+ that was microiontophoretically applied to single units markedly increased the discharge rates of about 50% of PONmg neurons in the eel brain (Sugita and Urano, 1986).

3. PLASMA VERSUS PITUITARY LEVELSOF VT Plasma levels of VT were measured by RIA in several euryhaline and stenohaline species (Perrott et al., 1991; Balment et al., 1993). In flounder and rainbow trout that were acclimated to FW or SW at least for 14 days, the FW-acclimated animals had about two-fold higher plasmaVT concentrations compared to SW-acclimated fish. Such differences in plasma VT concentrations were not detected in FW- and SWacclimated eel, although a transient increase in the plasma level was found during the initial transfer from FW to SW. Plasma VT levels in carp, as a representative ofstenohaline fish, did not vary between fish maintained in FW and 40% SW. In correlation with the plasma VT level, the pituitary VT contents rose within 1 day, and remained at higher levels for up to 1 week in FW-acclimated compared to SW-acclimated flounder, whereas the VT contents were significantly decreased within 1 day after FW to SW transfer. The pituitary VT accumulation with higher plasma level may indicate highly elevated VT gene expression that overcomes VT release in FW-acclimated compared to SW-acclimated flounder.

4. CHANGES IN VT-IROF PONMGNEURONS In medaka (Oryzias latipes),changes in pituitary VT contents were contradictory to those reported in the flounder. When FW-adapted medaka were transferred to SW, pituitary VT contents decreased within 2 hr, attained the lowest level by 1 day, and thereafter returned to near the initial level 1 week after the transfer (Haruta et al., 1991). The decrease in the number of VT-ir PONmg neurons was accompanied by an increase in the cell nuclear size in these animals, suggesting an elevation in synthetic and transport activity following the initial increase in VT release during the first day of SW adaptation. Transfer of SW-adapted medaka to FW induced a transient but significant increase in pituitary VT content 1 to 2 hr after the transfer. This elevated VT content rapidly returned to the normal FW level

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within 1 day. The number of VT-ir neurons simultaneously began to increase to about 2.5 times the initial value 2 hr after the transfer, although the cell nuclear size remained unchanged. These results indicate a rapid inhibition of VT release, but not synthesis, immediately after SW to FW transfer.

5. ELECTRICAL VERSUS MOLECULAR REPONSES

The electricophysiological response of PONmg neurons to osniotic stimuli, either excitation or inhibition of firing activity, is acute and immediate. Most histological and RIA studies, however, examined effects of osmotic stimuli after a day or even a week. It is thus probable that many previous studies failed to detect early changes by osmotic stimuli in morphological features and also neurohypophysial hormone inimunoreactivity in the hypothalamic neurosecretory system. What we have to examine is immediate and/or early responses in the PONmg neurons, as well as more long term influences, to clarify physiological responsiveness, such as those reported by Haruta et al. (1991) in medaka. Otherwise we will not be able to realize the function of fish neurosecretory system in homeostatic osmoregulation. B. Effects of Transfer Experiments on VT and I T mRNAs An early effect of osmotic stimulation on the expression of neurohypophysial hormone genes in mammals was the replacement of VP mRNA with new mRNA that had a longer poly(A) tail. An increase in the amount of both VP and O T mRNAs occurred within 2 days (see Hyodo and Urano, 1991b).Hence, it was expected that transfer offish from FW to SW or vice versa would induce a change in the expression of the V T and IT genes in the hypothalamic neurosecretory system. Such information on the responsiveness of neurosecretory neurons is a prerequisite for understanding the physiological control mechanisms of VT and I T gene expression.

1. FW-SW TRANSFER EXPERIMENT IN RAINBOW TROUT

The effects of environmental hyper- and hypo-osmotic stimuli on the expression of the VT and I T precursor genes were investigated in rainbow trout by use of an in situ hybridization technique (Hyodo and Urano, 1991a). The molecular probes used in this study were 46mer synthetic oligonucleotides with sequences corresponding to the chum salmon mRNAs encoding the V T and IT precursors (-5 to 11) (Hyodo

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et al., 1991). The VT probe was highly similar to chum salmon VT-I (97.8%) and VT-I1 (93.5%), and to masu salmon VT (100%) mRNAS, whereas the IT probe was homologous to chum salmon IT-I (93.5%) and IT-I1 (91.3%), and to masu salmon IT (93.5%) mRNAs. Because the identity between V T and IT probes was 67.4%, it was certain that the VT probe would hybridize with salmonid VT mRNAs, but not with IT mRNAs, and would not discriminate among species and types. The intensity of autoradiographic hybridization signals, which reflected the amounts of mRNA, was determined as the number of silver grains in magnocellular neurons of the PONmg. The conspicuous finding was a decrease in hybridization signals for VT mRNA within 1 day after transfer from FW to 80% SW, although the amount of IT mRNA remained unchanged. The low levels of VT mRNA in SW trout were consistently maintained for up to 2 weeks. After a transfer back to FW from 80% SW, the VT mRNA in SW-acclimated fish returned to the initial FW level (Fig. 7), whereas IT mRNA did not change significantly. The decrease in the level of VT mRNA in the magnocellular PONmg neurons of SW-acclimated trout coincided with the change in the pituitary content and the plasma level of V T in rainbow trout and flounder (Perrott et al., 1991). Secretion of VT may be enhanced by FW transfer of SW-acclimated fish. Thus VT may have an important physiological role in adaptation to hypo-osmotic environments in these euryhaline species.

EXPRESSION VP and OT are involved in osmotic regulation in mammals (see Urano et al., 1988). Hyperosmotic stimulation depleted both VP and OT from the NSC somata and the neurohypophysis, elevated their plasma levels, and also increased the amount of VP and O T mRNAs in the rat hypothalamus within a few days of the start of the stimuli. Rats given 2% NaCl orally showed polydipsia and polyurea with a rapid and significant increase in the plasma sodium level. The VP mRNA level was gradually elevated by this sodium loading, whereas the change in OT mRNA level was a phasic increase. The magnitude of the increase was more significant in the supraoptic (SON) than the paraventricular (PVN) neurons. In contrast, water-deprived rats showed severe antidiuresis in maintaining the plasma osmolarity and sodium level fairly stable. The increase in the VP mRNA level was more rapid and conspicuous in the PVN than the SON neurons, whereas the elevation of the OT mRNA level in the SON was more significant than that in the PVN. Thus the response of mammalian

2 . MAMMALIAN VP AND OT

GENE

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the level of VP and OT mKNAs, hypertrophy ofVP-ir and OT-ir NSCs, and a decrease in immunoreactivity with VP and OT antisera. However, in the hypothalami of developing mice, the gradual elevation of VP immunoreactivity in PVN neurons did not accompany any changes in the intensity of hybridization signals for VP mRNA, whereas those in the SON neurons increased in parallel (Hyodo et al., 1992). Thus, in the magnocellular NSCs, the intensity of hybridization signals for VP and OT mRNAs is not necessarily associated with immunoreactivity of the hormones. The same can be true also in the fish PONmg neurons. In the foregoing case, the intensity of the hybridization signals may represent the amount of mRNA but not transcriptional or translational activity, and the magnitude of immunoreactivity may show the amount of stored hormone. Dissociation of such parameters from releasing activity is another problem in a study of hormonal gene expression. I n the pituitary of the rainbow trout in serum-free culture, synthesis and release of growth hormone (GH) were highly elevated, but those of prolactin (PRL) declined within 2 days in culture. The GH mRNA levels in individual pituitary cells were maintained at around the same level, but the stainability of GH-ir cells was decreased. In contrast, the stainability of PRL-ir cells remained unchanged, although the PRL mRNA levels decreased within 8 days (Yada et al., 1991). Information on changes in transcriptional and translational activities is what we seek in the study of gene expression. Hence, it is crucial to clarify changes in the turnover rates ofVT/IT mRNAs and the hormones. In such studies, the possibility of seasonal variation in the responsiveness to osmotic stimulation should be taken into account. This is because gene expression and the release of GH are activated by SW transfer in smolts with adequate seawater adaptability, which is seasonally variable (Yada et al., 1992). In addition to the rather technical problems mentioned here, it is possible that VT and/or IT regulate the pituitary-adrenal system. This system is also related to the osmotic environment, although neither regulation of this system by VT and IT nor feedback control of the PONmg neurons by corticosteroids is clear in salmonid fish.

V. CONCLUSION The structures of cDNAs encoding neurohypophysial hormone precursors in catostomid and salmonid fishes are homologous to those in tetrapods. The precursors are composed of a signal peptide, hormone,

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and neurophysin. Comparisons of deduced amino acid sequences of precursors among vertebrates suggest an independent evolutionary pathway of fish and tetrapod neurohypophysial hormones. The biological roles of fish neurohypophysial hormones, for example, VT and IT in teleosts, are still ambiguous, probably because of a lack of appropriate bioassays. Application of molecular techniques in combination with physiological and immunological methods is expected to improve this situation. Recent evidence indicates that a role of VT in water and salt metabolism is not uniform among fish species. In euryhaline fish, such as salmonids and flounder, VT seems to be involved in freshwater adaptation. Hybridization signals for VT mRNA in PONmg neurons were increased b y FW transfer of SW-acclimated rainbow trout. Biologically meaningful stimuli to the NSCs are presumed to result in changes in the expression of neurohypophysial hormone genes. Such changes include various aspects of secretory activity, including transcription, translation, and release. In addition to clarifying the changes in secretory activity, elucidation of the regulatory mechanism of gene expression, unknown in fish, will be crucial for understanding the molecular basis of VT/IT function in fish.

ACKNOWLEDGMENTS Part of our study referred to in this article was supported by research grants froni the Ministry of Education, Science and Culture, and the Fisheries Agency, Japan.

REFERENCES Acher, R. (1985).Biosynthesis, processing, and evolution of neurohypophysial hormone precursors. In “Neurosecretion and the Biology of Neuropeptides” (H. Kobayashi, H . A. Bern, and A. Urano, eds.), pp. 11-25. Japan Science Society Press, Tokyo/ Springer-Verlag, Berlin. Acher, R., Chauvet, J., and Chauvet, M. T. (1970).A tetrapod neurohypophysial hormone in African lungfishes. Nature (London) 227, 186-187. Acher, R., Chauvet, J . , Chauvet, M. T., Michel, G . , and Rouille, Y. (1991). Evolution of the oxytocin-vasopressin superfarnily duplication events: Two new neurohypophysial hormones in cartilaginous fish. Znt. Syrnp. Neurosci., 1 1 th, Amsterdam, Netherlands, 13. Adachi, J.. and Hasegawa, M. (1992a).Amino acid substitution of proteins coded for in mitochondria1 DNA during mammalian evolution. J p n . J. Genet. 67, 187-197.

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Adachi, J., and Hasegawa, M. (1992b). MOLPHY: Programs for molecular phylogenetics, I. PROTML: Maximum likelihood inference of protein phylogeny. Computer Science Monographs. No. 27, The Institute of Statistical Mathematics. Minato-ku, Tokyo. Adan, R. A. H., Cox, J. K., Beischlag, T. V., and Burbach, J. P. H. (1993). A composite hormone response element mediates the transactivation of the rat oxytocin gene by different classes of nuclear hormone receptors. Mol. Endocrinol. 7, 47-57. Ashihara, M., Suzuki, M., Kobayashi, M., Urano, A., and Aida, K. (1992). Presence of two different precursors for salmon type gonadotropin.-releasing hormone in salmonid. Proc. J p n . Soc. Comp. Endocrinol. 7, 36. Balment, R. J., Warne, J. M., Tierney, M., and Hazon, N. (1993). Arginine vasotocin and fish osmoregulation. Fish Physiol. Biochem. 11, 189-194. Batten, T. F. C., Cambre, M. L., Moons, L., and Vandesande, F. (1990). Comparative distribution of neuropeptide-immunoreactive systems in the brain of the green molly, Poecilia latipinnu. J. Comp. Neurol. 302, 893-919. Cumming, R., Reaves, T. A,, and Hayward, J. N. (1982).Ultrastructural immunocytochemical characterization ofisotocin, vasotocin and neurophysin neurons in the magnocellular preoptic nucleus of the goldfish. Cell Tissue Res. 223, 685-694. Du Vigneaud, V., Ressler, C. H., arid Trippet, S. (l953a). The sequence of amino acids in oxytocin, with a proposal for the structure of 0xytocin.j. Biol. Chem. 205,949-957. Du Vigneaud, V., Lawler, H. C., and Popenoe, E. A. (1953b). Enzymatic cleavage of glycinaniide from vasopressin and a proposed structure for this pressor-antidiuretic hormone of the posterior pituitary. J. Am. Chem. Soc. 75, 4880-4881. Dyball, R. E. J., Barnes, P. R. J., and Shaw, F. D. (1985). Significance of phasic firing in enhancing vasopressin release from the neurohypophysis. In “Neurosecretion and the Biology of Neuropeptides” (H. Kobayashi, H. A. Bern, and A. Urano, eds.), pp. 239-246. Japan Science Society Press, TokyoiSpringer-Verlag, Berlin. Figueroa, J., Morley, S. D., Heierhorst, J., Krentler, C., Lederis, K., and Richter, U. (1989). Two isotocin genes are present in the white sucker Catostomus comtnersoni both lacking introns in their protein coding regions. EMBO J. 8, 2873-2877. Garlov, P. E., and Khutinaev, A. S. (1991). Ecological and histophysiological investigation of the preoptico-posthypophysialneurosecretory system in the pink salmon Oncorhynchus gorbuscha during spawning. Zh. Eool. Biokhim. Fiziol. 27,41-48. Goossens, N., Dierickx, K., and Vandesande, F. (1977). Immunocytochemical localization of vasotocin and isotocin in the preopticohypophysial neurosecretory system of teleosts. Gen. Comp. Endocrinol. 32, 371-375. Guibbolini, M. E., Henderson, I. W., Mosley, W., and Lahlou, B. (1988). Arginine vasotocin binding to isolated branchial cells of the eel: Effect of salinity. /. Mol. Endocrinol. 1, 125-130. Guibbolini, M. E., and Lahlou, B. (1992).G(i)-protein mediates adenylate cyclase inhihition by neurohypophyseal hormones in fish gill. Peptides 13, 865-871. Haruta, K., Yamashita, T., and Kawashima, S. (1991). Changes in arginine vasotocin content in the pituitary of the niedaka (Oryzias Zutipes) during osmotic stress. Gen. Comp. Endocrinol. 83, 327-336. Heierhorst, J., Morley, S. D., Figueroa, J., Krentler, C., Lederis, K., and Richter, D. (1989). Vasotocin and isotocin precursors from the white sucker, Catostomus conzersoni: Cloning and sequence analysis of the cDNAs. Proc. N a t l . Acad. Sci. U.S.A. 86,5242-5246. Heierhorst, J., Mahlmann, S., Morley, S. D., Coe, I. R., Sherwood, N. M., and Richter, D. (1990). Molecular cloning of two distinct vasotocin precursor cDNAs from chum

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salmon (Oncorhynchus ketu) suggests an ancient gene duplication. F E B S Lett. 2, 301-304. Heierhorst, J., Lederis, K., and Richter, D. (1992). Presence of a member of the Tcllike transposon family from nematodes and Drosophila within the vasotocin gene of a primitive vertebrate, the Pacific hagfish Eptutretus stouti. Prod Natl. Acad. Sci. U.S.A.89, 6798-6802. Hiraoka, S., Suzuki, M., Yanagisawa, T., Iwata, M., and Urano, A. (1993). Divergence of gene expression in neurohypophysial hormone precursors among salmonids. Gcn. Coinp. Endocrinol. 92, 292-301. Hyotlo, S., Kato, Y., Ono, M., and Urano, A. (1991). Cloning and sequence analyscs of cDNAs encoding vasotocin and isotocin precursors of chum salmon, O n c o rh ~ / i ~ c h u s keta: Evolutionary relationships ofneurohypophysial hormone precursors../. ~ o m p . Physiol. B . 160, 601-608. Hyodo, S., and Urano, A. (199la).Changes in expression of provasotocin and proisotocin geries during adaptation to hyper- and hypo-osmotic environments in rainbow trout. J . C o m p . Physiol. B . 161, 549-556. Hyodo, S., and Urano, A. (199lb). Expression of neurohypophysial hormone precursor genes in the mammalian hypothalamus. Zool. Sci. 8, 1005-1022. IIyodo, S., Yamada, C., Takezawa, T., and Urano, A. (1992).Expression ofprovasopressin gene during ontogeny in the hypothalamus of developing mice. Neuroscience (Ox. f o r d )46, 241-250. Kandel, E. R. (1964). Electrical properties of hypothalamic neuroendocrine cells. J . G e n . Physiol. 47, 691-717. Land, H., Schutz, G., Schmale, H., and Richter, D. (1982). Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin I1 precursor. Nature (London)295, 299-303. Land, H., Grez, M., Ruppert, S., Schmale, H., Rehbein, M., Richter, D., and Schutz, G. ( 1983). Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA. Nature (London)302,342-344. Lane, T . F., Sower, S. A,, and Kawauchi, H. (1988).Arginine vasotocin from the pituitary gland of the lamprey Petromyzon murinus: Isolation and amino acid sequence. Gen. C o m p . Endocrinol. 70, 152-157. hlaetz, J., and Lahlou, B. (1974). Actions of neurohypophysial hormones in fishes. I n “Handbook of Physiology,” Section 7: Endocrinology, Vol. 4, Part 1, pp. 521-544. A m . Physiol. SOC., Washington, D.C. Xlichel, G., Chauvet, J., Joss, J. M. P., and Acher, R. (1993a). Lungfish neurohypophysial hormones: Chemical identification of mesotocin in the neurointermediate pituitary of the Australian lungfish Neoceratodus forsteri. Gem C o m p . Endocrinol. 91, 330-336. Michel, G., Chauvet, J., Chauvet, M.-T., Clarke, C., Bern, H., and Acher, H. (19931,). Chemical identification of‘ the mammalian oxytocin in a holocephalian fish, the ratfish (Hydrolagus coliei). Gen. Comp. Endocrinol. 92, 260-268. Mivata, T., Hayashida, H., Kikuno, R . , and Yasunaga, T. (1985). Computer analysis of‘ homology between genes. In “Methods for Gene Research” (M. Takanami, S. Nishimura, and M. Matsumura, eds.), pp. 381-425. Tokyo Kagaku Dojin, Tokyo. \loons, L., Cambre, M.,Batten, T. F. C . , and Vandesande, F. (1989). Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass Dicentrurchus labrax. Neurosci. Lett. 100, 11-16. \loore, F. L. (1992). Evolutionary precedents for behavioral actions of oxytocin and vasopressin. Ann. N.Y. Acad. Sci. 652, 156-165. llorgan, J. I . , and Curran, T. (1991). Stimulus-transcription coupling in the nervorts

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system: Involvement of the inducible proto-oncogenes fos and jun. Annu. Hec. Neurosci. 14, 421-451. Morley, S. D., Schonrock, C., Heierhorst, J., Figueroa, J., Lederis, K., and Richter, D. (1990). Vasotocin genes o f t h e teleost fish Catostomus cornmersoni: G e n e structure, exon-intron boundary, and hormone precursor organization. Biochemistry 29, 2506-251 1. Nojiri, H., Ishida, I., Miyashita, E., Sato, M.,Urano, A,, and Deguchi, T. (1987). Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus. Proc. Natl. Acad. Sci. U.S.A.84, 3043-3046. Numachi, K. (1984).A study on the divergence and phylogeny of salmonids by isozymes. Heredity (Tokyo) (Zden)38, 4-11. Ohno, S., Wolf, U., and Atkin, N. B. (1968). Evolution from fish to mammals by gene duplication. Hereditas 59, 169-187. Olivereau, M., Moons, L., Olivereau, J., and Vandesande, F. (1988). Coexistence of corticotropiri-releasing factor-like immunoreactivity and vasotocin in perikarya of the preoptic nucleus in the eel. Gen. Comp. Endocrinol. 70,41-48. Ono, M . , Wada, C., Oikawa, I., Kawazoe, I., and Kawauchi, H. (1988). Structures o f t w o kinds of mRNA encoding the chum salmon melanin-concentrating hormone. Gene 71,433-438. Parent, A. (1983).T h e monoamine-containing neuronal systems in the teleostean brain. I n “Fish Neurobiology” (R. E. Davis and R. G. Northcutt, eds.), Vol. 2, pp. 285-315. T h e Univ. Michigan Press, Ann Arbor. Perrott, M. N., Carrick, S., and Balment, R. J. (1991). Pituitary and plasma arginine vasotocin levels in teleost fish. Gen. Comp. Endocrinol. 83, 68-74. Reaves, T . A., Jr., and Hayward, J. N. (1980). Functional and morphological studies of peptide-containing neuroendocrine cells in goldfish hypothalamus.]. Comp.Neurol. 193,777-788. Rouille, Y., Chauvet, J., Chauvet, M. T.,Acher, R., and Polenov, A. L. (1991).Neurohypophysial hormones as molecular evolutionary tracers: Investigations on the sturgeons Acipenser stellatus and Acipenser guldenstadti. C o m p . Biochem. Physiol. B: C o m p . Biochem. 100,721-726. Ruppert, S., Scherer, G., and Schutz, G. (1984).Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence. Nature (London)308, 554-557. Saitou, N., and Nei, M. (1987).T h e neighbor-joining method: A new method for reconstructing phylogenetic trees. M o l . Biol. Eu01. 4, 406-425. Sausville, E., Carney, D., and Battey, J. (1985). T h e human vasopressin gene is linked to the oxytocin gene and is selectively expressed in a cultured lung cancer cell line. /. Biol. Chem. 260, 10236-10241. Sharrer, E. (1928).Untersuchungen uber das Zwischenhirn der Fische. I. D i e Lichtempfindlichkeit blinder Elritzen. Z . Velgl. Physiol. 7, 1-38. Scharrer, E., and Scharrer, B. (1954). Hormones produced by neurosecretory cells. Recent Prog. Horm. Res. 10, 183-240. Subhedar, N., Rama Krishna, N. S., and Deshmukh, M. K. (1987). The response of nucleus preopticus neurosecretory cells to ovarian pressure in the teleost, Clarias hatrachus Linn. Gen. Comp. Endocrinol. 68, 357-368. Sugita, R., and Urano, A. (1986). Responses of magnocellular neurons in in oitro eel preoptic nucleus (PONmg) to acetylcholine, catecholamines, vasotocin, isotocin, angiotensin, and Na’. Zool. S c i . 3, 1081. Suzuki, M . , Hyodo, S., and Urano, A. (1992).Cloning and sequence analyses of vasotocin

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and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: Evolution of neurohypophysial hormone precursors. Zool. Sci. 9, 157-167. Suzuki, M., Nagasawa, H., and Urano, A. (1993). Cloning and sequence analysis of‘ cDNAs encoding vasotocin precursors in the cyclostomes. Zool. Sci. lO(suppl.), 133. Uemura, H. (1965). Histochemical studies on the distribution of cholinesterase and alkaline phosphatase in the vertebrate neurosecretory system. Annot. Zool. J p n . 38, 79-96. Urano, A. (1971). Monoamine oxidase in the hypothalamo-hypophysial region of the teleosts, Anguilla japonicu and Oryzias latipes. Z. Zellforsch. 114, 83-94. Urano, A. (1988). Neuroendocrine control ofanuran anterior preoptic neurons and initiation of mating behavior. Zool. Sci. 5 , 925-937. Urano, A., Hyodo, S., and Sato, M. (1988). In situ hybridization study ofneurohypophysial hormone mRNAs. In “Neurosecretion” (B. T. Pickering, J . B. Wakerley, and J. S. Summerlee, eds.), pp. 43-51. Plenum, New York. Urano, A., Hyodo, S., and Suzuki, M. (1992). Molecular evolution of neurohypophysial hormone precursors. Prog. Brain Res. 92, 39-46. Yada, T., Urano, A., and Hirano, T. (1991). Growth hormone and prolactin gene expression and release in the pituitary of rainbow trout in serum-free culture. Endocrinology 129, 1183-1192. Yada, T., Kobayashi, T., Urano, A., and Hirano, T. (1892). Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of amago salmon (Oncorhynchus rhodurus).J . E x p . Zool. 262,420-425. Yamada, C., Kaneko, T., and Kobayashi, H. (1985). Co-localization of angiotensin-11 and arginine vasotocin in the same neurons of the preoptic nucleus of the goldfish, Carassius auratus. Zool. Sci. 2, 975. Yulis, C. R., and Lederis, K. (1987). Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni. Cell Tissue Res. 247, 267-273.

I1 PITUITARY H O R M O N E S

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4 CONTROL OF TELEOST GONADOTROPIN GENE EXPRESSION FEI XIONG, KUNlMASA SUZUKI, A N D CHOY L. HEW Department of Biochemistry, Research Institute, Hospital for Sick Children, Toronto, and Departments of Clinical Biochemistry and Biochemistry, University of Toronto, Toronto, Ontario, Canada

I. Introduction 11. Duality of Teleost Gonadotropins A. Duality of Teleost Gonadotropins B. Function of GTHI and GTHII C. Structure of Teleost Gonadotropins D. Gonadotropin Cell Types and the Temporal Appearance of GTHI and GTHII 111. Genoinic Organization of Teleost Gonadotropins A. Structure of the a Subunit Gene B. Structure of the p Subunit Gene IV. Control of Gonadotropin Gene Expression A. General Overview B. Pituitary-Specific Expression of GTH Subunit Genes C. Regulation of Gonadotropin Gene Expression by Gonadal Steroids D. Regulation of Gonadotropin Gene Expression by GnRH V. Conclusion References

I. INTRODUCTION The teleost pituitary contains polypeptide hormones vital for somatic growth, reproduction, metabolic regulation, environmental adaptation, and many other important biological activities. Gonadotropins (GTH), in particular, regulate sexual maturation and the reproductive process and are therefore essential for the maintenance and preservation of the species. The synthesis and secretion of GTH is temporally regulated. Its regulation is complex and includes the participation and feedback 135 k I \ H PIlISIOLOGY VOL XI11

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controls of the hypothalamus-pituitary-gonadal axis. With the advent of molecular biology, our understanding of the regulation of manimalian GTH is substantial. Several relevant reviews are available (Gharib et al., 1990; Miller, 1993; Jameson and Hollenberg, 1993). In comparison, studies on teleost GTH gene expression are limited. However, because of the large number of fish species with different reproductive strategies as well as their high fecundity, teleosts represent a useful model to study the various aspects controlling gonadotropin synthesis. In the present chapter, we will summarize some ofthe more recent studies on the cloning, characterization, and regulation of teleost GTH genes. Where appropriate, comparisons will be made with what is currently known about mammalian GTH.

11. DUALITY OF TELEOST GONADOTROPINS A. Duality of Teleost Gonadotropins Gonadotropins in mammals, including both follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are secreted by the anterior pituitary gland. In addition, the placenta of primates and Equidae secretes chorionic gonadotropins (CG).Together with thyroidstimulating hormone (TSH),they belong to a glycoprotein hormone family that is structurally and evolutionarily related (Pierce and Parsons, 1981). These hormones are heterodimers consisting of two different noncovalently linked polypeptides, the a and p subunits. Within a given species, the a subunits are identical. However, the p subunit is distinct and provides each hormone with a unique biological specificity (Pierce and Parsons, 1981). The nature of teleost GTH has been the subject of many investigations over the past two decades. One major controversy focuses on the number of GTHs required to carry out the many concerted activities associated with reproduction. These activities include vitellogenesis, steroidogenesis, maturation of oocytes, ovulation, and spermiation. A single GTH, often referred to as maturational GTH, was isolated from several teleost species (Swanson et al., 1991, and references therein). This GTH preparation stimulated almost all aspects of the reproductive process, even though the level of hormone(s) was low or nondetectable until just prior to the final reproductive maturation (Fontaine and Dufour, 1987).This observation suggests that more than one hormone is involved in controlling fish reproduction.

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Definitive characterization of two GTHs, namely, GTHI and GTHII, was provided by Kawauchi and colleagues using chum salmon pituitary (Suzuki et al., 1988a,b: Itoh et al., 1988; Kawauchi et al., 1989). Both GTHI and GTHII have subsequently been identified from coho salmon (Swanson et al., 1991), bonito (Kawauchi et al., 1991), common carp (Van Der Kraak et al., 1992),and several others. Molecular biological approaches were used to isolate cDNAs encoding both GTHI and GTHII from several teleost fishes (Trinh et al., 1986; Sekine et al., 1989; Lin et al., 1992).

B. Function of GTHI and GTHII In the teleost, gonadal function is controlled primarily by pituitary gonadotropins, which by binding to specific receptors in the testes and ovary regulate steroidogenesis and gametogenesis. The function of the individual GTHs has been studied extensively using purified hormones. For example, GTHI and GTHII are equally potent in stimulating estradiol-17p production from midvitellogenic oocytes of amago salmon. However, GTHII is more active than GTHI in stimulating 17a,20p-dihydroxy-4-pregnene-3-one (the maturation inducing factor of 17a,20@-DHP)production from postvitellogenic oocytes, and in stimulating 17a-hydroxyprogesterone (precursor of 17a,20@-DHP)production by thecal layers. GTHII also enhances 20P-hydroxysteroid dehydrogenase (the enzyme catalyzing the conversion of 17a,20P-DHP from 17a-OHP) activity in the granulosa layer of the postvitellogenic oocytes (Suzuki et al., 1 9 8 8 ~ )In . the male, the relative potency of GTHI versus GTHII changes depending on the maturity of the testes. In early spermatogenic testes, both GTHI and GTHII are equipotent in stimulating 11-ketotestosterone production. However, GTHII is superior in stimulating 17a,20P-DHP production in late spermatogenic testes (Planas et al., 1993). The effects of GTHI and GTHII on vitellogenin incorporation were observed by Tyler et al. (1991). In both i n vivo and i n zjitro studies, salmon GTHI was more potent in stimulating radiolabeled vitellogenin incorporation. Rodriguez et al. (1993) observed similar effects of carp GTHl and GTHII on vitellogenin incorporation by goldfish oocytes. The serum GTHI level increases during the gametogenic period, whereas the GTHII level is low. Furthermore, GTHI appears to promote the production of estradiol-17p (in the female), 1l-ketotestosterone (in the male), and vitellogenin incorporation into the oocytes. These observations suggest that GTHI regulates gametogenesis.

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I n contrast, the GTHII level begins to increase around sexual maturation and reaches a maximum during spawning. Furthermore, GTHII has a higher potency than GTHI in stimulating 17a,20p-DHP production, which is required for final maturation of the oocytes and sperm. Therefore, GTHII stimulates the maturation of the gametes and is involved in ovulation and spermiation. The mode of action of GTHs on oocyte maturation will be discussed in Chapter 13 b y Nagahama et u1. (1994).

C. Structure of' Teleost Gonadotropins Like their tetrapod counterparts, teleost gonadotropins contain two noncovalently linked a and P subunits with 113aa and 119 aa residues, respectively. The amino acid sequences of the (Y and P subunits of several teleost fish are shown in Fig. 1. The subunit composition is GTHI ( a , IP) and GTHII ( a , IIP). The a polypeptides are common to both GTHs, whereas IP and IIP are specific for GTHI and GTHII, respectively. GTHI and GTHII are distinctly different from each other, but are homologous to the tetrapod FSH and LH hormones, respectively. When compared to the p subunit of bovine FSH and LH, GTHIP shows slightly greater identity to FSHP (41%) than to LHP (35%). In contrast, GTHIIP is more homologous to LHP (42%) than the FSHP (38%). Similar observations can be derived using the cDNA sequence coding for these polypeptides (Kawauchi et al., 1989; Sekine et ul., 1989). It is generally suggested that GTHIp is niore FSH-like, whereas GTHIIP is inore LH-like. Based on numerous studies from mammalian GTHs, the protein domains important for receptor binding and subunit interaction have been identified. For receptor binding, two regions in the himian a subunit, that is, residues 30 to 40 (R1 in Fig. 1) and 81 to 92 (82 in Fig. l),were identified (Charlesworth et ul., 1987; Reed et ul., 1991). Two intercysteine loops, the determinant loop (R4, residues 93 to 100 in hLHICGP and 33 to 53 in hFSHp) and the large loop (R, residues 93 to 100 in hLHICGP and 33 to 53 in hFSHP), in the p subunit were studied for their role in receptor hinding (Keutinann, 1992; and references within). The CAGYC sequence (86 to 96 in hLHICp) is found to be invariant among almost all mammalian subunits and is implicated in subunit interaction. However, teleost hormones show significant divergence in this sequence. For example, salmon GTHIP and IIp contain CSGLC and CSGHC sequences, respectively.

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-TN-Y-SA-N-IIl)-Y-T-LYAHI---Tg-H-G--DS-S---NRGLGPSY-SFGEWKQYPTALSY

C R 4 -

Fig. 1. Comparison ofthe protein structure for several teleost and human gonadotropins. Based on studies in mammalian GTH, the protein domains important for receptor binding in cr subunits (R1 and R2) and p subunits (R3, the large loop; R4, determinant loop) and subunit interaction(s) are indicated. Abbreviations: Cm, chum salmon; Ck, chinook salmon; Cp, common carp; Ct, African catfish; Bon, bonito; el, European eel; h, human; Fun, fundulus. For the cr subunit, cr2 is a and a1 is the minor component observed in chum salmon and carp.

In addition to the common a subunit, a minor and distinct subunit, a l , has been identified in salmon and carp (Suzuki et al., 198811; Lo et al., 1991) (Fig. 1).In salmon, a 1 associates only with GTHI. The function of this minor GTHI ( a 1 I@) , as compared to GTHI and GTHII is currently unknown.

D. Gonadotropin Cell Types and the Temporal Appearance of GTHI and GTHII Research from several laboratories suggests that teleost GTHI and GTHII are synthesized in different gonadotropes. Using electron microscopy, Olivereau (1976, 1978) and Naito et d.(1988, 1991) have demonstrated the presence of two types of gonadotropes differing in their location within the proximal pars distalis (PPD) and their biosynthetic activity during the reproductive cycle. This was con-

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firmed b y Nozaki et al. (1990a) using immunohistochemical methods with antisera against coho salmon GTHIP and GTHIIP. In rainbow trout, immunoreactive (ir) GTHII was localized in cells located mainly in the central region of the glandular cords of the PPD, whereas irGTHI was localized in the periphery of the glandular cords of PPD. Furthermore, the biosynthetic activity of these two cell types varied with the reproductive cycle. In previtellogenic and prespermatogenic rainbow trout, only irGTHI cells were present, but irGTHII cells were not found. In the spawning trout, however, the number of irGTHII cells exceeded that ofthe GTHI cells (Nozaki et aZ., 199Ob).Consistent with the histological studies, the plasma level of GTHI was the predominant GTH in juvenile trout. The surge of GTHII was late, just before spawning (Swanson et al., 1991). The temporal synthesis of GTHI and GTHII in the pituitary, therefore, is both cell type specific and developmentally regulated.

111. GENOMIC ORGANIZATION OF TELEOST GONADOTROPINS The a and P subunits ofteleost GTHI and GTHII, like the mammalian hormones, are encoded by separate genes. Since the first report of the cloning of chinook salmon GTHIIP cDNA by Trinh et al. (1986), the cDNA sequences coding for the a and /3 subunits of both GTHI and GTHII have been reported in several fish, including chum salmon (Sekine et al., 1989),chinook salmon (Trinh et al., 1986; Suzuki et al., 1994), European eel (Querat et al., 1990), carp (Huang et al., 1992), and several others. Genomic Southern analysis was used to estimate that the genes for the a and IIP subunit are single copies (Xiong and Hew, 1991; Suzuki et ul., 1994). However, the gene copy for ID subunit is presently unknown. Preliminary evidence from our laboratory suggests that it might have multiple copies (K. Suzuki and C. L. Hew, unpublished).

A. Structure of the a Subunit Gene The genomic organization for the a subunit gene fi-om the human, cow, mouse, and rat (Burnside et al., 1988, and references within) has been elucidated. In each species, a single gene composed of four exons and three introns encodes the a subunit. The positions of the introns are highly conserved. The size of the a subunit gene in different

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species varies considerably (from 8 to 16.5 kilo bases, kb) which can be attributed to differences in the size of the first intron (6.4, 13, 10, and 5.4 kb in the human, bovine, mouse, and rat genes, respectively). The structure of two fish GTH a genes is available. The genomic organization of carp GTH a subunit (cGTHa) is very similar to that of the tetrapod GTH a subunit genes. However, it spans only 1.2 kb because its introns are much shorter; the first intron is 177 bp, the second 82 bp, and the third 108 bp, respectively (Huang et al., 1992). Our current data indicate that the salmon GTHa subunit gene (sGTHa) also has a similar length and organization as the cGTHa subunit gene (Suzuki et al., 1994).

B. Structure of the P Subunit Gene The genomic sequences encoding the LHP and FSHP genes from several mammalian species have been isolated and characterized (Gharib et al., 1989, and references within). All the P subunit genes possess three exons and two introns. Unlike the a subunit gene, P subunit genes are smaller, with a total length of approximately 1.5 kb for LHP and 4kb for FSHP, which is attributable to the considerably smaller sizes of their introns. The location of the introns, like those of the a subunit gene, however, is highly conserved, with the first intron interrupting the region of either the signal peptide or the 5’untranslated sequences. The second intron is located three amino acids downstream from the fifth cysteine residue of the P subunit, a position strictly conserved in all the P subunit genes identified so far, including those encoding TSHP from various species (Tatsumi et al., 1988). The first genomic sequence for a teleost GTH subunit gene, GTHIIP, was isolated from the chinook salmon (Xiong and Hew, 1991). The salmon GTHIIP (sGTHIIP) gene also contains three exons interrupted by two introns. The locations of the exodintron junctions are also conserved. The 3’-untranslated tract of the sGTHIIP gene contains AATAAA, the putative signal for polyadenylation. Because this sequence is followed by a poly(A) tract in sGTHIIP cDNA (Trinh et al., 1986), it most probably represents the 3‘ end of the transcriptional unit. Y. S. Chang et al. (1992) isolated the GTHIIP gene from common carp (cGTHIIP). Both the sGTHIIP and cGTHIIP genes are similar to each other and to previously identified tetrapod GTHP genes judged by the number of introns and exons, the location of exodintron junctions, and the length of the exons and introns.

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One ofthe interesting observations concerning the genomic organization of the teleost GTHP is that, as a result of alternate splicing, the mRNA of the chinook salmon GTHIIP is heterogeneous and contains at least two isoforms differing by 12 nucleotides (Xiong and Hew, 1991). The multiplicity of GTHIIP mRNAs raises the question of the functional role(s) of alternate splicing. These functions could involve RNA stability, intracellular RNA transport, or translational efficiency. It is possible that gonadotropes produce two sets of mRNAs that are preferentially regulated by, for example, steroids or GnRH at the posttranscriptional level. Although the data from chinook salmon demonstrate the multiplicity of GTHIIP mRNAs, the majority being the shorter form, it remains unclear whether the pituitary ofother members of the salmonid family produces a similar splicing pattern.

IV. CONTROL OF GONADOTROPIN GENE EXPRESSION A. General Overview Initiation of messenger RNA (mRNA) synthesis is a primary control point in the regulation of differential gene expression. Frequency of initiation of mRNA synthesis, in responding to intra- and extracellular cues, depends ultimately on protein factors that interact with specific regulatory regions of the genes. The transcriptional control of gene expression is a complex mechanism that requires both. specific protein-DNA and protein-protein interactions. The synthesis and secretion of gonadotropin are regulated by both positive and negative mechanisms in a complex fashion with the involvement of the central nervous system (CNS), gonads and the GTH-secreting tissues, the pituitary, and placenta (Fink, 1988; Gharib et al., 1990). The pituitary-gonadal system is controlled by the brain through gonadotropin-releasing hormone (GnRH), a decapeptide that is released from hypothalamic neurons into the hypophysial portal vessels (Sherwood et al., 1993, and references within). The release of GnRH from hypothalamic neurons is influenced by external and interrial factors acting by way of central nervous pathways. Both the pituitary and hypothalamus are, in turn, regulated by positive or negative feedback control from the gonads. The use of a molecular biological approach has made it feasible to examine the effects of these factors on gonadotropin release and synthesis at the pretranslational level (Gharib et al., 1990).

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B. Pituitary-Specific Expression of the GTH Subunit Genes 1. EXPRESSION OF THE a SUBUNIT GENE The a subunit gene is expressed in both the thyrotropes and gonadotropes. Until recently, investigation of the molecular basis of the gonadotrope-specific expression of the a subunit gene was impeded because of the lack of a suitable a-subunit-producing cell line. The lack of a P-subunit-expressing cell line remains the major difficulty in studying its expression (see the following). Using transgenic mice, Fox and Solter (1988) have shown that an 18-kb DNA fragment carrying the entire human a subunit gene was expressed in a tissue-specific manner in the pituitary. Similarly, the a subunit gene with only a 1.8 kb of the 5’ flanking sequence produced the same result (Bokar et al., 1989). Kendall et al. (1991) have further demonstrated that a 313bp fragment of the bovine a subunit promoter directed the expression of the diphtheria toxin-A chain specifically to the gonadotropes in transgenic mice. Animals carrying this transgene generally exhibit reproductive failure and lack of gonadal differentiation, consistent with gonadotropic ablation. Windle et al. (1990) demonstrated that 1.8 kb of the 5’ flanking region of human a subunit gene directed the pituitary-specific expression of the simian virus 40 (SV40) T-antigen oncogene (Tag) in transgenic mice. More importantly, the tumors derived from these mice were cultured to derive clonal cell lines. One of these cell lines (aT3-1) continues to produce the endogenous mouse a subunit and expresses the human a-Tag transgene. Unfortunately, these cells do not produce endogenous P subunit and have thus been proposed to represent a precursor to the gonadotrope lineage (Windle et al., 1990). However, using this cell line (aT3-l), the same group demonstrated that the expression of the (Y subunit gene in pituitary gonadotropes is at least partially due to a DNA-binding protein (GSEB) found uniquely in these cells; this protein binds to a gonadotropespecific element (GSE) bearing the sequence 5’-TGACCTTGT-3’ (Horn et al., 1992). The 2.0-kb 5’ flanking sequence of the chinook salmon GTHa subunit gene has been elucidated (Suzuki et al., 1994). I n addition to the presence of TATA and CAAT motifs located at -23 and -97 bp, respectively, several upstream regulatory sequences (URS) were identified (Fig. 2). Among these URS, a perfect GSE motif is present at 274 bp. Other URSs also include the responsive elements for GnRH, AP1, and AP2 sites. The interaction of these binding factors and other .novel factors in response to different hormonal and physiological sig-

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ll2ERE GnRR-RE AP-l -1991 -1825 -1643

GnRR-RE 1/2CRE I/PCRE AP-2 TRB-RE 1/2ERE -1381 -1267-1058 -1003 -867 -752

Salmon GTH

AP-1

CRE CRE TRE AP-1 AP-1 CAAT TATA -223 -204-154-132-105-53 -26

-556

TSE

CRE -135

-197

CAAT -84

CRE -117

L

--220 GSE

Porcine G T H a

GSE

CAAT AP-1 TATA -191 -97 -23

-274 GSE

a

AP-2

-1046

I/ZTRE -277

TATA -24

m

-150 GATA

H u m a n GTH

a

Fig. 2. Comparison of the organization of regulatory elements in chinook salmon GTHa promoter with human and porcine a subunit promoter. The solid bases represent putative cis-acting elements, and the numbers above the boxes indicate the location. GnRH-RE: gonadotropin-releasing hormone response element; TRH-RE: TRH response element; 1/2 ERE; half-palindrome of estrogen response element; 1/2 CRE: half-palindrome of CAMP response element; GSE: gonadotrope-specific element; 1/2 TRE: half-palindrome of thyroid hormone response element; TSE: trophoblast specific element.

nals will provide a mechanism to dictate the expression ofthe a subunit gene. Research is now in progress to examine the role of these URS using a pituitary primary cell culture.

2. EXPRESSION OF THE

GONADOTROPIN

p SUBUNITGENE Studies on gonadotropin p subunit gene expression have been limited owing to the lack of an appropriate cell line. Attempts to transfect primary pituitary cell cultures with the gonadotropin p subunit promoter linked to reporter genes have had limited success. Kumar et d. (1992) have introduced a 10-kb fragment encompassing the gene for human FSHP into the germline of transgenic mice. The human FSHp gene was expressed exclusively in mouse pituitary gonadotropes as demonstrated b y Northern blots, immunofluorescence histochemistry, and radioimmunoassays. This 10-kb fragment, which contains approximately 4 and 2 kb of 5‘ and 3’ flanking sequences, is apparently sufficient to dictate the correct cell type expression of the /?subunit gene.

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Brown et al. (1993) have demonstrated that a 1.9-kb sequence of the bovine LHP promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene was sufficient to target gonadotrope-specific expression in transgenic mice. The chimeric bLHP-CAT reporter gene was expressed in the pituitary of transgenic mice (two out of three lines). Immunostaining showed that the CAT protein was localized in the gonadotrope cells. The transgene approach, although direct and successful in demonstrating the minimum length of the 5’-DNA sequences required for tissue-specific expression, is insufficient in providing detailed identification and mapping of the URS. Unlike the mammals, where the GTH-producing cells account for approximately 10% of the total pituitary cells (Brown et al., 1993), the GTHII-producing gonadotropes are abundant in spawning rainbow trout (-60-80%) and can be further enriched with proper dissection of the pars distalis. We have used these pituitary cells enriched with gonadotropes to examine the salmon GTHIIP gene expression. A region that is approximately 3.2 k b and upstream of the sGTHIIP gene has been characterized (Xiong and Hew, 1993). This sequence and several of its deletion fragments were fused to the CAT gene and transfected into the primary pituitary cells derived from spawning trout. A summary of the sGTHIIp-CAT activity is shown in Fig. 3. It is evident that the transcriptional regulation of sGTHIIP is complex and involves the participation and interaction of both positive and negative control mechanisms (Xiong et al., 1994a,b). Some ofthe pertinent features of the sGTHIIP promoter are shown in Fig. 4. a. Presence of a Strong Minimal Promoter in the Salmon GTHIIP Gene. The minimal promoter of the sGTHIIP gene (Active Region 1, AR-I; i.e., -39CAT in Fig. 3) exhibits strong activity only in the pituitary primary cells, but not in a collection of heterologous cell lines. The

sequence of the sGTHIIP minimal promoter was compared with the minimal promoters of other pituitary-specific hormones in an attempt to uncover the information concerning the cell type specificity. In the sGTHIIP gene, a short DNA sequence of TGCCCA is repeated twice between the TATA box and the CAP site, with another short sequence AAACAC positioned in between. Interestingly, in the rat LHP gene, the same TGCCCA sequence is also present between the TATA box and the CAP site; in the bovine LHP gene an AAACAC sequence is located in a similar position. Although exact matches were not observed in human, ovine, and bovine LHP, equine CGILHP, and carp GTHIIP gene promoters, a sequence similar toTGCCCA was found in an analogous position. A consensus was thus derived bearing the

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146

CAT construct

pBLCAT3 +18 -39 -95 -258 -289 -447

-533 -563 -728 -1 189 -1260

-3500 -3500+LHRH

RSV Fig. 3. Expression of the sCTHIIPICAT fusion genes in primary pituitary cells prepared from spawning male trout. The 5' flanking sequence of sGTHIIP was fused to the bacterial CAT gene, and the individual construct was transfected in primary pituitary cells. The CAT activity was monitored by thin-layer chromatography. The numbers indicated are the lengths of the 5' flanking sequence of the sCTHIIP gene. Control plasmids, pBLCAT3, RSVCAT, are indicated. LHHH, mammalian M. From gonadotropin-releasing hormone (GnRH), was used at a concentration of Xiong et al. (1994a). Copyright The Endocrine Society.

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-88 (CH~RE) -74 -70 (CHS ~ I - 1 ) -52 5'- G'ITG'ITGTGAAGGGGGAGMTCATGAGCAGAGCATCCAACCCTGAAT

4 ~ y . o z y r n c ~ - ~-32 .~n~ +1 ACTAGCCTCCTCCACTG TATATGCCCAAACACACTGCCCATCAAGA- 3'

Fig. 4. Organization of the structural and regulatory domains of the chinook salmon GTHIIP gene. Schematic representation of the structural (1.0 kb) and regulatory (3.5kb) regions of the sGTHIIp gene. The exons ( E l A , E l B , E2, and E3) are shown in striped boxes. The initiation of transcription ( + 1) is indicated by a bent arrow. The black boxes represent the positive (AR-I, AR-2) and negative (pSil) regulatory regions. The sequence of psi1 ( - 95 to - 35) can be roughly divided into three parts as indicated by solid lines and nucleotide positions. The two distal sequences are similar to growth hormone silencers, GH pRE and GH Sil-1; the proximal sequence is similar to that of a lysozyme silencer. The estrogen responsive elements are shown as either one open box (pERE) or three smaller open boxes (dERE).

sequence 5'-TNCCCA/T-3' ( N = any nucleotide). Moreover, a similar AC-rich sequence is also located in the minimal promoter region of these genes. A search of corresponding regions of several other pituitary hormone promoters, including FSHP, glycoprotein hormone a subunit, and PRL, failed to reveal such similarities (Xiong et al., 1994a). The highly active sGTHIIP minimal promoter is reminiscent of the minimal promoter of the TSH subunit genes (Chin et al., 1993). TSH, another member of the glycoprotein hormone family, is specifically expressed by the thyrotropes of the anterior pituitary and its synthesis is directly regulated by thyroid hormones in a negative fashion (Chin et al., 1993). The negative regulation occurs at the transcriptional level of the subunit genes. Analysis of 5' deletion mutants of the rat a subunit gene showed that a region located just upstream of the TATA box ( - 80 to + 33) was necessary for the thyroid hormone effect. This sequence contains a set of direct repeats of the consensus

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TRE hexamer half-site [AGGT(C/A)A] defined as nTREs (negative TREs). Interestingly, in the human TSH a gene, the nTREs are located downstream of the TATA box. Moreover, using a similar approach, a set of overlapping repeats of nTREs downstream of the TATA box were also characterized in the TSHP genes of rat, mouse, and human. It has been proposed that the TR (thyroid hormone receptor)/TRE complex exerts a negative effect over TSH subunit gene expression through interference with the general transcription factors. By analogy, it is also possible that factor(s) capable of binding to repeats of the sGTHIIP minimal promoter region would facilitate the function of this promoter, perhaps b y enhancing the general transcription machinery. The function of these repeats is currently under investigation in our laboratory.

h. Presence of a Silencer Sequence Upstream of the Minimal Promoter. From transfection studies, it was evident that the sequence between - 95 and - 39 had an inhibitory effect on the minimum promoter (Fig. 3 and Xiong et al., 1994a). The minimum length of sequence required for the silencing function contained only 56 bp. A homology search revealed that this region shares extensive sequence identity with several previously identified silencer sequences in other genes (Fig. 4). This sequence, called GTHIIP proximal silencer (pSil), can be roughly divided into three parts; the distal two parts share extensive identity with the GH silencers, GH Sil-1 and GH pRE (Pan et al., 1990; Roy et al., 1992), and the proximal part is similar with the consensus silencer sequence (ANCCTCTCT/C or ANCTCTCCTCC) derived from the genes of the avian lysozyme and several mammalian genes (Baniahmad et al., 1987 and references within). The role of the tripartite silencer psi1 was further confirmed by the demonstration that psi1 placed in either orientation next to the T K promoter inhibited the TK-directed CAT activities in the heterologous cell systems (Xiong et al., 1994a). The location of pSil, which is imniediately upsteam of the strong minimal promoter, may be functionally significant. It might be responsible for maintaining the quiescent endocrine state of the GTHIIP gene during the early stages of fish development, perhaps by inhibiting the minimal promoter, which is not only intrinsically strong but is also insensitive to reproductive development. The activity of this silencer could be minimized or abolished when transcription factors capable of interacting with upstream regulatory sequences are synthesized or activated in the mature fish, or when the inducers (e.g., GnRH, gonadal steroids) are present (see the following).

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C . Presence of G S E Homologues. Another active region (ARII), based on transfection studies, appears to be located from -1260 to - 1189. Within AR-11, a 9-bp sequence (ACACCTTGT, antisense strand) is present between - 1232 and - 1224, and this sequence is imperfectly repeated (GCACCTTGA) 24 bp downstream from - 1200 to - 1192. Interestingly, Mellon and colleagues identified a very similar 9-bp sequence that is located from -216 to -220 in the human GTH a subunit gene (Horn et al., 1992). Designated GSE, the sequence is at least partially responsible for the gonadotrope-specific expression of the a subunit gene. GSE binds to a 54-kDa protein (GSEB) present only in aT3-1 cells. Transfection studies indicate the involvement of GSE in determining aT3-1cell specificity of the human and murine a subunit genes. At present, it is unclear why a distal region of salmon GTHIIP promoter should exhibit structural and functional properties similar to those of a region located in the proximal promoter area ofthe tetrapod a subunit genes. In all the LHP genes characterized so far, a single GSE homologous sequence is located around - 130 b p (Brown et al., 1993). Clearly, the mere presence of a single GSE motif is not enough to achieve gonadotrope specificity. It would be interesting to determine if the duplication of GSE in the sGTHIIP AR-I1 region would generate new enhancer activity capable of functioning in mature gonadotropes. Furthermore, single GSE-like sequences are also present in other parts of the sGTHIIP promoter and are yet to be linked with any activity.

C. Regulation of Gonadotropin Gene Expression by Gonadal Steroids In mammals, the mechanism, site of action, and integrative cooperation of steroid hormones in the regulation of gonadotropin secretion and biosynthesis have been under intensive investigation (Fink, 1988; Gharib et al., 1990; Miller, 1993). Gonadal steroids have diverse effects on gonadotropin secretion and synthesis. For decades it has been appreciated that the secretion of LH and FSH appears to be controlled by gonadal steroids via a classical negative feedback loop. Gonadectomy, for example, by removing gonadal steroids, leads to a prompt increase in plasma gonadotropin level that can be substantially decreased by the administration of gonadal steroids (Gay and Bogdanove, 1969). Paradoxically, in addition to their negative regulatory effect on gonadotropin secretion, steroids are also capable of increasing the secretion and synthesis of GTH (Shupnik et al., 1989). Controversy remains, however, regarding the mechanism by which sex steroids

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regulate gonadotropin synthesis and secretion. This regulation (both positive and negative) could occur as the result of alterations in the pattern of GnRH secretion, direct effect at the level of the pituitary gland, or both. The conflicts can be explained, for the most part, by the use of different animal models, in vivo versus in vitro systems, and the mode of hormone delivery (Gharib et al., 1990 and references therein). In teleosts, two opposite steroid effects on the reproductive axis have also been reported. In adult fish, gonadal steroids have an inhibiting effect on GTH synthesis and release by a negative feedback mechanism comparable to that in mammals (Billard, 1978; Peter, 1982). For example, castration of mature rainbow trout resulted in an increased level of circulating immunoreactive GTH (Billard et al., 1977) that can be effectively reversed b y either testosterone or estrogen treatment (Billard, 1978). The positive effect of steroids on GTH release and synthesis has been extensively documented (Crim et al., 1981;Trinh et al., 1986;Counis et al., 1987).In reproductively inactive fish, this positive feedback of gonadal steroids is generally considered to be part of the mechanisms for the development and differentiation of pituitary GTH cells, which in turn lead to the onset of sexual maturation or gonadal recrudescence. In the following sections, some of our recent data on the role of gonadal steroids on sGTHIIP gene expression are summarized. In brief, we have demonstrated the presence of functional estrogen response elements (ERE) in both the proximal and distal regions of the sGTHIIP promoter. In the pituitary cells o f t h e juvenile trout, the activity of these EREs could effectively antagonize the action of the pSil. The activity ofthe EREs, as judged by transient expression studies, correlates well with the responsiveness of GTHIIP gene to gonadal steroids (Xiong et al., 1994a,b). 1. PHESENCE OF BOTHPROXIMAL AND DISTAL ERE

To localize potentially functional steroid responsive elements in the sGTHIIP gene, the 5’ flanking sequence was inspected and two potential EREs were located (Fig. 5). The proximal ERE (pERE), which is located from -273 to -260, bears the sequence 5’-ATGTCAATCTGACCC-3’. It is an imperfect palindrome, but shares 12 identical nucleotides with the consensus ERE (5’-G/ AGGTCANNNTGACC-3’) identified from Xenopus vitellogenin A 1 and A2 genes (Klein-Hitpass et al., 1988). Another potential ERE, distal ERE (dERE), located from -2736 to -2659, is composed of three tandemly linked half ERE palindromes (GGTCA), separating from one another by 31 bp. These two EREs will be discussed in a later section.

4.

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-2736

dERE

GGAGGCCAGGTCATCTGATGCAGCACTCTA

-

TCACTCTCCTTCTTGGTCAAATAGCCCTTA -2659

CACAGCCTGGAGGTGTGTTGGGTCATTGTC

-280

ERE

-

L

-250

ACAATTATGTCAATCTGA~CCTATAAAGCC

AGGTCANNNTGACC Fig. 5. The D N A sequences and positions of the estrogen responsive elements (EREs) in the 5'-flanking region of'the chinook salmon GTHIIp gene. The sequences for the proximal ERE (pERE) and the distal ERE (dERE) are shown. A nearly perfect palindrome (pERE) is indicated by two arrows of opposite direction; overlapping CAAT boxes are underlined. The half-palindromes are indicated by single arrows.

2. FUNCTIONAL ANALYSISOF THE sGTHIIP GENE

THE

PROXIMAL ERE

IN

We have examined the effect of gonadal steroids on GTHIIp gene expression using in vitro cultured primary pituitary cells derived from the juvenile trout. Both estrogen (E2) and testosterone (T) stimulate GTHIIP gene expression. Only GTHIIP mRNA increased dramatically in response to steroid treatment based on RNA blotting analysis. The response was both specific and dose dependent, with no effect on the mRNA levels of GTHIP, GTHIa, and GH (Xiong et d.,199413). Gene transfer experiments using primary pituitary cell cultures were subsequently performed to determine whether the pERE could initiate an estrogen-mediated response. These transfection studies indicate that indeed the pERE can mediate an estrogen-dependent response that is abolished by mutating either half of the palindrome. Furthermore, b y using footprinting analyses, pERE was shown to bind to the estrogen receptor (ER) specifically (Xiong et al., 1994b). These experiments established the functionality of the pERE. 3. ACTIVITYOF THE pERE AT DIFFERENT REPRODUCTIVE STAGES The positive E2/T responsiveness of the GTHIIP promoter cannot be attributed solely to the pERE, because the pERE seems to function only in pituitary cells prepared from juvenile trout. As fish mature,

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the pituitary starts to synthesize GTHIIP mRNA, which can still be up-regulated b y steroid hormones. However, this regulation did not involve the pERE, as the shorter version of the pERE containing CAT gene failed to respond to either T or E2 induction in pituitary cells derived from these fish (Xiong et al., 1994b). The distal ERE is composed of three tandemly linked half ERE palindromes separated from each other by exactly 31 bp, with the central half-site also part of an imperfect ERE palindrome (5’-GGTCAAATAGCCC-3’; Fig. 5). We propose that the dERE might be responsible for the positive regulation by the gonadal steroids for the expression of the GTHIIP gene in the pituitary of the maturing and mature fish. The location and organization of the dERE are reminiscent of those of one of the functional EREs ( D H 3 ERE) in the chicken ovalbumin gene located 3.3 k b upstream from the transcription initiation site. The DH3 ERE is composed of four half-palindromic ERE motifs separated from each other by approximately 100 bp (Kato et al., 1992). It is suggested that widely spaced half-palindromic EREs could act synergistically to achieve estrogen inducibility. Such a synergism is the result of interactions of either ER-ER or ER with other transcriptional factors. Our investigations suggest that there is a synergistic interaction between dERE and pERE (up to 200-fold) in regulating the estrogen response of the GTHIIP gene expression (Xiong et al., 1994b). D. Regulation of Gonadotropin Gene Expression by GnRH The actions of GnRH and various synthetic analogs on pituitary LH and FSH cells have been extensively studied in mammals, mainly in rat, sheep, rhesus monkey, and human (Fink, 1988; Gharib et al., 1990).GnRH is secreted from the hypothalamus in a pulsatile fashion that parallels the pulsatile release of LH and FSH. One of the most notable aspects of GnRH action in mammals is the observation that the frequency and amplitude of GnRH pulses can affect GTH secretion and synthesis (Shupnik, 1990).The biochemical response of gonadotropins to GnRH is a complex signaling process involving an increase in phosphoinositide (PI)turnover, a rise in the concentration of intracellular free Ca2++,and activation of protein kinase C (Conn et al., 1987; Naor, 1990). GnRH action on GTH secretion has been extensively documented using both in uiuo (mammalian model) and in uitro (pituitary cells) approaches (Gharib et al., 1990).Subsequently,many investigators have studied the effects of GnRH on GTH biosynthesis. The results are still the subject of debate and the detailed molecular events of the effect remain largely unknown (Gharib et al., 1990).

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GnRH action in the teleost has been extensively studied in goldfish (Peter et al., 1990), and to some extent in several members of the salmonids (Crim et al., 1988; Weil and Marcuzzi, 1990). Similar to the studies of the GnRH action in mammals, stimulation of GTH release by GnRH in goldfish is dependent on both Ca2+ and protein kinase C activation (Chang et al., 1991). Other evidence also suggests the involvement of CAMP in GTH release ( J . P. Chang et al., 1992). Schoderbek et al. (1993) have reported the presence of two different DNA sequences that could mediate the GnRH effect on the mouse glycoprotein hormone a subunit gene. One of the motifs is glycoprotein hormone basal element (PGRE, GCTAATTA); the other motif, GnRHresponse element [GnRH RE, TT(C/T)CT(A/G)(A/C/T)(C/T)],is responsive to GnRH and phorbol myristate acetate (PMA) treatment. These sequences are also found in both the salmon GTHa (at - 1267 and - 1991 bp) and the GTHIIP subunit genes. In addition, the third GGTCA in the dERE of the sGTHIIP promoter is preceded by a TG dinucleotide resulting in the sequence 5‘-TGGGTCA-3’,which differs in only one nucleotide from the AP-1 recognition site (5’-TGAGTCA3’). Gaub et al. (1990) demonstrated that the same sequence (5’TGGGTCA-3’) is responsive to both E2- and phorbolester-induced transcription. As most evidence suggests that GnRH receptor signaling is coupled to the phosphatidyl inositol pathways, with concomitant changes in intracellular calcium and PKC activation, it is tempting to speculate that the TGGGTCA motif in the sGTHIIP may represent another example of convergence of steroid and peptide hormone (GnRH) induction at the transcriptional level. Evidence for cross-talk between steroid receptor and the signal transduction pathways has been described (Ponta et al., 1992). +

V. CONCLUSION

Studies of teleost gonadotropins have progressed tremendously in the past few years. Many teleost GTH genes have been cloned and characterized, and the studies of their transcriptional control mechanisms are being initiated in several laboratories. The regulation of GTH gene expression involves a complex interplay of the tissuespecific factors, GnRH, and the gonadal steroids. The organization of the salmon GTHa and P subunit genes provides a useful model for study of the molecular basis of the cell type specific, developmentally regulated, and coordinated synthesis of these subunit polypeptides.

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154 ACKNOWLEDGMENTS

We thank Linda Gardiner for the preparation of the manuscript. This investigation is supported by the Medical Research Council of Canada (to C.L.H). We also thank Coolwater Trout Farm, Pickering, Ontario, and Rainbow Spring Hatchery, Thaniesford, Ontario, for the provision of experimental animals. The financial assistance of a MRC Studentship and an Ontario Graduate Fellowship (to F.X.), and a Research Trainee Fellowship, Hospital for Sick Children (to K.S.) is gratefully acknowledged.

REFERENCES Baniahmad, A., Muller, M., Steiner, C., and Renkawitz, R. (1987).Activity oftwo different silencer elements of the chicken lysozyme gene can be compensated by enhancer elements. E m b o ] . 6 , 2297-2303. Billard, R. (1978). Testicular feedback on the hypothalamo-pituitary axis in rainbow trout (Salmo gairdneri R.). Annu. B i d . Anim. Biochem. Biophys. 18, 813-818. Billard, R., Richard, M., and Breton, €3. (1977). Stimulation of gonadotropin secretion after castration in rainbow trout. Gen. Comp. Endocrinol. 33, 163-165. Bokar, J. A., Keri, R. A., Farmerie, T. A , , Fenstermaker, R. A,, Andersen, B., Hamernik, D. L., Yun, J., Wagner, T., and Nilson, J. H. (1989). Expression of the glycoprotein hormone alpha-subunit gene in the placenta requires a functional cyclic AMP response element, whereas a different cis-acting element mediates pituitary-specific expression. Mol. Cell. Biol. 9, 5113-5122. Brown, P., McNeilly, J. R., Wallace, R. M., McNeilly, A. S., and Clark, A. J. (1993). Characterization ofthe ovine LHP-subunit gene: The promoter directs gonadotropespecific expression in transgenic mice. Mol. Cell. Endocrinol. 93, 157-165. Burnside, J., Buckland, P. R., and Chin, W. W. (1988). Isolation and characterization of the gene encoding the alpha-subunit of the rat pituitary glycoprotein hormones. Gene 70, 67-74. Chang, J. P., Jobin, R. M., and de Leeuw, H. (1991). Possible involvement of protein kinase C in gonadotropin and growth hormone release from dispersed goldfish pituitary cells. Gen. Comp. Endocrinol. 81,447-463. Chang, J. P., Wong, A. O., van der Kraak, G., and van Goor, F. (1992). Relationship between cyclic AMP-stimulated and native gonadotropin-releasing hormonestimulated gonadotropin release in the goldfish. Gen. Comp. Endocrinol. 86, 359-377. Chang, Y. S., Huang, F. L., and Lo, T. B. (1992). Isolation and sequence analysis of carp gonadotropin p-subunit gene. Mol. Mar. Biol. Biotechnol. 1, 97-105. Charlesworth, M. C., McCormick, D. J , , Madden, B., and Ryan, R. J. (1987). Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach. J . Biol. Chem. 262, 13409-13416. Chin, W. W., Carr, F. E., Burnside, J., and Darling, D. S. (1993). Thyroid hormone regulation of thyrotropin gene expression. Recent Prog. Horm. Res. 48, 393-414. Colin, P. M., Huckle, W. R., Andrews, W. V., and McArdle, C. A. (1987). The molecular mechanism of action of gonadotropin releasing hormone (GnRH) in the pituitary. Recent Prog. Horm. Res. 43,29-68.

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Counis, R., Dufour, S., Ribot, G., Querat, B., Fontaine, Y. A,, and Jutisz, M. (1987). Estradiol has inverse effects on pituitary glycoprotein hormone alpha-subunit messenger ribonucleic acid in the immature European eel and the gonadectomized rat. Endocrinology (Baltimore) 121, 1178-1 184. Crim, L. W., Peter, R. E.,and Billard, R. (1981).Onset ofgonadotropic hormone accumulation in the immature trout pituitary gland in response to estrogen or aromatizable androgen steroid hormones. Gen. Comp. Endocrinol. 44, 374-381. Crim, L. W., Nestor, J., Jr., and Wilson, C. E. (1988). Studies of the biological activity of LHRH analogs in the rainbow trout, landlocked salmon, and the winter flounder. Gen. Comp. Endocrinol. 71, 372-382. Fink, G. (1988).Gonadotropin secretion and its control. In “The Physiology ofReproduction” (E. Kriobil and J . Neill, eds.), pp. 1349-1377. Raven, New York. Fontaine, Y. A,, and Dufour, S. (1987). Current status of LH-FSH-like gonadotropin in fish. In “Proceedings of the Third International Symposium on Reproductive Physiology of Fish” (D. R. Idler, L. W. Crim, and J. W. Walsh, eds.), pp. 48-56. Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. Fox, N., and Solter, D. (1988). Expression and regulation of the pituitary- and placentaspecific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice. Mol. Cell. Biol. 8, 5470-6. Gaub, M. P., Bellard, M., Scheuer, I., Chambon, P., and Sassone, C. P. (1990).Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex. Cell (Cambridge, Mass.) 63, 1267-1276. Gay, V. L., and Bogdanove, E. M. (1969). Plasma and pituitary LH and FSH in the castrated rat following short-term steroid treatment. Endocrinology (Baltimore)84, 1132. Gharib, S. D., Roy,A., Wierman, M. E., andchin, W. W. (1989).Isolation andcharacterization of the gene encoding the beta-subunit of rat follicle-stimulating hormone. DNA 8,339-349. Gharib, S. D., Wiernian, M. E., Shupnik, M. A,, and Chin, W. W. (1990). Molecular biology of the pituitary gonadotropins. Endocr. Reu. 11, 177-199. Horn, F., Windle, J. J,, Barnhart, K. M., and Mellon, P. L. (1992). Tissue-specific gene expression in the pituitary: The glycoprotein hormone alpha-subunit gene is regulated by a gonadotrope-specific protein. M o l . Cell. Biol. 12, 2143-2153. Huang, C. J., Huang, F. L., Wang, Y. C., Chang, Y. S., and Lo, T. B. (1992).Organization and nucleotide sequence of carp gonadotropin alpha subunit genes [published erratum appears in Biochim. Biophys. Acta 1129(3),3471; Biochim. Biophys. Actu 1129,239-242. Itoh, H., Suzuki, K., and Kawauchi, H. (1988).The complete amino acid sequences of beta-subunits of two distinct chum salmon GTHs. Gen. Comp. Endocrinol. 7 1 , 438-451. Jameson, J . L., and Hollenberg, A. N. (1993).Regulation of chorionic gonadotropin gene expression. Endocr. Reu. 14, 203-221. Kato, S., Tora, L., Yamauchi, J., Masushige, S., Bellard, M., and Chambon, P. (1992). A far upstream estrogen response element ofthe ovalbumin gene contains several halfpalindromic 5’-TGACC-3‘ motifs acting synergistically. Cell (Cambridge,Mass.) 68, 73 1-742. Kawauchi, H., Suzuki, K., Itoh, H., Swanson, P., Nozaki, M., Natio, N., and Nagahama, Y. (1989). Iluality of salmon pituitary gonadotropins. Fish Physiol. Biochem. 7, 29-38. Kawauchi, H., Itoh, H., and Koide, Y. (1991). Additional evidence for duality of fish

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gonadotropins. In “Proceedings of the Fourth International Symposium on Reproductive Physiology of Fish” (A. P. Scott, J . P. Sumpter, D. E. Kime, and M. S. Rolfe, eds.), pp. 19-21. Univ. East Anglia Printing Unit, Norwich, UK. Kendall, S. K., Saunders, T. L., Jin, L., Lloyd, R. V., Glode, L. M., Nett, T. M., Keri, R. A,, Nilson, J. H., and Camper, S. A. (1991). Targeted ablation of pituitary gonadotropes in transgenic mice. M o l . Endocrinol. 5 , 2025-2036. Keutmann, H. T. (1992). Receptor-binding regions in human glycoprotein hormones. Mol. Cell. Endocrinol. 86, C1-C6. Klein-Hitpass, H. L., Ryffel, G. U., Heitlinger, E., and Cato, A. C. (1988). A 13 bp palindrome is a functional estrogen responsive element and interacts specifically with estrogen receptor. Nucleic Acids Res. 16, 647-663. Kumar, T. R., Fairchild, H. V., and Low, M. J. (1992). Gonadotrope-specific expression of the human follicle-stimulating hormone beta-subunit gene in pituitaries of transgenic mice. Mol. Endocrinol. 6, 81-90. Lin, Y. W. P., Rupnow, B. A., Price, D. A., Greenberg, R. M., and Wallace, R. A. (1992). Fundulus heteroclitus gonadotropins. 3. Cloning and sequencing of gonadotropic hormone (GTH) I and I1 p-subunits using the polymerase chain reaction. M o l . Cell. Endocrinol. 85, 127-139. Lo, T. B., Huang, F. L., Liu, C. S., Chang, Y. S., Chang, G. D., and Huang, C. J. (1991). Studies on pisine gonadotropin-Structure, function, molecular cloning, expression and gene structure. Bull. Inst. Zool. Acad. Sin. Monogr. 16, 37-59. Miller, W. L. (1993). Regulation of pituitary gonadotropins by gonadotropin-releasing hormone, estradiol, progesterone, inhibin and activin. In “Genes in Mammalian Reproduction,” pp. 247-269. Wiley-Liss, New York. Sagahama, Y., Yoshikuni, M., Yamashita, M.,and Tanaka, M. (1994). Regulation of oocyte maturation in fish. In “Fish Physiology” (N. Sherwood and C . Hew, eds.), L’ol. 13, Chapter 13. Academic Press, San Diego. Naito, N., Naki, Y., Nagahama, Y., Suzuki, K., and Kawauchi, H. (1988). Immunoelectron microscopy of two distinct gonadotropes in teleost pituitary. Zool. Sci. 5 , 1302. Naito, N., Hyodo, S., Odumoto, N., Urano, A., and Nakai, Y. (1991). Differential production and regulation of gonadotropins (GTHI and GTHII) in the pituitary gland of rainbow trout, Oncorhynchus mykiss, during ovarian development. Cell Tissue Res. 266,457-467. Naor, 2. (1990). Signal transduction mechanisms of Ca2+ mobilizing hormones: The case of gonadotropin-releasing hormone. Endocr. Reo. 11, 326-353. Nozaki, M., Naito, N., Swanson, P., Miyata, K., Nakai, Y., Oota, Y., Suzuki, K., and Kawauchi, H. (1990a).Salmonid pituitary gonadotrophs. I. Distinct cellular distributions of two gonadotropins, GTH I and GTH 11. Gen. Comp. Endocrinol. 77, 348-357. Nozaki, M., Naito, N., Swanson, P., Dickhoff, W. W., Nakai, Y., Suzuki, K., and Kawauchi, €1. (l99Ob). Salmonid pituitary gonadotrophs. 11. Ontogeny of GTH I and GTH I1 cells in the rainbow trout (Salmo gairdneri irideus). Gem Conap. Endocrinol. 77, 358-367. Olivereau, M. (1976). Les cellules gonadotropes hypophysaires du Saumon de 1’Atlantique: Unicite our dualite? Cen. Comp. Endocrinol. 28, 82-95. Olivereau, M. (1978). Les cellules gonadotropes chez les Salmonides. Ann. B i d . Anim. Biochem. Biophys. 18, 793-798. Pan, W. T., Liu, Q. R., and Bancroft, C. (1990). Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNAbinding proteins. ]. Biol. Chem. 265, 7022-7028.

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Peter, R. E. (1982). Xeuroendocrine control of reproduction in teleosts. Can. J . Fish. Aquat. Sci. 39, 48-55. Peter, R. E., Habibi, H. R., Chang, J . P., Nahorniak, C. S., Yu, K. L., Huang, Y. P., and Marchant, T. A. (1990). Actions of gonadotropin-releasing hormone (GnRH) in the goldfish. In “Progress in Comparative Endocrinology” (A. Epple, C. G. Scanes, and M. H. Stetson, eds.), pp. 393-398. Wiley-Liss, New York. Pierce, J. G., and Parsons, T. F. (1981). Glycoprotein hormones: Structure and function. Annu. Rev. Biochem. 50,465-495. Planas, J . V., Swanson, P., and Dickhoff, W. W. (1993). Regulation of testicular steroid production in vitro by gonadotropins (GTHI and GTHII) and cyclic AMP in coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 91,8-24. Ponta, H., Cato, A. C. B., and Herrlich, P. (1992). Interference of pathway specific transcription factors. Biochirn. Biophys. Acta 1129, 255-261. Querat, B., Moumor, M., Jutisz, M., Fontaine, Y. A., and Counis, R. (1990). Molecular cloning and sequence analysis of the cDNA for the putative p subunit of the type I1 gonadotropin from the European eel. J . Mol. Endocrinol. 4,257-264. Reed, D. K., Ryan, R. J . , and McCormick, D. J. (1991). Residues in the alpha subunit of human choriotropin that are important for interaction with the lutropin receptor. J. Biol. Chem. 266, 14251-14255. Rodriguez, J. N., Suzuki, K., Peter, R. E., and Itoh, H. (1993). Effects of common carp gonadotropin-I and -I1 on vitellogenin synthesis and uptake and 17p-estradiol secretion in goldfish, Carassius auratus. Fr. J. Physiol. (submitted). Roy, R. J., Gosselin, P., Anzivino, M. J., Moore, D. D., and Guerin, S. L. (1992). Binding of a nuclear protein to the rat growth hormone silencer element. Nucleic Acids Res. 20,401-408. Schoderbek, W. E., Roberson, M. S., and Maurer, R. A. (1993). Two different DNA elements mediates gonadotropin releasing hormone effects on expression of the glycotropin releasing hormone effects on expression of the glycoprotein hormone a subunit gene. J . B i d . Chem. 268,3903-3910. Sekine, S., Saito, A,, Itoh, H., Kawauchi, H., and Itoh, S. (1989). Molecular cloning and sequence analysis of churn salmon gonadotropin cDNAs. Proc. Natl. Acad. Sci. U.S.A. 86, 8645-8649. Sherwood, N. M., Lovejoy, D. A,, and Coe, I. R. (1993). Origin ofmammalian gonadotropin releasing hormones. Endocr. Rev. 14, 241-254. Shupnik, M. A. (1990). Effects of gonadotropin-releasing hormone on rat gonadotropin gene transcription in vitro: Requirement for pulsatile administration for luteinizing hormone-beta gene stimulation. Mol. Endocrinol. 4, 1444-1450. Shupnik, M. A,, Gharib, S. D., and Chin, W. W. (1989).Divergent effects of estradiol on gonadotropin gene transcription in pituitary fragments. M o l . Endocrinol. 3,474-480. Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988a). Isolation and characterization of two distinct gonadotropins from chum salmon pituitary glands. G e n . Comp. Endocrinol. 71, 292-301. Suzuki, K., Kawauchi, H., and Nagahama, Y. (1988b). Isolation and characterization of subunits from two distinct salmon gonadotropins. Gen. Comp. EndocrinoZ. 71, 302-306. Suzuki, K., Nagahama, Y., and Kawauchi, H. (1988~).Steroidogenic activities of two distinct salmon gonadotropins. Gen. Comp. Endocrinol. 71, 452-458. Suznki, K., Liu, D., and Hew, C. L. (1994). Gene structure coding for the a subunit of chinook salmon gonadotropin (submitted). Swanson, P., Suzuki, K., Kawauchi, H., and Dickhoff, W. W. (1991). Isolation and charac-

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terization of two coho salmon gonadotropins, GTH I and GTH 11. Biol. Reprod. 44, 29-38. Tatsumi, K., Hayashizaki, Y., Hiraoka, Y., Miyai, K., and Matsubara, K. (1988). The structure of the human thyrotropin beta-subunit gene. Gene 73, 489-497. Trinh, K.-Y., Wang, N. C., Hew, C. L., and Crim, L. W. (1986). Molecular cloning and sequencing of salmon gonadotropin P-subunit. Eur. J . Biochem. 159,619-624. Tyler, C. R., Sumpter, J . P., Kawauchi, H., and Swanson, P. (1991). Involvement of gonadotropin in the uptake of vitellogenin into vitellogenic oocytes of rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 84,291-299. Van Der Kraak, G., Suzuki, K., Peter, R. E., Itoh, H., and Kawauchi, H. (1992). Properties of common carp gonadotropin I and gonadotropin 11. Gen. Comp. Endocrinol. 85, 2 17-229. Weil, C., and Marcuzzi, 0. (1990). Cultured pituitary cell GtH response to GnHH at different stages of rainbow trout spermatogenesis and influence of steroid hormones. Gen. Comp. Endocrinol. 79, 492-498. Windle, J. J., Weiner, R. I., and Mellon, P. L. (1990). Cell lines of the pituitary gonadotrope lineage derived by targeted oncogenesis in transgenic mice. MoZ. Endocrinol. 4,597-603. Xiong, F., and Hew, C. L. (1991). Chinook salmon gonadotropin I1 p-subunit gene encodes multiple messenger ribonucleic acids. Can. J . 2002.69, 2572-2578. Xiong, F., and Hew, C. L. (1993). Role of gonadal steroids in salmon gonadotropin IIp gene expression, p16, in Symposium on “Advances in the Molecular Endocrinology of Fish,” May 23-25, Toronto, Canada. Xiong, F., Liu, D., Elsholtz, H. P.,and Hew, C. L. (1994a).The chinook salmon gonadotropin IIP subunit gene contains a strong minimal promoter with a proximal negative element. M o l . Endocrinol. 8, 771-781. Xiong, F., Liu, D., Le Drean, Y., Elsholtz, H. P., and Hew, C. L. (1994b). Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin IIp subunit gene during salmon maturation. M o l . Endocrinol. (in press).

5 THE SOMATOLACTIN GENE MASAO ONO* Department of Molecular Biology, School of Medicine Kitasato University, Sagamihara, Kanagawa 228, Japan

HIROSHI KAWAUCHI Laboratory of Molecular Endocrinology, School of Fisheries Sciences Kitasato University, Sanriku, Iwate 022-01, Japan I. Somatolactin A. Historical Background B. cDNA Cloning and Structural Analysis C . Distribution and Possible Functions 11. Somatolactin Gene A. Structural Features B. Evolutionary Relationships to Growth HormonelProlactin Family 111. Regulation of Somatolactin Gene Expression A. Pit-1 €3. Somatolactin Gene and Pit-1 IV. Conclusion References

I. SOMATOLACTIN A. Historical Background The vertebrate pituitary in humans is a very small endocrine organ the size of a pea and attached at the bottom of the brain. It consists of the adenohypophysis and neurohypophysis, which have developmental origins distinct from each other. The neurohypophysis stores and releases neurohypophyseal hormones such as oxytociii and vasopressin in most mammals; they are produced in neuroendocrine cells in the hypothalamus. The adenohypophysis, mainly divided into ante-

* Send correspondence to: Dr. Masao Ono, Department of Molecular Biology, School of Medicine, Kitasato University, Sagamihara, Kanagawa 228, Japan. 159 FISH PHYSIOLOGY, VOL. XI11

Copyright 0 19% b y Academic Prrss, Inc. All rights of reproduction in any form reserved.

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rior and intermediate lobes, produces and secretes several adenohypophyseal hormones. In the anterior lobe, growth hormone (GH), prolactin (PRL), two kinds of gonadotropins (GTHs), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), and lipotropin (LPH) are produced. Proopiomelanocortin (POMC)-derived peptides, such as melanocyte-stimulating hormone (MSH), are the main products in most tetrapod intermediate lobes. Pituitary hormones have significant roles in growth, maturation, and maintenance of homeostasis in vertebrates and thus have been extensively studied in mammalian systems. Pituitary hormones other than these may be absent from the mammalian pituitary. A novel pituitary protein, somatolactin (SL), belonging to the GH/ PRL family, was discovered in the teleost pituitary. At the start of this research, glycoproteins obtained from Atlantic cod (Gadus rnorhua) and flounder (Paralichthys oliuaceus) pituitaries during GH purification were found to be present in the intermediate lobe (Rand-Weaver et al., 1991a). As in mammals, the teleost pituitary is divided into anterior, intermediate, and posterior (neural) lobes. In contrast to mammals, the intermediate lobe is well developed in the teleost and forms a neurointermediate lobe by complexing with the neural lobe. MSHproducing cells were the only endocrine cells that could be found in the tetrapod intermediate lobe. The teleost intermediate lobe, however, has been shown to contain at least two types of endocrine cells distinguishable by staining with lead-hematoxylin and periodic acid-Schiff (PAS) reagent. Lead-hematoxylin-positive cells produced peptides such as MSH, whereas the product of PAS-positive cells was an unidentified hormone. Histological studies indicate that PASpositive cells in the intermediate lobe are activated under a black background (Baker and Ball, 1970; van Eys, 1980; Ball and Batten, 1981), acidic p H (Wendelaar Bonga et al., 1986), low calcium (Olivereau et al., 1981; Olivereau and Olivereau, 1981; Ball et ul., 1982), or low osmolarity (Olivereau et al., 1980) of ambient water. They may thus produce hormones essential for adaptation to environmental changes. Based on preliminary studies, the glycoprotein in the intermediate lobe of Atlantic cod and flounder pituitaries may be a novel pituitary hormone. cDNA cloning and structural analysis on this glycoprotein have been conducted.

B. cDNA Cloning and Structural Analysis From the flounder pituitary cDNA library, two groups of clones corresponding to 1.2-kilobase (kb) and 1.8-kb mRNAs were obtained (Fig. 1) (On0 et al., 1990). Two different mRNAs were generated by

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Fig. 1. Structures ofAtlantic cod (A),chum salmon (B),and flounder (C)somatolactin (SL) cDNA clones (From Takayama et al., 1991a). The thick box represents the protein coding region. The position of the signal peptide is shown by the striped box. A vertical line indicates a Cys residue; a line with a solid circle indicates a Cys residue diagnostic for SL; arrowheads indicate a potential N-glycosylation site; A, polyadenylation signal. The thin box indicates the 3‘ untranslated region conserved between salmon and flounder SL cDNAs. Part C also shows 1.2- and 1.8-kb flounder SL mRNAs generated by the alternative polyadenylation (Ono et al., 1990).

alternative polyadenylation. Subsequently, chum salmon (Oncorhynchus keta) and Atlantic cod cDNA clones were isolated using flounder cDNA (Takayama et al., l99la). Salmon and cod mRNA size was 2.5 and 1.3 kb, respectively (Fig. 1). These mRNAs encoded a protein having signal peptides consisting of 24 to 26 amino acids (aa). The mature forms posessed 207 aa for flounder and 209 residues for salmon and cod. The latter forms are two residues longer than in the case of flounder at the C terminus and have a molecular mass of about 24,000 Da. The amino acid sequence of the mature form deduced from cod cDNA was identical with that of the cod 26-kDa glycoprotein determined by protein sequencing (Rand-Weaver et al., 1991b). A similarity search was consequently conducted by computer and, surprisingly, the proteins were found to be distantly and similarly related to (average 24% identity) but clearly distinct from GH and PRL. The name somatolactin was thus proposed for these proteins in consideration of their relation to GH (somatotropin) and PRL. Because lactin means “easy” in Japanese, somatolactin thus means that cDNA clones are characterized by much less somatic (physical) effort. The three SLs each possessed seven common Cys residues, four of which are at the C-terminal portion and correspond to those in GH and PRL. Two Cys residues at the N terminus were aligned with those in tetrapod PRL. No vertebrate GH or fish PRL has this Cys-containing

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region. Neither GH nor PRL had a Cys residue corresponding to the third Cys residue from the N terminus, and thus this Cys may be considered characteristic of SLs and is unrelated to disulfide bonding (Rand-Weaver et al., 199lb). For each SL, the number of potential N-glycosylation sites was different (Fig. 1).There is only one site for flounder SL (On0 et d., 1990) but two for cod (Rand-Weaver et al., 1991b; Takayama et d., 1991a). These N-glycosylated SLs should certainly correspond to PASpositive molecules in the pars intermedia and react with the anti-cod SL antibody. Such salmon pars intermedia cells were PAS-negative and somewhat chromophobic (Rand-Weaver et al., 1991a), and thus carbohydrate molecules would not likely be associated with salmon SL. Should the three SLs have the same physiological roles, carbohydrate molecules associated with cod and flounder SLs may have less significant functions. The amino acid sequences of the three SLs were more colinear with each other than those of the corresponding three GHs (Takayama et al., 1991a). Amino acid sequence identity in these GHs was assessed as 62% (cod vs. salmon), 57% (cod vs. flounder), and 58% (flounder vs. salmon). Sequence identity in SLs was 18%higher on the average. It should be pointed out that SL alignment was possible without gap introduction. The alignment of salmon, cod, and flounder GH sequences required gaps for maximum homology. The significance of SL conservation remains to be clarified. Expression of the SL gene in flounder tissue was examined by Northern hybridization (Ono et al., 1990). As in the case of pituitary hormone mRNAs, SL mRNA was specifically expressed in the pituitary, but not in the stomach, liver, heart, intestine, spleen, or brain. A comparison has been made of’the nucleotide sequences of the three SL cDNAs (Takayama et al., 19Yla). For cod versus salmon and cod versus flounder, sequence similarity was noted only in the region coding for the SL precursor protein. Neither the 5’ nor 3’ untranslated region showed significant similarity. In addition to the conserved protein coding region, a flounder versus salmon comparison indicated similar sequences of about 200 bases in the 3’ untranslated region. The significance of this similarity remains to be determined. Nucleotide sequence similarity in the coding region for mature SL protein was 77% (cod vs. salmon), 80% (cod vs. flounder), and 79% (flounder vs. salmon). A short 5’ untranslated region was found in flounder (<40 bases) (On0 et al., 1990)and salmon (31 bases) (Takayama et nl., 1991b) SL mRNAs. Cod SL mRNA had a longer region (>250 bases). In 3’ untranslated regions, the three SL cDNAs differed in size, the shortest

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region being in cod (about 140 bases), followed by 1.2- and 1.8-kb flounder SL mRNAs possessing 0.3- and 0.9-kb sequences, respectively (On0 et al., 1990). Salmon had the longest region (1.9 kb) (Fig. 1). C. DISTRIBUTION AND POSSIBLE FUNCTIONS 1. DISTRIBUTION Cells reactive with the antibody raised against cod SL have been shown to be present in the intermediate lobe of all fish pituitaries so far examined (Rand-Weaver et al., 1991a). From the pituitary cDNA libraries of flounder, Atlantic cod, and chum salmon, cDNA clones for SL have been obtained at a frequency of about 1% (Takayama et al., 1991a), and more than 1 mg of SL has been prepared from 1 g of pituitaries of Atlantic cod, flounder (Rand-Weaver et al., 1991b), and gilthead sea bream (Sparus aurata) (H. Kawauchi, 1992). Thus, the content of SL in these pituitaries is comparable to that of GH, although SLs have been isolated from several other fish, such as Atlantic salmon (Salmosalar),coho salmon (Oncorhynchus kitsutch),bonito (Katsuwonus pelamis),channel catfish (Zctalurus punctatus),common carp (Cyprinus carpio),and tilapia (Sarotherodon mossambicus), in yields less of than 200 p g per gram weight of pituitaries (H. Kawauchi, 1992). The reason for the low yield remains to be elucidated but the time of SL production, particularly the developmental stage and/or environmental conditions, may be important. Several tetrapod pituitaries (rat, pigeon, and frog) have been examined for the presence of SL immunohistochemically by the antibody against Atlantic cod SL but the results were negative (Rand-Weaver et al., 1991a). Of the many antibodies raised against human, ovine, and bovine PRLs, some have been shown to react with PAS-positive cells in the intermediate lobe along with lactotrophs in the teleost pituitary (Rawdon, 1979). Following the adsorption of these antibodies by the corresponding fish anterior lobes, only antibodies reactive with the PAS-positive cells remained. In certain human PRL-producing tumor cells, the presence of PRL-like protein with an apparent molecular mass of 25 kDa and distinct from human PRL has been reported (Sinha et al., 1987). The presence of sequences hybridizable with the flounder SL cDNA has been demonstrated in tetrapod genomes such as mouse, rat, human, and bull frog (On0 et al., 1990). These observations appear to be evidence for the universal presence of SL in vertebrates. Tetrapod SL is now being characterized.

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2. POSSIBLE FUNCTIONS SL proteins, cDNAs, and antibodies are available and the biological activity of SL is being studied. SL is a member of the GH/PRL family and thus the growth-promoting activity of the chum salmon SL to rainbow trout was examined first. However, no significant effect that would promote body weight or length has been observed (H. Kawauchi, 1992). In euryhaline teleost fish, PRL is essential for freshwater adaptation. For seawater adaptation in salmonids, GH prevents an increase in salt concentration in body fluid. Based on these observations and the morphology of PAS-positive cells in the teleost intermediate lobe (i.e., SL-producing cells), in response to changes in pH, calcium concentration, and osmolarity in the ambient water, SL may be concluded to participate in the regulation of certain ions. Should this be the case, ion regulation may be an indispensable function of the GH/PRL/SL family in teleosts. To determine the functions of SL based on fluctuation in its concentration in plasma, radioimmunoassay systems for Atlantic cod and coho salmon have been established (Rand-Weaver et al., 1992). Preliminary examination indicated that the plasma concentration of SL in Atlantic cod is higher in mature than in immature fish. SL was thus measured during maturation of coho salmon. A gradual increase in SL was observed with gonadal development. The concentration was maximum at final maturation and spawning stages. Coho salmon SL was shown to stimulate gonadal steroidogenesis in a culture system ofcoho salmon ovarian follicles and testicular fragments (Planas et al., 1992). The roles of SL in reproduction should thus be investigated. 11. SOMATOLACTIN GENE A. Structural Features

To elucidate the structure and organization ofthe SL gene, genomic clones coding for chum salmon SL were isolated (Fig. 2) (Takayama et al., 1991b). Salmon SL gene length was 16 kb, the largest among mammalian G H (about 2 kb) (Barta et al., 1981; DeNoto et al., 1981) and PRL (about 10 kb) (Cooke and Baxter, 1982; Truong e t al., 1984) and salmonid GH (about 4 kb) genes (Fig. 2). As also observed for mammalian GH/PRL and carp GH genes, the salmon SL gene possessed five exons in contrast to rainbow trout (Salmo gairdneri) (Agellon et nl., 1988)and Atlantic salmon (Johansen et al., 1989) GH genes, in which six exons are present.

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Fig. 2. Organization of GH/PRL/SL genes (from Rand-Weaver et al., 1993). Boxes represent exons, and typical restriction sites are shown (B, BamHI; E, EcoRI; H , HindIII; P, PstI). The position of the fifth intron in salmonid GH genes is indicated by an arrowhead. Reported structures of chum salmon SL (sSL: Takayama et ul., 199lb), 1988), human GH (hGH: DeNoto et al., 1981), rainbow trout CH (tCH: Agellon et d., rat <:H (rGH: Barta et al., 1981), human PRL (hPRL: Truong et al., 1984), and rat PHL (rPRL: Cooke and Baxter, 1982) were used.

The sizes of the five exons and four introns of the chum salmon SL gene were found to be as follows: exon I, 47 bp; 11, 191 bp; 111, 114 bp; IV, 180 bp; and V, 1807 bp; intron A, 631 bpi €3, 4.6 kb; C, 8.0 kb; and D, 183 bp. After the 5' untranslated region of 31 bases, exon I encodes the first 5 amino acids and the first letter of the sixth amino acid of the signal peptide. Exon I1 codes for the remaining signal peptide and the N-terminal 45 residues of mature SL. Exons I11 and IV encode 38 and 60 residues, respectively. Exon V encodes 66 amino acids in addition to the 3' untranslated region of 1609 bases. The exon I-intron A junction was separated between the first and second letters of the codon (class 1 splice site). Other junctions were separated between codons (class 0 splice site) (Sharp, 1980). In the upstream region, a TATA box identical to those of mammalian PRL genes and four consensus sequences corresponding to the Pit-1 binding element (Nelson et al., 1988) were found. Pit-1 was originally isolated as a transcription factor contributing to the pituitaryspecific expression of mammalian GH and PRL genes (Bodner et al., 1988; Ingraham et al., 1988).

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tGH hGH rGH hPRL rPRL

. 90 SSL

tGH hGH rGH hPRL rPRL

I

. .

100

II

110

120

130

111

140

150

160

170

180

190

*

200

210

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B. Evolutionary Relationships to Growth Hormone/Prolactin Family A comparison of the amino acid sequences of SL with GH/PRL indicates that the SL gene is apparently derived from an ancestor common to GH and PRL genes (Ono et al., 1990). For confirmation of this point, the amino acid sequences of GH/PRL/SL precursors in each exon were aligned (Fig. 3) (Takayama et al., 1991b). Exon I coded for fewer than 10 amino acids, with virtually no sequence similarity being noted. The longest exon, exon 11, with 64 residues, exceeded the length of the shortest one of rainbow trout GH b y 17 amino acids (Agellon et nl., 1988). The protein coding capacity of the other three exons was basically the same (exon 111, 36-40 residues; IV, 52-60; V, 63-69). Exon-intron junction patterns were the same as those in the SL gene. The first junction was separated between the first and second letters of a codon and all others between codons. This again shows exon organization to be similar in GH/PRL/SL genes. It follows from the four internally similar amino acid sequences in human GH/placental lactogen and ovine PRL that an ancestral gene for the GH/PRL family may have been produced by the duplication of a small primordial gene (Niall et al., 1971). Figure 3 shows exons I1 and IV of human GH to each possess one such region. Two others were found in exon V. Gene structure analysis supports internal similarity and generation of an ancestral gene by primordial exon duplication (Miller and Eberhardt, 1983; Slater et al., 1986). These evolutionary models are based on analysis of mammalian genes only. In the structure of the rainbow trout GH, no significant degree of internal similarity has been determined (Agellon et al., 1988). For assessment of internal similarity, fish SLs more than GHs should perhaps be used. SLs may be much more conserved between species than teleost GHs

Fig. 3. Alignment of amino acid sequences of SLiGHlPRL genes. Amino acids are represented by one-letter codes. Gaps (marked by asterisks) have been inserted to make the similarity more apparent. Amino acid residues common with SL are indicated in the black boxes. s, chum salmon; t, rainbow trout; h, human; r, rat. solid triangle, Cys residue common in GHIPRLISL; open triangle, Cys residue common in tetrapod PRL and SL; open circle, Cys residue characteristic for SL; open oval, N terminus of mature protein. Short arrows indicate the position of the fifth intron in salmonid GHs. Four internally similar regions as described by Niall et al. (1971) in hGH are enclosed by rectangles. The amino acid residues are numbered from the N terminus of the mature sSL. Reported amino acid sequences of rainbow trout G H (tGH), human GH (hGH), rat GH (rGH), human PRL (hPRL), and rat PRL (rPRL) are the same as shown in the legend of Fig. 2. From Takayama et u1. (1991b). Copyright The Endocrine Society.

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that diverged more than tetrapod GHs (Kawauchi and Yasuda, 1989). Internal similarity in the three fish SLs has thus been sought (Takayama et al., 1991b), but none was found even in regions studied by Niall et al. (1971). Three internally similar regions were shown to be actually present in significantly diverged regions in GH/PRL/SL (Fig. 3). One was observed in a conserved region close to the C-terminal side. The internally similar regions noted by Niall et al. may thus be attributed to some coincidence, as suggested b y Nicoll et al. (1986). Figure 3 indicates that the amino acid sequences of GH/PRL/SL are mutually similar in exons II-V, thus demonstrating a common origin of these exons in the GH/PRL/SL gene family. Nucleotide sequences around the exon I-intron A boundary were compared and the completely conserved AG/GTA was present at the splicing junction in all GH/PRL/SL genes reported so far. The common origin model of all five exons in the GH/PRL/SL gene family is thus applicable. The common origin of the regulatory region located upstream from exon I should also be applicable in that four consensus sequences (Nelson et al., 1988) corresponding to the Pit-l/GHF-l binding element were noted in the 5' flanking region of exon I. An evolutionary model for the GHIPRLISL gene family has been made based on these considerations (Takayama et al., 1991b).This model differs principally with the former one (Miller and Eberhardt, 1983) in the following respects: (1)exons I-V are not produced by primordial gene duplication because none of the five is mutually similar; (2) an ancestral gene common to the GH/PRL/SL gene family can be obtained by shuffling five prototype exons and a regulatory element; and ( 3 ) duplication of the five-exon-type ancestral gene takes place prior to divergence between fish and tetrapods.

111. REGULATION OF SOMATOLACTIN GENE EXPRESSION A. Pit-1 GH and PRL are produced in somatotrophs and lactotrophs, respectively, and both are differentiated from Rathke's pouch, which arises from the oral ectoderm at the roof of the embryonic mouth by invagination. The expression of GH and PRL in these tissues has been studied as a model system to elucidate the mechanism of cell-typespecific gene expression. A transcription factor Pit-l/GHF-l (we use Pit-1 from now on) that participates in the pituitary-specific expression

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T H E SOMATOLACTIN GENE

Pr ( - )

sal SL (0.8)

169

rat GH (0.3) sal GH (0.5) rat LH B (0.6)

Fig. 4. Stimulation of somatolactin gene expression by Pit-1. Pica Gene basic vector, PCB-B (Toyo Inki, Tokyo), having the firefly luciferase gene was used as a reporter. As an effector plasmid, pRc/RSV (Invitrogen, San Diego) was used. Transfection into HeLa cells was carried out by the calcium phosphate coprecipitation method. Using the Pica Gene Luminescence kit, PKG-100 (Toyo Inki, Tokyo), the luciferase activity was measured by a luminophotometer TD-4000 (NDS, Tokyo). Numbers in parentheses indicate the size (kb) of the 5' upstream region inserted into the reporter plasmid. Pr ( - ) , promoter minus; sal SL, chum salmon SL promoter; rat GH, rat GH promoter; sal GH, chum salmon GH promoter; rat LHP, rat luteinizing hormone @chain promoter. From Ono et al. (1994). Copyright T h e Endocrine Society.

of GH and PRL genes has been isoIated and anaIyzed (Bodner et al., 1988; Ingraham et d., 1988). Rat and bovine Pit-1 contains 291 aa and belongs to the POU family of proteins, each possessing two conserved DNA binding domains, the POU-specific domain (about 75 aa) and the POU homeodomain (60 aa) (Herr et al., 1988). The POU homeodomain near the carboxy-terminus of Pit-1 is similar to the homeobox region observable in gene products essential to pattern formation in Drosophila. The POU-specific domain found about 20 aa upstream from the POU homeodomain is specific for members of the POU family such as Oct-1, Oct-2, and Unc-86. Pit-1 stimulates the transcription of the GH gene by binding to two specific sites situated within 200 bp upstream from the transcription start site (Ingraham et al., 1990; Karin et al., 1990). Several rat Pit-1 binding sites, four of which are situated within 250 bp upstream from the transcription start site of the rat PRL gene and another four are located between 1.5 to 1.8 kb upstream region, are involved in PRL

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gene expression (Ingraham et al., 1990). In addition to somatotrophs and lactotrophs, rat and mouse Pit-1 proteins are present in thyrotrophs, where the thyroid-stimulating hormone is produced (Simmons et al., 1990).Pit-1 protein, originally identified as a transcription factor, has been shown to be indispensable to the development of somatotrophs, lactotrophs, and thyrotrophs, (Li et al., 1990). In contrast to the localization of GH- and PRL-producing cells in the anterior lobe derived from the anterior wall of Rathke’s pouch, SL-producing cells are present in the intermediate lobe (Rand-Weaver et al., 1991a), which develops from the posterior wall of the pouch. Pit-1 protein is involved not only in the regulation of GH and PRL gene expression but also in the development of GH- and PRLproducing cells. There is thus the question of whether Pit-1 participates in the expression of the SL gene and/or development of SLproducing cells, although the localization and development of SLproducing cells are distinct from those of GH- and PRL-producing cells. For clarification of this point, cDNA cloning and characterization of the chum salmon Pit-1 were carried out. Using rat Pit-1 cDNA, chum salmon clones were isolated from the pituitary cDNA library at a fequency of 0.02% (On0 and Takayama, 1992). Major chum salmon Pit-1 mRNAs were 2 and 3 kb in size and specifically expressed in the pituitaries. Several forms of Pit-1 mRNAs having different poiyadenylation sites were found. Chum salmon Pit1mRNAs encoded a protein having 365 amino acids, 74 residues larger than mammalian Pit-1. The amino acid sequence identity between salmon and rat Pit-1 was 69%. In rat and salmon Pit-1 proteins, the POU-specific domain and POU homeodomain were well conserved with 88% identity for the former and 83% for the latter. The remaining was less conserved with 54% identity. From the POU-specific domain to the POU homeodomain, the aniino acid sequences of rat and salmon Pit-1 are colinear, whereas neither is colinear upstream from these regions. In addition to the Cterminal 11aa, salmon Pit-1 has three additional regions corresponding to aa 47 to 72 (26 aa), 97 to 129 (33 aa), and 144 to 150 (7 aa) of salmon Pit-1. This is the main reason for the difference in the size of salmon and rat Pit-1 proteins. Twenty-six amino acids specific for salmon Pit1 were found exactly between exons I and I1 of the rat Pit I gene and 33 aa were in exons I1 and 111. Rat Pit-1 having an extra 26 aa between exons I and I1 has been shown to be a minor isoform, although its functions and significance are not clear (Konzak and Moore, 1992; Morris et a,?., 1992; Theill et al., 1992). Turkey Pit-1 (327 aa) (Wong et nl., 1992) had three extra segments (26, 38, and 7 aa) at exactly the

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same positions as those observed in the salmon Pit-1, indicating that the Pit-1 having these regions is the prototype.

B. Somatolactin Gene and Pit-1 The presence of Pit-1 protein in SL-producing cells and participation of Pit-1 in the expression of the SL gene were investigated. To characterize salmonid Pit-1 protein, antibodies against chum salmon Pit-1 were prepared (On0 et al., 1994). Chum salmon Pit-1 protein was produced in E . coli as a fusion protein. Because the POU domain of Pit-1 protein is conserved in POU family proteins, a portion characteristic to salmon Pit-1 (aa number 84 to 180, 97 aa) was conjugated to the C-terminal end of glutathione S-transferase (GST) to form a fusion protein (37 kDa). We next characterized antibodies raised against the fusion protein. Two individual antisera against the fusion protein reacted with common antigens having a molecular mass of 40 kDa in the total extract of rainbow trout pituitaries. Based on the nucleotide sequence of salmon Pit-1 cDNA, the molecular mass of salmon Pit-1 protein was estimated as 40 kDa. Thus, the 40-kDa bands may be rainbow trout Pit-1. Based on densitometrical analysis, the amount of rainbow trout Pit-1 was calculated as 1/5000 of the total pituitary protein. This value agrees well with the content of chum salmon Pit-1 mRNA in the pituitary as determined by cloning frequency in the screening of the cDNA library (On0 and Takayama, 1992). Rainbow trout pituitaries were separated into anterior and neurointermediate lobes and examined for the presence of Pit-1 by Western blotting analysis. In the neurointermediate lobe fraction, a slightly fainter 40-kDa band was detected, compared with that in the anterior lobe fraction. Because the two lobes were separated sufficiently judging from the localization of GH, PRL, and SL, Pit-1 protein was concluded to be present in the neurointermediate lobe of rainbow trout pituitaries. Immunohistochemical studies demonstrate rainbow trout Pit-1 protein to be located in the nuclei of SL-producing cells present in the well-developed neurointermediate lobe of the teleost pituitary (On0 et al., 1994). Pit-1 is essential to the development of somatotroph, lactotroph, and thyrotroph cells and thus may be indispensable to the development of SL-producing cells. If SL is present in tetrapods, it should also be present in Pit-l-producing cells. In adult rat pituitary, Pit-1 protein is localized exclusively in the anterior lobe, where somatotrophs, lactotrophs, and thyrotrophs are present, but not in the poorly

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developed intermediate lobe, where cells producing MSH are predominant (Simmons et al., 1990). Assuming that a significant number of SL-producing cells (more than several percent) are present in the tetrapod pituitary, they should be located in the anterior pituitary and produce Pit-1 but not GH, PRL, or TSH. Cells having these phenotypes can be detected by antibodies against Pit-1, GH, PRL, and TSH. In the anterior lobe of the mammalian pituitary, Pit-1 protein is present in somatotroph, lactotroph, and thyrotroph cells (Simmons et al., 1990), so that it is difficult to compare amounts of Pit-1 with that of the specific hormone in each cell type during development or environmental change. Pit-1 is exclusively present in SL-producing cells in the neurointermediate lobe of the teleost pituitary and thus the relations between the amount of Pit-1 and that of SL in certain situations can be found by dividing the pituitary into anterior and neurointermediate lobes. The content of SL in the pituitary of immature rainbow trout was much less than that of GH or PRL. The amount of Pit-1 in the neurointermediate lobe was slightly less than that in the anterior lobe, where GH and PRL are extensively produced. In coho salmon, plasma SL increases gradually following maturation and reaches a maximum at the time of final maturation (Rand-Weaver et al., 1992). In contrast, the content of Pit-1 in the neurointermediate lobe of rainbow trout pituitary is virtually constant during maturation, so that factor(s) other than the Pit-1 may be important for increased SL production following maturation, although the contribution of posttranslational modifications such as phosphorylation remains a possibility. Accordingly, Pit-1 in GH-, PRL-, TSH-, and SL-producing cells may be a tissue-specific transcription factor required not only for the development of these cells but for the maintenance of the differentiated state. Rat Pit-1 protein produced in GH- and PRL-producing cells binds to Pit-1 binding elements located in the upstream region of GH and PRL genes and stimulates the expression ofthese genes (Ingraham et al., 1990). The effects of Pit-1 on the expression of the chum salmon SL gene, a member of the GH/PRL gene family, are thus being investigated (On0 et al., 1994). When the luciferase gene was linked with the 0.8-kb upstream region of the chum salmon SL gene and cotransfected into HeLa cells with rat Pit-1 effector DNA, there was 10 times as much stimulation of luciferase gene expression (Fig. 4). Stimulation by rat Pit-1 of the SL promoter was similar to that of the 0.3-kb rat G H promoter used as the positive control. Compared with rat Pit-1, salmon Pit-1 showed less but significant stimulation to rat and chum salmon GH promoters. Its effect on the SL promoter was much less.

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In rat and chum salmon Pit-1, the amino acid sequences of the D N A binding domain, POU, were colinear and highly conserved, having an amino acid sequence identity of 86%. Upstream of the POU domain, the two sequences differed greatly in size, mostly because the salmon Pit-1 had two additional sequences each consisting of about 30 aa. The two sequences also were less conserved with 54% identity (On0 and Takayama, 1992).The region responsible for transcriptional activation is located upstream of the POU domain (Theill et al., 1989). Because a mutant form of rat Pit-1 that has most of the chinook salmon (Oncorhynchus tschawytscha) POU homeodomain, where the two sequences diverge in the conserved POU domain, stimulated the activity of rat and salmon PRL promoters (Elsholtz et al., 1992; Elsholtz, 1994), the POU domain of rat and salmon Pit-1 is thus functionally similar in transactivation activity to PRL promoters when introduced into HeLa cells. Accordingly, the reason for the lesser transactivation activity of the salmon Pit-1 than rat Pit-1 to SL and GH promoters must be due to the inability of salmon Pit-1 to interact with transcriptional machinery in HeLa cells owing to structural differences in the transactivation domain between mammalian and fish Pit-1. If we had used fish cells corresponding to HeLA cells in this study, salmon Pit-1 may have shown higher transactivation activity than rat Pit-1 for SL and GH promoters. The upstream region of the SL gene required for transactivation by rat Pit-1 was examined by changing it from 0.5 kb to 7 kb. Activation by rat Pit-1 to the SL promoter region from 0.5 kb to 7 kb was relatively constant, indicating that the 0.5-kb upstream region of the SL gene was sufficient for transactivation in HeLa cells. Four segments corresponding to the Pit-1 binding site are in this region (Takayama et al., 1991b) and they should mediate the functions of rat Pit-1. As in the case of mammalian GH and PRL gene expression (Ingraham et al., 1990), expression of the SL gene in SL-producing cells must surely be regulated by many transcription factors including Pit-1. Thus the 0.5-kb upstream region may not be adequate for the complete expression of the SL gene in SL-producing cells. Pit-1 protein in SLproducing cells may quite likely be a transcription factor participating in the pituitary-specific expression of SL gene. IV. CONCLUSION A presumed pituitary hormone, somatolactin, which belongs to the growth hormone prolactin family, was isolated from fish. The chum salmon SL gene was 16 kb in length and comprised five exons. Similar-

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ity in organization of the SL gene with GH and PRL genes indicates that the SL gene is quite likely produced from an ancestral gene common to the GH and PRL genes. Although no conclusive data on the functions of SL are available, SL may participate in ion regulation and/or maturation in fish. We still do not know whether tetrapod SL is present. Within 500 bp upstream from the transcriptional start site of the salmon SL gene, four segments were found corresponding to the binding element of the transcription factor Pit-1, which participates in the pituitary-specific expression of mammalian GH and PRL genes. To determine the possible involvement of the Pit-1 protein in the expression of the SL gene, chum salmon Pit-1 cDNA clones were isolated and characterized. Chum salmon Pit-1 protein consisted of 365 amino acids (40 kDa), this being 74 amino acids larger than mammalian Pit-l. The D N A binding domain of these proteins was highly conserved. The antibody against recombinant chum salmon Pit-1 reacted with a 40-kDa protein present in the anterior and neurointermediate lobes of rainbow trout pituitary. Using this antibody, Pit-1 protein in SL-producing cells was shown to be present by immunohistochemical study. Rat Pit-1 protein was found to activate SL gene expression when cotransfected into HeLa cells. Pit-1 protein in SL-producing cells would thus appear to participate in the expression of the SL gene. Pit-1 is essential for the development of somatotroph, lactotroph, and thyrotroph cells, where it is produced and thus should also be required for the development of SL-producing cells.

ACKNOWLEDGMENTS The present study was supported by the Ministry of Education, Science, and Culture and the Fisheries Agency, Japan.

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Nelson, C., Albert, V. R., Elsholtz, H. P., Lu, L. I.-W., and Rosenfeld, h4. G. (1988). Activation of cell-specific expression of rat growth hormone and prolactin genes by a cominon transcription factor. Science 239, 1400-1405. Niall, H. D., Hogan, M. L., Sauer, R., Rosenblum, I. Y., and Greenwood, F. C. (1971). Sequences of pituitary and placental lactogenic and growth hormones: Evolution from a primordial peptide by gene reduplication. Proc. Natl. Acud. Sci. U.S.A. 68, 866-869. Nicoll, C . S., Mayer, G. L., and Russell, S. M. (1986). Structural features of prolactins and growth hormones that can be related to their biological properties. Endocr. Reo. 7, 169-203. Olivereau, M., and Olivereau, J.-M. (1981).Calcium-sensitive cells of the pars intermedia in the teleost Carassius auratus to acid water. Cell Tissue Res. 222, 231-241. Olivereau, M., Aimar, C., and Olivereau, J.-M. (1980). PAS-positive cells of the pars intermedia are calcium-sensitive in the goldfish maintained in a hyposniotic milieu. Cell Tissue Res. 212, 29-38. Olivereau, M., Olivereau, J.-M., and Aimar, C. (1981). Specific effect of calcium ions on the calcium-sensitive cells of the pars intermedia in the goldfish. Cell Tissue Res. 214, 23-31. Ono, M.,and Takayama, Y. (1992).Structures ofcDNAs encodingchum salmon pituitaryspecific transcription factor, Pit-l/GHF-I. Gene 226, 275-279. Ono, M., Takayama, Y . , Rand-Weaver, M., Sakata, S . , Yasunaga, T., Noso, T., and Kawauchi, H. (1990). cDNA cloning of somatolactin, a pituitary protein related to growth hormone and prolactin. Proc. Natl. Accid. Sci. U.S.A. 87, 4330-4334. 0 1 1 0 , hl., Harigai, T., Kaneko, T., Sato, Y., Ihara, S., and Kawauchi, H. (1994). Pit-l/GH factor-1 involvement in the gene expression of somatolactin. MoZ. Endocrinol. 8, 109-115. Planas, J. V., Swanson, P., Rand-Weaver, M.,and Dickhoff, W. W. (1992). Soinatolactin stimulates in uitro gonadal steroidogenesis in coho salmon, Oncorhynchus kiutch. Gen. Contp. Eiidocn‘nol. 87, 1-5. Rand-Weaver, M., Baker, B. I., and Kawauchi, H. (1991a).Cellularlocalization of somatolactin in the pars intermedia of some teleost fishes. Cell Tissue Res. 263,207-215. Rand-Weaver, M., Noso, T., Muramoto, K., and Kawauchi, H. (199lb). Isolation and characterization of somatolactin, a new protein related to growth hormone and prolactin from Atlantic cod (Gadus morhua) pituitary glands. Biochemistry 30, 1509-1515. Rand-Weaver, M., Swanson, P., Kawauchi, H., and Dickhoff; W. W. (1992). Somatolactin, a novel pituitary protein: Purification and plasma levels during reproductive maturation of coho salmon. J . Endocrinol. 133, 393-403. Rand-Weaver, M., Kawauchi, H., and Ono, M. (1993). Evolution of the structure ofthe growth hormone and prolactin family. I n “The Endocrinology of Growth, Development, and Metabolism in Vertebrates” ( M . P. Schreibman, C . G. Scanes, and P. K. Pang, eds.), pp. 13-42. Academic Press, San Diego. Rawdon, B. B. (1979). Immunostaining of TJ cells in the rostra1 pars distalis and PASpositive cells in the pars intermedia of teleost (Sarotherodon mossambicus) by antisera to mammalian prolactins. Gen. Cornp. Endocrinol. 37, 374-382. Sharp, P. A. (1980).Speculations on RNA splicing. Cell (Cambridge,Mass.) 23,643-646. Simmons, D. M., Voss, J . W., Ingraham, H. A,, Holloway, J. M., Broide, R. S., Rosenfeld, M. G., and Swanson, L. W. (1990).Pituitary cell phenotypes involve cell-specific Pit1 mRNA translation and synergistic interactions with other classes of transcription factors. Genes Deo. 4, 695-711.

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Sinha, Y. N., Ott, K., and Vanderlaan, W. P. (1987). Detection of multiple PRL- and GH-like proteins in human pituitary by Western blotting analysis. Am. J. Med. Sci. 294, 15-25. Slater, E. P., Baxter, J. D., and Eberhardt, N. L. (1986).Evolution ofthe growth hormone gene family. Am. Zool. 26, 939-949. Takayania, Y., Ono, M., Rand-Weaver, M., and Kawauchi, H. (1991a).Greater conservation of somatolactin, a presumed pituitary hormone of the growth hornioneiprolactin family, than growth hormone in teleost fish. Gen. Comp. Endocrinol. 83,366-374. Takayama, Y., Rand-Weaver, M., Kawauchi, H., and Ono, M. (1991b). Gene structure of chum salnion somatolactin, a presumed pituitary hormone ofthe growth hormone/ prolactin family. Mol. Endocrinol. 5 , 778-786. Theill, L. E., Castrillo, J.-L., Wu, D., and Karin, M. (1989). Dissection of functional domains of the pituitary-specific transcription factor GHF-1. Nature (London)342, 945-948. Theill, L. E., Hattori, K., Lazzaro, D., Castrillo, J.-L., and Karin, M. (1992). Differential splicing ofthe G H F l primary transcript gives rise to two functionally distinct homeodomain proteins. E M B O J. 11, 2261-2269. Truong, A. T., Duez, C., Belayew, A., Renard, A., Pictet, R., Bell, G. I., and Martial, J. A. (1984). Isolation and characterization of the human prolactin gene. E M B O J. 3,429-437. van Eys, G . J. J , M. (1980). Structural changes in the pars intermedia o f t h e cichlid teleost Sarotherodon mossambicusas aresult ofbackground adaptation and illumination. 11. The PAS-positive cells. Cell Tissue Res. 210, 171-179. Wendelaar Bonga, S. E., van der Meij, J. C . A,, and Flik, G . (1986).Response of PASpositive cells of the pituitary pars intermedia in the teleost Carussius uuratus to acid water. Cell Tissue Res. 243, 609-617. Wong, E. A., Silsby, J . L., and el Halawani, M . E. (1992).Complementary DNA cloning and expression of Pit-IiGHF-1 from the domestic turkey. DNA Cell B i d . 11, 65 1-660.

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6 STRUCTURE AND EVOLUTION OF FISH GROWTH HORMONE AND INSULINLIKE GROWTH FACTOR GENES THOMAS T . CHEN, ADAM MARSH, M I K E SHAMBLOTT, K . - M . CHAN,* Y.-L. TANG,? CLARA M . C H E N G , A N D B.-Y. YANG Center of Marine Biotechnology, University of Maryland Biotechnology Institute, and Department of Biological Sciences, University of Maryland at Baltimore County, Baltimore, Maryland

I. 11. 111. IV. V. VI. VII. VIII. IX.

Introduction Conserved Domains of Fish Growth Hormones Conserved Domains of Fish Prolactins and Somatolactins Genomic Organization of Fish GH, PRL, and SL Genes Ancestral Gene of the Fish Growth Hormone Gene Family A Functional Model of Fish Growth Hormone Gene Family Fish IGF I and IGF I1 mRNAs Age- and Tissue-Specific Levels of Five IGF niRNAs Concluding Remarks References

I. INTRODUCTION Growth hormone (GH), prolactin (PRL), placental lactogen (PL), and somatolactin (SL) form a family of polypeptide hormones with common structural and overlapping biological characteristics. GH and PRL are synthesized mainly by somatotrophs and lactotrophs, respectively, in the anterior pituitary gland of all vertebrates, whereas PL is

* Current address: Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Current address: American Red Cross, 15601 Crabs Branch Way, Rockville, MD 20855. 179 FISH PHYSIOLOGY, VOL. XI11

Copyright 0 l Y Y 4 by Academic Pre\\, Inc. All right5 of reproduction in any fnrm reserved.

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synthesized by syncytiotrophoblasts in the mammalian placenta and SL is only synthesized by cells in the pars interniedia of the fish pituitary glands. GH is involved in the regulation of somatic growth primarily through the induction of the insulinlike growth factor (IGF). Maintenance of nitrogen, lipid, carbohydrate, and mineral metabolic levels in vertebrates is also a property of GH (Bentley, 1982). PRL functions as a reproductive hormone to initiate and maintain lactation in mammals, or to maintain intracellular osmolarity in modern bony fish (teleosts) (Bern, 1983). Mammalian GH can exhibit both somatic growth and lactogenic actions. It has been demonstrated, for example, that administration of bovine GH stimulates lactation in dairy cows (Peel et al., 1981). The biological activities of PL and SL are the least understood of these four hormones. One of the possible functions of mammalian PL is to augment the carbohydrate-regulating properties of GH during pregnancy (Simpson and MacDonald, 1981). Hirano (1993) has suggested that fish SL may be involved in the osnioregulation of calcium. The primary structures of GH, PRL, and PL (Bewley et al., 1972; Niall et al., 1971), the genomic sequence of these genes (Miller and Eberhardt, 1983; Slater et al., 1986),and the regulation oftheir expression (Moore et al., 1985)in mammals have been studied in detail over the last few years. On the basis of the primary structures of these hormones and the structures of their genes, it has been postulated that they evolved from a common ancestral gene by duplication and divergence, and the ancestral gene, in turn, arose b y repeated duplication of a smaller gene or coding domain and insertion of additional domains (Niall et ul., 1971; Barta et ul., 1981; Miller and Eberhardt, 1983). However, this hypothesis was developed entirely based on mammalian species data. Thorough characterization of homologous hormones at many phylogenetic levels will allow a better assessment of the general applicability of this hypothesis to the GH gene family in all vertebrates. By applying sophisticated technologies of protein purification and recombinant DNA technology, the primary structures of GH, and PRL for lower vertebrates such as birds, amphibians, reptiles, and fish, and the primary structures of SL for several fish species have been determined. Either the purified polypeptides or their cDNA were directly sequenced (Kawauchi and Yasuda, 1989 for review). Furthermore, the nucleotide sequence and the intron and exon organizations of GH, PRL, and SL genes have also been determined for several fish species (Agellon et al., 1988a,b; Chen et al., 1991; Takayama et al., 1991; Tang et al., 1993). Part of this chapter will be devoted to re-

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viewing the present knowledge concerning the amino acid sequences of GH, PRL, and SL, the structures of the respective genes, and the evolutionary implications of these genes in lower vertebrates and bony fish. The insulin gene family is a highly diverse group that includes insulin and insulinlike growth factor 1 ( I G F I ) from a variety of vertebrate species, insulinlike growth factor I1 ( I G F 11), relaxin, insect prothoracicotrophic hormone (PTTH), and molluscan insulin-related peptide (MIP) (Blundell and Humbel, 1980; Smit et al., 1989). Both IGF I and IGF I1 are mitogenic peptide hormones that play an important role in the regulation of growth, differentiation, regeneration, and metabolism. Mammalian IGFs are translated as a preprohormone and, in this form, resemble preproinsulin. Insulin and IGFs are divided into distinct regions or domains, including an N-terminal prepeptide leader followed, from the N to C termini, by a B domain, a C domain, and an A domain. The IGFs contain an additional D domain and C terminal E domain. Both the I G F signal peptide and E domain are proteolytically removed from the preprohormone to form the mature hormone, as is the insulin signal peptide and the internal C domain. I G F I has been identified, at either the protein or nucleic acid level, in a number of nonmammalian vertebrates, including chicken (Kajimoto and Rotwein, 1989),Xenopus laevis (Kajimoto and Rotwein, 1990), coho salmon (Duguay et al., 1992), Atlantic hagfish (Chan et al., 198l), and amphioxus (Chan et al., 1990).Because IGF I1 cDNA clones were not identified from the aforementioned studies, it is commonly believed that IGF I1 evolved after the divergence of mammals from other chordates. Isolation ofrainbow trout I G F cDNAs was undertaken by Shainblott and Chen (1992) iis a first step toward examination of the physiology, biochemistry, and phylogeny of nonmammalian IGFs. Both IGF I and I G F I1 cDNA sequences were identified. The second part of this chapter will be devoted to reviewing the current knowledge of nonmammalian lGFs and their evolutionary implications.

11. CONSERVED DOMAINS OF FISH GROWTH HORMONES Substantial structural information is now available on GH from mammalian and nonmammalian species. By the use of newly developed protein purification technology, GH has been isolated from birds

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FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQ~SFLQ~QTSLCFSESIPTPSN~ET~KSNLELLRISLLLIQSWLEPVQFL~VFAN AFPAMsLSGLFANAVLRAQHLHQLTFKEFERTYIPEGQRYSI.QNTQVAFCFSETIPAPTGKAENQQKSDLELLRISLLL1QSWLGPLQFLSRVFTN TFPAMPLSNLFANAVLRAQHLHLLAAETYKEFERTYIPEWRYT.M(NSQAAFCYSETIPADTGKDDAQQKSDMELLRSLVLRQSWLTPVQYLSKVFTN FPQMPLSSLFANAVLRAQHLHQMVADTYRDYERTYIPEEQRHS.NKISQSAFCYSETIPAPTGKEDAEQKSDMELLRSLIL1QSWLNPVQFLSRVFTN

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SLVYGASDSNVYDLLKDLEEGIQT~~RLEDGSPRTGQIF.KQ..TYSKFDTNSHM)DALLKNYGLLYCFRKDMDKVETFLRIVQCR.SVEGSCGF SLVFGTSD.RVYEKLKDLEEGILALHRELEDGTPRAGQIL.KQ..TYDKFDT~DDALLKNYGLLSCFRKDLHKTETYLRVMKCF NLVFGTSD.RVFEKLKDLEEGIQALMRELEDRSPRGPQLL.R..PTYDKFDIHLINEDALLIGLLSCFKKDLH~TYLKVGESNCTI SLVFGTSD.RVYEKLRDLEEGIQALMRELEDGSLRGFQVL.R..PTYDKFDINLRNEDALLKNYGLLSCFKKDLHKVETYLKLMGESNCTI

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SLMFGTSD.GIFDKLEDLNKGINELMKWGDGGIYI.EDV.R.NLRYENFDVHLRM)AGLMKNGLLACFKKD~KVETYLMIVESNCTL SFAVRT ...QVTSKLSFLKMG ..KLIEANQDGAGGFSESSVLQLTPYGN SELFACFKKDMHKVETYLmAKCRLFPEANCTL

..............

GSSLRLRN.QISPRLSELKTGILLLIRANQDEAENYPDTDTLQHAPYGNYYQSLGGNESLRQTYELSPEANCTL

GSAPRPRN.QISPKLSELKTGIHLLIRANQD~~ADSSALQ~PYG~YQSL~ESL~~ELLACFK~~~ETYLTVAKCRLS~~CTL SLMVRNAN.QISEKLSDLKVGINLLITGSQDGVLStDDNDSQQLPPYGNYQ~~DG~~EL~CFKKD~~TYLmAKCRKSL~CTL ...NP..N.HISEKLADLKMGIGVLIEGCVDGQTGLDEM)SL.APPFEDFYQTL.SEGNLRKSFRLLSCFKKD~KVETYLSVAKCRRALDSNCTL

-------

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--------------

DGE

-____---__---

(1) Human GH; (2) Bovine GH; (3) Chicken GH; (4) Turtle GH; (5) Eel GH; (6) Flounder GH; (7) Tilapia GH;

(8) Tuna GH; (9) Rainbow Trout GH; (10) Catfish GH.

6. GROWTH HORMONE AND INSULINLIKE GROWTH FACTOR GENES

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(Farmer et al., 1974; Scanes et al., 1975; Harvey and Scanes, 1977), reptiles (Papkoff and Hayashida, 1972; Farmer et al., 1976a; Chang and Papkoff, 1985), amphibians (Farmer e t al., 1977a; Yamamoto and Kikuyama, 1981), primitive bony fish (Lewis et al., 1972; Farmer et al., 1981),and modern bony fish (teleosts) (Farmer et al., 1976b,1977a, Cook et al., 1983; Wagner e t al., 1985; Kawauchi et al., 1986; Kawazoe et al., 1988; Rand-Weaver et al., 1989; Watanabe et al., 1992). The complete amino acid sequence of GH has also been determined for the polypeptides from many teleosts (Noso et al., 1988; Yamaguchi et al., 1987) and two phylogenetically important species, namely, sea turtle (Yasuda et al., 1990) and blue shark (Yamaguchi et al., 1988a). Moreover, by employing recombinant DNA technology, the G H complementary DNA (cDNA) and its nucleotide sequence have been determined for birds (Lamb et al., 1988; Foster et al., 1990),channel catfish (Tang et al., 1993), coho salmon (Nicoll et al., 1987; GonzalezVillasenor et al., 1988), chum salmon (Sekine et al., 1985), common carp (Chao et al., 1989), flounder (Watahiki et al., 1989), rainbow trout (Agellon and Chen, 1986; Agellon et al., 1988b), red sea bream (Momota et al., 1988), striped bass (Cheng et al., 1991),tilapia (RentierDelrue et al., 1989a), yellowfin porgy (Tsai et al., 1993),and tuna (Sato et al., 1988). Amino acid sequence comparisons were carried out for GHs by aligning the conserved half-cystine residues and by introducing several deletions to maximize the homology (Fig. 1).Thus far, 196 amino acid residues were identified for GHs of different origins. From these sequence alignments, it was clear that GH is a single-chain polypeptide of about 21K Da and contains two conserved disulfide bonds. By amino acid sequence alignment, four highly conserved domains (A,,, B,,, CGH,and DGH)have been identified for all vertebrate GHs (Kawauchi and Yasuda, 1989). These four domains are at alignment positions 13-33, 54-94, 113-132, and 157-187 and are separated by variable domains and deletions. Abdel-Meguid et al. (1987) determined the three-dimensional structure of a recombinant methionyl-porcine GH Fig. 1. Multiple amino acid level alignment ofgrowth hormone ofvarious vertebrate species. Period indicates that a deletion was added to obtain maximum homology among various G H sequences; an asterisk indicates identical and highly conserved residues. , DG”. The Four highly conserved domains for GHs are marked AG”, BGH, C G ~and sequences are catfish G H (Tang et al., 1993), chum salmon G H (Sekine et al., 1985), tuna GH (Sato et al., 1988), tilapia G H (Yamaguchi et al., 1991), flounder G H (Momota et al., 1988), eel G H (Yamaguchi et al., 1987), turtle GH (Yasuda et al., l990), chicken G H (Tanaka et d., 1991), bovine G H (Woychik et al., 1982), and human GH (Martial et al., 1979).

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b y X-ray crystallography; they showed that the regions corresponding to A,,, B,,, and C,, domains are a-helical structures and arranged toward the outside of the GH molecule, whereas the D,, domain is located in the inside. These structural features are likely maintained in all GH molecules studied to date. A comparison of structural characteristics between the fish GHs and hGH was further executed using the Wisconsin GCG program package. The PEPTIDESTRUCTURE routine uses estimates of hydrophilicity and flexibility to calculate a surface probability statistic (Fig. 2). The profiles of this statistic are nearly identical for the fish GHs and hGH. The key feature to note is the terminal a-helix of the A,, domain that emerges from the protein's interior toward the surface at the amino acid motif of AV x RV x HLH x LA. This is consistent with the observation that some amino acids between position 40-45 are involved in receptor binding (Y42 and Q46 in hGH). There is another terminal a-helix motif in the BGWdomain that appears to begin at the surface and move into the protein's interior at the amino acid motif of E x QKSSVLK. Just upstream from this region, there are two more amino acids that are important for GH receptor binding (P61 and N63 in hGH). The largest structural differences between hGH

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Fig. 2. Surface mobabilitv d o t of fish and human growth hormones. The estimates ,~ were obtained from the Wisconsin GCG program package, which based the calculations on the method of Emini. In general, this approach considers hydrophilicity and flexibility in determining the amino acid groups that are most likely to be located at the surfiace of' the protein molecule. The location of the four evolutionary conserved domains of GH is indicated above the plots along with the position of their a-helixes, as estimated b y the method of Garnier Osguthorpe, and Robson.

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GROWTH H O R M O N E A N D 1NSULlNLIKE GROWT€I FACTOR G E N E S

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and fish G H appears to arise in the CGH domain. There is a short ahelical motif in this region in all members, but no consistent localization as to internal or external positioning. In tuna and rainbow trout, this domain has a high relative probability of contacting the surface, whereas in tilapia and catfish there is a high probability that this domain is internalized. The most dominant structural feature among the fish GHs in terms of the number of amino acid residues involved is a lengthy a-helix in the Dc;H domain. In rainbow trout, this structural motif is propagated over 30 amino acids. In the other fish, the D G H domain a-helix is composed of about 20 amino acids, whereas in hGH it involves only 10. The most interesting point to note on the surface probability plot is that in all cases, the a-helix appears to begin in the protein core, emerges at the surface around . . . CFKKDMH , . . , and then returns toward the protein’s interior. It is in this region at the surface where there are several amino acids involved in receptor binding (K168, D171, T175, C182 in hGH). Overall, it appears that the a-helix motifs in the A, B, and D domains are important for establishing a structural configuration that maintains some amino acids at the surface of the protein. In all cases, the a-helices indicate a definite tendency to span from the surface to the core of the protein.

111. CONSERVED DOMAINS OF FISH PROLACTINS AND SOMATOLACTINS The primary structures of lower vertebrate prolactins have been determined by sequencing the purified polypeptides directly (Farmer et al., 197713; Kawauchi et al., 1983; Prunet and Houdebine, 1984; Specker et al., 1984, 1985; Yamaguchi et al., 198813; Yasuda et al., 1986, 1987) or their respective cDNAs (Song et al., 1988; RentierDelrue et al., 198913). Amino acid sequence comparison was carried out for PRLs of mammals and lower vertebrates by aligning the conserved half-cystine residues and by introducing several deletions to maximize the homology (Fig, 3 ) . Thus far, a total of 202 amino acid residues are identified for PRLs. From the sequence alignment, it is clear that teleostean PRLs are single-chain polypeptides of about 21 I( Da and contain two conserved disulfide bonds similar to those of GHs, but PRLs of tetrapods have a third disulfide bond in their Nterminal portions. Similar to GHs, there are three highly conserved domains, ApRL, BpR,, and DpR,, identified in PRLs of all vertebrates characterized to date. The CpR,2 domain has a significant conservation within tetrapod

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VNLNDLLDRASQLSDKMHSLSTSLTNDLDSHFSSVGGKLM.RPSMCHTSSLQIPM)I(DQALSVPEGELLSLVL~WSDPLALLSS VGLNDLLERASELSDKLHSLSTSLTNDLDSHFPPVGR~RPSMCHTSSLQVP~~~~EDPLLSLARSLLLAWSDP~LSS IGLSDLMERASQRSDKLHSLSTSLTKDLDSHPPP~R~~SMCHTSS~TP~~QAL~SE~LIS~LL~WNDPLLLLSS VPINELLERASQHSDKLHSLSTTLTQELDSHFPPIGRVIMPRPAMCHTSSLQTPIDKDQALQVSESDLMSLARSLLQAWSDPLWLSS VPINDLIYRASQQSDKLHALST~TQELGSEAFPIDRV LA..... CHTSSLQTPTDKEQALQVSESDLLSLARSLLQAWSDPLEVLSS

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(6) QPICPNGGTNCQIPTSALFDRAVKLSHYIHSLSSEMFNEFDERFTPGRELAKSSGISCHTSSLNTPEDKEQARQIQHEDLLNLVLKLRSWNDPLVHWS (7) LPVCPSGSVGCQVSLENLFDRA~SHYIHHLSSEMENEFDERYAQGRGFLTKAINGCHTSSLTTPEDKEQAQQIHHEDLLNLVLGVLRSWNDPLLHLVS (8) LPICPIGSVNCQVSLGELFDRAVKLSHYIHYLSSEIENEFDERYAQGRGFITKAVNGCHTSSLTTPEDKEQAQQIHHEDLLNLWGVLRSWNDPLIHLAS (9) TPVCPNGPGDCQVSLRDLFDRAVMVSHYIHNLSSEMFNEFDKRYAQGKGFITMALNSCHTSSLPTPEDKEQAQQTHHEVLMSLILGLLRSWNDPLYHLVT (10) L P V C . . S G G D C Q T P L P E L F D R V V M L S H Y I H T L Y T D M F I E F D K Q W Q D P L F Q L I T (11) LPICPGGAARCQVTLRDLFDRAWLSHYIHNLSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQ~QKDFLSLIVSILRSWNEPLYHLVT

____----_-__-_-_-BPRL ___----_--________-

--_-_-------ApRL ________--_

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EATSLPH.PERNSINTKTRELQDHTNSLGAGLERLGRKMGSSPESLSSLPFNSN.DL.GQDNISRLVNFHFLLSCFRSHKIDSFLKVLRCDAAKMG EASSLAH.PERNTIDSKIKELQENINSLGAGLEHVF"LSSLPFYTN.SLGE.DKTSRLVNFHGLLSCFRRDSHKIDSFLKVLRCRAKK-RPE EAPTLPH.PSNGDISSKIRELQEYSKSLGDGLDIMVNKMGPSSQYISSIPFK~D.LGN.DKTSRLINFHFLMSCFRRDSHK1DSFL~LRC~T~ETC SASTLPH.PAQSSIFNKIQEMQQYSKSLKDGLEVLSSKMGSPAQAITSLPYR~TNLGH.DKITKLINFNFLLSCLRRDSHKIDSFL~LRC~~QP~C STNVLPY.SAQSTLSKTIQ~EHSKELKEGLDILSSKMGPRAQTITSLPFIETNEIGQ.DKITK...... LLSCFRRDSHKIDSFLKVLRCRAANMQPQVC

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EVQDIREAPDTIL.KTVEVEEQTKRLLEGMER.IIGRIQPGDLENEIYSPWPGPASIPG-DENSRLFAFFNLL~CL~SHKID~LKLLKC~..IHEGN~ EVQSIKEAPDTIL.KAVEIEEQDKRLLEC;MEK.IVGQVHPGEIENELYSPWSGLESLQQ~EDSRLFAFYNLLHCLRRDSHKIE~LKLLKC~..IHDNNC EVQRIKEAPDPILWKAVEIEEQNKRLLE~K.IVGRVHgwHAGNEIYSHSDGLPSLQ~EDS~FAFYNLLHCHRRDSHKID~L~LKC~..IHDSNC EVRC;MKGVPDAILSRAIEIEEENKRLLEC;MEM.IFGQVIPGAKETEPYPWlSGLPSLQTKDEDARHSAFYNLLHCLRRDSSKIDTYLKLLNCRI..IYN"C GLGGIHEAPDAIISRAKEIEEQNKRLLEGIEK.IIGQAYPEAKGNEIYLVWSQLPSLQG~EESKDLAFY"IRCLRRDSH~~LKFLRCQI..VHK"C (11) EVRGMQEAPEAILSKAVEIEEQTKRLLEGMEL.IVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCL~SHKID~LKLLKCRI..IHN"C

(6) (7) (8) (9) (10)

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(2) Carp PRL; (3) Chum Salmon PRL; (4) Tilapia 24K PRL; (5) Tilapia 20K PRL; (6) Bullfrog PRL; (7) Sea Turtle PRL; (8) Chicken PRL; (9) m i n e PRL; (10) Rat PRL; (11) Human PRL.

(1) Catfish PRL;

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PRLs, but not between tetrapod and teleost PRLs. Because the locations of A, B, and D domains in GH and PRL are nearly identical, the domain C,, may be GH specific and responsible for the biological activity of GH. This assumption is supported by the observation that a tryptic peptide of bovine GH, composed of amino acid residues 96-134, retains about 10% of the biological activity of the intact hormone (Yamazaki et al., 1972).This fragment ofbovine GH polypeptide is included in the C,, domain. As mentioned earlier, the presence of only two disulfide bonds in vertebrate GHs and teleostean PRLs, but a third disulfide bond in the N-terminal portions of tetrapod PRLs suggests that PRLs may have had more structural divergence than GHs during the evolution of vertebrates. The amino acid sequence of a newly identified pituitary hormone, SL, has also been determined for chum salmon (Takayama et al., 1991), flounder (On0 et al., 1990), cod (Rand-Weaver et al., 1991), catfish (Tang et al., 1993), lumpfish and halibut (Iraqi et al., 1993) by directly sequencing the purified polypeptide or cDNA clones. The somatolactin precursor is a single-chain polypeptide of approximately 231 amino acid residues. Although the amino acid homology among SL, GH, and PRL is low (22-28%), they are structurally related to one another because four ofthe six cysteine residues are present in positions similar to those in GH and PRL polypeptides. Figure 4 shows the alignment of SL amino acid sequences for six fish species: flounder, Atlantic cod, chum salmon, channel catfish, lumpfish, and halibut. Like GHs and PRLs, four highly conserved domains, A,,, B,,, C,,, and D,,, are also identified in SLs. The patterns and the relative locations of these four conserved domains in SLs are similar to those identified for GHs and PRLs.

Fig. 3. Multiple amino acid level alignment of prolactin of various vertebrate species. A period indicates that a deletion was added to obtain maximum homology among

various PRL sequences; an asterisk indicates identical and highly conserved residues. Four highly conserved domains for PRLs are marked A,,,, BpRL, CPRL,and D p R L . The sequences are catfish PRL (Tang et al., 1993), carp PRL (Yasuda et a / . , 1987), chum salmon PRL (Yasuda et al., 1987),tilapia PRL (Yamaguchi et al., 1988b), bullfrog PRL (Yasuda et al., 1991),sea turtle PRL (Yasuda et al., 1990), chicken PRL (Li et d.,1976), ovine PRL (Li et al., 1974), rat PRL (Cooke et al., 1980), human PRL (Cooke et al., 1981).

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---_------- As, ----------

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..ADLQDSLDMYDNVPSSLISKTRWMSTKLMNLKQGVLVLMSKMLDEGSVELE"ESMLR.PAMAEHVLRDYAVLSCFKKDAHKMETFLKLLRCRQTDNPTCSLF ..VYLQTTLDRYDNAPDMLLNKTKWVSDKLISLEQGVWLIRKMLDE(;MLTATYNEQGLFQYDAQPDMLESVMRDYTLLSCFKKD~~IFLKLLKCRQTDKYNCA.. ..VYLQTTLDRYDDVPDVLLNKTKWMSEKLISLEQGVWLIRKMLDGAILNSSYNEYSAVQLDVQPEVLESILRDYNVLCCFKKDAHKIETILKLLKCRQIDKYNCALY ..VYLQTTLDRYDDAPDTLLKKTKWVSEKLLSLEQGVWLIRKMLDDDMLTTSYYEQGVAPYALQPEVLESVLRDYTLLSCFKKDAHKMETFLKLLKLLKCRQTDKYSCFLH P L V Y L Q T S L D R Y N A A P E M L L N K T K W V S E K L I S L E Q G V W L I T D R Y N C S . .

PLVYLQTTLDRYDNASEMLLNKTKWVSDKLISLEQGVWL1RKMLDEC;MLTATYNEQGLFQYDVLPD~ESVMRDYTLLSCF~AH~IFLKLLKCRQTDKYNCP..

(1) Catfish SL; (2) Flounder SL;

(3) Atlantic Cod SL; (4) Chum Salmon SL; ( 5 ) Lumpfish SL;

( 6 ) Halibut S L -

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IV. GENOMIC ORGANIZATION OF FISH GH, PRL, AND SL GENES The genomic sequences of GH and PRL genes have been determined for human, rat, and bovine species. Mammalian GH genes span a region of about 2.5 kb, comprising five exons and four introns (Barta et al., 1981; DeNoto et al., 1981; Woychik et al., 1982), whereas the mammalian PRL genes also contain five exons, but four long introns to make the genes about 10 kb or longer (Truong et aZ., 1984). In the last five years, GH genes for chicken (Tanaka et al., 1991), rainbow trout (Agellon et al., 1988a), Atlantic salmon (Johansen et al., 1989), chinook salmon (Du et al., 1993),Chinese grass carp (Zhu et al., 1992), tilapia (Ber and Daniel, 1992), and channel catfish (Tang et al., 1993) have also been isolated and characterized. The exon-intron organization of GH and PRL genes in mammals, birds, and fishes are presented in Fig. 5A. In comparison with their counterparts in mammals, the GH genes of rainbow trout, Atlantic salmon, and chinook salmon contain an additional intron in exon 1 7 to make the total size of the gene about 4.5 to 5 kb. On the contrary, the GH genes of channel catfish and carp span a region of 3.5 kb with the same exon-intron organization as that of mammalian GH genes. Although the tilapia GH gene is in the same size range as that of carp or channel catfish, it contains six exons and five introns similar to salmonid species. Fish PRL genes have been isolated and characterized for common carp (Chen et al., 1991), chinook salmon (Xiong et al., 1992), channel catfish (Tang et al., 1993),and tilapia (Swennen et al., 1992).In contrast to the human PRL gene, the fish counterpart is a small gene of about 2.5 to 3 kb, comprising five exons and four introns (Fig. 5B). The genomic sequence for the fish SL polypeptide is only characterized in chum salmon (Takayama et al., 1991). Although the exon-intron organization of the salmon SL gene is similar to that of mammalian GH or fish PRL genes, it contains very large introns like those of the human PRL gene.

Fig. 4. Multiple amino acid level alignment of somatolactin of various vertebrate species. A period indicates that a deletion was added to obtain maximum homology among various SL sequences; an asterisk indicates identical and highly conserved residues. Four highly conserved domains for SLs are marked A,,, BSL, C,,, and DSL. T h e sequences are catfish SL (Tang et al., 1993),flounder SL (Ono et d . , l990), cod SL (Rand-Weaver et al., 1991), chum salmon SL (Takayama et al., 1991), lumpfish SL and halibut SL (Iraqi et al., 1993).

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2 3. 4.

5. 6. 7.

8. 9.

(I) Iiumm PRL: (2) Rat m,(3) Cnp I% (4) clt68hpRL;(5) Tqi.PRL; (6) S.haon H1L. (7) chum s.Lmaa -ladin

(SL)

Fig. 5. Exon-intron organization of GH, PRL, and SL genes.

In mammals, tissue-specific expression of GH and PRL genes has been shown to be regulated by a trans-acting factor encoded by the Pit-1 gene and a cis-acting element residing in the 5' flanking region of the GH or PRL gene (Bodner et al., 1988; Nelson et al., 1986, 1988). The consensus sequence of the Pit-1 binding site is AAITAI TTANCAT. Furthermore, thyroid hormone (T3 or T4) and glucocorticoid hormone are known to regulate the transcription of mammalian G H genes (Evans et ul., 1982). Although it is not certain whether thyroid hormone and/or glucocorticoid hormone regulate the transcrip-

6.

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1

I

Regulatory Region

GH

PRL

Structural Gene Region

PL

I

SL

Conserved Domains

Fig. 6. Structure of the proposed ancestral gene of the G H gene family. The five conserved domains are shown in solid boxes.

tion of fish G H genes, several Pit-1 binding site consensus sequences have been identified in the 5' flanking regions of fish GH, PRL, and SL genes (Fig. 6). The biological significance of these Pit-1 consensus sequences requires further investigation by a functional assay. V. ANCESTRAL GENE OF THE FISH

GROWTH HORMONE GENE FAMILY On the basis of the presence of four regions with internally similar amino acid sequences in human GH/PL and bovine PRL, Niall et al. (1971) proposed that a small primordial domain was repeatedly duplicated to form a larger gene that became the ancestral gene for the GH/PRL/PL gene family. It was further proposed that this ancestral gene went through duplication and divergence and gave rise to the current GH gene family. In the human GH gene, for instance, the first two of the four internally similar regions are each encoded b y separate exons (exon I1 and exon IV), but the last two regions are encoded by the last exon (exon V). Furthermore, analysis of the structures of rat GH, rat PRL, human GH, and human PL genes revealed

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the presence of imperfect direct repeat sequences flanking exons, I, 111, and V of these genes (DeNoto et al., 1981; Selby et al., 1984; Seeburg, 1982). These results were taken as evidence that exons I and I11 of the ancestral GH gene arose by separate insertion events via a mechanism analogous to DNA transposition. Although this evolutionary model was supported by observations from the mammalian system, studies on the primary structures of fish GH polypeptides (Sekine et ul., 1985; Agellon and Chen, 1986) as well as the genomic sequences of rainbow trout (Agellon et al., 1988a,b), Atlantic salmon (Johansen et al., 1989), tilapia (Ber and Daniel, 1992), and chinook salmon (Du et al., 1993)GHs failed to reveal either the presence ofany internally similar amino acid sequences or imperfect direct repeat nucleotide sequences. Alignment of amino acid sequences of GH polypeptides of lower and higher vertebrates resulted in the identification of four highly conserved domains, that is, A,,, B,,, C,,, and DGH.Similarly, four highly conserved domains are also found in PRL ofvertebrates. Hence, instead of internally repeated sequences in the common ancestral gene of the GH/PRL family, there are domains that are conserved throughout evolution (Fig. 6). This conclusion was further supported b y the finding that fish SLs also possess four highly conserved domains (Ono et al., 1990; Takayama et al., 1991; Rand-Weaver et al., 1990; Y . Tang e t al., 1993 unpublished results; Iraqi et al., 1993; Takayama et al., 1991). These SL polypeptides have been considered as members ofthe fish G H gene family because they share a high degree of amino acid sequence homology with fish GHs and PRLs. It is further believed that the internal repeat sequences observed by Niall et al. (1971) may have resulted from some sort of coincidence, as pointed out by Nicoll et nl. (1986). The concept of a common ancestral gene for the fish GH gene family is further supported by the observation of consensus sequences analogous to the Pit-1 binding elements of the mammalian GH/PRL genes (Fig. 7). These sequences were localized in the flanking region 5’ to exon 1 of the fish GH, PRL, and SL genes studied to date (K. M . Chan and T. T. Chen, 1993, unpublished results; Zhu et al., 1992; Takayama et al., 1991; Swennen et al., 1992; Xiong et al., 1992). Thus, the presence of Pit-1 consensus elements in the regulatory region of GH, PRL, and SL genes suggests that a protein homologous to the mammalian Pit-1 may be produced by fish pituitary cells to regulate the tissue-specific transcription of the fish GH gene family. The GH genes in mammals span a region o f 2 . 5 kb, comprising five exons and four introns (DeNoto et al., 1981; Woychik et al., 1982;

-

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... . .

-122 -124 -106 -52 -170 -543 -31 -143 -341 -443 -612 -52 -170 -404 -114 -445 -112 -287 -308

TAATATGCAT ATTAAAACAT AAAAATTCAT TATAATACAT AAATAATCAT ATTTAAACAT CTAAATACAT ATTTTTACAT TATTTTCCAT

-114 -115 -97 -43 -161 -534 -22 -134 -332 -434 -603 -43 -161 -395 -105 -436 -103 -218 -299

Common carp GH

-50 ATTAAAACAT

-41

Tilapia GH

- 5 1 ATTTAAACAT

-42

ACTTATGCAT AAAATACCAT TAAAAATCAT AAATAAACAT TTTTATTCAT ATATTTTCAT TTTTATTCAT AATTTTACAT

-57 -165 -265 -433 -310 -614 -628 -983

Human GH Rat GH Chicken GH Rainbow trout GH2

Atlantic salmon GH1

Atlantic salmon GH2 Catfish GH Grass carp GH

Chinook salmon PRL

Tilapia PRLl

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-66 -114 -214 -442 -319 -623 -637 -992

-460 AATTATCCAT - 4 5 1

193

TT

ATGNATAAAT -81 ATGCATAAAT -87 ATGAATAAAT

-78 -78

-174 ATGGAAAAAT

-165

-96 ATCTATAAAA

-617 ATGAATAATA

-81 -137 -229 -608

-154 ATGGAAAAAT

-165

-146 ATGAAAATCT -238 ATGGAAAAAT

- 5 5 ATGCATTAAA

-46

-91 -169 -219 -858 -54 -113 -55 -242

-56 ATGCATCAAA ATGCATATTA ATGGAAATTG ATGGAAAGAA ATGAATTATC ATGCATTAAA ATGCATAATC ATGAATTTAA ATGTAAATAT

-47 -88 -150 -210 -849 -45 -104 -46 -233

-293 ACGAATAAAT

-284

-607 ATGAATATTT - 5 9 8 1 1 3 2 ATCAATAAAA -1123

- 3 1 ATCCATATAA - 2 6 1 ATGTATTATG

-22 -252

Fig. 7. Consensus sequence of Pit-1IGHF-1 binding sites in the 5' flanking regions of' GH, PRL, and SL genes.

Barta et al., 1981). In comparison with their counterparts in mammals, the GH genes in rainbow trout (Agellon e t al., 1988a), chinook salmon (Du e t al., 1993), and Atlantic salmon (Johansen et al., 1989) contain an additional intron in exon V to make the total size of the gene about 4.5 kb. To our surprise, the GH genes of Chinese grass carp (Zhu e t al., 1992) and channel catfish (Tang e t al., 1993) have a size and intron-exon structure similar to those of the mammalian GH genes. One possibility for the origin of the fifth intron in salmonid G H genes is that it may have been present in the ancestral GH gene after its divergence from PRL and SL genes, or alternatively it may have been the result of an insertion during the tetraploidization of the salmonid genome. To confirm the former, a fifth intron should be present in GH genes of primitive fish. Although modern tilapia diverged from a

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common ancestral line at a time later than that of the salmonids, Ber and Daniel (1992) have reported the observation of a small intron V (79 bp) in the GH gene of this fish species. If the presence of intron V in salmonid GH genes was the result of tetraploidization, the same fifth intron should have been observed in PRL and SL genes in rainbow trout or salmon unless the event of tetraploidization occurred subsequent to the divergence of GH, PRL, and SL genes (Takayama et al., 1991). Furthermore, the fifth intron should also be present in the GH genes of other ancestral polyploid fish species like common carp.

VI. A FUNCTIONAL MODEL OF FISH GROWTH HORMONE GENE FAMILY From the structural similarities described in the previous section among GH, PRL, and SL, it is highly probable that these hormones have evolved from a common ancestral gene. However, for any evolutionary model of a gene family to be considered complete, a plausible description of some physiological function of the ancestral gene must also be included. The purpose of such a functional model is to provide a reasonable selective mechanism that would describe some impetus for the ancestral gene to have given rise to the current members of this hormone family. In trying to identify a physiological function for the GH-family ancestral gene, there are three critical considerations. First, we know that no homologs of GH, PRL, and SL have been currently identified in any invertebrates (i.e., 95% of the animal kingdom species), suggesting that the common ancestral forerunner of these hormones may have appeared after the chordate ancestors diverged from the main stem of deuterostome invertebrates. Second, in looking for an ancestral physiological function, we are restricted to considering the fish that are the oldest extant group of chrodates in which these hormones are found. Third, when looking at what is currently known about the physiological functions of these hormones in fish, we find that one common function does emerge: GH, PRL, and SL are all known to be actively involved in the osmoregulatory responses of teleosts (Sakamot0 et al., 1990; Hirano, 1993). These observations suggest that the GH-family ancestral gene may have been involved in the osmoregulation response of the deuterostome-invertebrate ancestors of the chordates. Thus, a detailed consideration of osmoregulation is warranted in establishing a functional model of GH-family gene evolution.

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Focusing on osmoregulation as a potential area of physiological activity for the common ancestral gene, we note that modern deuterostome invertebrates (relatives of the ancestral invertebrate chordate) generally exhibit an isosmotic cellular response to changes in environmental salinity. After hyperosmotic exposure (i.e., a sudden increase in external salinity), cellular metabolism is shifted toward the catabolism of large molecules to increase the number of intracellular osmolytes, thereby increasing the osmotic pressure of the cytoplasm. After hypoosmotic exposures, cellular metabolism is shifted toward the anabolism of macromolecules to reduce the number of intracellular osmolytes. The metabolites used for this metabolic shuffling are generally in flux between the cytoplasm and short-term nutritional reserves, with the net mass balance shifted appropriately to maintain cell volume. Animals also have structural deposits that serve as long-term sources or sinks of metabolites, but these are generally not used for short-term, immediate metabolic responses. The flux of metabolites to structural components is reserved for long-term allocations, like growth and development, or dire metabolic needs, like starvation. If the deuterostome-invertebrate ancestors of the chordates were osmoconformers and regulated their cell volume by changing the levels of intracellular osmolytes, then we propose that the GH-family ancestral gene produced a protein product that regulated the intracellular flux of metabolites to and from the pool of short-term nutritional reserves (Fig. 8). In this hypothetical scenario, the ancestral protein was adapted to rapidly increase the net flux of monomeric organic metabolites from storage nutrients in response to a hyperosmotic stress. Conversely, this activity of the ancestral protein would suppress the flux of metabolites into storage reserves under hypoosmotic stress, thereby increasing the deposition of those metabolites into long-term structural components. In this case, because the total flux rate between nutritional storage and intracellular metabolite pools is generally high, the removal of metabolites to structural components under hypoosmotic conditions would reduce the total, short-term flux rate by eliminating those metabolites from circulation. The probability of survival for an osmoconformer is dependent on the speed and efficiency with which organic solutes can be mobilized or sequestered to maintain normal cell volume and function. The source and allocation of these metabolites would be coordinated by this ancestral GH-family protein. Thus, we hypothesize that the common ancestral gene of this hormone family could have evolved to rapidly adjust the overall flux of intracellular metabolites under the selective pressure of coordinating an isosmotic response to changes in environmental salinity.

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Ancestral GH-Family Forerunner

Metabolite

Fig. 8. A functional model of the physiological role of a hypothetical ancestral CH family forerunner. Following a hyperosmotic stress, w e propose that this ancestral protein was adapted to regulate the rapid release of metabolic osmolytes from nutritional reserves to increase intracellular osmotic pressures. Conversely, under hypoosmotic stress, this ancestral protein may have inhibited the flux of intracellular metabolites into nutritional reserves, forcing them instead to be deposited into more permanent structural components to reduce levels of intracellular osmolytes.

As this ancestral gene began to give rise to a group of structurally related genes in the early chordate ancestors, the protein products of these new genes may have begun to develop more specialized controls over cellular metabolism. For instance, it is easy to imagine an early lineage of GH-like proteins arising to become specialized in the rapid mobilization of metabolites from short-term nutritional reserves during a hyperosmotic stress. As this lineage of GHs developed, its efficiency in controlling the mobilization of nutrients from storage reserves became so dominant that it eventually became the primary regulator of organismal growth rate, as evident in modern vertebrate GHs. Likewise, an early lineage of PRL-like proteins could have developed to specifically promote the reduction of intracellular metabolites under hypoosmotic stress, by promoting their sequestration in germinal tissues rather than structural components. As this lineage of PRLs developed, its efficiency in controlling the mobilization of nutrients to reproductive tissues became so dominant that it eventually became a primary regulator of reproduction, as evident in modern vertebrate PRLs. In summary, we hypothesize that the ancestral gene from which GH, PRL, and SL were derived was active in regulating the flux of

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metabolites necessary to maintain cell volume in response to changes in environmental salinity in the early deuterostome ancestors of today’s modern chordates. This functional model of the early evolution of the GH family suggests that the common ancestral gene was pivotal in determining the flux of intracellular metabolites by regulating their mobilization from short-term nutritional reserves and/or their net deposition into long-term structural components. Although conjectural, this functional model provides a selective mechanism to account for the evolution of modern GH, PRL, and SL from an ancestral metabolic regulator that is consistent with what we know about the physiological activities of this hormone family.

VII. FISH IGF I AND IGF I1 mRNAs

To examine the phylogeny, biochemistry, and physiology of nonmammalian IGF, we have undertaken studies to isolate and characterize JGF cDNAs for rainbow trout. By employing the polymerase chain reaction (PCR), we amplified an internal portion of I G F cDNA from the total cDNA of rainbow trout liver RNA and confirmed its identity by nucleotide sequence determination (Shamblott and Chen, 1992). Using this DNA fragment as a hybridization probe, recombinant clones encoding the I G F cDNA sequence were isolated from a rainbow trout liver cDNA library. Nucleotide sequence determination confirmed the isolation of two distinct IGFs. The alignment of the predicted amino acid sequences of these two IGFs with the amino acid sequences of human I G F I and I1 is shown in Fig. 9. On the basis of a 98.7% nucleotide sequence homology with coho salmon I G F I (Cao et al., 1989), one of the cDNA sequences was identified as trout I G F I. The second cDNA sequence shared 43.3%identity with trout I G F I at the predicted amino acid level and 53.6% identity with human IGF 11, and was thus identified as trout IGF 11. Reverse transcription (RT)/ PCR analysis revealed that both I G F I and I G F I1 mRNAs were present in the livers of rapidly growing yearling rainbow trout. This is the first report o f a nonmammalian IGF I1 cDNA sequence. Furthermore, this result suggests that I G F I1 diverged from I G F I at the time or earlier than salmonid fish evolved from their ancestral species. As a result of differential splicing in the 5’ untranslated region, signal peptide, E domain, and 3’ untranslated region, as well as transcription from multiple promoters, multiple size forms of I G F I and I1 mRNA had been detected in mammals (Bell et al., 1985; Rotwein et ul., 1986). To detect the presence of multiple size forms of IGF I

I B I --cI ___ MSSGHFFQWHLCDVFKSAMCCVSCTHTLSLLLCVLTLTSAATGAGPETLCGAELVDTLQFVCGERGFYFSKPTGYGPSSRRSHNRGIVDECCF MITPTV. .HTM.SS.LFY.A..L..F..S.. .............A ......D.....N.......S.....QT........ c METQKRHEYHSVCHTCRRTENTRMKVKHM SS . . . .A . ....ED ...... d MGIPHGKS ASC ...........D......

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I

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and I1 mRNA in rainbow trout, a RT/PCR method was developed (Shamblot and Chen, 1993). This assay employed two sets of primers each for I G F I and I1 so that small PCR product size differences could be resolved on high-concentration agarose gels. The identity of each product was confirmed by nucleotide sequence determination. The primer sets were designed to separately amplify the 5’ region (from the predicted start codon to the C domain) or 3’ region (from the C domain to approximately 100 b p beyond the predicted stop codon) of both I G F I and 11. Although only one size form of IGF I and I1 mRNA resulted from RT/PCR with the 5’ I G F I and both 5’ and 3’ I G F I1 primer sets, four size forms of I G F I mRNA resulted from the 3’ I G F I primer set. Results of nucleotide sequence determination of the four size forms of I G F I mRNA showed that the size differences were due to insertions or deletions in the E domain. These four forms of I G F I mRNA, in increasing nucleotide length, are designated as I G F IEa1, -2, -3, -4 in accordance with suggested revisions of IGF I nomenclature (Holthuized et al., 1991; Duguay et al., 1992),and have E-domainpredicted amino acid residues of 35,47,62, and 74, respectively (Fig. 10). The entire nucleotide sequence for I G F IEa-2 and Ea-3 mRNA have been determined from their respective intact cDNA clones. cDNA clones corresponding to the two remaining RT/PCR generated I G F I forms ( I G F 1Ea-1 and Ea-4) were not detected. By the use of a RT/PCR assay, Duguay et al. (1992) detected three forms of I G F I mRNA for coho salmon, and these three mRNA forms are equivalent to rainbow trout I G F IEa-1, Ea-3, and Ea-4. By using the same approach, Wallis and Devlin (1993) have also detected three size forms of I G F I mRNA for chinook salmon. These three size forms correspond to rainbow trout I G F IEa-4, Ea-2, and Ea-1. The reasons for the absence of one form of I G F I in the livers of coho salmon and chinook salmon, which lack analogues to rainbow trout I G F IEa-2 and IEa-3, respectively, are unknown. It is conceivable that the analogues were not resolved and, therefore, not recognized after agarose gel electrophoresis. Alternatively, the I G F I mRNA form absent in these two reports may not have been present or detectable in these fish, in

Fig. 9. Multiple predicted amino acid level alignment of trout and human IGFs (a) Trout IGF I, (b) human IGF I, (c) trout IGF 11, and (d) IGF 11. Predicted amino

acids are indicated in capitalized one-letter code. Stop codon is indicated as an asterisk. Identity with (a) is indicated as dots. Identity between only (c) and (d) is indicated by shading. Gaps are introduced by absence of the one-letter code. The putative IGF domain boundaries are indicated in boldface above (a) for IGF I and below (d) for IGF 11. From Shamblott and Chen (1992) with permission from the authors.

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THOMAS T. CHEN ET AL. PreDeDtide Leader B

C

A

D

E

Ea-4

Ea-2

Ea-1

Fig. 10. Schematic representation of the four subforms of rainbow trout IGF I mRNAs. T h e shaded line open box indicates that the nucleotide sequence of the molecule has not been confirmed from the cDNA clones yet. B, C, A, D, and E indicate different domains of the IGF preprohormone.

which case it is surprising that the two salmonid species are missing different analogues of rainbow trout IGF I. In mammals, two of the forms of IGF I mRNA, I G F Ia and Ib, differ mainly in the length and amino acid sequence ofthe E domain. On careful analysis of the rainbow trout I G F RT/PCR products and cUNA clones, IGF mRNA analogues similar to the mammalian IGF Ib were not identified. It is of interest to note that a number of single- or double-nucleotide substitutions are present throughout the two intact cDNAs (Ea-2 and -3)and the PCR products (Ea-1 and -4) of trout I G F I (Shamblott and Chen, 1993). However, none of these substitutions leads to the differences in the predicted amino acid sequence of the mature I G F I polypeptide. These results suggest the existence of at least three I G F I genes (or allelic forms); one encodes IGF IEa-3, one encodes I G F IEa-4, and the third one encodes I G F IEa-1 and -2. The multiple I G F I nucleotide substitution phenomenon was also reported in chinook salmon, leading Wallis and Devlin (1993) to suggest that there are four IGF-I genes.

VIII. AGE- AND TISSUE-SPECIFIC LEVELS OF FIVE IGF mRNAs An KNase protection assay (KPA) was established to determine the mKNA level of each of the four I G F I forms and IGF I1 in the liver, skeletal muscle, spleen, pyloric ceca (pancreatic tissue), heart, brain,

1

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Fig. 11. Levels of five forms of IGF mRNA in the tissues of juvenile and adult trout. The forms of I C F I mRNA are abbreviated as Ea-I, Ea-2, Ea-3, and Ea-4. Total IGF I, IGF 11, and total I C F levels are abbreviated as I, 11, and IGF, respectively.

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and gill of rapidly growing juveniles (7-8 months old) and sexually mature adults and in testes and ovaries from the sexually mature adult rainbow trout (Shamblott and Chen, 1993). Probe templates for the RPA were constructed by cloning the 3' region (from the C domain to approximately 100 bp into the 3' untranslated region) of each I G F I or I G F I1 into the Bluescript plasmid vector in order to generate radiolabeled antisense cRNA probes and unlabeled sense cRNA concentration standards respectively by in witro transcription with T7 or T3 RNA polymerase. The level of 18s rRNA in each tissue was also determined to serve as an internal standard for normalization. Each of the four I G F I mRNA forms and the I G F I1 mRNA were readily distinguishable and determined by this RPA, and the protected fragments for the four I G F I mRNA forms are 354 bp, 390 bp, 438 bp, and 471, and for I G F I1 mRNA 496 bp. The results of I G F mRNA RPA are summarized in Fig. 11. At least one form of I G F I and IGF I1 mRNA is expressed in all the tissues examined in both developmental stages. Liver is the site of the greatest I G F mRNA abundance ( P < 0.01), and the level of total IGF I and I1 mRNA is one to two orders of magnitude higher than that in other tissues examined. Furthermore, it is interesting to note that the level of total I G F I and I1 mRNA is twofold higher in the adult liver than the juvenile liver ( P < 0.01). In mammals, IGF I mRNA has been detected primarily in the postnatal liver, kidney, spleen, pancreas, lung, and testis of the mouse (Mathews et al., 1986), the brain and several other regions of the central nervous system of the rat (Rotwein et al., 1988), and placenta and whole premenopausal ovary of the human (Hernandez et al., 1992). In chicken, I G F mRNA has been detected in the eye, skeletal muscle, and brain prior to hatching, and in the liver only after hatching (Kikuchi et al., 1991).IGF I1 mRNA has been detected in muscle, skin, lung, intestine, thymus, heart, kidney, brain, and spinal cord of fetallneonatal rats and in the brain and spinal cord of adult rats. Except in the liver, levels of rainbow trout I G F I1 mRNA are much higher than those of the total I G F I mRNA in gill, kidney, heart, spleen, brain, muscle, pyloris, testis, and ovary. These results suggest that, in addition to IGF I, I G F I1 may play an important role in growth enhancement of fish.

IX. CONCLUDING REMARKS Our laboratory is primarily interested in understanding how animals regulate their growth rates. In this chapter, we have addressed two imporant aspects of growth regulation. First, we have proposed a

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structural model for the evolution of GH family genes from a common ancestral gene, as well as a functional model to provide a selective mechanism for this evolution. Second, we have discussed the recent progress in identifying insulinlike growth factor members in teleosts. The obvious next step is to relate these two areas into a single, unified perspective on growth regulation. Although our structural and functional models for GH family evolution are only hypothetical, their utility at this point in time is to provide us with specific research objectives for further investigations regarding the evolution of growth regulation: (1) Are there any GH, PRL, or SL related genes in any other deuterostomes, particularly the hemichordates? ( 2 )Did the regulatory relationship between GH and insulin family members coevolve as a mechanism for metabolic activation in response to osmotic stress? ( 3 ) Can a coevolutionary relationship between GH and insulinlike growth factors be described from examining the relationship between GH-receptor and I G F promoter domains? (4) Are any insulin family members involved in the osmoregulatory response of teleosts or invertebrates? (5) Can any phylogenetic distinctions be made between the metabolic activation of insulins and the mitogenic stimulation of IGFs? Overall, it is fair to say that we still have a long way to go before we understand why vertebrates are so unique in terms of the genes they use to regulate growth, and why those specific mechanisms evolved in only a small subset of the animal kingdom.

ACKNOWLEDGMENTS This work was in part supported by grants from NSF (DCB-91-05719) and Maryland Sea Grant College (R/F 63) to T.T.C. This is contribution No. 215 of the Center of Marine Biotechnology, University of' Maryland Biotechnology Institute.

REFERENCES Abdel-Meguid, S. S., Shieh, H.-S., Smith, W. W., Dayringer, H. E., Violand, B. N., and Bentle, L. A. (1987). Three-dimensional structure of genetically engineered variant of porcine growth hormone. Proc. Natl. Acad. Sci. U.S.A. 84, 6434-6437. Agellon, L. B., and Chen, T. T. (1986). Rainbow trout growth hormone: Molecular cloning of cDNA and expression in E . coli. DNA 5, 463-471. Agellon, L. B., Davies, S. L., Chen, T. T., and Powers, D. A. (1988a). The nucleotide sequence of a gene encoding rainbow trout growth hormone (GH): Implication on the evolution of the GH gene structure. Proc. Natl. Acud. Sci. U.S.A.85,5136-5140.

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I11 OTHER HORMONES

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7 STRUCTURE AND EXPRESSION O F INSULINLIKE GROWTH FACTOR GENES IN FISH SHUJIN CHAN AND DONALD F . STEINER T h e Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois

I. Introduction 11. I G F Activity in Fish 111. Cloning of Fish I G F cDNAs and Genes IV. Expression and Regulation of I G F V. Summary and Perspective References

I. INTRODUCTION The insulinlike growth factors IGF-I and -11 together with insulin are three members of the insulin superfamily that share close similarities in primary structure and biological activity. Insulin is a wellcharacterized pancreatic peptide hormone that plays an important role in regulating metabolism, particularly in carbohydrate utilization, and is found in all vertebrates. Genes encoding two-chain insulinlike molecules have also been characterized in several invertebrate species, including molluscs (Smit et al., 1988) and insects (Iwami et al., 1989; Lagueux et al., 1990). In contrast, until recently IGF-I and -11 genes have been characterized only in mammalian species. However, facilitated by advances in recombinant DNA techniques, IGF genes have now been identified in birds (Kajimoto and Rotwein, 1989)andamphibians (Kajimoto and Rotwein, 1990; Shuldiner et al., 1990). This objective of this review is to present recent advances in our knowledge of the structure and expression of I G F genes in fish. Additional information on related topics, including the characterization of mammalian IGF genes, I G F receptors, bindingproteins, and evolution213 FISH PHYSIOI.OC.Y, \'OL XI11

Cop)right D 1994 Iry Academic Press, Inc. All rights of reprirdirctiorr iri any form rrwr\ed.

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ary aspects, can be found in several other reviews (Chan et al., 1992; Chen et al., 1994; Cohick and Clemmons, 1993; Daughaday and Rotwein, 1989; Rechler and Nissley, 1985).

11. IGF ACTIVITY IN FISH

Earlier reports of I G F activity in fish presented conflicting data. Using radioimmunoassay (RIA) and receptor binding assay (RRA),several laboratories reported detecting IGF-I and -11 activities in tissues and sera from a number of nonmammalian species, including teleosts (Daughaday et al., 1985; Zangger et al., 1987; Drakenberg et al., 1989; Bautista et al., 1990). However, several negative findings were also reported using similar experimental techniques (Furlanetto et al., 1977; Wilson and Hintz, 1982). There are several possible explanations for these discordant results. First, both RIA and RRA were heterologous assays based on IGFs and receptors isolated from mammalian sources. The use of heterologous assays often results in a decreased sensitivity that may have hampered detection. The tissue and serum levels of IGF in nonmammalian species could also vary widely in response to different physiological conditions. Furthermore, other studies have confirmed that fish contain serum proteins that bind I G F with high affinity (Upton et al., 1993). It has been shown that the binding proteins must be removed to obtain accurate assay results (Bowsher et al., 1991). However, Moriyama et al. (1993) have reported the development of a RIA using antisera to recombinant coho salmon IGF-I. The availability ofa homologous RIA should result in more accurate quantitation of IGF-I levels in salmonids and related fish species. Although direct assays for I G F have yielded ambiguous results, there is good evidence that exogenously added I G F is bioactive in teleosts. Duan and Hirano (1990) and McCormick et al. (1992b) have shown that recombinant mammalian IGFs stimulated [35S]sulfateuptake in cartilage cultured from Japanese eel and coho salmon. Injections of IGF-I also stimulated somatic growth in salmon and gobys (Gray and Kelley, 1991; McCormick et al., 1992a). Funkenstein et al. (1989) have reported that injected bovine growth hormone stimulated a twofold increase in serum IGF-I level in the sea bream. Taken together, these results indicate that a principal feature of the somatomedin hypothesis, in which the action of growth hormone is mediated by IGF-I (Daughaday, 1972), has remained operative in teleosts.

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111. CLONING OF FISH IGF cDNAs AND GENES

The first fish I G F cDNA cloned from coho salmon was reported by Cao et al. (1989). Using the polymerase chain reaction (PCR) technique and degenerate oligonucleotide primers, these workers first amplified a highly conserved segment of the IGF-I A domain from salmon liver cDNA. The complete coding sequence, including 175 nucleotides (nt)of the 5' untranslated region and 1317 nt ofthe 3' untranslated sequence, was then obtained by screening a liver cDNA library. The deduced sequence of coho salmon preproIGF-I reported by Cao et al. contained 176 amino acids (aa) and consisted of a 44-aa signal peptide, 70-aa mature IGF-I, and 62-aa E peptide. In addition to coho, preproIGF-I cDNA or gene sequences have been reported for Atlantic salmon (Duguay et al., 1992), chinook (Wallis and Devlin, 1993),chum (Kavsan et al., 1993), and rainbow trout (Shamblott and Chen, 1992). The salmonid preproIGF-I sequences are all very similar to each other, although multiple E peptides are generated via alternative RNA splicing. Amino acid sequence comparisons indicated that preproIGF-I has been highly conserved between teleosts and mammals (Fig. 1).Thus, like mammalian preproIGF-I, the salmon precursor contains an unusually long signal peptide that is 44% identical with the human signal peptide. In the mature hormone, salmon IGF-I is identical to human IGF-I in 56 out of 70 aaand within the structurally important B and A domains the identity is 90% (45 of 50 aa). The sequence similarity between the E domains is also quite high, being 77% identical (27 of 35 aa) if a 27-39 aa internal segment in the salmon E peptide is deleted. In humans and rodents, two IGF-I precursors, designated proIGFIA and proIGF-IB, are synthesized by alternative RNA processing that generates different carboxyl-terminal E peptide sequences (Daughaday and Rotwein, 1989). Alternative splicing of the IGF-I mRNA also occurs in salmonids. Using reverse transcriptase-linked PCR (RTPCR), Duguay and co-workers (1992) demonstrated that at least three IGF-I transcripts were expressed in different tissue RNAs isolated from coho and Atlantic salmon. One transcript, which they labeled proIGF-1A-1, encoded a 35-aa E peptide, which is the same length as found in human proIGF-IA. A second transcript, proIGF-1A-2, encoded a 62-aa E peptide that contained the 27-aa insert as reported by Cao et al. (1989). The third transcript, proIGF-1A-3, encoded a 74-aa E peptide in which the 27-aa insert was followed by an additional

216

S H U J I N C H A N AND D O N A L D F. S T E I N E R

Human Coho Chum Chinook Trout Human Coho Chum Chinook Trout Human Coho Chum Chinook Trout Human Coho Chum Chinook Trout

Fig. I. Comparison of human and salmonid preproIGF-I. 1G . preciirsoi- scquences from coho (Cao et ul., 1989; Duguay et ul., 1992),chinook (Wallis and Devlin, 199:3), and chum (Kavsan et ul., 1993) salmon are aligned with human preproIGF-IA (Jaiisen et u l . , 1983).T h e asterisk indicates the position where an additional 0-39 aa E peptide segment in salmon is inserted by alternative RNA splicing.

l2-aa segment. As shown in Fig. 1,the salmon E peptide sequences are all clearly homologous to human proIGF-IA. A homologue to human or rodent proIGF-IB sequences has not been identified in teleosts. The findings of Duguay and co-workers have been confirmed and extended by Wallis and Devlin (1993) in their analysis of IGF-I transcripts in another salmonid species, the chinook salmon. Using PCR, these workers showed that the 27-aa E peptide insert was produced by utilizing an alternative intron donor site located downstream within the same exon, whereas the 12-aa insert was encoded by a distinct 36lip exon in the salmon IGF-I gene. They also identified a fourth IGFI transcript that encoded a 47-aa E peptide. Wallis and Devlin demonstrated that chinook salmon contained two nonallelic IGF-I genes. This is consistent with the fact that salmonid species are partially tetraploid (Allendorf and Thorgaard, 1984). Two IGF-I genes have also heen reported in the frogxenopus laevis, which is also tetraploid (Shuldiner et al., 1990). However, it is not known whether expression of these genes is regulated differently.

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STRUCTURE AND FUNCTION OF INSULINLIKE GROWTH FACTOR

217

A possible functional role for the IGF-I E peptides in salmon has not been demonstrated. Siegfried et al. (1992) reported that a fragment of the E peptide derived from human proIGF-IB appeared to bind to specific receptors in bronchial epithelial cells and had mitogenic activity. However, as noted earlier, the homologous proIGF-IB sequence has not been found in teleosts. Alternatively, Duguay and coworkers (1992)have noted that the E peptides contain a preponderance of basic acids to yield calculated PIS in the 11.0-13.0 range. It is possible that this characteristic basic charged peptide may play a role in facilitating the correct folding of proIGF-I or possibly in regulating posttranslational processing. The nucleotide sequence of the IGF-I gene from chum salmon has been reported b y Kavsan and co-workers (1993). The cloned gene spans over 22,000 b p and is organized into four exons (Fig. 2). Mature IGF-I is encoded in exons 2 and 3 , and the E peptide coding sequence is split between exons 3 and 4. Interestingly, the chum salmon IGFI gene contains a point mutation in exon 3, which precludes RNA splicing to generate the short proIGF-1A-1 transcript that is found in Atlantic, coho, and chinook salmon. In addition, the small exon that encodes the 12-aa E peptide insert has been shown to be located

L

Fig. 2. Organization of the salmon IGF-I gene. Exons represented by open boxes indicate untranslated regions and shaded boxes contain coding sequences. Four transcripts generated by alternative RNA splicing are shown.

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SHU JIN CHAN A N D DONALD F. STEINER

between exons 3 and 4 in chinook salmon IGF-I gene (Wallis and Devlin, 1993),but has not been identified in chum salmon. Interestingly, the putative 5' untranslated sequence in chum salmon IGF-I gene was shown to share considerable identity with the 5' untranslated segments from human and chicken IGF-I mRNAs (Fig. 3 ) . In evolutionary comparisons the 5' and 3' untranslated regions of RNA transcripts are often the most divergent. This unusual conservation of noncoding nucleotide sequence suggests that this region may have a regulatory function, perhaps as a site for interacting with growth hormone-related factors that increase the expression of IGF-I. The sequence similarities observed between mammalian and salmonid IGF-I cDNAs strongly suggested that teleosts might also contain a homologue to IGF-11. Shamblott and Chen (1992)have characterized two I G F cDNAs cloned from rainbow trout. One clone was 98% identical to coho salmon preproIGF-I; however, in the second cDNA

1

Human Rat Chicken

salmon Human Rat Chicken Salmon

20

40

GO

MGGG-----AAAAAIUTGCTTCTGTGCTCTAGTTTTAAAATGCAAAGGTATGATGTATTTGTCACCAT AAGGGGGGGAAAAAAA4CGCCTCTGTGCTCCAGTTmAAAAATTTGTCA~GG~ A A A G G G G - - A A A A A A T A T G C " C T G T G C T C T A G T T T T C A C C A T AAG---------------------------------AAAAGTATAAATGATGACGTATTTGAATATGT

80 100 * 120 * 140 GCCCAAAAAAGTCCTTACTCMTMCTTTGCCAGMGAGGGAGAGAGAGAGMGGC~TGTTCCCCCAG GCCCAAAAAAGTCCTTACTCGATMC~TGCCAGMGAGGGAGAGAGAGAGMGGCGMTGTTCCCCCAG GCCCAAAAAAGTCCTTACTCGGTM~GCCAGMGAGGGAGAGAGAGAGMGGCAAATGCTCCCCCAG

GCCCAAAA---TCCTTMTGMTM-TTT--------AGGATGAG---GAGMGGC~TGCTGCCCCAG

Chicken Salmon

160 180 200 CTGTTTCCTGTCTACAGPGTCTGTGTTTTGTAGATAGATAAATGTGA~-ATTTTCTCT--~TCCCTCTTCT CTGTTTCCTGTCTACAGM;TCTGTGmTGTAGATAAATGTAGATAAATGTGAffi-A~CTCT--~TCCCTC~CT CTGTTTCCTGTCTACAGKTCTGTGT~ATGTAGATAAATGTGAGG-ATITCTCT- -AAATCCCTCTTCT CTGTTTCCn;TTGAAAATGTCTGAGTM~TAGATACA~TGA~ATITCTCTCT~TCCGTCTCCA

Human Rat Chicken Salmon

GTTTGCTAAATCTCACPGTCCTGCTAAATTCAGAGCAGA~GATAGAGCCTGCGCMTGGM-TAAAGTCCTC GC~AMTCTCACn;TCGCTGCTAAATTCAGACCAGACCAGATAGAGCCTGCGCMTCGAAAT~GTCCTC G'RTGCTAAATCTCACPGTCACTGCTAMATCAGAGCAGATAGAGCCTGCGCMTGGAA-TAAAGTCCTC GTTCGCTAMTCTCAeP-TC--T-CCMMCGAGCCPGCGAGC~CG----GC~GCMTGGMC-AAAGTCG-G

Human Rat Chicken Salmon

CAAAATTGAAATGTGACATTGCTCTCAACATCTCCCATCTCCCAT~CTG--GA~CTTTTTGCTTCATTATTCCT CAAAATXAMTGTGACITTGCTCT-MCATCTCCCATCTCTCKGATT-C"TTTGCCTCATTATTCCT CMTATEAAATGTGACATTGCTCTCAACATCTCACATCTCTCTGGAlTl'CTl"l'l"T€-TCATCA~ACT GMTATTGAGATGTGACATTGCCTGCATCTTATCCACITTCTCACTG~MTGA~CAAACMG~

Human Rat Chicken Salmon

GCTMCCMTTCATTlTCAGACTPTGTACTT--CAGMGCA&XGGGAAAAATCAGCAGTC-TTCCAACCC

Human R a t-

220

240

260

300

280

360

340

320

380

400

GCCCACCM~CAlTl'CCAGACTTTGTACTT--CAGMGCG~~TCAGCAGTC-TTCCMCTC GCTAACAAATTCATTTCCAGACTlTXACTITTAAGMGCA&EG~TCMCAGTC-~CMCAC

CATlTTTGCM;GGGCT'ITGTCGG~AGACCCGT-GGG-~T-----CT-AGCGGTCA~Cl'TC-C

Fig. 3. A I i g i i i r i r i i t of thc. p i i t ; i t i \ . c . 5' i i i i t r . i i i s h t r d d i i d f l m k i i i g r c * . x i o i i \ o t \ . c i t c . l i r . i t c * IGF-I gc'iic's. Secliic~iiccsl o r I i i i i i i ~ i i ~rat. . ctiickchir, a i i t l chum s a l i l w i i l(;F-l g t ~ .IN. i ~ >IIIJ\VII(lia\.saii ef u l . , 1UY.3\. 'l'hc t r m i . ; l i i t i o i i start >ite h r i i i c t h i o n i i i c . i s i i i i t l c . r l i r i c * t l . :\stc-risk.; indicate, t r a i i . ; c . r i p t i o i i start h i t v s clc.tc.riiiinc.tl l o r t h c c d i i c k e i i 1c;F-l C C I I C * .

~

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7.

STRUCTURE AND FUNCTION OF INSULINLIKE GROWTH FACTOR

219

a 642-nucleotide open reading frame encoding a 214-aa precursor was obtained. The deduced sequence of mature IGF in this clone was 82% identical to human IGF-11. Additional sequence homology was found when the trout E peptide was compared to human proIGF-I1 E peptide (Fig. 4). However, the sequences were not as well conserved as was the case for human and fish proIGF-I E peptides. The gene organization for rainbow trout IGF-I1 has not yet been reported. Nonetheless, these results indicate that teleosts contain homologues to both IGF-I and -11. In more primitive fish, an IGF cDNA has been cloned from the Atlantic hagfish, which is a representative agnathan or jawless vertebrate (Nagarnatsu et al., 1991).Although the signal peptide was incomplete, predicted sequences for a 71-aa mature IGF and 30-aa E peptide were obtained. Sequence comparisons revealed that hagfish IGF was 60% identical to either human IGF-I or -11, but no significant homology was found when the E peptides were compared. The lack of a specific sequence bias in hagfish IGF toward either IGF-I or -11 raised the possibility that it is descended from a putative common ancestral IGF gene that subsequently duplicated to form the IGF-I and -11 genes. At present, IGF genes in species more primitive than hagfish have not been identified and it has been proposed that IGF may have emerged early in vertebrate evolution from a hybrid insulin/IGF gene, similar to the gene found in amphioxus (Chan et al., 1990).

Signal peptide Human Trout

MGIPMGKSMLVLLTFLAFASCCIA MERQRKHEYklSVCHTCRRTENTRMKVKM-SS-NR--VIA--LTLTY-V

Human Trout

AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRV*SRRS*'R

0 domain

Human Trout

C domain

EVASA----------A-----E---------T--SN----QNA domain D domain GIVEECCFRSCDLALLETYCATPAKSE -------------N---Q---K-----

E peptide

Human Trout

RDVSxxx**~xXTPPTVLPDNFPRYPVGKFFQYDTWx~QST~RL~RGLP ----ATSLQIIPMV--1KQ-VPRKHVTV-YSK-EA-Q-KAA------V-

Human Trout

ALLRARRGEVLAKELEAFREAKRHRPLIALPTQDPAHGGAPPEMASNRL -I----KFRRQ-VKIKAQEQ-MF-----T--SKL-PVLPPTDNYV-HN Fig. 4. Comparison of human and trout preproIGF-I1 sequences.

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SHU TIN CHAN AND DONALD F. STEINEK

IV. EXPRESSION AND REGULATION OF IGF In mammals the expression of IGF-I and -11 genes is regulated both temporally and in a tissue-specific manner. IGF-I1 is expressed in multiple tissues predominately during embryogenesis. In contrast, IGF-I is also expressed in many tissues and during embryogenesis but is synthesized predominately in the postnatal liver under the control of growth hormone. The expression of IGF-I in teleosts appears to follow a similar pattern. In coho salmon, IGF-I mRNA has been detected by the RTPCR technique in embryos and in multiple tissues from juvenile and adult fish, including adipose tissue, brain, heart, kidney, liver, muscle, ovaries, testes, and spleen (Duguay et al., 1992). Ribonuclease protection assays showed that the IGF-I mRNA level is highest in the liver (Duan e t al., 1993). As stated previously, multiple IGF-I transcripts were found in all tissues owing to alternative RNA splicing. However, the structure of the secreted hormone is unaffected because the coding sequence for mature IGF-I is identical in all the transcripts. There is also good evidence that growth hormone regulates the expression of IGF-I in teleosts. Cao et al. (1989) showed that the injection of bovine growth hormone into coho salmon induced a sixfold increase in liver IGF-I mRNA level. In contrast, Duan et al. (1993) and Duguay et al. (1994) showed that the relative amount of IGF-I mRNA in heart, fat, brain, kidney, ovaries, and spleen was not affected b y growth hormone. They also found that two other pituitary hormones, prolactin and somatolactin, had no effect on the hepatic IGF-I mRNA level. In salmonid fishes, growth hormone appears to facilitate the adaptive osmoregulation from seawater to fresh water and this action is independent of its effects on somatic growth (Bolton et al., 1987). Sakamoto and Hirano (1993) reported that the osmoregulatory action of growth hormone may be mediated by an increased IGF-I mRNA level in gills and body kidney in rainbow trout. However, it should be noted that the effect was relatively small in that the stimulated IGF-I mRNA levels in kidney and gills were still 20 times less than that found in liver. I n contrast to IGF-I, a teleost IGF-I1 mRNA has only recently been cloned from rainbow trout and little has been reported on its expression. Shamblott and Chen (1992) indicated that liver RNA isolated from rapidly growing juvenile rainbow trout contained higher levels of IGF-I1 than IGF-I mRNA based on Northern blot analysis.

7.

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221

This is similar to the situation found in adult hiinian liver, which expresses high levels of IGF-I1 mRNA, whereas adult rats and mice contain very low levels of hepatic IGF-11. The expression of IGF-I1 in other teleost tissues has not been reported. In the hagfish, Nagamatsu et al. (1991) investigated the tissue expression of I G F mRNA using RNA blot analysis and the more sensitive RT-PCR technique. IGF mRNA was detected in hagfish liver, but not in brain, heart, muscle, or islet tissue. Since growth hormone has not been identified in hagfish, it is not known whether I G F expression is regulated by this hormone. Although the restricted tissue expression of IGF was surprising, it should be noted that only RNAs isolated from adult hagfish tissues were assayed. It is possible that the IGF mRNA may be expressed in different hagfish tissues in the younger larval stage. To further investigate the structure and regulation of IGF in agnathans, we have recently cloned an I G F cDNA from the sea lamprey Petromyzon murinus and assayed for tissue expression by RT-PCR. Preliminary analysis indicates that lamprey I G F mRNA is expressed predominately in the liver but it can also be found in other tissues, notably brain and pancreas (S. J. Chan and J. Youson, unpublished results, 1994).

V. SUMMARY AND PERSPECTIVE The IGFs have long been suspected of playing important roles in promoting tissue growth and development. The recent results obtained with “knock-out’’ mice models have now provided definitive evidence that this is, indeed, the case in mammals. Transgenic mice with null mutations in IGF-I or -11 expressed deficiencies in normal growth, whereas double mutants as well as mice with a mutation in the type I IGF receptor were found to be nonviable (DeChiara et al., 1990; Liu et al., 1993).The identification of I G F genes in teleosts and agnathans, reviewed here, extend these studies and filrther suggests that I G F may play an essential developmental role in all vertebrate species. To define this role, however, it is important to further characterize the physiology and biological actions of IGF in fish. In particular, the relative expression of IGF-I and -11 teleosts during embryogenesis and their actions in specific tissues need to be clarified. It would also be interesting to determine whether primitive vertebrates, such as hagfish and lamprey, contain only a single IGF gene.

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ACKNOWLEDGMENTS We thank Florence Rozenfeld for expert assistance in preparing the manuscript. Research from our laboratory was supported by the Howard Hughes Medical Institute and USPHS Grants DK13914 and DK20595.

REFERENCES Allendorf, F. W., and Thorgaard, G. H. (1984).Tetraploidy and the evolution of salmonid fishes. In “The Evolutionary Genetics of Fishes” (B. J. Turner, ed.), pp. 1-53. Plenum, New York. Bautista, C. M., Mohan, S., and Baylink, D. J. (1990).Insulin-like growth factors I and I1 are present in the skeletal tissues of ten vertebrates. Metabolism 39, 96-100. Bolton, J. P., Young, G., Nishioka, R. S., Hirano, T., and Bern, H. A. (1987). Plasma growth hormone levels in normal and stunted yearling coho salmon, Oncorhynchus kisutch. /. Erp. Zool. 242, 379-382. Bowsher, R. R., Lee, W.-H., Apathy, J. M., O’Brien, P. J., Ferguson, A. L., and Henry, D. P. (1991). Measurement of insulin-like growth factor-I1 in physiological fluids and tissues. Endocrinology (Baltimore)128, 805-814. Cao, Q.-P., Duguay, S. J., Plisetskaya, E. M., Steiner, D. F., and Chan, S . J. (1989). Nucleotide sequence and growth hormone-regulated expression of salmon insulinlike growth factor I mRNA. M o l . Endocrind. 3, 2005-2010. Chan, S. J., Cao, Q.-P., and Steiner, D. F. (1990).Evolution ofthe insulin superfamily: Cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus. Proc. Natl. Acad. Sci. U.S.A. 87, 9319-9323. Chan, S. J., Nagamatsu, S., Cao, Q,-P., and Steiner, D. F. (1992). Structure and evolution of insulin and insulin-like growth factors in chordates. Prog. Brain Aes. 15-24. Chen, T. T., Marsh, A,, Shamblott, M., Chan, K. M., Tang, Y. L., Cheng, C. M., and Yang, B. Y. (1994).Structure and evolution of fish growth hormone and insulinlike growth factor genes. In “Fish Physiology” (N. Sherwood and C. Hew, eds.), Vol. 13, Chapter 6 . Academic Press, San Diego. Cohick, W. S., and Clemmons, D. R. (1993).The insulin-like growth factors. Annu. Rec. Physiol. 55, 131-153. Daughaday, W. H. (1972). Somatomedin: Proposed designation for sulphation factor. Nature (London)235, 107. Daughaday, W. H., and Rotwein, P. (1989). Insulin-like growth factors I and 11. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocr. Reu. 10,68-91. Daughaday, W. H., Kapadia, M., Yanow, C. E., Faraick, K., and Mariz, I. K. (1985). Insulin-like growth factors I and I1 of nonmammalian sera. Gen. Comp. Endocrinol. 59, 316-325. DeChiara, T. M., Efstratiadis, A,, and Robertson, E. J. (1990). A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor I1 gene disrupted by targeting. Nature (London) 345, 78-80. Drakenberg, K., Sara, V. R., Lindahl, K. I., and Kewish, B. (1989). The study of insulinlike growth factors in tilapia, Oreochromis mossambicus. Gen. Comp. Endocrind. 4, 173-180.

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Duan, C., and Hirano, T. (1990).Stimulation of=S-sulfate uptake by mammalian insulinlike growth factor I and I1 in cultured cartilages of the Japanese eel, Anguilla japonica. J. E x p . Zool. 256,347-350. Duan, C., Duguay, S. J., and Plisetskaya, E. M. (1993). Insulin-like growth factor I (IGFI ) mRNA expression in coho salmon, Oncorhynchus kisutch: Tissue distribution and effects of growth hormoneiprolactin family proteins. In “Fish Physiology and Biochemistry,” pp. 371-379. Kugler, Amsterdam. Duguay, S. J., Park, L. K., Samadpour, M., and Dickhoff, W. W. (1992). Nucleotide sequence and tissue distribution of three insulin-like growth factor I prohormones in salmon. Mol. Endocrinol. 6, 1202-1210. Duguay, S. J . , Swanson, P., and Dickhoff, W. W. (1994). Differential expression and hormonal regulation of alternatively spliced IGF-I mRNA transcripts in salmon. J. Mol. Endocrinol. 12, 25-37. Funkenstein, B., Silbergeld, A., Cavari, B., and Laron, Z. (1989). Growth hormone increases plasma levels of insulin-like growth factor I (IGF-I) in a teleost, the gilthhead seabream (Sparus aurata).J. Endocrinol. 120, R19-21. Furlanetto, R. W., Underwood, L., Van Wyk, J. J., and D’Ercole, A. J. (1977). Estimation of somatomedin C levels in normals and patients with pituitary disease by radioimmunoassay. J. Clin. Invest. 60, 648-657. Gray, E. S., and Kelley, K. M. (1991).Growth regulation in the gobiid teleost, Gillichthys mirabilis: Roles of growth hormone, hepatic growth hormone receptors and insulinlike growth factor-I. J. Endocrinol. 131, 57-66. Iwami, M., Kawakami,T., Ishizaki, H.,Takahashi, S. Y.,Adachi, R., Susuki, Y., Nagasawa, H., and Suzuki, A. (1989). Cloning of a gene encoding bombyxin, an insulin-like brain secretory peptide of the silkmoth Bombyx mori with prothoraciocotropic activity. Dev. Growth Differ. 31, 31-37. Jansen, M., vanSchaik, F. M. A., Ricker, A. T., Bullock, B., Woods, D. E., Gabbay, K. H., Nussbaum, A. L., Sussenbach, J. S., and Van den Brande, J. L. (1983).Sequence of cDNA encoding human insulin-like growth factor I precursor. Nature (London) 306,609-611. Kajimoto, J., and Rotwein, P. (1989). Structure and expression of a chicken insulin-like growth factor I precursor. Mol. Endocrinol. 3, 1907-1913. Kajimoto, Y., and Rotwein, P. (1990).Evolution of insulin-like factor I (IGF-I): Structure and expression of an IGF-I precursor from Xenopus laevis. Mol. Endocrinol. 4, 217-226. Kajimoto, Y., and Rotwein, P. (1991). Structure of the chicken insulin-like growth factor I gene reveals conserved promoter elements. J. Biol. Chem. 266, 9724-9731. Kavsan, V. M., Koval, A. P., Grebenjuk, V. A., Chan, S. J., Steiner, D. F., Roberts, C. T., Jr., and LeRoith, D. (1993).Structure of the chum salmon insulin-like growth factor I gene. DNA Cell Biol. 12, 729-737. Lagueux, M., Lwoff, L., Meister, M., Gotzent., F., and Hoffnian, J. A. (1990). cDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulins. Eur. J. Biochern. 187, 249-254. Liu, 1.-P., Baker, J., Perkins, A. S., Robertson, E. J., and Efstratiadis, A. (1993). Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igflr). Cell (Cambridge, Mass.) 75, 59-72. McCormick, S. D., Kelley, K. M., Young, G., Nishioka, R. S., and Bern, H. A. (1992a). Stimulation of coho salmon growth by insulin-like growth factor I. Gen. Comp. Endocrinol. 86. 398-406.

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XlcCormick, S. D., Tasi, P. I., Kelley, K ., Yonng, G., Nishioka, K. S., and Bern, H. A. (1992b). Hormonal control of sulfate incorporation in hranchial cartilage of coho salmon: Role of IGF-I. J. E x p . Zool. 262, 166-171. Aloriyama, S., Swanson, P., Nishi, M., Takahashi, A,, Kawauchi, H . , Dickhoff, W. W., and Plisetskaya, E. M. (1993). Development of coho sahnon insulin-like growth factor-I radioimmunoassay. A m . Zool. 33, 11A. Nagarnatsu, S., Chan, S. J., Falkmer, S., and Steiner, D. F. (1991). Evolution of the insulin gene superfamily: Sequence of a preproinsulin-like growth factor cDNA from the Atlantic hagfish. J. B i d . Chem. 266,2397-2402. Rechler, M. M., and Nissley, S. P. (1985). The nature and regulation of the receptors for insulin-like growth factors. Annu. Rec. Physiol. 47, 425-442. Sakamoto, T., and Hirano, T. (1993). Expression of insulin-like growth factor 1 gene in osmoregulatory organs during seawater adaptation of the salmonid fish: Possible mode of osmoregulatory action of growth hormone. Proc. Natl. Acud. Sci. U . S . A . 90, 1912-1916. Shamlilott, M. J., and Chen, T. T. (1992). Identification of a second insulin-like growth factor in a fish species. Proc. Natl. Acad. Sci. U.S.A. 89, 8913-8917. Shuldiner, A. R., Nirula, A., Scott, L. A., and Roth, 3. (1990). Evidence that Xenopus laeuis contains two different nonallelic insulin-like growth factor-I genes. Biochem. Biophysic. Res. Commun. 166,223-230. Siegfried, J. M., Kasprzyk, P. G., Treston, A. M., Mulshine, J. L., Quinn, K. A., and Cuttitta, F. (1992). A mitogenic peptide amide encoded within the E peptide domain of the insulin-like growth factor IB prohormone. Proc. Natl. Acad. Sci. U.S.A. 89, 8109-8111. Smit, A. B., Vreugdemjo, E., Ehberink, R. H. M., Gaeraerts. W. P. M.,Klootwijk, j., and Joosse, J. (1988). Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide. Nature (London)331, 535-538. Upton, Z., Chan, S. J., Steiner, D. F., Wallance, J. C., and Ballard, F. J . (1993). Evolution of insulin-like growth factor binding proteins. Growth Regul. 3, 27-30. Wallis, A. E., and Devlin, R. H. (1993). Duplicate insulin-like growth factor 1 genes in salmon display alternative splicing pathways. Mol. Endocrind. 7, 409-422. Wilson, D. M., and Hintz, R. L. (1982). Inter-species comparison of somatomedin structure using immunological probes. J . Endocrind. 95, 59-64. Zangger, I., Zapf, J., and Froesch, E. R. (1987). Insulin-like growth factor I and I1 in 14 animal species and man as determined by three radiohgand assays and two bioassays. Actu EndocrinoL 114, 107-112.

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES STEPHEN 1.DUGUAY The Howard Hughes Medical Institute and Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois

THOMAS P. MOMMSEN Depaitment of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canadz1

I. Introduction 11. Insulin A. Gene Structure B. Messenger RNA Transcripts and cDNA Sequences C. Peptide Sequences D. Biosynthesis E. Secretion F. Physiological Actions 111. Glucagon and Glucagonlike Peptide A. Gene Structure and cDNA Sequences €3. Gene Expression C . Glucagon Processing and Message Transduction D. Physiological Actions of Glucagon E. Glucagonlike Peptide Processing and Message Transduction F. Physiological Actions of Glucagonlike Peptides G. Conclusion IV. Somatostatin A. Gene Structure B. Messenger RNA Transcripts and cDNA Sequences C . Biosynthesis D. Secretion E. physiological Actions V. Pancreatic Polypeptide and Related Peptides A. The Pancreatic Polypeptide Family B. cDNA and Peptide Sequences 225 Copyright 0 1994 by Academic Presq, Inc. All right* of reproduction i n any fomm rerewed.

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S T E P H E N J. DUGUAY A N D T H O M A S P. M O M M S E N

C. Prohormone Processing D. Immunohistochemical Identification of Peptides E. Physiological Actions References

I. INTRODUCTION As the field of molecular biology has developed and matured in recent years, the repertoire of experimental approaches and techniques available to investigators studying piscine systems has been expanded enormously. Many laboratories engaged in fish research are now utilizing molecular methods, and others will certainly adopt these techniques in the future. The purpose of this chapter is to review the current knowledge of the molecular biology of insulin, glucagon, glucagonlike peptide, somatostatin, and pancreatic peptide in piscine systems. Gene and messenger RNA structures will be emphasized when available. Other molecular aspects that are particularly well characterized in piscine systems, such as biosynthesis, or exciting developments such as glucagon receptor studies or structure-function relationships of glucagons and glucagonlike peptides (GLPs) will also be discussed. It is hoped that this review will be a useful resource for those investigators wishing to initiate research and expand our knowledge on molecular aspects of pancreatic peptides in fish.

11. INSULIN A. Gene Structure The chum salmon (Oncorhynchus keta) insulin gene has been shown to consist of three exons separated by two introns with a total length of approximately 1560 base pairs (bp) (Fig. 1). Exon 1 codes for most of the 5' untranslated (5' UT) region. Exon 2 codes for the remaining 5' UT region as well as the signal peptide, B-chain, and first six amino acids of the C-peptide. Exon 3 encodes the remainder of the C-peptide as well as the A-chain and 3' UT region. Intron 1 occurs in the 5' UT region and is 393 b p long. Intron 2 interrupts the codon for the seventh amino acid of the C-peptide and is 287 bp in length (Koval et al., 1989a,b).This gene structure has been remarkably well conserved during evolution. In fact, with the exception of the special case of the rat I and mouse I insulin genes, all known vertebrate insulin genes consist of three exons separated by two introns. The

8.

227

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES Insulin Gene Structure

\

\

\ \

Insulin mRNA

SP

B

I

C

A

Fig. 1. Structure of salmon insulin gene, insulin mRNA, and preproinsulin. Exons are indicated by numbered boxes and introns 1 and 2 are represented by thin lines connecting exons. The TATA box and putative Nir and Far boxes are shown in the 5’ end of the gene. Dashed lines indicate the exon splicing pattern used to generate the insulin mRNA. The start codon (AUG) and poly-A tail ofthe mRNA are labeled. Regions ofthe mRNA coding for the signal peptide (SP) and B-, C-, and A-chains are indicated. The processing sites for preproinsulin are labeled by an open arrow for the site of signal peptide cleavage and solid arrows for cleavage are dibasic residues to remove the Cpeptide.

positions of the introns have been highly conserved as well, interrupting the 5' UT region and the C-peptide in all cases (Chan et ul., 1992). Even the amphioxus (Brunchiostoma californiensis) insulinlike peptide gene structure is similar, with an intron interrupting the seventh codon of the C-peptide (Chan et al., 1990). Considerable allelic polymorphisms can be found in chum salmon gene sequences. Kashuba et al. (1986) identified 16 point mutations in the untranslated and translated regions of three cDNA clones. Also, the gene sequence reported by Koval e t al. (1989b) differs by more than 30% in the 3' UT region with the chum salmon cDNA sequence reported by Sorokin et al. (1982),suggesting the presence oftwo genes. It has been shown by genomic analysis with Southern blotting and polymerase chain reaction (PCR) that there are indeed two insulin genes in this species, probably as a result of chromosomal duplication (Kavsan et al., 1993).

228

STEPHEN J. DUGUAY A N D THOMAS P. MOMMSEN

B. Messenger RNA Transcripts and cDNA Sequences Insulin cDNAs have been cloned from anglerfish (Lophius ainericanus), hagfish (Myxine glutinosa), chum salmon (Oncorhyncus kitsutch), and carp (Cyprinus carpio) (Hobart et al., 1980b; Chan et al., 1981; Sorokin et al., 1982; Hahn et al., 1983). Sequence analysis indicates that the insulin molecule has been well conserved during evolution. For instance, the B- and A-chains of hagfish insulin share 65% amino acid sequence identity with human counterparts. In contrast, C-peptides vary greatly in sequence and length, but are always flanked b y dibasic residues that serve as processing sites (see Fig. 1 and following). Fish preproinsulin signal peptides also differ considerably in amino acid sequence but retain features considered essential for function, such as a positively charged amino terminus and a hydrophobic core. Northern blot analysis of Brockmann body RNA from anglerfish, hagfish, and salmon indicates a single mRNA transcript of 840 nucleotides (nt), 1050 nt, and 760 nt, respectively, for insulin of each species (Hobart et al., 1980b; Chan et al., 1981; Sorokin et al., 1982).

C. Peptide Sequences Insulin peptides have been purified and sequenced from over 60 species of vertebrates. All have a two-chain structure consisting of a B-chain of approximately 30 residues linked to an A-chain of about 21 amino acids by two cystine bridges. An intramolecular A-chain disulfide linkage has also been found in all insulin molecules. A total of' 17 amino acid residues have been identified as invariant (Chan et al., 1992).A comparison of insulin sequences from representative teleosts, holocephalans, elasmobranchs, and agnathans can be found in Mommsen and Plisetskaya (1991).

D. Biosynthesis 1. REGULATIONOF SYNTHESIS a. Gene Transcription. The promoter region of the salmon insulin gene contains a standard TATA box transcription initiation site at -34 bp (Fig. 1) (Koval et al., 1989b). Although promoter analysis has not been conducted on fish insulin genes, mammalian insulin promoters have been studied extensively and binding sites for both

8.

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES

229

positive and negative regulatory factors have been identified. Two important enhancer elements that have been identified are the Nir and Far boxes, also referred to as I E B l and IEB2, which confer celltype specific expression of the insulin gene. The Nir and Far box sequence CANNTG appears to define the binding site for members of the basic helix-loop-helix family of transcription factors (Moss et al., 1988; Karlsson et al., 1989; Aronheim et al., 1991; German e t al., 1991; Clark and Docherty, 1992). Inspection of the chum salmon insulin gene sequence reveals potential Nir and Far motifs at -124 bp and -143 bp of the promoter region (Fig. 1).

h. Protein Translation. In general, translation of proteins destined for the secretory pathway is initiated by ribosomes at an AUG codon of the mRNA and proceeds until the nascent signal peptide interacts with the signal recognition particle (SRP), leading to SRP-mediated translational arrest. The SRP-signal peptide complex then interacts with SRP receptors on the surface of the rough endoplasmic reticulum (RER),at which point translation resumes and the nascent polypeptide is translocated into the lumen ofthe RER. Translation initiation, elongation, and SRP-mediated arrest have been shown to be important regulatory points in the biosynthesis of proinsulin in mammalian systems and can be influenced by glucose and other nutrients (Itoh, 1990; Steiner, 1990). In experiments on hagfish ( M y x i n e glutinosa), neither glucose nor amino acids stimulated proinsulin biosynthesis (Emdin and Falkmer, 1977). 2. CONVERSION OF PREPROINSULIN TO INSULIN a. Signal Peptide Cleavage. Detailed studies using anglerfish (Lophius americanus) and sea raven (Hemitripterus americanus) Brockmann body mRNA in cell-free translation systems have shown that an 11.5-kDa protein can be immunoprecipitated by anglerfish insulin antisera. When cell-free translation is performed in the presence of pancreatic microsomes containing signal peptidase activity, the signal peptide is cleaved after 23 (anglerfish) or 25 (sea raven) residues to produce proinsulin (Shields and Blobel, 1977). Likewise, a 12- to 14-kIla protein that is immunoreactive with carp insulin antisera was produced in a cell-free translation system utilizing carp ( Cy p r i n u s carpio) Brockmann body mRNA (Rapoport et al., 1976). This preproinsulin molecule was converted to the 9-kDa proinsulin molecule during in uitro translation with pancreatic microsomes (Prehn e t al., 1980).

230

STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN

b. Endoproteolytic Removal of C-Peptide. Proinsulin is the biosynthetic precursor of insulin and has the primary structure NH,-Bchain-C-peptide-A-chain-COOH. Inter- and intrachain disulfide bonds are formed after proinsulin enters the endoplasmic reticulum. Proinsulin is then transported through the Golgi compartment and packaged into secretory granules, where it is converted to insulin. Conversion to mature insulin is accomplished by endoproteolysis of dibasic residues at the B-chain/C-peptide and C-peptide/A-chain junctions (Fig. 1)(Steiner, 1990). This cleavage is mediated by the prohormone convertases PC2 and PC3 (also known as PC1). PC2 and PC3 are calcium-dependent serine proteases that cleave only at dibasic residues (Steiner et a/., 1992). Cleavage occurs on the carboxyl side of the second basic residue of the pair. The basic residues remaining on the carboxy terminus of the B-chain are then removed by a carboxypeptidase (Steiner, 1990). The available evidence indicates that the proinsulin processing pathway described here, which has been studied extensively in mammals, is also utilized in fish. Proinsulin molecules have been detected in islets of hagfish (Myxine glutinosa), carp (Cyprinus carpio), and anglerfish (Lophius americanus) (Yamaji et al., 1972; Steiner et al., 1973; Lukowsky et al., 1974), and conversion of proinsulin to insulin has been demonstrated in hagfish (Steiner et al., 1973).Also, PC2-like and carboxypeptidaselike processing enzymes capable of converting proinsulin to insulin have been isolated from anglerfish secretory granules (Mackin and Noe, 1987a; Mackin et d., 1991b). An interesting variation on this processing theme may be found in the ratfish, Hydrolagus colliei. Four insulin peptides have been isolated from this species that are probably generated by multiple cleavages of a single proinsulin and/or degradation by carboxypeptidase. The dibasic sequence normally present at the B-chain/C-peptide junction has been replaced by isoleucine-arginine. This cleavage site is apparently still recognized by a processing enzyme in the ratfish islet (Conlon et al., 1989). E. Secretion Secretion of insulin from the secretory granules of islets is stimulated by nutrients and may be modulated by hormones and neurotransmitters. For many fish species, amino acids are more effective than carbohydrates as secretagogues, and these agents often elicit a typical mammalian-type biphasic response. Although amino acids and glucose also stimulate insulin release from mammalian islets, there appear to

8. MOLECULAR ASPECTS

OF PANCREATIC PEPTlDES

231

be important differences in the mechanism of action of secretagogues between fish and mammals. For example, arginine stimulates insulin secretion from mammalian islets indirectly by stimulating glucagon release. In salmon, arginine-stimulated insulin release operates independently of any effect on glucagon (cf. Mommsen and Plisetskaya, 1991). F. Physiological Actions Insulin is the major anabolic hormone in fish. It stimulates the uptake of glucose and amino acids by skeletal muscle and liver and increases the rate of protein synthesis in these tissues. Insulin also acts to suppress hepatic gluconeogenesis and glycogenolysis. In addition to these actions on carbohydrate and protein metabolism, insulin exerts a positive effect on the flux of fatty acids into hepatic lipids. The physiological actions of insulin in fish have been reviewed in detail by Mommsen and Plisetskaya (1991) and Plisetskaya and Duguay (1993).

111. GLUCAGON AND GLUCAGONLIKE PEPTIDE In contrast to the situation just described for insulin and for somatostatin in the following, comparatively little is known about the molecular biology ofpiscine glucagons or glucagonlike peptides (GLP). Generally, for members of the glucagon family of hormones, attention has been focused on evolutionary aspects of peptide occurrence and peptide sequence, sites, and modes ofaction, as well as immunocytochemical localization of production sites, rather than on gene structure, processing, or posttranslational modification. Therefore, to present an overview of the glucagon complex in fishes, we give examples from mammals and other vertebrate groups. We also incorporate specific aspects of physiological and other functions of these peptides, especially where piscine systems offer new and exciting views on aspects of these somewhat neglected pancreatic and gut hormones. Glucagon is the best-known member of a constantly expanding superfamily of peptide hormones that includes secretin, vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP), growth hormone-releasing hormone, peptide histidine methionine, helospectin, helodermin, pituitary adenylyl cyclase-activating peptide (PACAP), PACAP-related peptide and closely related peptides. Many of these have been described for several groups of vertebrates includ-

232

STEPHEN J. DUGUAY AND THOMAS P . hlOhlhlSEh

ing fishes (see Chapter 1, this volume) (Conlon, 1988; Plisetskaya, 1990a; Jonsson, 1991; Parker et al., 1993a). In addition, a number of related peptides are co-encoded in the preproglucagon gene of all vertebrates. These include, starting from the 5' end of the gene, at times with overlapping sequences, glicentin-related polypeptide (GRPP), glicentin, glucagon (preproglucagon 33-61), oxyntomodulin (preproglucagon 33-70), and one or two glucagonlike peptides. Fulllength GLP-1 in mammals spans from residue 72 to residue 108 in the preproglucagon sequences, whereas GLP-2, if present, is from 126 to 159. Glucagon also displays some sequence homology with prealbumin (Jornvall et al., 1981).

A. Gene Structure and cDNA Sequences The human proglucagon gene, deduced from a genomic library, contains a total of six exons separated by five introns (White and Saunders, 1986), a pattern that is found in numerous other mammals (cf. White and Saunders, 1986) (Fig. 2A). Exons 2 to 5 encode the following four regions of the preproglucagon sequence: exon 2 encodes the signal peptide and part of the N-terminal region; exon 3 covers the remainder of the N-terminal peptide and glucagon, as well as the first four amino acids of an intervening peptide; the remaining six amino acids of the intervening peptide and the full-length glucagonlike peptide 1 are encoded by exon 4; and exon 5 covers the intervening peptide between GLP-1 and GLP-2, as well as GLP-2. In mammals, but not necessarily in the fishes (see the following), glucagon and the two glucagonlike peptides show strong sequence conservation and are considered to have arisen from two independent duplications of an ancestral gene coding for glucagon (Lopez et al., 1984). The rat glucago11 gene contains three DNA control elements in the 5' flanking sequence of the glucagon gene: a promoter, which accounts for A-cellspecific expression, as well as two enhancerlike elements (Philippe and Rochat, 1991). The Brockmann bodies of the anglerfish (Lophius americanus) express two nonallelic preproglucagon genes, leading to the production ofthree proglucagon transcripts of630,650, and 670 bases, respectively (Lund et al., 1983), coding for two closely related glucagons plus some related peptides (see the following). The single mRNA with 650 bp was shown to code for the so-called glucagon I, whereas the other two sequences were detected using a probe coding for glucagon 11. It is not known whether the length difference in the two transcripts for gliicagon I1 is due to different genes encoding this glucagon precursor,

8.

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES

233

differential splicing of the mRNA, or the degree of polyadenylation of the 3’ end of the mRNAs. Nevertheless, compared with the proglucagon mRNA transcripts of mammals, which contain around 1300 bases, the fish transcript is small (Fig. 2B). Part of the size difference can be explained through the absence of regions coding for glucagonlike peptide 2 (GLP-2) and the intervening peptide (between GLP-1 and GLP-2) in the fish transcripts, corresponding to exon 5 in mammals (cf. Fig. 2A). Because GLP-2 (34residues), plus the preceding intervening peptide (6 residues), accounts for only about 120 base pairs, the bulk of the size difference between anglerfish and mammal is due to an extended untranslated 3’ sequence in the mammals. The size of the untranslated region in mammals exceeds 400 bases, but amounts to only 186 bases from the stop codon immediately following GLP to the consensus polyadenylation motif AATTAAA at the 3’ end of anglerfish glucagon I. In the case of anglerfish glucagon 11, the same region is only 92 bases long (from stop codon to AATAAA sequence). The role of the comparatively larger untranslated region in the mammals is under debate because deletion of variable amounts of the 3‘ untranslated region (plus all 3’ flanking regions) will produce read-through mRNA transcripts with compromised 3’ ends (Lee and Drucker, 1990). Although these deletions have little effect on the relative amounts of rat proglucagon transcripts measured in BHK fibroblast or islet cell lines, the study by Lee and Drucker (1990) shows quite conclusively that as few as 50 bases of the 3’ flanking region are essential for accurate formation of the glucagon mRNA 3’ end and polyadenylation. In all teleostean and elasmobranch fishes analyzed to date, peptides corresponding to GLP-2 have not been found (Fig. 2B). This holds true for peptides isolated from Brockmann bodies or the intestinal tract of numerous species of fishes as well as for the preproglucagon cDNAs analyzed for the anglerfish. The anglerfish cDNAs from proglucagon gene I and gene I1 contain stop codons immediately following the sequence of GLP. Interestingly, the intron connecting exons 4 (GLP-1, see earlier) and 5 (GLP-2) in preproglucagon genes of mammals (human and rat) contains a stop codon (Bell, 1986). Therefore it can be imagined that faulty processing of this intron, that is, retaining this stop codon, would result in glucagon precursors lacking peptides corresponding to GLP-2. Unfortunately, to date an analysis of fish genomic DNA to support or refute this idea has not been published. A similar situation may exist in the birds, which also appear to lack GLP-2, but in contrast to the fishes, the chicken contains an exceptionally long intervening peptide (28 amino acid residues, including the

234 STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN h

8.

235

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES

B 3 UT

KR

Signal peptide

RK

Glucagon

RR

KR

KR

IP

UT

GLP

Fig. 2. (A) Structure of human preproglucagon gene and proglucagon mRNA. Exons are indicated by numbers. Modified from Bell (1986) and White and Saunders (1986). Processing sites (K or R) are indicated by vertical bars. For reasons of clarity, the dibasic (RR) processing site within glucagon is indicated by the amino acids only. Glucagon is stippled. The N-terminal region of GLP-1, which is removed to produce the biologically active form, is indicated by striping. IP, intervening peptides. (2B) Structure ofanglerfish (Lophius arnericanus) proglucagon. Modified from Lund et al. (1983) and Andrews and Ronner (1985). Abbreviations as in Part A. UT, untranslated regions. Numbers on top of the translated box indicate residue number of the proglucagon.

two flanking, dibasic processing sites) joining glucagon and GLP-1 (Hasegawa et al., 1990). The presence of GLP-2 in all vertebrates, excepting fishes and birds, appears to be a bit of an enigma. Whenever present, the sequence of GLP-2 is highly conserved, which is usually taken as an indication of increased evolutionary pressure and important physiological function. Alas, to date no clear functions and/or targets for GLP-2 have been identified, with the exception of the apparent ability of GLP-2 to regulate DNA synthesis under specialized conditions (Lund et al., 1993) and to stimulate adenylyl cyclase in rat hypothalamus and pituitary (Hoosein and Gurd, 1984).Future research will undoubtedly charify whether these activities constitute generalized features of GLP-2 peptides in higher vertebrates (except birds). In spite of the strong sequence homology with GLP-1 (cf Table I11 for the human GLPs), GLP-2 neither binds to the GLP-1 receptor nor interferes with the binding of GLP-1 or glucagon to their respective membrane receptors. In spite of the many similarities in proglucagon cDNA between mammals and fishes, there are a number of differences in the processing and sequence of GLP-1 (mammals) or piscine GLP. Exon 4 of the mammalian proglucagon gene encodes a short intervening peptide of 10amino acids, followed by the full-length glucagonlike peptide 1 with 37 residues. However, during posttranslational processing at sites removed from the endocrine pancreas, inactive GLP-1 is cleaved at Arg' to produce a biologically highly potent truncated GLP-l(7-37) (tGLP). Therefore, the exons encode a 16-residue spacer (a 10-residue intervening peptide and a 6-residue N-terminal truncation) between functional glucagon and functional (truncated) glucagonlike peptide

236

STEPHEN J. DUGUAY AND THOMAS P . MOMMSEN

Table I Comparison of Base and Amino Acid Sequences of' Intervening Peptide between Human Glucagon and GLP-1 and Anglerfish Gliicagon and GLP" 0.ryntomodulin

1 Intervening peptide

N-terminal esten\ion of GLP-I

GLP

C A C GAT GAA TTT GAG ACA

CAC His'

Arg

G AAT AAC AcnAsn

ATT GCC I AAA CGT IleAld LysArg

An)rlerfish I

AGC Ser

GGT GTC; GI) Val

GCA GAA AAG CGT A l a C l u Lys A r g

N o t present

c 4c

Anylrrfi\h I1

AAT Asn

GGT TTA GlyLeu

T T T - - - AGA CGC Phe - - - A r g A r g

Uot p r r w n t

C.4T Hi\'

Humm

AG

I

H I \ AspGlnPheGInAiy

HIS'

B a w nratches human \.erru* anglerfish intervening peptide. ,Anglmfidi 1

**

.Atipl?rfi\h I 1

*

* *

* I *

1

* I

*

***

* **

" 1 Indicates the 3' e n d of exon 3. 1 signifies the C-terminal end of oxyntomodulin, the extended form of glucagon (glucagon 1-37). Modified from White and Saunders (1986) and Lund et al. (1983).

1. In the anglerfish proglucagon cDNAs, the intervening peptide is even shorter (gene I: 7 residues; gene 11: 6 residues), but apart from the two basic amino acids critical for processing (Lys-Arg),little homology is discernible between fish and mammalian intervening peptides. Since the time that the existence of glucagonlike peptide(s) was first demonstrated from the cDNAs of anglerfish and mammalian proglucagon genes, the sequences for numerous GLPs have been published for fishes, including cyclostomes, elasmobranchs, and teleosts. In all cases, a sequence corresponding to the 6-residue N-terminal extension of the mammalian GLP-1 was absent (cf. Table I). It is removal of this extension that appears to be critical in developing the insulinotropic activity of GLP-1 in all mammalian (and amphibian) systems; this proteolytic processing does not occur at the site of GLP-1 production. Again, if the intervening peptide found in the two anglerfish glucagons can serve as a general example for the situation in fishes, few similarities, if any, exist between the intervening peptide and the amino acids truncated from the N-terminal His' of the biologically active GLP-1. The notion that this situation has wider application finds full support in amino acid sequences described for numerous species of teleostean fishes. Homology of fish GLPs with mammalian GLP-1 is greatest if aligned with His' of the truncated mammalian peptide (Table 111).

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237

B. Gene Expression In the rat, as in most other mammals analyzed to date, the glucagon gene is a single-copy gene that is expressed in a selective, cell-specific manner. The gene is expressed preferentially in the A-cells of the endocrine pancreas in the L-cells of the intestine, and in selected neurons of the brain. These tissues produce a single identical transcript. As demonstrated for the rat, expression differs substantially between tissues owing to processing at different dibasic amino acid processing sites. Intestinal L-cells, on the one hand, produce glicentin, oxyntomodulin (glucagon, 1-37), and GLP-1 and GLP-2, but not glucagon. The postulated, intestine-specific element for the glucagon gene has been located between -2000 and -1300 upstream from the start of transcription. In addition, the glucagon gene promoter has been located in an area together with numerous cis-acting domains. On the other hand, glucagon, full-length GLP-1, and a proglucagon fragment prevail as products of proglucagon processing in mammalian pancreatic tissue. Proglucagon and the major proglucagon fragment encompassing the two GLPs in mammals can be retained on lectin columns, suggestive of the fact that these peptides could occur in glycosylated forms (Patzelt and Weber, 1986). A number of fishes possess two proglucagon genes, which are nonallelic in the case of the anglerfish. Without invoking differential processing, at least five species (gar, anglerfish, daddy sculpin, eel, flounder) express two closely related glucagons and glucagonlike peptides (Tables I1 and 111).The similarities of glucagon sequences within the fishes and between elasmobranches and mammals have been used to develop an unrooted phylogenetic tree (Cutfield and Cutfield, 1993). As pointed out in the lower part of Table 11, the glucagon sequences of the primitive cartilaginous fishes reveal greater similarities to human glucagon than those of the other fishes. This fact has been interpreted to indicate a higher rate of molecular evolution of the gene in the teleosts than in other vertebrate groups. This accelerated rate of evolution is linked to the morphological development of the endocrine pancreas into the well-defined endocrine Brockmann bodies in these animals (Conlon and Thim, 1985). Tissue-specific expression in fishes differs from the mammalian picture in several ways. First, fish pancreas releases only one form of GLP, corresponding to the truncated GLP-1. Second, proglucagon gives rise to glucagon in gut tissue, at least in the dogfish (Scyliorhinus canicula). Little evidence has been found for the presence ofglicentinlike peptides in either tissue. Third, considering the gene structure

238

STEPHEN J. DUGUAY A N D THOMAS P. MOMMSEN

Table I1 Primary Structures of Fish Glucagons" Cvclostome 5

10

15

HSEGT

FTSDY

SKYLE

20 NKQAK

25 DFVRW

20 LMNA

HSEGT HSEGT

FTSDK FTSDY

SKYMD SKYLD

NRRAK NRRAK

DFVQW DFVQW

LMST LMNT

HTDGI HSEGT

FSSDY FSSDY

SKYLD SKYLD

NRRTK TRRAQ

DFVQW DFVQW

LLSTK LKNS

RNGAN

HSQGM HSQGM HSQGT HSQGT

FTNDK FTNDY FTNDY FTNDY

SKYLE SKYLE SKYMD SKYLD

EKRAK EKSAK TRRAQ TRRAQ

EFVEW EFliEW DFVQW DFVQW

LKNGK LKNGK LMST LMST

S

HSECT HSEGT HSEGT HSEGT HSQGT HSQGT HSEGT HSEGT HSEGT HSEGT

FSNDY FSNDY FSNDY FSNDK I T NDY FTNDY FSNDK FSNDY FSNDY FSNDY

SKYLE SKYLE SKYLE SKYQE SKYLE SKYQE SKY L E SKY L E SKY L E SKY L E

DRKAQ TRRAQ TRRAQ E RMAQ TRRAQ MKQAQ DRKAQ TRRAQ TRRAQ TRRAQ

E FVRW DFVQW DFVQW DFVQW DFVQW DLVQW DFVQW DFVQW DFVQW DFVQW

LMNN LKNS LM(NS) LMNS LMNS LMNSK LMNS LKNN LKNS LKNS

** *

* ***

* * I

HSQGT

FTS DY

SKY L D

1 Lamprey

Elasmohranchs Dogfish Ray

Holocephalans Ratfish Elephantfish Actinopterygians Paddlefish I Paddlefish I1 Bowfin Gar

T

S

Teleosts Anglerfish I

Anglerfish I1 Catfish Coho salmon Eel 1 Eel 11 Sculpin I Sculpin I1 Flounder Tuna I n \ ariant residues

a

h

*

a**

RNGSS

*

Xlammalian 1Iuman

S RRAQ

DFVQW

LMNT(KRNRNN1A)

' The eight-residue C-terminal extension of human glucagon has been included to indicate the full sequence of oxyntomodulin. Species: common dogfish, Scyliorhinus canicula (Conlon et al., 1987d); ray, Torpedo m a m o r a t a (Conlon and Thim, 1985); ratfish, Hydrolagus colliei (Conlon et al., 1989);elephantfish, Callorhynchus rnilii (Berks et al., 1989); paddlefish, Polyodon spathula (Nguyen et al., 1994); bowfin, Amia caloa (Conlon et al., 1993); gar, Lepisosteus spatula (Pollock et al., 1988); anglerfish, Lophius umericanus (Lund et al., 1983; Andrews et al., 1986; Nichols et al., 1988); channel catfish, lctalurus punctatus (Andrews and Honner, 1985); coho salmon, Oncorhynchus kitsutch (Plisetskaya et al., 1986); European ee1,Anguilla rostrata (Conlon et al., 1988b); flounder, Platichthys Jesus (Conlon et al., 1987b); daddy sculpin, Cottus scorpius (IIConlon et al., 1987c; I-Cutfield and Cutfield, 1993); tuna, Thunnus obesus (Navarro et al., 1991). a, acidic residue (glutamate or aspartate); b, basic residue (arginine or lysine).

8.

239

MOLECULAR ASPECTS OF PANCREATIC PEPTIDES

Table I11 Primary Structure of Fish Glucagonlike Peptides" Cycloctomes 1 5 HADGT

10 FTNDM

15 TSYLD

20 AKAAR

25 DFVSW

Elasmnhranchs Dogfish Ratfish

HAEGT HADGI

YTSDV YTSDV

DSLSD ASLTD

YFKAK YLKSK

RFVDS RFVES

LKSY LSNYN

RKQND

Actinnpterygians Bowfin* Gar Paddlefish

YADAP HADGT HADGT

YISDV YTSDV YTSDA

YSYLQ SSYLQ SSFLQ

DQVAK DQAAK EQAAR

K--- W KFCTW DFISW

LKSGQ LKQGQ LKKGQ

DRRE DRRE

Teleosts Anglerfish 1 Anglerfish I1 Catfish Coho salmon Eels Soulpin

HADGT HADGT HADGT HADGT HAEGT HADGT

FTSDV YTSDV YTSDV YTSDV YTSDV FTSDV

SSYLK SSYLQ SSYLQ STYLQ SSYLQ SSYLN

DQAIK DQAAK DQAAK DQAAK DQAAK DQAIK

DFVDR DFVSW DFITW DFVSW EFVSW DFVAK

LKAGQ LKAGR LKSGQ LKSGR LKTCR LKSKV

V(RRE) GRRE P A

**a**

f***

HAEGT HADGS

FTSDV FSDEM

Lamprey

Invariant residues (all fish,except bowfin) Mammals truncated human-I hunran-2

1,

SSYLE NTILD

GQAAK NLAAR

30

LARSD

*

EFIAW DFINW

KS

I,

LVKGRG LIQTK

I

a American eel, Anguilla rostrata; European eel, Anguilla anguilla. For other species names and references, refer to Table 111. f, aromatic amino acid ( Y or F); dash indicates deletions; a, acidic residue (glutamate or aspartate); b, basic residue (arginine or lysine).

and the low resemblance of the intervening peptide of fishes to the C-terminal extension of mammalian glucagon (i.e., the portion making up oxyntomodulin), the absence of an oxyntomodulin-type peptide in fishes does not come as a surprise. Possible exceptions to this hypothesis are a glucagonlike structure isolated from the pancreas of the holocepalan ratfish and an extended, unprocessed glucagon (possibly a storage form of glucagon) in an eel (cf. Table 11). The 36-residue peptide, which is thought to be a storage form of a 29-residue glucagon rather than a hormone in its own right, bears limited homology to oxyntomodulin in its 8-residue C-terminal extension. Obviously, an analysis of the gene structure of the proglucagon gene in this ancient group of fishes might give interesting comparative insights into the evolution of exon assembly and intron splicing because, in human and rat, part of the short C-terminal extension of oxyntomodulin is encoded by exon 3, whereas the remaining 6 amino acids are contributed by exon 4.

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The mechanism of glucagon gene transcription is multifaceted and seems to differ substantially depending on the experimental system used. In a rat pancreatic cell line, for instance, activation of the gene appears to involve a protein kinase C pathway (Philippe et al., 1987), whereas in isolated rat islets, rat intestinal cells, and a mouse neuroendocrine cell line, a CAMP-dependent pathway is thought to prevail (Gajic and Drucker, 1993). As usual, the CAMP-dependent pathway involves a CAMPresponsive element (CRE) as well as the appropriate CRE binding protein. A new family of activating transcription factors has been identified that may bind specifically to CRE sequences. The appropriate sequence (5’-TGACGTCA-3‘) has been found to be a common theme in CAMP-activated genes (Meyer and Habener, 1993), including the glucagon gene (Drucker et al., 1991). It is the selectively phosphorylated form of this binding protein (or family members) that mediates increased rates of gene transcription, although CRE activity is further regulated by nucleotides flanking the core CRE octamer (hliller et al., 1993). In pancreatic A-cells, membrane depolarization can lead to induction of glucagon gene transcription. In this case the process is thought to depend on calcium influx and calciumicalmodulin-dependent protein kinase (Philippe et al., 1987; Schwaninger et ul., 1993). Although similar analyses on genomic D N A are sorely lacking, these results open up promising lines of inquiry for fish researchers interested in regulatory and evolutionary aspects of hormone action.

C. Glucagon Processing and Message Transduction In mammals glucagon is a highly conserved peptide of 29 amino acids with identical sequences in most species (Epple and Brinn, 1987). During the last few years, an impressive body of literature has accumulated dealing with structure-function analysis of mammalian glucagons. Unfortunately, the same cannot be said about their piscine counterparts, although the natural variability of peptide sequences found in a group of vertebrates as heterogeneous as the fishes is a powerful tool to analyze structure-function relationships. Such an analysis will also help identify conserved areas of the peptide as well as areas incurring larger species-dependent variability. In those cases where glucagon acts through a cell-surface receptor, as in hepatocytes, two processes combine to convey glucagon’s message to the interior of the cell. Hormone binding to the receptor is followed b y receptormediated changes in intracellular messengers. Although it is now estab-

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24 1

lished that in many mammalian cell types, glucagon regulates intracellular targets through different, likely interacting, mechanisms, including CAMP, inositolpolyphosphates, and intracellular calcium (Bygrave and Benedetti, 1993), most attention has been devoted to the analysis of receptor binding in conjunction with activation of adenylyl cyclase. By this route, selected areas of the glucagon molecule have been assigned different roles in receptor binding and adenylyl cyclase activation. Positions 1 through 5 are most critical to receptor recognition and binding, whereas Aspg is crucial for effective message transduction. One publication points out that it is the interaction between Asp' and His' that produces optimal receptor binding together with adenylyl cyclase activity of the peptide, whereas the positive charge of His' is essential for the activation of the adenylyl cyclase (Unson et al., 1993). Ultimately, most of glucagon's 29 residues are important to receptor binding, and not all biological functions of the hormone are localized to the N-terminal region of the peptide. C-terminally altered glucagons possess altered receptor-binding properties compared with the native hormone. Similarly, oxyntomodulin, the C-terminally extended glucagon (1-37) with specific actions directed toward the oxyntic cells (Jarrousse et al.,1985), will bind to the hepatic glucagon receptor, albeit with considerably reduced affinity. It should be kept in mind, however, that such structure-function studies have almost exclusively focused on receptor binding and activation of adenylyl cyclase, whereas other routes of message transduction and cross-talk between message transduction systems have been necessarily ignored given the experimental approach. The potential importance of non-CAMP message transduction systems to glucagon actions cannot be overstated. Working with three different species of teleostean fishes (American eel, Anguilla rostrata; rainbow trout, Oncorhynchus mykiss; brown bullhead, Zctalurus nebulosus),we have shown that a relatively poor correlation exists between cAMP increases and the concentrations of glucagon, or GLP. The rate of glucose output through endogenous glycogenolysis in isolated liver cells is activated significantly at low nanomolar (0.5 to 2) hormone concentrations, whereas significant increases in intracellular CAMP cannot be picked up until the hormone concentrations reach the mid-nanomolar range (20-100 nM) (Mommsen and Moon, 1990). This implies that a CAMPindependent route of message transduction may be involved at low (physiological) hormone concentrations. However, it is also possible that the route of analysis for determining cAMP masks hormonedependent changes in cAMP levels. Researchers normally determine total cAMP in the presence of a phosphodiesterase inhibitor to foil

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STEPHEN J. DUGUAY A N D T H O M A S P. M O M M S E N

degradation of the cAMP formed and, thus, rely on detecting relatively large increases in total CAMPto study hormone effects. What is convenient for the researcher, however, may be irrelevant to the cell. It is likely that the fractional amount of free cAMP or the fraction bound to the regulatory subunit of protein kinase A constitutes the regulatory principle and not the total amount ofthis compound stashed on nonspecific or specific binding sites. For all vertebrates, many intracellular proteins are known that bind CAMP, ranging from CAMP-dependent regulatory elements on the nuclear DNA and their associated binding proteins, through phosphodiesterases to CAMP-dependent protein kinase A. These and similar proteins are likely to make up the bulk of total CAMP, with a considerably smaller fraction of the compound existing in the unbound form or sequestered by the regulatory subunits of protein kinase A. For instance, upon hormonal stimulation of adrenal cells isolated from the rat, the fraction ofcAMP bound to protein kinase A incurred the largest percentage increase of all cAMP pools assayed 1979).In conclusion, a role ofcAMP as intracellular messen(Sala et d., ger at low concentrations of glucagon cannot be excluded until further experimentation has been conducted on different intracellular cAMP fractions. As shown in Table 11, fish glucagons are relatively variable in sequence, but key amino acids are invariant in all species. Among these are, as expected, His' and Aspg, and the bulk of the N-terminal region. Ten of the first 13 amino acids are identical, giving credence to the idea of the overall importance of the N-terminal region of the peptide. By the same token, 60% of the last 10 amino acids are invariant (including an exchange of Asp2' for G1uZ1),pointing to the crucial role of the C-terminal region to glucagon's biological activity. Some interesting differences exist in the specific responses to glucagon and its synthetic analog. His' has been identified as essential to the biological activity of the peptide, both to receptor binding and to activation of adenylyl cyclase. Another critical role has been assigned to Aspg owing to chain length more than its charged environment. Deletion of His' and replacement of Asp9 with Glug yields a potent glucagon analog that retains some of the native peptide's receptor binding, measured by the ability of radioiodinated glucagon to displace analog binding, but is unable to activate adenylyl cyclase (Unson et al., 1991). As shown in Table 11, both His' and Aspg flank a rather conserved region even in the comparatively heterogeneous teleostean glucagons (cf. Table 11). In such potency and binding experiments, activation of adenylyl cyclase is normally measured with isolated liver membranes. When we tested the function of mammalian des-His'-

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243

Glu9 in fish liver cells, a different picture emerged. Whereas mammalian and teleostean glucagons are equipotent in their ability to activate gluconeogenesis and glycogenolysis in hepatocytes isolated from numerous species of teleostean fishes, the alleged antagonist behaved as a weak agonist. Its dose-response curve was right-shifted compared with unmodified glucagons by about two orders of magnitude (T. P. Mommsen, unpublished results). However, the two assay systems may not be directly comparable because the observed activation of glycogenolysis involves both receptor binding and activation of intracellular message transduction systems. Although glucagon action in fish systems is thought to involve mainly the activation of adenylyl cyclase, the activation of alternative pathways cannot be excluded. Mammalian glucagon possesses a dibasic processing site at Arg17Arg". In the rat liver it has been shown that glucagon processing by a specific endopeptidase (Blache et al., 1993) can generate a fragment (glucagon 19-29) with its own distinctive biological activity, that is, inhibition of the liver Ca2+ pump (Mallat et al., 1987). In addition, processing of oxyntomodulin at this dibasic site will also generate an active peptide with biological function similar to that of the full-length oxyntomodulin (Jarrouse et al., 1993). Although Arg" is conserved in all glucagons analyzed, with the exception of the lamprey, position 17 is variable. Some of the fish glucagons sequenced to date possess a dibasic processing region (KR, cf. Table 11),requiring a similar enzymatic process to produce the piscine equivalent of the mammalian mini" glucagons, whereas the remaining species require endopeptidases capable of recognizing a single Arg residue flanked by a number of different resides. However, when we tested the biological activity of different fish-derived miniglucagons, we failed to detect any activity in fish hepatocyte systems; the activity was measured by analyzing flux through glycogenolysis or gluconeogenesis (T. P. Mommsen and G. A. Cooper, unpublished). We did not attempt to determine the activity of the hepatocyte Ca2+pump in response to hormone exposure. Judging by the relative amounts of processed products (in this case glucagon) available in Brockmann bodies (exceeding about 7 nmol/g) and gut (less than 1 nmol/g) (Andrews and Ronner, 1985), one can assume that pancreatic cells contribute the bulk of glucagon present in the circulatory system. The pancreas also contains some 85 nmol of GLP/g. Although the ratio for the endocrine pancreas indicates that GLP outnumbers glucagon by over 10-fold, a much reduced ratio (about 3) is determined in plasma in the hepatic vein (Plisetskaya and Sullivan, 1989).This discrepancy may be due to (a) differential release ofthe two peptides, (b)different turnover ofthe peptides, (c) underesti"

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STEPHEN J. DUGUAY A N D THOMAS P. MOMMSEN

mates for glucagon owing to determination of only one of the two nonallelic glucagons, or (d) postpancreatic processing of oxyntomodulin (where it exists) into glucagon. At any rate, the discrepancy is suprising, considering the similar role of the two peptides in fish metabolism and the origin of the two peptides from the same proglucagon sequence. In addition to the endocrine pancreas (A-cells), cells or cell groups with glucagonlike immunoreactivity have been detected in fish stomach (elasmobranch) and CNS (cyclostomes, elasmobranchs) (Conlon, 1988; Jonsson, 1991). The relative contributions of these different potential sources of glucagon in fish plasma are not known, and the actual release of the active hormone from the cells cannot be assumed. The regulation of glucagon secretion from the endocrine pancreas of fishes bears resemblance to that described for mammals: basic amino acids and KCI, all applied in supraphysiological concentrations, are potent secretagogues, whereas glucose is an inhibitor of glucagon release (Ince and So, 1984; Ronner and Scarpa, 1987; Plisetskaya et al., 1989).Further, epinephrine exerts a relatively strong glucagonotropic action in fish Brockmann bodies, albeit at pharmacological concentrations of the catecholamine (Mazeaud, 1964).

D. Physiological Actions of Glucagon The best-known and principal function of glucagon in fishes and mammals is a strong glycogenolytic action on liver resulting in hyperglycemia. It largely opposes the glucose-oriented actions of insulin. In addition, the hormone has numerous other functions, such as activation of lipolysis and other indirectly linked functions in fishes. With regard to the physiological roles of glucagon in fishes, attention has largely been focused on the hepatic action. However, if mammalian work can serve as a rough guide for future research, the brain and the endocrine pancreas, displaying some degree of glucagon binding capacity and expressing glucagon receptors, are worthy of attention. In the absence of data on receptor binding and glucagondependent gene expression in nonhepatic tissues of fishes, we will briefly summarize aspects of glucagon’s action on parenchymal hepatocytes. Glucagon injection results in a pronounced hyperglycemia in most species of fishes, a process brought about by activation of hepatic glycogenolysis and gluconeogenesis. Season, reproductive state, and temperature influence the targets and effectiveness of the hormone in piscine systems. At supraphysiological concentrations of glucagon, activation of glycogenolysis involved activation of adenylyl cyclase

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(Ottolenghi et al., 1990) and protein kinase A-mediated phosphorylation of glycogen phosphorylase; the latter process increases the proportion of the enzyme in the active @-form(Brighenti et al., 1991; Foster and Moon, 1990; Janssens and Lowrey, 1987). At the same time, glycogen synthase is inactivated by similar phosphorylation (T. Moon, G. Foster, and M. Vijayan, unpublished results). Flux through gluconeogenesis is enhanced through phosphorylation-dependent inhibition of pyruvate kinase and, in long-term experiments, through increases in the activity of phosphoenolpyruvate carboxykinase (PEPCK). Incidentally, the short-term induction observed for mammalian PEPCK is found wanting in fish liver. Exposure of fish systems to high concentrations of glucagon (largely bovine) increases lipolysis mediated via enhanced triglyceride lipase activity, the rate of amino acid uptake by the liver, and, finally, ureagenesis (reviewed in Mommsen and Moon, 1990). In addition to the hyperglycemia mentioned, in wiwo effects of the hormone are an accumulation of plasma unesterified fatty acids and glycerol as well as increases in ammonia excretion by treated fish. In fishes the hormone does not seem to change the rate of mitochondrial respiration as it does reproducibly in mammalian test systems. E. Glucagonlike Peptide Processing and Message Transduction Fish Brockmann bodies synthesize only one-short-GLP from a comparatively shorter proglucagon. If two GLPs or two glucagons are found, these are products of two nonallelic genes. Transcription of the proglucagon genes in the anglerfish will lead to the production of GLPs with 34 residues, terminating in G-R-G-R-R-E in the case of gene I1 and G-Q-V-R-R-E for gene I. Both peptides contain additional dibasic processing sites. As shown for gene I1 (Andrews et al., 1986), but not ruled out for gene I (Nichols et al., 1988), such processing indeed takes place in wiwo, leading to the production of two C terminally truncated GLPs. One of these has 31 residues and terminates in an amidated arginine; the amidation is likely produced via a stable 32-mer intermediate, derived from proteolytic processing of the original GLP at the additional dibasic processing site. As described next for fishes and as shown for GLP-1 in mammalian systems, the amino acid sequence at the C terminus and amidation do not compromise the biological activity of these peptides. Piscine glucagonlike peptides are not nearly as conserved in their amino acid sequences as glucagon (Table 111),with 10 invariant residues out of 31. With the exception of the unusual bowfin peptide,

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STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN

which bears some resemblance to GIP and has a notably reduced biological potency (Conlon et al., 1993), the N-terminal region of the peptide is highly conserved. Of the first 9 amino acids, 8 positions are occupied by invariant residues and the C-terminal region is highly variable, with only 2 invariant positions for the entire remainder of the 31-residue peptide. The importance of the N terminus is supported by our observation for fish systems (T. P. Mommsen and A. Jardim, unpublished) and by those of others for mammals (Suzuki et al., 1989) that His' is essential to the biological action of GLP, be it glycogenolytic action in fishes or insulinotropic action in mammals. Further, the obvious variability in the C-terminal region of GLP in fishes can be taken as an indication that selection pressure is less strong to maintain the charge and lipophilic characteristics of the C terminus. That is exactly what was found experimentally when C-terminally altered mammalian GLPs were analyzed for their insulinotropic activity: large tracts of the C-terminal region were dispensible (Suzuki et al., 1989). There is some indication that CAMP-dependent pathways are involved in GLP message transduction in some species of fishes (Zctalurus sp., Anguilla), albeit only at supraphysiological concentrations of peptide. At physiological concentrations of GLP in these species and in other species (Oncorhynchus mykiss, Sebastes sp.), CAMPindependent routes are likely operative at all times (Mommsen and Moon, 1990). Contrary to numerous earlier reports, fish liver cells have been found to respond to selected hormones with a redistribution of intracellular calcium stores (Zhang et al., 1992a,b) and with liberation of' inositolpolyphosphates (T. W. Moon, personal communication). Thus, the stage is finally set for an in-depth analysis of intracellular message transduction systems in fish systems. A similar heterogeneous picture is emerging for mammals. In a pancreatic cell line, truncated GLP-1 was found to increase CAMP levels, whereas receptor affinity sensitivity to guanine nucleotide was taken to indicate the involvement of G-proteins in transduction (Goke et al., 1989). In marked contrast to glucagon binding to these cells, GLP exposure failed to alter intracellular Ca2+ levels and membrane potential remained unaffected by GLP treatment. However, in the presence of glucose, GLP may lead to membrane depolarization of subpopulations of pancreatic islet cells, likely through closing of ATPgated K+-channels (Holz et al., 1993), whereas in other experimental systems adenylyl cyclase as well as protein kinase C have been implicated as intracellular message transduction systems (Wheeler et al., 1993; Yada et al., 1993).

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F. Physiological Actions of Glucagonlike Peptides With regard to the function of GLPs in fishes and mammals, diametrically opposing roles have been described in which there is a predominant endocrine role in mammals as opposed to a clear metabolic role in fishes. In all mammals (Schmidt et al., 1985; Mojsov et al., 1987), amphibians, and reptiles (T. P. Mommsen and E. M. Plisetskaya, unpublished), the primary site of action of truncated GLP is in the pancreas. Nevertheless, judging from the expression of tissue receptors, some physiological role for tGLP in brain, stomach, kidney (removal site), and lung can be predicted. In pancreatic cells, the hormone increases insulin synthesis and secretion, while also suppressing glucagon gene transcription and glucagon secretion. In the course of the last year, GLP-selective receptors have been expression cloned and sequenced for different tissues and in different mammals (Thorens, 1992; Dillon et al., 1993) (human brain: S. Mojsov, personal communication). These receptors show sufficient resemblance to the receptors for parathyroid hormone, calcitonin, secretin, and glucagon ( Jelinek et al., 1993) to form a new subgroup of the family of G-protein coupled receptors with seven transmembrane domains. From different angles it has been confirmed that the liver is not a target for GLP action: first, liver shows no GLP binding; second, the tissue fails to express GLP receptors; third, intracellular message transduction systems are not recruited after GLP exposure; fourth, liver does not degrade GLP or remove it from the circulation; and finally, no biological action of GLP could be identified (Ruiz-Grande et al., 1990; Murayama et al., 1990; Blackmore et al., 1991; Thorens, 1992). On a metabolic level, the mammalian truncated GLP functions to accentuate insulin’s action. GLP does so by increasing insulin availability and concentration and by removing glucagon, one of insulin’s major antagonists. This situation illustrates the surprising principle that two peptide hormones with directly countering actions are derived from genes that evolved by duplication of a single gene. In contrast, fish liver has been identified unequivocally as the main target of GLP action (Mommsen and Moon, 1990; Brighenti et al., 1991), with little or no action of the peptide on endocrine pancreatic cells (Mommsen and Plisetskaya, 1993). Nonhepatic tissues (e.g., brain) cannot be entirely excluded as potential target tissues. In addition to being the site of strong metabolic rather than endocrine action, fish liver is also the primary site of GLP removal from the circulation. GLPs activate hepatic gluconeogenesis, glycogenolysis and lipolysis.

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STEPHEN J. DUGUAY AND THOMAS P. MOXIMSEN

Thus the peptides have identical or similar targets to glucagon, but as implied earlier, they may differ in their intracellular message transduction pathways. It should be mentioned at this point that fish GLPs and mammalian truncated GLPs are entirely interchangeable in their specific actions: fish GLPs act as powerful insulinotropins in isolated mammalian islets (Plisetskaya and Duguay, 1993),and truncated GLPs are equipotent to fish GLPs in their glycogenolytic action in fish hepatocytes. Although both glucagon and GLPs act on fish liver in a similar fashion, they do not involve the same hepatic receptors. Radiolabeled glucagon that is bound to highly specific glucagon receptors on fish hepatocytes will only be minimally displaced by 1000-fold higher concentrations ofGLP (Navarro and Moon, 1994). However, the physiological actions of the two peptides can be discerned at identically low peptide concentrations (Mommsen and Moon, 1990; T. P. Mommsen and G. A. Cooper, unpublished). Exposure to GLP (or glucagon) changes metabolic output of liver cells immediately. Within 30 sec, increases in glucose output by the cells can be detected, and within a few minutes of the first exposure to hormone, the cells respond less and less readily to the hormone. After about 45 min the cells produce glucose at a slower rate than untreated control cells (Fig. 3 ) and are unresponsive even to much higher concentrations of the agonist (Mommsen and Plisetskaya, 1993). If a rat system can serve as a model, the obvious rapid decrease in fish hepatocyte responsiveness to GLP (Fig. 3 ) or glucagon (not shown) is due to desensitization of postreceptor mechanisms (Houslay et al., 1992).Down-regulation (decreased availability) of receptors can likely be ruled out as an explanation because internalization of glucagon is a relatively slow process in fish hepatocytes (Navarro and Moon, 1994). Nevertheless, fish liver has been shown to remove 75% of hepatic vein glucagon and more than 50% of its GLP in a single pass (Plisetskaya and Sullivan, 1989).

G. Conclusion Apart from the untranslated regions, preproglucagon gene structure appears to be similar in fishes and mammals, but small, physiologically important differences exist in the primary transcript and in processing sites. The glucagon sequence is highly conserved throughout all vertebrates, and in fishes the peptide assumes the same pivotal position opposite of insulin in carbohydrate and lipid metabolism. The hormone may do so b y slightly different intracellular routes and using

8.

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MOLECULAK ASPECTS OF PANCREATIC PEPTIDES

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L Q)

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40

60

80

100

120

Time (minutes) Fig. 3. Time course of glucose production in response to truncated mammalian GLP in isolated rockfish (Sebastes caurinus) hepatocytes demonstrating the rapid desensitization of the cells to the hormone. Cells respond similarly to fish GLPs, mammalian glucagon, and fish glucagons. Cell behavior is tested as glucose release from endogenous glycogen. Glucose release is presented as an arbitrary rate per time interval (10 or 15 min), that is, a slope approaching zero indicates a constant rate of glucose production. Solid circles: control treated with vehicle; open circles: cells exposed to 5 nmol/liter of GLP1-37at 10 rnin. Data recalculated from Momrnsen and Plisetskaya (1993).

different intracellular targets, but ultimately by targeting the same tissues. The same cannot be said for the other important processing product of the proglucagon gene in fish pancreas-the GLPs. One gene product (GLP-2) is missing from the fish altogether. Fish GLP is structurally similar to the truncated GLP-1 of mammals, but different routes and sites of processing lead to the mature gene product. The fish gene is devoid of a region corresponding to the N-terminal sixamino-acid extension found in the mammalian proglucagon. Also, the mammalian pancreas secretes the full-length GLP-1, which is largely inactive. Fish proglucagon processing leads to storage and secretion of a biologically fully active short GLP from the pancreas. In the final analysis, GLP is an endocrine hormone and a powerful antagonist to

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glucagon via its action on insulin synthesis and release. In fishes the same gene product (piscine and mammalian GLPs are freely interchangeable in their actions) functions as a true “glucagonlike” hormone. IV. SOMATOSTATIN

A. Gene Structure Although somatostatin genes have not been cloned from any fish species, there are several lines of evidence indicating that teleosts contain two somatostatin genes. Two cDNAs coding for different preprosomatostatins have been cloned and sequenced from anglerfish (Lophius americanus) (Goodman et al., 1980a; Hobart et al., 1980a; Goodman et al., 1982) and channel catfish (Zctalurus punctatus) (Taylor et al., 1981; Magazin et al., 1982; Minth et al., 1982). Southern blot analysis of Zctalurus genomic DNA using probes specific for each ofthe cloned cDNAs suggests that the corresponding genes are located on different fragments ofthe genome (Minth et al., 1982).Furthermore, multiple somatostatin peptides with different amino acid sequences have been isolated from coho salmon (Oncorhynchus kitsutch) (Plisetskaya et al., 1986), as well as daddy sculpin and flounder (Cottus scorpius and Platichthys flesus) (Conlon et al., 1987a).

I3. Messenger RNA Transcripts and cDNA Sequences Two anglerfish (Lophius americanus) somatostatin cDNAs have been sequenced and designated SST gene I and SST gene I1 cDNAs (Goodman et al., l980a, 1982; Hobart et al., 1980a). The SST gene 1 cDNA encodes a 121-amino-acid precursor containing a 25-amino-acid signal peptide, an 82-amino-acid propeptide region, and SST-14 at the carboxy terminus (Fig. 4). The sequence of anglerfish gene I SST-14 (aSST-14 I ) is identical to mammalian SST-14 and is preceded by the dibasic processing signal of arginine-lysine. There is also a single arginine residue upstream from the aSST-14 sequence that could be utilized to generate aSST-28 (see Section IV,C,2). The anglerfish gene I preprosomatostatin (aPPSS-I) sequence reported by Hobart et al. (1980a) differs at one nucleotide from the corrected sequence reported by Goodman et al. (1982). This nucleotide change is a glycine to glutamic acid substitution at residue 58 of the propeptide and the

8.

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MOLECULAR ASPECTS OF PANCREATIC PEPTIDES

Producing Cell

aSST-14

aPPSS

I

121 amino acid precursor -25

I

- 1

20

60

02

I

R

R

RK

4

96

I

t t

t

aSST-14

I

1-

7 10

Producing Cell

aSST-28

aPPSS

II

125 amino acid precursor

-24

I

- 1

I

4

30

73

07

KR

R

RK

101

t aSST-28

II

(h Fig. 4. Processing of preprosomatostatin precursors in SST-14 and SST-28 producing cells of the anglerfish (Lophius americanus) Brockmann body. Negative numbers refer to amino acid residues in the signal peptide. Positive numbers refer to residues of the prohormone and mature hormone. Open arrows indicate the location of signal peptide cleavage by signal peptidase. Large solid arrows indicate the major processing sites utilized to generate mature somatostatins. Small solid arrows indicate minor cleavage sites. The residues indicative of gene I somatostatins (Phe', Thr'") and the substitutions that are the hallmark of gene I1 somatostatins (Ty?', Gly24)are indicated in the respective mature aSSTs. The lysine hydroxylation site on aPPSS-I1 is indicated by OH.

discrepancy may be attributed to allelic variation or sequencing errors. Peptide sequencing data indicates that residue 58 is glutamic acid (Andrews and Dixon, 1987). Anglerfish gene I1 cDNA codes for a 125-amino-acid preprosomatostatin (aPPSS-11) containing a 24-residue signal peptide, an 87-amino-

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STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN

acid propeptide, and aSST-14 at the carboxy terminus. Like aPPSS-I, this cDNA codes for mono- and dibasic residues that could be utilized to generate both aSST-14 and aSST-28 (see Section IV,C,2). However, anglerfish gene I1 SST-14 (aSST-14 11) contains two amino acid substitutions relative to aSST-14 I. Phenylalanine-7 has been replaced by tyrosine and threonine-10 has been replaced by glycine, which generates (Tyr7Gly")SST-14 (Hobart et al., 1980a). aPPSS-I and aPPSS-I1 share approximately 45% amino acid sequence identity. Northern blot analysis of Brockmann body RNA with probes specific for aPPSS-I and aPPSS-I1 indicate that gene I and gene I1 mRNAs comigrate with markers of approximately 700 nucleotides. However, gene I and gene I1 mRNAs have been localized to different cell populations in the Brockmann body. Gene I mRNAs were found in large clusters of cells that were distributed throughout the islet whereas gene I1 mRNAs were present in smaller clusters of cells. Gene I and gene I1 mRNAs do not appear to be colocalized to the same regions of the Brockmann body (Sevarino et al., 1989). Two cDNAs coding for distinct somatostatin peptides have been isolated from the channel catfish (Zctalurus punctatus). One cDNA codes for a 114- amino-acid precursor containing a signal peptide, a propeptide of approximately 75 amino acids, and SST-14, which is identical in sequence to aSST-14 I and the mammalian SST-14 peptides. This cDNA also codes for a pair of basic residues immediately preceding the SST-14 sequence. The catfish SST-14 cDNA lacks a monobasic processing site upstream of the SST-14 sequence and it is therefore not possible to produce catfish SST-22 from this product (Taylor et al., 1981; Minth et al., 1982). The second catfish somatostatin cDNA encodes a 105-amino-acid precursor containing a typical signal peptide, a pro region of approximately 57 amino acids, and the somatostatin sequence analogous to the SST-22 peptide previously isolated by Oyama et al. (1980). The SST-22 sequence is preceded by a single arginine residue. Catfish SST-22 and SST-14 differ in amino acid sequence at 7 out of 14 residues. The catfish SST-22 precursor cannot be processed to SST-14 owing to the replacement of the dibasic processing signal argininelysine with lysine-proline (Magazin et al., 1982). Northern blots of catfish Brockmann body RNA with a gene I (SST14) probe reveal a strong hybridization signal at 1000 nucleotides and two minor bands at 1375 and 810 nucleotides (Taylor et al., 1981).A single band of 880 nucleotides is detected using a probe of catfish gene I1 (SST-22) (Magazin et al., 1982).

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C . Biosynthesis 1. COTRANSLATIONAL PROCESSING

a . Signal Peptide Cleavage. The functionality of anglerfish preprosomatostatin signal peptides has been verified using in vitro translation systems. When anglerfish Brockmann body RNA is used in cell-free translation reactions, peptides of 14 to 18 kDa molecular mass are generated that can be immunoprecipitated with antisera to SST-14. When translation is performed in the presence of pancreatic microsoma1 membranes containing signal peptidase activity, the molecular weight of SST-14 immunoprecipitable proteins decreases by about 2 kDa (Goodman et al., 1980b; Shields, 1980). Signal peptide cleavage sites have been determined by sequence analysis of metabolically labeled aPPSS-I peptides (Noe et al., 1986a) and fast atom bombardment mass spectrometry (FABMS) of isolated aPPSS-I and aPPSS-I1 peptides (Andrews and Dixon, 1987; Andrews et al., 1987).Results indicate that the signal peptide cleavage ofaPPSS1occurs at the C ~ S ” - S ~bond. ? ~ The aPPSS-I1 signal peptide is cleaved at the Se?4-Gln25 bond (Fig. 4). 12. Lysine Hydroxylation. Anglerfish prosomatostatin I1 (aPSS-11) has been found to contain hydroxylysine at residue 23 of the SST-28 sequence (Fig. 4). The significance of this modification in terms of aSST-28 function is unknown. Hydroxylation of lysine residues is thought to be a cotranslational modification. The hydroxylation signal, X-Lys-Gly, is unique to anglerfish SST-28 because of a glycine substitution for the canonical threonine residue that is found in the analogous position of gene 1 and mammalian SST-14 (Andrews et al., 1984a; Spiess and Noe, 1985).Approximately 40% of the SST-28 in anglerfish Brockmann body contains hydroxylysine (Morel et al., 1984).

2. POSTTRANSLATIONAL PROCESSING u. Anglerfish Prosomatostatins. As described earlier, both aPSS-I and aPSS-I1 contain dibasic residues that could be cleaved to generate aSST-14 I and aSST-14 11, respectively. Both precursors also contain a monobasic cleavage site located 15 residues on the amino-terminal side beyond the dibasic processing site that could be utilized to produce aSST-28 I and aSST-28 11. aPSS-I and aPSS-I1 share 66% amino acid sequence identity between the carboxy-terminal 29 residues, which contain aSST-14, aSST-28, and putative processing signals. Despite this high degree of conservation, it has been shown that aPSSI and aPSS-I1 are processed to generate different products (Fig. 4).

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STEPHEN J. DUGUAY AND THOMAS P. MOMMSEN

High-pressure liquid chromatography (HPLC)and sequencing analysis of metabolically labeled peptides isolated from anglerfish Brockmann body indicate that aPSS-I is cleaved at Arga1Lys8' to generate aSST-14 I. aSST-28 I was not detected (Noe, 1981; Noe et al., 1986a). FABMS analysis of aPSS-I-derived peptides confirmed this observation; aSST-14 I, but not aSST-28 I, was present in Brockmann body extracts (Andrews and Dixon, 1987). Other peptides detected in these experiments were aPSS-I (1-27), (1-67), and (69-80). aPSS-I (1-27) is generated by cleavage at the monobasic residue Arg28.aPSS-I (1-67) is produced after cleavage at ArgW,the monobasic cleavage site that must be utilized to liberate aSST-28. Because aSST-28 I is apparently not produced, it is likely that cleavage at the dibasic site necessary to release aSST-14 I precedes cleavage at Arg68.Cleavage at both ofthese sites generates aPSS-I (69-80). Several studies indicate that, relative to aPSS-I, aPSS-11 is processed in a reciprocal manner. aSST-28 I1 has been isolated from Brockmann body extracts, but aSST-14-11 was not detected during the purification procedure (Morel et al., 1984). HPLC analysis of labeled Brockmann body peptides revealed that gene I1 SST is larger than aSST-14 (Noe and Spiess, 1983). FABMS experiments showed that aPSS-I1 is cleaved at Arg73to generate aSST-28 11. A minor cleavage site is which produces aPPSS-I1 (1-36). aSST-14 I1 was not detected (Andrews et al., 1987). In addition to differential processing, it is also apparent that aPPSSI and aPPSS-I1 are expressed in different cells of the Brockmann body. McDonald et al. (1987) showed by immunohistochemistry that aSST14 and aSST-28 are present in distinct areas of the islet and are not produced by beta, alpha, or pancreatic polypeptide cells. aSST-14 cells were found in large clusters distributed evenly throughout the islet. aSST-28 immunopositive cells were found individually or in small clusters and were often associated with glucagon-producing cells. The physiological significance ofthis association is not clear, and the mechanism responsible for cell-specific expression of SST gene I and gene I1 are unknown. These results have been corroborated by in situ hybridization with probes for anglerfish SST gene I and gene I1 mRNAs (Sevarino et al., 1989). The fact that aPPSS-I and aPPSS-I1 are posttranslationally processed at different sites, although both mono- and dibasic processing sites are present in each precursor, and that they are expressed in different cells raises the possibility that processing specificity may be dictated by (a) secondary structure surrounding the mono- and dibasic cleavage signals or (b)cellular factors, that is, the presence or absence

8.

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255

of appropriate processing enzymes. Sevarino et al. (1989) were able to distinguish between these factors by expressing cDNAs coding for aPSS-I and aPSS-I1 in mammalian cell lines. It had been previously determined that mouse corticotroph AtT20 cells generate both SST14 and SST-28 from transfected rat SST cDNA (rats have only one SST gene that can be processed to SST-14 or SST-28). The rat RIN 5F insulinoma cell line generates only SST-14 from transfected rat SST cDNA. When expressed in AtT2O cells, aPSS-I and aPSS-I1 were both processed to aSST-14 and aSST-28. In RIN 5F cells, both anglerfish precursors were processed to aSST-14, but aSST-28 was not detected. These results indicate that there are no inherent properties in the anglerfish prosoniatostatins molecules per se that preclude them from being processed to both aSST-14 and aSST-28. It is therefore likely that cellular factors are responsible for determining the patterns of aPSS processing. It is also interesting to note that the studies described here (Noe et al., 1986a; Andrews and Dixon, 1987; Andrews et al., 1987) provide evidence that both aPSS-I andaPSS-I1 are cleaved at mono- and dibasic sites to some extent (Fig. 4). This implies that regulation of processing might be achieved by (a) controlling expression of a repertoire of enzymes capable of distinguishing one monobasic site (or one dibasic site) from another by subtle differences in the context of the cleavage site or (b) closely regulating the level of expression of monobasic and dibasic processing enzymes.

b. Catfish Prosomatostatins. Both SST-14 and SST-22 have been isolated from channel catfish (Ictalurus punctatus) Brockmann bodies (Oyama et al., 1980; Andrews and Dixon, 1981). SST-14 and SST-22 share 50% amino acid sequence identity and cDNA analysis indicates that they are the products of different genes. The SST-14 prohormones contains a proline residue in place of the arginine residue necessary for generation of SST-22 and, therefore, cannot be processed to yield the larger peptide (Minth et al., 1982). In the SST-22 prohormone, the dibasic processing site utilized to produce SST-14 has been replaced by lysine-proline, eliminating conversion of this precursor to SST-14 (Magazin et al., 1982). SST-22 has been shown to be O-glycosylated on the threonine residue at position 5 (Andrews et al., 1984b). c . Other Fish Species. Four somatostatin peptides have been isolated from coho salmon (Oncorhynchus kitsutch). The predominant islet somatostatin is SST-25, which contains the T y P , GlyZ1sequence characteristic of anglerfish SST-28 11. In contrast to the situtation in anglerfish, salmon islets appear to be capable of producing SST-14 I1

256

STEPHEN J . DUGUAY AND THOMAS P. MOhlMSEN

from SST-25 I1 or its precursor. There is also evidence for very low levels of SST-28, an amino-terminal extension of SST-25. The fourth somatostatin present in salmon Brockmann body is SST-14 I, which is identical to anglerfish SST-14 I (Plisetskaya et al., 1986).Immunohistochemical studies revealed that SST-14 is located in the central part of the islet, in close association with beta cells, and SST-25 I1 is localized to the periphery of the islet and juxtaposed with alpha cells (Nozaki et al., 1988). Somatostatin peptides have been isolated from islet tissue of daddy sculpin (Cottus scorpius) and flounder (Platichthys jiesus). Both species contain SST-14 I peptides that are identical to anglerfish SST-14I (Conlon et al., 1987a; Cutfield et al., 1987). In addition, sculpin and flounder also produce SST-28 peptides that have 92% sequence identity to each other and 86% identity with anglerfish SST-28 11. These peptides contain the conserved dibasic residues necessary to produce an SST-14 I1 peptide, but they do not appear to be utilized as SST-14 11 was not detected. Both sculpin and flounder SST-28 I1 peptides contain Ty?' and Gly24residues, as does aSST-28 I1 (Conlon et al., 1987a). Two somatostatins have been isolated from the bowfin (Amia cuZva) and they appear to be derived from the same precursor. Bowfin SST14 is identical to aSST-14 1and bowfin SST-26 is an amino-terminally extended form of SST-14. The antisera used to identify somatostatin peptides during purification does not recognize gene Il-type SSTs (Tyr7Gly"). Therefore, the existence of additional SSTs in bowfin cannot be ruled out (Wang et al., 1993). Using the antisera specific to gene I-type SSTs, Conlon and colleagues have also identified SST-14 in the Pacific ratfish (Hydrolagus colliei),a holecephalan, and the elasmobranch (Torpedo marmoratu). Torpedo SST-14 is identical to aSST-14 I, whereas the ratfish SST-14 contains a serine substitution for asparagine at position 5 (Conlon et al., 1985; Conlon, 1990). Somatostatins have also been isolated from the oldest class ofvertebrates, the agnathans. The islet of the lamprey Petromyzon marinus contains three SSTs that appear to be derived from a single precursor. The largest and least abundant form is SST-37. SST-34 is generated from SST-37 or a common precursor by cleavage at a single arginine residue. SST-14 is produced by additional cleavage at a dibasic site to produce a gene 1-type molecule that differs from aSST-14 I by substitution of serine for threonine at position 12. SST-14 and SST34 are apparently produced by the same cells of the islet (Andrews et al., 1988).

8.

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257

The most abundant somatostatin in Atlantic hagfish (Myxine glutinosa) islet tissue is SST-34. Monobasic and dibasic cleavage sites for the generation of SST-28 and SST-14 are present in this precursor. However, it appears that only the dibasic site is utilized to generate SST-14, which is identical to aSST-14 I (Conlon et al., 1988a).

3. PROHORMONE CONVERTASES The nature of the enzyme involved in converting prohormones to biologically active mature hormones has been the subject of extensive investigation for many years (Steiner et al., 1992), and the anglerfish (Lophius americanus) Brockmann body has been a valuable model system for studying these enzymes (Mackin et al., 1990). Two putative prohormone convertases (PCs) have been isolated from anglerfish Brockmann body secretory granules. One enzyme converts aPSS-I to aSST-14 I and can also process aPSS-I1 to the unnatural product aSST-14 11. The second enzyme converts aPSS-I1 to aSST-28 11. The aSST-14 I generating enzyme also converts anglerfish proinsulin to insulin by cleavage at dibasic residues flanking the C-peptide. The aSST-28 I1 generating enzyme does not recognize proinsulin as a substrate. This indicates that mono- and dibasic cleavages are performed by distinct PCs (Mackin and Noe, 1987b). The aSST-14 generating enzyme migrates as a doublet band of 67,65 kDa and at 57 kDa on SDS-polyacrylamide gels. Activity of this enzyme is calcium dependent, and the amino-terminal sequence is identical to that of the mammalian dibasic prohormone convertase PC2 in 11 of 17 residues (Mackin et aZ., 1991b). PC2 is also known to be calcium dependent and is activated b y removal of an aminoterminal pro region to produce the smaller, functional protease (Steiner et al., 1992). The aSST-28 I1 generating enzyme has been identified as a singlechain 39-kDa protein. This enzyme displays maximal activity toward monobasic sites at pH 4.2 and this activity is abolished by aspartyl protease inhibitors. Amino-terminal sequence analysis reveals significant homology to other known aspartyl proteases (Mackinet al., 1991a). An apparent homolog to the anglerfish monobasic aspartyl protease has been identified in the yeast Saccharomyces cerevisiae. When aPSS-I1 is expressed in yeast it is processed exclusively to aSST28 in both normal and kex2-deficient yeast strains (Bourbonnais et al., 1991). This is significant because the Kex2 enzyme is the yeast homolog of vertebrate serine proteases with specificity toward dibasic residues, that is, furin, PC1/3, and PC2. Bourbonnais et al. (1993) succeeded in isolating yeast mutants that were deficient in

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somatostatin-28 expression (sex mutants) and identified the defective gene as yeast aspartyl protease 3 (YAP3). They showed that yeast strains with a functional YAP3 gene could process aPSS-I to aSST-28 I and aPSS-I1 to aSST-28 11.

D. Secretion Regulation of secretion of somatostatin from fish islets has not been studied extensively. However, it appears that fish islets respond to secretagogues in a fashion similar to that of mammalian islets. Glucose evokes a biphasic release of SST-14 from perfused catfish (Ictalurus punctatus) Brockmann body whereas arginine has only a minor effect (Ronnerand Scarpa, 1982,1984). Mannose was also an effective secretagogue in this system; fructose and a-ketoisocaproate had minor effects; and alanine and leucine were ineffective (Ronner and Scarpa, 1987). Glucose also stimulates a biphasic release of SST-14 from perfused Brockmann body of the European silver eel (Anguilla anguilla) (Ince and So, 1984).

E. Physiological Actions Most of the physiological studies of Brockmann body somatostatins have focused on SST-25 in coho salmon (Oncorhynchus kitsutch). When injected into salmon, SST-25 decreases plasma insulin, glucagon, and GLP levels, depletes liver glycogen content, and causes hyperglycemia. Administration of SST-25 antiserum results in SST25 deficiency with associated increases in plasma insulin and liver glycogen content. The reader is referred to several reviews for further details (Plisetskaya, 1989; 1990a,b; Plisetskaya and Duguay, 1993).

V. PANCREATIC POLYPEPTIDE AND

RELATED PEPTIDES A. The Pancreatic Polypeptide Family The pancreatic polypeptide (PP) family is composed of three peptides that are 36 amino acids long, contain an amidated carboxy terminus, and share extensive sequence similarities. The common feature that defines this family, however, is the unique tertiary structure known as the PP-fold. Structural analysis using techniques such as model building, circular dichroism, secondary structure prediction

8.

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259

algorithms, and X-ray crystallography indicate that members of the PP family contain an amino-terminal polyproline helix and a carboxyterminal a-helix joined by a type-I1 p-turn (Glover et al., 1985). In mammals, three members of the PP family have been characterized. PP is expressed in the pancreas. Peptide Y (PYY) is found in the intestine and the name reflects the fact that the first and last residues of the amino acid sequence are tyrosine (single-letter code = Y). Neuropeptide Y (NPY) is localized to the nervous system and its primary sequence also begins and ends with tyrosine (Larhammar et al., 1993). B. cDNA and Peptide Sequences PP family peptides and cDNA clones have been characterized from several piscine species. The sequences are shown in Table IV. All piscine sequences have been determined from peptides isolated from islet tissue and are therefore referred to as “PP” except the ray (Torpedo rnarrnorata) and goldfish (Carassius auratus) NPY sequences, which were deduced from cDNA clones obtained from libraries of nervous tissue. Also, the anglerfish peptide isolated from the Brockmann body has been named aPY by Andrews et al. (1985).On the basis of sequence comparisons of PP family proteins from fish, amphibians, birds, and mammals, Larhammar et al. (1993) have argued that Torpedo NPY may resemble the ancestral peptide of the PP family. Torpedo NPY is 94% identical to both porcine and goldfish NPY, indicating that the primary structure has been extremely well conserved during evolution. The pancreatic polypeptides isolated from several fish species are actually more similar in structure to NPY than PP (Table IV). C. Prohormone Processing NPY cDNA sequences have been determined for goldfish (Carussius auratus) and ray (Torpedo marmoratn). Both cDNAs encode a

28-amino-acid signal peptide followed by the 36-amino-acid sequence of mature NPY. The Torpedo cDNA codes for a carboxy-terminal extension propeptide that is 30 amino acids long. The Carussius propeptide is 28 residues in length. The mature peptide and the propeptide are separated by the sequence Gly-Lys-Arg. The Lys-Arg residues serve as the dibasic processing site, which may be cleaved by a member of the PC family of serine prohormone convertases. The carboxy-terminal glycine residue then donates the amide group to produce tyrosineamide (Blomqvist et aZ., 1992).

Table IV Primary Structure of PP Family Peptides" ~~

Porcine PP

-VY -

Porcine PYY Alligator NPY

-EA-

TP-Q SP- E

__ __

MAQ - SR-

- ML- -v-

AAE -AS -

(47%) (64%)

M-R- -R-

Porcine NPY Kay NPY

PAED ---E

Goldfish NPY

(92%) (94%)

LAKY

( 100%)

____

(94%)

-P- E -P- E

(86%) (86%) (83%)

Salmon PP

-P-E -P- E

Skate PP

AP- E

Eel PP

SP- E SP- -

Dogfish PP Gar PP Bowfin PP

Sculpin PP Anglerfi\h PY Invariant residues

SP1

*

*

*

*

*

*

(83%) (81%) (78%) (64%) (64%)

-

*

h

*

*

*

*

* *36

'I Amino acid alignment of PP family peptides. Residues are represented by single-letter code. Dashes indicate residues identical to ray sequence. Sequence identity relative to the ray sequence (in bold) is indicated in parentheses at right. Invariant residues are labeled with an asterisk. For porcine sequence references, see Larhammar et ul. (1993); ray, Torpedo rnarrnoruta (Blomqvist et ul., 1992); alligator, Alligcitor r,iississippiensis (Parker et ul., 1993b); goldfish, Curussius ciuratus (Blomqvist et ul., 1992); dogfish, Scqliorhinus cuniculu (Conlon et d . ,19911)); alligator gar, Lepisosteus spatula (Pollock et al., 1987); bowfin, Amiu culuu (Conlon et al., 1 991 ~)coho ; salmon, Oncorhynchus kitsutch (Kimmel et ul., 1986); skate, Ruju rhina (Conlon et ul., 1 9 9 1 ~ ); American eel, Anguilla rostruta (Conlon et ul., 1 9 9 1 ~ )daddy ; sculpin, Cottus scorpius (Conlon et al., 1986); anglerfish, Lophius urnericanus (Andrews and Dixon, 1986).

8.

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261

In addition to mature PY, the putative proregion of this molecule has been isolated from anglerfish (Lophius americanus) Brockmann bodies and identified b y homology to the pro region of human proNPY (Andrews and Dixon, 1986). D. Immunohistochemical Identification of Peptides

PP family peptides have been isolated from the agnathan intestine (Conlon et al., 1991d), and there is immunohistochemical evidence for the existence of these peptides in islets of the lamprey. Using antisera to mammalian NPY and PP, and anglerfish PY, immunoreactivity was observed in the pancreas of Petromyzon marinus. All antisera stained the same cells and these cells were distinct from B- and Dcells (Cheung et al., 1991). However, Youson and Potter (1993) found cells that were immunopositive for NPY and aPY in the intestine but not the islet of two other lampreys (Geotria australis and Mordacia mordax). In the dogfish (Squalus acanthias) and the coho salmon (Omorhynchus kitsutch), PP immunoreactive cells are localized to the periphery of the islet. PP cells are associated with A-cells in the salmon (ElSalhy, 1984; Yi-Qiang et al., 1986). PP and glucagon are found in the same cells in small and intermediate islets of Sparus auratus but are not colocalized in the principal islet (Abad et al., 1988). In anglerfish (Lophius americanus),aPY is expressed in the islet and NPY immunoreactivity has been localized to islet nerves (Noe et al., 1986b). E. Physiological Actions

The physiological function of PP in fish is unknown. When tested in mammalian systems, piscine PPs exert NPY-like effects. These include increasing blood pressure and decreasing heart rate in rats, as well as stimulating appetite (Balasubramaniam et al., 1990). Injection of the dogfish (Scyliorhinus canicula) pancreatic NPY-like peptide into dogfish causes an increase in blood pressure (Conlon et al., 1991b).

ACKNOWLEDGMENTS We thank Erika Plisetskaya for stimulating discussion. S. J . D. is supported b y a Post-Doctoral Fellowship from the Howard Hughes Medical Institute. T. P. M. acknowledges the continued support through a research grant from the Natural Sciences and Engineering Research Council of Canada.

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Blackmore, P. F., Mojsov, S., Exton, J. H., and Habener, J. F. (1991).Absence ofinsulinotropic glucagon-like peptide-I (7-37) receptors on isolated rat liver hepatocytes. F E B S Lett. 283, 7-10. Blomqvist, A. G., Soderberg, C., Lundell, I., Milner, R. J., and Larhammar, D. (1992). Strong evolutionary conservation of neuropeptide Y: Sequences of chicken, goldfish, and Torpedo marmorata DNA clones. Proc. Natl. Acad. Sci. U.S.A. 89, 2350-2354. Bourbonnais, Y., Danoff, A., Thomas, D. Y., and Shields. D. (1991).Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cereoisiae. J . Biol. Chem. 266, 13203-13209. Bourbonnais,Y.,Ash, J . , Daigle, M., andThomas, D.Y. (1993).Isolationandcharacterization of S. cerevisiae mutants defective in somatostatin expression: Cloning and functional role of a yeast gene encoding an aspartyl protease in precursor processing at monobasic cleavage sites. E M B O J . 12, 285-294. Brighenti, L., Puviani, A. C., Gavioli, M. E., Fabbri, E., and Ottolenghi, C. (1991). Interaction of salmon glucagon, gulcagonlike peptide, and epinephrine in the stimulation of phosphorylase a activity in fish isolated hepatocytes. Cen. Comp. Endocrinol. 82, 131-139. Bygrave, F. L., and Benedetti, A. (1993). Calcium: Its modulation in liver by cross-talk between the actions of glucagon and calcium-mobilizing agonists. Biochem. J . 296, 1-14. Chan, S. J., Edmin, S. O., Kwok, S. C. M., Kramer, J. M., Falkmer, S., and Steiner, D. F. (1981).Messenger RNA sequence and primary structure of preproinsulin in a primitive vertebrate, the Atlantic hagfish. J . Biol. Chem. 256, 7595-7602. Chan, S. J., Cao, Q.-P., and Steiner, D. F. (1990). Evolution of the insulin superfamily: Cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus. Proc. Natl. Acad. Sci. U.S.A. 87, 9319-9323. Chan, S. J., Nagamatsu, S., Cao, Q.-P., and Steiner, D. F. (1992). Structure and evolution of insulin and insulin-like growth factors in chordates. Brain Res. 92, 15-24. Cheung, R., Anclrews, P. C., Plisetskaya, E. M., and Youson, J. H. (1991).Immunoreactivity to peptides belonging to the pancreatic polypeptide family (NPY, aPY, PP PYY) and to the glucagon-like peptide in the endocrine pancreas and anterior intestine of adult lampreys, Petromyzon marinus. An immunohistochemical study. Gen. Comp. Endocrinol. 81, 51-63. Clark, A. R., and Docherty, K. (1992).The Insulin gene. Zn “Insulin: Molecular Biology to Pathology” (F. M. Ashcroft and S. J . H. Ashcroft, eds.), pp. 36-63. IRL Press, New York. Conlon, J. M. (1988). Proglucagon-derived peptides: Nomenclature, biosynthetic relationships and physiological roles. Diabetologia 31, 563-566. Conlon, J. M. (1990).[Ser’l-Somatatostatin-14: Isolation from the pancreas ofaholocephaIan fish, the Pacific ratfish (Hydrolaguscolliei).Gen. Comp. Endocrinol. 80,314-320. Conlon, J . M., and Thim, L. (1985). Primary structure of glucagon from an elasmobranchian fish, Torpedo marmorata. Gen. Comp. Endocrinol, 60, 398-405. Conlon, J. M., Agoston, D. V., and Thim, L. (1985). An elasmobranchian somatostatin: Primary structure and tissue distribution in Torpedo marmorata. Gen. Comp. Endocrinol. 60,406-413. Conlon, J. M., Schmidt, W. E., Gallwitz, B., Falkmer, S., and Thim, L. (1986). Characterization ofan amidated form ofpancreatic polypeptide from the daddy sculpin (Cottus scorpius). Regul. Pept. 16,261-268. Conlon, J . M., Davis, M. S., Falkmer, S., and Thim, L. (1987a).Structural characterization of peptides derived from prosomatostatins I and I1 isolated from the pancreatic

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Schmidt, W. E., Siegel, E. G., and Creutzfeldt, W. (1985). Glucagon-like peptide-1 but not glucagon-like peptide-2 stimulates insulin release from isolated rat pancreatic islets. Diabetologia 28, 704-707. Schwaninger, M., Lux, G., Blume, R., Oetjen, E., Hidaka, H., and Knepel, W. (1993). Membrane depolarization and calcium influx induce glucagon gene transcription in pancreatic islet cells through the cyclic AMP-responsive element. 1.B i d . Chem. 268,5168-5177. Sevarino, K. A., Stork, P., Ventimiglia,-R., Mandel, G., and Goodman, R. H. (1989). Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity. Cell (Cambridge, Mass.) 57, 11-19. Shields, D. (1980). In vitro biosynthesis of fish islet preprosomatostatin: Evidence of processing and segregation of a high molecular weight precursor. Proc. N a t l . Acad. Sci. U.S.A. 77,4074-4078. Shields, D., and Blobel, G. (1977). Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. Proc. N a t l . Acad. Sci. U.S.A. 74, 2059-2063. Sorokin, A. V., Petrenko, 0. I., Kavsan, V. M., Kozlov, Y. I., Dababov, V. G., and Zlochevskij, M. L. (1982).Nucleotide sequenceanalysis ofthe cloned salmon preproinsulin cDNA. Gene 20,367-376. Spiess, J . , and Noe, B. D. (1985). Processing of an anglerfish somatostatin precursor to a hydroxylysine-containing somatostatin 28. Proc. N a t l . Acad. Sci. U . S . A . 82, 277-281. Steiner, D. F. (1990).The biosynthesis of insulin. Handb. E x p . Pharmacol. 92,67-92. Steiner, D. F., Peterson, J . D., Tager, H., Edmin, S., Ostberg, Y., and Falkmer, S. (1973). Comparative aspects of proinsulin and insulin structure and biosynthesis. A m . Zool. 13,591-604. Steiner, D. F., Smeekens, S. P., Ohagi, S., and Chan, S. J. (1992).The new enzymology of precursor processing endoproteases. 1.Biol. Chem. 267,23435-23438. Suzuki, S., Kawai, K., Ohashi, S., Mukai, H., and Yamashita, K. (1989). Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas. Endocrinology (Baltimore) 125, 3109-31 14. Taylor, W. L., Collier, K. J., Deschenes, R. J., Weith, H. L., and Dixon, J . E. (1981). Sequence analysis of a cDNA coding for a pancreatic precursor to somatostatin. Proc. N a t l . Acad. Sci. U.S.A. 78, 6694-6698. Thorens, B. (1992). Expression cloning of the pancreatic beta cell receptor for the gluco-incretin hormone glucagon-like peptide 1. Proc. N a t l . Acad. Sci. U.S.A. 89, 8641-8645. Unson, C. G., Macdonald, D., Ray, K., Durrah, T. L., and Merrifield, A. B. (1991). Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding. J. Biol. Chem. 266,2763-2766. Unson, C. G., Macdonald, D., and Merrifield, R. B. (1993). The role of histidine-1 in glucagon action. Arch. Biochem. Biophys. 300, 747-750. Wang, Y., Youson, J . H., and Conlon, J . M. (1993). Prosomatostatin-I is processed to somatostatin-26 and somatostatin-14 in the pancreas of the bowfin, Amia calvu. Regul. P e p t . 47,33-39. Wheeler, M. B., Lu, M., Dillon, J. S., Leng, X.-H., and Boyd, A. E. (1993). The rat glucagon-like peptide-I (GLP-I)receptor can couple to adenylate cyclase, phospholipase C and the free cytosolic calcium level ([CA2']i). Digestion 54, 348-349. White, J. W., and Saunders, G. F. (1986). Structure of the human glucagon gene. Nucleic Acids Res. 14,4719-4730.

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Yada, T., Itoh, K., and Nakata, M. (1993). Glucagon-like peptide-l-(7-36)amide and a rise in cyclic adenosine 3',5'-monophosphate increase cytosolic free Ca" in rat pancreatic p-cells by enhancing Ca2+channel activity. Endocrinology (Baltimore) 133, 1685-1692. Yamaji, K., Tada, K., and Trakatellis, A. C. (1972). On the biosynthesis of insulin in anglerfish islets. J . Biol. Chem. 247, 4080-4088. Yi-Qiang, W., Plisetskaya, E., Baskin, D. G., and Gorbman, A. (1986). Immunocytocheniical study of the pancreatic islets of the Pacific salmon, Oncorhynchus kisutch. Zool. S c i . 3, 123-129. Youson, J. H., and Potter, I. C. (1993).An immunohistochemical study ofenteropancreatic endocrine cells in larvae and juveniles of the Southern-Hemisphere lampreys Geotria uustrulis and Mordacia mordax. Gen. Comp. Endocritlol. 92, 151-167. Zhang, J., Desilets, M., and Moon, T. W. (1992a). Evidence for the modulation of cell calcium by epinephrine in fish hepatocytes. Am. J . Physiol. 263, E512-E519. Zhang, J,, DCsilets, M., and Moon, T. W. (1992b).Adrenergicmodulation ofCa2+homeostasis in isolated fish hepatocytes. Gen. Comp. Endocrinol. 88, 267-276.

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9 THE M O L E C U L A R BIOLOGY OF THE C O R P U S C L E S OF STANNIUS A N D R E G U L A T I O N OF STANNIOCALCIN G E N E EXPRESSION GRAHAM F . WAGNER Department of Physiology, Faculty of Medicine University of Western Ontario London, Ontario, Canada

I. Introduction 11. A Brief History of Discovery 111. Molecular Cloning of Eel and Salmon Stanniocalcin IV. Structural Comparisons of Eel and Salmon Stanniocalcin V. Studies on Tissiie-Specific Expression of the Stanniocalcin G e n e VI. Localization of Stanniocalcin mRNA in CS Cells by i n Situ Hybridization VII. Calcium Regulation of Stanniocalcin Cell Activity A. Regulation of Stanniocalcin Secretion by Calcium B. Regulation of Stanniocalcin mRNA Levels by Calcium VIII. Conclusions References

I. INTRODUCTION For over 150 years, zoologists have been intrigued by the corpuscles ofstannius, endocrine glands that were first described by Stannius (1839) on the kidneys of teleostean and holostean fishes. The glands are unique to bony fishes as they have not been identified in other vertebrates and are derived embryologically from kidney tubule cells (Garrett, 1942; Kaneko et al., 1992). In salmon and trout, the corpuscles of Stannius are readily apparent as oval, cream-colored bodies situated midway along the ventral surface of each kidney. On average, a typical salmonid has 2-6 glands of varying size. In other fish, however, the glands can vary both in location (i.e., ureter) and number. Species such as the bowfin (Amin culua) can have more than 300 individual 273 FlSH P H Y S I O I L K Y , VOL XI11

Cop\nght 0 1994 hv Academic Pre\\, IIK 411 right, of reproductinn in dnv form rewrvcd

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glands scattered throughout the posterior half of the kidney (Youson et al., 1976). But there is no evidence that variations in anatomical distribution have any bearing on their physiology. The corpuscles of Stannius (CS) play a major role in regulating calcium homeostasis through the synthesis and secretion ofstanniocalcin, a homodinieric, glycoprotein hormone with a novel primary structure. Stanniocalcin performs a function not unlike that of calcitonin in mammals, as one of its main roles is the prevention ofhypercalcemia. However, the two hormones acconiplish this through entirely different mechanisms. Whereas calcitonin inhibits osteoclastic bone resorption (Friedinan and Raisz, 1965; Milhaud et nl., 1965; Aliapoulios ct ul., 1966), stanniocalcin lowers the rate of gill calcium transport from the aquatic environnient (So and Fenwick, 1977, 1979). This underscores the differences between fish and mammals in the maintenance of calcium homeostasis. Unlike mammals, whicli rely on bone as a calcium reservoir, fish rely on the environnient as their principal source o f calcium and use the gills to draw from it according to metabolic needs. Despite the long passage oftinie since the CS discovery, the science o f their physiology has come of age only in the last 30 years beginning with Fontaine (1964), who first established a relationship between the C S and calcium homeostasis. The consequence of his discovery was renewed interest in these glands in laboratories all over the world. The CS have been extensively studied a s a result and are the subject of several reviews (Krishnamurthy, 1976; Wendelaar Bonga and Pang, 1986, 1991; Hirano, 1989; Wagner, 1993). In spite ofall that has been learned, however, the stanniocalcin field is still relatively unknown in comparison to other endocrine systems. The purpose of this chapter is to focus on recent developments concerning the molecular biology of the CS and stanniocalcin (STC), ’ specifically as they relate to tlie molecular cloning of STC, the loc,‘1I 1zation of STC mRNA in CS cells by i i i ,situ hybridization, and tlie regulation of STC gene expression by calcium. Because the niolecular biology of STC is a comparatively new field, there is not a large body of literature on the subject. Stanniocalcin has been cloned and sequenced from only two species of fish, salmon and eel. Therefore, readers should bear in mind that the perspective presented herein on comparative structure will be limited by the paucity of available data. Finally, few laboratories are presently engaged in studies on the regulation of STC gene expression, making it impossible to generalize with respect to salmonids, our chosen experiiiiental model, or bony fishes in gen-

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eral. As a consequence future reviews will be needed for periodic updates on this rapidly expanding and exciting field of research.

11. ,4 BRIEF HISTORY OF DISCOVERY Before Fontaine (1964) established a connection between the CS and calcium homeostasis, the glands were studied sporadically and by only a few laboratories around the world. Stannius (1839)believed that these structures were adrenal glands and, consequently, there were repeated efforts up until the inid-196Os to identify and characterize steroids and steroidogenic enzymes in CS tissue (Krishnamurthy, 1976). However, the ontogeny of CS cells was clearly different from that of fish interrenal tissue (Garrett, 1942), making it highly unlikely that they produced adrenal steroids at all. Ultrastructural studies then finally disclosed an extensive network of rough endoplasmic reticulum and Golgi and secretory granules in CS cells (Ogawa, 1967), all of which suggested that they synthesized polypeptides, not steroids. In a classical fashion, Fontaine (1964)demonstrated that surgically removing the CS in the European eel (Anguilln wnguilla) caused a form of hypercalceniia that could be alleviated simply by injecting CS extracts back into the animal. His findings established a connection between the CS and calcium homeostasis and inferred that the glands were the source of an antihypercalcenlic hormone, now known as stanniocalcin. Setting aside for the moment all queries as to the chemical nature of this active principle, efforts were focused on the cause ofthe hypercalceniia and the organs involved, again using the eel a s an experimental model. Originally it was believed that the hypercalcemia might be due to either decreased urinary calcium excretion or to increased mobilization of calcium froin bone. However, several laboratories independently concluded that removing the CS (stanniectomy) had no effect on the renal handling of calcium (Butler, 1969; Fenwick, 1974) or, for that matter, bone resorption (Fontaine et al., 1972). The environment was finally implicated as the source of calcium when it was observed that hypercalcemia did not develop if eels were transferred to low-calcium water following stanniectomy (Fenwick and So, 1974; Fontaine et al., 1972; Pang et nl., 1973). The gills were then pinpointed as the affected target organ when it was shown that the rate of gill calcium transport increased dramatically following stanniectomy, thereby revealing the true cause of the hypercalcemia. It was

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thus concluded that the CS were the source of an inhibitor of gill calcium transport ( Fenwick and So, 1974; So and Fenwick, 1977,1979). The active principle, STC, has since been purified from salmon and trout CS tissue and proven to be a potent inhibitor of gill calcium transport (Lafeber et al., 1988a,b; Wagner et al., 1986, 1988a, 1993). 111. MOLECULAR CLONING OF EEL AND

SALMON STANNIOCALCIN Literature searches turn up few studies of any kind dealing with nucleic acids in CS tissue prior to the molecular cloning of STC from the Australian eel, Anguillu australis (Butkus et al., 1987). There had been no attempts to even quantify KNA levels in CS cells, for example, or to identify the products of CS cells through in vitro translation of CS RNA. The few reports that existed at the time were mainly histological in nature. Tinctorial stains such as toluidine blue had been used to simply localize KNA in fixed tissue sections (Krishnamurthy, 1976). Furthermore, when Butkus and her colleagues began their quest for eel STC, the partial sequence of salmon STC had not yet appeared in press (Wagner et ul., 1986). As a consequence, they had little information at the outset regarding the size or structure of the hormone that could assist them in their cloning strategy. Accordingly, they devised an approach whereby the electrophoretic patterns of various eel tissue extracts were compared to that obtained with a CS extract. In this way they could identify proteins that were CS specific. Because of its sheer abundance in CS tissue, the STC band was selected for sequence analysis and proven to have a unique primary structure. On the basis of this sequence, an oligonucleotide probe (75-mer) was synthesized for screening an eel CS cDNA library and several positive clones were obtained. DNA sequence analysis of the largest clone yielded the complete primary structure ofeel STC, which consisted ofa 17-residue hydrophobic leader sequence, a 15-residue prosequence, and 231 amino acids comprising the mature protein core of the hormone. Northern blot analysis revealed that the eel STC message was 3.5 kilobases in length. Nothing further was accomplished in the field until the inolecular cloning of salmon STC (Wagner et al., 1992). Corpuscles of Stannius were collected from upstream migrating coho salmon (Oncorhynchus kisutch) as a source of RNA for library construction. Thereafter, the processes of oligonucleotide probe synthesis (50-mer) a n d library

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screening were made easier by knowledge ofthe N-terminal sequence of the coho salmon STC protein (Wagner et al., l988a). The entire salmon message was finally obtained in two cDNA clones that encompassed most of the 5' untranslated region, the complete protein coding region, and the entire 3' untranslated region. Northern blot analysis revealed that the salmon message was similar in size among representative salmonids ( 2 kb; Fig. l), but considerably smaller than in the Australian eel (3.5 kb).

-28s

-18s

Fig. 1. The rnRNA encoding STC is the same size ( 2 kb) among representative salmonids. Total CS RNA (30 w g per lane) from three representative salmonids-coho salmon (Oncorhynchus kisutch), arctic char (Salvelinus d p i n u s ) , and rainbow trout (0. mykiss)-was subjected to electrophoresis in 1%agarose/formaldehyde gels. The RNA was transferred to nitrocellulose and probed under conditions of high stringency with a "P-labeled cDNA corresponding to nucleotides 174-274 of coho salmon STC (see Fig. 2). Adapted from Wagner et u1. (1992).

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GRAHAM F. LVAGNER

IV. STRUCTURAL COMPARISONS OF EEL AND SALMON STANNIOCALCIN The molecular cloning of eel STC was an important achievement in the field simply because it provided the first complete primary structure of the hormone. Knowledge of the sequence from various species prompted Genbank searches for possible homologues, but revealed instead that the STC sequence was unique among vertebrate proteins (Butkus et al., 1987; Wagner et nl., 1986, 1992) and put to rest years of speculation that STC was structurally related to parathyroid hormone (Lopez et al., 1984).With the cloning and complete character-

10 20 30 40 50 GATATCAACA GCCCAACTGT TCTCCACCAA CAATTCAAGC CGACCTGTCC 60 70 80 AACCTATCCC ATCGAAGAAC ATCACCATCT GACAAG

90 100 110 120 130 ATG CTC GCA AAA TTC GGC CTG TGC GCG GTC TTC CTC GTC CTG GGA MET LEU ALA LYS PEE GLY LEU C Y S ALA VAL PHB LEU VAL LEU GLY -19 ILE LEU THR (-) VAL ARG MET S E R

140 150 160 170 ACT GCC GCC ACC TTC GAC ACC GAC CCG GAG GAA GCT TCT CCT CGC THR ALA ALA THR PHE A S P THR ASP PRO GLU GLU ALA S E R PRO ARG TYR GLU GLN ASP GLU S E R PRO LEU

-4

180 190 200 210 220 CGT GCA CGC TTC TCA TCC AAC AGC CCC TCG GAT GTG GCT AGG TGT ARG ALA ARG pHE S E R S E R ASN SER PRO SER ASP VAL ALA ARG CYS +12 THR A L A SER 230 240 250 260 TTG AAT GGC GCT CTA GCC GTG GGA TGT GGT ACG TTT GCC TGC CTG LEU ASN GLY LEU ALA VAL GLY C Y S GLY THR PHE ALA CYS LEU +27 GLN SER ALA

270 280 290 300 310 GAG AAT TCT ACC TGT GAC ACT GAT GGC ATG CAT GAT ATC TGT CAA GLU A S N S E R THR C Y S ASP T?IR ASP GLY MET HIS ASP I L E CYS GLN +42 ASP GLU ARG

Fig. 2. T h e complete cDNA and deduced amino acid sequence of coho salmon STC niRNA. T h e mRNA encodes 256 residues, 223 ofwhich comprise mature stanniocalcin. T h e amino acid residues that differ or that are missing entirely (-) in the Australian eel are shown beneath the salmon sequence. Underlined amino acid residues include the initiator methionine, the N-terminal phenylalanine of mature salmon STC, the asparagine-linked glycosylation consensus sequence, potential dibasic and tribasic cleavage sites, and the polyadenylation signal. The cleavage site between pre and proSTC, which occurs between Ala.,6-Tyr-l, in the Australian eel, has not been determined in salmon. From Wagner et a / . (1992).

320

330

340

350

CTG TTC TTT CAC ACC GCA GCT ACC TTT AAC ACA CAG GGT AAG ACA LEU PHE PHE HIS THR ALA ALA THR PHE ASN THR GLN GLY LYS THR +57 SER LEU GLY LYS 360

370

380

390

400

TTT GTA AAG GAG AGT CTG AGG TGT ATT GCC AAC GGT GTC ACG TCT PHE VAL LYS GLU SER LEU ARG CYS ILE ALA ASN GLY VAL THR SER +72 LYs ILE 410

420

430

440

AAA GTC TTT CAG ACC ATC AGG CGC TGT GGA GTC TTC CAG AGA ATG +87

LYS VAL PHE GLN THR ILE ARG ARG CYS GLY VAL PHE GLN ARG MET LEU SER SER LYs 450

460

470

480

490

ATT TCT GAG GTC CAG GAG GAG TGT TAC AGT AGA CTG GAC ATC TGT ILE SER GLU VAL GLN GLU GLU CYS TYR SER ARG LEU ASP ILE CYS +lo2 LYS LEU 510

500

520

530

GGT GTG GCT CGC TCT AAC CCT GAG GCC ATT GGA GAG GTG GTG CAG GLY VAL ALA ARG SER ASN PRO GLU ALA ILE GLY GLU VAL VAL GLN +117 SER GLN MET ALA 540 550 560 570 580 GTC CCT GCA CAC TTC CCC AAC AGG TAC TAC AGC ACT CTG CTC CAG VAL PRO ALA HIS PHE PRO ASN ARG TYR TYR SER THR LEU LEU GLN +132 SER GLN 590

600

610

620

TCC CTG CTA GCC TGT GAT GAG GAG ACA GTG GCT GTG GTC AGG GCA SER LEU LEU A I A CYS ASP GLU GLU THR VAL ALA VAL VAL ARG ALA +147 THR ASP GLU GLN 630

640

650

670

660

GGG CTT GTT GCT AGG CTG GGG CCA GAC ATG GAA ACT CTC TTC CAG GLY LEU VAL ALA ARG LEU GLY PRO ASP MET GLU THR LEU PEE GLN +162 SER GLU GLU GLY VAL 680

690

700

710

TTG CTG CAG AAC AAA CAC TGC CCC CAG GGT TCT AAC CAG GGT CCT LEU LEU GLN ASN LYS HIS CYS PRO GLN GLY SER ASN GLN GLY PRO +177 THR ALA PRO SER ALA ALA GLY THR 720

730

750

740

760

AAC TCA GCC CCC GCT GGC TGG CGC TGG CCA ATG GGG TCG CCT CCT ASN SER ALA PRO ALA GLY TRP ARG TRP PRO MET GLY SER PRO PRO +192 GLY PRO VAL GLY GLY SER ARG CYS PRO TRP GLY 770

780

790

800

TCC TTC AAG ATC CAG CCC AGC ATG AGA GGA AGA GAC CCC ACC CAC SER PHE LYS ILE GLN PRO SER MET ARG GLY ARG ASP PRO THR HIS PRO CYS SER ARG SER SER PRO THR CYS ALA PRO GLY THR PRO PRO 810

820

830

840

850

CTA TTC GCT AGG AAA CGC TCT GTG GAG GCA TTG GAG AGA GTG ATG LEU PHE ALA ARG LYS ARG SER VAL GLU ALA LEU GLU ARG VAL MET THR SER LEU LEU ARG ASN ALA ARG PRO PRO ASN TYR H I S PRO GAG GLU +223 PRO ARG LEU ALA LEU MET ASP CYS PRO 864

a74

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+222

+231

884

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TAGATTGGAG AAGAGGAGGC AGACATACAC ACCACTTATA CCTTAAGCAT ACATTCACAT

Fig. 2. Continued

280

GKAIIAXI F. \VAG;ZIER 974

GTACACACAC ACATACACAC ACCACAGCTA CCTTAAACAC AAAACACACT CATGATAGCT 1034

TTGCTCACAC ACACACTGAC TCACACGCAC ACTGACACAC ACACATTTTC ACACACATGC 1094

ACACACACAT AGCTTTACCC TCAAATGATT AAGGCTAAAT TATTAATGGA AGTTTGGGGC 1154

TGTTGTAATG TAGTATTTGA TTTGGGGAAG CATCTCTCTG TAAATGCTGT TGAAGGTATT 1214

TCTGTGTGGG TTGATACPTG ATGAAGGGGA GATGAAACCT GTTACCTAGA GCTTGAATGT 1274

GGAGGATTAT ATCTCCTCAG AATAGACTCG ACTAAACATG AGAGCTATTG AAAAGTCTGA 1334

ACATTTAATA TTAACAAGTG AAACA'MTCA AATGCCACCT AAGAAAACGA ACCATCACTG 1394

TAGTTCCATT GGATTTCAAC GTGGCCACTA CGGCCATAAC ATCCCCGTTT GGACCAGTCA 1454

TTAAAGACCG ATGGGTATAT TATTATAATA ATATTATTGA TATTTATTTT CTTACAGAAT 1514

G-ATTAAT

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CACCTCAGAG GGACAGTGTG ATGTCACCAT GCTGGTTGTG GGACTCACCG CGTCCCTCTC 1694

TGTCTl'CATC

TGTGTTAATG TAAGATCCTG TAGTGTGTAA AGACATTATA GAGTGATCCT 1754

TTGCTGTGTT CCTCAGATGT GGTTATGTGG TGTATGTTTT GAGATCCTGT GTGAGAATGT 1814

GTGCTAGTCA GGTACTATAC ACCTTGGGGC TTGGGGTCAT TCTCTCTGCT ATGAATACAT 1874

W G T G A C C T T C A T A A m T GTCTGAGGTG ATTTGTGTCG AGACTCACTG GTGATTAAAC 1934

GCTCACAGTT TCAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

Fig. 2. Continiicd

izatiori of salmon STC, it was finally possible to make some structural comparisons between species and pinpoint regions of the moleci1le that were more or less conserved. One such comparison is shown in Fig. 2, which illustrates the complete niicleotide and deduced protein sequence of coho salmon STC and amino acid substitutions a s they occur in the eel. Figure 3 , on the other harid, is a comparison of their nucleotide sequences, minus the 5' and 3' untranslated regions, which are completely divergent between the two species.

9.

28 1

R E G U L A T I O N OF S T A N N I O C A L C I N G E N E E X P R E S S I O N Coho Eel

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ATGCTCGCAAAATTCGGCCTGTGCGCGGTCTTCCTCGTCCTGGGAACTGC 1 3 6 I I I I I I I I I I

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TGCCTACGAGCAGGATGAGAGCGAGCCCTTATCTCCAAGGACA&G&&& 2 18

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TCTCATCCAACAGCCCCTCGGATGTGGCTAGGTGTTTGAATGGCGCTCTA 2 3 6

Eel

219

TCTCCGCCAGCAGCCCATCTGATGTTGCACGCTGTCTGAACGGGGCCCTG 2 6 8

Coho 237

GCCGTGGGATGTGGTACGTTTGCCTGCCTGGAGAATTCTACCTGTGACAC 2 8 6

Eel

269

CAGGTGGGCTGCAGTGCATTTGCCTGTCTTGACAACTCCACCTGCAACAC 3 1 8

Coho 287

TGATGGCATGCATGATATCTGTCAACTGTTCTTCTTTCACACCGCAGCTACCT 3 3 6

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TTAACACACAGGGTAAGACATTTGTAAAGGAGAGTCTGAGGTGTATTGCC 3 8 6

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&GACACACAGGGCAAGACTTTTGTGAAGGAGAGCCTGAAGT&&A&&

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468 AATGGCATCACCTCCAAAGTGTTC~TTACCATCCGCCGC

I 1

419

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II

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II

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CCAGAGAATGATTTCTGAGGTCCAGGAGGAGTGTTACAGTAGACTGGACA 486

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ccAGAAGATG AT&

469

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TCTGTGGTGTGGCTCGCTCTAACCCTGAGGCCATTGGAGAGGTGGTGCAG 5 3 6

Eel

519

TCTGCTCTGTTGCC~AGAGCAACCCAGAGGCCATGGGGGAGGTGGCCAA~ 5 6 8

Coho 5 3 7

GTCCCTGCACACTTCCCCAACAGGTACTACAGCACTCTGCTCCAGTCCCT 5 8 6

Eel

569

GTG~~CAGCCAGTTTCCCAACAGGTACTACAGCACCCTGCTGCAGAGTCT 618

Coho 5 8 7

GCTAGCCTGTGATGAGGAGACAGTGGCTGTGGTCAGGGCAG~CTTGTTG6 3 6

Eel

T&GA~GTGTGATGAGGACACCGTGGAGCAGGTGAGGGCCGGGTTGGTGT

IIII

I1 I 1

I 1

619

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I 1 I 1

668

Fig. 3. Comparative nucleotide sequences of coho salmon and Australian eel preproSTC. T h e DNA sequence encoding eel preproSTC is derived from Butkus et al. (1987) and nucleotides are numbered as in the original article. T h e nucleotide sequence of salmon STC is numbered as in Fig. 2. On the basis of a 774-bp overlap (minus the stop codon and including a 6-bp gap in the salmon sequence) and after introducing two gaps in the eel sequence to maximize alignment, there was 66.8% sequence similarity between the two species. The initiator methionine, N-terminal phenylalanine of mature STC, and the termination codons are shown in boldface in both species. From Wagner st

(11.

(1992).

Salmon and eel STC share certain structural features that are common to the other characterized STCs as well. In all known species, for instance, phenylalanine occupies the N terminus of the mature hormone. There is also a single glycosylation consensus sequence at the same position in all known species: Asn,,-Sei-30-Thr:,, (Butkus et

282

GRAHAM F. \VAGNER

Coho 637

CTAGGCTGGGGCCAGACATGGAAACTCTCTTCCAGTTGCTGCAGAACAAA 686

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~CCACCTGGAGCCAGAGATGGGGGTGCTCTTCCAGCTCCTCCAGACCAAG

I

669

Coho 687 Eel

719

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GCCTGCCCCCCAAGCG&GCCGGTGGCA&G&C&ATAGGGGCAGGAGG

7 18 732 768

Coho 733

..GCTGGCGCTGGCCAATGGGGTCGCCTCCTTCCTTCAAGATCCAGCCCA 780

Eel

769

CAGCTGGCGCTGCCCATGGG....GCCCCCCATGTTCAAGATCCAGCCCA 8 14

Coho 781

GCATGAGAGGAAGAGACCCCACCCACCTATTCGCTAGGAAACGCTCTGTG 830

815

ACCTGCGCTCCCGGGACCCCACCCACCTCTTTGCTAAGAAACGCTCGACC 864

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Fig. 3. Continued

u l . , 1987; Lafeber et al., l988a; Sundell et d., 1992; Wagner et al., 1986, 1988a, 1992). Whether or not nonglycosylated or more heavily glycosylated forms of the hormone occur in other fishes is unknown. However, it is interesting to note that STC from the bowfin (Arnia caluti) is incapable ofbinding to the plant lectin concanavalin A, distinguishing it from all other known species of the hormone (Marra et al., 1992). There are also numerous disulfide bonds in salmon and eel STC as a result ofthe large number ofhalf-cysteine residues (15in eel, 11in salmon), one of which must be unpaired and therefore involved in the formation of STC dimers. This has been conclusively proven in salmonids, where the native hormone is a dimer of identical subunits (Lafeber et al., 198811; Sundell et al., 1992; Wagner et ul., 1986,lYSSa) and is likely to be true in the eel as well, although definitive proof is lacking. The most salient differences between salmon and eel STC lie in their nucleic and amino acid sequences, particularly at the extreme ends ofthe molecule (Figs. 2 and 3 ) . In the prepro region for instance, encoded b y nucleotides 87-185 in salmon, the two species share only 49% identity in amino acid sequence and 51% identity at the nucleotide level. The eel hormone also has one less residue than s''I 1mon STC in this region. An even sharper divergence occiirs in the Cterminal region, STC171-223,encoded b y nucleotides 696-8.54 in salmon. Here there is 59% identity at the iiucleotide level (but only after introducing gaps in the sequences of both species to maximize alignment) and just 7.5% identity at the amino acid level. In comparison to the eel, salmon STC also has eight fewer residues on the C terminus. Yet there is reasonably high homology in the N-terminal

9.

REGULATION OF STANNIOCALCIN G E N E EXPRESSION

283

and midmolecule regions at both the nucleotide (72%)and amino acid level (78%);this region is STC,-,,, encoded by nucleotides 186-695 in salmon. Because this latter region is highly conserved, it likely contributes more than the C terminus does to STC receptor binding and subsequent biological activity. This has been born out in bioassays where N-terminal fragments of eel and salmon STC (STC,-,,,) are capable of inhibiting gill Ca2+transport (Milliken et al., 1990; Verbost et ul., 1993). However, these fragments are less potent on a molar basis than native STC. Hence, full biological activity clearly requires more than the first 20 amino acids of the hormone. Salmon and eel STC also share a dibasic pair (Arg,,-Arg,,) that may be used for bringing about posttranslational modifications to the hormone. There are two additional sites in salmon, one dibasic (Lys167-His168) and one tribasic (Arg,ll-Lys212-Arg213), which also appear accessible to proteolytic attack based on a hydropathy profile of the salmon hormone. There are nuinerous monobasic sites in both species that also could be cleaved. Posttranslational modifications to salmon STC do occur. Numerous truncated forms of the hormone are found in CS tissue (Wagner et d., 1988b, 1992) and the circulation (Wagner et al., 1991, 1993) and are released by CS cells in vitro (Wagner, 1993). Interestingly, there is no evidence of this occurring in the eel (Butkus et nl., 1987, 1989; Wagner et al., 1992).

V. STUDIES ON TISSUE-SPECIFIC EXPRESSION OF THE STANNIOCALCIN GENE Southern blot analysis reveals that there is more than one copy of the STC gene in salmonids (Fig. 4). This is not particularly surprising considering the tetraploid karyotype of salmonids; multiple genes have already been described for most salmonid hormones. But it has not been established that all gene copies are functionally expressed in CS tissue or whether they might also be expressed at other loci. As yet, there is little evidence for ectopic STC production in fish. However, the possibility always exists that one or more gene copies may be ectopically expressed and perhaps have novel functions at these loci, as in the case of placental growth hormone and decidual prolactin in primates. There are reports of STC immunoreactivity in fish brain and pituitary (Fraser et al., 1991), but they have not been characterized further. We have been unable to identify STC-immunoreactive cells in any tissue other than the corpuscles of Stannius. Northern blot and

A

S

C

T

S

B C

T

23

99

=4

-2

Fig. 4. Evidence that there is inore thaii one cop>-of the STC gene in salnionids. Swmples of DN.4 (15 pgilane) froin three salmonids-chinook salmon, Oiicorliyt~cl~us t.cliciiclyt.rclzcr (S), arctic char, S a l c e l i n u ~cilj~inus (C), and rainbow trout, 0. myki.c.s (T)-were digested with Pst 1 and subjected to Southern blot analysis. T h c blot w a s first prol)ed with a 100-bp fragment encoding the N terminus of salmon STC (panel A ) , then stripped and probed a second time (panel B) with a near full-length cDN.4 (1.7 kb). Panel A illustrdtes that the smaller cDNA probe hybridized to three 01-four fragments in each species following Pst 1 digestion. The arrows refer to fragnrents that 1iyl)ridized uniquely to this probe. Panel B illustrates the same blot probed instead \\it11 the 1.7-kb cDNA clone. The larger probe hybridized to many ofthe sanie fragments a\ the smaller probe, but additional fragments were revealed that hybridized uniquely to the larger probe (arrows and arrowheads). T h e key evidence for multiple gene copies is shown in panel A . Pst 1 digestion of salmon and trout DNA yielded four genornic fragments that hybridized to the 100-bp prohe. If there is only one copy of the gene in salmon and trout then it must contain tlirec. Pst 1 sites in the region encoded b, the probe. However, there is only o ~ i ePst 1 site in the entire e D N A sequence of salmon STC and it lies outside the region encoded b y the 100-lip probe. If there are three Pst 1 sites in the gene within the sanie 100-bp sti-etch encoded by the probe, this short region o f t h e gene would have to be interrupted I)>- three introns, each containing a Pst 1 site. This is highly improbable and argues for nriiltiple copies of the gene. From Wagner ct a / . (1992).

9. HEGULATION

OF STANNIOCALCIN G E N E EXPRESSION

285

in situ hybridization analyses of a wide range of salmon tissues (brain, pituitary, urophysis, pancreatic islets, thyroid, digestive tract, spleen, gonads, and heart) have likewise yielded negative results (Sterba et al., 1993; Wagner et al., 1992). And yet we still cannot rule out the possibility of the gene being expressed in other tissues, perhaps at some early stage in the life history of the fish. Future studies should probably approach this question with more sensitive methods of detection such as polymerase chain reaction technology. But until conclusive evidence is forthcoming, we should proceed on the assumption that the STC: gene is expressed exclusively in CS cells.

VI. LOCALIZATION OF STANNIOCALCIN mRNA IN CS CELLS BY I N SZTU HYBRIDIZATION An unusual histological feature of CS tissue is that the secretory activity of STC cells varies in different parts of the gland. In the CS of Colisa M i a , for example, successive rounds of depletion and subsequent repletion of stored hormone occur in select regions ofthe gland (Krishnamurthy, 1976). The availability of purified STC and specific antisera has allowed us to corroborate this phenomenon with greater precision using immunocytochemistry. We have found that it also occurs in winter flounder and sockeye salmon, for instance, and is manifested simply as low levels of immunoreactive hormone in specific regions of the gland (G. F. Wagner, unpublished observations). We have explored this phenomenon further by in situ hybridization using "S-labeled probes with interesting results (Sterba et al., 1993). First, there is always evidence of STC gene expression in every region of the gland. STC mRNA levels are often barely detectable, yet there is always a constitutive level of expression throughout the gland. Seeond, in some cases the level of' gene expression is equal throughout the gland (Figs. 5A and 5F),but typically it varies widely and is highest on the CS perimeter (Figs. 5C-5E), abutting either kidney tissue or the intraperitoneal cavity. There are statistically higher message levels in lobules of cells on the perimeter as compared to the center of the gland (Table I). Lastly, the level of STC gene expression is obviously a good indicator of STC synthesis. Throughout all regions of the gland, the levels of STC mRNA are closely correlated with the levels of immunoreactive hormone (Table I ) ; this is illustrated most convincingly in Fig. 6. It is apparent from these findings and those of Krishnamurthy (1976) that all CS cells are not in synchrony, synthesizing and secreting STC together at the same rate, and that this phenomenon is

Fig. 5. Dark-field illumination of sockeye salmon (Oncorhynchus nerku) CS following i n situ hybridization with "S-labeled STC cRNA probes. The positive hybridization signal appears as small silver grains, whereas the large white spots are pigment granules i n the kidney to which sense and antisense probes bound nonspecifically. (A) Silver grains are densely localized over CS tissue and much less evident over surrounding kidney tissue following the use of antisense probes. The level of STC gene expression is evenly distributed throughout the gland. (B) Specific hybridization is not evident o n a tissue section adjacent to A following the use of sense probes. (C) An example of variable STC gene expression in salmon CS tissue. Note the higher level of expression o n the perimeter ofthe gland adjacent to kidney tissue. (D)A second example ofvariable STC gene expression. In this case, the highest level ofexpression occurs on the perimeter

9.

REGULATION OF STANNIOCALCIN GENE EXPRESSION

287

Table I STC mRNA and Immunoreactive STC Levels in Sockeye Salmon CS Cells as Assessed by Morphometric Analysis"

CS region

STC mRNA (grains per cell)

Immunoreactive STC (optical density)

All glandular cells Peripheral lobular cells Central lobular cells

27.8 5 11.1 74.7 2 14.2 11.5 2 10.9

0.038 ? 0.008 0.067 2 0.01 0.025 5 0.007

~~~~

"

N = 40 CS, data expressed as means

~

?

S.E.M. From Sterba et al. (1993)

widespread among fishes. How and why it occurs is uncertain, but it may be ficilitated by varying CS regional blood flow and is possibly a strategy for placing the burden of secretion on one region of the gland, while allowing the remainder of the gland to concentrate on renewed hormone synthesis. A disadvantage to using radioactive probes for in situ hybridization as illustrated in Figs. 5 and 6 is the lack of resolution inherent in autoradiography. As a consequence, it is difficult to pinpoint the exact cellular location of the mRNA using this technique. To circumvent this problem, we have turned to a nonisotopic method that uses digoxigenin-labeled cRNA probes for more precise localization of mRNA. The ability to localize STC mRNA within the cell has allowed us to visualize the sites of hormone production and furthered our understanding of structure-function relationships in CS cells. In sockeye salmon for instance, the CS glands are coniposed of individual lobules of concentrically arranged cells (Fig. 7). There is also a welldefined polarity to the organelles in these cells, whereby secretory granules are tightly packed on the lobule perimeter nearest the surrounding capillaries and cell nuclei are found at the opposite pole near the center of the lobule (Wagner et al., 1988b).The concentration of secretory granules on the lobule perimeter against the basolateral

of the gland facing the intraperitoneal cavity. (E) A third example of variable gene expression. In this case, the highest level of expression occurs in all the lobules on the perimeter of the gland. ( F ) As in panel A, the level of STC gene expression is evenly distributed throughout this particular gland. CS = corpuscle of Stannius; k = kidney; tfe = tissue-free environment or intraperitoneal cavity; calibration bar = 100 pm.From Sterba et al. (1993).

288

GRAHAM F. [L’AGSER

Fig. 6 . Correlative in situ hybridization (A) and immunocytocheniistry (B) in sockeye salmon corpuscles of Stannius. (A) Note the high levels of STC mRNA in half of the gland. Nonetheless, a low but discernable level of gene expression is evident i r i the other half as well. (B) Note that the levels of imniunoreactive STC are highest i n the region exhibiting the highest level of gene expression. CS = corpuscle of Stannius; k = kidney; calibration bar = 100 pni. Adapted from Sterba et u1. (1993).

9.

REGULATION OF STANNIOCALCIN GENE EXPRESSION

289

membrane ensures rapid release of STC into the circulation upon the appropriate stimulus. It now appears that STC mRNA also is polarized within the cell. The use of high-resolution, digoxigenin probes has revealed that STC mRNA is concentrated at the apical cell membrane nearest the center of the lobule, so that cell nuclei lie between the secretory granules at the one pole and STC mRNA at the other (Fig. 7). Ultrastructural studies on salmon CS cells have revealed that the apical pole is also rich in rough endoplasmic reticuluni (Carpenter and Heyl, 1974; Meats et nl., 1978).Therefore, it appears that newly synthesized message is preferentially released on the apical side of the nuclear envelope and becomes associated here with ribosomal RNA and the endoplasmic reticulum for the initiation of new hormone synthesis.

VII. CALCIUM REGULATION OF STANNIOCALCIN CELL ACTIVITY A. Regulation of Stanniocalcin Secretion by Calcium

The notion that STC cells might be responsive to calcium was first deduced on the basis of histological evidence. Beginning in the 1960s, it was commonly observed that transferring fish from fresh water to seawater altered the appearance of CS cells. Among the noted cytological changes were increased protein synthesis, nucleolar and nuclear hypertrophy, increased amounts of endoplasmic reticuluni and Golgi, cellular hypertrophy, glandular hypertrophy, and increased secretory activity of CS cells (Krishnamurthy, 1976, Wendelaar Bonga and Pang, 1986).The cause of these changes, in particular the secretory response, was subsequently identified as the calcium content of the water (Pang Fig. 7. I n situ localization of STC mRNA in sockeye salmon corpuscles of Stannius using a digoxigenin-labeled antisense cRNA probe. T h e black deposits throughout the tissue correspond to STC mRNA. Note how the cells are arranged into lobules that together form an individual corpuscle. Capillaries are found primarily in the clear regions between the lobules. T h e large arrow points to a cluster of three cell nuclei. T h e sinall arrows point to the lobule perimeter and site of the basolateral cell membrane, where secretory granules are concentrated and poised for release into the perivascular space. STC mRNA is localized for the most part at the opposite pole, against the apical cell membrane. Note that in most cases, CS cell nuclei lie between secretory granules and STC mRNA on the basolateral and apical cell poles, respectively. From T . Sterba and G . F. Wagner, unpublished.

290

GRAHAM F. LVAGXEH

et al., 1973, 1974; Pang and Pang, 1974; Cohen et al., 1975),which is much higher in seawater (10 m M ) compared to fresh water (0.11.0 mM). It was concluded that the movement of calcium across the gills, gut, and integument after seawater transfer raised plasnia calcium levels to an extent that stimulated CS cells. It was subsequently shown that inducing hypercalcemia in 2jiz)o prompted a secretory response as well (Lopez et al., 1984), as did exposing glands in u i tr o to high calcium levels (Aida et al., 1980). From a physiological standpoint these findings made sense; cells that secreted a calcium-regulating hormone were in turn responsive to calcium levels in the extracellular compartment. Cytophysiological studies on CS cells were rendered obsolete with the purification of STC and the subsequent development ofimmunoassays (Gellersen et al., 1988; Mayer-Gostan et al., 1992; Wagner et al., 1Y9l), which now made it possible to quantify hormone release and assess the actions of reputed secretagogues (i.e., calcium). Both in c i t r o and in 2jiz;o model systems have since been used to explore the effects of calciuni on STC secretion, employing species such as rain1)ow trout (0.mykiss), coho salmon, Atlantic salmon (Salino salar), and, of course, the European eel. We have relied solely on primary cultured trout CS cells for our own in vitro studies because of their wide availability and ease of culture in a variety of media forniulations (Gellersen et al., 1988; Wagner et al., 1989). Our findings suggest that trout CS cells are extremely sensitive to changes in ionic calcium levels within the physiological range. Between 0.3 and 2.4 mM calcium, these cells undergo stepwise increases in STC secretion with each successive rise in calcium concentration (Fig. 8). The calcinmresponse curve is steepest around the physiological set point (- 1.2 mM Ca”), where CS cells are most responsive, and levels off’ at higher and lower calcium concentrations. This is precisely how these cells should respond given their role in preventing hypercalcemia and is wholly reminiscent of‘ calcium regulation of calcitonin secretion in mammals (Anast and Conway, 1972; Gage1 et ul., 1980). There is a l s o a temporal aspect to the secretory response as its magnitude increases with increasing length of exposure to calcium (Fig. 9A). These effects of calcium are not mimicked by magnesium (Fig. 9B), or by the principal monovalent ions in plasma, sodium and chloride (Wagner et al., 1989). Conflicting findings have been reported in the European eel, where cultured CS glands are reputedly unresponsive to changing calcium levels within the physiological range (Hanssen et aZ., lYYl), prompting the authors ofthis study to challenge the notion that calcium is a regulator of STC secretion. However, as their findings are com-

9.

R E G U L A T I O N OF STANNIOCALCIN CENE EXPHESSION

400

I

300

I

200

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291

3 4

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0.7

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1.9

m M IONIC CALCIUM Fig. 8. Stanniocalcin secretion by primary cultured rainbow trout CS cells is positively regulated by ionic calcium within the physiological range. Trout CS cells were maintained for 4 h r in serum-free RPMI media containing increasing concentrations of ionic calcium. Between 0.3 and 1.9 mM calcium, CS cells exhibited stepwise increases in stanniocalcin secretion. T h e secretory response was steepest around the set point (-1.2 m M ) and leveled off at higher and lower calcium concentrations. Each data point represents the mean ? S.E.M.ofthree replicate cultures (0.5 x 106cells/well).Redrawn from Wagner et al. (1989).

pletely at odds with previous work on this species (Fenwick and Brass e w , 1991; Lopez et al., 1984), an alternative explanation may lie in their use of a heterologous, salnionid STC immunoassay to quantify eel STC release. Salmon and trout CS cells respond similarly i n civo to elevations in plasma calcium levels, delivered via intraperitoneal or intra-arterial injections (Glowacki et al., 1990; Hanssen et al., 1991; Wagner et nl., 1991). There is a defined time course to the secretory response (Fig. 10). The response is dose-related in the sense that larger dosages of calcium result in more sustained elevations in hormone levels and magnesium has no effect on hormone release (Wagner et a,?., 1991). The similarities in the i n vivo and in citro responses to calcium support the notion that CS cells are finely tuned calcium sensors, capable of modulating secretory activity in accordance with changing levels of extracellular calcium. A rise in plasma calcium provokes a measured

292

GRAHAM F. \VAGhER

80

70

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60

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W

C

50

Y

3z

40

z

6 cn

r

30

20

1

10

0.5

1

2

4

HOURS of EXPOSURE to 1.8mM Ca2+

0

0.62 1.25 1.88 2 5 3.12

MAGNESIUM (mM)

Fig. 9. (A) Stanniocalcin secretion by primary cultured CS cells increases with length of exposure to calcium. Rainbow trout CS cells were maintained for up to 4 hr in serum-free RPMI media containing 0.3 rnM (solid bars) or 1.6 nlk! ionic cakiunl (open bars). STC secretion increased 5-fold after a 30-min exposure to 1.6 tnM calcitini. T h e rate of STC secretion then rose progressively between 1 hr (&fold) and 2 hr (10fbld) and had leveled off by 4 hr (4-fold). Each data point represents the mean 2 S.E.M. of three replicate cultures (0.5 x loficellsiwell). (B) Magnesium has no effect on STC secretion. Trout CS cells were exposed for 4 hr to increasing amounts of magnesium. Each data point represents the mean t S.E.M. of three replicate cultures (0.5 x lo6 cellsiwell). From Wagner et ul. (1989).

increase in STC secretion, which causes a corresponding reduction in the rate of gill calcium transport. As plasma calcium levels decline, there is a gradual drop in the rate of STC secretion until norniocalcemia (or the set point) has been reestablished. The regulation of STC secretion b y calcium resembles that of mammalian calcitonin, which is also

9.

293

REGULATION OF STANNIOCALCIK GENE EXPKESSION

IS

Fig. 10. Plasma levels of stanniocalcin and total calcium in free-swimming, adult rainbow trout after an intra-arterial infusion of calcium (5 mgikg). Blood samples were withdrawn before and after infusing calcium chloride through a dorsal aorta cannula. Note the quick rise in plasma STC levels within 5 min of infusing calcium (sixfold) and the rapid restoration of nonnocalcemia. T h e reason for the comparatively slow restoration in plasma hormone levels is unknown. From Wagner et al. (1991).

positively regulated b y calcium (Anast and Conway, 1972; Gagel et al., 1980),but is in contrast with parathyroid hormone (PTH), which is negatively regulated b y calcium (Brown et d., 1987). B. Regulation of Stanniocalcin m K N A Levels b y Calcium Little is known about the regulation of STC synthesis at either the transcriptional or posttranscriptional level. In the case of PTH, calcium regulates the biosynthetic pathway at two different levels. Low levels of plasma calcium stimulate PTH gene transcription and discourage newly synthesized hormone from entering a degradative pathway. Meanwhile, low levels of plasma calcium are also a stimulus for PTH secretion. Hence, the regulation of' PTH secretion is tightly coordinated with renewed hormone synthesis, thereby ensuring that a constant supply of PTH is always available for release. In view of the

294

GKAIIAM F. \VAGNER

regulatory effects of calcium on STC secretion, it would make sense for calcium to have a regulatory role in hormone biosynthesis as well. There is, in fact, one study in rainbow trout which has shou711 that administering repeated calcium injections over several days (presumably to deplete the glands of STC) has a significant effect (1.7-fold) on the rate of hormone synthesis (Flik et al., 1990). The notion that calcium stimulates STC biosynthesis is supported as well b y histological observations. For instance, the CS are more active in fish adapted to seawater or water that is simply high in calcium content. The CS cells in marine fishes have a more extensive endoplasmic reticulum and Golgi apparatus and a higher content of secretory granules, and generally have increased nuclear and cytoplasmic volumes in comparison to their freshwater counterparts (Krishnamurthy, 1976, Wendelaar Bonga and Pang, 1986, 1991). Stages in life history can also influence the activity of STC cells, especially ifthey involve changes in calcium metabolism. Reproduction in the Indian catfish ( M y s t u s uittatus), for instance, is correlated with large increases in both serum calcium and the mean nuclear diameter of CS cells (Ahmad and Swarup, 1990). The nuclear hypertrophy that occurs in CS cells may be indicative of increased STC gene expression to accommodate higher levels of hormone secretion. Although STC gene expression has not been nionitored in fish under different environmental conditions, calciiim does have direct effects on steady-state mRNA levels in primary cultured, rainbow trout CS cells. Moreover, as in the case of calcium-stimulated~ secretion, the effects ofcalciiirn on STC me ge levels are dependent ou both concentration and length of exposlire (Wagner and Jaworski, 1994). Short exposure times have only modest effects. For instance, exposing trout cells to calcium for 24 hr produces small, stepwise increases in STC mRNA levels between 0.7 and 1.9 mizl calcium and a maximum %fold induction in comparison to controls (Fig. 111. However, 3-day exposures produce steeper calciuni-response c u r \ ~ s (Fig. 12). There is also a greater induction of message levels following 3-day exposures to calcium (1.7-to 3-fold). Even longer exposure times, in this case 6 days, have the most pronounced effects on gene expression, resulting in steeper response curves and inducing message levels a s much as 14-fold in comparison to controls (Fig. 13).What is most interesting about these findings is that STC secretion and STC gene expression in trout CS cells are both subject to regulation over the same range of calcium concentrations, and that the maximum response in both cases occurs around 1.9-2.3 mM calcium (compare Figs. 8 and 12).That both STC secretion and mRNA levels are siinilarly regulated by calcium makes sense from a physiological standpoint. Above all,

9.

295

REGULATION OF STANNIOCALCIN GENE EXPRESSION

0.7

1.1

1.5

1.9

2.3

2.1

0

I

I

I

1

50

100

150

200

STCIActin, % of Control Fig. 11. STC mRNA levels in primary cultured rainbow trout CS cells following a 1-day exposure to calcium. STC mRNA levels were progressively stimulated between 0.7 and 1.9 mM calcium but were inhibited by higher calcium levels. Message levels were maximally induced 1.3- to 2-fold over controls (1.1 mM Ca”) in three separate experiments. Cell cultures were maintained in Leibovitz media (0.4x lo6 cells/well). Total RNA was harvested from each well of cells and subjected to Northern blot analysis as described in Fig. 1. The blot was then stripped and reprobed with a cDNA encoding carp beta actin. After X-ray film exposure, the STC and actin bands were quantified by densitometry and expressed as STC/actin mRNA ratios. For statistical analysis, all data were expressed as a percentage of controls and subjected to arcsine transformation. Each data point represents the mean rt S.E.M. ofthree replicates (*P< 0.05 in comparison to controls; two-tailed ANOVA and Dunnet’s test). Adapted from Wagner and Jaworski ( 1994).

it ensures a continuous supply of template for hormone synthesis, which would be especially important in high-calcium, marine environments where greater secretory demands are placed on CS cells (Glowacki et al., 1990; Mayer-Gostan et al., 1992). Oddly enough, STC mRNA levels in cells from seawater-adapted salmon are regulated

296

GRAHAM F. If'AGXER

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0.7

1.1

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1.5

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.-E

1.9

0 I

2.3

L

m**

2.7

I

I

1

I

I

1

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50

100

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250

STC/Actin, % of Control B

STC

c*

I

0.7 mM

I

1 . 1 mM

I

1.5 mM 1 1.9 mM Ionic Calcium

I

2.3 mM

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2.7 mM

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Actin

Fig. 12. (A) STC mRNA levels in rainbow trout C 5 cells after a 3-dav exposuie to ~alciuiii Three-day exposure5 result in steeper response curves between 0 7 a i d 1 9 ink' cakiuni and more pronounced effects o n message levels STC mRNA le\el\ were maximally \timulated between 1 9 and 2.3 mM calciuin and inhibited I>\ higher calciiiin levels. Message levels were maximally induced 1.7- to 3-fold over controls (1 l inM Ca2+)in three separate experiments. Each data point represent\ the mean ? S.E.Xl of three replicate5 ( * P < 0.05, **P < 0 01 in comparison to controls, two-tailed ANOVA and Dunnet'\ te\t) Cell culture conditions, Northern blotting, arid data analv\i\ were a\ described in Fig 11 (B) Autoradiographs of STC and actin mHNA from replicate \veil\ of cell\ in the dewribed experinlent Adapted from Wagner and Tauorski (1994)

9.

297

REGULATION OF STANNIOCALCIN GENE EXPRESSION

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.-E,

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STC/Actin. % of Control

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SIC

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Actin 1.5 mM 1.9 mM Ionic Calcium

I 2.3 mM I

Fig. 13. (A) STC niRNA levels in rainbow trout CS cells after a 6-day exposure to calcium. Six-day exposures produced the steepest response curves and the greatest induction of message levels. STC mRNA levels were inaximally stimulated between 1.9 and 2.3 m M calcium and again inhibited by higher calcium levels. Message levels were maximally induce 3-, 11-, and 14-fold over controls (1.1 m M Ca") in three separate experiments. Each data point represents the mean 2 S.E.M. of three replicates (**P < 0.01 in comparison to controls; two-tailed ANOVA and Dunnet's test). Cell culture conditions, Northern blotting, and data analysis were as described in Fig. 11. (B) Autoradiographs of STC and actin mRNA from replicate well ofcells in the described experiment. Adapted from Wagner and Jaworski (1994).

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GKAHAhl F \f’AGNEK

over the same range as in trout (0.3-l.Y inM calcium) and stimulated

by calcium to roughly the same extent (1.6-fold; Fig. 14). One might expect CS cells from a marine fish to be more sensitive and exhibit greater responses to calcium, which may indeed be the case. However, any differences that do exist between marine and freshwater fishes may be apparent only under in cico conditions and may be lost in

0.3

0.7

2.7 1.9 mM Mgz‘

I

I

0

50

I

100

I

150

200

STCI 18s RNA; % of Control Fig. 14. STC m R S A levels in sea\vater-adaijted Atlantlc salnlon C S cells tollo\~~inq :i-day exposurc to calcium. The responses of salmon and trout CS cells were similar. There was a progressive rise iri STC mHNA levcls of salmon cclIs between 0.3 a ~ ~ d 1.0 mhf calciunr, after which message levels declined. Notice that 1.9 mA1 magncsiiuir ( i n the presence of 1.2 milf calcium) had no eftrct on message level\. Cell culture were a s described in Fig. 11, except contfitions, Northern blotting, and data anal) that a rabbit 18s probe was used in lieu of carp actin to normalize the data. Each data point represents the mean t S.E.RI. of three replicates (*P < 0.01 in comparison t o controls in 1.1 inM Cii” ; two-tailed ANOVA and Dunnet’s test). From C;. F. \\’agner, uii~mblished. ;i

9.

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REGULATION OF STANNIOCALCIN GENE EXPRESSION

the primary cultured cell. Magnesium is not a regulator of STC gene expression in either species of salmonid (Figs. 14 and 15), which is expected because it also has no effects on secretion (Wagner et ul., 1989). It also strengthens the notion that of the major plasma electrolytes, calcium alone is a regulator of CS cell and STC gene activity.

1.2 m M Ca"

2.3 m M Mgz'

2.3 m M Ca"

I

I

I

I

I

1

0

50

100

150

200

STC/Actin, % of Control Fig. 15. Magnesium does not stimulate STC mRNA levels in rainbow trout CS cells. Cultured cells were exposed to 2.3 niM Ca" or 2.3 m M Mg'+/l.2 niM Ca" for 3 days and analyzed for STC mRNA content as described in Fig. 11. Calcium prompted a 1.8-fold induction of message levels over controls (1.2 m M Ca2+)whereas magnesium had no effkct. In additional experiments, &day exposures to magnesium were also without effect. Cell culture conditions, Northern blotting, and data analysis were as described in Fig. 11. Each data point represents the mean 2 S.E.M. of three replicates ( * P < 0.01 in comparison to controls in 1.2 m M Ca"; two-tailed ANOVA and Dunnet's test). Adapted from Wagner and Jaworski (1994).

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CS cells are unique in their ability to modulate STC mRNA levels bidirectionally in accordance with ambient calcium levels (Figs. 11-14). This is a quality that is not shared by PTH and calcitonin cells. The PTH gene, for example, is regulated in only one direction by calcium i n vioo and i n oitro (Brookman et al., 1987; Heinrich et al., 1983; Naveh-Many and Silver, 1990; Russel et al., 1983; Mouland and Hendy, 1991), whereas the calcitonin gene is not influenced by calcium at all (Naveh-Many et al., 1989, 1992). Therefore, primary cultured CS cells are exceptional by comparison, as STC mRNA levels are increased and decreased, respectively, by calcium concentrations about and below the physiological set point (-1.2 mM). Nevertheless, it is crucial that the regulation of STC gene expression is also characterized under in cico conditions. Whether or not the gene is siniilarly regulated b y calcium in the whole animal will be of particular interest. We have conducted only one i n vivo study to date, in this case on

CaC4 NaCl

9080

-

70

-

I

I

24

48

TIME AFTER INJECTION (hrs) Fig. 16. STC mRNA levels in calcium-challenged sockeye salmon as quantified by .situ hybridization. Juvenile, freshwater salmon (50 2 10 g) were given intraperitoneal injections of NaCl ( 0 )and CaC1, (V)equivalent to 30 mg/kg body weight of sodium and calcium. Five animals were sacrificed from each group at different times postinjection and CS glands were processed for in situ hybridization using 3sSS-labeledcRNA probes. All slides were developed at the same time and subjected to grain counting as previously described (Sterba et ul., 1993).Calcium prompted a rise in STC mRNA levels at the 5-hr mark that was statistically insignificant. Each data point represents the mean S.E.M.of five corpuscles ofStannius. From T. Sterbaand G. F. Wagner, unpublished. it1

*

9.

RE(:LJL.AI‘lON OF STANNIOCALCIN GENE EXPRESSION

30 1

juvenile, freshwater-adapted sockeye salmon that were given intraperitoneal injections of sodiuni or calcium chloride (equivalent to 30 mg/ kg of Na’ or Ca”). The fish were sacrificed at different times postinjection (5, 24, and 48 hr) and because of the small size of the CS glands in these animals, STC mRNA levels were quantified by in situ hybridization and grain counting (Sterba et al., 1993). As expected, the calcium-injected salmon had elevated plasma STC levels at the 5-hr mark in comparison to sodium-injected controls. However, there were no statistically significant effects of calcium on STC mRNA levels at any time postinjection, though a small rise was apparent after 5 hr (Fig. 16). It is possible that minor hypercalcemic challenges do not require increased STC gene transcription and can be accommodated merely by increasing the rate of hormone synthesis from preexisting message. Increased rates of transcription may only be required in the event of a continuous calcium challenge such as that afforded by the marine environment.

VIII. CONCLUSIONS

The purpose of this chapter has been to provide a current perspective on the molecular biology of the corpuscles of Stannius and stanniocalcin. It should be readily apparent to the reader that the STC field, in spite of 30 years of progress, is still in its infancy in comparison to most other areas of endocrinology. This is true not only with respect to gene structure and function, but also as it applies to basic hormone physiology. Over the last 20 years, for instance, the only function definitively shown to be regulated by STC has been gill calcium transport. Only more recently have the intestinal transport of calcium (Sundell et al., 1992) and the renal handling of phosphate (Lu et al., 1994) been identified as being under the influence of stanniocalcin. Similarly, little is known ahout the regulation of STC secretion by other hormones, the nervous system, the life history of the fish, diet, or season, to name just a few factors ofpotential influence. In all fairness, part of the problem has to do with STC itself. The molecule is notoriously difficult to iodinate without causing irreparable damage to both its receptor binding and antibody binding properties. Consequently, there are grave difficulties inherent in identifying new targets and/or actions of STC and in studying the regulation of STC secretion using the traditional methods of radioreceptor assay and radioimmunoassay, respectively. Fortunately, these problems do not apply to studies on gene regulation, which are nonetheless still in the developmental

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stages. STC has been cloned and fully characterized in only two species of fish. However, several new clonings are currently in progress and the sequence information that will soon be forthcoming should broaden our perspective on the evolution of hormone structure and function even further. This is vitally important because unlike most other fish hormones, STC lacks an evolutionary perspective owing to its apparent absence in other vertebrates. The studies described here on calcium regulation of STC mRNA levels represent only the first in a series of steps by our laboratory to further our understanding of how this gene is regulated in salmon. Our next step is to determine the mechanisms by which calcium alters message levels; these include increased mRNA stability, an increased rate of gene transcription, or perhaps a combination of the two processes. Given the high level of induction that occurs after 6-day exposures to calcium (Fig. 13), it would appear that the gene is, in part, transcriptionally regulated by calcium. Beyond this, the next important objective will be to characterize the STC gene, which is uncharted territory at present. The exciting possibilities that the STC gene encodes more than one product, as in the case of the calcitonin-CGRP gene (Breimer et al., lYSS),and that novel transcription fixtors control the ontogeny and regulation of STC gene expression should keep us busy in the laboratory for years to come.

ACKNOWLEDGMENTS I am especially grateful to Henry G. Friesen, M.D., for giving me the opportunity of learning the basics of molecular biology. Salmon stariniocalcin was cloned and partially sequenced in his laboratory. I am also indebted to H . E. Ann MacPhail, l l S c . , for reviewing the manuscript. T h e contributions of my colleagues to the work discussed in this review are greatly appreciated as well. Grant and Scholarship support were provided by T h e Natural Sciences and Engineering Council of Canada and T h e Medical Research Council of Canada.

REFERENCES Ahmad, N., and Swarup, K. (1990).Seasonal changes in structure and behavior ofcorpuscles of Stannius in relation to the changes in serum calcium level and the reproductive cycle of a freshwater female catfish-Mystus oittatus (BLOCH). E u r . Arch. B i o l . 101, 285-294.

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Aida, K., Nishioka, R. S., and Bern, H. A. (1980).Degranulation ofthe Stannius corpuscles of coho salmon (Oncorhynchus kisutch) in response to ionic changes in citro. Gen. C o m p . Endocrinol. 41, 305-313. Aliapoulios, M . A., Goldhaher, P., and Munson, P. L. (1966). Thyrocalcitonin inhibition of bone resorption induced by parathyroid hormone in tissue culture. Science 151, 330-33 1. Anast, C. S., and Conway, H. H. (1972). Calcitonin. Cliiz. Orthop. 84, 207-262. Breimer, L. H., Maclntyre, I., and Zaidi, M. (1988). Peptides from the calcitonin genes: hlolecular genetics, structure and function. Biochern. J . 255, 377-390. Nicholson, L., O’Riordan, J. L. H., and Hendy, G . N. Brookman, J. J., Farrow, S. &I., (1987). Regulation b y calcium of parathyroid hormone mRNA in cultured parathyroid tissue. J . Bone Miner. Res. 6, 529-537. Brown, E. M.,LeBoff, M. S., Oetting, M., Possilico, J. T., and Chen, C. (1987). Secretory control in normal and abnormal parathyroid tissue. Recent Prog. Horm. Res. 43, 337. Butkus, A,, Roche, P. J., Fernley, R. T., Haralambidis, J., Penschow, J. D., Ryan, G. B., Trahair, J. F., Tregear, C:. W., and Coughlin, J. P. (1987). Purification and cloning of a corpuscles of Stannius protein from Anguillu uustralis. Mol. Cell. Endocrinol. 54, 123-134. Butkus, A,, Yates, N. A,, Copp, D. H., Milliken, C., and McDougall, J. G. (1989). Processing and bioactivity of the corpuscles of‘ Stannilis protein of the australian eel. Fish Pliysiol. Biochem. 7, 359-365. Butler, D. G. (1969). Corpuscles of Stannius and renal physiology in the eel (Anguillu rostrutu).J . Fish. Res. Bourd Can. 26, 639-654. Carpenter, S. J . , and Heyl, H . L. (1974). Fine structure of the corpuscles of Stannius ofAtlantic salmon during the freshwater spawningjourney. Gen. C o m p . Endocrinol. 23, 212-223. Colien, R. S., Pang, P. K. T., and Clark, N. B. (197.5). Ultrastructure of the Stannius corpuscles ofthe killifish, Fundulus heteroclitus, and its relation to calcium regulation. Gen. Comp. Endocrinol. 27, 413-423. Fenwick, J. C. (1974). The corpuscles of Stannius and calcium regulation in the North American eel (Anguillu rostrutu LeSueur). Gen. C o m p . Endocrinol. 29, 127-135. Fenwick, J. C., and Brasseur, J. G. (1991). Effircts of stanniectomy and experimental hvpercalcemia on plasma calcium levels and calcium influx in American eels, Anguilla rostrutu, LeSueur. Gen. Comp. Endocrinol. 82, 459-465. Fenwick, J. C., and So, Y. P. (1974). A perfusion study of the effect ofstanniectoniy on the net influx of calcium-45 across an isolated eel gill.]. E x p . Zoo/. 188, 125-131. Flik, G., Labedz, T., Neelissen, J. A. hl., Hanssen, R. G. J. M.,Wendelaar Bonga, S . E., and Pang, P. K. T . (1990). Rainbow trout corpuscles of Stannius: Hypocalcin synthesis in citro. Am. J . Physiol. 258, R1157-1164. Fontaine, M. (1964). Corpuscules de Stannius et regulation ionique (Ca, K, et Na) du milieu interieur d’un poisson l’arrguille. C.R. Acud. Sci. Ser. D 529, 875-878. Fontaine, M., Delerue, N., Martelly, E., Marchelidon, J., and Milet, C. (1972). Role des corpuscule de Stannius dans les echanges d e calcium d’un poisson teleosteen (Anguille unguille L.) avec le milieu anibiant. C.R. Acud. Sci. Ser. D 275,1523-1528. Fraser, R. A., Kaneko, T., Pang, P. K. T., and Harvey, S. (1991). Hypo- and hypercalcemic peptides in fish pituitary glands. A m . J. P h y s i o l . 260, R622-626. Friedman, J., and Raisz, L. G. (1965). Thyrocalcitonin: Inhibitor of bone resorption in tissue culture. Science 150, 1465-1467. Gagel, R. F.. Zeytinoglu, F. N., Voelkel, E. F., and Tashijian, Jr., A. H. (1980). Establish-

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inent of a calcitonin-producing rat medullary thyroid carcinoma cell line. 11. Secretory studies of the tumor and cells in culture. Endocrinology (Baltimore) 107,

5 16-523. Garrett, F. D. (1942). The development and phylogeny of the corpuscles of Stannius in ganoid and teleostean fishes. J . Morphol., 70, 41-67. Gellersen, B., Wagner, G. F., Copp, D. H., and Friesen, H. G . (1988).Developnient of a primary culture system for rainbow trout corpuscles of Stannius and characterization of secreted teleocalcin. Endocrinology (Baltimore) 123, 913-921. Glowacki, J., Milhaud, G., Benson, A., Wagner, G., Cox, K., Fargher, R. C., and Copp, D. H . (1990). Effect of calcium challenge on secretion of stanniocalcin (teleocalcini hypocalcin) in adult seawater coho salmon: A preliminary study. I n “Calcium Regulation and Bone Metabolism” (D. V. Cohn, F. H. Glorieux, and T. J. Martin, eds.), pp. 74-79. Elsevier Science Publ., Amsterdam. Hanssen, H. G. J. M.,Aarden, E. M., van der Venne, W. P. H. G., Pang, P. K. T., and Wendelaar Bonga, S. E. (1991).Regulation of secretion of the teleost fish hormone stanniocalcin: Effects ofextracellular calcium. Gen. C o m p . Endocrinol. 84,155-163. Heinrich, G., Kronenburg, H. M., Potts, Jr., J . T., and Habener, J. F. (1983).Parathyroid hormone messenger ribonucleic acid: Effects of calcium on cellular regulation in citro. Endocrinology (Baltinzore) 112, 449-458. Hirano, T. (1989).The corpuscles of Stannius. In “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and X I . P. Schreibman, eds.), Vol. 3 , pp. 139-169. Academic Press, San Diego. Kaneko, T., Hasegawa, S., and Hirano, T. (1992). Embryonic origin and development of the corpuscles of Stannius in chum salmon (Oncorhynchus ketci). Cell Tissue Res. 268, 65-70. Krishnamurthy, V. G. (1976). Cytophysiology of corpuscles of Stannius. Znt. Rec. Cytol. 46, 177-249. Lafeber, F. P. J. G., Flik, G., Wendelaar Bonga, S. E., and Perry, S. F. (1988a).Hypocalcin from Stannius corpuscles inhibits gill calcium uptake in trout. A m . J . Physiol. 254, K891-R896. Choy, Y. M.,Flik, G., Hermann-Erlee, M.P. Lateher, F. P. J. G., Hanssen, R. G. J . M,, XI., Pang, P. K. T., and Wendelaar Bonga, S. E. (1988b). Identification of hypocalcin (teleocalcin) isolated from trout corpuscles of Stannius. Gen. Comp. Endocrinol. 69, 19-30. Lopez, E., Tisseran-Jochem, E. M., Eyquem, C., Milet, C., Hillyard, C., Lallier, F., Vidal, B., and MacIntyre, I. (1984). Immunocytochemical detection in eel corpuscles of Stannius of a mammalian parathyroid-like hormone. Gen. Comp. Endocrinol. 53, 28-36. Lu, X f . , Wagner, G. F., and Henfro, J. L. (1994). Stanniocalcin stimulates phosphate reabsorption by flounder renal proximal tubule in primary culture. A m . J . Physiol. 267 (Regul. Integr. Comp. Physiol. 36), in press. hlarra, L. E., Youson, J. H., Butler, D. G., Friesen, H. G., and Wagner, C . F. (1992). Stanniocalcin-like immunoreactivity in the corpuscles of Stannis of the bowfin, Amia calva L. Cell Tissue Res. 267, 283-290. Xlayer-Gostan, N., Flik, G., and Pang, P. K. T. (1992).An enzyme-linked immunosorbent assay for stanniocalcin, a major hypocalcemic hormone in teleost. Gen. Comp. Endocririol. 86, 10-19. hleats, M., Ingleton, P. M., Chester-Jones, I., Garland, H. O., and Kenyon, C . J. (1978). Fine structure of the corpuscles of Stannius of the trout, Sulnlo gciirdneri: Structural changes in response to increased environmental salinity and calcium i o n s . Gen. Camp. Eiidocrinol. 36, 451-461.

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Milhaud, G., Perault, A.-M., and Moukhtar, M . S. (1965). Etude du mecanisme d e l'action hypocalcemiante d e la thyrocalcitonine. C . R .Hebd. Seances Acad. Sci. 261, 813-816. Milliken, C., Fargher, R. J . , Butkus, A,, McDonald, M., and Copp, D. H. (1990). Effects of synthetic peptide fragments of teleocalcin (hypocalcin) on calcium uptake in juvenile rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 77, 416-422. Mouland, A. J., and Hendy, G. H. (1991). Regulation of synthesis and secretion of chromogranin-A by calcium and 1,25-dihydroxycholecalciferolin cultured bovine parathyroid cells. Endocrinology (Baltimore) 128, 441-449. Naveh-Many, T.,and Silver, J. (1990). Regulation of parathyroid hormone gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J . Clin. Inoest. 86, 1313- 1319. Naveh-Many, T., Friedlaender, M. M., Mayer, H., and Silver, J. (1989).Calcium regulates parathyroid hormone messenger ribonucleic acid (mRNA), but not calcitonin mRNA in cioo in the rat. Dominant role ofl,25-dihydroxyvitaininD. Endocrinology (Baltimore) 125,275-280. Naveh-Many, T.,Raue, F., Grauer, A,, and Silver, J. (1992). Regulation of calcitonin gene expression by hypocalcemia, hypercalcemia, and vitamin D in the rat. J. Bone Miner. Res. 7, 1233-1237. Ogawa, M.(1967). Fine structure of the corpuscles of Stannius and the interrenal tissue in goldfish (Curussius auratus). Z . Zellerforsch. 81, 174-189. Pang, P. K.T., and Pang, R. K. (1974). Environmental calcium and hypocalcin activity in the Stannius corpuscles of the channel catfish, Ictulurus punctatus (Rafinisque). Gen. C o m p . Endocrinol. 26, 179-185. Pang, P. K.T., Pang, R. K., and Sawyer, W. H. (1973).Effect ofenvironmental calcium and replacement therapy on the killifish, Fundulus heteroclitus, after surgical removal of the corpuscles of Stannius. Endocrinology (Baltimore) 93, 705-710. Pang. P. K. T., Pang, H. K., and Sawyer, W. H.(1974). Environmental calcium and sensitivity of killifish (Fundulus heteroclitus) in bioassays for the hypocalcemic response to Stannius corpuscles from killifish and cod (Gadus rnorhuu).Endocrinology (Baltimore) 94,548-555. Russel, J., Lettieri, D., and Sherwood, L. M. (1983). Direct regulation by calcium of' cytoplasmic ribonucleic acid coding for pre-proparathyroid hormone in isolated bovine parathyroid cells. 1.Clin. Inaest. 72, 1851-1855. So, Y. P.,and Fenwick, J. C.(1977). Relationship between net "calcium influx across a perfused isolated eel gill and the development of post-stanniectomy hypercalcemia. J. Exp. Zool. 200, 259-264. So,Y.P.,and Fenwick, J. C.(1979).The in ciao and in citro effects ofStannitis corpuscles extract on the branchial uptake of "Ca in stanniectomized North American eel (Anguilla rostruta). Gen. Comp. Endocrinol. 37, 143-149. Stannius, H. (18.39). Ueber Nebenniere bei Knochenfischen. Arch. An&. Physiol. 6, 97- 101. Sterba, T.,Wagner, G. F., Schroedter, I. C., and Friesen, H.G. (1993). In situ detection arid distribution of stanniocalcin mRNA in the corpuscles of Stannius of sockeye salmon, Oncorhynchus nerka. Mol. Cell. Endocrinol. 90, 179-185. Sundell, K.,Bjornsson, B. Th., and Kawauchi, H.(1992). Chum salmon (Oncorhynchus keta) stanniocalcin inhibits in citro intestinal calcium uptake in Atlantic cod (Gadus morhua).1.Comp. Physiol. B 162, 489-495. Verbost, P. M., Butkus, A,, Atsma, W., Willems, P., Flik, G., and Wendelaar Bonga, S. E . (1993). Studies on stanniocalcin: Characterization of hioactive and antigenic domains of the hormone. Mol. Cell. Endocrinol. 93, 11-16.

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LVagner, G. F. (1993). Stanniocalcin: Structure, function and regulation. I n “Rioclicnristry and Molecular Biology of Fishes” (P. W. Hochachka and T. P. Moiiinisen, cds.), Vol. 2, Chap. 21, pp. 419-434. Elsevier Science Pirbl., Anisterdani. \Vagner, G. F., and Jaworski, E. (1994). Calcium regulates stanniocalcin niKNA levels in primary cultured rainbow trout corpuscles of Stannius. M o l . Cell. Encfocririol. 99,315-322. \Vagner, C. F., Hampong, M., Park, C. M., andCopp, D. H. (1986).Purification, characterization and bioassay of teleocalcin, a glycoprotein from saliiion corpuscles of Stanrrius. Cen. Comp. Endocrinol. 63, 481-491. \Vagner, G. F., Fenwick, J. C., Milliken, C., Park, C. XI., Copp, D. H., and Friesen, H. (2. (1988a). Comparative biochemistry and physiology of teleocalciii froni sockeye and coho salmon. Gen. Cornp. Endocrinol. 72, 237-246. Wagner, 6. F., Copp, D. H., and Friesen, H. G. (19881)). Imtiiurrological studics on teleocalcin and salmon corpuscles of Stannius. Erzdocririology (Baltimore) 122, 2064-2070. \Vagner, G. F., Gellersen, B., and Friesen, H. G. (1989). Primary culture of teleocalcin cells from rainbow trout corpuscles of Stannius; Regulation of teleocalcin secretioii b y calcium. M o l . Cell. Endocrinol. 62, 31-39. \\’agner, G. F., Milliken, C., Friesen, H. G., and Copp, D. H. (1991). Studies on tlic regulation and characterization of plasma stanniocalcin in rainbow trout. Mol. Cell. Endocrinol. 79, 129-138. Wagner, G. F., Di Mattia, G. E., Davie, J. R., Copp, D. H., and Friesen, H. G. (1992). Rfolecular cloning and cDNA sequence analysis of coho salmon stanniocalcin. M o l . Cell. Endocrinol. 90, 7-15. fi‘agiier, G. F., Fargher, R. C., Milliken, C., McKeown, B. A,, and Copp, U. H. (lYS3). The gill calcium transport cycle in rainbow trout is correlated with plasma levels ofbioactive, not immunoreactive, stanniocalcin. Mol. Cell. Endocrinol. 93,185- 191. \Vendelaar Bonga, S. E., and Pang, P. K. T., eds. (1986). Stannius corpuscles. Z t i “Vertebrate Endocrinology, Fundamentals and Biomedical Implications,” i’ol. 1, pp. 439-464. Academic Press, Orlando, Florida. \Vendelaar Bonga, S. E., and Pang, P. K. T. (1991). Control of calciuni regulating hormones in the vertebrates: Parathyroid hormone, calcitonin, prolactin, and stanniocalcin. I n t . Hec. Cytol. 128, 139-213. l’ouson, J. H., Butler, D. C., and Chan, A. T. C. (1976). Identification arid distri1)utioir of the adrenocortical homolog, chroniaffin tissue and the corpuscles of Stannius in Arnici ccilau L. Geri. Conip. Endocrinol. 29, 198-21 1 .

IV HORMONE REGULATION

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10 COMPARATIVE ASPECTS OF PITUITARY D E V E L O P M E N T A N D Pit-1 F U N C T I O N SONALI MAJUMDAR AND HARRY P . ELSHOLTZ Department of Clinical Biochemistry and Banting %I Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada M5G 1L5

I. Introduction 11. Comparative Organization of the Pituitary Gland 111. Differentiation of Adenohypophysial Cell Types A. The Rat Adenohypophysis B. The Fish Adenohypophysis IV. Transcription Factor Pit-1 A. Role in Mammalian Pituitary Development B. Expression of Pit-I during Mammalian Pituitary Development C. DNA Binding and Target Gene Specificity D. POU Domain: Structure and Function E. N-Terminal Sequences: Multiple Isoforms F. Pit-1 Dimerization and Interaction with Other Proteins V. Comparison of Pit-1 in blanimals and Teleost Fish: Studies on the PRL Target Gene A. Conservation of Pit-1 POU Domain Function in Fish B. Species Differences in Alternative RNA Splicing C. N-Terminal Sequences of Rat and Salmon Pit-1 VI. Conclusion References

I. INTRODUCTION The pituitary gland or hypophysis is a critical endocrine regulator

of vertebrate growth, metabolism, reproduction, ion balance, and behavior, and accordingly it was once described as the “master gland.” Closely associated with the brain, the pituitary is under predominant neural control although multiple feedback signals from target organs 309 F I S l l I ’ l T l b l O L O G ~ 01. XI11

Copbiight 0 I994 In A c a d e m ~Pie\\ Inc A l l right, of Irprodnctmn i n arw form iewrved

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SONALI MAJUMDAR A h D €IARRY P ELSHOLTZ

can play equally important regulatory roles. The pituitary displays remarkable morphological variation, not only among different vertebrate classes but even within the same species (e.g., different breeds of dogs). Yet the secretory cell types and polypeptide hormone products first identified in eutherian mammals have also been observed in distantly related vertebrates, suggesting that common mechanisms may dictate pituitary organogenesis. Inimunohistological, biochemical, and molecular studies have provided new insights into the mechanisms of pituitary differentiation (Voss and Rosenfeld, 1992). Factors required for the regulation of pituitary-specific genes have been identified or cloned and their fiinctional domains characterized. This chapter focuses on the transcription factor Pit-1, which plays a pivotal role in the differentiation of specific pituitary cell lineages and in the activation of a subset of endocrine genes. Structural and functional comparisons of mammalian and fish Pit-1 are discussed.

11. COMPARATIVE ORGANIZATION OF THE PITUITARY GLAND The pituitary can be divided into two principal structures, the adenohypophysis or anterior pituitary and the neurohypophysis or posterior pituitary. During embryogenesis the pituitary derives from two different sources. The adenohypophysis develops from the ectoderma1 cells growing out from the roof of the oral cavity, an embryonic structure called Rathke’s pouch. This pharyngeal evagination ultimately separates to associate with an outpouching of the diencephalon, partitioned from the oral cavity by the sphenoid bone of the skull. In teleost fish the adenohypophysis is further organized into rostra1 and proximal portions that are distinguishable on the basis of specific endocrine cell types (described in the following). The vertebrate neurohypophysis, which is also of ectodermal origin, develops from the downward outgrowth of the diencephalon and contains both neural and glial cell types. Cells of the embryonic adenohypophysis that contact the neurohypophysis give rise to a third pituitary structure, the intermediate lobe (pars intermedia), which separates the anterior and posterior lobes of the pituitary. This lobe may he either well defined (e.g., rodents, reptiles, teleost fish), poorly defined (e.g., primates, birds), or completely absent (e.g.,whales) depending on the species. Another pituitary structure subject to species differences is the pars tuberalis, which arises from lateral extensions of Rathke’s pouch. In some species, particularly in birds, it forms a prominent collar around the u p p e r

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pituitary stalk, whereas in others, including teleost fish and a number of mammals, it is well defined during development but difficult to observe in the mature organism. In most tetrapods stimulatory or inhibitory factors from the hypothalamus that regulate pituitary secretory function are transported by the hypothalamohypophysial portal plexus. In teleosts a similar portal vascular system is lacking and arterial blood passes directly to the pituitary; blood vessels within the neurohypophysis are associated with neurosecretory fibers from the hypothalamus and transport neural regulatory factors to the anterior lobe. Aminergic fibers from the hypothalamus (and in some cases peptidergic fibers) can also interact directly with cells of the adenohypophysis. In some cyclostomes such as the hagfish, the pituitary is a loosely organized tissue in which hypothalamic fibers project to the neurohypophysis and regulatory factors reach the cells of the anterior lobe by simple diffusion (Holmes and Ball, 1974; Batten and Ingleton, 1987).

111. DIFFERENTIATION OF ADENOHYPOPHYSIAL CELL TYPES The mature anterior pituitary contains five major endocrine cell types characterized by their polypeptide hormone product (Chetelain et al., 1979; Watanabe and Daikoku 1979; Hoeffler et al., 1985). These horniones are critical to homeostatic regulation, growth, and reproduction. Adrenocorticotropin (ACTH), synthesized by the corticotrophs, regulates steroid hormone production by the adrenal cortex; thyroidstimulating hormone (TSH) from the thyrotrophs promotes synthesis and release of T3 and T4 from the thyroid; luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the gonadotrophs regulate ovarian or testicular function; and growth hormone (GH) from somatotrophs enhances physical growth. Prolactin (PRL) is the most functionally diverse of the adenohypophysial hormones, regulating milk production and lactation in mammalian species, osmoregulation in teleost fish, and reproductive and behavioral functions in certain birds and mammals. A. The Rat Adenohypophysis In the rat embryo the a-subunit ofthe glycoprotein hormones (FSH, LH, TSH) serves as the earliest marker for anterior pituitary development, detectable by the eleventh day in ectodermal cells beneath the neural tube (Simmons et al., 1990). Corticotrophs are observed by

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Day 13 or 14 and, based on organ culture studies, appear to arise independently of exogenous cues (Begeot et al., 1982). By contrast, cell types arising at later stages of pituitary development are more dependent on paracrine or endocrine factors. Thyrotrophs are detectable by about embryonic (e) Day 14 in the rostral tip of the anterior lobe and are followed by gonadotrophs (Day e l 6 to e17) and somatotrophs (Day e l 7 to e18).Somatotrophs are located caudally and proliferate dorsally around Day e18. Prolactin-producing lactotrophs are only weakly detectable by Day el7 to el8 but undergo a dramatic expansion in cell number during the early postnatal period. Unlike the endocrine cells of the fish anterior pituitary (see the following), mature rat adenohypophysial cells are distributed in a random manner, a characteristic that may be determined by local migratory factors or intercellular recognition signals (Voss and Rosenfeld, 1992). In rodents the appearance of lactotrophs is largely dependent on the differentiation of the somatotroph lineage. Transgenic studies have demonstrated a dramatic reduction in lactotroph number in animals carrying a toxin gene specifically targeted to embryonic somatotrophs (Behringer et al., 1988; Borrelli et al., 1989). These data suggest that all lactotrophs or at least the majority (Behringer et al., 1988) derive from a somatotroph stem cell. In the mature animal the anterior pituitary retains a population of cells that coexpresses growth hormone and PRL; the ratio of growth hormone to PRL produced b y these cells may be altered depending on physiological requirements (Frawley and Boockfor, 1991). B. The Fish Adenohypophysis The pattern of pituitary cell development in teleost fish is distinct from that ofmammals. Even within the same family (e.g., chum salmon vs. coho salmon) the chronological appearance of distinct cell types varies. In chum salmon, 5 weeks postfertilization and prior to hatching the dorsal half of the adenohypophysis contains columnar cells packed tightly, whereas the ventral portion contains cells that are more randomly and loosely arranged. Prolactin-producing cells are the first cell type to appear and are located in the rostroventral portion o f t h e adenohypophysis as follicular structures. Somatotrophs also appear early followed by corticotrophs and thyrotrophs. Whereas soniatotrophs are centrally positioned, corticotrophs are observed dorsally from the rostral to the caudal region, and thyrotrophs are interspersed with somatotrophs. The gonadotrophs appear late in development, becoming detectable at 3 weeks after hatching (Naito et nl., 1993). In

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the coho salmon, prolactin-producing cells are again the first cell type to appear but are followed by thyrotrophs and then corticotrophs. Somatotrophs in this species develop between 5 and 6 weeks postfertilization, that is, within a week of hatching. As in chum salmon, gonadotrophs appear last-gonadotrophin (GTH) I cells at about 2 weeks after hatching and GTH I1 cells shortly before gametogenesis (Ma1 et az., 1989). The early appearance of PRL-secreting cells is not restricted to salmonid species but is also observed in the euryhaline fishes such as tilapia (Hwang, 1990) and certain marine species. In a report of pituitary development in sea bream (Power and Canario, 1992),somatotrophs are first detectable on Day 1 after hatching. PRL-producing cells are detectable on Day 4. The number of PRL-producing cells increases in the rostral pars distalis up to Day 12. Gonadotrophs are observed by the sixth day progressively up to Day 12, at which time they project into the pars intermedia. GTH-producing cells in the sea bream occupy almost 30%of total pituitary volume. Two populations of ACTH-producing cells are observed on Day 8, one in an anterior location in the pars distalis and a second in a posterior location in the pars intermedia (Power and Canario, 1992). The early appearance of PRL cells in a number of teleost fish is an interesting distinction from the pattern of lactotroph development in rodents. Whether developmental differences are in any way related to phylogenetic changes in PRL function has not been determined. Furthermore, in certain fish species PRL cells appear late during pituitary ontogeny. In the sea bass, for example, the predominant cells on Day 1 after hatching are corticotrophs, with lesser numbers of somatotrophs and thyrotrophs detectable in the rostral portions of the pituitary. PRL-producing cells are observed on Day 9 posthatching, whereas gonadotrophs are not detected until after Day 26 (Cambr6 et az.,

1990).

IV. TRANSCRIPTION FACTOR PIT-1 A. Role in Mammalian Pituitary Development

The coordination of developmental processes that determine the appearance of specific cell types is dependent on the interaction of transcriptional regulators with specific target genes. One critical class of developmental regulators is encoded by the homeobox genes, originally identified in invertebrate species and characterized by genetic

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SONALI h l A J U h l D A R AhLD IIAHRY P. ELSHOLTZ

analyses (Akam, 1987; Gehring, 1987). Multiple homeobox genes have also been identified in vertebrate species (Kessel and Gruss, 1990; McGinnis and Krumlauf, 1992) and targeted knock-out approaches have revealed their developmental functions in several cases (McGinnis and Krumlauf, 1992 and references therein; Joyner et al., 1991; Mouellic et al., 1992; Ramirez-Solis et ul., 1993). In the manimalian pituitary a homeodomain-containing factor, Pit-1 or GHF-1 (Ingraham et al., 1988,Bodner et al., 1988),plays a pivotal role in the development of specific adenohypophysial cell lineages. Although Pit-1 was first described in biochemical experiments as a pituitary-specific DNAbinding protein capable of activating the growth hormone (Bodner and Karin, 1987, Nelson et al., 1988) and PRL (Nelson et nl., 1988) gene promoters, genetic evidence has now established the importance of Pit-1 in vivo during embryogenesis. Dwarf mouse strains (Snell, Jackson, Ames) that underexpress the pit-1 gene (Li et al., 1990) share a common phenotype, that is, hypoplastic pituitaries deficient in three specific cell types-somatotrophs, lactotrophs, and thyrotrophs. Serum levels of GH, PRL, and TSH are essentially undetectable. In the case ofthe Jackson and Snell mutants the pit-1 gene is rearranged or pointmutated, respectively, whereas in the Ames mouse the pit-1 gene is apparently normal but underexpressed because of a defect at a separate chromosomal locus. A variety of mutations in the human p i t - l gene have been reported (Tatsumi et ul., 1992; Radovick e t al., 1992; Pfiiffle et al., 1992; Ohta et ul., 1992) and linked to combined pituitary hormone deficiency (Winter et al., 1974; Kogol and Kahn, 1976). The endocrine abnormalities, which included cretinism and dwarfism, vary in severity depending on the position and nature of the mutations in the p i t - l gene. Examples of such mutations are discussed in Section IV,D. B. Expression of Pit-1 during Mamm,a 1.ian Pituitary Development The earliest detectable expression ofpit-l RNA transcripts is found in the neural tube of the embryonic mouse on Day 10 (He et ul., 1989, Sininions et al., l99O), although the absence of neural tube defects in Pit-1 deficient dwarf mice may question the developmental significance ofthis event. In the anterior pituitary, expression of p i t - 1 transcripts and protein precedes the appearance of GH- and PRL-secreting cells. Both pit-l niRNA and protein are observed by Days e l 5 to e l 6 (Simmons et ul., 1990; Dolle et al., l990), and levels of'immunoreactive Pit-1 increase gradually until Day 10 after birth (Day p10). This in-

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crease in the level of Pit-1 protein probably reflects the expansion of cell populations (e.g., lactotrophs) that express p i t - 1 . Pit-1 protein is not detectable in pituitary corticotrophs or gonadotrophs, being restricted to somatotroph, lactotroph, and thyrotroph cell types (i.e., the cell types absent in Pit-1-deficient animals). The early appearance of‘ the thyrotroph cell lineage on Day e l 4 (see earlier) prior to expression of pit-1 would appear to suggest a maintenance function for Pit-1 in this cell type. Other evidence, however, indicates that the developing pituitary may produce two populations of thyrotrophs-an early transient and Pit-l-independent population that is subsequently replaced by a permanent population of Pit-l-dependent cells (Lin e t d . , 1994). Although in rodents expression of pit-1 protein is restricted to cells producing GH, PRL, or TSH, the level of pit-1 mRNA was found to be similar in all five endocrine cell types (Simmons et uZ., 1990). These data suggest that a translational mechanism may determine the distribution of Pit-1 in the anterior pituitary. Similar observations have not yet been reported for other mammalian species. We have shown in human pituitary adenomas and nontumorous pituitary tissue that the pattern of Pit-1 protein expression correlates well with that of Pit1niRNA (Asa e t al., 1993).The human data, therefore, are more readily explained by a “pretranslational” regulatory model for Pit-1 express ion.

C. DNA Binding and Target Gene Specificity Pit-1 binds with high affinity to specific regulatory elements of pituitary target genes to activate transcription. Although Pit-1 likely regulates multiple genes in somatotroph, lactotroph, and thyrotroph cells, the PKL and GH genes have been studied most extensively. In the rat, both the PRL and GH gene contain proximal Pit-1 sites within 250 base pairs 5‘ to the transcription start site. The PRL gene contains an additional Pit-1 binding enhancer region located 1.5 to 1.8 kb upstream of the proximal region. The sequence of this distal enhancer appears to be about 80% identical among mammalian PRL genes (e.g., rat, human, cow) and the proximal region nearly 90% identical. In the case of the rat PRL gene, deletional analysis demonstrated that the conserved proximal and distal regions are required for activation in pituitary cells (GH4, GC) but lack a stimulatory cis-activity in heterologous cells, such as fibroblasts or HeLa cells. In cultured pituitary cells, the relative activity of the distal enhancer and proximal region can be variable (Nelson e t ul., 1986, 1988; Lufkin and Bancroft, 1987), due

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in large part to the particular cell line used (Jones and Catanzaro, 1991). Based on transgenic mouse studies, however, both the proximal and distal Pit-1 binding regions are required for optimal high-level expression of the PRL transgene in pituitary cells (Crenshaw et al., 1989). Interestingly, PRL genomic sequences between the two Pit1 binding regions appear to be necessary to prevent inappropriate expression of the transgene in Pit-l-containing thyrotrophs. Alignment of multiple DNase I footprints from GH and PRL genes has revealed a consensus binding site for Pit-1-(A/T),TATNCAT (Nelson et al., 1988). The specificity of this consensus sequence has been examined in the most proximal site of the rat PRL promoter using a series of clustered mutations or point transversions in the TATNCAT core and flanking bases (Elsholtz et al., 1990). Individual mutations at most positions in the core reduce Pit-1 binding by >70%. Flanking mutations 5' of the core also reduce the binding of Pit-1 albeit to a lesser degree, whereas mutations 3' to the core have little effect. Interestingly, phosphorylation of Pit-1 by protein kinase A or C can decrease the ability of Pit-1 to bind to certain DNA sites by a mechanism dependent on nucleotides immediately upstream of the TATNCAT box (Kapiloff et al., 1991). These nucleotides could therefore determine the efficiency of Pit-1 sites to function as hormone response elements.

D. POU Domain: Structure and Function The cloning of Pit-1 (Ingraham et al., 1988; Bodner et al., 1988) and two other mammalian transcription factors, Oct-1 and Oct-2 (KO et al., 1988; Muller et al., 1988; Scheidereit et al., 1988; Sturni et ul., 1988), revealed a novel conserved sequence N-terminal to the homeodomain that was also present in the product of a Caenorhabditis elegans developmental gene, unc-86 (Finney et al., 1988). This sequence was about 80 amino acids in length and was separated from the homeodomain by a nonconserved linker sequence of 15 to 25 residues. The bipartite sequence was named the POU domain and is composed of an N-terminal POU-specific domain (POU,) and a Cterminal POU homeodomain (POU,,) (Fig. 1). Many POU domain proteins have been identified over the past few years in both vertebrates and invertebrates (Rosenfeld, 199 1;Verrijzer and Van der Vliet, 1993). The POU protein family appears to be of particular importance in the development ofthe CNS (Treacy and Rosenfeld, 1992). Classification of POU proteins into groups POU-I to POU-VI (Rosenfeld, 1991; Okamoto et al., 1993, Johansen et al., 1993) is based on overall

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POU-specific domain-

89% High affinity DNA binding Enhanced site specificity Protein/protein interaction

317

LPOU-homeodomain71%

85% Low affinity DNA binding Relaxed site specificity

Fig. 1. Schematic representation of the bipartite POU domain indicating the POUspecific and POU homeodomains. Boxes depict the predicted a-helices. Positions of basic ( + ) and acidic ( - ) amino acid residues, and the percent conservation in the two domains between salmon and rat, are indicated at bottom.

sequence similarity, especially in the basic amino acid cluster at the N terminus of the POUHDand in the spacer region separating the POU, and POUHD. The Pit-1 POUHI,is about 20-30% identical to the classic homeodomains encoded by the Drosophila developmental genes such as Antennapedia and Ultrabithorax (Gehring, 1987; Scott and Carroll, 1987). Of the nine amino acid residues invariant among Drosophila homeodomains, seven are conserved in Pit-1. The POU,, contains three ahelices, ofwhich the the third helix (also called the recognition helix) is most highly conserved among POU proteins. Based on crystallography studies of the Drosophila engrailed homeodomain (Kissinger et al., 1990), the POUHDrecognition helix, KXV(V/I)RVWFCN(R/Q)RQ (K/ R)KR, is likely to form base contacts within the major groove of the DNA site. The functional importance of the Trp(W) residue in the Pit1 POUIiDis well demonstrated by the Snell mouse, whose pit-l gene contains a single-nucleotide change that causes a Trp to Cys substitution in the recognition helix. The mutation abolishes Pit-1 binding to DNA, resulting in a dwarf phenotype. Pit-1 binding to DNA is also abolished by substitution of a Gly residue at a conserved Arg position (i.e., W F C N G R Q ) in helix 3 (Ingraham et al., 1990). Interestingly, conversion of the highly conserved Cys(C) residue to a Gln(Q) (found in many “classic” homeodomains) does not impair Pit-1 binding but reduces by three- to fourfold the ability of Pit-1 to activate the P R L promoter. This demonstrates that the Pit-1 POU,, functions not only in DNA recognition but also in transactivation. A similar loss-of-function mutation has been reported in a patient with combined pituitary hormone deficiency (Radovick et al., 1992); in this case disruption of a basic amino acid near the C terminus of the Pit-1 POUHD (Arg to Trp substitution) yielded a dominant negative Pit-1 mutant that bound DNA but failed to activate transcription.

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SONALI MAJUMIIAH AND HAKHY P. ELSWOLTZ

In experimental Pit-1 mutants that lack the POUS domain, the POUF1, is sufficient for low-affinity interactions with AIT-rich sequences (Ingraham e t al., 1990). Studies with the POU protein Oct-1 indicate that the POUs domain may also be capable of autonomous low-affinity DNA binding (Verrijzer et al., 1992). Together, however, the POU, and POU,, cooperate to facilitate recognition ofthe specific consensus element and enable high-affinity interactions with the D N A site (Ingraham e t al., 1990; Sturm and Herr, 1988; Verrijzer et al., 1990). Although the POUS domain is critical for POU protein function and its sequence is highly conserved, its structure has only recently been characterized. Using nuclear magnetic resonance analysis, AssaMunt et al. (1993) and Dekker et a1. (1993) have determined the solution structure ofthe POU, domain of Oct-1. The POUS consists of four a-helices packed around a core of hydrophobic residues. Helices two and three form a helix-turn-helix structure with striking similarity to the DNA-binding doniains of certain prokaryotic proteins, including bacteriophage A repressor and 434 Cro. Helix 3 contributes numerous contacts within the DNA major groove and is the most highly conserved sequence of the POU, domain. Accordingly, inversion of a short peptide sequence within the third helix of the Pit-1 POUSdomain disrupts Pit-1IDNA interactions (Ingraham e t al., 1990). Introduction of two Pro residues into POUS helix 2, or rearrangement of the amino acid sequence in POU, helix 1, also interferes strongly with Pit-1 binding to DNA. It is noteworthy that POU, helix 2 may have a transactivating function in addition to its role in DNA binding. An interesting clinical case was reported in which the a-helical structure of the Pit-1 POUS helix 2 was perturbed by an Ala to Pro substitution; the mutant protein in this patient retained DNA-binding activity but failed to activate the PRL or GH promoter (Pfaffle et ul., 1992), as in the case of certain POUIIDmutations discussed earlier.

E. N-Terminal Sequences: Multiple Isoforms

The major transactivating function of Pit-1 has been localized to sequences N-terminal to the POU domain (Theill et al., 1989; Ingraham et al., 1990). Deletion of aniino acids 8-80 resulted in an 85% decrease in reporter gene activation without a concomitant loss in DNA-binding activity. Furthermore, when fused to the DNA-binding domain of the E . coli repressor, LexA, N-terminal pit-1 sequences strongly activate promoters containing LexA binding sites. The Nterminal region of pit-1 is rich in serine and threonine residues that are likely to be important for the transactivating function. A similar

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content of hydroxylated amino acids is found in the transactivation domains of certain other POU proteins, such as Oct-2. As a result of alternative RNA processing and dual translational start sites, rat Pit-1 occurs in a number of isoforms having unique Nterminal sequences. Voss et al. (1991b) have demonstrated that translational use of an internal methionine residue (position 26) produces the characteristic doublet (33 and 31 kDa) observed on Western blots of purified Pit-1 protein. Functional differences or differential regulation of these two variants has not yet been established. Alternative processing of the rat Pit-1 primary transcript results in an isoform referred to as Pit-lp (Konzak and Moore, 1992), GHF-2 (Theill et al., 1992), or Pit-la (Morris et al., 1992). Pit-lp contains an insertion of26 amino acids resulting from use of an alternate RNA splice acceptor in the first intron. Although the ratio of the Pit-la to Pit-lp isoforms in rat pituitary is 7:1, Pit-lp has been shown in some transfection studies to be a more potent activator of the rat GH promoter than Pit-la (Konzak and Moore, 1992; Theill et al., 1992; Morris et al., 1992). In contrast, the PRL promoter is preferentially activated by the Pit-la isoform. Another isoform of Pit-1 has been reported that utilizes an unusual "AT" splice acceptor in the first p i t - l intron between the aand /3-specific "AG" acceptor sites; this variant, called Pit-lT, contains 14 C-terminal amino acids of the @insert. The expression of Pit-1T appears to be restricted to pituitary thyrotrophs and transfection studies have demonstrated the ability of this variant to activate the TSHp promoter (Haugen et al., 1993).

F. Pit-1 Dimerization and Interaction with Other Proteins In addition to its DNA-binding functions, the POU domain of Pit1mediates protein/protein interactions required for activation of transcription. Gel mobility shift and protein cross-linking experiments support a model in which Pit-1 binds to DNA as a dinier although it exists as a monomer in solution (Ingraham et wl., 1990). A qualitative difference in Pit-1 dimerization has been proposed as the basis for inefficient transactivation by the human Pit-1 (A1a'"Pro) mutant (PfAffle et al., 1992; described in Section IV,D), in which the a-helical structure of' the POUS domain was disrupted. Heterodimerization of normal and mutant Pit-1 has also been proposed as one possible mechanism for the dominant negative effect of' the human Pit-1 Arg'"Trp mutant (Radovick et ul., 1992; described in Section IV,D). Because the products of both the mutant and normal allele bound DNA with

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high affinity, the severe pituitary deficiency phenotype in this case is consistent with an effective block of normal Pit-1 function by the mutant at the level of the target gene. The function of Pit-1 is also determined by interactions with other proteins. These include potential interactions with other POU proteins such as Oct-1. The native rat PRL promoter and a promoter construct containing an individual Pit-1 binding site are each activated more efficiently by cotransfection of Pit-1 and Oct-1 than by transfection of Pit-1 alone (Voss et al., 1991a). Protein binding studies support a model in which Pit-1 and Oct-1 interact synergistically by formation of heterodimers. Pit-1 can also interact with structurally unrelated transcription factors. The distal enhancer ofthe PRL gene, for example, contains an estrogen response element adjacent to one of the Pit-1 sites. In nonpituitary cells, expression of an estrogen receptor construct alone has little effect on basal activity of the PRL promoter, but when coexpressed with Pit-1, the estrogen receptor becomes strongly stimulatory (Day et al., 1990; Simmons et al., 1990). In the case of the GH promoter, cooperative interactions between Pit-1 and the thyroid hormone receptor have been observed (Schaufele et al., 1992). A novel zinc finger protein Zn15 has been identified that binds a conserved “Z-box” sequence in the rat GH promoter between positions - 94 and - 113 (Lipkin et al., 1993); this binding site is located between two proximal Pit-1 binding sites. Mutations in the Z-box resulted in >loofold decrease in GH promoter function when assessed in transgenic mice, demonstrating that the two Pit-1 sites were insufficient for full function of this pituitary-specific promoter. In cultured cells, cotransfection of Zn15 and Pit-1 indicated a strong synergism between these two factors in activation of the GH promoter. Lastly, Pit-1 has also been shown to interact with heterologous factors in the regulation of‘ its own gene. Binding of Pit-1 at a distal enhancer element appears to be critical for retinoic acid responsiveness ofthe pit-1 gene (Rhodes et al., 1993).

V. COMPARISON OF PIT-1 IN MAMMALS AND TELEOST FISH: STUDIES ON THE PRL TARGET GENE In marked contrast to its lactogenic role in mammals, PRL in teleost fish regulates transport of ions across the gill epithelia. Despite functional differences among distantly related vertebrates, the PRL gene is conserved within coding regions and at all intronlexon splice junctions

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(Xiong et al., 1992). Interestingly, sequence conservation in the 5’flanking regions of mammalian and salmon PRL genes is observed only in the TATA box. Sequences similar to the mammalian proximal activating region and distal enhancer appear to be absent in the salmon PRL gene. Accordingly, we examined the ability of divergent salmon PRL 5’ sequences ( - 2.4 kb) to direct gene expression in heterologous and PRL-secreting cell lines of mammalian or fish origin. Similar to the rat PRL gene, 5’ sequences of the salmon PRL gene were unable to activate expression of a CAT reporter in nonpituitary cells. In PRLsecreting rat GH4 cells, 5’ sequences of the salmon PRL gene activated transcription significantly, although at levels 90-fold lower than similar constructs containing rat PRL 5’-flanking sequences (3.0 kb) (Elsholtz et al., 1992). These data indicated that pituitary-specific factors are required for salmon PRL gene activation, but suggested also that species-specific differences in pituitary cell function may impede efficient use of the salmon PRL gene promoter. The species specificity of transcriptional regulation was further examined in salmonid primary pituitary cells. Interestingly, in these cells the salmon PRL/CAT constructs were expressed at very high levels, whereas rat PRL/CAT constructs were only weakly active (Elsholtz et al., 1992). These studies supported the argument that both the rat and salmon PRL promoters are species specific, requiring a homologous pituitary system for optimal expression. The restricted expression of the salmon PRL promoter in rat GH4 cells strongly suggested that rat Pit-1 might be involved in promoter activation. Moreover, the species differences observed in transfected GH4 cells and salmonid pituitary cells further suggested that a teleost fish Pit-1 may activate the salmon PRL promoter more efficiently than rat Pit-1. To perform a functional comparison of rat and salmon Pit-1 we first used a combination of polymerase chain reaction (PCR) and cDNA library screening (chinook salmon pituitary) to isolate the salmon homolog of rat Pit-1. Homology was confirmed in three fulllength clones on the basis of sequence similarity to mammalian Pit-1’s and the pituitary-specific expression of salmon p i t - l RNA transcripts. The chinook salmon pit-1 cDNA contains an open reading frame of 1074 nucleotides encoding a protein of 358 amino acids. In the Cterminal half of salmon p i t - l the POU domain exhibits 87% identity with mammalian pit-l POU domains. The highest conservation is observed in the N-terminal part ofthe POUSdomain and in the third helix of the homeodomain. Most amino acid substitutions were localized to the C-terminal portion of the POUS domain and to helix 1 and 2 of the POU,,. In the salmon POU domain there are 14 amino acid substi-

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SONALI MAJUMDAR AND HARRY 1'. ELSHOLTZ

tutions at positions highly conserved among mammalian Pit-1's. Nterminal to the POU domain, sequences of salmon Pit-1 are less than 60% identical to those of mammalian Pit-1's and contain numerous amino acid insertions, deletions, and nonconservative substitutions (Fig. 2). A p i t - l cDNA has been cloned from a second salmonid species, chum salmon (Oncorhynchus keta) (Ono and Takayama, 1992). Chinook and chum salmon Pit-1 sequences are highly conserved, although an insertion (or deletion) of four amino acids has occurred in the N terminus and a deletion of seven base pairs in the chum salmon 3' untranslated region extends the pit-1 open reading frame by 11codons, relative to chinook salmon and mammalian pit-1 ' s .

A. Conservation of Pit-1 POU Domain Function in Fish Because the POU domains of sahnon and rat p i t - l contain several amino acid differences, we tested whether these might contribute to functional differences in PRL gene activation. A chimeric pit-1 was constructed in which most ofthe salmon pit-1 POU domain was substituted for the rat pit-1 POU domain in a rat p i t - l c D N A expression vector. Transactivation b y the rat/salmon chimeric Pit-1 was then compared to wild-type rat Pit-1 in HeLa cells cotransfected with rat PRL

salmon rat

salmon

rat

salmon rat

gL:Ni'

'

QEMLSASISQTRILQT~SVPHPNMVNGANTL

143

______..______ ___----107

L 291 I 249

359 318

Fig. 2. Amino acid sequence comparison between salmon and rat Pit-1. The I~lack l ~ o s e srepresent residues conserved between salmon and rat Pit-1. Abbreviations for the aiiiino acid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H , His; I , I l r ; K, Lys; I,, Leu; M, Met; N, Asn; P, Pro; 0, Gln; R, Arg; S, Ser; T, Tlir; Y,\'id; \\'. Trp; :und Y, T y .

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or salmon PRL promoters. Over a range of vector concentrations, the chimeric rat/salmon Pit-1 transactivated the rat and salmon PRL promoters with comparable efficiency to wild-type rat Pit-1 (Elsholtz et al., 1992). These data suggested that any structural differences in the POU,,, of salmon p i t - 1 that result from amino acid substitutions do not significantly change its ability to regulate the PRL target gene. B. Species Differences in Alternative RNA Splicing Chinook salmon p i t - 1 encodes a protein of 358 amino acids in contrast to the rat pit-la, which encodes a protein of 291 amino acids. The greater length of the salmon Pit-1 polypeptide is due primarily to insertions of' 26 amino acids at a position corresponding to the junction of exon I and I1 (i.e., the /?-insert describe earlier) and also to a 33-amino-acid sequence (which we refer to as the y-insert) positioned at the junction of Pit-1 exons I1 and 111. Interestingly, although a similar y-insert has not been reported in mammalian Pit-l's, a sequence 76% identical to the salmon Pit-1 y-insert is present in a turkey pit-1 cDNA (Wong et al., 1992). In salmon the predominant form of Pit-1 contains both the p- and y-inserts. The a form of Pit-1, which lacks the /?-insert and is the major Pit-1 isoform in the rat, appears to be completely absent in chinook salmon. Even with a combination of PCR (exon I- and exon 111-specific primers) and Southern analysis, an a-specific Pit-1 splice was not detected in total salmon pituitary cDNA (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Sequence analysis indicates that in the salmon, the /?-specific splice site may be used by default, because the consensus splice acceptor dinucleotide AG used for a-specific splicing of mammalian Pit-1 pre-mKNA is replaced by CG in this teleost species. To determine whether the 33-amino-acid segment in salmonid Pit1 represents a novel alternatively spliced product of the p i t - 1 gene, we isolated a pit-1 genomic clone from an EMBL3 salmon genomic library. In contrast to the /?-insert described here, the y-insert is encoded by a distinct exon flanked by a consensus splice acceptor, polypyrimidine tract, and branch point at its 5' end, and by an intron splice donor consensus at its 3' end. Using degenerate PCR primers, designed to match conserved y-insert sequences in salmonid and turkey p i t - l , we have been unable to isolate related sequences from genomic DNA of'three divergent mammalian species (human, rat, cow). Furthermore, using PCR and Southern analysis, primers specific for rat Pit-1 in exon I and exon I11 did not amplify specific p i t - 1 fragments of

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SONALI MAJUMDAR AND HARRY P. ELSHOLTZ

greater mass than Pit-lp (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Our data suggest, therefore, that the Pit-1 y-insert may be restricted in its phylogenetic distribution to nonmammalian classes of vertebrates.

C. N-Terminal Sequences of Rat and Salmon Pit-1 In spite of structural differences in the N-terminal sequences of rat and salmon p i t - 1 , rat Pit-1 efficiently activates reporter constructs containing the salmon PRL promoter and 5'-flanking region (Elsholtz et al., 1992).To determine whether N-terminal sequences of salmon Pit-1 (which contain the p- and y-inserts) could activate the rat PRL promoter as efficiently as N-terminal sequences of rat Pit-1, a chimeric cDNA was constructed with N-terminal salmon pit-1 sequences fused to a rat pit-1 POU region. Rat Pit-1, salmon Pit-1, and the chimeric salmon/rat Pit-1 were functionally compared in several heterologous mammalian cell lines, including HeLa (cervical carcinoma), HepG2 (hepatoma),and Ltk- (fibrosarcoma), and in a salmonid hepatoma cell line, RTH. Although minor variations were observed among the different cell lines, each of the Pit-1 constructs strongly stimulated expression of the rat PRL/CAT construct (S. Majumdar and H. P. Elsholtz, unpublished results, 1994). Control reporter constructs indicated that activation was specific for the PRL gene. Therefore, phylogenetic changes in the structure of the Pit-1 N-terminal region do not prevent cross-species activation of PRL genes by Pit-1. Our data suggest that the dramatic species differences in PRL promoter function, observed using rat or salmonid pituitary cells (Elsholtz et al., 1992), are likely to depend on pituitary factors other than Pit-1. VI. CONCLUSION

In mammals the POU transcription factor Pit-1 has a critical role in anterior pituitary development and endocrine gene activation. Although the p i t - l gene is structurally conserved in teleost fish, both the DNA-binding POU domain and the N-terminal transactivation region have undergone a number of structural changes in these vertebrates. The rapid divergence of PRL gene 5' regulatory sequences and the species-specific pattern of PRL gene expression in mammalian and fish pituitary cells suggest that changes in Pit-1 function resulting from phylogenetic divergence might contribute to species-specific ex-

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pression of the PRL gene. Our studies suggest, however, that basal transcription of the PRL gene is activated with similar efficiency by Pit-1’s of distantly related vertebrates. Because these studies were performed in heterologous cell types, experiments are now needed to assess the impact of Pit-1 evolution on PRL gene regulation by other transcription factors in pituitary cells, including members of the steroid receptor family. An obligatory role for Pit-1 in teleost pituitary development has not been established. The early appearance of PRL cells during differentiation of Rathke’s pouch suggests that expression of the pit-1 gene may occur at the onset of pituitary organogenesis in certain families of fish. Lastly, transgenic approaches with targeted ablation of teleost pituitary cells will be necessary to establish whether a common Pit1-expressing progenitor cell can give rise to more than one cell type, as demonstrated in mammalian species.

ACKNOWLEDGMENTS We wish to thank Valdine Sundmark for proofreading the manuscript and Dr. Vladimir Lhotak for help with computer analysis of salmon and rat Pit-1. We would also like to thank Dr. C. L. Hew and members of his lab for providing the chinook salmon pituitary cDNA library, salmon genomic library, and salmon PRL/CAT constructs.

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S T R U C T U R E A N D R E G U L A T I O N OF G E N E S FOR E S T R O G E N R E C E P T O R S YVES LE DREAN, FARZAD PAKDEL, A N D YVES VA ,OTA RE Laboratoire d e Biologie Molkculaire URA, CNRS 256, UnicersitC d e Rennes I, 35042 Rennes Cedex, France

I. Introdnction A. Mechanism of'Estradio1 Action in Fish Physiology B. T h e Nuclear Estrogen Receptor 11. T h e Rainbow Trout (Oncorhynchus m y k i s s ) Estrogen Receptor A. Messenger RNA B. Protein 111. The Rainbow Trout Estrogen Receptor Gene .4. Expression and Regulation 13. Structure and Organization IV. Conclusion References

I. INTRODUCTION A . Mechanism of Estradiol Action in Fish Physiology In all vertebrate species, estradiol (E,) is involved in many regulatory steps controlling reproduction. In oviparous species, estradiol mainly controls egg yolk protein synthesis, whereas in viviparous species it regulates embryo implantation in the uterus. In fish, the main effect of E, is the control of vitellogenesis. Exogenous vitellogenesis is the period of egg yolk accumulation in the growing oocyte. As in all oviparous species, rainbow trout egg yolk protein synthesis is strictly controlled by estrogens. E, is synthetized in the ovary and released into the blood. In the liver, which is the main 33 1 PI5H PHYSIOLOGY, VOL. XI11

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target organ in salmonids, E, first increases its own receptor number, then induces vitellogenin synthesis. After protein maturation, vitellogenin is released into the blood, then incorporated into the oocyte after specific membrane receptor recognition (Fig. 1B). Estradiol also controls other physiological functions in fish. Many experiments have shown that E2 has feed-back regulatory effects on the hypothalamo-hypophysial axis (Fig. 1A). Peptidic hormones such as gonadotropin hormones (GtH) or gonadotropin-releasing hormone (GnRH) can be induced by E, (Dufour et al., 1985; Goos, 1987; Counis et al., 1987). Hence, the situation is unclear because this feedback regulation changes with the maturation stage of the fish (Weil and Marcuzzi, 1990)and, in some cases, the in vivo effect of estradiol may be indirect. Moreover, there is a wide variety of E, effects in organisms. However, most of these effects can be explained by specific modifications of gene expression. In the latter case, estradiol action is mediated by a specific nuclear receptor that binds E, with high affinity and recognizes specific DNA sites. The estrogen receptor is a transcription factor that belongs to the large nuclear receptor family.

B. The Nuclear Estrogen Receptor 1. NUCLEARRECEPTORSUPERFAMILY The nuclear receptor superfamily includes not only the steroid receptors (SR) but also receptors for retinoic acid (RAR), thyroid hormone (TR), vitamin D (VDR), and other proteins whose ligand is unknown (orphan receptors). cDNA cloning of some of these superfamily members in different species has revealed the primary structure of these proteins. In the steroid receptor family, structural comparison has shown the presence of six more or less conserved domains (A to F domains; Fig. 2A) (Weinberger et al., 1985). The definition of these specific domains has allowed the inclusion of other transcription factors that are not known as receptors in the family. The conserved amino acid domains of the steroid receptors have distinct functions that operate independently (Kumar et al., 1986, 1987).The most conserved domain in the steroid receptor family and in all animal species examined is the C domain, whose function is DNA binding. This region contains two zinc fingers that are directly involved in the recognition of specific DNA sequences. Region E is the largest domain; it is also well conserved for each member of the SR family. For the estrogen receptor, deletion experiments in the E

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functions _Main ___ ____________----Transcription Transactivation

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region have shown that it is essential for estradiol binding (Kumar et ul., 1986). The ligand-binding function of the E domain was later confirmed for other steroids (Giguere et al., 1986).Furthermore, other functions have been assigned to this domain. These functions include interaction with other proteins such as heat shock protein (hsp) (Beau-

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lieu, 1990; Howard et aZ., 1990), interaction with other receptor molecules resulting in the formation of homodimers (Fawell et al., l99Oa), and nuclear localization of the receptor (Picard et al., 1990). The E domain is also implicated in the most important function of the receptors, which is hormone-dependent transactivation of specific gene transcription (Webster et al., 1988). A transactivation function has also been detected in the poorly conserved A/B domain (Lees et al., 1989). This region activates transcription in a hormone-independent manner. The region located in the A/B domain is called activation function 1 (AFJ and the one located in the E domain is activation function 2 (AFJ. However, AF,, is repressed by the E domain in the absence of the ligand. The AF,, transactivation function is cell type and promoter dependent (Tora et al,, 1989a; Bocquel et al., 1989). 2. ESTROGEN RECEPTOR ACTIVATION In the absence of hormone, the estrogen receptor is part of an oligomeric complex (8.S complex). This complex contains various proteins, especially hsp 90 (Catelli et d., 1985), hsp 70, and P59/hsp 56 proteins (Sanchez et al., 1990). The SR-hsp associations are common to all steroid receptors, but the combinations are different according to animal species and receptor type (Smith and Toft, 1993). An early proposal for the functions of hsp 90 protein in the 8.S complex was that of a protease inhibitor (Housley et al., 1990) or a repressor receptor activity (Chambraud et al., 1990; Scherrer et uZ., 1990). Other results show that these proteins play more dynamic roles such as the maintenance of the receptor in an optimal conformation for hormone binding (Bresnick et al., 1989) or nuclear translocation of the receptor (Pratt, 1992). Hormone binding to the receptor allows the 8.S complex to dissociate and leads to receptor activation. The whole mechanism is not fully understood (for review see Bagchi et al., 1992a). During this activation receptor conformation is modified (Allan et al., 1992)and phosphorylation occurs (Bagchi et al., 1992b; Ali et al., 1993). The receptor forms a homodimer (Kumar and Chambon, 1988) before binding to a specific DNA sequence, that is, the estrogen responsive element (ERE), on the promoter (Tsai et al., 1988). This binding allows transcription to occur (Fig. 2B). AND TRANSCRIPTION 3. THE ESTROGEN RECEPTOR After activation by the ligand, the estrogen receptor modifies the transcriptional activity to various genes (for review see Green and Chambon, 1988; Briehl et al., 1990).

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YVES LE DREAN, FARZAD PAKUEL, AND YVES VALOTAIRE

Sequence comparison of estradiol-dependent promoters (Walker et al., 1984) and experiments with chimeric promoter constructions (Klein-Hitpass et al., 1986; Seiler-Tuyns et al., 1986) led to identification ofa specific D N A sequence, the ERE sequence, which is responsible for induction of transcription by E,. The E, receptor specifically recognizes the ERE structure and binds to it with high affinity. The ERE sequence (5’-GGTCANNNTGACC-3’) is similar but distinct from those recognized by other steroid receptors (Klock et nl., 1987; Martinez et al., 1987). The palindromic structure of the ERE, as well as that for other hormone-response elements (HRE),is in concordance with homodimer binding leading to dyad symmetry (Klein-Hitpass et d., 1989). After binding to an ERE, the receptor modulates transcriptional activity of the promoter by different mechanisms. The ERE is generally considered to be a hormone-dependent enhancer with an action that is independent of its location and orientation on the promoter (Martinez et aZ., 1987).Transcriptional activation by an E R is a complex event that is influenced by cell type and promoters. One general mechanism by which steroid receptors stimulate transcription is by facilitating the formation of a stable preinitiation complex on the promoter (Klein-Hitpass et al., 1990)(Fig. 2B). This stahilization occurs through direct contact between receptors and the preinitiation complex. For instance, hER transactivation functions (AFl-AF2) involve receptor association with the TATA-binding protein associate factors that form the general transcription factor TFIID (Brou et d . , 1993). Also an association of‘ the steroid receptors with TFIIB have been proposed (Ing et al., 1992), The nucleosome organization of chromatin is important for the regulation of gene expression (for review see Adams and Workman, 1993; Laybourn and Kadonaga, 1993).There are different mechanisms depending on the promoter. In some cases, the nucleosome structure is able to bring the receptor nearer to the preinitiation complex site. This proximity stabilizes the interaction between ER and other transfactors or RNA-polymerase I1 (Schild et al., 1993). In other cases the repression of the promoter exerted by the nucleosome is removed by the binding of the receptor, which is able to disorganize and dissociate the nucleosome (Reik et al., 1991). In addition, nuclear receptors are able to cooperate with other trans-factors that are not part of the general transcription factors (TF). Synergism between the E K and different members of the superfamily have been demonstrated (Muller et al., 1991). One classic example is the cooperative binding of two ER dimers with two tandem ERE in

11. STRUCTURE

AND REGULATION OF ESTROGEN RECEPTOR GENES

337

the Xenopus vitellogenin promoter (Martinez and Wahli, 1989). There is a possible cooperation between SR and other trans-factors such as SPI, OTF, or NFI (Schule et al., 1988; Wahli et al., 1989). These activations can be the result of nucleosome structural modifications that facilitate the binding of other factors or protein-protein interactions between different activating factors (Beato, 1991).These different possibilities of interaction create multiple pathways for control of promoter activation. For instance, the binding of SR alone to the HRE may not be sufficient to ensure transcription activation (Slater et al., 1989).Other cell-type (Corthesy et al., 1989)or developmental-specific factors (Ben-Or and Okret, 1993) are necessary to ensure a hormonal response. Finally, ER can be included in a multiprotein complex that will activate transcription after binding to a specific DNA sequence other than the ERE (Gaub et al., 1990). The steroid receptors are also capable of transcriptional repression. This repression is mediated by specific DNA sequences that are recognized by an inactive or repressive form of the receptor (Drouin et al., 1989,1993). Competition between different TF for binding to a specific DNA sequence is another possible repression mechanism. For instance, receptor binding to a HRE can prevent TFIID fixation (Chatterjee et al., 1989).

11. THE RAINBOW TROUT (ONCORHYNCHUS MYKISS) ESTROGEN RECEPTOR A. Messenger RNA

The complete human estrogen receptor was cloned in 1986 from a MCF-7 cDNA library (Green et al., 1986). Subsequently, estrogen receptor cDNAs of other species, such as chicken (Krust et al., 1986), Xenopus (Weiler et al., 1987), mouse (White et al., 1987), and rat (Koike et al., 1987), were cloned. The rainbow trout estrogen receptor (rtER) cDNA was cloned in our laboratory from a AgtlO cDNA library constructed from a female trout liver (Pakdel et al., 1989, 1990). The 3.5-kb rtER cDNA encodes a protein with 574 amino acids (AA). In other species, the ER cDNA varies from 6 to 7 kb. This difference in length is essentially due to a much shorter 3’untranslated region in the rtER (Fig. 3). By Northern blot, we have identified a 3.5-kb mRNA in liver, brain, pituitary, and pineal in trout. Also, two variant mRNA forms of 4.5 and 1.4 kb were detected. Interestingly, the 4.5-kb form was specific to the liver, whereas the 1.4-kb mRNA

338

YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIHE ATG

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Fig. 3. Schematic representation of the ER protein and mRNA. Comparison between rainbow trout ER and human ER proteins.

was detected only in the pituitary (Pakdel et al., 1990; Le Goff et ul., 1992). Of relevance to this, retinoic acid and thyroid receptors also have different isoforms that can be expressed in different tissues (Leid et nl., 1992). For steroid receptors, the number ofrnKNAs is generally lower. However, for the glucocorticoid receptor, two mRNAs were detected in rat (Miesfeld et al., 1986) and three in human (Hollenberg cct al., 1985). In Xenopus four ER mRNAs were observed (Weiler et ul., 1987). The cDNA clones corresponding to 4.5- and 1.4-kb mRNAs in trout have not yet been isolated. Thus, we do not know whether these isoforms encode functionally active estrogen receptors. Several hypotheses can be formulated for the origin of these mRNAs. They could be generated through alternative splicing and/or transcribed from distinct promoters of the same gene or from different genes. Southern blot experiments indicate that the rtER gene was duplicated in the trout genome (Valotaire et al., 1993). Thus, in addition to a common estrogen receptor for the different target tissues (encoded by the 3.5-kb ER mRNA), some cell-specific isoforms (encoded b y the 4.5and 1.4-kb mRNAs) could multiply cell responsiveness to estrogens.

B. Protein In trout and other species, DNA-binding (C)and hormone-binding (E)domains are highly conserved (Fig. 3). In contrast, the N-terminal region of rtER is different from that of other vertebrates because the A

11.

STRUCTURE AND REGULATION OF ESTROGEN RECEPTOR GENES

339

domain is absent and the B domain is poorly conserved. The sequence homology of the B domain with other ERs was found only in two small portions. Domains C and E show about 90 and 60% sequence homology, respectively among vertebrate species. The hormonebinding domain in the rtER is not as well conserved as among other species. This may explain the difference between dissociation constants for the E R in salmonids compared with other vertebrates. 1. DNA-BINDING DOMAIN u . Structure. The DNA-binding domain (C) contains two zinc finger structures that are implicated in the recognition and specific binding ofthe receptor to the DNA. The zinc finger motifwas first described in 1985 by Miller and collaborators for the transcription factor TFIIIA in Xenopus lueuis. Subsequently, a precise structure for the zinc finger motifs in TFIIIA was proposed (Berg, 1988; Jacobs, 1992). However, both zinc fingers in the steroid receptor family are quite different from the classic TFIIIA zinc finger family, reflecting different modes of binding to DNA (Hard e t ul., 1990a; Freedman, 1992). Although the 66 residues ofthe C domain in the ER are identical between Xenopus and human, 4 amino acids are different in trout (Fig. 4A). Nevertheless, it is unlikely that these changes in trout alter the structure or function of the DNA-binding domain of the receptor. Earlier studies on the structure of zinc fingers in steroid receptors were performed on the GR (Hard et ul., 1990b). Later, comparative studies on the E R arid the GR (Zilliacus e t ul., 1991) suggest that the structure of zinc fingers is similar in both receptor families. The DNA-binding domain binds two zinc atoms that are required for proper folding and DNA binding (Archer et ul., 1990). Each zinc atom is bound by four cysteine residues highly conserved in each zinc finger (Fig. 4B) that are encoded b y an individual exon in human as well a s trout ERs (Ponglikitmongkol et ul., 1988; Le Roux et d., 1993). The two zinc fingers differ slightly from each other. The first finger (C.1) at the N terminus of the domain consists of a 13-residue loop, whereas the second one ((2.11)is formed with only 9 residues (Fig. 4B). Also, the number of amino acids between the first two cysteines of each zinc finger differs with two amino acids in C.1 and five in C.11. Both zinc fingers exhibit a difference in the net charge of the amino acids. C.1 contains several hydrophobic amino acids, whereas C.11 contains more basic residues. This reflects different roles for each loop in DNA binding. Indeed, the first zinc finger is essential for recognition and specific DNA binding, whereas the second zinc finger is responsible for formation of a proper dimer.

340

YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIRE

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b. Role of Dimerization. The hormone-response element has a palindromic DNA sequence indicating that steroid receptors bind the HRE as a dimer so that each monomer of the receptor binds a HRE half-site. Kumar and Chambon in 1988 demonstrated that the E domain

11.

STRUCTURE AND REGULATION OF ESTROGEN RECEPTOR GENES

341

is required for dimerization, although other domains such as the DNAbinding domain are also implicated (Eriksson and Wranger, 1990). The receptor monomer can bind an ERE, but a dimer strongly enhances the binding affinity (Chalepakis et al., 1990; Cairns et al., 1991). DahlmanWright and collaborators in 1990 suggested that protein-protein interaction of the receptor dimer would stabilize the receptor-DNA complex allowing DNA binding cooperativity on each ERE half-site. In trout, most of the residues important for dimerization are conserved, indicating that the mechanisms of DNA binding are likely to be similar in all vertebrates. c. Specijicity in ERE Binding. Experiments carried out with sitedirected mutagenesis in the zinc fingers identified amino acids involved in the recognition and binding of the ERE (Danielson et ul., 1989, Mader et al., 1989), in receptor dimerization (Dahlman-Wright et al., 1991), and in transactivation functions (Schena et al., 1989). These studies show that three residues (Glu 203, Gly 204, and Ala 207 in hER) are important for discriminating between different responsive elements. In rtER these residues are also conserved as Glu 166, Gly 167, and Ala 170. It is interesting to note that substitutions of these amino acids to Gly, Ser, and Val, respectively, change the ER to a protein that binds a glucocorticoid responsive element (GRE) (Mader et al., 1989). These residues belong to the P-box characterized by Umesono and Evans in 1989 (Fig. 4B). This sequence forms an ahelix that fits into the major groove of DNA allowing specific contacts between amino acids ofthis helix and base pairs in HRE (Hard et al., l990a; Luisi et ul., 1991). The P-box also contains several residues that are conserved among all steroid receptors. These amino acids, namely, Lys 169, Lys 173, and Arg 174 of rtER or Lys 206, Lys 210, and Arg 211 of hER, make specific contacts with base pairs common in all HRE. Moreover, the P-box has highly conserved hydrophobic residues that are crucial for maintaining the structure of this region. In rtER these amino acids are Phe 171, Phe 172, Ser 175, and Ile 176. The second zinc finger contains a group of amino acids between the two first cysteines that forms a small loop called the D-box (Fig. 4B). These amino acids, absolutely identical in all ERs, are important for protein-protein interactions and receptor dimerization (Hard et al., 199Ob).This region imposes a proper three-dimensional structure for each zinc finger in the monomer receptor. Indeed, a spacing of three nucleotides between each half-site of the palindromic response element is required for the accurate positioning and proper binding of the dimer receptor to the DNA. Other residues in the C domain are

342

YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIRE

also important in stabilizing the ER-DNA complex. For instance, in the rtER, His 159 and Tyr 160 in the first zinc finger and Arg 197 and Lys 198 in the second zinc finger (His 196, Tyr 198, Arg 234, and Lys 235 in hER) were identified as residues that form interactions with phosphate groups in DNA. Through hydrogen bonds, residues located in an a-helix at the C-terminal position of the second zinc finger (Arg 206 and Lys 207 of rtER) can make contact with the minor groove and phosphate groups of the DNA double helix. To summarize, the highly conserved DNA-binding domain of the rtER suggests the same model of DNA binding as the one proposed for other SRs (Fig. 5). Thus, receptor dimerization due to interactions between residues in the D-box and E domain allows a specific and symmetrical recognition of the two half-sites of the ERE by the first zinc fingers. The amino acid sequence forming an a-helix in the first zinc finger functions as a recognition site for a specific DNA sequence and fits into the major groove of the DNA double helix. Through the interactions between phosphate groups of the DNA and different amino acids in both zinc fingers, the rtER-DNA complex is stabilized. This observation suggests that the ERE sequences are highly condyad symmetry axis

Monomer of rtER

1 half-site ERE

Monomer of rtER

half-site ERE

Fig. 5. Model of ERE recognition by the rtER zinc fingers. Adapted from Muller and Renkawitz (1991).

11.

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served in salnionids. Gel-shift assays carried out in our laboratory with the purified DNA-binding domain of rtER support this hypothesis. As shown in Fig. 6, a protein fragment produced from recombinant DNA in Escherichia coli, and containing the rtER DNA-binding domain, exhibits sequence-specific DNA binding to the consensus ERE (Pak-

del et al., 1994).

1

2

3

4

5

Fig. 6. Gel-shift experiment carried out with 5’-3’PP-end-labeled synthetic oligonucleotide containing a consensus ERE sequence. Lane 1: negative control with no added protein. Lane 2: 100 ng of purified rtER-DNA-binding domain (rtER-DBD) from fusion protein GST-rtER. Lane 3: 400 ng of purified rtER-DBD from fusion protein GST-rtER. Lane 4:control with 100 ng of GST protein. Lane 5 : control with 400 ng of GST protein.

344

YVES LE UREAN, FARZAD PAPDEL, A N D YVES VALOTAIKE

2. HORMONE-BINDING DOMAIN a . Structure and Homology. The hormone-binding domain is the most hydrophobic region in the receptor (Ojasoo et LIZ., 1991). Experiments with deletions showed that this domain is responsible for ligand binding (Kumar et al., 1986), although the precise sequence was not characterized for that function. Rather, the three-dimensional structure ofthis domain makes a hydrophobic pocket for hormone binding. This domain is also involved in other receptor functions, such as dimerization, transactivation, nuclear localization, and interaction with heat shock proteins. In this region several functional subdomains might overlap and serve separate functions. The hormone-binding domain of rtER exhibits less homology compared with other ERs. However, three subdomains that show more sequence similarity can be distinguished (regions, a,p, and y in Fig. 7). Note that all amino acids involved in estrogen binding lie in these portions of the receptor. The recognition of estradiol by its receptor probably depends on the aromatic rings as well as the hydroxyl groups at positions C 3 and C17 of estradiol (Vessi51-e~ et al., 1989). Earlier studies using affinity-labeling ligands, such as tamoxifen aziridine, defined regions at the carboxyl terminus of the receptor involved in estradiol binding (Ratajczak et al., 1989). These regions consist of amino acids between 470 and 480 and 498 and 514 in the rtER and lie in the conserved y region. More precisely, Harlow and colleagues in 1989 used affinity-labeled ligands to identify in the hER the specific amino acid Cys 530 that binds covalently to the aziridine group of an estrogen analog (ketanonestrol aziridine) and antiestrogen analog (tarnoxifen aziridine). This residue is conserved in the trout E K (Cys 496) as are adjacent residues, namely, Lys 495, Lys 497, and Asn 498 corresponding to Lys 529, Lys 531, and Asn 532 in the hEK. The latter amino acids were identified through site-directed mutagenesis as residues involved in discrimination between estrogens and antiestrogens (Pakdel and Katzenellenbogen, 1992). Also, other amino acids such as Ile 480 and Gly487 were conserved in the rtER [corresponding to Ile 518 and Gly 525 in the mouse E R (Fawell et al., 1990a)l or Gly 366 and Lys 415 [corresponding to Gly 400 (Tora et al., l989b) and Lys 449 in the hER (Pakdel et al., 1993)] and were identified as important in estradiol-binding function. It was also reported that the tyrosine residues in the hormone-binding domain could be implicated in ligand binding (Migliaccio et al., 1991; Koffnian et al., 1991).Among the five tyrosines in the E domain of the hER, there are only two conserved in the rtER. These residues at positions 492 and 503 are

11.

STRUCTURE AND REGULATION OF ESTROGEN R E C E P T O R G E N E S

345

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located in the y region, which contains a large number of amino acids implicated in hormone binding. Studies from different groups indicate that two close but distinct, ligand-binding sites could exist in the hormone-binding domain (Svec et al., 1989; Vegeto et al., 1992). According to these authors, the first pocket participates in the recognition and binding of estrogen, whereas the second one is responsible for antiestrogen binding. Nevertheless, both binding sites are very close and could interfere with each other during agonistlantagonist competition events (Bond et al., 1992).

346

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YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIRE 400

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Fig. 8. Estradiol binding to in citro translation products of rtER mRN.4. One-half milligram of in uitro synthesized rtER mRN.4 was translated in reticulocyte lysate (final volume 50 ml). T h e in oitro translation mix (25 ml) was incubated with 6 nM 3H-labeledE2 in either the presence or absence of increasing concentrations of unlabeled estradiol ( A ) or with 1 nM of various competitors (B). T h e nonspecific binding was determined through incubation of lysate (without mRN.4) with 6 nhl "H-labeled E, in either the presence or absence of 1m M unlabeled estradiol ( C ) .Steroid competitors are diethylstilbestrol (DES), testosterone (TEST), and cortisol (CURT). T h e values represent the duplicate determination mean of the total bound of .'H-labeled E,.

6 . Affinity and Specijicity of the rtER f o r Estrogens. The rtER exhibits a lower affinity for E, compared with other ERs, although it contains most amino acids defined to be involved in E, binding. Dissociation constants ( K d )for the E R in salmonids were about 1.,5to 4 nM (Maitre et ul., 1985; Lazier et al., 1985), whereas the K,, for the human E R was 4- to 10-fold lower (0.4 n M ) (Tom et ul., 1989b).This agrees with our previous studies in which the rtEK cDNA was transcribed and translated i n vitro. The translated products showed specific E, binding with a Kd of about Fj nM (Pakdel et d., 1990). This difference in E,-binding affinity may reflect the structure of the E domains in fish and mammals. Salmonids live at lower temperatures (10-12"C) compared with mammals and this may influence E,-binding affinity. This idea can be tested by investigating the Kd for tropical fishes. A receptor with higher affinity for its ligand may have been selected during evolution. This selection would result in a more sensi-

11.

STRUCTLJRE A N D REGULATION OF ESTROGEN R E C E P T O R G E N E S

347

tive receptor that responds to lower E, concentrations. The requirement for a diminished E, concentration could eliminate nonspecific hormone binding with proteins other than receptors and hence suppress interference events. Experiments carried out in our laboratory show that in addition to estradiol, the rtER binds diethylstilbestrol, estrone, and estriol. However, competition assays show that other steroids do not alter E,binding capacity of the receptor, leading us to conclude that the rtER exhibits specificity for estrogens (Fig. 8) (Pakdel et al., 1990). It is also interesting to note that the rtER, like other ERs, binds antiestrogens such as tamoxifen or ICI 164,384. Hence, the action of the rtER can be totally inhibited. The binding of the ligand (agonist or antagonist) induces a conformational change in the receptor that varies with the ligand (Fawell et al., 1990b; Weigel et al., 1992). This liganddependent conformational change is an important event in different steps of transactivation (Allan et al., 1992).However, the rtER can also bind with a low affinity other compounds like xenobiotics (lindane) or phytoestrogens (equol and coumestrol) that induce a conformational change sufficient to transactivate some hormone-dependent genes such as vitellogenin (Flouriot et al., 1992).

3. TRANSACTIVATION Two distinct regions are responsible for the transcriptional activity of the ER (Webster et al., 1988; Lees et al., 1989). These regions, called activation function AF, and AF,, are located in the A/B and E domains, respectively. The activity of AF, strictly depends on the presence of the hormone, whereas the AF, activity is constitutive. AF, activity, which exhibits a marked cell and promoter specificity, accounts for 5 to 50% of the total E R activity (Bocquel et al., 1989). The A F , activity is suppressed by the E domain in the absence of ligand, but AF, and AF, are able to act synergistically in the presence of estradiol (Tasset et al., 1990). The amino acid residues corresponding to AF, and A!?, have not yet been identified, but it is known that AF, is encoded by more than one exon (Webster et al., 1989). It is conceivable that these activation domains are formed from the three-dimensional organization of several peptides that are required for the transactivation function. It is interesting that neither AF, nor AF, is similar to previously identified activation sequence motifs such as the acidic regions (found in yeast transcription factor Gal 4), the glutamine-rich domains (found in Spl), or the proline-rich domains found in the transcription factor C T F (Carey et

348

YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIRE

al., 1991).Tasset and colleagues in 1990 showed that the two transactivation functions may interact with different factors depending on the cell type, and that the ability to activate transcription synergistically with other activators may be mediated by different mechanisms.

a . AF, Function. The N-terminal region of the rtER lacks the A domain and exhibits very low sequence similarity with other ERs. In this region we have found only a portion of amino acids, located between serine 69 and histidine 81, displaying a significant identity with other ER sequences. This serine-rich region has been shown to be phosphorylated in the hER (Ali et al., 1993). A deletion in this region abolished the AF, activity (Imakado et al., 1991). At the present time, we do not know whether an AF, function exists in the rtER, but current experiments in our laboratory should provide results on this question in the near future. 13. AF, Function. To study the ability of the rtER to activate gene transcription, we have cotransfected the rtER cDNA expression vector (pSG,-rtER) and an estrogen-dependent reported plasmid (ERE-TKCAT) into an embryonic trout cell line (STE-137). As shown in Fig. 9A, the transcriptional activation of the reporter gene (CAT activity) is markedly dependent on the presence of the receptor, estradiol, and the ERE. The E,-induced transcription is dose dependent and is inhibited in the presence of the antiestrogen tamoxifen (Le Drkan, 1993).We have also shown that the rtER induces CAT activity when the reporter plasmid is under the control of salmonid gene promoters. Moreover, using a simple reporter plasmid consisting of two EREs upstream of a TATA-box sequence fiised to the CAT gene (ERE,TATA-CAT), we showed that the rtER can recruit the general factors required for the preinitiation complex of transcription, and thereby induce transcription (Fig. 9B). A study on mouse ER has identified several amino acids located between residues 538 and 552, having an essential role in AF, activity (Danielan et ul., 1992). Based on the high level of sequence similarity in this region with the rtER (corresponding to residues between 500 and 517 in the y region of the rtER; see Fig. 7), it is conceivable that the hormone-dependent transactivation mechanism is similar in the rtER. However, preliminary experiments between the hER and rtER in different cells show that the trout estrogen receptor exhibits some differences in sensitivity to estrogen and promoter transactivation.

11.

STRUCTURE AND REGULATION OF ESTROGEN RECEPTOR GENES

A

349

B

60000

r

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Fig. 9. (A) Transfection in STE-137 cell line. 1.2 x lo6 cells were dispersed in a 60-mm-diameter dish in the presence of M E M medium supplemented with 10%steroidfree serum. Two days later, using the CaP0,IDNA coprecipitation method, transfection was carried out with 10 pg of total plasmid (5 p g of CAT-reporter plasmid, 1 pg of rtER expression vector, and 4pg of carrier plasmid). A glycerol shock was performed 12 hr later and the cells were stimulated or not by E, and left for 48 hr for transient expression. The cells were harvested and an extract was prepared for CAT assay. 1: ERE-TK-CAT plasniid with pSG5; 2: ERE-TK-CAT plasmid with pSG5-rtER expression vector; 3: TK-CAT plasmid with pSG5-rtER expression vector. (B) Transfection in CHO cell line. The same protocol was used to transfect 3 x 1O'cells with 1 p g ofCAT-reporter plasmid, 500 ng of rtER expression vector, and 8.5 pg of carrier. 4:(ERE)B-TATA-CAT plasniid with pSG5-rtER expression vector.

111. THE RAINBOW TROUT

ESTROGEN RECEPTOR GENE A. Expression and Regulation '4s noted in the foregoing, estradiol-dependent promoters are directly regulated b y the estrogen receptor. We and others have shown that vitellogenin gene expression in hepatocytes of male trout or Xenopus is strictly dependent on the quantity of estrogen receptor (Corth6sy et al., 1990; Pakdel et al., 1991). Other data demonstrate that a basic level of the receptor is required for cellular responsiveness and that a direct correlation exists between receptor concentration and biological response to the hormone (Bourgeois and Newby, 1979; Bloom et al., 1980). Several studies in salmonids show that during the reproductive cycle, the ER concentration varies in the liver (Smith and Thomas, 1991).In rainbow trout we have demonstrated that hepatic ER mRNA and protein are both induced by estradiol (Pakdel et al., 1991). This

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YVES LE DKEAN, FAHZAD PAKDEL, A N D YVES VALOTAIKE

marked increase in receptor concentration increases cell sensitivity to estrogen and thus induces vitellogenesis. Apart from estradiol, other hormones also appear to regulate the expression of ER genes (Ree et al., 1989, Freyschuss et al., 1991). Thus, E, treatment increases the concentration of ERs in rat liver and pitutuary and decreases the concentration in uterine cells (Shupnik et al., 1989). Other observations on human breast cancer cells (T47D and MCF-7) support the idea that E, action on receptor induction is cell specific (Read et al., 1989; Saceda et al., 1989). In rainbow trout the expression of the ER gene is also dependent on the target cell and its hormonal content. We have shown that E, induces both hepatic E R mRNAs (3.5 and 4.5 kb) but fails to stimulate E R gene expression in the pituituary (Pakdel et al., 1991; Le Goff et al., 1992). Our studies using rainbow trout hepatocyte cultures demonstrated that the expression of ER gene can be modulated by glucocorticoids, or xenobiotics, in addition to E, (G. Flouriot, 1994, unpublished data).

B. Structure and Organization To determine the molecular basis of rtER gene regulation, a trout genomic library was constructed in hEMBL3 and screened with rtER cDNA probes in the Molecular Genetic Laboratory at Rennes University (Le Roux et al., 1993). Six clones were isolated and analyzed. These clones were divided into two groups. The first clone, fully sequenced, represents 99% sequence identity with rtER cDNA, whereas the second clone that was partially sequenced exhibits only 67% sequence similarity. This implies the existence of at least two genes for the rtER. We do not know whether the second gene is functional. 1. EXON-INTRON ORGANIZATIOK The cloning of steroid receptor genes, as well as the rtER gene, indicates that the position of the introns is strictly conserved. The rtER gene encompasses 10 exons (Fig. 10) containing 462, 386, 222, 117, 194, 151, 139, 134, 184, and 1591 base pairs (bp) (Le Roux et al., 1993).The sum of these exons gives rise to a 3.5-kb mRNA corresponding to the isolated cDNA. The rtER gene structure resembles other ER genes cloned in the human (Ponglikitmongkol et al., 1988) and mouse (White et al., 1987). The rtER gene possesses one additional intron separating exons 4 and 5 that code, respectively, for the second zinc finger and the D domain. It is possible that during evolution this intron was lost in

Exons

1

3

2

4

5 6

7

8

9

1

0

rtER Protein 574 aa

AIB

C

D

E

F

Domains

Fig. 10. Schematic representation of the rtER gene structure and corresponding mRNA (cDNA) and protein.

352 Y\'ES L E DKEAN, FAKZAD PAKDEL, Ah11 YVES \'ALOTAlKE

x

0

C

r

W

52 r

0 X W

N u \-

L

11. STRUCTURE AND REGULATlON OF ESTROGEN RECEPTOR GENES 353 higher vertebrates. Exon 1, corresponding to a leader exon, is also found in the human and mouse ER (Keaveney et al., 1991; White et d.,1987). This exon does not encode the receptor and does not have sequence identity with other ERs. The A/B regions are mostly encoded by exon 2, whereas the DNA-binding domain is encoded by exons 3 and 4 (one exon per zinc finger). Exon 5 encodes the D domain and hormone-binding and transactivation regions are encoded by several exons (6, 7, 8, 9, and part of 10). Exon 10, which is the longest one, represents all the variable F domain and the 3' untranslated region of the mRNA.

2.

PROMOTER

To determine the position of the rtER gene promoter, primer extension experiments were done using oligonucleotides corresponding to the 5' end of the rtER cDNA and trout liver poly A + mRNA from E2stimulated male trout. One unique initiation site of transcription was detected (Le Roux et al., 1993), suggesting the existence of only one functional promoter. However, primer extension assays did not reveal an extension product with poly A + RNA from untreated male trout. It is therefore unknown whether the initiation site of transcription of rtER gene is similar in both control and E,-treated animals. Data from the glucocorticoid receptor (Strahle et al., 1992), progesterone receptor (Kastner et al., 1990),and human or mouse ER (Keaveney et al., 1991; Weisz, 1992) indicate that each of these genes has several promoters that function in different tissues generating distinct transcripts (Strahle et al., 1992; Grandien et al., 1993). It was therefore of interest to know the initiation site of the rtER gene for different target tissues. Figure 11 shows a sequence analysis of the rtER gene promoter by computer. The rtER gene promoter lacks a TATA box but it exhibits a TATA-like box sequence at position -58, which is not the usual position. A TATA box is generally positioned 30 bp upstream of initiation site of transcription. Near the cap site, two INR sequences are observed. These sequences are recognized by transcription factor TFIIi, which recruits general factors allowing formation ofthe preinitiation complex of transcription (Roeder, 1991). At - 71 a GC box corresponding to the SP1 factor and an enhancerlike sequence at -416 (Latchman, 1991a) are found. The rtER gene promoter contains three silencer sequences (Baniahmad et al., 1987) located at positions - 1751, - 595, and + 165. Two AP1 consensus sequences (Latchman, Fig. 11. Sequence analysis of the rtER gene promoter by computer comparison

354

Y\'ES LE DKEAN, FAKZAD PAKDEL, A N D YVES VALOTAIRE

+

+

199lb) are present at positions 54 (in exon 1)and 614 ( in intron 1). Interestingly, there are two sequences at - 1116 and - 187, presenting some homology with the hepatic nuclear factor HNF4 consensus sequence. Several imperfect palindromic GREs were detected. The rtER promoter presents several half estrogen responsive elements at positions -1155, -770, -576, -253, -208, +37, and +223, and an imperfect palindromic element at + 242 that shows a single point mutation (G T) compared with the consensus sequence. A similar single mutation was found in the functional ERE for salmonid GtH IIP (Xiong and Hew, 1991, 1993). It is known that half-sites or imperfect palindromic elements are able to confer hormone responsiveness (Kato et ul., 1992).

-

3 . PHOMOTER ACTIVITY A 2.5-kb genomic fragment of the EK gene ( - 1758 to

+ 740) containing several potential regulatory elements was inserted upstream of the bacterial chloramphenicol acetyl-transferase (CAT) gene in the reporter plasmid pBCO-CAT. Data from earlier experiments showed that when this vector was microinjected into the nuclei of Xenopus oocytes, the CAT activity was enhanced in the presence of this 2.5-kb fragment. However, the promoter activity was low, due perhaps to the presence of the silencer sequences or the absence of' consensus TATA and CAAT boxes. To characterize the activity of these putative cis-elements, we made a set of reported plasmid constructs containing different regions of the rtER promoter. These vectors were transfected into a Chinese hamster ovarian (CHO) cell line and the results are represented in Fig. 12. Data indicate that deletions in the 5' end of the 2.5-kb promoter increase transcriptional activity of the reporter plasmid. This is shown b y a comparison of' CAT activity among the 740 fragment), the 1.88-kb construct 2.5-kb construct ( - 1758 to ( - 1627 to +257), and the 0.74-kb construct (-481 to +257). These enhancements in CAT activity may be due to the deletion of silencer sequences in the promoter. We verified this by using the reporter constructs containing the CAT gene placed under the control of' the thymidine kinase promoter in the presence or absence of the silencer sequence. The silencer element efficiently diminished the constitutive transcriptional activity of the reporter plasmid in both mammalian and trout cell lines (Le Drean, 1993). These experiments prove that this potential silencer sequence is constitutively active and ubiquitous. In contrast, the 0.57-kb fragment (-318 to +257) resulted in a sinall increase in CAT activity. This is surprising because the deleted sequence corresponds to an enhancer element so that the CAT activity should have decreased when the 0.74-kb fragment was deleted. It is

+

11. STRUCTURE

AND REGULATION OF ESTROGEN RECEPTOR GENES

355

Controls TK-CAT BCo I

I

0

100

200

300

400

500

600

700

CAT Activity (dpm) / pg plasmid / 6-gal Activity

rtFR V I - s n r t r x t k m PER-2.50 K b PER-1.88 KB PER-0.74 K b PER-0.57 Kb

0

100

200

300

CAT Activity (dpm) / pg plasmid / Rgal Activity

Fig. 12. rtER gene promoter activity. DifT’erent constructions were tested in CHO cells by transfection, using the same protocol described in Fig. 9. Reporter plasmid TK-CAT (a positive control) and BCo-CAT (a promoterless CAT plasmid) were used to determine the nonspecific dpm value. T e n micrograms of the different rtER gene promoter constructions were cotransfected with 500 ng of pSG5-rtER expression vector and 500 ng of p C H l l 0 ( = SV40 promoter-Lac Z gene plasmid) as an internal control. PER-2.5 kb = - 1758 to + 740 promoter fragment; PER-1.88 kb = - 1627 to + 257; PER-0.74 kb = -481 to +257; PER-0.57 kb = -381 to +257. T h e cells were treated for 48 hr with E, (1 pA4).

thus possible that this cis-element is not fiinctional as expected from its sequence. To characterize different cis-elements responsible for estrogen responsiveness, we tested different promoter constructs in transient expression experiments. All the different constructs showed an E, induction. The shorter construct was transfected into C H O cells either alone or with the expression vectors encoding trout or human estrogen receptors (rtER or hER in Fig. 13B). We observed that CAT activity was induced only in the presence of the receptor when the cells were treated with estradiol. It is conceivable that the rtERactivates transcription of its own gene in the presence of hormone. As we described earlier, the ER acts by binding to an ERE. This sequence with one

356

YVES LE DREAN, FARZAD PAKDEL, AND YVES VALOTAIRE

rtER gene promoter Construct PER-0.57 Kb-CAl

A

TK

CAT

pERE-TK-CAT construction

PER-0.57 Kb-CAT

B

No Stimulation

SG5 0

10000

5000

15000

20000

CAT Activity (dprn)

pERE-TK-CAT

C rtER

1 E2 = 1 /JM No Stimulation

SG 5 0

10000

20000

30000

40000

50000

CAT Activity (dpm) Fig. 13. Characterization of the active ERE in the rtER gene promoter. (A) Schematic representation of the construct used. (B) Cotransfection in C H O cells of the smallest rtER gene promoter construct with or without ER expression vector. (C)Cotransfection in STE-137 cells o f t h e heterologous reporter plasmid ( = ERE of the rtER gene, upstream of the TK promoter).

11.

STRUCTURE AND REGULATION OF ESTROGEN RECEPTOR GENES

357

point mutation was found at +242 of the rtER gene. The location of functional ERE downstream of the initiation site of transcription has been reported elsewhere (Chauchereau et al., 1992). To investigate if this imperfect palindromic ERE without any other flanking sequences is sufficient to confer estrogen responsiveness, we inserted this element upstream of the TK-CAT plasmid. Data indicate that this sequence can confer E, inducibility to the T K promoter and enhance transcription (Fig. 13C) (Le Drkan, 1993).

IV. CONCLUSION Our aim is to better understand the structure-function relationship of the rtER and its action on the regulation of estrogen-dependent gene expression. Previous studies from this and other laboratories have shown important estrogen mechanisms in the reproductive biology of fish, but several points concerning the regulation of rtER gene transcription, the structure of domains, and function of the receptor remain unknown. We have characterized the specificity and the affinity of rtER and proved that this fish protein has the same DNA response sequence as its mammalian homolog. We showed that the rtER can act as a transcription factor and is able to E,-transactivate certain hormonedependent promoters. Concerning the regulation of the rtER gene, we found a silencer that maintains basal expression at a low level. The presence of an ERE sequence in the first exon allows an E2transcriptional enhancement of rtER synthesis. Moreover, transcription factors, other than the estrogen receptor were identified in the tissue-specific regulation of rtER gene transcription.

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tional domains of the human estrogen receptor. Cell (Cumbridge, MCLW.)51, 94 1-95 1. Latchman, D. S. (l99la).Transcription factors and constitutive transcription. Z t l “Eukaryotic Transcription Factors,” pp. 41-61. Academic Press, San Diego. Latchman, D. S. (19Ylb).Transcription factors and inducible gene expression. I n “Eukaryotic Transcription Factors,” pp. 62-86. Academic Press, Sari Diego. Laybourn, P. J., and Kadonaga, J. T. (1991).Hole ofnucleosoinal cores and histone H l in regulation of transcription by RNA polymerase 11. Science 254, 238-245. Lazier, C. B., Lonergan, K., and Mominsen, T . P. (1985).Hepatic estrogen receptos and plasma estrogen-binding activity in the Atlantic salmon. Gen. C o n i ) ~Etrdocrittol. . 57,234-245. Le Drean, Y. (1993). “Etude par cotransfection d e la regulation par le reccpteur aur estrogenes, d e I’expression d e genes hornmono-dependants chez la truite arc-en-ciel (Oiicorhynchus mykiss), dans des lignees cellulaires et e n culture primaire.” These dc l’universite d e Rennes I. Lees, J. A,, Fawell, S. E., and Parker, X I . G . (1989j. Identification o f t w o transactivation domains in the mouse oestrogen receptor. Niicleic Acids Res. 17, 5477-5488. Le Goff, P., Salbert, G., Prunet, P., Saligaut, C., Bjornsson, B. Th., Haux. C., and Valotaire, Y. (1992).Absence of direct regulation of prolactin cells by estradiol-171) i n rainbow trout. Mol. Cell. Etidocrinol. 90, 133-139. Leid, M., Kastner, P., and Chambon, P. (1992). Multiplicity generates diversity in the rctinoic acid signalling pathways. Trends in Biochetn. Sci. 17, 427-433. Le Houx, M. G., Thezk, N., Wolff; J., and Le Pennec, J. P. (1993).Organization of’a rainbow trout estrogen receptor gene. Biochini. B i o p h y s . Actu 1172, 226-231, Luisi, B. F., Xu, W. X., Otwinowski, Z . , Freedman, L. I’., Yamamoto, K. H., and Siglei-, P. B. (1991).Crystallographic airalysis ofthe interaction ofthe glucocorticoid receptor with DNA. Nature (London) 352, 497-50.5. Xlader, S., Kuniar, V., de Verenenil, hl., and Chambon, P. (1989). Three amino acids o f the oestrogen receptor are essential to its ability to distinguish an oestrogen ti-om a glucocorticoid-responsive receptor, Nnture (London)338, 271-274. ,\laitre, J. L., Mercier, L., Dolo, L., and Valotaire, Y . (1985).Caractt.risation de rtceptetirs spdcifiques Q l’oestradiol, induction de la vitellogenine c t de son inHiX.4 danr Ic> foie. Riochimie 67, 215-225. \lartinez, E., and Wahli, W. (1989).Cooperative binding of estrogen receptor to inipcrfeet estrogen-responsive DNA elements correlates with their synergistic Iiornionedependent enhancer activity. E M R O J . 8, 3781-3791. XIartinez, E., Givel, F., and Wahli, W. (1987). T h e estrogen-responsi\.~ elenicnt as an inducible enhancer: DNA sequence requirements and conversion to :t glncocorticoid-respo~isjveelement. EA4HO J . 6, 3719-3727. lliesfeld, R., Husconi, S., Godowski, P. I., Maler, B. A., Oknet, S., Wilstrom, A. C . , Gustafsson, J. A , , and Yamamoto, I(.H. (1986).Genetic complementatioti ofglucocorticoid receptor deficiency by expression of cloned receptor ”cDNA. Cell (Cumlit-itlge, Mass.)46, 389-399. lligliaccio, A,, Castoria, G . , D e Falco, A,, Di Donienico, l l . , C:aldiero, hf., Noln, E., Chambon, P., and Auricchio, F. (1991).111 citro phosphorylation arid hornione hinding activation of the synthetic wild type human estradiol receptor. ./. Steroid Biochern. Mol. H i o l . 38, 407-413. Xliller, J., Mc Lachlan, A. D., and Klug, A. (1985). Kepetitive zinc-binding domain in the protein transcription factor 111-A from Xenopus oocytes. E M B O /. 4, 16091614.

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Muller, hl., and Renkawitz, R. (1991). T h e glucocorticoid receptor. Biochini. B i o p l l ! / s . Actu 1088, 171-182. Muller, XI., Baniahmad, C., Kaltschmidt, C., Schiile, R., and Renkawitz, H. (1991).Cooperation transactivation of steroid receptor. I n “Nuclear Hornwne Receptors” (M.G. Parker, ed.). Academic Press, San Diego. ojasoo, T., DorC, J. C., Mornon, J. P., and Raynaud, J. P. (1991). Two approaches to structure-activity relationships in the field of sex-steroids and their analogs. I t 1 “Molecular Structure and Biological Activity of Steroids” (31. Bohl and W. L. Duax, eds.), pp. 157-207. Pakdel, F., and Katzenellenbogen, K. S.(1992). Human estrogen receptor mutants with altered estrogen and antiestrogen-ligand discrimination. J . B i d . Cheiri. 267,

3429-3447. Pakdel, F., Le Guellec, C., Vaillant, C., Le Roux, M. G., and Valotaire, Y. (1989). Identification and estrogen induction of two estrogen receptors (ER) messenger ribonucleic acids in the rainbow trout liver: Sequence homology with other ERs. hlol. E ridocrinol. 39, 44-51. Pakdel, F., Le Gac, F., Le Goff, P., and Valotaire, Y. (1990). Full-length sequence and i n citro expression of rainbow trout estrogen receptor cDNA. M o l . Cell. Endocritiol.

71, 195-204. Pakdel, F., Feon, S., L e Gac, F., L e Menn, F., and Valotaire, Y. (1991).I n G ~ O Oestrogen induction of hepatic estrogen receptor mHNA and correlation with vitellogenin mRNA in rainbow trout. M o l . Cell. Etrdocritiol. 75, 205-212. Pakdel, F., Reese, J. C., and Katzenellenbogen, B. (1993). Identification of charged residues in a N-terminal portion of the hormone binding domain of the human estrogen receptor important in transcriptional activity o f t h e receptor. Mol.Endocri~101.

7, 1408-1417.

Pakdel, P., Petit, F., Anglade, I., Hah, O., Delauney, F., Railhache, T., and Valotaire, Y. (1994). “Overexpression of rainbow trout estrogen receptor domains in E . coli: Characterization and utilization in the production of antibodies for imniunoblotting and imtnunocytochemistry.” Submitted. Picard, D., Kuniar, V., Chanihon, P., and Yan~amoto,K. H. (1990). Signal transductioil by steroid hormones: Nuclear localization is differentially regulated in estrogen and glucocorticoid receptors. Cell. Regzrl. 1, 291-299. Donglikitniongkol, M., Green, S., and Clianibon, P. (1988).Genonric organization o f t h e human oestrogen receptor gene. E M B O J . 7, 3385-3388. Pratt, W. B. (1992).Control ofsteroid receptor function and cytoplasmic-nuclear transport b y heat shock proteins. BioEssu!js 14, 841-848. Ratajczak, T., Wilkinson, S. P., Brockway, k l . J., Hiihnel, H., Lloritz, R. L., Begg, G . S., and Simpson, R. J. (1989).The interaction site for tamoxifen aziridine with the bovine estrogen receptor. /. B i d . Cheni. 264, 13453-13459. Read, L. D., Greene, G. L., and Katzenellenbogen, H. S. (1989).Regulation of estrogen receptor messenger ribonucleic acid and protein level in hunian breast cancer cell lines by sex steroid hormones, their antagonist and growth factors. Mol. Endocriiio/.

3, 295-3304, Ree, A. H., Landmark, B. F., Eskild, W., Levy, F. O., Lahooti, H., Johnsen, T., Aakvag, A , , and Hansson, V. (1989). Homologous down-regulation of messenger rihonucleic acid and protein levels for estrogen receptors in MCF-7 cells: An inverse correlation to progesterone receptor levels. Eridocritiolog(/ (Baltimore)124, 2.577-2583. Reik, A,, Schiitz, G., and Stewart, A. F. (1991).Glucocorticoids are required for establishment and maintenance of an alteration in chromatin structure: Induction leads to

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a reversible disruption of nucleosomes over an enhancer. E M B O J . 10, 25692576. Roeder, R. G . (1991).T h e complexities ofeukaryotic transcription initiation: Regulation of preinitiation complex assembly. Trends Biochern. Sci. TIBS 16, 402-408. Saceda, M., Lippman, M. E., Lindsey, R. K., Puenta, M . , and Beth Martin, hl. (1989). Role of an estrogen receptor-dependent mechanism in the regulation of estrogen receptor mRNA in MCF-7 cells. Mol. Endocrinol. 3, 1782-1787. Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990).T h e 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins. Biochemistry 29, 5145-5152. Schena, M., Freedman, L. P., and Yamamoto, X. R. (1989). Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA-binding arid transcriptional enhancement activities. Genes Deo. 3, 1590-1601. Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B . (1990). Structural and functional reconstitution of the glucocorticoid receptor-Hsp90 complex. 1.Biol. C h e m . 265, 21397-21400. Schild, C., Claret, F. X., Wahli, W., and Wolffe, A. P. (1993). A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in oitro. EMBO J. 12, 423-433. Schiile, R., Muller, M., Kaltschmidt, C., Renkawitz, R. (1988).Xlany transcription factors interact synergistically with steroid receptors. Science 242, 1418-1420. Seiler-Tuyns, A., Walker, P., Martinez, E., Merillat, A. hl., Givel, F., and Wahli, W. (1986).Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line. Nucleic Acids Res. 14, 8755-8770. Shupnik, M . A,, Gordon, M. S., and Chin, W. W. (1989). Tissue-specific regulation of rat estrogen receptor mRNAs. M o l . Endocrinol. 3, 660-665. Slater, E. P., Posseckert, G., Chalepakis, C . ,Redeuihl, G., and Beato, M . (1989).Binding of steroid receptors to the HREs of mouse maniniary tumor virus, chicken and Xenopus vitellogenin and rabbit uteroglobin genes: Correlation with induction. 1.Steroid Bioclzem. 34, 11-16. Smith, D. F., and Toft, D. 0. (1993). Steroid receptors and their associated proteins. Mol. Endocrinol. 1, 4-11. Smith, J. S., and Thomas, P. (1991).Changes in hepatic estrogeri-recepto~coiice~itr~~tions during the annual reproductive and ovarian cycles of a marine teleost, the spotted seatrout, Cynosciori nebulosus. Gen. Coinp. Endocrinol. 81, 234-245. Strahle, U., Schmid, A,, Kelsey, G., Stewart, F., Cole, T. J., Schmid, W., and Schutz, G. (1992). At least three promoters direct expression of the mouse glucocorticoid receptor gene. Proc. N u t . Acad. Sci. U S A 89, 6731-6735. Svec, F., Teubner, V., and State, D. (1989).Location of the second steroid-binding site on the glucocorticoid receptor. Endocrinology (Baltimore) 125, 3103-3108. Tusset, D., Tora, L., Fromental, C., Scheer, E., and Chambon, P. (1990). Distinct classes of transcriptional activating domains function by different mechanisms. Cell ( C a m bridge Muss.) 62, 1177-1187. Tora, L., White, J., Brou, C., Tasset, D., Webster, N., Scheer, E., and Chambon, 1’. (198%). T h e human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell (Cnmbridge Mass.) 59, 477-487. Tom, L., Mullick, A,, Metzger, D., Ponglikitmongkol, M., Park, I . , and Chambon, P. (1989b).T h e cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. E M B O ] . 8, 1981.

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Tsai, S. Y., Carlstedt-Duke, J., Weigel, N. L., Dahlrrian, K., Gustafsson, J. A,, Tsai, M.J., and O’Malley, B. W. (1988). Molecular interactions ofsteroid hormone receptor with its enhancer element: Evidence for receptor dimer formation. Cell (Cunibridge M a s s . ) 55, 361-369. Unresono, K., and Evans, R. M . (1989). Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell (Cui~ibridge Muss.) 57, 1139-1146. Valotaire, Y., Lt: Roux, hl. G., and Jego, P. (1993). Estrogen receptor gene: Structure and expression in rainbow trout. I n “Biochemistry and Molecular Biology ofFishes” ( P . Hochachka and Th. P. Mominsen, eds.), Vol. 2. Elsevier Science Publ. Anisterdam. Vegeto, E., Allan, G. F., Schrader, W. T., Tsai, \l. J., McDonnell, D. P., and O’Malley, B. W. (1992). T h e mechanism ofRU486 antagonism is dependent on the conforniatioir of the carboxy-terminal tail of the human progesterone receptor. Cell ( C o n bridge Jf.lnss.)69, 703-713. Vessi&res,A,, Vaillant, C . , Snlnrain, M., and Jaoueii, G. (1989). Organometallic derivatives ofestradiol as bioligands: Targetted binding ofthe estradiol receptor.]. Steroid Hioclwm. 34, 301-305. Wahli, W., Martinez, E., Corthesy, B., and Cardinaux, J . K. (1989). Cis and trans-acting elements ofthe estrogen-regulated vitellogeiiin gene B1 ofXenopu.7 laevis.]. Steroid Riochem. 34, 17-32. Walker, P., Gcrniond, J. E., Brown-Lriedi, M.,Givel, F., and Wahli, W. (1984). Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-\’LDLII gcnes. h’ucleic Acids Res. 12, 8611-8626. Webster, N . J. G . , Green, S., Hui Jin, J , , and Chamlion, P. (1988). T h e hormone-binding domains ofthe estrogen and glucocorticoid receptors contain an inducihle transcription activation function. Cell (Cartibridge Aluss.) 54, 199-207. Webster, N . J. G., Green, S., Tasset, D., Ponglikitmorrgkal, M., and Chanibon, P. (1989). T h e transcriptional activation function located in the hormone-binding domain of the hunran estrogen receptor is not encoded in a single exon. E M B O ] . 8,1441-1446. Weigel, N. L., Beck, C:. A,, Estes, P. A,, Prendergast, P., Altmann, M.,Christensen, K., and Ed\vards, D. P. (1992). Ligands indnce conforniational changes in the carboxylterminus ofprogesterone receptors which are detected by a site-directed antipeptide nionoclonal antibody. M o l . Endocrinol. 6, 1585-1597. Weil, C., and Marcuzzi, 0 . (1990). Cultured pituitary cell GtH response to GnRH at different stages of rainbow trout oogenesis arid infliience of steroid hormones. Gen. C o i ~ i pEndocrinol. . 79, 483-391. Weiler, I. J., Lew, D., and Shapiro, D . J. (1987). The Xetiopus laecis estrogen receptor: Sequence homology with human and avian receptors and identification of multiple estrogen receptor messenger ribonucleic acids. M o l . Endocrinol. 1, 355-362. Weinberger. C., Hollenberg, S. hl., Rosenfeld, hl. G., and Evans, R. h l . (1985). Domain structure of hrunan glucocorticoid receptor and its relationship to the v-erbA oncogene product. Nature (Lolidon)318, 670-672. Weisz, A. (1992).Transgenic mice as nrodels for steroid receptor function. Oral presentation in Third Course on hIolecular Biology of Iformone Action in Endocrinology and Pharmacology. hlilan. White, It., Lees, J. A , , Needham, M.,Ham, J., and Parker, M. (1987).Structural organisation and expression of the mouse estrogen rcceptor. MoI. Endocrinol. 1, 735-744. Xiong, F., and Hew, C. (1991).Chinook salmon (Oricorhpcliustshauytscha)gonadotropin I1 p snbiinit gene encodes multiple messenger ribonucleic acids. Can. J . Zool.

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12 PROLACTIN RECEPTORS PATRICK PRUNET AND BENOIT AUPERIN Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, 35042 Rennes Cedex, France

1. Introduction 11. Prolactin Receptors in Mainnialian Tissues 111. Prolactin Receptors in Fish

A. Characterization of PRL Receptors with '"I-Labeled Ovine Prolactin B. Characterization of Prolactin Receptors with Fish Hormones References

I. INTRODUCTION

Prolactin (PRL) is a polypeptide hormone that in all vertebrates SO far studied is synthesized mainly in the adenohypophysis. There is one known exception, the cyclostomes, where the pituitary gland differs from that of other fishes (Schreibman, 1986). The versatility of PRL and its wide spectrum of actions in vertebrates has led to numerous studies. These have been described in reviews (Clarke and Bern, 1980; Nicoll, 1982; Loretz and Bern, 1982; Bern, 1983; Hirano et al., 1987). Nicoll and Bern (1972) have listed more than 85 different effects of PRL. To highlight the common denominators among all of these actions, the effects can be grouped into seven categories (Kelly et al., 1991): (1)reproduction and lactation; (2) water and salt balance; (3) growth and morphogenesis; (4) metabolism; ( 5 )behavior; (6) immunoregulation; and (7) effects on the ectoderm and skin. Although this versatility of action is also observed in teleost fish, regulation of the hydromineral balance is the prevailing effect of PRL 367 FISH PtlYSIOL.OGY, VOL. XI11

Copyright 0 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

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in these animals. Since the pioneering work by Pickford and Phillips (1959),demonstrating that hypophysectomized Fundulus heteroclitus require PRL for survival in fresh water, PRL has been recognized as the principal hormone ensuring adaptation to fresh water, acting at the level of the gut, kidney, and gills (for reviews see Loretz and Bern, 1982; Hirano et al., 1987; Prunet et al., 1990).Numerous studies support the view that PRL has a Na +-retaining effect and probably acts by reducing the permeability to ions and water and by stimulating ion pumps at the level of osmoregulatory surfaces. However, studies on the osmoregulatory role of PRL in salmonids have failed to demonstrate an involvement of PRL in active Na+ transport (Prunet et al., 1990). In the Gillichthys urinary bladder, moreover, PRL reportedly has no effect on Na+ or C1- reabsorption, in contrast to reported observations on the urinary bladder of other fish species, where ion reabsorption was found to occur (Loretz and Bern, 1982). This suggests that PKL exerts significantly different actions in different fish species. Fortner and Pickford (1981)have likewise shown that in hypophysectomized Zctarus mellas, PRL restores plasma Na+ levels but not C1levels, whereas both are restored in several other fish species (Clarke and Bern, 1980). These results preclude automatic generalization to all fishes or any findings about the osmoregulatory role of PRL. Studies on the role offish PRL in other functions such as reproduction (Rubin and Specker, 1992), metabolism (McKeown et al., 1975), or behavior have received less attention, but here again no picture emerges that can be generalized to all fish species. In tilapia, for instance, PRL was shown to regulate gonadal steroidogenesis and to stimulate estradiol production (Tan et al., 1988), which by feedback also regulates pituitary PRL cell activity (Barry and Grau, 1986),a scheme that does not apply to rainbow trout (Le Goff et ul., 1992). Such versatility of PRL within a taxon implies that this hormone can have different mechanisms ofaction in different fish species (Bern, 1990).The first event in PRL action is its binding to a specific receptor located on the cell membrane. Multiplicity of PRL action should be reflected at the receptor level. Investigators faced with this new and complex situation will welcome the development of molecular approaches in this field of PRL endocrinology as an important means for elucidating these mechanisms and their physiological implications. After a survey of PRL receptors in higher vertebrates, the remainder of this chapter will deal with ovine PRL binding sites in lower vertebrates, the biochemical characteristics of fish PRL, the characterization and regulation of PRL receptors in tilapia, and finally the biological interpretations of these new data on PRL receptors in fish.

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11. PROLACTIN RECEPTORS IN MAMMALIAN TISSUES In mammals, PRL is primarily known for its involvement in the development of the mammary gland in lactogenesis and in the regulation of various aspects of reproductive function. However, binding sites for PRL are not limited to the corresponding organs and have been observed in many other tissues such as mammary gland, ovary, uterus, placenta, testis, liver, kidney, intestine, lymphatic and immune cells, and brain (Hughes et al., 1985). For some of these PRL binding sites, no direct association with a biological function has ever been firmly established. High PRL receptor levels have been detected in the liver, but the biological role of PRL in liver physiology remains unclear; PRL causes hepatic hypertrophy and increases ornithine decarboxylase activity (Richards, 1975; Buckley et al., 1985), thus suggesting its involvement in hepatocyte renewal. Both PRL and GH have been shown to be immunostimulatory factors (see review by Gala, 1991). These immunological effects of PRL are interesting; other studies have revealed direct production of PRL by a human lymphoblastoid cell line (Di Mattia et al., 1988),b y normal human lymphocytes, and by human lymphocyte cell lines (Pellegrini et al., 1992). Moreover, PRL binding sites and PRL receptor mRNAs have been detected in these lymphoid cells (Russel et al., 1985; Bellussi et al., 1987; Pellegrini et al., 1992). Together with the fact that PRL is produced at other extrapituitary sites such as the decidua, brain, and ovine and caprine mammary glands (Di Mattia et al., 1990; Emmanuele et al., 1992; Le Provost et al., 1993),these results suggest that it would be more appropriate to see PRL not only as an endocrine factor, but also as a paracrine or autocrine growth factor (Kelly et al., 1991). A4ost studies on PRL receptors have focused on liver or mammary gland receptors. Receptors have been found on plasma membranes, but the majority of PRL receptors have been located in intracellular membranes such as endosomes and Golgi and lysosomal structures (Bergeron et nl., 1978,1986; Djiane et al., 1981).This reflects the rapid turnover of the receptors that, after synthesis and targeting to the plasma membrane, are further internalized and degraded in lysosomes (Djiane et al., 1982). Moreover, the presence in the cytosol (liver, mammary gland, or kidney cells) of PRL receptors exhibiting a different binding affinity from that of the membrane receptor suggests that these cytosolic receptors may be involved in mediating the actions of intracellular PRL within cells (Ymer et al., 1987; Herington et al.,

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1988). Further support for this hypothesis lies in the identification of

PRL receptors and immunoreactive PRL in hepatic nuclei (Buckley et al., 1992). PRL binding proteins immunologically related to the PRL receptor were characterized in the serum of estradiol-treated female rats (Cohen et al., 1993) and in rabbit milk (Postel-Vinay et al., 1991). The biological roles of these binding proteins, which probably correspond with the extracellular domain of the membrane receptor, are still under investigation. PRL receptor structure was examined and multiple forms ofreceptors were identified after isolation of a first full-length cDNA coding for a PRL receptor in the rat liver (Boutin et al., 1988). From this initial study arose many publications on the molecular biology of this receptor, which were reviewed by Kelly et al. (1991). Identification of the cDNA made it possible to deduce the amino acid (aa) sequence of the mature receptor and revealed the presence of an extracellular region (210 aa), a single transmembrane domain (24 aa), and a cytoplasniic region of varying size, in which the first receptor isolated from rat liver contains a short cytoplasniic region (57 aa), whereas a second receptor form identified in the rabbit mammary gland has a long cytoplasmic domain (358 aa) (Edery et al., 1989). Other PRL receptor cDNAs have been further characterized in different tissues and appear to belong either to the long-form class, which includes the human PRL receptor from hepatoma cells (Boutin et al., 1989) and the PRL receptor from rat ovary (Zhang et al., 1990), or to the short-form class, which includes the receptor from the mouse liver (Davis and Linzer, 1989). In the Nb2 lymphoma cell line, a PRL receptor of intermediate size has been identified (Ah et nl., 1992). These different receptor forms arise from alternative splicings of a single gene located on chromosome 5p13-pl4 (Arden et al., 1990). The PRL receptor is a member of the hematopoietic/cytokine receptor superfamily, which, among other characteristics, exhibits sequence similarities in the 210-amino acid, amino-terminal, extracellular domain; two pairs of cysteines and a highly conserved WS x WS motif are consistently found in all niemhers of this receptor family (Bazan, 1990; Thoreau et al., 1991). Expression of the PRL receptor gene varies according to the species and organ studied (see review b y Kelly et al., 1991). In rabbit and humans, the only characterized niRNAs code for the long form generated by differential splicing (Boutin et al., 1989; Davis and Linzer, 1989; Dusanter-Fourt et al., 1991). In the rat and mouse, however, specific mRNAs encoding both the short and the long receptor form have been detected, each form being encoded by one or several transcripts resulting from alternative splicing and differing froni tissue

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to tissue (Shirota et al., 1990; Clarke and Linzer, 1993). Differential expression of the two respective transcripts coding for the long and short receptor forms was further confirmed by in situ hybridization, a technique that revealed receptor mRNA in unexpected tissues such as muscle and heart (Ouhtit et al., 1993). Structure-function relationships in the various receptor forms were further studied in a functional assay involving cotransfection of Chinese hamster ovarian (CHO) cells with a PRL receptor cDNA along with a fusion gene containing a target gene for PRL, the a-lactoglobulin or a-casein gene promotor linked to the CAT gene (see review b y Kelly et nl., 1991). When the cells were cotransfected and further tested in the presence of PRL, an increase in reporter gene activity was observed only when the long form of PRL receptor was used (Lesueur et al., 1990,1991). In a similar functional assay, the respective contributions of various domains of the PRL receptor to the mechanism of signal transduction were determined. Rozakis-Adcock and Kelly (1991,1992) have shown that the four cysteines found in the extracellular domain and the WS x WS motif proximal to the transmembrane domain are crucial to ligand binding and to the functional integrity of the PRL receptor. Signal transduction pathways for PRL, that is, the events occurring after binding of the hormone to its receptor, have remained an enigma until recently, when important new insights were provided by Witthuhn et al. (1993) and Argetsinger et al. (1993). Having shown that GH and erythropoietin (EPO) might function by coupling ligand binding with the activation of a tyrosine kinase, which in turn induces phosphorylation, these authors identified JAK2, a 130kDa tyrosine kinase, as the protein involved in GH receptor and EPO receptor signal transduction, a protein constitutively associated with these receptors. Because the PRL receptor belongs to the same cytokine receptor family as G H and EPO, it seems likely that a similar mechanism involving the JAK2 tyrosine kinase could apply to the PRL receptor (Witthuhn et al., 1993).However, the wide range ofbiological actions of PRL and the existence of multiple receptor forms suggest that we may face a complex situation with several JAK family kinases acting on several different phosphorylation substrates, thus activating various signaling pathways. The complexity and versatility of PRL action is also reflected in the hormonal regulation of PRL receptors. Implicated factors include glucocorticoids, sex steroids (estradiol, progesterone, testosterone), growth hormone (GH), and PRL itself (Kelly et al., 1991). The effects of these factors on PRL receptors depend, however, on the target organ. In the mouse mammary gland, for instance, adrenalectomy in

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nursing mothers reduces the number of PRL receptors, an effect preventable by hydrocortisone treatment (Sakai and Banerjee, 1979).Likewise, in mouse mammary cell cultures, cortisone effectively increases PRL binding (Sakai et al., 1979). On the other hand, adrenalectomy increases the number of PRL receptors in rat kidney and hydrocortisone effectively reduces PRL receptor binding in intact male rats (Marshall et al., 1978). Another example of the complexity of PRL receptor regulation is the difference in regulation of the receptor in rat liver and mammary gland during pregnancy and lactation (Jahn et al., 1991). A final illustration is regulation by PRL itself; whereas high levels of plasma PRL rapidly and reversibly down-regulate the PRL receptor owing to increased internalization and degradation of the hormone-receptor complex (Djiane et al., 1979, 1980), low doses of PRL cause long-term up-regulation ofthe receptor (Posner et al., 1979; Barkley et al., 1980). In conclusion, our present knowledge of PRL receptors in mammals highlights the complex functioning and regulation of PRL receptors, in keeping with prolactin’s known biological and functional versatility.

111. PROLACTIN RECEPTORS IN FISH A. Characterization of PRL Receptors with 1251Labeled Ovine Prolactin 1. OVINEPROLACTIN BINDINGSITES IN LOWER VERTEBRATES

The lack of purified PRL preparations from lower vertebrates and the initial finding that ovine PRL can specifically bind membranes isolated from adult bullfrog kidney (Posner et al., 1974) led to the use of this mammalian hormone as a tool for studying PRL receptors in lower vertebrates. Use of this heterologous ligand was justified in the specific biological effects that it induced in lower vertebrates (Clarke and Bern, 1980), indicating that it can bind to the endogenous PRL receptors. Thus, following the initial studies suggesting that ovine PRL is an almost universal ligand for prolactin receptors in vertebrates (Nicoll et ul., 1980), the hormone was used to characterize PRL receptors in various tissues of amphibians and reptiles. The most consistent results were obtained with kidney and skin preparations (D’Istria et al., 1987; Tarpey and Nicoll, 1987) and in epithelial cell lines from amphibian urinary bladder and kidney (Dunand et al.: 1985). Ovine

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PRL was also used to study the regulation of PRL binding sites in amphibians. Induction of PRL binding sites in tadpole kidney was shown to depend on the developmental stage and on the plasma thyroxine level, but induction was inhibited by ovine PRL (White and Nicoll, 1979; White et al., 1981). In Xenopus laevis, dehydrating conditions caused PRL binding to kidney and skin membranes to decrease (Muccioli et al., 1989). In teleost fish, similar attempts were made to characterize renal PRL receptors with ovine PRL. Only kidney membranes from tilapia (Oreochromis mossambicus), among several species tested, exhibited specific binding (Fryer, 1979). Our own attempts to detect specific binding of ovine PRL in various tissues of rainbow trout were unsuccessful (P. Prunet and 0.Sandra, unpublished data, 1993).By autoradiography, however, Gona (1984) was able to locate '"1-labeled ovine PRL specifically in the kidney tubules of another teleost, Colisa lalia. Specific binding was also found in white eel liver membranes (Ng et al., 1991). Further binding studies confirmed the presence of PRL binding sites in tilapia adapted to fresh water (FW): Edery et al. (1984) obtained significant specific binding (5-10%) with liver, testis, and ovary. Scatchard analysis of PRL binding in the liver revealed a single type of high-affinity binding site. In the same study, however, no consistent results were obtained with kidney preparations. Using a modified procedure for preparing microsomal membranes and a modified incubation protocol, Dauder et al. (199Oa) were able to confirm the presence of PRL binding sites in the liver, gill, and kidney of FWadapted tilapia. However, Scatchard curves derived from the competition data obtained with these various tissues were consistently curvilinear, leading the authors to conclude that two different PRL binding sites are present in the liver, gill, and kidney. Surprisingly, competition studies also indicated that the two tilapia PRL forms have less affinity than ovine PRL for these binding sites. This contradicts the biological activity data on these PRLs, obtained from the same osmoregulatory bioassay where ovine PRL appeared consistently less potent than the tilapia PRLs (Specker et al., 1985; Auperin et al., 1994a). Finally, the fact that tilapia GH and one form of tilapia PRL were in the same range of effectiveness in displacing labeled ovine PRL from liver membranes (but not from kidney or gill membranes) led the authors to question the specificity of the binding observed in their study (Dauder et ul., 1990a). The same authors also studied the regulation of ovine PRL binding sites in tilapia; adaptation to seawater led to a significant decrease in the hormone's specific binding to gill, kidney,

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and liver membranes (Dauder et al., 1990a). Binding in the gill and kidney, but not in the liver, was also shown to be under the positive control of PRL (Dauder et al., 1990b). 2. CRITICAL APPRAISAL OF THE U S E OF '251-LABELED OVINEPRL TO STUDYPRL RECEPTORSIN FISH

Given their structural relatedness and common biological properties, the hormones PRL, growth hormone, and placental lactogen are considered members of the same hormone family (Nicoll et al., 1986). Because of their structural similarities, there have been questions since the early studies on PRL receptors in lower vertebrates regarding the specificity of binding of ovine PRL, which can also bind to GH receptors in fish tissues (Nicoll et al., 1980). Currently, several arguments suggest that use of ovine PRL may lead to erroneous or inaccurate characterization of PRL receptors in fish. First, the availability of recombinant tilapia GH and PRL has enabled us to further characterize

150 125 100 0

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HORMONE (ng/tube) Fig. 1. Competition curves for ""I-labeled ovine PRL binding to liver nienibrane preparations from freshwater tilapia (Oreochromis niIoticus) by increasing concentrations of various hormone preparations. Binding is expressed as a percentage of 12,'1labeled ovine PRL specific binding in presence of competition (B) divided by lZ5Ilabeled ovine PRL specific binding in absence of competition (B").

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LABELLED HORMONE Fig. 2. Percentage of specific binding (9’0 BIT: B, specific binding, T, amount of radioactivity added to the tube) of differently labeled hormones to the same liver membrane preparation from freshwater tilapia (Oreochrornis niloticus).

the specific binding of ‘“I-labeled ovine PRL to liver membranes from tilapia (Oreochrornisniloticus).As shown in Fig. 1, recombinant tilapia GH was more potent than either form of tilapia PRL in its ability to displace ovine PRL from its binding site. These results agree with the data reported by Dauder et al. (1990a). Moreover, specific binding of the tilapia prolactins to liver membranes was low (<6%), whereas tilapia GH exhibited much higher specific binding to the same membranes (Fig. 2). Altogether, these results suggest that ovine PRL is probably unable to distinguish PRL from GH receptors in tilapia. This would lead to ambiguous interpretations when both receptors are present in the same tissue (e.g., in the liver) and might explain why two binding sites were characterized for ovine PRL (Dauder et al., 1990a). Nicoll et al. (1980) have suggested that a similar situation exists in amphibians. However, it might not be possible to apply this conclusion to all lower vertebrates because ovine PRL fails to bind

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PATRICK P R U N E T 4 N D BENOIT AUPERIN

to the GH receptors of rainbow trout liver or gill (Yao et al., 1991; J. M. E. Almendras, B. Auperin, and P. Prunet, unpublished data, 1992). Second, in all studies published so far as well as in our experiments, specific binding of '"I-labeled ovine PRL was usually quite low. The highest reported value was 10% and a more usual value was about 2-4%. This generally leads to higher values for nonspecific than for specific binding. Classical methods for estimating nonspecific binding (addition of excess unlabeled hormone) have often proven inadequate, resulting in an inaccurate estimate of binding affinity and of the number of binding sites (Mendel and Mendel, 1985). In the case of ovine PRL binding, where a significant proportion of the total radioactivity is nonspecifically bound, large errors are made in the estimate of specific binding, leading to inaccurate characterization of this binding (Brooks et al., 1982). Such problems could easily explain the discrepancy between the data of Edery et al. (1984), who reported a single high-affinity binding site for ovine PRL in tilapia liver, and those of Dauder et al. (1990a),who found two binding sites in the same tissue. Although such studies have contributed useful information on the target tissues of PRL in fish, ovine PRL does not appear to be an adequate ligand for characterizing fish PRL receptors at the molecular level or for studying their physiological regulation. The foregoing survey of PRL receptors in mammals has clearly shown that various receptor forms exist. There is currently no reason to believe that a similar situation does not exist in fish. Homologous hormones should be used to accurately characterize the different receptor forms in fish (if they exist), especially because different PRL forms have been isolated and biologically characterized in fish species (see the following section).

B. Characterization of Prolactin Receptors with Fish Hormones

1. DIFFERENT PRL FORMS IN TELEOST FISH PRL belongs to a family of structurally and functionally related polypeptide hormones that includes growth hormone, placental lactogen, proliferin, and the recently isolated somatolactin. Among teleost fish, PRL has been isolated in different species, including the tilapia species Oreochromis mossambicus (Specker et al., 1985); chum, chi1978; Kawauchi et d . , 1983; nook, and Atlantic salmon (Idler et d, Prunet and Houdebine, 1984; Andersen et al., 1989);carp (Yasuda et al., 1987); and eel (Suzuki et al., 1991). These fish PRLs show 60-80%

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amino acid sequence identity among themselves and only 20-30% with mammalian PRLs. As compared with other vertebrate PRLs, their major characteristic is the absence of one disulfide loop in the Nterminal region. During purification of several fish PRLs, two forms were isolated and their differences further analyzed after partial or complete amino acid sequencing. In salmon (Yasuda et al., 1986), carp (Yasuda et al., 1987), and eel (Suzuki et al., 1991),the two forms are very similar and present strong homology, whereas those isolated from tilapia are quite different. In tilapia, the larger PRL molecule (named tiPRLlB8or tiPRL,) has a molecular mass of 20,836 Da and contains 188 amino acids; the smaller, 19,584-Da PRL (named tiPRL,,, or tiPRL,,) has an 11-amino-acid deletion. The two forms have different isoelectric points 8.7 and 6.7, respectively and migrate as different bands in SDS-PAGE experiments (Specker et al., 1985; Yamaguchi et al., 1988). Interestingly, they are only 69% similar and the sequence of tiPRL,, the tilapia form most closely resembling the other fish PRLs, is 69% identical to the salmon PRL sequence. From a comparison of the corresponding polypeptide or nucleotide sequences, it clearly appears that the two PRL forms characterized in tilapia species are products of distinct genes (Yamaguchi et al., 1988; Rentier-Delrue et al., 1989). The biological significance of the two PRL forms has only been studied in tilapia, where the structural differences are sufficiently marked to suggest that the two forms may have specific functions. In initial studies, both tilapia PRLs exhibited a similar ability to restore plasma Na' levels in hypophysectomized tilapia specimens (Oreochromis mossambicus) transferred to fresh water (Specker et al., 1985; Young et al., 1988).With the isolation ofcDNA clones encoding tiPRL, and tiPRL,, (Rentier-Delrue et al., 1989), it has been possible to produce and purify each form of biologically active recombinant tilapia PRL (Swennen et al., 1991). Using these preparations, we have shown in Oreochromis niloticus that the two tilapia PRL forms have different biological functions in fish during adaptation to a hyperosmotic environment (Auperin et al., 1994a).After transfer to brackish water (BW), plasma tiPRL, and tiPRL,, levels, measured in specific radioimmunoassays, exhibit different patterns of change: the plasma tiPRL, level drops rapidly to below the detection threshold, whereas a measurable and significant level of tiPRLIl persists. Moreover, repeated injections of recombinant tiPRL, in fish adapted to brackish water induce a clear, dose-dependent ion-retaining effect, whereas the effects produced by tiPRL,, are markedly smaller and not dose-related.

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PATRICK P K U N E T A N D BENOIT AUPERIN

Similar biological differences between the two tilapia PRL forms have been observed in hypophysectomized tilapia kept in fresh water, where tiPRL, is significantly more potent than tiPRL,, in its ability to restore plasma C1- levels to normal (Auperin et al., 1994a). It is important to note that the two recombinant tilapia PRL forms exhibited similar potencies in three different bioassays and showed the same potency as natural tilapia PRL in binding to PRL receptors (Swennen et al., 1991; Auperin et al., 1993).In hypophysectomized specimens of 0. niloticus, moreover, the tilapia prolactins are equally potent in their ability to restore plasma Na+ levels (Auperin et al., 1994a), in agreement with the results of Specker et al. (1985). Altogether, these results clearly indicate that the observed differences do not merely reflect differences in the biological quality of the two recombinant proteins. It appears, rather, that the two PRL forms in tilapia have different roles in osmoregulation. Furthermore, these findings raise interesting questions of whether these osmoregulatory effects are linked with different PRL receptors.

2. CHARACTERIZATION OF TILAPIA PRL RECEPTORS IN GILL AND KIDNEYOF TILAPIA (Oreochromis niloticus) In Auperin et al. (1994b) the two recombinant tilapia PRL forms were used as ligands. Specific binding of '"I-labeled tiPRL, and lzSIlabeled tiPRL,, to various tilapia tissues is shown in Fig. 3. Kidney and gill tissues exhibited the highest levels of specific binding, the level always being higher for tiPRL, than for tiPRL,,. The other tissues, including the liver, displayed significant but low specific binding. The tiPRL, and tiPRL,, receptors were further characterized in the gill and kidney, two major osmoregulatory organs. A necessary prerequisite to such a study was to improve specific hormone binding. This was achieved with enriched cellular membrane preparations and by determining the optimal incubation conditions. For our experiments, the optimum was 12°C for 20-22 hr in a buffer with low Tris-HC1 and divalent ion concentrations that were 10 times lower than that currently used in the mammalian PRL radioreceptor assay. A final major improvement involved collecting tissues from fish maintained in brackish water for 36-48 hr. This procedure was shown to significantly increase the specific binding obtained with both tiPKL forms. Further analysis of tiPRL, and tiPKL,, binding sites on gill and kidney membranes revealed only one class of high-affinity tilapia PRL receptors to which tiPRL, binds with a higher affinity than tiPRL,,.

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0: tiPRL,

50

: tiPRL,,

40

. I-

co 30 M 20

1 10

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I

Gills

Kidney

Gut

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Fig. 3. Percentage of specific binding (% BIT: B, specific binding, T, amount of' radioactivity added to the tube) of 1251-labeledtiPRL, (open) and lZ5I-labeledtiPRL,, (hatched) to tilapia (Oreochromis niloticus) organs.

Several arguments support this conclusion. First, Scatchard analysis oftiPRL, and tiPRL,, binding data yields straight-line plots, suggesting a single class of binding sites that both hormones recognize (Fig. 4). However, the association constant ( K , ) calculated from saturation data was always higher for tiPRL, than for tiPRL,, in both kidney and gill preparations (in gill, 2.9 and 1.9 x 10'" M - ' , respectively, and in the kidney, 2.3 x 10'" and 5.0 x 10' M - ' , respectively). Second, experiments with various competing hormones also show tiPRL, to be more effective at displacing both '251-labeledtiPRL, and '251-labeledtiPRL,, bound to kidney and gill membranes, being usually 8 to 15 times more potent than tiPRL,,. Interestingly, neither tilapia PRL form was significantly displaced by tilapia or bovine GH, indicating that the receptor characterized in this study is PRL specific. PRLs from other fish species were able to displace the '251-labeled tiPRL, tracer from kidney receptors. As competitors for the kidney PRL receptors, some of these hormones (trout PRL) were as effective as tiPRL,; others were less effective (carp PRL) (Fig. 5). Mammalian lactogenic hormones such as ovine PRL and human GH were also able to bind to the tilapia kidney PRL receptor, but were less potent than any fish PRL studied.

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PATRICK P R U N E T AND B E N O I T AUPERIN

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BOUND (pMol) Fig. 4. Scatchard plot of specific 12'I-labeled tiPRL, and '251-labeledtiPRLIlbinding to gill (A) and kidney (B) membrane preparation of brackish water-adapted tilapia.

It was also interesting to compare the characteristics of gill and kidney PRL receptors. Renal and branchial PRL receptors exhibited a similar specificity range, tiPRL, being the most potent competitor, followed by tiPRL,,, ovine PRL, and, as very weak competitors, tilapia

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HORMONE (ng/tube) Fig. 5. Competition curves for '"1-labeled tiPRL, binding to kidney membrane preparation by increasing concentrations of various hormone preparations. Binding is expressed as a percentage of '"I-labeled tiPRL, specific binding in the presence of competition (B) divided by '251-labeledtiPRL, specific binding in absence ofcompetition

@,,I. GH and ovine GH. In both tissues, the K , values obtained with 12jIlabeled tiPRL, were similar as were the capacity values expressed per gram of initial fresh tissue. However, the K , values were significantly different when calculated from the '"1-labeled tiPRL,, binding data. Are the receptors characterized in gill and kidney tissues identical? More complete characterization of the PRL receptor is needed. The observation that tiPRLI binds gill and kidney tilapia PRL receptors with a higher affinity than tiPRL,, is in agreement with the biological effects of these hormones in BW-adapted tilapia, where tiPRL, is significantly more potent than tiPRLII (Auperin et d., 1994a). However, two results make it difficult to explain these biological differences on the sole basis of the higher affinity of tiPRLI binding to tilapia PRL receptors in osmoregulatory organs: (1) tiPRL,, does not produce the dose-related effects observed with tiPRL, in BW-adapted tilapia; and (2) in hypophysectomized tilapia reared in FW, the effects of tiPRL,, and tiPRLI are similar in maintaining plasma Na' levels, but their

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PATRICK PRUNET AND BENOIT AUPEHIN

effects on C1- levels differ (B. Auperin, F. Rentier-Delrue, J. A. Martial, and P. Prunet, unpublished data, 1993). At present, the most reasonable hypothesis seems to be that the two tilapia PRL forms bind to the same receptors in gill and kidney, but use different signal transduction pathways to transmit their effects inside the cell (Auperin et al., 1994b).

3 . REGULATION OF TILAPIAPRL RECEPTORS IN TILAPIA (Oreochromis niloticus) GILL a. Effects of Transfer from Fresh Water to Bruckish Water on Tilapia Receptor Regulation. Direct transfer of tilapia from fresh water

to brackish water causes a readjustment of the hydromineral balance, a ftinction performed mainly by PRL in tilapia species. This is demonstrated by the necessary presence of PRL in FW-adapted tilapia (Dharmamba et al., 1967),in the sharp decrease in plasma tiPRL, and tiPRL,, levels observed a few hours after a salinity increase, and in the low levels of prolactin maintained in fish adapted to the hyperosmotic environment (Auperin et al., 1994a).Considering the different biological functions carried out by the two tilapia PRL forms during adaptation to brackish water (see earlier discussion), it was interesting to study changes induced by the transfer in the PRL receptors of the gill (a major osmoregulatory organ important in adaptation to a hyperosmotic environment). After an initial hydromineral imbalance observed as early as 3 hr after direct transfer to brackish water, the fish appeared to adapt to the hyperosmotic environment within 3 days (Auperin et ul., 1994). Specific binding oftiPRL, and tiPRL,, to gill membranes was found to increase four- to sevenfold and two- to fivefold, respectively, within 24 hr after transfer and the levels remained high until the end of the experiment (28 days in brackish water). To further characterize the binding of both forms, Scatchard analysis and specificity studies using tiPRL, as a tracer were carried out . points emerge from this analysis. First, (Auperin et al., 1 9 9 4 ~ )Several whatever the salinity and even in BW-adapted fish, we still observed only one class of tilapia PRL receptors in the gill. Moreover, tiPRLl always displayed a higher affinity than tiPRL,, for tilapia PRL receptors. The evolution of tilapia PRL receptor affinity and capacity after transfer into brackish water showed a similar pattern whichever ligand was used, tiPRL, or tiPRL,,. This supports the view that both tilapia PRL forms bind to the same receptor. Second, the binding affinities significantly increased as early as 24 hr after transfer and remained high in tilapia adapted to the hyperosmotic environment. We cannot totally reject the hypothesis that this change reflects the appearance of a new class of tilapia PRL receptors characteristic of the BW situation.

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Should this be the case, however, we should have temporarily observed two binding sites in the time course of our study (FW receptors disappearing while brackish water receptors were developing). Because we did not, it seems more likely that the increase in binding affinity was due to a change in the fluidity of the gill membranes resulting from the salinity change. Such effects have been reported (Hazel and Williams, 1991). Lastly, transfer to brackish water results in a large increase in the number of tilapia PRL receptors measured with the tiPRLI form. By analogy to observations in mammals, it seems that the increase could be partially linked with a simultaneous drop in plasma tiPRL, and tiPRL,, levels, which would slow down internalization of the hormone-receptor complexes and their degradation in lysosomes (Djiane et al., 1982). This would result in a short-term rise in the free membrane receptors, although the plasma tilapia PRL levels (2-5 ng/ml) measured in FW-adapted fish (Auperin et al., 1994a, 1 9 9 4 ~seem ) too low to allow occupancy of' a large proportion o f t h e PRL receptors. Thus, the drop in plasma tilapia PRL levels explains neither the sixfold increase in the number of PRL receptors nor the duration (at least 4 weeks) of this effect. Rather, these results suggest an increase in the rate of synthesis of gill tilapia PRL receptors under the control of an undetermined hormonal factor or factors (see the following section). Alternatively, cryptic tilapia PRL receptors might be present in the gill tissue. These would be unmasked after transfer from FW to BW, a situation causing profound changes in gill physiology. Such cryptic receptors have been described in incompletely differentiated rat mammary cells (mammary tumor cells and normal proliferating mammary cells), in which case they can be unmasked by ATP depletion (Costlow and Hample, 1982, 1984).At the present stage, it is difficult to exclude any of these mechanisms of' PRL receptor regulation. Several of the mechanisms may even occur simultaneously. Further study is needed before a conclusion can be drawn.

b. Hormonal Regulation of Gill PRL Receptors. Direct transfer of 0. riiloticus from FW to BW is associated with a rapid and sharp decrease in plasma tilapia PRL levels. The drop is temporally related to the rise in the gill tilapia PRL receptor content (Auperin et al., 1994c), which suggests an inhibitory control by PRL on its own receptors in the gill. Cortisol, however, was also shown to play an important role in the adaptation of' tilapia to a hyperosmotic environment and plasma cortisol levels were found to rise after direct transfer of 0. mossambicus from FW to SW (Assem and Hanke, 1984). To clarify this problem, the tilapia PRL receptor content in gill tissue was studied

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PATRICK P K U N E T A N D BENOIT A U P E K I N

after hypophysectomy of FW-adapted 0. niloticus, a situation that mimics the drop in plasma tilapia observed after transfer to BW but not the rise in plasma cortisol. As shown in Fig. 6, within 4 days after hypophysectomy the tilapia PRL receptor content underwent a significant threefold rise. Although tilapia PRL levels were found to drop to undetectable levels over the same period, the tilapia PRL receptor content did not rise as markedly as after transfer to BW. This suggests that a factor or factors other than tilapia PRL are involved in PRL receptor regulation. 4. TILAPIAPRL RECEPTORS IN OSMOREGULATORY A PICTUREOF THE BIOLOGYOF ORGANS: ADAPTATIONOF THE FISH? A similar attempt to characterize tilapia PRL receptors was performed in salmonid fish using purified salmon PRL (sPRL) (Prunet et al., 1984). Although this hormone allowed us to develop a specific radioimmunoassay for salmon PRL, all our attempts to demonstrate consistent specific binding were unsuccessful. Various incubation con-

0:tiPRLl 10.0

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s

8 5.0

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0 SHAM

HYPEX

Fig. 6. Percentage of' specific binding (% B/T: B, specific binding, T, amount of radioactivity added to the tube) of '"I-labeled tiPRL, (open) and 1251-labeledtiPRL11 (hatched) on membrane preparations from gill of sham-operated and hypophysectomized tilapia (Oreochrornis niloticus).

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ditions were tested, as were various membrane preparations from potential target organs, but the experiments were negative. Interestingly, 12sI-labeledsPRL was able to significantly bind to tilapia kidney preparations, suggesting that the labeling technique we used did not result in degraded tracer. This led us to hypothesize that, in salmonid fish, PRL receptors showing high affinity and low concentration might occur in osmoregulatory organs. Such a picture would be clearly different from what we observed in Oreachromis niloticus, which showed a significant concentration of tilapia PRL receptors in the gills and kidney. Transfer of tilapia into brackish water resulted in a large increase of PRL receptors in the gills. This was an unexpected result as PRL has been clearly shown to control adaptation to fresh water and to inhibit adaptation to hyperosmotic environment in tilapia species (Dharmamba and Maetz, 1972, 1976; Young et al., 1988). However, we must remember that tilapia is characterized by a general tolerance to a wide range of environmental conditions, including fresh water and salinities that exceed that of seawater (Stickney, 1986). Many tilapia species are broadly euryhaline, whereas others are restricted to fresh water or low-salinity water. A tilapia species such as Oreochromis niloticus is frequently reported to live in brackish water, estuaries, or lagoons. In such situations fish have to adapt quickly to salinity changes. The necessity of prolactin for fresh-water adaptation implies that the presence of a large number of tilapia PRL receptors in brackish water-adapted fish might be a benefit for the fish in adapting quickly to environmental changes, such as a decrease in salinity. For example, juvenile tilapia Oreochromis niloticus directly transferred from brackish water to fresh water are able to regulate their plasma ion levels within a few hours (B. Auperin, unpublished data, 1993). Similar studies of fish during adaptation to various salinities or to low-quality water may provide further arguments to confirm the importance of the PRL receptor as a measure of the capacity of this fish species to adapt. ACKNOWLEDGMENTS This work was partially supported by grants from CIRADIIMVT. The authors also appreciate the continuous support from F. Rentier-Delrue and from J. Martial, who provided recombinant tilapia prolactins.

REFERENCES Ah, S., Edery, M., Pellegrini, I., Lesueur, L., Paly, J., Djiane, J . , and Kelly, P. A. (1992). The Nb2 form of prolactin receptor is able to activate a milk protein gene promoter. Mol. Endocrinol. 6, 1242-1248.

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Le Goff, P., Salbert, G., Prunet, P., Saligaut, C., Bjornsson, B. Th., Haux. C., and Valotaire, Y. (1992).Absence of direct regulation of prolactin cells by estradiol-17P in rainbow trout (Oncorhynchus mykiss). Mol. Cell. Endocrinol. 90, 133-139. Le Provost, F., Leroux, C., Martin, P., Gaye, P., and Djiane, J . (1993).The gene encoding prolactin is expressed in ovine and caprine mammary gland. Proceedings of the Sixth International Prolactin Congress, Paris p. 9 (abstract). Lesueur, L., Edery, M., Paly, J., Clark, J., Kelly, P. A,, and Djiane, J. (1990). Prolactin stimulates milk protein promoter in CHO cells cotransfected with prolactin receptor cDNA. Mol. Cell. Endocrinol. 71, R7-Rl2. Lesueur, L., Edery, M., Ah, S., Paly, J., Kelly, P. A,, and Djiane, J . (1991). Comparison of long and short forms of the PRL receptor on PRL induced milk protein gene transcription. Proc. Natl. Acad. Sci. U.S.A. 88, 824-828. Loretz, C. A., and Bern, H. A. (1982). Prolactin and osmoregulation in vertebrates. Neuroendocrinology 35,292-304. McKeown, B. A,, Leatherland, J. F., and John, T. M . (1975).The efrectofgrowth hormone and prolactin on the mobilization of free fatty acids and glucose in the kokanee salmon, Oncorhynchus nerka. Comp. Biochem. Physiol. 50,425-430. Marshall, S., Huang, H. H., Kledzik, G. S., Campbell, G. A,, and Meites, J. (1978). Glucocorticoid regulation of prolactin receptors in kidneys and adrenals of male rats. Endocrinology (Baltimore) 102, 868-875. Mendel, C. M., and Mendel, D. B. (1985).“Non-specific” binding: The problem, and a solution. Biochem. J. 228, 269-272. Muccioli, G., Guardabassi, A., Pattono, P., and Genazzani, E. (1989).Further study on the changes in the concentration of prolactin-binding sites in different organs of Xenopus laevis male and female, kept under dry conditions and then returned to water (their natural habitat). Gen. Comp. Endocrinol. 74, 411-417. Ng, T. B., Hui, T. Y., and Cheng, C. H. K. (1991). Presence of prolactin receptors in eel liver and carp kidney and growth hormone receptors in eel liver. Cornp. Biochem. Physiol. 99A, 387-390. Nicoll, C. S. (1982). Prolactin and growth hormone: Specialists on the one hand and mutual mimics on the other. Perspect. Biol. Med. 25, 369-381. Nicoll, C. S., and Bern, H. A. (1972). On the action of prolactin among the vertebrates: Is there a common denominator? In “Lactogenic Hormones” (G.E. W. Wolstenholm and J. Knight, Eds.) pp. 299-324. Churchill Livingstone, London. Nicoll, C. S., White, B. A,, and Leung, F. C . (1980).Evolution ofprolactin, its functions, and its receptors. In “Central and Peripheral Regulation of Prolactin Function” (R. M. McLeod and U. Scapagnini, eds.), pp. 11-25. Raven, New York. Nicoll, C. S., Tarkey, J. F., Mayer, G. L., and Russell, S. M. (1986). Similarities and differences among prolactins and growth hormones and their receptors. A m . 2001. 26,965-983. Ouhtit, A., Morel, G., and Kelly, P. A. (1993).Visualization of gene expression of short and long forms of prolactin receptor in the rat. Endocrinology (Baltimore) 133, 135- 144. Pellegrini, I., Lebrun, J.-J., Ali, S., and Kelly, P. A. (1992). Expression of prolactin and its receptor in human lymphoid cells. Mol. Endocrinol. 6, 1023-1031. Pickford, G. E., and Phillips, J. G . (1959). Prolactin, a factor in promoting survival of hypophysectomized killifish in fresh water. Science 130, 454-455. Posner, B. I., Kelly, P. A,, Shiu, R. P. C., and Friesen, H. G. (1974). Studies on insulin, growth hormone, and prolactin binding, tissue distribution, species variation and characterization. Endocrinology (Baltimore) 95, 521-531. Posner, B. I., Kelly, P. A,, and Friesen, H. G . (1979). Induction of a lactogenic receptor

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in rat liver: Influence of estrogen and the pituitary. Proc. W a t l . A c c d Sci. U.S.A.

71,2407-2410. Postel-Vinay, M.-C., Belair, L., Kaiser, C., Kelly, P. A,, and J . Djiane (1991).Identification of prolactin and growth hormone binding proteins in rabbit milk. Proc. Nutl. Acud.

Sci. U.S.A. 88, 6687-6690. Prunet, P., and Houdebine, L. M. (1984). Purification and biological characterization of chinook salmon prolactin. Gen. Comp. Endocrinol. 53, 49-57. Prunet, P., Avella, M., Fostier, A,, Bjornsson, B. Th., Boeuf, C., and Haux, C. (1990). Roles of prolactin in salrnonids. I n “Progress in Comparative Endocrinology” (A. Epple, C. G. Scanes, and M . T. Stetson, eds.), pp. 547-552. Wiley-Liss, New York. Rentier-Delrue, F., Swennen, D., Prunet, P., Lion, M., and Martial, J. A. (1989).Tilapia prolactin: Molecular cloning of two cDNA and expression in Escherichia coli. V N A

8, 261-270. Richards, J. F. (1975). Ornithine decarboxylase activity in tissues of prolactin-treated rats. Biochem. Biophys. Res. Commun. 63, 292-295. Rozakis-Adcock, M., and Kelly, P. A. (1991). Mutational analysis of the ligand-binding domain of the prolactin receptor. J . Biol. Chem. 266, 16472-16477. Rozakis-Adcock, M., and Kelly, P. A. (1992).Identification of ligand binding determinants of the prolactin receptor. J , Biol. Chem. 267, 7428-7433. Hubin, D. A., and Specker, J. L. (1992). I n oitro effects of homologous prolactins ou testosterone product by testes of tilapia (Oreochromis mossamhicus). Gen. C o m p .

Endocrinol. 87, 189-196. Russel, D. H., Kibler, R. K., Matrisian, L., Larson, 1).F., Poulos, B., and Magun, B. E. (1985).Prolactin receptors on human B and T lymphocytes: Antagonism of prolactin binding by cyclosporine. J . Immunol. 134,3027-3031. Sakai, S., and Banerjee, M.R. (1979). Glucocorticoid modulation of prolactin receptors ou mammary cells of lactating mice. Biochim. Biophys. Acta 582, 78-88. Sakai, S., Bowman, P. D., Yang, J . , McCormick, K., and Nandi, S. (1979).Glucocorticoid regulation ofprolactin receptors on mammary cells in culture. Endocrinology (Bulti-

more) 104, 1447-1449. Schreibman, M. P. (1986).Pituitary gland. I n “Vertebrate Endocrinology: Fundamentals and Biomedical Implications” (P. K. T. Pang and M. P. Schreibman, cds.), Vol. 1, pp. 11-55. Academic Press, Toronto. Shirota, M., Banville, D., Ali, S., Jolicoeur, C., Boutin, J. M., Edery, M., Djiane, J.. and Kelly, P. A. (1990).Expression of two forms of prolactin receptor in rat ovary and liver. M o l . E t s d o c r i d . 4, 1136-1143. Specker, J . L., King, D. S., Nishioka, R. S., Shirahata, K., Yamaguchi, K., and Bern, H. A. (1985). Isolation and partial characterization of a pair of prolactins released in citro by the pituitary of a cichlid fish, Oreochromis mossamhicus. Proc. N a t l .

Acad. Sci. U.S.A.82, 7490-7494. Stickney, H. R. (1986). Tilapia tolerance of salinity waters: A review. Progressice FishCulturist 48, 161-167. Suzuki, H . , Yasuda, A , , Kondo, J , , Kawauchi, H., and Hirano, T. (1991). Isolatiou and characterization of‘ Japanese eel prolactins. Gen. Comp. Enclocrinol. 81,

39 1-402. Swennen, D., Rentier-Delrue, F., Auperin, H., Prunet, P., Flik, G., Wendelaar Bonga, S. E., Lion, M., and Martial, J. A. (1991).Production and purification of biologically active recombinant tilapia (Oreochrontis niloticus) prolactins. J . Endocrinol. 131, 2 19-227. Tan, C . I{., Wong, L. Y., Pang, M. K., and Lam, T. J. (1988).Tilapia prolactin st~niulates

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39 1

estradiol-17p synthesis in oitro in vitellogenic oocytes of the guppy Poeciliu reiiculata. J. Exp. Zool. 248, 361-364. Tarpey, J. F., and Nicoll, C. S. (1987). Characterization of renal prolactin-binding sites oftwo amphibians (Ambystomatigrinum and Rana catesbeiuna) and a reptile (Pseudemys scripta elegans).J. Exp. 2001.241, 317-325. Thoreau, E., Petridou, B., Kelly, P. A,, Djiane, I., and Mornon, J. P. (1991). Structural symmetry of the extracellular domain of the cytokineigrowth hormone/prolactin receptor family and interferon receptors revealed by hydrophobic cluster analysis. F E B S Lett. 282, 26-31. White, B. A., and Nicoll, C. S. (1979). Prolactin receptor in Rana catesbeianu during development and metamorphosis. Science 204, 851-853. White, B. A., Lebovic, G. S., and Nicoll, C. S. (1981). Prolactin inhibits the induction of its own renal receptors in Ranu catesbeiana tadpoles. Gen. Comp. Endocrinol. 43, 30-38. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N. (1993). JAKZ associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell (Cumbridge, Mass.) 74, 227-236. Yao, K., Niu, P. D., Le Gac, F., and Le Bail, P. Y. (1991). Presence of specific growth hormone binding sites in rainbow trout (Oncorhynchusmykiss) tissues: Characterization of the hepatic receptor. C e n . Comp. Endocrinol. 81, 72-82. Yamaguchi, K., Specker, J. L., King, D. S., Yokoo, Y., Nishioka, R. S., Hirano, T., and Bern, H. A. (1988). Complete amino acid sequences ofa pair of fish (Tilapia)prolactins, tPRL,,, and tPRL,a. J . Biol. Chem. 263, 9113-9121. Yasuda, A., Itoh, H., and Kawauchi, H. (1986). Primary structure ofchum salmon prolactins: Occurrence of highly conserved regions. Arch. Biochem. Biophys. 244, 528-541. Yasuda, A,, Miyazima, K. I., Kawauchi, H., Peter, K. E., Lin, H. R., Yamaguchi, K., and Sano, H. (1987). Primary structure of common carp prolactins. Gen. Conip. Endocrinol. 66,280-290. Ymer, S. I., Kelly, P. A., Herington, A. C., and Djiane, J. (1987). Studies on the relationship between the membrane bound and cytosolic lactogen receptor of rabbit mammary gland. Mol. Cell. Endocrinol. 53, 67-73. Young, P. S., McCormick, S. D., Demarest, J. R., Lin, H. J., Nishioka, R. S . , and Bern, H. A. (1988). Effects of salinity, hypophysectomy, and prolactin on whole-animal transepithelial potential in the tilapia Oreochronais mossnmhicus. Gem C o m p . E n docrinol. 71, 389-397. Zhang, R., Buczko, E., Tsai-Morris, C. H., Hu, 2. Z., and Dufau, M. (1990). Isolation and characterization of two novel rat ovarian lactogen receptor cDNA species. Biochetn. Biophys. Res. Commun. 168, 415-422.

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13 R E G U L A T I O N OF OOCYTE MATURATION I N FISH YOS HlTAKA NAGAHAMA, M ICHl YA S U Y 0sH I K UNI , MASAKANE YAMASHITA, AND MINORU TANAKA Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444, Japan

I. Introduction 11. Phenomenology

111. Structure of Follicles IV. Gonadotropin: Primary Mediator of Oocyte Maturation A. Chemistry of Fish Gonadotropin B. Gonadotropin Surge C. Induction of Oocyte Maturation by Gonadotropin V. Maturation-Inducing Hormone (MIH): Secondary Mediator of Oocyte Maturation A. Identification of Fish MIH B. Synthesis of MIH (17a,2OX)P-DP):Two-Cell Type Model C. Mode of Action of Gonadotropin on MIH Production D. Steroidogenic Shift in Postvitellogenic Follicles E. Gene Cloning of Steroidogenic Enzymes F. Changes in mHNA Levels of Steroidogenic Enzymes in Hainbow Trout Ovarian Follicles during Oocyte Growth and Maturation G. Surface Site of MIH Action V1. Xlaturation-Promoting Factor (MPF): Tertiary Mediator of Oocvte Maturation A. Existence of MPF in Fish Eggs U. Generality of MPF Activity C. Purification and Characterization of Fish MPF and Histone H1 Kinase VII. Conclusions References

I. INTRODUCTION Like most other vertebrates, teleost fishes have full-grown postvitellogenic oocytes in the ovary that are physiologically arrested at the G2/M border in first meiotic prophase and cannot be fertilized. For 393 b I\H PH1\lOLOGl VOL XI11

Cop\iiyht 0 1YO1 In A c . t d e n , ~ P i t \ \ l u c I n ‘tnv t m m ~ e w t ~ e d

All iigbt\ d ieproducttim

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the oocytes to be fertilizable, they must complete the first meiotic division. Under an appropriate hormonal stimulation, full-grown 00cytes resume their first meiotic division, which involves breakdown of the germinal vesicle (GVBD), chromosome condensation, assembly ofthe first meiotic spindle, aiid extrusion ofthe first polar body. Meiosis is again arrested at the second metaphase. Shortly thereafter, in’‘It ure fertilizable oocytes are ovulated.The meiotic process leading to extrusion of the second polar body is resumed again at the time of fertilization, immediately after sperm penetration. The time period between the resumption of meiosis and the second meiotic rnetaphase has been referred to as the period of oocyte maturation. Thus, the process of oocyte maturation is a prerequisite for successful fertilization. Hormonal regulation of oocyte maturation has been investigated intensively in teleost fishes (Goetz, 1983; Nagahama, 1 9 8 7 ~Jalabert ; et uZ., 1991; Nagahama et al., 1993; Redding and Patino, 1993). Oocyte maturation in teleosts is usually quite rapid and accomplished within 24 hr (depending on species, temperature, etc.).The ovaries ofteleosts, in general, contain large numbers of oocytes that are relatively easy to maintain in citro. Thus, the development of an in vitro system, using GVBD as a biological indicator of hormone action, was a great stiinuliis to work in this field. Accumulating evidence suggests that oocyte maturation in teleost fishes is regulated b y a series ofinterdependent hormonal actions. Three regulators have been described: gonadotropin, maturation-inducing hormone (MIH; maturation-inducing steroid or substance, MIS), and maturation-promoting factor (MPF) (Goetz, 1983; Nagahama, 1 9 8 7 ~Jalabert ; et LIZ., 1991; Nagahama et al., 1993; Redding and Patino, 1993).This article attempts to siimmarize our current understanding of the regulation of oocyte maturation in teleost fishes.

11. PHENOMENOLOGY

The full-grown oocyte of teleosts possesses a large nucleus (germinal vesicle, GV) in meiotic prophase. The GV ofthis stage is generally located centrally or halfway between the center and the oocyte periphery. In most species, the GV cannot be seen by external observ,a t’1011 because ofthe opaque cytoplasm. The use of clearing solution (Goetz aiid Bergman, 1978) increases yolk transparency and the CTV appears dark brown under transmitted light after this treatment. In the terminal phase ofvitellogenesis, goldfish (Cnrassius auratus) oocytes lose their spherical shape and become slightly flattened. The animal pole, on

13.

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one of the flattened surfaces, is located around a small depression in the follicle and zona radiate, the micropyle. Under the dissecting microscope this area can be easily distinguished, appearing as a number of small furrows in the follicle radiating out from the micropyle. In some species such as the yellow perch (Percaflaoescens) and the Indian catfish (Heteropneustesfossilis), the animal pole is quite conspicuous with an elevated cap (Goetz, 1983; Goswami and Sundararaj, 1971a). In other species, particularly marine teleosts, there is an enormous volume increase due to rapid water uptake, which occurs concomitant with oocyte maturation (Fig. 1).

111. STRUCTURE OF FOLLICLES Each full-grown postvitellogenic oocyte of teleosts is entirely siirrounded by the follicle layer. The ovarian follicle layer of teleosts, as in other vertebrates, consists of two major layers: the thecal layer, containing fibroblasts, capillaries, collagen fibers, and large glandular cells designated as special thecal cells, and the granulosa layer, composed of a single population of granulosa cells (Fig. 2). The special thecal cells possess features that characterize steroidogenic cells in general, that is, mitochondria with tubular cristae and a tubular agranular endoplasmic reticulum (Nagahama, 1983). Histochemically, 3pand 17P-hydroxysteroid dehydrogenase (3P-HSD and 17p-HSD) have been demonstrated in the special cells of several teleosts (Nagahama, 1983). Recently we raised a polyclonal antibody against a synthetic peptide (TCALRPMYIYGE) corresponding to an amino acid sequence in 3P-HSD that is completely conserved between mammals (human, bovine, rat, and mouse) and rainbow trout (Oncorlaynchus m y k i s s ) (M. Kobayashi and Y. Nagahama, unpublished). Immunocytochemical studies using this antibody revealed that in tilapia (Oreochrornisniloti-

'0 D Fig. 1. 1 7 o l , 2 0 P - D i h y d r o x y - 4 - p r e ~ ~ ~ e 1 ~ - ~ -oocyte ~ ) ~ ~ e maturation - i 1 ~ ~ ~ ~ l cof' ~ ~Pngrtl.y l in(ijor. T h e bar represents 200 p n . Oocytes were incul)ated with the steroid (0.1 /LR/

nil) for 0 (A), 8 (B), 10 (C), and 12 (D) hr. Arrow indicates an oil drop.

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Fig. 2. Electron micrographs ofovarian follicles ofPagrus mujorduring early vitellogenesis (A) and oocyte maturation (B). Ovarian follicles consist oftwo major cell layers, the thecal cell layer (STC) and the granulosa cell layer ( G C ) .Grarlulosa cells during

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oocvte maturation (migrated nucleus stage) contain dilated granular endoplasmic reticul r i m filled with amorphous materials in the stretched granulosa cell and wide interccllular spaces. BM, basement membrane.

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YOSHITAKA NAGAHAMA ET AL.

3P-HSD is located in special thecal cells, but not in granulosa cells of the ovarian follicles (M. Nakamura and Y. Nagahama, unpublished). Histochemical studies have indicated that steroidogenic enzyme activities are localized in the granulosa cells of some teleosts. However, these histochemical observations on granulosa cells do not seem to be supported by ultrastructural studies because these cells contain features suggestive of protein synthesis, but lack organelles associated with steroid-producing cells (Nagahama, 1983; Dodd, 1987). During oocyte maturation of the mummichog (Fundulus heteroclitus),the granulosa cells undergo specific cytological alterations (Wallace and Selman, 1981).These changes include an enormous proliferation o f t h e Golgi apparatus with accumulated secretory material, and an increase in the number of cisternae of the granular endoplasmic reticulum. Similarly, Matsuyama et al. (1991)observed an extremely dilated granular endoplasmic reticulum with an amorphous intracisternal substance appearing between the lamellae (Fig. 2). In the medaka (Oryzias Zati p s ) , “special granulosa cells” having mitochondria with tubular cristae appear 14.5 hr after the beginning of the light phase in oocytes destined for maturation (lwamatsu and Oota, 1981). These observations suggest a possible contribution of the granulosa cells to the production of MIH (see the following sections).

CILS),

IV. GONADOTROPIN: PRIMARY MEDIATOR OF OOCYTE MATURATION A. Chemistry of Fish Gonadotropin l t is well established that in teleosts, as in other vertebrates, pituitary gonadotropins are the major hormones that stimulate gonadal activities. Although a number of biochemical studies have been conducted to purify fish gonadotropins, the number and identity of fish gonadotropins have been controversial. A glycoprotein-rich gonadotropin with a molecular weight of25,000-40,000 (glycoprotein-rich “maturational” gonadotropin) has been purified in several teleosts (BurzawaGerard, 1982; Fontaine and Dufour, 1987). This type of gonadotropin has been reported to stimulate almost all gonadal activities including gametogenesis and steroidogenesis. However, there is now biochemical evidence that teleosts, similar to other vertebrates, possess two gonadotropins. Idler and his colleagues isolated two gonadotropins from pituitaries of four teleost species (Idler and Ng, 1983). One ad-

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sorbed to concanavalin-A Sepharose and stimulated gonadal steroidogenesis, whereas the other did not adsorb to concanavalin-A Sepharose and stimulated in vivo vitellogenin uptake by ovarian follicles. Subsequently, two distinct carbohydrate gonadotropins, designated as GTH I and GTH 11, were identified in chum salmon (Oncorhynchus keta) (Suzuki et al., 1988a,b; Itoh et al., 1988; Kawauchi et al., 1989). These two gonadotropins are distinctly different from each other in chemical characteristics and structurally homologous to tetrapod folliclestimulating hormone (FSH) and luteinizing hormone (LH). Each gonadotropin consists of a and p subunits; the p subunits have only about 31% amino acid sequence identity to each other. GTH I and GTH 11, with chemical characteristics similar to those of chum salmon, were also purified from coho salmon (Oncorhynchus kisutch) pituitaries (Swanson and Dickhoff, 1990; Swanson, 1991; Swanson et al., 1991). In both chum salmon and coho salmon, GTH I and GTH I1 exhibited similar in vitro steroidogenic potencies (Suzuki et al., 1 9 8 8 ~ ; Swanson et al., 1991).Blood and pituitary levels ofthese two gonadotropins vary significantly during reproductive development. GTH I was the predominant gonadotropin in the plasma and pituitary of vitellogenic females, whereas GTH I1 was the predominant gonadotropin at the time of final oocyte maturation (Suzuki et al., 1988d; Swanson and Dickhoff, 1990; Swanson 1991). Obviously, more detailed investigations of the specific roles of GTH I and GTH I1 in salmonid reproduction are necessary. In this chapter, the term gonadotropin is taken to refer to the GTH I1 (“maturational”) gonadotropin.

B. Gonadotropin Surge The first demonstration of the possible involvement of endogenous gonadotropin in the induction of oocyte maturation and ovulation in teleosts was obtained by an ultrastructural study on the gonadotrophs in the goldfish pituitary (Nagahama and Yamamoto, 1969). Gravid female goldfish were induced to undergo oocyte maturation and ovulation by an increase in ambient water temperature. This temperatureinduced oocyte maturation and ovulation was preceded by a dramatic decrease in the number of secretory granules in the pituitary gonadotrophs. Later, this observation was confirmed b y several physiological studies in which changes in plasma levels of gonadotropin were directly monitored by radioimmunoassay. Thus, an preovulatory increase in blood levels of gonadotropin was demonstrated in goldfish (Stacey et al., 1979; Kobayashi et al., 1987a) and carp (Cyprinus carpio) (Santos et al., 1986). Preovulatory changes in blood levels of gonadotro-

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YOSHITAKA NAGAHAMA ET AL.

pin have also been studied in several other teleost fishes (Fostier et w l . , 1981, 1983; Scott et al., 1983; Shimizu et al., 1985; Fitzpatrick et d.,1986; Kobayashi et al., 1987b). As a result, it has become apparent that the profiles of preovulatory changes in gonadotropin differ according to species. In rainbow trout, for example, increased gonadotropin levels are evident during the 1- to 2-week period before ovulation, with an initial peak of 10-20 ng/ml just prior to the time of final oocyte maturation and ovulation (Fostier et al., 1981).After this peak, gonadotropin levels decline slightly, and then increase even further to approximately 20-30 ng/ml following ovulation (Fostier et w l . , 1981). The functional significance of the difference in postovulatory gonadotropin profiles of salmonids and cyprinids is not known, but may be related to the extended period ofpostovulatory oocyte viability in salmonids.

C . Induction of Oocyte Maturation by Gonadotropin

I n a number of teleost species, the eggs of mature females can be induced to mature and ovulate by injection of a variety of gonadotropin preparations. Similarly, follicle-enclosed, full-grown, postvitellogenic oocytes of several teleosts undergo GVBD in oitro when they are incubated with a number of gonadotropin preparations. However, denuded oocytes are incapable of responding to gonadotropin. Cyanoketone, a specific inhibitor of 3P-HSD, completely abolished the maturational effects of gonadotropin and pregnenolone, but not of A‘-steroids (l’ia,BOpsuch as progesterone or 17a,20P-dihydroxy-4-pregnen-3-one UP) (Young et al., 1982; Iwamatsu and Onitake, 1983). These results indicate that the action of gonadotropin in inducing oocyte maturation is dependent on the synthesis of a second A4-steroidal mediator of meiotic maturation (Wasserman and Smith, 1978; Masui and Clarke, 1979; Nagahama, l987b; Jalabert et w l . , 1991). V. MATURATION-INDUCING HORMONE (MIH): SECONDARY MEDIATOR OF OOCYTE MATURATION A. Identification of Fish MIH In teleosts, a variety of‘ C21-steroids have been shown to be potent initiators of GVBD in uitro; these include progesterone, 17ahydroxyprogesterone (17a-P), 17a,20P-DP, 17a,20P,2I-trihydroxy-4-

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pregnen-3-one (2Op-S), cortisol, and deoxycorticosterone (Hirose, 1972; Yamauchi and Yamamoto, 1973; Jalabert, 1976; Iwamatsu, 1978; Duffey and Goetz, 1980; Epler, 1981; Goetz, 1983;Theofan and Goetz, 1983; Nagahama et al., 1983; Greeley et al., 1986; Upadhyaya and Haider 1986; Scott and Canario, 1987; Canario and Scott, 1988; Trant and Thomas, 1988; Haider and Inbaraj, 1989; Matsuyama et al., 1990; Truscott et al., 1992; Kime, 1993). Among them, however, only two steroids (17a,20P-DP and 2Op-S) have been identified as the naturally occurring MIH in fish (see the following sections). It has also been reported that a wide range of reduced and conjugated steroid metabolites that are biologically active can be synthesized by ovarian tissue during oocyte maturation (Lambert and van Bohemen, 1979; Suzuki et al., 1981; Theofan and Goetz, 1983; Lin et al., 1989; Schoonen et al., 1989; Trant and Thomas, 1989b; Petrino et al., l989b; Canario and Scott, 1989; Canario et al., 1989; Kime, 1990; Lessman, 1991; Scott and Canario, 1992; Kime et al., 1992). Testosterone as well as other CIS-steroids induce GVBD only at high concentrations. Estradiol-l7p and other C18-steroids are generally not effective in inducing oocyte maturation in fish oocytes.

1. 17a,2Op-DIHYDROXY-4-PREGNEN-3-ONE (17a,20@-DP) The MIH of a salmonid, the amago salmon (Oncorhynchus rhodurus), was identified from media in which immature but full-grown folliculated oocytes had been incubated with gonadotropin (Nagahania and Adachi, 1985). In this study, reversed-phase high-performance liquid chromatography (HPLC) was employed to fractionate medium in which oocytes had been induced to mature with gonadotropin. Of the fractions prepared by HPLC, only one exhibited maturationinducing activity as assessed by an in vitro GVBD assay. This fraction had a retention time that coincided exactly with an authentic 17a,20/3DP standard. The purity and final characterization of the residue of' this fraction were further confirmed by a comparison with authentic 17a,2OP-DP using thin-layer chromatography (TLC) and mass spectroscopy. In addition, 17a,20P-DP levels in the plasma were low in vitellogenic amago salmon, but were strikingly elevated in mature and ovulated females. The increase in plasma 17a,20p-DP levels correlated well with a dramatic rise in plasma gonadotropin levels (Young et ul., 1983b). Of the nine pregnene derivatives tested, 17a,2O@-DPwas found to b e the most effective inducer of GVBD in four species of teleosts including amago salmon (Nagahama et al., 1983).Taken together, these results indicate that 17a,20p-DP is the major naturally occurring MIH in amago salmon.

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Petrino et al. (1993) provided strong evidence that 17a,20@-DP plays the major role as a MIH in Fundulus heteroclitus. In this study MIH was identified by incubating ovarian follicles with radioactive precursors and comparing the maturation-inducing activity among the various metabolites produced. Among all the metabolites produced by the follicles, 17a,20/3-DP was the most potent and short-acting inducer ofoocyte maturation. In addition, follicles were also incubated with a F . heteroclitus pituitary extract. After steroids obtained from such incubations were separated by HPLC, there were several fractions that were able to induce GVBD. The highest activity, however, was confined to two fractions corresponding with 17a,20@-DPand 5apregnan-3/3, 17a,20p-triol. Our study also showed that 17a,20p-DP plays ainajor role in mediating the gonadotropic action of maturation in the inedaka (Oryzias latipes) (Fukada et al., 1994).This fish, under a constant long photoperiod (14 hr light-10 hr dark) at 26"C, usually spawns daily within 1 hr ofthe onset of light for a number of consecutive days. By this method the sequence of events leading to spawning such as the completion of vitellogenesis, GVBD, and ovulation can be timed accurately (Iwamatsu, 1978). Follicles isolated at 12 different stages (from 30 to 2 hi- before the expected time of spawning) were incubated with "C-labeled pregnenolone, progesterone, and 17a-P for 4 hr in the presence of NADPH. The radioactive metabolites produced were separated by TLC and tested for their ability to induce GVBD in oocytes using an in vitro homologous bioassay. Among all the metabolites produced, the most effective metabolite that induced GVBD was the one comigrating with 17a,20p-DP. A marked increase in the production ofthis metabolite was observed in follicles collected prior to or during oocyte maturation (between 10 and 6 hr before spawning). This metabolite was identified as 17a,ZOp-DP by recrystallization to constant specific activity. Immediately after its formation, 17a,20P-DP was converted to a much less biologically active metabolite, 17a,20/3dihydroxy-5P-pregnane-3-one. The timely synthesis of 17a,20p-DP in medaka at the onset of oocyte maturation, together with the demonstration that this progestogen was the most potent inducer of oocyte maturation in vitro, provides strong evidence that 17a,20P-DP is the naturally occurring MIH in the medaka. These findings also suggest that the conversion of 17a,20/3-DP to its 5p-reduced metabolite may be an inactivation process. 17a,20/3-DP has also been shown to be the most effective steroid in many nonslamonid fishes (Scott and Canario, 1987).This steroid was first identified in the blood of postspawning females of Oncorhynchus

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nerkn (Idler et al., 1960). Since then, high plasma levels of 17a,20PD P during final oocyte maturation have been reported in several fish species (reviews, see Goetz, 1983; Fostier et al., 1983; Nagahama, 1987a; Scott and Canario, 1987; Jalabert et al., 1991). Sundararaj and Goswami (1977)have proposed that MIH in the Indian catfish is interrenal in origin and is probably cortisol. However, subsequent in zjizjo and in oitro studies on the African catfish, Clarias gariepinus (Lambert and Van den Hurk, 1982)and the Malaysian catfish Clarias macroceplzalus (Suzuki et al., 1987) have unequivocally demonstrated 17a,20@DP production by the ovaries of gonadotropin-stimulated females. Furthermore, although Goswami and Sundararaj’s earlier works indicated that corticosteroids were the most effective steroid in inducing oocyte maturation i n vitro in the Indian catfish (Goswami and Sundararaj, 197la,b, 1974), their later studies appear to indicate that 17a,20PDP is more effective (Sundararaj et al., 1985). 17a,2O@-DPwas the most effective steroid in inducing oocyte maturation in the Indian catfish Mystus vittatus (Upadhyaya and Haider, 1986). Finally, Haider and Kao (1992) provided direct evidence that 17a,20P-DP is the major naturally occurring MIH in another Indian catfish, Clarias batruchus. In their studies, the medium in which oocytes had been induced to mature by gonadotropin was subjected to extraction and HPLC fractionation. Chromatography ofthe methanol phase revealed several substances including 17a,20/3-DP, but none of them coincided with the retention time of deoxycorticosterone.

2. 17a,20p,21-TRIHYDROXY-4-PRECNEN-3-ONE(20p-s) 20p-S has been identified as a naturally occurring MIH ofthe Atlantic croaker (Micropogonias undulatus) (Trant et ul., 1986; Trant and Thomas, 1989a,b). Iodated ovarian tissue in the process of oocyte maturation was incubated with human chorionic gonadotropin (HCG) and pregnenolone in tissue culture medium for 8 hr. Steroids were extracted from the medium and fractionated by HPLC and TLC. Fractions were hioassayed for their potency to induce GVBD of Atlantic croaker oocytes in zjitro. 20p-S was identified as the predominant steroid product and the major MIH produced by the ovary of Atlantic croaker in oitro. Although a small amount ofanother steroid with equal potency to induce GVBD was identified as 17a,20P-DP, over 10 times more 20p-S than 17a,20/3-DP accumulated in the incubation medium. The involvement of 20p-S in the induction of oocyte maturation was also demonstrated in a closely related species, spotted seatrout ( C ~ ~ O S cion nebulosus) (Thomas and Trant, 1989). However, 2Op-S does not appear to be involved in the induction of oocyte maturation in salmo-

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nids, as there is no evidence for the presence of significant amounts of this steroid in the blood of female salmonids undergoing maturation or ovulation (Scott and Canario, 1987).

B. Synthesis of MIH (17a,BO@-DP):Two-Cell Type Model The primary site of MIH production in fish ovaries is the ovarian follicle layer, which surrounds the oocyte. Development of a simple dissection technique to separate the ovarian follicles of salmonids into two cell layers, the thecal and granulosa cell layers, has made it possible to elucidate the relative contributions of each cell layer and salmon gonadotropins (partially purified chinook and chum salmon gonadotropins, SG-G100 and SGA, respectively) in the process of 17a,2OP-DP production. Using different follicular preparations obtained from postvitellogenic amago salmon and rainbow trout, we examined the role of' each follicular layer in gonadotropin-induced 17a,20P-DP production. Salmon gonadotropins (SG-G100 and SGA) stimulated 17a,20@-DP production b y intact follicles and co-cultures of thecal and granulosa layers, but not by isolated thecal or granulosa layers alone, indicating that both cell layers are necessary for gonadotropin-stimulated 17a,20P-DP production (Young et al., 1986). Experiments examining the effects of conditioned media from incubates of one follicular layer on steroidogenesis by the other layer revealed that the thecal layer produced a steroid precursor that was metabolized to 17au,20P-DP in the granulosa layer. We further identified 17a-P as the steroid precursor produced b y thecal layers in response to gonadotropin. Gonadotropin greatly stimulated 17a-P production by thecal layers, hut not by granulosa layers. Levels of l7a-P in media from intact follicles and coculture incubations peaked at 12 hr and rapidly decreased concomitant with a rapid rise in 17a,BO@DP. Incubation of granulosa layers with exogenous 17a-P resulted in elevated 17a,20@DP levels, indicating the presence of BOP-hydroxysteroid dehydrogenase (BOP-HSD), the key enzyme involved in the conversion of 17a-P to 17a,BOP-DP. Based on the results of these in vitro studies, a two-cell type model was proposed for the first time in any vertebrate for the follicular production of MIH (Young et nl., 1986; Nagahama, 1987a). In this model, the thecal cell layer produces 17a-P, which traverses the basal lamina and is converted to 17a,BOP-DP by the granulosa cell layer, where gonadotropin acts to enhance the activity of2OP-HSD (Fig. 3 ) .A similar interaction of thecal and granulosa layers also occurs for the production of estradiol-17p by ovarian follicles of amago salmon during vitellogen-

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, -

Gonadotropin GTH-receptor

THECAL CELL P-45oscc

Cholesterol +?-CAMP

0 $.

I

Pregnendone JP-HSLJ

0t

Progesterone P-45017a

17a-Hydroxyprogesterone

-

[

GTH-rrtor GRANULOSA CELL 170 -Hydroxyprogesterone

0 $.

~ O ~ H S D

[I

-CAMP

17a ,20p-Dihydroxy-4-pregner3-one

Fig. 3. Two-cell type model for the production of 17a,20P-dihydroxy-4-pregnen-3one by salmonid postvitellogenic follicles. P-450,,,,cholesterol side-chain cleavage cytochrome P-450;3P-HSD, 3P-hydroxysteroid dehydrogenase-isomerase; P-45017a,P450 17a-hydroxylase, 2Op-HSD, 20P-hydroxysteroid dehydrogenase.

esis (Kagawa et d., 1982) (see also Section V,E). It is of particular interest that the granulosa layer is the site of production of both estradiol-l7/3 and 17a,20/3-DP,but production of these steroids by the ovarina follicle depends on the provision of precursor steroids by the thecal layer. In the ovary of F . heteroclitus, the follicle (granulosa) cells immediately adjacent to the oocyte are the primary source of steroids, including 17a,20/3-DP, produced by the follicle in response to gonadotropin stimulation (Petrino et al., 1989a). Complete removal of the follicular wall eliminated steroid accumulation induced by gonadotropin treatment. In contrast, removal of the thecdepithelial layer did not compromise the steroidogenic response of the follicle to gonadotropin. Thus, in this species the production of 17a,20/3-DP does not require the involvement of two cell types as shown for salmonid fishes. Similar to these observations for F . heteroclitus, Onitake and Iwamatsu (1986)

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reported that in medaka, progestogens are synthesized in the follicle cells without the involvement of thecal layers. It is noteworthy that in these two species, typical steroidogenic “special thecal cells” are not evident. C. Mode of Action of Gonadotropin on MIH Production 1. GONADOTROPIN RECEPTORS

In the two-cell type model for the production of 17a,20P-DP just described, gonadotropin has at least two sites of action, thecal and granulosa layers. This suggests the existence of gonadotropin receptors in these layers. Specific gonadotropin receptors were demonstrated in both thecal and granulosa layers from amago salmon postvitellogenic follicles (Kanamori and Nagahama, 1988a). Scatchard analysis showed that both layers exhibit essentially the same characteristics of binding to labeled gonadotropin. The values of the dissociation constants ( K , ! ) were in the range of 0.2-0.8 nM. These results suggest that amago salmon preovulatory follicles contain a single population ofgonadotropin receptors in both thecal and granulosa layers. We also showed that the number of gonadotropin receptors per follicle in both thecal and granulosa layers increases during oogenesis (Kananiori and Nagahama, 1988a). Subsequently, Yan et al. (1992) provided evidence suggesting the existence of at least two types of gonadotropin receptors, designated as type I and type I1 receptors, in coho salmon. The type I receptor binds both GTH I and GTH 11, but with higher affinity for GTH I, whereas the type I1 receptor binds GTH I1 specifically and may have only limited interaction with GTH I. The type I receptor exists in both thecal layers and granulosa cells, whereas the type I1 receptor exists in granulosa cells. The characterization of the molecular nature of the two gonadotropin receptors awaits future study. Certainly these studies will lead to valuable insights concerning the role of the gonadotropin receptors in the action of gonadotropin on steroidogenesis in thecal layers and granulosa cells in fish ovarian follicles.

2. THECALCELLLAYERS Because the major role of thecal layers in gonadotropin-induced 17a,20P-DP production by follicular layers during oocyte maturation is to secrete the immediate precursor ofthis steroid, 17a-P, we investigated the mechanism of gonadotropin action on 17a-P production. A dose-dependent increase in 17a-P production was observed when

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thecal layers obtained immediately prior to or during oocyte maturation were incubated with agents known to increase intracellular CAMP concentrations, such as forskolin, an adenylyl cyclase activator, and dibutyryl CAMP(dbcAMP) (Kanamori and Nagahama, 1988b).Furthermore, SGA and forskolin (a direct activator of adenylyl cyclase) stimulated CAMP formation by thecal layers in a dose-dependent manner. A significant increase in 17a-P production by thecal layers in response to SGA and forskolin could be detected within 1 hr after the onset of incubation. These findings are consistent with the view that gonadotropin acts on thecal layers to stimulate 17a-P production through a mechanism involving one or more receptor-mediated adenylyl cyclase-CAMP-dependent steps. Additional evidence is provided by the observation that steroidogenic responses induced by submaximal concentrations of gonadotropin, forskolin, and dbcAMP are consistently enhanced by the presence of the phosphodiesterase inhibitors, theophylline and 3-isobutyl-1-methylxanthine (IBMX) (Kanamori and Nagahama, 198813). Furthermore, gonadotropin- and CAMP-induced 17a-P production b y the thecal layer is abolished by cycloheximide and puromycin, but not by actinomycin D and cordycepin, suggesting that the stimulatory effects of gonadotropin in the thecal layer require the synthesis of a new protein (Nagahama, 1987a).

3. GRANULOSA CELLLAYERS In addition to specific gonadotropin receptors, crude membranes of amago salmon granulosa cells contain guanine nucleotide-binding regulatory proteins (G-proteins) and adenylyl cyclase (Mita et d., 1994).Although cholera toxin-catalyzed ADP ribosylation occurred in 45- and 58-kDa proteins, only the 45-kDa protein was recognized by an antibody against the a subunit of the stimulatory G-protein (Gs). With pertussis toxin, only 41-kDa protein was ADP-ribosylated. This 41-kDa protein was recognized by an antibody against the CY subunit of the inhibitory G-protein (Gi).SGA stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells only in the presence of pertussis toxin in the incubation medium. These results provide evidence that both stimulatory (Gs) and inhibitory (Gi) regulation ofadenylyl cyclase operate in the granulosa cells of amago salmon postvitellogenic ovarian follicles. It is possible that although a stimulatory gonadotropin receptor interacts with Gs, its activity is influenced by the functional state of Gi. Neither 17a-P nor 17a,20P-DP was produced when granulosa layers were incubated with gonadotropin alone. Our earlier cell-free studies indicated that, unlike thecal layers, granulosa layers lack the

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side-chain cleavage enzyme systems. We then examined the effects of various agents on the activity of 20p-HSD in granulosa layers. 20PHSD was found to be stimulated by both salmon gonadotropins and forskolin in a dose-dependent manner. The action of gonadotropin on SOP-HSD enhancement in granulosa cells was also mimicked by dbcAMP, but not by dbcGMP, or by theophylline and IBMX (Nagahama et al., 1985a). Furthermore, gonadotropin and forskolin caused a rapid accumulation ofcAMP in granulosa cells with maximum levels at 30-60 min. These findings suggest that gonadotropin enhances the activity of granulosa cell BOP-HSD through a receptor-mediated Gprotein-adenylyl cyclase-CAMP-dependent step. Although cAMP is viewed as the principal intracellular messenger mediating the actions of gonadotropins on ovarian steroidogenesis in fish, there is evidence that calcium ions also play an important role (Nagahama 198713; Van Der Kraak, 1991). Calcium ionophore A23187 and the phorbol ester PMA, a protein kinase C activator, were shown to attenuate the stimulatory effects of gonadotropin and forskolin on 17a,20P-DP production by postovulatory ovarian follicles of goldfish (Van Der Kraak and Jacobson, 1991). We found that calmodulin inhibitors such as trifluoroperazine (TFP), N-(6-aminohexyl)-l-naphthalenesulfonamide hydrochloride (W5), and N-(8aminohexyl)-5chloro-l-naphthalenesulfonamide(W7) prevented gonadotropin-stimulated BOP-HSD activation in granulosa cell layers in a dose-dependent manner. The inhibitors also blocked forskolin- and dbcAMP-stimulated 20P-HSD activation, suggesting an action distal to cAMP generation. These results emphasize the importance of calcium as a regulator of gonadotropin-induced 20P-HSD activation and suggest that multiple signaling pathways may participate in the regulation of 20P-HSD activation. A very important question is the gene regulation of gonadotropininduced 20P-HSD activation by the granulosa layer. It was shown that inhibition of RNA synthesis with actinomycin D, cordycepin, and aamanitin, or protein synthesis with cycloheximide and puroniycin, completely abolished the stimulatory effects of SGA and dbcAMP on the production of 17a,20P-DP by granulosa cells. Furthermore, the stimulatory effects of gonadotropin and cAMP on 20P-HSD activity by granulosa layers required at least 12 hr. These results are consistent with the suggestion that one of the major actions of gonadotropin and CAMP on BOP-HSD activation in amago salmon granulosa cells is to enhance new KNA and protein synthesis (Nagahama 1987a; Nagahama et al., 1985b). Our time course studies further suggest that de novo synthesis of20P-HSD occurs in vitro in response to gonadotropin and

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dbcAMP and consists of gene transcriptional events within the first 6 hr of exposure to gonadotropin and dbcAMP and translational events 6-9 hr after the exposure to gonadotropin and CAMP. Thus, these results suggest that gonadotropin causes the de novo synthesis of 20pH S D in the amago salmon granulosa cell through a mechanism dependent on RNA synthesis. The induction of 2OP-HSD activity b y gonadotropin in amago salmon granulosa cells is a good example of differentiated functions expressed by targets cells in response to peptide hormone stimulation (Fig. 4).

Precursor 17a-Hydmxyprogesterone

Gonadotropin

1 Receptor

t Plasma membrane

G-protein

t

Adenylyl cyclase

/

*

Granulosa cell ATP

17aaSly&oxy-

CAMP

t

Ca

PfOgeSterOne

Oocyte maturation Fig. 4. Diagram of BOP-hydroxysteroid dehydrogenase (2Op-HSD) activation of salmonid granulosa cells via the receptor-G-protein-adenylylcyclase-CAMP mechan i s m .

YOSHITAKA NAGAHAhlA ET AL.

410 D. Steroidogenic Shift in Postvitellogenic Follicles

It is well established that vitellogenesis in teleosts is promoted b y a two-step mechanism in which gonadotropin increases ovarian secretion of estradiol-l7P, which in turn stimulates the hepatic synthesis and secretion of vitellogenin (Ng and Idler, 1983; Wallace, 1985). In all the teleost species studied so far, elevated levels of estradiol17p have been reported in females during active vitellogenesis. The primary site of estradiol-l7p in the teleost ovary is the follicles that surround the vitellogenic oocytes (Nagahama, 1983).Preovulatory follicles of most teleost species, unlike postovulatory follicles, produce predominantly estradiol-17p in uitro in response to gonadotropin stimulation (Kagawa et aZ., 1983).Using incubation techniques of isolated salmonid follicular preparations similar to those used for studies on follicular production of 17a,20p-DP production, we proposed a twocell type model for the follicular production of estradiol-17P (Kagawa et ul., 1985; Nagahama, 1987a). In this model, the thecal cell layer, under the influence of gonadotropin, secretes the androgen substrate (probably testosterone) that traverses the basal lamina and is converted to estradiol-17p by the granulosa cell layer where aromatase, the key enzyme involved in the conversion of testosterone to estradiol-l7p, is localized exclusively. The capacity of amago salmon intact follicles to produce estradiol17p in response to gonadotropin stimulation increased during oocyte growth, but rapidly decreased in association with oocyte maturation in response to gonadotropin (Kagawa et al., 1983). The capacity of the thecal cell layer to produce testosterone in response to gonadotropin gradually increased during the course of oocyte growth and peaked during the postvitellogenic period (Kanamori et al., 1988).Aromatase activity in granulosa cell layers increased during vitellogenesis and decreased rapidly in association with oocyte maturation in response to gonadotropin (Young et al., 1983a; Kanamori et al., 1988). This decrease in aromatase activity appears to coincide with a decrease in estradiol-17p production by intact follicles in response to gonadotropin. Testosterone production in thecal cell layers does not decline during this time, suggesting that the reduced production of estradiol17p by postvitellogenic follicles is due, in part, to decreased aroniatase activity in granulosa cell layers. 17a,20p-DP production b y intact follicles at different stages of development showed that the potential to secrete 17a,20p-DP in response to gonadotropin by synthesizing this steroid is acquired immediately prior to oocyte maturation. Similarly, thecal cell layers did not

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H E G U L A T I O N OF OOCYTE MATURATION

develop the ability to produce 17a-P in response to gonadotropin until inmiediately prior to oocyte maturation. However, granulosa cell layers first acquired the ability to convert 17a-P to 17cq20P-DP in response to gonadotropin about 2 months prior to oocyte maturation. Nevertheless, increased endogenous BOP-HSD activity occurs in granulosa cells only during oocyte maturation. Thus, a decrease in 17,20-lyase and/or 17pHSD activity in thecal cells and an increase in BOP-HSD in granulosa cells appear to be the two major factors responsible for the rapid increase in 17a,20P-DP production b y intact follicles during oocyte maturation.

E. Gene Cloning of Steroidogenic Enzymes Our findings described in the foregoing have provided evidence that a distinct shift in steroidogenesis, from estradiol-17P to 17a,20PUP production, occurs in the follicular layer immediately prior to final oocyte maturation (Fig. 5).The rates of production of steroid hormones

r

THECAL CELL r GRANULOSA CELL. P-45oscc C hI)o lfe s t e r o l 1 0 Pregnenolone

I I

Progesterone

I

c21

y

3

HO-C-H

3

Testosterone

____)

@-I

Estradiol-17p

+5 0

Fig. 5. Pathway of steroid biosynthesis i n the ovarian follicle of salmonids during oocyte growth and maturation, showing the relative contribution of thecal and granulosa cell layers in the production of estradiol-l7p and 17a,20p-dihydroxy-4-pregnen-3-one (17a,20p-DP).P-450scc,cholesterol side-chain cleavage cytochrome P-450; 3p-HSD, 3phydroxysteroid dehydrogenase-isomerase; P-45017,, P-450 l7a-hydroxylase; 17P-HSD, 17p-hvdroxysteroid dehydrogenase; P-450arom, aromatase cytochrome P-450; 200HSD, 20P-hydroxysteroid dehydrogenase.

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YOSHITAKA NAGAHAMA E T AL.

are related to the follicular content of' various steroid hormones. To understand more fully the mechanisms by which gonadotropins or other hormones regulate the contents of steroidogenic enzymes, we have initiated studies on the cloning of genes that encode several steroidogenic enzymes from cDNA libraries constructed from rainbow trout ovaries. Five steroidogenic enzymes involved in the production of estradiol-17P and 17a,20P-DP were selected for cloning (Fig. 5);these are cholesterol side-chain cleavage cytochrome P-450 (P450scc), 30-hydroxysteroid dehydrogenase-isomerase (3P-HSD), 17ahydroxylase/ 17,20-lyase cytochrome P-450 (P-450~17), aromatase cytochrome P-450 (P-4SOarom), and BOP-HSD. Using mammalian cDNAs (kindly provided by Drs. W. L. Miller, S. Chen, and J. I. Mason, U.S.A.) and synthetic oligonucleotides, we have so far isolated and sequenced cDNAs encoding rainbow trout P-450scc, 3P-HSD, P-450~17,and P450arom. In addition, BOP-HSD cDNA has been cloned from a pig

J. + COS - 1 cells

control 437f15.31

+ Transfected COS-1 cells

pSVL 457f19.53

RIA

pEST4 6760f680.59

Fig. 6. (Top)Schematic illustration of the procedures used to express rainbow trout P-450arom cDNA in COS-1 monkey kidney tumor cells. The amount of estradiof-176 formed by COS-1 cells were detected hv the conversion of exogenous testosterone to estradiol-l7,8 by radioimmnnoassay. (Hottom) Aromatase activity of rainbow trout 1'450aroni in COS-1 cells. COS-1 cells harboring pEST4 (containing the 2.3-kb EcoRI insert) produced significantly more estradiolL17p than did COS-1 cells having pSVL (no insert) or no vector (control).

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REGULATION OF OOCYTE MATURATION

Table I Rainbow Trout Ovarian Steroidogenic Enzymes: The Number of Amino Acid Residues and Percentage Sequence Homology with Those of Mammals Sequence homology

(%I

Amino acid number

(troutimammals)

Cholesterol side-chain cleavage cytochrome P-450 (P-450scc)

514

46-48

3P-Hydroxysteroid dehydrogenaseisomerase (3P-HSD) 17a-Hydroxylase/l7,20-lyase cytochrome P-450 (P-450~17) Aroniatase cytochrome P-450 (P-450arom)

374

48

514

46-48

522

52

Enzyme

testis c D N A library. The c D N A inserts were confirmed to encode each steroidogenic enzyme by introducing a plasmid with the insert into nonsteroidogenic COS-1 monkey kidney tumor cells (Fig. 6). The complete nucleotide sequences and deduced amino acid sequences of these enzymes were determined. The number of amino acids and percentage sequence identity between trout and mammals is shown in Table 1 for the enzymes.

a. P-45Oscc. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone. This reaction is catalyzed by P-45Oscc. A c D N A clone encoding P-45Oscc was isolated from a rainbow trout ovarian follicle c D N A library (Takahashi et al., 1993).The c D N A contained an open reading frame of 1542 nucleotides encoding a protein of 514 amino acids. The predicted amino acid sequence of trout P-45Oscc shows 48% homology with that of human and 46% homology with that of rat, bovine, and pig. P-45Oscc activity was confirmed by transfected COS-1 cells with an expression vector for trout P-45Oscc c D N A and subsequent detection of conversion from 25-hydroxycholesterol to pregnenolone by radioimmunoassay. The c D N A only hybridized to a single 1.8-kilobase (kb) RNA transcript.

b. 3P-HSD.A c D N A clone encoding3P-HSD,the enzyme responsible for the oxidation and isomerization of A5-3P-hydroxysteroid precursors into A4-3-ketosteroids, was isolated from a c D N A library of rainbow trout ovarian thecal cells (Sakai et al., 1993).The c D N A contained an open reading frame of 1122 nucleotides encoding a protein of 374 amino acid residues. Comparison of the deduced amino acid sequence of trout 3P-HSD with that of mammalian 3P-HSD revealed at least five

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YOSHITAKA NAGAHAMA ET AL.

conserved regions, including a putative pyridine-nucleotide binding domain. Southern hybridization analysis of trout genomic D N A with the cDNA suggested the presence of a single gene encoding 3P-HSD in the rainbow trout showing a total genomic size of less than 4-kb. The cDNA hybridized only to a single 1.4-kb transcript isolated from rainbow trout ovaries. The trout 3P-HSD expressed in COS-1 cells showed a unique enzymatic 3P-HSD activity. Dehydroepiandrosterone was a more favored substrate ofthe trout 3P-HSD than 17ahydroxypregnenolone. Interestingly the trout 3P-HSD expressed in COS-1 cells exhibited minimal ability to convert pregnenolone to progesterone. This activity profile was distinct from that of rat and human forms of 3P-HSD, which catalyze the conversion of pregnenolone to progesterone at similar rates as the conversion of other A5-3P-hydroxysteroids to A4-3-ketosteroids.

c. P - 4 5 0 ~ 1 7A. full-length cDNA encoding P-450~17,the enzyme that is involved in the l7a-hydroxylation of both pregnenolone and progesterone as well as their conversion to C 19-steroids, was cloned from a rainbow trout ovarian thecal cell layer cDNA library (Sakai et al., 1992). The cDNA contained an open reading frame of 1542 nucleotides encoding a protein of 514 amino acid residues. The amino acid sequence of trout P-450~17shows a much greater homology with chicken P-450~17than with that of human, bovine, and rat. The trout P-450~17expressed in COS-1 cells showed both 17a-hydroxylase and 17,20-lyase activities, a finding similar to those reported for mammalian P-450~17in which P-450~17is a single enzyme mediating both 17a-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones (Nakajin and Hall, 1981; Zuber et al., 1986). The cDNA hybridized only to a single 2.4-kb transcript isolated from rainbow trout ovaries. As discussed earlier, a dramatic switch in salmonid steroidogenesis from testosterone to 17a-P occurs only in thecal layers immediately prior to oocyte maturation. The formation of testosterone requires the activities of both l7a-hydroxylase and 17,20-lyase, whereas 17a-P requires only 17a-hydroxylase. Therefore, it is of great interest to investigate the molecular mechanisms responsible for the differential regulation of these two enzymatic activities of P-450~17 in thecal layers immediately prior to oocyte maturation. d. P-450arorn. P-450arom is responsible for catalyzing the conversion of C 19-steroids to C18-steroids. A full-length cDNA encoding P450arom was cloned from the rainbow trout ovary (Tanaka et d., 1992a). The insert of aromatase was sequenced and found to contain

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REGULATION OF OOCYTE MATURATION

an open reading frame predicted to encode a protein of' 522 amino acid residues. The deduced polypeptide is 52% homologous with aromatase of' human, mouse, and rat and 53% homologous with that of chicken. The insert was confirmed to encode aromatase by introducing it into COS-1 cells and detecting the conversion of testosterone to estradiol-17p by radioimmunoassay. The cDNA hybridized to a single 2.6-kb RNA transcript only in the trout ovary during vitellogenesis (Fig. 7). We also isolated the structural gene encoding P-450arom for the first time from a nonmammalian vertebrate, medaka, using the rainbow trout P-450arom cDNA as a probe (Tanaka et al., 1994). The medaka P-450arom gene is composed of nine exons but spans only 2.6 kbp,

Aromatase mRNA

Aromatase activity

A

B

C

D

E

Fig. 7. Changes in aromatase activity and mRNA levels in rainliow trout during oocyte growth and maturation. Aromatase activity was assessed b y incubating intact follicles in Ringer without (open bars) or with 100 ng/ml testosterone (hatched bars). Levels of estradiol-17p were measured by radioininiunoassay. Poly(A)+ HNAs were extracted from the same ovaries as used for determining aromatase activity. HNA transcripts (2.6kb) are present in ovaries during vitellogenesis. A, early vitellogenic stage; B, late vitellogenic stage; C , migrated nucleus stage; D, matnre stage; E, postovnlatory stage.

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YOSHITAKA NAGAHAMA E T AL.

being much smaller than the human P-450arom gene (at least 70 kbp) as the result of extremely small introns (medaka has 73-213 b p VS. human with 1.3-10 kbp). The splicing junctions are located exactly at the same positions as those found in the human P-450arom. The deduced amino acid sequence is 51-52% identical to those of mammals and chicken, and 75% identical to the rainbow trout amino acid sequence. Genomic Southern blots revealed the presence of a single medaka gene. S1 nuclease mapping and primer extension indicated two major transcription initiation sites 60 and 61 bp upstream from a putative initiation codon. The promoter region of medaka P-450arom gene also contains potential Ad4 binding protein (Ad4BP) sites (Morohashi et al., 1993) and estrogen responsive element (ERE) half-sites. These results suggest that the basic structural organization of P450arom genes as well as their regulatory mechanisms of expression are well conserved throughout the vertebrates.

e . BOP-HSD. Unfortunately, unlike most of the ovarian steroidogenic enzymes, a cDNA BOP-HSD has not been cloned in any animal species. Therefore, we have begun to isolate cDNA encoding pig testis 20P-HSD because purified preparations of 20P-HSD are available only from the pig (Nakajin et al., 1988). Using synthetic oligonucleotides deduced from the partially determined amino acid sequences, for the first time we have isolated and cloned a cDNA encoding 20P-HSD from a pig testis cDNA library (Tanaka et d., 1992b). The c D N A contained an open reading frame predicted to encode 289 amino acid residues. Surprisingly, it had 85% amino acid homology with human carbonyl reductase. We are now using this porcine cDNA clone to isolate and clone cDNAs encoding BOP-HSD from a rainbow trout ovarian cDNA library.

F. Changes in mRNA Levels of Steroidogenic Enzymes in Rainbow Trout Ovarian Follicles during Oocyte Growth and Maturation The mHNA levels ofP-450scc, 3P-HSD, and P-450~17were barely detected in follicles during the midvitellogenic stage and were abundant in follicles during the postvitellogenic stage and oocyte maturation (Takahashi et al., 1993; Sakai et al., 1992, 1993).In contrast, the 2.6-kb P-450arom RNA transcripts were found in the ovary during active vitellogenesis but could not be found in the ovary in the stage of oocyte maturation or in the ovary containing postovulatory follicles (Fig. 7) (Tanaka et al., 1992a). These results are consistent with the

13.

REGULATION OF OOCYTE MATURATION

417

rapid decrease in aromatase activity in the granulosa cell layers during the postvitellogenic period (Young et al., 1983a). It is thus concluded that the ability of the granulosa cells to produce estradiol-17p is regulated b y the amount of the 2.6-kb RNA transcript present (Tanaka et al., 1992a). Our preliminary results indicated that forskolin-induced 17q20p-DP production is accompanied by a dramatic decrease in P450arom mRNA levels by granulosa cells isolated from postvitellogenic follicles. Also, a two- to threefold increase in P-450scc and 3pHSD mRNAs and a slight decrease in P-450~17mRNA were observed during forskolin-induced 17a,20/3-DP production (M. Tanaka and Y. Nagahama, unpublished). G. Surface Site of MIH Action

1. MIH RECEPTORS Previous in vitro studies in fish have shown that the mechanism of action involved in the steroid stimulation of GVBD has special characteristics not typical of the classical steroid mechanism of action. Steroid-induced oocyte maturation was prevented by puromycin, a translational inhibitor, but not by actinomycin D, a transcriptional inhibitor, indicating that the steroid mechanism of action in final maturation is nongenomic (Dettlaff and Skoblina, 1969; Jalabert, 1976; DeManno and Goetz, 1987). It was also reported that CAMP, activators of adenylyl cyclase such as forskolin and cholera toxin, and phosphodiesterase inhibitors such as IBMX, theophylline, and l-ethyl4-hydrazino-1H-pyrazolo[3,4-6]pyridine-5-carboxylic acid ethyl ester hydrochloride, which increase the intracellular levels of CAMP, inhibit steroid-stimulated maturation in vitro in several species (Goetz and Hennessy, 1984; Jalabert and Finet, 1986; DeManno and Goetz, 1987; Finet et al., 1988). These results suggest that CAMP is involved in the mechanism of action of MIH-induced oocyte maturation in vitro. We also demonstrated that 17a,20p-DP was ineffective in inducing oocyte maturation when microinjected into the immature oocytes of goldfish, but external application of the steroid is effective (Nagahama, 1987a). Taken together, these in v i t r o results suggest that the site of MIH action in inducing meiotic maturation in fish oocytes is at or near the oocyte surface. It is known from amphibian studies that the steroid apparently acts via a receptor on the oocyte surface plasma membrane and not through cytoplasmic or nuclear receptors (Maller, 1985). More direct evidence for the existence of MIH receptors in oocyte plasma membranes has been obtained by binding studies using la-

418

YOSHITAKA NAGAHAhlA ET AL.

beled MIH. We identified and characterized specific binding of "Hlabeled 17a,200-DPto plasma membranes prepared from the defolliculated oocytes of rainbow trout (Yoshikuni et al., 1993). Binding was rapid and reached equilibrium in 30 min. 17a,20P-DP strongly inhibited 'H-labeled 17q20P-DP binding in a competitive manner. Scatchard analysis revealed two different binding sties: a high-affinity binding site with Kd of 18 nM and a binding capacity (B,,, of 0.2 pmol/ mg protein, and a low-affinity binding site with a Kd of 0.5 p M and a BlllitX of 1 pmole/mg protein. Maneckjee et aZ. (1989) also reported, without full characterization, 17a,20P-DP binding activity in the zona radiata-oocyte membrane complex of rainbow trout and brook trout. We also demonstrated a specific 17a,20P-DP binding to flounder (hirame, Paralichthys olivaceus) oocyte cortices. The specific binding increased and reached an equilibrium within 1 hr. Scatchard analysis revealed a single binding site with a Kd of 63 nM and a B,,,,, of 25 fmol/cortex (M. Yoshikuni and Y. Nagahama, unpublished). The 17a,20P-DP binding was found in oocyte cortices from postvitellogenic oocytes (500-600 p m in diameter) and ovulated eggs, but neither midvitellogenic oocytes (425-500 pm) nor early vitellogenic oocytes (<425p m ) showed the binding. A specific binding site for 20p-S exists in plasma membranes from spotted sea trout ovaries (Patino and Thomas, 1990b). The association of20P-S to saturable binding sites was extremely fast; maximum binding was achieved after 5 min of incubation at 0°C. The binding site with a Kd of 1 nM and a B,,,, of 10~'3-10-'2 mol/g ovary in sea trout follicles was several orders of magnitude higher than the affinity of progesterone to its receptor on the amphibian oocyte (Kostellow et d., 1982; Sadler and Maller, 1982). In agreement with these observations, the apparent in vitro ED,, for 20p-S (approximately 0.1-1 nM) in the sea trout GVBD bioassay appears to be several orders ofmagnitude lower than the reported ED,, for progesterone (100 nM) in the Xenopus bioassay (Thomas and Trant, 1989; Sadler and Maller, 1982). Thomas and Patino (1991) further investigated the physiology of the 2Op-S receptor. 20p-S concentrations were significantly elevated in ovarian plasma membranes of spotted sea trout undergoing final oocyte maturation relative to the concentrations seen in ovaries of vitellogenic females. The threefold increase in receptor concentrations in fish collected during their natural spawning cycle is similar to that previously observed in sea trout undergoing final maturation following injections of luteinizing hormone-releasing hormone (LH-RH) analog (PatThese observations suggest that changes in ino and Thomas, 1990~). the concentration of MIH receptors in the ovaries are of physiological importance during natural oocyte maturation.

13.

REGULATION OF OOCYTE MATURATION

419

2. DEVELOPMENT OF 0ocu.m MATURATIONAL COMPETENCE Newer studies with several teleost species, such as kisu (Sillago japonica) (Kobayashi et al., 1988),dragonet (Repomucenusbeniteguri) (Zhu et al., 1989), Atlantic croaker (Patino and Thomas, 1990a), and hirame (M. Yoshikuni and Y. Nagahama, unpublished), have shown that there are two distinct stages of final oocyte maturation: an early MIH-insensitive phase in which oocytes can mature in vitro with gonadotropin but not with MIH alone, followed by a MIHsensitive stage. The MIH-insensitive oocytes respond to MIH if they are previously primed with gonadotropin. Thomas and Patino (1991) examined the relationship between 2Op-S receptor concentrations and the development of maturational competence using an ovarian incubation system. Treatment of spotted sea trout ovarian follicles with gonadotropin caused a twofold increase in 2Op-S receptor concentrations and, concomitantly, full-grown oocytes matured in response to 2Op-S. In contrast, 2Op-S did not induce an increase in receptor concentration or the development of oocyte maturational competence. These results suggest that a gonadotropin-induced increase in MIH receptor concentrations is essential for the development of oocyte maturational competence in sea trout and is not mediated by increases in the production of the MIH.

VI. MATURATION-PROMOTING FACTOR (MPF): TERTIARY MEDIATOR OF OOCYTE MATURATION A. Existence of MPF in Fish Eggs

The existence of MIH receptors at the surface ofoocytes suggests that there is a cytoplasmic factor that mediates the action of MIH on the induction of oocyte maturation. This factor, designated maturationpromoting fiactor (MPF), was first detected in unfertilized amphibian eggs, from which cytoplasm was withdrawn and microinjected into full-grown amphibian oocytes, causing the oocytes to mature into unfertilized eggs (Masui and Clark, 1979). It was therefore anticipated that a cytoplasmic factor similar to amphibian MPF is also produced in fish oocytes under the influence of 17cq20p-DP. MPF activity was extracted from goldfish oocytes matured by human chorionic gonadotropin treatment in vivo or naturally and was injected into immature Xenopus oocytes (Yamashita et al., 1992a).The

420

YOSHITAKA NAGAHAMA ET AL.

mature oocytes were crushed by ultracentrifugation at 100,OOOg for 1 hr in a buffer containing phosphatase inhibitors and EGTA. MPF activity was not detected when a buffer without EGTA and phosphatase inhibitors was used. After centrifugation, the five major layers obtained, from centripetal to centrifugal pole, were: supernatant, green layer, yolk, chorion, and cortical alveoli. The green layer was enriched with cellular organelles such as mitochondria and endoplasmic reticulum. MPF activity was detected in the supernatant and the green layer. However, microinjection of the green layer into goldfish immature oocytes caused cell lysis. A similar MPF activity was also detected in extracts from oocytes matured in vitro by 17a,20/3-DP, but was not present in those from either immature oocytes or activated eggs. MPF activity extracted from goldfish was also effective when injected into immature goldfish oocytes under conditions of inhibited protein synthesis. The injected oocytes matured much faster than oocytes induced to mature in vitro by incubation with 17a,2OP-DP. GVBD usually occurred at the center of the MPF-injected oocytes, because the movement of the GV to the animal pole did not take place. In contrast, GV migration always occurs in the oocytes matured naturally, in vivo by HCG, or in vitro by 17a,20/3-DP. During oocyte maturation induced in vitro by 17cu,2O,&DP, M P F activity (assessed by histone H1 kinase activity) oscillated according to the cell cycle of the oocytes. The activity increased before GVBD, peaked at the first meiotic metaphase, then decreased transiently when the first polar body was eliminated. It then increased again and remained at a high level until insemination (Fig. 8) (Yamashita et al.,

l992b). B. Generality of MPF Activity MPF transfers were carried out between oocytes of goldfish and a frog (Xenopus laevis) (Yamashita et al., l992a; M. Yamashita, unpublished). MPF from mature oocytes of either source induced maturation in oocytes of the other species. Furthermore, it was previously shown that goldfish MPF induced maturation of immature oocytes of the starfish Asterina pectinifera (Kishimoto, 1988).We showed that injection of extracts of pachytene microsporocytes of a higher plant (lily, Lilium longiflorum) induced GVBD and chromosome condensation in Xenopus oocytes (Yamaguchi et nl., 1991). These results suggest that MPF is similar among vertebrates and invertebrates. It has also been shown that MPF activity is present in mitotic somatic cells and that this factor is not species specific (Nagahama, 1987b; Kishimoto,

13.

42 1

REGULATION OF OOCYTE MATURATION I PE

Fig. 8. Oscillation of M P F activity during goldfish oocyte maturation induced by 17a,20P-dihydroxy-4-pregnen-3-one (17a,20P-DP).M P F activity was assessed by nieasuring histone H1 kinase activity using histone H 1 as a substrate. Schematic drawings on the figure indicate the maturational stage of the oocytes. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, first meiotic metaphase; 1PB, first polar body; MII, second meiotic metaphase.

1988). Thus, MPF is not merely a maturation-promoting factor, but may be a more general factor responsible for the initiation of nuclear membrane breakdown and subsequent cell division of both mitosis and meiosis. C. Purification and Characterization of Fish MPF and Histone H1 Kinase Because of its instability, MPF has been found to be very elusive. However, Lohka et al. (1988) were able to purify MPF as a 200-kDa complex containing 32- and 45-kDa proteins from mature oocytes of Xenopus and later it was also purified in starfish (Labbe et al., 198Ya,b). In Xenopus and starfish, MPF consists of two components (Lohka et

422

YOSHITAKA NAGAHAMA ET AL.

d., 1988; Gautier et al., 1988, 1990; Labbe et al., 1989a,b). A catalytic subunit of MPF is the homolog of the serine/threonine protein kinase, encoded by the fission yeast (Schizosaccharomyces pombe) cdc2+ gene (cdc2 kinase) (Simanis and Nurse, 1986). This kinase can use histone H 1 as an exogenous substrate (Brizuela et al., 1989). A regulatory subunit of MPF is cyclin (Gautier e t al., 1990), which was first discovered, independently of MPF, in the early embryos of marine invertebrates (Rosenthal et al., 1980; Evans et al., 1983). 1. PURIFICATION OF CARPMPF

Carp (Cyprinus carpio) is an excellent source of MPF, as MPF has been successfully extracted from carp unfertilized eggs and a large number of eggs (300-1000 ml/fish) arrested in a metaphase I1 can be easily obtained by an i n vivo injection of HCG into gravid females. MPF was purified from the 100,OOOg supernatant of crushed, naturally spawned carp oocytes using four chromatography columns (QSepharose Fast Flow, p13’”” -affinity Sepharose, Mono S, and Superose 12) (Yamashita et al., 1992a). MPF activity was assayed by injecting the sample into full-grown immature Xenopus oocytes in the presence of cycloheximide, a protein synthesis inhibitor. GVBD was detected by a white spot on the animal pole ofthe oocyte and confirmed by manually dissecting oocytes after boiling. H 1 kinase activity was also determined using histone H 1 and a synthetic peptide (SP-peptide, KKAAKSPKKAKK) as exogenous substrates (Yamashita et al., 1992b). MPF activity comigrated with histone H 1 kinase activity throughout purification. Analyses by SDS-PAGE showed that the most active fraction after Mono S contained four proteins, with molecular masses of 33-, 34-, 46-, and 48-kDa (Fig. 9). When the active fractions after Mono S were applied to Superose 12, MPF and kinase activity coeluted as a single peak with an apparent molecular mass of about 100-kDa, indicating that these four proteins form complex(es) of about 100-kDa in their native form. The 34-, 46-, and 48-kDa proteins corresponded well to MPF and H1 kinase activities, but the peak of 33-kDa protein seemed to be different from the peak of MPF and H 1 kinase activities. The 46- and 48-kDa proteins were labeled when y-32P-labeledATP was applied in the fractions, indicating that these proteins are one of the endogenous substrates for the kinase. The final preparation of MPF was purified over 1000-fold with a recovery ofabout 1%.The final preparation was also purified 5000-fold with a recovery of 5%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP-peptide was used.

13.

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REGULATION OF OOCYTE MATURATION

kD

cdc2 kinase

Cyclin 6

Serine 14.4 > Fig. 9. (Left) Silver-stained SDS-PAGE of the fraction corresponding to the Mono S peak of carp MPF activity. Arrowheads on the right of the gel show 33-, 34-, 46-, and 48-kDa proteins. (Right) Diagram of two subunits of fish MPF. P, phosphorylation.

2. CHARACTERIZATION OF CARPMPF Because the 34-kDa protein and the 46- and 48-kDa proteins found in purified carp MPF appeared to correspond to cdc2 kinase and cyclin B of Xenopus MPF, respectively, we then attempted to isolate cDNA clones encoding fish homologs of cdc2 kinase and cyclin B from goldfish, a species closely related to carp. u. cdc2 Kinase. A full-length cdc2 kinase cDNA was isolated from a AgtlO library constructed from full-grown immature goldfish oocytes. The isolated clone had an insert of 1284 bp containing a poly (A) tail with an open reading frame encoding 302 amino acids. Northern blot analysis showed a hybridization signal at the 1.3-kb position, indicating that the clone is nearly full length. The PSTAIR sequence motif (EGVPSTAIREISLLKE)is a hallmark of cdc2, cdk2, and cdk3, but the isolated c D N A clone encodes a PSTAVR sequence in which isoleucine changes to valine. To examine whether the PSTAVR sequence is a genuine sequence in goldfish cdc2 or merely an artifact obtained during the cloning procedures, we also used another cDNA library in AZAPII vector, which was independently constructed from mRNA different from that used to construct +

424

YOSHITAKA NAGAHAMA ET AL.

the hgtl0 library. Nevertheless, we obtained the same cDNA clone with the PSTAVR sequence. Furthermore, a 2.2-kbp cdc2 genomic DNA fragment isolated from goldfish blood cells also had the PSTAVR sequence. Therefore, the PSTAVR sequence is not an artifact but an authentic sequence present in goldfish cdc2. The predicted molecular weight of the protein encoded b y this gene is 34,499. This clone has higher homology with cdc2 (85% for Xenopus, 85% for human, and 84% for mouse) than cdk2 (67% for goldfish, 66% for Xenopus, and 67% for human) at the amino acid sequence level.

b. Cyclins. Two species of cyclin 13 clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. One clone encoded 397 amino acids covering the entire coding region. The predicted molecular weight ofthe protein encoded by this gene is 44,763. The goldfish cyclin B contained "cyclin box," a hallmark of cyclins (Pines, 1991), and resembled cyclin B more closely than cyclin A. At the amino acid level, the goldfish cyclin B cDNA showed homology of 66% for Xenopus B1 and 50% for Xenopus B2, whereas it showed homology of 33% for Xenopus A. C . Antibodies. Anti-goldfish cdc2 kinase monoclonal antibodies were raised against the peptide CPYFDDLDKSTLPASNLKI, which corresponds to the C-terminal sequence of goldfish cdc2 cDNA with an additional cysteine in the N terminus (Kajiura et nl., 1993). Anticyclin B monoclonal antibodies were raised against E. coli-produced fiill-length goldfish cyclin B (Katsu et al., 1993). We also raised a monoclonal antibody against the niost conserved amino acid sequence, the PSTAIR sequence of cdc2 kinase (Yamashita et nl., 1991). This antibody recognizes 31- to 35-kDa proteins b y immunoblotting in all species examined so far. The proteins recognized by the anti-PSTAIR antibody are probably either cdc2 kinase itself or proteins highly homologous to cdc2 kinase in the given species, because, in all species studied to date, they are all precipitated with p13""", the fission yeast suc1+ gene product, which binds to cdc2 kinase with high specificity.

d . M P F Characterixntion. Various monoclonal antibodies raised against the components of goldfish MPF were used to characterize the purified carp MPF, which contained 33-, 34-, 46- and 48-kDa proteins. Both the 33- and 34-kDa proteins were recognized by anti-

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PSTAIR antibody, indicating that they are the cyclin-dependent kinases. A monoclonal antibody against the C-terminal region of goldfish cdc2 kinase reacted with the 34-kDa but not the 33-kDa protein. The latter was recognized with the anti-cdk2 C-terminal antibody (Hirai et al., 1992b). The 46- and 48-kDa proteins were recognized by the anti-full-length goldfish cyclin B antibody but not by an anti-cyclin A antibody (Yamashita et al., 1992a; Katsu et al., 1993). These findings indicate that carp MPF is a complex of cdc2 kinase (34 kDa) and cyclin B (46 and 48 kDa) (Fig. 9). 3. CHANGES IN C D C ~ KINASEAND CYCLINB PROTEIN LEVELSDURING OOCYTEMATURATION In immature oocytes of Xenopus and starfish, cdc2 kinase forms a complex with cyclin B as pre-MPF. In contrast, we found that there is no detectable cyclin B in immature goldfish oocytes (Fig. 10). This finding strongly suggests that cdc2 kinase in immature goldfish oocytes is monomeric. To confirm this, we examined the consecutive fractionation of immature and mature oocyte extracts eluted from a gel filtration column by immunoblotting with anti-cdc2 C-terminal and anti-cyclin B antibodies. When immature oocyte extracts were applied on the gel filtration column, cdc2 kinase was eluted as a single peak at the

0

A

0

0

0 65.5 80 88.6 100100

-9

0

, ~ ~ -

-KUd

* 35 * 34

2 4 6 8 Time after hormone treatment (hr)

Fig. 10. Changes in cdc2 kinase (A and D) and cyclin B (B and C) protein levels during goldfish oocyte maturation induced by 17~~,2OP-dihyroxy-4-pregnen-3-one. Anticdc2 (A and D) and anti-cyclin B (B and c)immunoblots of p13""' (A and 8)and anticyclin B (C and D) precipitates. GVBD, germinal vesicle breakdown.

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YOSHITAKA NAGAHAMA ET AL.

monomeric position around 35-kDa. However, cdc2 kinase in mature oocyte extracts was eluted as two peaks at around 100- and 35-kDa. The 34-kDa cdc2 kinase was found only in the first peak at 100 kDa, where cyclin B was also detected. The vast majority of the 35-kDa cdc2 kinase in mature oocytes migrated at the monomeric position. These results demonstrate that most, if not all, cdc2 kinase (inactive 35-kDa form) in immature oocytes is monomeric, but when oocytes mature, a part of cdc2 kinase forms complexes with cyclin B and is activated (active 34-kDa form). It is also notable that a minor fraction ofthe 35-kDa cdc2 kinase was also detectable at 100-kDa. This means that not all cdc2 kinase that binds to cyclin B is activated. Taken together, it is most likely that inactive 35-kDa cdc2 kiiiase binds to cyclin B first, then it is activated, which is associated with an electrophoretic mobility shift from 35- to 34-kDa. To further investigate cdc2 kinase and cyclin B protein levels during oocyte maturation, oocyte extracts at various times after the addition of 17a,20/3-DP were precipitated with either p13””“ or anti-cyclin B antibody and immunoblotted with anti-cdc2 kinase and anti-cyclin B antibody. As described earlier, immature oocytes contained the 35-kDa inactive cdc2 kinase but no cyclin B, and mature oocytes contained both the 35-kDa inactive and the 34-kDa active cdc2 kinases arid cyclin B. The appearance of the 34-kDa active cdc2 kiiiase coincided with the appearance of cyclin B just before GVBD. Anti-cyclin B immunoblots of the p13”““ precipitates and anti-cdc2 kinase imniunoblots of anti-cyclin €3 immunoprecipitates showed that the binding of cdc2 kinase and cyclin B coincided with the appearance of cyclin B and the 34-kDa active cdc2 kinase. The cyclin B that appeared during oocyte maturation was marked with 35S-labeled methionine (Hirai et al., l992a), demonstrating de nouo synthesis during oocyte maturation. On the other hand, anti-cyclin B immunoprecipitates from mature oocyte extracts sometimes contained the 35-kDa inactive cdc2 kinase (Hirai et al., 19924, and the 35-kDa cdc2 kinase, as well as the 34-kDa form, can bind to cyclin €3 in a cell-free system (M. Yamashita et a l . , unpublished). Therefore, it is most likely that the 35-kDa inactive cdc2 kinase binds to de n o w synthesized cyclin B at first, then is rapidly converted into the 34-kDa active form.

4. INDUCTION OF MPE’ ACTIVATION BY SYNTHETIC CYCLIN B PHOTEIN The preceding results indicate that the appearance of cyclin B is required and is sufficient for inducing oocyte maturation in goldfish. To confirm that the appearance of cyclin B is sufficient for inducing

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KEGULATION OF OOCYTE MATURATION

427

oocyte maturation, E . coli-produced fdl-length goldfish cyclin B protein was injected into immature oocytes. Even under conditions of inhibited protein synthesis, injected cyclin B induced oocyte maturation within 1 hr after injection in a dose-dependent manner. Injection of 1 ng cyclin B fully induced GVBD in the recipient oocytes. The concentration of cyclin B within the injected oocyte was estimated to be 2 pg/ml. This is about equal to the cyclin B concentration in mature oocytes. MPF activation was also induced when cyclin B protein was introduced into immature oocyte extracts (which contained the 35-kDa inactive cdc2 kinase but no cyclin B), occurring in an almost “all or nothing” manner. The threshold concentration ofcyclin B for inducing the activation was around 2 pg/ml, equivalent to that for inducing oocyte maturation by injection. These results demonstrated that the presence of’ 2 pglml cyclin B, corresponding to the concentration in mature oocytes, is sufficient for inducing oocyte maturation. Introduction of E. coli-produced cyclin B into immature oocyte extracts also induced the activation of cdc2 kinase concurrent with the change in apparent molecular weight from 35,000 to 34,000, as found in oocytes matured with 17a,20P-DP. Phosphoamino acid analysis showed that threonine phosphorylation of the 34-kDa cdc.2 kinase and serine phosphorylation of cyclin B were associated with the activation. The same phosphorylation was also found in oocytes matured by 17a,20P-DP. Cyclin B-induced cdc2 activation was not observed when threonine phosphorylation of cdc2 kinase and serine phosphorylation of cyclin B were inhibited by protein kinase inhibitors, although the binding of the 35-kDa cdc2 kinase to cyclin B occurred even i n the presence of the inhibitors. On the other hand, cdc2 kinase was activated by mutant cyclins that underwent no serine phosphorylation during the activation. These results indicate that the threonine phosphorylation of cdc2 kinase, but not serine phosphorylation of cyclin B, is required for cdc2 kinase activation (M. Yamashita et al., unpublished). In Xenopus oocytes, dephosphorylation of threonine (Thr 14) and tyrosine (Tyrl5) is required for cdc2 kinase activation (Maller, 1991). However, it is doubtful that tyrosine (Tyrl5) dephosphorylation of cdc2 kinase is a prerequisite for its activation during goldfish oocyte maturation because the activation was not inhibited by vanadate, a protein phosphatase inhibitor. Furthermore,anti-phosphotyrosine antibodies reacted with neither the 35-kDa cdc.2 kinase nor the 34-kDa form bound to cyclin B. Therefore, the 35-kDa cdc2 kinase found in immature oocytes may already be dephosphorylated on tyrosine and

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YOSHITAKA NAGAHAMA ET AL.

the threonine phosphorylation may be required only for the activation. Taken together, it strongly suggests that 17a,2OP-DP induces oocytes to synthesize cyclin €3, which in turn activates preexisting 35-kDa cdc2 kinase through its threonine (probably Thrl61) phosphorylation, producing the 34-kDa active cdc2 kinase (Fig. 11).These mechanisms of MPF activation in fish apparently differ from those in Xenopus and starfish, in which cyclin B is present in immature oocytes and forms a complex with cdc2 kinase (pre-MPF).

VII. CONCLUSIONS This chapter briefly reviews the current status of investigations on the regulation of oocyte maturation in teleosts (Fig. 12). Pituitary gonadotropin is of primary importance in triggering meiotic maturation in teleost oocytes. However, the maturational action of gonadotropin is not direct but is mediated by the follicular production of steroidal MIH. Two progesterogens (17a,20@-DPand 2Op-S) have been identified as the MIH in several teleost species. Production of 17a,20/3-DP occurs via the interaction of two distinct cell layers, the thecal and granulosa cell layers (two-cell type model). The first step ofthe stimulating effect of gonadotropin in both layers is the receptor-G-proteinGonadotropin I

17a,2Op-Dihydroxy-4-pregnen-3-one

v

/

~

Immature oocyte

I I

?

I I

Mature oocyte (M-phase) 161 @ t

cdc2 kinase

de novo synthesis

Inactive MPF

Active MPF

b

Fig. 11. Current model of the formation and activation of MPF during fish oocytc maturation. P, phosphorylation; S, serine; T, threonine; Y, tyrosine.

13.

429

KEGULATION OF OOCYTE MATURATION ( r Pituitary

Gonadotropin Ad'

i7a-Hydroxyprogesterone 1 0 ,

b

t' \ -

Thecal cell

4-pregnen-d-one

Oocyte maturation

Fig. 12. Hormonal regulation of oocyte maturation in teleosts. Three major mediators, gonadotropin, maturation-inducing hormone, and maturation-promoting fkctor, are involved.

mediated activation of adenylyl cyclase and formation of CAMP. Our findings indicate that the major stimulating action of gonadotropin on 17au,20P-DPbiosynthesis is due to the stimulation of 17a-P production by the thecal layer and the selective induction ofthe de novo synthesis of BOP-HSD in the granulosa layer. A distinct steroidogenic shift from estradiol-17p to 17a,20p-D€' occurring in salmonid ovarian follicles immediately prior to oocyte maturation is a prerequisite for the growing oocytes to enter the maturation stage. The cDNAs for the most of' the steroidogenic enzymes responsible for estradiol-170 and 17a,20PDP biosynthesis have been cloned from rainbow trout ovaries. This opens an entirely new area of research on steroidogenesis in fish ovaries, that is, investigations on the levels of steroidogenic enzymes and their transcripts as well as the regulation of the genes encoding steroidogenic enzymes. To this end, it will be particularly important to establish fish cell lines that are representative of differentiated

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steroidogenic ovarian cells. In addition to these hormonal regulators, a number of other substances are produced within the ovary that may act locally to regulate oocyte maturation. Molecular biological technologies should be applied to identify these substances. Unlike other steroid hormones, MIH acts at the surface of the oocyte. Further studies on the molecular characterization of the MIH receptors and their associated signaling pathways within the oocytes should lead to the discovery of a new mechanism for steroid hormone action. The early steps following MIH action involve the formation of the major cytoplasmic mediator of MIH, MPF. Fish MPF, like that of Xenopus and starfish, consists of two components, cdc2 kinase and cyclin B. Nevertheless, the mechanism of MIH-induced MPF activation in fish oocytes differs from that in Xenopus and starfish in that the appearance of cyclin B protein is a crucial step for 17a,20P-DPinduced oocyte maturation in fish. Future work should focus on determining the mechanisms of de novo synthesis of cyclin B and phosphorylation of threonine (probably 161), both of which are induced by 17cq20P-DP. The continued application of molecular biology should hasten our understanding of regulation of meiotic maturation of fish oocytes.

ACKNOWLEDGMENTS The authors wish to thank the many wonderful colleagues who have contributed to the work described herein, especially G. Young, M. Nakamura, H. Kagawa, S. Adachi, N. Sakai, T. Hirai, M. Matsuyama, M . Takahashi, H. Kajiura, S. Fukada, and Y. Katsu. A special thanks to Mrs. N. Nagahama and K. Noda for their assistance in the preparation of this manuscript. The research reported here was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture in Japan (02102010 to Y.N.).

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Kinie, D. E., Scott, A. P., and Canario, A. V. M. (1992). In uitro biosynthesis of steroids, by ovaries of the including 11-deoxycortisol and 5a-pregnane-3P,7a,17,20p-tetrol, goldfish Carassius auratus during the stage of oocyte final maturation. Gen. Comp. Endocrinol. 87, 375-384. Kishimoto, T. (1988). Regulation of metaphase by a maturation-promoting factor. Deti. Growth Differ. 30, 105-115. Kobayashi, M., Aida, K., and Hanyu, I. (1987a). Hormone changes during ovulation and effects of steroid hormones on plasma gonadotropin levels and ovulation in goldfish. Gen. C o m p . Endocrinol. 67, 24-32. Kobayashi, M., Aida, K., Sakai, H., Kaneko, T., Asahina, K., Hanyu, I., and Ishii, S. (1987b). Radioimmunoassay for salmon gonadotropin. Nippon Suisan Gakkaishi 53, 995- 1003. Kobayashi, M., Aida, K., Furukawa, K., Law, Y. K., Moriwaki, T., and Hanyu, I. (1988). Development of sensitivity to maturation-inducing steroids in the oocytes of the daily spawning teleost, the kisu Sillago japonicu. Gen. C o m p . Endocrinol. 72, 264-271. Kostellow, A. B., Weinstein, S. P., and Morrill, G. A. (1982).Specific binding ofprogesterone to the cell surface and its role in the meiotic divisions in Rana oocytes. Biochem. Biophys. Acta 720, 356-363. Labbe, J. C., Picard, A,, Peaucellier, G., Cavadore, J. C., Nurse, P., and Doree, hl. (1989a). Purification of MPF from starfish: Identification as the H1 histone kinase p34rdc2 and a possible mechanism for its periodic activation. Cell (Cambridge,Mass.) 57,253-263. Labbe, J.-C., Capony, J.-P., Caput, D., Cavadore, J.-C., Derancourt, J., Kaghad, M., Lelias, L.-M., Picard, M., and Doree, M. (1989b). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO 1.8, 3053-3058. Lambert, J. G. D., and van Bohemen, Ch. G. (1979). Steroidogenesis in the ovary of the rainbow trout, Salmo gairdneri, during the reproductive cycle. Proc. Indiun Natl. Acad. B 45,414-420. Lambert, J. G. D., and Van Den Hurk, R. (1982). Steroidogenesis in the ovaries of the African catfish, Clarias Zuzeru, before and after an HCG induced ovulation. In “Proceedings of the International Symposium on Reproductive Physiology of Fish” (C. J. J. Richter and H. J. Th. Goos, eds.), pp. 99-102. Pudoc, Wageningen. The Pietherlands. Lessnian, C . A. (1991). Metabolism of progestogens during in tiitro meiotic maturation of follicle-enclosed oocytes of the goldfish (Carussius ouratus).1.Exp. Zool. 259, 59-68. Lin, Y.-W. P., Petrino, T. R., and Wallace, R. A. (1989). Metabolic and developmental aspects of steroidogenesis in teleost ovaries. C o m p . Endocrinol. (Life S c i . A d o . ) 8, 33-45. Lohka, M. L., Hayes, M. K., and Maller, J. L. (1988).Purification ofmaturation-promoting factor, an intracellular regulator of early mitotic events. Proc. Natl. Acad. Sci. U.S.A. 85,3009-3013. Maller, J. L. (1985).Regulation ofamphibian oocyte maturation. CellDiffeer. 16,211-221. Xlaller, J. L. (1991). Mitotic control. Curr. Opin. Cell B i d . 3, 269-275. hlaneckjee, A., Weisbert, M., and Idler, D. R. (1989).The presence of 17a,20p-dihydroxy4-pregnen-3-one receptor activity in the ovary of the brook trout, Saloelirius funtinalis, during terminal stages of oocyte maturation. Fish Physiol. Biochem. 6,19-38. Masui, Y., and Clarke, H. J. (1979). Oocyte maturation. Int. Rev. Cytol. 57, 185282.

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AUTHOR INDEX

Numbers in italics refer to pages on which the complete references are listed A

Aakvag, A., 349,363 Abad, M. E., 261,262 Abdel-Meguid, S. S., 183, 203 Abe, K. I., 185, 206, 376, 389 Acher, R., 29,51, 102-103,128,130-131 Ackland, J. F., 12, 36, 51, 66 Adachi, J., 115,128-129 Adachi, R., 213,223 Adachi, S., 401-402, 404-405, 408, 410, 413-414, 416-417,431,433, 435-439 Adams, C . C., 336,357 Adan, R.A.H., 112, 129 Adashi, E. Y., 202, 205 Adelman, J. P., 30-32, 35-36, 44-45, 51, 53-54, 57, 64 Agellon, L. B., 164-165, 167, 174, 180, 183, 189, 192-193,203-204 Agoston, D. V., 256, 263 Aguilera, G., 77, 94 Ahmad, N., 294, 302 Aida, K., 31-32,35,37,39-40,45-47, 51-52, 58, 62, 65, 117, 129, 290, 303, 399-400,419,434,436-437,439 Aimar, C., 160, 176 Akam, M., 314, 325 Akasaka, A., 183,206 Alarcon, C., 247, 269 Albert, V. R., 165, 168-170, 172-173, 175, 190, 206,314-319,327-328 Alestrom, P., 31-32, 35, 44-45, 58 Ali, S., 335, 347, 357, 369-371, 386-387, 390

Aliapoulios, M. A., 274, 303 Alkema, M. J., 318, 329 Allan, G. F., 335-336, 346, 348, 357, 361,364 Allendorf, F. W., 19, 51, 216, 222 Almendras, J. M. E., 376 Altmann, M., 346, 365 Amano, M., 37, 39-40,47, 51,58,62 Amino, N., 314, 329 Amoss, M., 30, 53 Anast, C. S., 290, 293, 303 Andersen, B., 143, 154 Andersen, O., 31-32, 35, 44-45, 58, 376, 386 Anderson, E., 31, 45, 47, 52 Andrews, P. C., 230, 235, 238, 243, 245, 251,253-256,259-261,262-264, 268 Andrews, W. V., 152,155 Andria, G., 46, 52 Anglade, I., 40, 57 Anthony, E.L.P., 33, 38, 58 Anzivino, M. J., 148, 157 Apathy, J . M., 214, 222 Appelbaum, S. W., 71, 97 Arai, Y., 46, 61 Archer, T. K., 339, 358 Arden, K. C., 370, 386 Argetsinger, L. S., 371, 386 Argos, P., 337,360-361 Arias, P. E., 312, 326 Arimura, A,, 6-7, 9-12, 14-15, 17-20, 24, 26, 29-30, 50, 51, 51, 52, 57-58, 60-62,65-66 Arnold, R., 246, 265

44 1

AUTHOR INDEX

442 Arnold-Reed, D. E., 71, 94 Arnt, L.R.G., 82,94 Aronheim, A., 229,262 Asa, S. L., 315, 325 Asahina, K., 400, 434 Asai, M., 78-80, 91, 95 Ash, J., 257, 263 Ashihara, M., 31-32, 45, 52, 117, 129 Ashmead, B. J., 31, 38, 59 Askensten, U., 257, 264 Assa-Munt, N., 318, 325 Assem, H., 383, 386 Atkin, N. B., 92, 97, 108, 114, 117, 131 Atsma, W., 283, 306 Aubert, M., 372,388 Audousset-Puech, M.-P., 241, 243, 266 Auerbach, B. A,, 314,327 Auperin, B., 367, 373, 376-378, 381-383, 385,386,391 Auricchio, F., 344, 362 Aurora, R., 318, 325 Avella, M., 368, 390 Azad, N., 30, 52 B

Baba, Y., 5, 30, 60, 63 Bagchi, M. K., 335, 358 Bagnato, A., 17-18, 50-51, 52, 61 Baird, A., 10, 21-22, 52, 58, 70, 95, 97 Baker, B. I., 160, 162-163, 170, 174,176 Baker, J., 221, 223 Balasubramaniam, A., 260-261, 262,264 Baldino, F., 94 Ball, A. K., 39, 45, 65 Ball, J. N., 62, 160, 174-175, 311, 327 Ballabio, A., 46, 52, 55 Ballard, F. J., 214, 224 Balmaceda, C.-G., 316,329 Balment, R. J., 71, 94, 122-123, 125, 129,131 Baloche, S., 49, 55 Baltimore, D., 169, 175 Bancroft, C., 148, 157, 170, 175, 315, 319,328 Banejee, M. R., 372,390 Baniahmad, A., 148,154,353,358 Baniahmad, C., 336,362 Banno, K., 399, 436

Banville, D., 371, 390 Barannikova, I., 47 Bardoni, B., 46, 52, 55 Barkley, R. J., 372, 386 Barlow, D. J., 259, 265 Barnes, P.R.J., 106, 129 Barnhart, K. M., 143, 149, 155 Barry, T. P., 368, 386 Barta, A,, 164-165, 175, 180, 189, 192-193,204,208 Barzilai, D., 372, 386 Bashirelahi, N., 344, 361 Baskin, D. G., 261, 271 Bass, A. H., 39, 56 Bataille, D., 241, 243, 262, 266-267 Bates, 0. J., 252, 255, 268 Batten, T.F.C., 40, 52, 104-105, 129-130, 160,174,311,325 Battenberg, E.L.F., 94 Battey, J., 108-109, 114,131 Bauer, G. E., 254,267 Baulieu, E. E., 334-335,358-359 Bauman, D. E., 180,207 Bautista, C. M., 214, 222 Baxter, J. D., 164-165, 167, 175-176, 180, 183, 187, 189, 192-193, 204, 207-208,320,329,349, 358 Baylink, D. J., 214, 222 Bazan, J. F., 370, 386 Beach, D., 422, 430 Beato, M., 337, 341, 358, 364 Beauvillain, J. C., 10, 52 Beck, C. A., 346, 365 Beckerman, T., 344,361 Beekman, J. M., 336,361 Begeot, M., 312,325 Begg, G. S., 344, 363 Behringer, R. R., 312, 326 Beischlag, T. V., 112, 129 Belair, L., 370, 372, 388-390 Belayew, A,, 164-165,177, 189, 192,208 Belkhole, S., 185, 208 Bell, G. I., 19, 52, 164-165, 177, 189, 192, 197,204,208,233, 235,262 Bellard, M., 152-153, 155, 337, 354, 360-361 Bellussi, G., 369, 386 Benedetti, A., 241,263 Benfey, B. G., 68,98 Benfey, T. J., 74, 99

AUTHOR INDEX

Ben-Jonathan, N., SO, 63 Bennett, H.P.J., 73, 99 Benoit, R., 10, 52, 70, 95 Ben-Or, S., 337, 358 Benrubi, O., 183,207 Bensch, P. A., 247,266 Benson, A., 291,295,304 Bentle, L. A,, 183, 203 Bentley, P. J., 180, 204 Ber, R., 189, 192, 194,204 Berelowitz, M., 52 Berg, J . M., 339,358 Berg, L.-V.A., 314, 317,319,328 Bergeron, J.J.M., 369, 386 Bergman, H. L., 394,432 Berkenstam, A., 353,360 Berks, B. C., 238, 262 Bern, H., 103, 130 Bern, H. A., 9, 59, 61, 77, 94, 180, 183, 185, 187,204-205,208-209,214, 220,222-224,290,303,367-368, 372-378,382,385,386-387, 389-391 Berry, M., 332, 335, 359, 361 Berry, S. A., 10, 17, SO, 52, 63, 65 Beth Martin, M., 350, 363 Bewley, T. A,, 180, 183, 185,204-205 Bhatt, R. S., 5-6, 14, 56 Bick, D., 46, 52, 64 Biggs, S., 247, 266 Billard, R., 39-40, 53, 150, 154-155, 400, 403,431 Binart, N., 335,358 Birnberg, N. C., 190,205 Bjenning, C., 261, 264 Bj@rnholm,B., 261, 264 Bjornsson, B. Th., 282, 301, 305, 338, 350,362,368,389-390 Blache, P., 243, 262, 266 Blackburn, M. B., 71, 94 Blackmore, P. F., 247, 263 Blackwell, R., 30, 53 Blahser, S., 40-41, 56 Blanar, M. A., 229,265 Blobel, G., 229, 270 Bloch, B., 7, 10, 52-53 Blomqvist, A. G., 259-260, 263,266 Bloom, E., 349,358 Bloom, F., 10, 52 Bloom, F. E., 94

443 Bloom, S. R., 18, 63 Bliim, V., 40, 63 Blume, R., 240,270 Blundell, T. L., 181,204,259, 265 Bocquel, M. T., 335,358 Bodner, M., 14, 52, 165, 169, 175, 190, 204, 314,316,326 Bodwar, W., 71, 94 Boelens, R., 318, 326, 339, 341, 360 Boeuf, G., 368,390 Bogdanove, E. M., 149,155 Bogerd, J., 31-32, 35,45,47,52-53, 64 Bohlen, P., 5-7, 10, 14, 21-22, 52-53, 55-56,59,70,95,97 Bohler, H.C.L., Jr., 59 Bohnet, H. G., 369,387 Bokar, J. A., 143, 154 Bolduc, T. C., 34,53 Bolton, J. P., 220, 222, 367-368, 388 Bolton, N. T., 183, 207 Bond, C. T., 30-32, 36, 44-45,53 Bond, J. P., 348, 358 Bond, M., 29, 63 Boockfor, F. R., 311-312,326-327 Borg, B., 40, 53 Bornert, J. M., 335,337, 347,357, 360-361 Boronat, A,, 5 , 7, 17, 56 Borrelli, H. R., 312, 326 Bosma, P. T., 47, 63 Bouamoud, N., 154 Bourbonnais, Y., 257, 263 Bourgeois, S., 349, 358 Boutin, J., 370, 373, 388 Boutin, J. M., 370-371, 386-387,390 Bowman, P. D., 372,390 Bowsher, R. R., 214, 222 Boyd, A. E., 246, 271 Boyd, A. E., 111, 247, 264 Bradford, C. S., 77, 98 Bradley, A., 314, 328 Bradshaw, R. A., 252,255, 268 Brady, H., 169, 175 Brar, A., 16, 22, 55 Brasseur, J. G., 291, 303 Brazeau, P., 5-7, 10, 14, 21-22, 52-53, 55-56,58 Brazeau, P. E., 5, 55 Breathnach, R., 29, 53 Breimer, L. H., 302, 303

AUTHOR INDEX

444 Bresnick, E. H., 335,358 Bresson, J . L., 10, 53 Breton, B., 39-40, 53, 57, 62, 150, 154, 394,400, 403,431,433 Briehl, M. M., 335, 358 Brighenti, L., 245, 247, 263,268 Brinn, J. E., 240, 265 Brinster, R. L., 55, 312, 326 Brizuela, L., 422, 430 Brockmann, G., 59 Brockway, M. J., 344,363 Broide, R. S., 170, 172, 176, 311, 314-315,320,329 Brookman, J. J., 300, 303 Brooks, C. L., 376,387 Brou, C., 335336,358, 364 Brown, C. J., 46, 55 Brown, E. M., 293,303 Brown, J. C., 183,208 Brown, J. H., 77, 94 Brown, M. R., 314, 318-319, 328 Brown, P., 145, 149, 154 Brown-Luedi, M., 336, 365 Brownstein, M., 31, 35, 64 Brownstein, M. J., 31, 34-36, 64 Bruhaker, P. L., 240, 264 Bruce, B. D., 5-6, 14, 60 Bruhn, T. O., 10, 12, 51, 53 Briilet, P., 314, 328 Buckland, P. H., 140, 154 Buckley, A. R., 369-370, 387 Biickley, D. I., 180, 206 Buczko, E., 370, 391 Biignon, C., 10, 53 Bukovskaya, O., 47 Bulet, P., 419-420, 422, 425, 439 Bullock, B., 216, 223 Bullock, T. H., 38, 55 Buonomo, F. C., 202, 206 Burbach, J.P.H., 112, 129 Burd, G., 39, .56 Burgess, S. K., 202, 207 Burgus, R., 30, 53 Burnside, J., 140, 147, 154 Burrin, J. M., 9, 18, 50, 56 Burton, M . P., 376, 389 Biirzawa-Gerard, E., 398, 430 Burzio, L. O., 69, 77-78, 90-91, 97 Butcher, M., 30, 53

Butkus, A., 276, 278,281-283, 303, 305-306 Butler, D. G., 274-275, 282,303-304, 306 Bygrave, F. L., 241,263 C

Cairns, C., 341, 358 Cairns, W., 341, 358 Calame, K., 29, 63 Calder, D. R., 401, 432 Caldwell, J. D., 96 Calvin, J. L., 34, 53 Cambell, G. S., 371, 386 CambrC, M., 105, 130, 313, 326 CambrC, M. L., 40, 52, 104, 129 Camerino, G., 46, 52, 55 Campbell, G. A., 372,389 Campbell, L., 46, 52 Campbell, R. M., 18, 22, 26, 53 Camper, S. A., 143,156, 183, 189,209 Campos, R., 240,264 Canario, A.V.M., 313, 328, 401-404, 430-431,433,436 Cao, Q.-P., 181,204,214-216, 219-220, 222,227-228, 263 Cao, X., 341, 358 Capony, J.-P., 421-422, 434 Caput, D., 421-422,434 Cardinaux, J. R., 337, 349, 365 Carey, M., 347, 358 Carles-Bonnet, C., 243, 266 Carlson, S. S., 93, 100 Carlstedt-Duke, J., 335, 339, 341, 359-360,364-365 Carlstrom, A., 232, 266 Carne, A,, 238, 256, 262, 264 Carney, D., 108-109, 114,131 Carney, R. L., 71, 96 Carpenter, S. J., 289, 303 Can, F. E., 147, 154 Carrick, S., 122-123, 125, 131 Carrillo, M., 40, 57 Carroll, S. B., 317, 329 Carrozzo, R., 46, 52, 55 Carter, C., 29, 63 Castano, J. P., 5, 18, 59 Castilla, C., 247, 269

AUTHOR INDEX

Castoria, G., 344, 362 Castrillo, J.-L., 165, 169-170, 173, 175, 177, 190,204,314,316,318-319, 326,329 Catanzaro, D. F., 316, 327 Catelli, M. G., 335, 358 Cater-Su, C., 371, 386 Cato, A. C., 150, 156 Cato, A.C.B., 153, 157 Catt, K. J., 17-18, 49-51, 52, 59, 61, 77, 94,242,270 Cavadore, J. C., 421-422,434 Cavari, B., 214, 223 Cavenee, W. K., 370,386 Cerelli, G., 338, 360 Cerelli, G. M., 5-6, 14, 60 Chadowick, A,, 183, 207 Chalepakis, A., 341, 358 Chalepakis, G., 337, 364 Chamberland, M., 337,359 Chambon, P., 25, 29, 53, 57, 152-153, 155,332,334-341,344,346-347, 349-350, 353-354,357-365 Chambraud, B., 335,359 Chan, A.T.C., 274, 306 Chan, K.-M., 179, 214, 222 Chan, S. J., 181, 192,204,213,214-221, 222-224,227-228,230,257,263, 2 70 Chance, W. T., 261,262 Chang, G. D., 139,156 Chang, J. P., 7, 9, 12, 18, 50, 59, 65, 141, 153, 154,157 Chang, J.-Y., 253-254,268 Chang, K. J., 24, 57 Chang, W.-C., 180, 183, 189, 197,204 Chang, Y. S., 139-141, 154-156, 183, 204 Chao, S. C., 183, 197,204 Charlesworth, M. C., 138, 154 Chartrel, N., 7, 10, 18, 54 Chasin, L. A., 170, 175, 319, 328 Chatterjee, V. K. K., 337,359 Chauchereau, A,, 357, 359 Chaudhary, S., 336,358 Chauvet, J., 102-103,128,130-131 Chauvet, M. T., 102-103,128,130-131 Chi., C., 369,386 Chen, C., 293,303 Chen, H.-T., 180, 189, 192,204

Chen, L.H.J., 247, 266 Chen, R., 165, 169-170, 172-173, 175, 314, 316, 320, 327-328 Chen, S., 414,416-417,437 Chen, T. T., 31-32,56, 164-165, 167, 174, 179, 180-181, 183, 187, 189, 192-193, 197, 199-200, 202, 203-205,208-209,214-215,218, 220,222,224 Cheng, C., 183,204 Cheng, C. M., 179,214,222 Cheng, C.H.K., 373,389 Chester-Jones, I., 289, 304 Chetelain, A., 311, 326 Cheung, R., 261,263 Chiang, Y.-C., 30-31, 34, 48, 54, 65 Chin, R. A,, 189, 192,209, 321,330 Chin, W. W., 136, 140-142, 147, 149-150, 152,154-155,157,250, 265, 350,364 Chino, N., 31, 60 Chiou, C.-S., 180, 189, 204 Chohan, K. S., 39, 45, 65 Chomczynski, P., 5, 7, 16, 18, 22, 55 Choy, Y. M., 276,282,304 Christensen, K., 346, 365 Christodoulou, C., 86, 96 Chu, L., 52 Ciofi, P., 24, 54 Claas, B., 39-40, 61 Claret, F. X., 336-337, 349, 359, 364 Clark, A. J., 145, 149, 154 Clark, A. R., 229, 263 Clark, J., 371, 389 Clark, J. H., 336, 361 Clark, N . B., 290, 303 Clarke, C., 103, 130 Clarke, D. L., 371, 387 Clarke, H. J., 400, 419, 434 Clarke, W. C., 367-368, 372, 387 Clauser, H., 372, 388 Clavequin, M. C., 10, 53 Clemens, A., 19, 57 Clemmons, D. R., 214, 222 Clerc, R. G., 169, 175 Coast, G. M., 71, 96 Coe, I. R., 7, 9-10, 14, 31-32, 35-36, 38, 40-41, 46, 49, 54, 57, 59, 62, 65, 108, 110, 117, 129, 142, 157, 232, 268

AUTHOR INDEX

446 Coghlan, J. P., 80, 89-90, 98 Cohen, H., 370,387 Cohen, P., 253-254,268 Cohen, R. S., 290, 303 Cohen-Solal, A., 243, 262 Cohick, W. S., 214, 222 Coit, D., 187, 204 Collard, M. W., 50, 65 Collie, N. L., 367-368, 388 Collier, K., 250, 252, 255, 267 Collier, K. J., 5-6, 14, 56, 250, 252, 270 Collin, F., 30-31, 54 Conlon, J. M., 7, 10, 18, 30-31, 34, 48, 54, 65, 71, 94, 230, 232, 237-238, 244,246,250,256-257,260-261, 263-264,271 Conn, P. M., 152,155 Conway, H. H., 290,293, 303 Cook, A. F., 183,204,399,437 Cooke, N. E., 164-165,175, 187,204 Cooper, G. A,, 248 Cooper, G. A,, 243 Copeland, N. G., 316,328 COPP,D. H., 276-279,281-285, 287, 290-291,293,295,303-306 Corcoran, L. M., 169, 175 Corneillie, S., 313, 326 Corrigan, A., 39-40, 57 Corthesy, B., 337, 349,359,365 Costlow, M. E., 383, 387 CBtC, J., 73, 76, 99 Coughlin, J. P., 276, 278, 281-283, 303 Counis, R., 140, 150, 154-155, 157, 332, 359 Coutelle, C., 228, 265 Cowan, J., 75 Cox, H. M., 19,54 Cox, J. K., 112, 129 Cox, K., 291, 295, 304 Cox, M., 318,326 COY,D. H., 7, 9, 11, 18, 22, 50-51, 52, 54, 60 Craig, A. G., 31, 34-35, 50, 59, 61 Crawford, R., 228, 250, 252, 265 Crawford, R. J., 80, 89-90, 98 Crenshaw, E. B., 111, 169-170, 172-173, 175, 190,206,314-316, 326-328 Creutzfeldt, W., 19, 57, 247, 270 Crim, J. W., 33-34, 54 Crim, L. W., 137, 141, 150, 153, 155, 158, 401,439

Croix, C., 82, 99 Croix, D., 24, 54 Cromlish, J. A., 316, 329 Cronin, M. J., 5-6, 55 Cross, R. B., 19, 63 Cruijsen, P.M.J.M., 73, 99 Cuet, P., 73, 76, 99 Culler, M. D., 7, 9, 18-19, 50, 54, 60 Cumming, R., 107, 129 Curlewis, J. D., 19, 63 Curran, T., 106, 130 Currie, R. A., 316, 329 Cutfield, J. F., 237-238, 256, 262, 264 Cutfield, S. M., 237-238, 256, 264 Cutler, G. B., Jr., 30, 63 Cuttitta, F., 217,224 D

Dababov, V. G., 227-228, 270 Dahl, R. D., 7, 10, 50, 60 Dahl, R. R., 7, 9-10, 18, 58, 60 Dahlman, K., 335, 339, 341,360,364 Dahlman-Wright, K., 341, 359,365 Daigle, M., 257, 263 Daikoku, S., 46, 54, 311, 330 Daikoku-Ishido, H., 46, 54 Dallemagne, C. R., 19, 63 Dalman, F. C., 335, 358, 363 D’Angio, L. A., Jr., 89-90, 98 Daniel, V., 189, 192, 194, 204 Danielan, P. S., 348, 359 Danielsen, M., 335, 360 Danielson, M., 341, 359 Danoff, A,, 257,263 Danville, D., 370, 386 Dargan, C., 30, 62 Darling, D. S., 147, 154 Das, G., 329 Dauder, S., 373-376,387 Daughaday, W. H., 214-215,222 Dave, J. R., 77, 94 Davidson, I., 336, 358 Davie, J. R., 276-279,281-285,306 Davies, S. L., 164-165, 167, 174, 180, 183, 189, 192-193,203-204 Davis, C. A., 314, 327 Davis, J. A,, 370, 387 Davis, M. S., 238, 250, 256, 263-264 Dawson, M. T., 350, 353,361

AUTHOR INDEX

Day, R. N., 320,326 Dayhoff, M. O., 49,54 Dayringer, H. E., 183, 203 Deacon, C. F., 238,264 DeChiara, T. M., 221, 222 Dee, P. C., 232, 235-236,238, 250,265, 267 Deerinck, T., 165, 169,175, 190,204, 314, 316,326 De Falco, A,, 344, 362 DeGroot, G. W., 314, 330 Deguchi, T., 78-79,89, 91, 96, 108-109, 114, 131 De Jesus, E. G., 312,328 Dekker, N., 318,326 de Lean, A., 337,359 de Leeuw, R., 40-41,56,153,154 Delerue, N., 275, 303 Dellovade, T. L., 44, 54 Delouis, C., 369, 383, 388 Delrio, G., 372, 387 DeManno, D. A., 417,431 Demarest, J. R., 377, 385, 391 Demski, L. S., 38-39,54-55, 66 DeNoto, F. M., 164-165, 175, 189, 192, 204 Denver, R. J., 5, 18, 55 Derancourt, J., 421-422,434 D’Ercole, A. J., 214, 223 Deschenes, R., 250,252,255,267 Deschenes, R. J., 250,252,270 Deshmukh, M. K., 107, 131 Desilets, M., 246, 271 DeSouza, E. B., 76-77,95 Dettlaff, T. A., 417,431 Deuben, R. R., 5, 55 de Vereneuil, M., 341, 362 Devereux, J., 79-80, 95 Devlin, R. H., 189, 192-193, 199,204, 208,215-216,218,224 de Zoeten-Kamp, C., 40-41,56 Dhariwal, A.P.S., 5, 55, 65 Dharmamba, M., 382,385,387 Di Carlo. R.. 369, 386 Dickhoff,W: W., 136-137, 140,156-158, 164, 172, 176, 181, 199, 204, 214-217, 220,223-224, 313,328, 399, 406,437,439 Didier, D. K., 197, 207 Di Domenico, M., 344,362 Dierickx, K., 104, 129

447 Dillon, J. S., 246-247, 264, 271 Di Mattia, G. E., 276-279,281-285,306, 314, 318-320,328,369,387 Dings, A., 73, 99 Distel, D., 422, 431 Distelhorst, C. W., 335, 360 D’Istria, M., 372, 387 Dixon, G. H., 7, 9-10, 14, 62, 232, 268 Dixon, J. E., 250-255, 259-261,262, 267-268,270 Dizon, J. S., 180, 204 Djiane, J., 367,369-373,383,386-391 Dmitrenko, V. V., 226-228,266 Dobado-Berrios, P. M., 5, 18, 59 Docherty, K., 229,263 Dodd, J. M., 398,431 Dolk, P., 314, 326 Dolo, L., 349, 362 Donaldson, E. M., 9, 18, 55 Dong, K. W., 45, 56 Dore, J. C., 344, 362 Doree, M., 421-422, 434 Doroshov, S., 34, 48, 64 Douglass, J. O., 89, 98 Downs, T. R., 5, 7, 16-18, 22, 55, 61 Draetta, G., 422, 430 Drakenberg, K., 214, 222 Drouin, J., 337, 359 Drucker, D. J., 233, 240,264-266,269 Du, S. J., 189, 192-193, 204 Du, Y., 314, 317, 319,328 Duan, C., 214,220,223 Dubois, M. P., 49, 56, 311-312, 325326 Dubois, P., 312, 325 Dubrasquet, M., 241,266 Duckworth, M. L., 369, 387 Duckworth, W., 243,262 Duez, C., 164-165,177, 189,208 Dufau, M., 370, 391 Dufau, M. L., 242,270 Duffey, R. J., 401,431 Dufour, M., 243,262,267 Dufour, S., 34, 48-49, 55, 58, 136, 150, 155, 332,359,398,431 Duguay, S. J., 181, 199, 204, 215-217, 220,222-223,225,231, 248, 258, 269 Dulka, J. G., 39-40, 57 Dumont, D., 73, 97 Dunand, M., 372,388

AUTHOR INDEX

448 Dunham, R. A., 180, 183, 187, 189, 192-193,208-209 Dupuoy, J. P., 311,326 Durrah, T. L., 242, 270 Dusanter-Fourt, I., 370,388 Dusantier, F. I., 370, 373, 388 Dutlow, C., 45, 56 Du Vigneaud, V., 102, 129 Dyball, R.E.J., 106, 129 Dye, H. M., 74,99 E

Early, P., 29, 63 Ebberink, R.H.M., 181,208,213,224 Eberhardt, N. L., 167-168, 175-176, 180, 183, 189, 192-193,204, 206-208 Ebner, K. E., 238, 260,266,269 Edery, M., 367,369-373,376,386-390 Edlund, T., 229, 262, 266 Edmin, S., 230, 270 Edmin, S. O., 228, 263 Edwards, D. P., 335, 346, 357, 365 Eelen, C., 24, 60 Efstratiadis, A,, 221, 222-223 Egly, J, M., 336, 358 Eiden, L., 31, 35, 64 Eiden, L. E., 35-36, 64 Eiders, L. E., 77, 94 Eidne, K., 45, 56 Elde, R. P., 254,267 El Halawani, M. E., 170, 177, 323, 330 Elisman, M., 314, 316, 326 Elkabes, S., 40, 46, 66 Elliot, W. M., 256, 262 Ellisman, M., 165, 169, 175, 190, 204, 314,326 El-Salhy, M., 261, 264 Elsholtz, H. P., 145-148, 150, 152, 158, 165, 168-170, 172-173,175, 190, 206,309,314-316,321,323-324, 325-328, 369,388 Enianuele, N. V., 30, 52 Emdin, S. O., 181, 204, 229, 265 Enimanuele, N. V., 369, 388 Emson, P. C., 86, 96 Endo, F., 314, 328 Enyeart, J. J., 183,205

Epler, P., 401, 431 Epple, A., 240, 265 Eriksson, P., 341, 359 Eriksson, H. A,, 349, 360 Eriksson, P., 337, 359 Erspamer, V., 71, 97 Ertan, A., 19, 61 Esch, F., 5-7, 10, 14, 21-22, 52-53, 56, 59, 70, 95, 97 Esch, F. S., 5-6, 55 Eskay, R. L., 77, 94 Eskild, W., 349, 363 Estes, P. A., 346, 365 Evans, R., 312, 326 Evans, R. M., 5-6, 14, 60, 190,205-206, 315,328,332, 334,360,364-365 Evans, T., 422,431 Everard, B. A,, 37, 57 Exton, J. H., 247, 263 Eyquem, C., 278,290-291,304 F

Fabbri, A., 10, 61 Fabbri, E., 245,247, 263,268 Faber, L. E., 335, 363 Fagerlund, U.H.M., 9,55,403,432 Fahrenkrug, J., 24, 57 Faiman, C., 314, 330 Fairchild, H. V., 144, 156 Falkmer, S., 181, 204, 228-230, 238, 250, 256-257,260,263-265,270 Faraick, K., 214, 222 Fargher, R. C., 183, 208, 276, 283, 291, 295, 304,306 Fargher, R. J., 283, 305 Farkash, Y., 316, 327 Farmer, S. W., 183, 185, 205 Farmerie, T. A., 143, 154 Farrow, S. M., 300, 303 Fasano, S., 34, 58, 372, 387 Fast, P., 316, 327 Fawell, S. E., 335, 344, 346, 359-360, 362 Fehmann, H.-C., 246,265 Felix, A. M., 22, 50, 53, 55, 63 Fellmann, D., 10, 53 Fellows, R., 30, 53 Fenstermaker, R. A., 143,154

449

AUTHOR INDEX

Fenwick, J. C., 274-277, 282,291,303, 305-306 Feon, S., 349-350,363 Feramisco, J. R., 335, 358 Ferewz, H.-J., 71, 97 Ferguson, A. L., 214, 222 Ferguson-Smith, M. A., 46, 52 Fermin, C . D., 11, 66 Fernald, R. D., 31-32, 36, 45, 53 Fernley, R. T., 80, 89-90, 98, 276, 278, 281-283 Ferris, S. D., 72, 95 Fields, R. D., 38, 55 Figueroa, J., 89, 93, 95, 97, 108, 111-112, 114, 117,129, 131 Finet, B., 417, 431, 433 Fink, G., 68,95, 142, 149, 152,155 Finney, M., 169,175,316,326 Firestone, L. A., 52 Fischer, W. H., 47 Fischer, J. E., 261, 262 Fischer, W. H., 30-31, 34-35, 50, 59, 61, 260,268 Fisher, A.W.F., 81-82, 100 Fitzpatrick, M. S., 400, 431 Fitzpatrick-McElligott, S., 94 Fjose, A., 316, 327 Flanagan, C. A,, 42, 60 Flik, G., 160, 177, 276, 282-283, 290, 294-295,303-304,306,377-378, 391 Flint, A.P.F., 401, 431 Flomerfelt, F. A., 335, 358 Flood, J. F., 19, 61 Flouriot, G., 346, 350, 360 Flynn, S . E., 165, 169, 175, 314, 316-319,327 Fong, N. M., 197,204 Fontaine, M., 274-275, 303 Fontaine, Y. A., 34, 48-49, 55, 58, 136, 140, 150, 155, 157, 332, 359, 398, 431 Forde, 350, 361 Fortner, N. A., 368, 388 Foster, D., 247, 266 Foster, D. M., 183, 205 Foster, D. N., 183, 206 Foster, G., 245 Foster, G. D., 245, 265 Foster, L. K., 183, 205

Fostier, A,, 368, 390, 394, 400, 403, 431, 433 Fourt, I., 370, 386 Fox, N., 143,155 Fraccaro, M., 46, 52 Frajese, G., 17-18, 50-51, 52, 61 Francis, R. C., 31-32, 36, 45, 53 Franco, B., 46, 55 Franco, R., 190,206,315,328 Fraser, B. A., 31, 34-35, 64 Fraser, R. A., 283, 303 Frawley, L. S., 311-312, 326-327 Frazier, M. L., 232, 267 Freedman, L. P., 339, 341,360,362-363 Freyschuss, B., 349, 360 Fridkin, M., 14, 52 Friedlaender, M. M., 300, 305 Friedman, J., 274, 303 Friesen, H., 276, 306 Friesen, H. G., 276-279, 281-285, 287-288,290-293,299-300, 304-306,369,372,387-388,390 Froesch, E. R., 214,224 Frohman, L. A., 5, 7, 10, 16-18, 22, 52, 55, 57, 61, 65 Frohman, M. A., 5, 7, 17, 22, 55, 61 Fromental, C., 347, 364 Fryer, J., 73, 75, 91, 96 Fryer, J. N., 67, 69, 72-75, 77, 81-82, 85-86,88,91,94-96,98-100,373, 388 Fujimoto, S., 314, 328 Fujino, M., 6-7, 10, 12, 14-15, 17, 26, 29, 57-58,60,62 Fukada, S., 402,414-417,419-420,422, 424-425, 431, 437-439 Funckes, C. L., 250, 252,255,267 Funeya, K., 71, 97 Funkenstein, B., 214, 223 Furlanetto, R. W., 214, 223 Furukawa, K., 399, 419,434,436, 439 Furutani, Y., 70, 78-80, 89, 91, 95-96,98

G Gabbay, K. H., 216, 223 Gaeraerts, W.P.M., 213, 224 Gage, L. P., 5-6, 14, 56

450 Gagel, R. F., 290, 293,303 Gagnon, J., 370,386 Gaillard, R., 7, 53 Gaillard, R. C., 52 Gainer, H., 40, 46, 66 Gait, M. J., 86, 96 Gajewski, T. C., 73, 96 Gajic, D., 240, 265 Gala, R. R., 369, 388 Galdiero, M., 344, 362 Galehouse, D. M., 183,206 Galloway, S. M., 238, 262 Gallwitz, B., 260, 263 Cannon, F., 25, 57, 350, 353, 361 Garcia Ayala, A., 40, 56 Garcia-Navarro, F., 5, 18, 59 Garcia-Navarro, S., 5, 18, 59 Garg, S. K., 417, 431 Garland, H. O., 289, 304 Garlov, P. E., 107, 129 Garrett, F. D., 273, 275, 304 Gaub, M. P., 153,155,337,360 Gauthier, Y., 337, 359 Gautier, L., 422, 431 Gautvik, K. M., 376, 386 Gavioli, M. E., 245, 247, 263, 268 Gay, V. L., 149, 155 Gaye, P., 369-370,388-389 Gehring, W. J., 314, 317, 326 Gellersen, B., 290-292, 299, 304, 306, 369, 387 Genazzani, E., 373, 389 Geoffre, S., 39-40, 53 Georges, D., 49, 56 Geraets, W.P.M., 181, 208 Gerhardt, D. S., 197, 204 German, M. S., 229, 265 Germond, J. E., 336, 365 Gerster, T., 316, 329 Gharib, S. D., 136, 141-142, 149-150, 152,155,157 Ghatei, M. A., 18, 63 Giguere, V., 334, 360 Gilbert, D. J., 316, 328 Gimelli, G., 46, 52 Givel, F., 336, 364-365 Glode, L. M., 143, 156 Glotzer, M., 422, 431 Glover, I. D., 259, 265 Glowacki, J., 291, 295, 304

AUTHOR INDEX

Gnessi, L., 10, 61 Godowski, P. J., 338, 362 Goetz, F. W., 394-395, 401, 403, 417, 431 -432,438 Goke, B., 246,265 Goke, R., 230, 238,246,264-265 Goldhaber, P., 274,303 Goldsworthy, G. J., 71, 96 Gona, O., 373,388 Gong, Z., 173, 175, 187, 189, 192, 205, 321,323-324,326 Gonzalez-Crespo, S., 5, 7, 17, 56 Gonzalez-Villasenor, I. L., 183, 205 Goodman, H. M., 164-165,175, 180, 183, 189, 192,204,206 Goodman, R. H., 232,235-236,238,250, 252-255,265,267,270 Goodwin, E. C., 183, 189,209 GOOS,H., 31-32,35,45,47,52-53 COOS,H.J.Th., 40-41, 47, 53, 56, 63-64,66 Goos, M.J.Th., 332, 360 Goossens, N., 104, 129 Gorbman, A., 33, 54, 238, 244, 250, 256, 261,268-269,271 Gorcs, T. G., 11, 58 Gordon, D. F., 30, 57,319,326 Gordon, M. S., 350, 364 Gorewith, R. C., 180, 207 Gosselin, P., 148, 157 Goswami, S. V., 395,403,432,437 Gothilf, Y., 31-32, 56 Gottschall, P. E., 24, 65 Gotzeni., F., 213, 223 Could, D., 370, 387 Gouteux, L., 73, 76, 99 Gowing, H., 9, 18, 50, 56 Gozes, I., 14, 52 Grandien, K.F.H., 353, 360 Granneman, J.C.M., 401,436 Grant, F. J., 247, 266 Grant, P., 40, 46, 66 Grau, E. G., 9, 61, 368, 386 Grauer, A., 300, 305 Gray, E. S., 214, 223 Grebenjuk, V. A., 215-218,223 Creeley, M. S., Jr., 401, 432, 436 Green, S., 332, 334-335, 337, 339, 344, 346-347,350,360-361,363,365 Greenberg, R. M., 137, 156

451

AUTHOR INDEX

Greene, G. L., 336, 350,360-361,363 Greenwood, F. C., 167-168,176, 180, 191-192,206 Greiner, F., 254, 261, 267-268 Grez, M., 108, 130 Crier, H. J., 31, 36, 40, 54, 64 Griffiths, S. L., 248, 265 Grigoriadis, D. E., 76, 95 Grino, M., 59 Grober, M. S., 39, 48, 56 Gronemeyer, H., 335, 353, 358,361 Gross, P., 14, 29, 57 Grnss, P., 314, 327 Guardabassi, A., 373, 389 Gubler, U., 5-6, 14, 56 Guenzi, D., 183, 206 Guerin, S. L., 148, 157 Guibbolini, M. E., 105, 129 Guillaumot, P., 370, 387 Guillemin, R., 5-7, 10, 21-22, 30, 52-53,55-56,59,70,95,97 Guioli, S., 46, 52, 55 Gurd, R. S., 235, 265 Gustafsson, J. A,, 335, 338-339, 341, 353,359-360,362,364-365

H

Habener, J. F., 14,29,57,232, 235-236, 238,240,246-247,250,253,263, 265,267,269,300,304 Habibi, H. R., 42, 56, 153, 157 Hagesawara, S., 367-368, 388 Hahn, V., 228-229,265,269 Haider, S., 401, 403, 432, 438 Hall, P. F., 414, 435 Halloran, M. M., 30, 52, 369, 388 Halpern, L. R., 40, 63 Halpern-Sebold, L., 39-40, 45, 63 Halpern-Sebold, L. R., 56 Ham, J., 337, 350, 365 Hamel, B., 46, 52 Hamernik, D. L., 143, 154 Hamilton, J. W., 238, 250, 256, 260, 266, 269 Hamilton, P. L., 75, 97 Hammer, R. A., 19, 61 Hample, A,, 383, 387

Hampong, M., 276,278, 282,306 Handa, H., 416,435 Handin, R. I., 382, 387 Hane, S., 72, 98 Hanke, W., 383,386 Hanssen, R.G.J.M., 276, 282, 290-291, 294,303-304 Hansson, V., 349, 363 Hanyu, I., 399-400,419,434,436437, 439 Hara, A., 183, 206 Hardambidis, J., 276, 278, 281-283, 303 Hard, T., 339, 341, 360 Hardiman, P. A., 400, 436 Harlow, K. H., 360 Harris, G. W., 68, 95 Harris, N., 45, 56 Hart, G. R., 9, 18,50,56 Haruta, K., 122-124, 129 Harvey, B., 35-36, 64 Harvey, D., 183, 205 Harvey, S., 5, 18, 57, 283, 303 Harwood, J. P., 77, 94 Hasegawa, M., 115, 128-129 Hasegawa, S., 185, 206, 235, 265, 273, 304, 376,389 Hasegawa, Y., 30-31, 37, 39-40, 47, 51, 58, 60, 62 Hass, L., 373-376, 387 Hattori, K., 170, 177, 319, 329 Haugen, B. R., 319, 326 Hauger, R. L., 77, 94 Haus, E., 246,265 Haux, C., 338, 350,362,368,389-390 Hawke, D., 253, 259,262 Hawke, D. H., 238,245,262 Hayashi, K., 242, 270 Hayashida, H., 78-80, 91, 95, 113-114, 130 Hayashida, T., 183, 185,205,207 Hayashizaki, Y., 140-141, 158 Hayes, M. K., 421,434 Hayflick, J. S., 30, 51, 53, 57, 60 Hayward, J. N., 104, 107, 129, 131 Hazel, J. R., 383, 388 Hazon, N., 123,129,238, 260, 264 He, X., 169-170, 172-173,175,314,327 Heierhorst, J., 89, 93, 95, 97, 108-112, 114, 117,129-131 Heilig, R., 25, 57

452 Heimer, E. P., 22, 53, 55 Heinrich, G., 14, 29, 57, 300, 304 Heitlinger, E., 150, 156 Heldwein, K. A,, 71, 99 Henderson, I. W., 105, 129, 238,264 Hendrix, M.J.C., 370, 387 Hendy, G. H., 300,305 Hendy, G. N., 303 Hennessy, T., 417,432 Henry, D. P., 214, 222 Henschen, A., 71, 97 Henzel, W. J., 335, 363 Herbert, E., 8 0 , 8 9 , 9 9 Herington, A. C., 369, 388, ,391 Hermann-Erlee, M.P.M., 276,282, 304 Hermodson, M. A., 255,262 Hernandez, E. R., 202,205 Herr, W., 169, 175, 316, 318, 325, 329 Herrlich, P., 153, 157 Herrup, K., 314, 327 Hew, C. L., 135, 137, 140-148, 150-152, 158, 173, 175, 187, 189, 192-193, 204-205,209,321,323-324,326, 330, 354, 365 Heyl, H. L., 289,303 Hidaka, H., 24, 65, 240, 270 Higgins, S. J., 349, 358 Higgs, D. A., 9, 55 Hillyard, C., 278, 290-291, 304 Hinck, L., 341, 359 Hintz, R. L., 214, 224 Hirai, T., 419-420, 422, 424-426, 432, 439 Hirai, Y., 425, 432 Hiramatsu, H., 183, 206 Hirano, T., 127, 132, 180, 183, 185, 187, 194,205-207,209,214,220, 222-224,273-274,304-305,312, 328,367-368,376-377,388-389, 391 Hiraoka, S., 101, 108, 120, 130 Hiraoka, Y., 140-141, 158 Hirose, K., 401, 432, 435, 437 Hirose, T., 78-80, 91, 95 Hobart, P., 228, 250, 252,265 Hodgson, T., 39, 56 Hoeffler, J . P., 311, 327 Hoffman, B. J., 5-6, 14, 56 Hoffman, J. A., 213, 223 Hogan, M. L., 167-168, 176, 180, 191-192,206

AUTHOR INDEX

Hohne, W. E., 229,269 Hokfelt, T., 24, 5 7 Hollenberg, A. N., 136, 155 Hollenberg, S. M., 332, 334, 338, 360, 365 Holley, S. J., 335, 360 Holloway, J. M., 170, 172, 176, 311, 314-315, 320, 329 Holm, T., 316, 327 Holmes, R. L., 311, 327 Hols, H., 401, 432 Holthuizen, E., 199, 205 Holz, G. G., 246, 265 Honda, S., 416, 435 Hontela, A., 77, 88, 100 Hood, L., 29, 63 Hoosein, N. M., 235, 265 Horger, G. L., 339, 358 Horikawa, S., 70, 78-80, 89, 91, 98 Horn, F., 143, 149, 155 Horowitz, M., 19, 61 Horowitz, S., 183, 189, 209 Horton, Y. M., 248,265 Horvath, J., 68, 71, 91, 98 Horvitz, H. R., 169, 175, 316, 326 Hosokawa, Y., 183, 189,208 Hosoya, M., 6-7, 10, 12, 14-15, 17, 26, 2 9 , 5 7 4 8 , 62 Hotta, Y., 420, 439 Houdebine, L. M., 207, 369, 372, 376, 384, 388,390 House, S., 346, 360 Houslay, M. D., 248, 265 Housley, P. R., 335, 360 Howard, K. J., 335, 360 Hoyt, E., 235, 267 Hsu, S.-M., 50, 63 Hsueh, A.J.W., 30, 62 Hu, Z. Z., 370,391 Huang, C . J., 139-141,155-156 Huang, F. L., 139-141,154-156 Huang, H. H., 372,389 Huang, Y. P., 153, 157 Huckle, W. R., 152, 155 Hughes, J. P., 369, 388 Huhnel, R., 344, 363 Hui, T. Y., 373, 389 Hulting, A.-L., 24, 5 7 Humbel, R. E., 181, 204 Hunt, D. F., 71, 94 Hunt, T., 422, 431, 436

AUTHOR INDEX

Hutwitz, A., 202, 205 Hwang, P. P., 313, 327 Hwang, S. J., 185, 208 Hyde, E. I., 339,341,360 Hyodo, S., 31-32, 35, 39, 45-47, 65, 86, 96, 104-105, 107-110, 112-115, 117-119, 121-122, 124-127,130, 132, 139,156

I

Ichikawa, 69, 91, 96 Ichikawa, M., 40, 62 Ichikawa, T., 69, 71, 78-79, 89, 91, 96 Ichimiya, Y., 86, 96 Idler, D. R., 37, 57, 185, 208, 376, 389, 398, 401, 403, 410, 418, 432, 434-435,438 Igarashi, A., 183, 192, 209 Igarashi, M., 30-31, 60 Ihle, J. N., 371, 386,391 Imakado, S., 347,361 Imura, H., 78-80,90-91, 96 Inagaki, H., 314,328 Inayama, S., 78-80, 91, 95 Inbaraj, R. M., 401, 432 Ince, B. W., 244,258,266 Incerti, B., 46, 55 Ing, N. H., 336, 361 Ingleton, P. M., 289, 304, 311, 325 Ingraham, H. A., 165, 169-170, 172-173, 175-1 76,311,314-320,327-329 Inomata, Y., 416, 435 Iraqi, F., 187, 189, 192, 205 Isakson, P. C., 24, 65 Ishida, I., 78-79, 89, 91, 96, 108-109, 114, 131 Ishii, S., 400, 434 Ishikawa, K., 10,57 Ishioka, H., 183, 206 Ishizaki, H., 213, 223 Itoh, H., 137-138, 140, 155-158, 183, 185, 192, 207, 209, 377, 391, 399, 432-433 Itoh, K., 246, 271 Itoh, N., 14, 57, 229, 266 Itoh. S., 137-138, 140, 157, 206, 399, 433 Itoh, Y., 7, 10, 58

Iwamatsu, T., 398, 400-402, 405, 432-433,435 Iwami, M., 213,223 Iwata, M., 108, 120, 130, 367-368, 388 J

Jackson, B. C., 30-31,34, 59 Jackson, I.M.D., 10-12, 36, 51, 62, 66 Jacobs, G. H., 339, 361 Jacobs, J. W., 250,253,265 Jacobson, P. M., 408,438 Jacobsson, B., 232,266 Jahn, G. A., 372,389 Jalabert, B., 394,400-401,403,417,431, 433 Jameson, J. L., 136, 155, 337, 359 Jansen, M., 216,223,314,318-319,328 Janssen-Dommerholt, C., 31, 35,53 Janssens, P. A,, 245, 266 Jansson, J.-O., 10, 57 Jaouen, G., 344,364 Jardim, A., 246 Jarrousse, C., 241, 243, 266 Jaworski, E., 294-297, 299,306 Jego, P., 338, 364 JBgou, S., 73, 76, 99 Jelinek, L. J., 247, 266 Jenkins, N. A,, 316, 328 Jenks, B. G., 73,99 Jennes, L., 39-40, 61, 66 Jensen, E. V., 376, 387 Jiang, L., 7, 9-10, 18, 50, 58, 60 Jin, J. R., 332, 361 Jin, L., 143, 156 Jingami, H., 78-80,90-91,96 Jirikowski, G. F., 96 Jobin, R. M., 153, 154 Johansen, B., 164,175, 189, 192-193, 205 Johansen, T., 316, 327 John, T. M., 368, 389 Johnsen, 0. C., 164, 175 Johnsen, T., 349,363 Johnson, L. K., 183, 207 Johnson, 0. C., 189, 192-193,205 Jolicoeur, C., 370-371, 373,386-388, 390 Jones, G. J., 316,327 Jonsson, A.-C., 232,244, 266

AUTHOR INDEX

Joosse, J., 181, 208, 213, 224 Jergensen, F. S., 261, 264 Jornvall, H., 232, 266 Josefsberg, Z., 369, 386 Joss, J.M.P., 103, 130 Joyner, A. L., 314, 327 Judo, A. M., 24, 65 Jung-Testas, I., 335, 358 Jurgens, J. K., 369,388 Jutisz, M., 140, 150, 155, 157, 332, 359 K

Kaattari, S. L., 77, 99 Kabayashi, T., 187,209 Kadonaga, J. T., 336,362 Kagawa, H., 37, 62,400-401, 405, 410, 417,433,435,439 Kaghad, M., 421-422,434 Kah, O., 39-40,53,57, 62 Kahn, C . R., 314,329 Kaibe, K., 314, 329 Kaiser, C., 370, 390 Kajimoto, J., 213, 223 Kajimoto, Y., 181, 202, 205-206, 213, 223 Kajiura, H., 424-425, 432-433 Kal, A. J., 318, 329 Kalla, K., 316, 326 Kalla, K. A., 320, 327-328 Kaltschmidt, C., 336-337, 362, 364 Kambegawa, A,, 401,439 Kanamori, A., 406-407,410, 433 Kandel, E. R., 123,130 Kaneko, T., 104,132, 273, 283,303-304, 400,434 Kangawa, K., 30-31, 60 Kanse, S. M., 18, 63 Kapadia, M., 214,222 Kapiloff, M. S., 169-170, 172-173, 175, 316-319,327 Kapler, G., 180, 206 Kaptein, R., 318, 326, 339, 341, 360 Karin, M., 165, 169-170, 173, 175, 177, 190,204,314,316,318-319,326, 329 Kariya, Y., 183, 207 Karlsson, O., 229, 266 Kashio, Y., 16, 22, 55 Kasprzyk, P. G., 217, 224 Kastner, P., 338, 353, 361-362

Kashuba, V. I., 227, 266 Katakami, H., 10, 57 Kataoka, H., 71, 96 Kato, S., 152, 155, 354, 361 Kato, Y., 108-110, 113-115, 117-118, 124-125,130 Katsoulis, S., 19, 57 Katsu, Y., 424-425, 433 Katz, S. H., 5, 55 Katzenellenbogen, B., 344, 363 Katzenellenbogen, B. S., 350, 360, 363 Katzenellenbogen, J. A., 360 Katzenellenbogen, K. S., 344, 363 Kavsan, V., 227, 266 Kavsan, V. M., 215-218,223,226-228, 266,270 Kawai, K., 246-247, 268,270 Kawakami, K., 316,329 Kawakami, T., 213,223 Kawashima, S., 40, 47, 51, 122-124, 129 Kawauchi, H., 29,57, 102, 117, 130-131, 136-140,155-159, 160-165, 167-168,170, 172-173,175-177, 180, 183, 185, 187, 189, 192-194, 205-209,214,224,282, 301,305, 376-377,389,391,399,432-433, 437 Kawauchi, K., 399,437 Kawazoe, I., 117, 131, 183,207 Kawazoe, L., 183, 206 Kay, I., 71, 96 Keaveney, M., 350, 353,361 Kellenbach, E., 339, 341, 360 Kelley, K. M., 9, 59, 214, 223-224 Kelley, M. R., 30, 52, 369, 388 Kelly, P., 369-370, 373, 386, 388 Kelly, P. A,, 367, 369-372, 383, 386-391 Kelsall, R., 49, 57 Kempe, T., 71, 94 Kendall, S. K., 143, 156 Kenyon, C. J., 289, 304 Kepa, J. K., 30, 57 Kerdelhue, B., 49, 55, 332, 359 Keri, R. A., 143, 154, 156 Kervran, A,, 243, 262 Kervran, A. P., 243, 266 Kessel, M., 314, 327 Keutmann, H. T., 138,156 Kewish, B., 214, 222 Khan, I. A,, 40, 58

AUTHOR INDEX

Khan, M. N., 369,386 Khutinaev, A. S., 107,129 Kibler, R. K., 369, 390 Kikuchi, K., 202, 206 Kikuno, R., 113-114,130 Kikuyama, S., 46, 61, 183, 187,209 Kim, S. U., 183,205 Kime, D. E., 401,433 Kimmel, J. R., 238, 250, 256, 259-260, 265-266,269 Kimura, A., 183,207 Kimura, C., 6-7, 10, 12, 14-15, 17, 26, 29,57-58, 62 Kimura, S., 183, 207 Kindsvogel, W., 247,266 King, D., 261, 262 King, D. S., 71, 97, 185, 187, 208-209, 373,376-378,390-391 King, J . A., 34-37, 42, 44, 48, 54, 58, 60,63 King, J. C., 33, 38, 58 King, S. D., 183, 207 Kingan, T. G., 71,94 Kioke, S., 320, 326 Kisen, G., 44, 58 Kisen, G. O., 31-32, 35,45, 58 Kishida, M., 183, 209 Kishimoto, T., 420,434 Kiss, J. Z., 86, 97 Kissinger, C. R., 317, 327 Kitada, C., 7, 10-12, 51, 52, 60, 62 Kiyama, H., 96 Klatt, D., 229, 269 Kledzik, G. S., 372, 389 Klein-Hitpass, H. L., 150, 156 Klein-Hitpass, L., 336, 361 Klock, G., 336, 361 Klootwijk, J., 181, 208, 213, 224 Kloss, B., 170, 175, 319, 328 Klug, A., 339, 362 Klug, J., 350, 353, 361 Klungland, H., 31-32,35,44-45, 58 Knepel, W., 240,270 KO, D., 86,91,97-98 KO, H.-S., 316, 327 Kobayashi, H., 33, 38-41, 61-62, 69, 71, 96, 104, 132 Kobayashi, M., 31-32, 35,45-47, 52,58, 65, 117,129,395,399-400,419,434, 436

Kobayashi, T., 127,132 Kobayashi, Y., 75, 77, 88, 91, 96, 100 Kobel, H. R., 72, 96 Kobukawa, K., 52 Koffman, B., 344, 361 Kohno, H., 314,329 Koide, Y., 137, 156 Koike, S., 337, 347, 361 Kondo, J., 376-377, 391 Kondo, S., 347, 361 Konzak, K. E., 170,175,319, 327 Kornberg, T. B., 317,327 Kosaka, T., 12, 62 Kostellow, A. B., 418, 434 Kosugi, R., 183, 206 Koval, A., 227, 266 Koval, A. P., 215-218,223,226-228, 266 Koves, K., 11,58,66 Koyama, E., 316,328 Kozlov, Y. I., 227-228, 270 Kraehenbuhl, J. P., 372, 388 Kraicer, J., 73, 96 Kramer, J. M., 181, 204,228,263 Kramer, S. J., 71, 96 Krause, J. E., 202,207 Krauss, S., 316, 327 Krentler, C., 89, 93, 95, 108, 111-112, 117,129 Kriebel, R. M., 40, 60 Krishnamurthy, V. G., 274-276, 285, 289, 294,304 Krivi, G. G., 197, 207 Kronenburg, H. M., 300, 304 Krulich, L., 5, 55 Krumlauf, R., 314, 328 Krust, A., 337,353,360-361 Kubo, K., 7, 10, 60 Kubokawa, K., 31-32,45, 52,101 Kubota, J., 183, 206 Kuga, T., 206 Kugoh, H., 6, 10, 12, 15, 17, 29, 57 Kuhtreiber, W. M., 246, 265 Kuijper, J. L., 247, 266 Kumar, T. R., 144, 156 Kumar, V., 332, 334-335, 337, 340-341, 344,358,360-363 Kuriyama, D., 183, 206 Kuwana, Y., 183, 192,206-207 Kwok, S.C.M., 181,204,228,263

AUTHOR INDEX

456 L

Labbe, J. C., 421-422,434 Labedz, T., 294, 303 Labrie, F., 73, 97 Lafeber, F.P.J.G., 276, 282, 304 Lagueux, M., 213,223 Lahav, M., 372,386 Lahlou, B., 102, 104-105, 107, 122, 129-1 30 Lahooti, H., 349, 363 Lallemand, Y., 314, 328 Lallier, F., 278, 290-291, 304 Lam, T. J., 368,391 Lamacz, M., 73, 76, 99 Lamb, I. C., 183,206 Lamba, V., 403,437 Lambed, J.G.D., 401,403,434,436 Lambros, T. J., 50, 63 Lan, N. C., 349,358 Lance, V., 31, 34, 48, 59, 64,238, 260, 269 Lance, V. A., 22,54 Land, H., 108,130 Landmark, B. F., 349, 363 Lane, T. F., 102,130 Larhammar, D., 259-260, 263, 266 Laron, Z., 214, 223 Larson, B. A,, 77, 94 Larson, D. F., 369, 390 Latchman, D. S., 353, 362 Laudano, A. P., 34, 53 Laudon, M., 50,63 Law, Y. K., 419, 434 Lawler, H. C., 102, 129 Lawrence, A. M., 369,388 Lawrence, C., 46,55 Laybourn, P. J., 336,362 Lazier, C. B., 349, 362 Lazzaro, D., 170, 177, 319, 329 Leatherland, J. F., 368, 389 Le Bail, P. Y., 376, 391 Lebo, R., 338,360 Lebo, R. V., 5-6, 14, 60 LeBoff, M. S., 293, 303 Lebovic, G. S., 373, 391 Lebrun, J.-J., 369, 390 Lechan, R. M., 10-12, 62 Lederis, J., 67, 69, 71-73, 75, 96

Lederis, K., 69, 71-73, 75, 77-78, 80-82, 85,87-91,93,94-97,99-100,104, 108-109, 111-112, 114, 117, 129-1 32 Lederis, K. P., 69, 86, 91, 98 Le Drean, Y., 347 Le Drean, Y., 145-146, 150-152, 158, 331, 354, 357 Lee, J . K., 337, 359 Lee, S., 71, 99 Lee, T., 30-31, 34,59 Lee, T. D., 238,245,262,268 Lee, W.-H., 214,222 Lee, Y., 22, 53 Lee, Y. C., 233, 266 Lees, J. A., 335, 337, 344, 346, 348, 350, 359,362,365 Lefevre, G., 73, 97 Le Gac, F., 337-338,345,349-350,363, 376,391 Legesse, K., 238, 245, 262 Le Goff, P., 337-338,345, 349-350, 362-363,368,389 Le Guellec, C., 337, 363 Lehman, M. N., 46, 61 Lehmberg, E., 71, 97 Leid, M., 338, 362 Leinen, J. G., 376, 387 Lelias, L.-M., 421-422, 434 Le Menn, F., 40,62,346,349-350,360, 363 Leng, X., 335, 346,357 Leng, X.-H., 246-247, 264,271 Le-Nguyen, D., 243,262 Leonard, C. M., 46, 66 Le Pennec, J. P., 339, 350, 353,362 Le Provost, F., 369, 389 LeRoith, D., 15, 58, 71, 97, 202, 205, 215-218,223,227,266 Leroux, C., 369, 389 Le Roux, M. G., 337-339,350,353, 362-364 Lerrant, Y., 154 Lescheid, D. W., 3, 9-10, 34, 47 Lesniak, M. A., 71, 97 Lessman, C. A., 401, 434 Lesueur, L., 370-371, 373,386-389 Letter, A,, 69, 71-72, 96 Lettieri, D., 300, 305

457

AUTHOR INDEX

Leung, E., 72, 95 Leung, F. C., 372, 374-375,389 Levi-Meyrueis, C., 370, 373, 388 Levy, F. O., 349,363 Lew, A. M., 315,325 Lew, D., 337-338,365 Lewis, U. J., 183, 206, 209 L’hoir, C. P., 183, 207 Li, C. H., 180, 187,204,206 Li, J. P., 71, 96 Li, K. W., 31, 35, 53 Li, S . , 170, 175, 314, 327 Li, W.-H., 25, 58, 232, 267 Licht, P., 5, 18, 55, 183, 205 Liddle, G . W., 99 Liebscher, D.-H., 228, 265 Ligon, B. B., 247, 264 Lin, B., 327 Lin, C . M., 180, 183, 187, 189, 192-193, 204,208 Lin, C. R., 165, 169, 175, 314, 316, 327 Lin, H. D., 10-12, 52, 62 Lin, H. R., 185, 187,209,376-377, 391 Lin, J. C., 240, 267 Lin, K.-L., 183, 208 Lin, R. J., 377, 385, 391 Lin, S.-C., 320, 328 Lin, Y.-W.P., 137, 156,401-402, 405, 434,436 Lindahl, K. I., 214, 222 Lindorfer, H. W., 38, 60 Lindsey, R. K., 350, 363 Ling, H., 189, 192-193, 209 Ling, N., 5-7, 10, 14, 21-22, 30, 36, 52-53, 55-56,58-59, 66, 70, 73, 76, 95, 97, 99 Linzer, D.I.H., 370-371,387 Lion, M., 183, 185, 207, 377-378, 390-391 Lipkin, S. M., 320, 327 Lippmann, M. E., 350,363 Lira, S. A., 190, 206, 315, 328 Liu, B., 317, 327 Liu, C . S., 139, 156 Liu, D., 140-141, 143-148, 150-152, 158 Liu, J.-P., 221, 223 Liu, Q . R., 148, 157 Livingstone, C., 248, 265

Lloyd, R. V., 143, 156 LO,T. B., 139-141,154-156 Lobban, M., 248,265 Lohka, M., 421-422,431 Lohka, M. L., 434 Lok, S., 247, 266 Lomedico, P. T., 5-6, 14, 56 Lonergan, K., 349, 362 Lopez, E., 278,290-291,304 Lopez, L. C., 232,267 Lopez-Novoa, J. M., 247,269-270 Lorens, J. B., 31-32, 35, 45, 58 Loretz, C. A., 367-368, 389 Lotersztajn, S., 243, 267 LOU,Y.-H., 419-420,422,425-426,432, 439 Loumaye, E., 49,59 Lovas, S., 31, 34, 48, 65 Lovejoy, D. A., 30-31, 34-35, 38-39,41, 46,50, 59, 61,64-65, 142,157 Low, M. J., 144,156 Lowrey, P., 245, 266 Lu, L.1.-W., 165, 168, 175, 190, 206, 314-316,328 Lu, M., 246, 271, 301, 304 Lufkin, T., 315, 328 Luisi, B. F., 341, 362 Lukowsky, A., 230,267 Lund, P. K., 232, 235-236, 238, 250, 253, 265,267 Lundell, I., 259-260, 263 LUO,C.-C., 25, 58, 232, 267 Luo, D., 9-10, 18, 50, 59 Luts, A,, 11, 65 Lutz, Y., 336, 358 Lux, G., 240,270 Lwoff, L., 213, 223 Lyons, R. H., 183, 189, 209 M

Ma, M . C., 71, 94 MacCannell, K. L., 75, 91, 96-97 Macdonald, D., 241-242,270 Macdonald, F., 248, 265 MacDonald, P. C., 180,208 MacIntyre, I., 278, 290-291, 302, 303-304

AUTHOR INDEX

Mackin, R. B., 230, 257, 267 MacLeod, R. M., 24, 65 Madden, B., 138,154 Mader, S., 341, 362 Maestrini, E., 46, 55 Maetz, J., 102, 104-105, 107, 122, 130, 385,387 Magazin, M., 250, 252, 255, 267 Magun, B. E., 369,390 Mahlmann, S., 108, 110, 117, 129 Maitre, J. L., 349, 362 Majumdar, S., 309,323-324 Majumdar-Sonnylal. S., 173, 175, 321, 323-324,326 Majzoub, J. A., 89-90, 98 Mal, A. O., 313, 328 Malagon, M. M., 5, 18, 59 Malamed, S., 5, 18, 63 Maler, B. A., 338-339, 341, 360,362 Maliyakal, E. J., 414, 439 Mallat, A., 243, 267 Maller, J., 422, 431 Maller, J. L., 417-418, 421-422, 427, 431,434,436 Mallet, A. I., 71, 96 Mafianos, E., 40,57 Mandel, G., 252,254-255,270 Mandel, J. L., 25, 57 Maneckjee, A., 418,434 Mangalam, H. J., 165, 169-170, 172-173, 175,314,316,327 Maramatsu, M., 337, 361 Maran, J. W., 5, 55 Maraschio, P., 46, 52 Marchant, T. A., 9, 18, 50, 59, 153, 157 Marchaterre, M. A,, 39, 56 Marchelidon, J., 275, 303 Marcuzzi, O., 153, 158, 332, 365,400, 431 Mareels, G., 313, 326 Margioris, A. N., 59 Margolis-Kazan, H., 39-40, 63 Margolis-Nunno, H., 38, 55 Marivoet, S., 10, 24, 60 Mariz, I. K., 214, 222 Marks, A. R., 180, 206 Marra, L. E., 282, 304 Marsh, A,, 179, 214, 222 Marsh, J. A,, 5, 18, 57 Marshak, D. R., 31, 34-35, 64

Marshall, C . J., 238, 262 Marshall, S., 372, 389 Martelly, E., 275, 303 Martial, J. A,, 164-165, 177, 183, 185, 189, 192,206-208,373,377-378, 381-383,386,390-391 Martin, P., 369, 389 Martin-Blanco, E., 317,327 Martinez, E., 336-337,362,364-365 Martinez, J . , 241,243,266 Martin-Myers, A,, 50, 63 Masahara, S., 347, 361 Mason, A. J., 30, 51, 60 Mason, R. T., 10, 53 Mason-Garcia, M., 68, 71, 91, 98 Massa, E., 335, 363 Masui, Y., 400, 419, 434 Masushige, S., 152, 155, 354, 361 Mathews, L. S., 202, 206, 312, 326 Matrisian, L., 369, 390 Matsubara, K., 140-141, 158 Matsuda, I., 314, 3.28 Matsukara, S., 90, 96 Matsuo, H., 30-31, 60 Matsuura, S., 398, 401, 434-435 Matsuyama, M., 398, 401, 415, 434-435, 438 Matthias, P., 316, 328 Matulich, D. T., 349, 358 Maule, A. G., 77, 99 Maurer, R. A., 153,157,320,326 Mayer, G. L., 168, 176, 183, 192,207, 374, 389 Mayer, H., 300,305 Mayer-Gostan, N., 290, 295, 304 Mayo, K. E., 5-7, 14, 17, 22, 60, 65 Mazeaud, F., 244, 267 Mazzuca, M., 10, 52 McArdle, C. A., 152, 155 McBride, J. R., 9, 55 McBride, W., 316, 327 McCann, S. M., 5, 55 McChesney, R. E., 170, 175, 319,328 McCormick, A., 169, 175 McCormick, D. J., 138, 154, 157 McCormick, K., 372, 390 McCormick, S. D., 9, 59, 214, 223-224, 377, 385,391 McDonald, J. K., 254, 261, 267-268 McDonald, M., 283, 305

AUTHOR INDEX

McDonnell, D. P., 348, 364 MeDougall, J. G., 283,303 McGinnis, W., 328 McKeown, B. A,, 9-10, 18,50, 59, 183, 208,276, 283,306,368,389 McKernan, P. A,, 247,266 McLachlan, A. D., 339, 362 McMaster, D., 69, 71-72, 91, 96-97 McNeilly, A. S., 145, 149, 154 McNeilly, J. R., 145, 149, 154 McRow, J. E., 3, 9-10, 18, 31, 50, 59, 260,268 Meats, M., 289, 304 Meigan, G., 10, 60 Meister, M., 213, 223 Meites, J., 5, 55, 372, 389 Mellon, P. L., 143, 149, 155,158 Mendel, C. M., 376,389 Mendel, D. B., 376,389 Mercier, L., 349, 362 MCrillat, A. M., 336, 364 Merrifield, R. B., 241-242, 270 Meshinchi, S., 335, 363 Metzger, D., 335,344, 347,349,357,364 Meunier, H., 73, 97 Meyer, D. L., 38, 60 Meyer, T. E., 240,267 Mezey, E., 86, 97 Michel, G., 102-103,128,130 Miesfeld, R., 338, 362 Miesfeld, R. L., 335, 358 Migliaccio, A,, 344, 362 Milbrandt, J. D., 202, 207 Milet, C., 275, 278, 290-291,303-304 Milgrom, E., 357,359 Milhaud, G., 274, 291, 295, 304 Millan, M. M., 77, 94 Millar, R., 45, 56 Millar, R. P., 34-37, 42, 44, 48, 54, 56, 58, 60, 63 Miller, C. P., 240, 267 Miller, J., 11, 58, 339, 362 Miller, J. A,, 183, 207 Miller, K. E., 40, 60 Miller, W. L., 136, 149, 156, 167-168, 175, 180,206,413-414,416, 436-437 Milliken, C., 276-277, 282-283, 290-291,293,303,305-306 Milner, R. J., 259-260,263

Milton, R.C. de L., 42, 56, 60 Minamino, N., 7, 9-10, 18, 50, 60 Minshull, J., 422, 431 Minth, C . D., 250,252,255,267 Mita, M., 407, 435 Mitsuhashi, N., 50, 62 Miura, O., 371, 391 Miyai, K., 140-141, 158 Miyajima, K., 185, 187, 209 Miyamoto, K., 10, 15, 30-31, 60 Miyamoto, Y., 6, 12, 17, 29, 57 Miyashita, E., 108-109, 114, 131 Miyata, A., 6-7, 9-11, 14, 18, 26, 50-51, 52, 60, 62 Miyata, K., 140, 156, 256, 268 Miyata, T., 78-80, 91, 95, 113-114, 130 Miyazima, K. I., 376-377, 391 Miyoi, K., 314, 329 Mizobuchi, M., 17, 61 Mizukami, T., 183, 192,207 Mizuno, K., 11, 24, 51, 52, 65 Mizuno, M., 50, 62 Mizuno, N., 78-80,91,96 Mizuno, Y., 314, 329 Moclarress, K. J., 344, 361 Moens, U., 316,327 Mohan, S., 214,222 Mohr, E., 93, 97 Mojsov, S., 247, 263, 267 Mommsen, T. P., 225,228,231, 238, 241,243-249,264,267-269,349, 362 Momota, H., 183, 206 Monahan, J. J., 5-6, 14, 56 Monahan, M., 30, 53 Monniot, C., 49, 55 Monniot, F., 49, 55 Montecucchi, P. C., 71,97 Montgomery, D. W., 370, 387 Montminy, M. R., 232,235-236, 238, 267 Moon, T., 245 Moon, T. W., 241,245-248, 260-261, 264-265,267-268,271 Moons, L., 10, 40, 52, 60, 82, 98, 104-105,129-131,313, 326 Moore, D. D., 148,157, 164-165, 170, 175, 180, 189, 192, 204, 206, 319, 327 Moore, F. L., 46, 61, 105, 130

460 Moore, G., 69, 71-73, 96 Morel, A., 253-254,268,371 Morel, G., 389 Morell, J., 77, 94 Moretti, C., 10, 17-18, 50-51, 52, 61 Morgan, J. I., 106, 130 Morimoto, Y., 70, 78-80, 89, 91, 95, 98 Moritz, R. L., 344, 363 Moriwaki, T., 419, 434 Moriyama, S., 183, 206, 214, 224 Morley, J. E., 19, 61 Morley, P.M.K., 19, 61 Morley, S. D., 69, 71, 77-78, 80, 85-91, 93, 95, 97-98, 108, 110-112, 114, 117,129,131 Mornon, J. P., 344, 362, 370, 391 Morohashi, K., 416, 435 Morrell, J. I., 46, 64 Morri11, G. A., 418, 434 Morris, A. E., 170, 175, 319, 328 Morris, N., 248, 265 Xiortishire-Smith, R. J., 318, 325 Mosley, W., 105, 129 Moss, J. B., 229, 268 hloss, L. G., 229, 265, 268 Motin, A., 39-40, 53 Mouellic, €I. L., 314, 328 Monkhtar, M. S., 274, 304 Mouland, A. J., 300, 305 Moumor, M., 140,157 Mount, S. M., 90, 97 Mowles, T. F., 22, 53 Muccioli, G., 369, 373, 386, 389 Mukai, H., 246, 270 Muller, M., 148, 154, 316, 328, 336-337, 342, 353,358,362,364 hlullick, A,, 344, 349, 364 Mulshine, J. L., 217, 224 Mnnegumi, T., 21-22,58 blungan, Z., 19, 61 Munson, P. L., 274, 303 Munz,H., 39-40, 61 Murakami, S., 46, 61 Muramatsu, M., 316, 320, 326, 328, 347, 361 hluramoto, K., 161-163, 176, 187, 189, 192,207 hlurata, K., 183, 207 Murathanoglu, O., 40, 56 Murayama, Y., 247, 268

AUTHOH INDEX

Murphy, W. A., 22, 54 Muske, L. E., 46, 61 Mutt, V., 24, 57, 232,266 N

Naar, A. M., 320, 327 Nagahania, Y., 398 Nagahama, Y., 137-139, 155-157,393, 394-395,398-408,410,413-420, 422, 424-426,431-433,435-439 Nagamatsu, S., 219, 221, 224 Nagamatsu, S., 214, 222, 227-228, 263 Nagasawa, H., 108,132,213, 223 Nagler, J. J., 401, 438 Nahorniak, C. S., 9, 18, 31, 34-35, 42, 50, 56, 59, 61, 63, 153, 157 Nair, R.M.G., 5, 30, 60, 63 Naito, N., 139-140, 156, 185, 206, 312, 328, 376,389,399,433 Nakai, Y., 139-140, 156, 185,206,312, 328, 376, 389,399,433 Nakajin, S., 414, 416, 435, 438 Nakamura, M., 398 Nakashima, K., 183, 189,208-209 Nakata, M., 230, 246, 271 Nakayama, Y., 30, 63 Nanaki, Y., 401, 434 Nandi, J., 382, 387 Nandi, S., 372, 390 Naor, Z., 152, 156 Naruse, S., 19, 61 Nata, K., 235, 265 Natio, N., 137-138, 155 Nations, M., 314, 317, 319, 328 Natsuo, N., 314, 328 Navarro, I., 238, 248, 268 Naveh-Many, T., 300, 305 Needham, M., 337,350,365 Neeley, C. I., 30, 57 Neelissen, J.A.M., 294, 303 Nei, M., 115, 131 Neilan, J. G., 350, 353, 361 Nelson, C., 165, 168, 175, 190, 206, 229, 265,314-316,328 Nemer, M., 337,359 Nemeroff, C. B., 77, 98 Nestor, J., Jr., 153, 155 Nestor, P. V., 350, 353, 361

AUTHOR INDEX

Netherton, J. C., 402, 436 Nett, T. M., 143,156 Newby, R. F., 349, 358 Ng, T. B., 373, 389,398, 410,432,435 Ngamvongchon, S., 31, 34-35, 50, 59,61 Nguyen, T. M., 238,268 Niall, H. D., 19, 61, 72, 97, 167-168, 176, 180, 191-192,206 Nichols, R., 238, 245, 253-255, 262, 268 Nicholson, L., 300, 303 Nicoll, C . S., 168, 176, 183, 192, 207, 367,372-375,389,391 Nieburgs, A., 40, 46, 66 Niel, H., 241, 243, 266 Nikolics, K., 30, 60 Nilson, J. H., 143, 154, 156 Nilsson, S., 353, 360 Nirula, A., 213, 216, 224 Nishi, M., 214, 224 Nishi, T., 183, 192, 207 Nishioka, R. S., 9, 59, 61, 77, 94, 183, 185, 187,205,207-209,214, 220, 222-224,290,303,373,376-378, 385,390-391 Nissley, S. P., 214, 224 Nitsubuchi, H., 314,328 Niu, P. D., 376, 391 Nobukuni, Y., 314,328 Noda, M., 78-80,91,95 Noe, B. C., 253,255,268 Noe, B. D., 230,238,245,253-254,257, 261,262,267-268,270 Noji, S., 316, 328 Nojiri, H., 108-109, 114, 131 Nokihara, K., 19, 61 Nola, E., 344, 362 Nomura, M., 30, 60 Nonaka, M., 183,207 Norbury, S., 422, 431 Norgren, R. B., Jr., 46, 61 Norsted, G., 202, 206 NOSO,T., 160-163, 167,176, 183, 187, 189, 192,206-207,209 Notake, M., 70, 78-80, 89, 91, 98 Notides, A. C., 348, 358 Notomi, T., 314, 329 Nozaki, M., 33, 38-41, 61-62, 137-138, 140,155-156,256,268, 399,433 Nurna, S., 70,78-80,89-91,95-96,98 Numachi, K., 120, 131

Nunez-Rodriguez, J,, 39-40,57, 62 Nurse, P., 420-422, 431,434,436, 439 Nussbaurn, A. L., 216,223 Ny, T., 30, 62

0 Obata, K., 14,57 O’Brien, P. J., 214, 222 Odumoto, N., 139, 156 Oetjen, E., 240,270 Oetting, M., 293,303 Ogasawara, T., 194,207,367-368,388 Ogawa, M., 275,305 Ogi, K., 6-7, 10, 12, 14-15, 17, 26, 29, 57-58, 62 Ohagi, S., 230, 257, 270 O’Hara, P. J., 247, 266 Ohashi, S., 246-247, 268, 270 Ohba, Y., 420,422,439 Ohgai, H., 183, 206 Ohkubo, S., 6-7, 10, 12, 14, 26, 58, 62 Ohlsson, H., 229, 262 Ohnishi, J., 17, 50, 52 Ohno, S., 92, 97, 108, 114, 117, 131, 416, 435,438 Ohta, K., 314, 328 Oikawa, I., 117, 131 Oikawa, M., 30, 62 Ojasoo, T., 344, 362 Oka, Y., 39-40,47, 51,62 Okamoto, H., 14, 57, 235, 265 Okamoto, K., 316, 328 Okarnura, H., 370, 386 Okamura, T., 414,439 Okamura, Y., 46, 54 O’Kane, T. M., 94 Okawara, Y., 67, 69, 71, 77-78, 80, 85-91,97-98 Okazaki, K., 6, 10, 12, 14, 26, 62 Okhubo, S., 6, 10, 12, 15, 17, 29, 57 Oknet, S., 338, 362 Okret, S., 337, 341, 358 Okuamura, Y., 54 Okumoto, N., 39-40, 47, 51 Okuzawa, K., 37, 47, 58, 62 Olivereau, J., 10-11, 62, 82, 98, 104, 131 Olivereau, J.-M., 160, 176

AUTHOR I N D E X

462 Olivereau, M., 10-11, 62, 82, 98, 104, 131, 139,156, 160,176 Ollevier, F., 313, 326 O’Malley, B. W., 335-336, 346, 348, 357-358,361,364 Omichinski, J. G., 339, 358 Omura, T., 416, 435 Onda, H., 6-7, 10, 12, 14-15, 17, 26, 29, 57-58, 62 O’Neill, P. A,, 39, 63 Ong, E. S., 338, 360 Onitake, K., 400, 405, 432, 435 Ono, M., 108-110, 113-115, 117-118, 124-125,130-131,159, 160-165, 167-171, 173,176-177, 180, 187, 189, 192, 194,207-208,322, 328 Oota, T., 398, 433 Oota, Y., 140, 156, 256, 268 O’Riordan, J.L.H., 300,303 Oro, A., 338, 360 Oshimura, M., 6, 10, 12, 15, 17, 29, 57 Ostberg, Y., 230, 270 Osuga, Y., 50, 62 Ota, R. B., 71, 97 O’Toole, L., 238, 264 Ott, K., 163, 176 Ottolenghi, C., 244-245, 247, 263, 268-269 Otwinowski, Z., 341, 362 Ouhtit, A,, 371, 389 Owens, M. J., 77, 98 Oyama, H., 252,255, 268 Ozaki, T., 19, 61 P

Pabo, C. O., 317,327 Page, M., 346,360 Pakdel, F., 331,337-338,343-345, 349-350,363 Pakoff, H., 183, 185, 205 Palmiter, R. D., 202, 206, 312, 326 Paly, J., 370-371, 386, 389 Pan, F. W., 183, 197, 204 Pan, J. X., 10-12, 62 Pan, W. T., 148,157 Pang, M. K., 368,391 Pang, P.K.T., 160, 175,274-276, 282-283,289-290,294-295, 303-306

Pang, R. K., 275,289-290,305 Papkoff, H., 183, 187,204,207,209 Parent, A,, 107, 131 Park, C. M., 276-278, 282,306 Park, C. W., 229, 262 Park, I., 344, 349, 364 Park, L. K., 181, 199,204,215-217, 220, 223 Park, M., 31, 59, 260, 268 Parker, D. B., 50 Parker, D. B., 3, 7, 9-10, 12, 14, 18-19, 31, 59, 62, 232, 260, 268 Parker, M., 337, 350, 365 Parker, M. G., 335, 344, 346, 348, 359-360,362 Parks, J. S., 314, 318-319, 328 Parsons, T. F., 136, 157 Paschall, C. S., 19, 50, 54 Pasieka, K. B., 89-90, 98 Pasquier, L. D., 72, 96 Patel, M., 71, 96 Patino, R., 418-419,435-436,438 Patthy, M., 68, 71, 91, 98 Pattono, P., 373, 389 Patzelt, C., 237, 268 Pavoine, C., 243, 267 Payvar, F., 180,206 Pearson, D., 77, 94 Peaucellier, G., 421-422, 434 Pecker, F., 243,267 Peel, C. J., 180, 207 Pelissero, C., 346, 360 Pellegrini, I., 369-370, 386, 390 Pelletier, G., 73, 76, 99 Pellicer, A., 202, 205 Peng, C., 7, 9, 12, 18, 50, 65 Penschow, J. D., 276,278, 281-283, 303 Pente, J., 56 Perault, A.-M., 274, 304 Peredo, M. J., 50, 65 Perez, F. M., 5, 18, 63 Perkins, A. S., 221, 223 Permutt, A., 252,255,268 Permutt, M. A., 247,264 Perrin, F., 25, 57 Perrott, M. N., 122-123, 125, 131 Perry, S. F., 276, 282,304 Persico, M. G., 46, 55 Persson, M., 232, 266 Pescovitz, 0. H., 10, 17, 50, 52, 63, 65

463

AUTHOR INDEX

Peter, R. E., 7, 9, 12, 18, 31, 34-35, 39-40, 42, 46-47, 50, 55-57, 59, 61, 63, 65-66, 69, 82, 85, 95, 137, 150, 153, 158, 183, 185, 187,204,209, 376-377,391,399,437 Peterson, J. D., 230, 270 Petrenko, A. I., 226-228,266 Petrenko, O., 227, 266 Petrenko, 0. I., 228, 270 Petridou, B., 370,373, 388, 391 Petrino, T. R., 401-402, 405, 434, 436 Pettersson, T., 232, 266 Peute, J.. 36, 40-41, 47, 56, 64, 66 Pfaff, D. W., 46, 64 Pfaffle, R. W., 314, 318-319, 328 Philiphart, J . C., 183, 207 Philippe, J., 232, 240, 269 Phillips, H. S., 30, 60 Phillips, J. G., 368, 390 Picard, A., 421-422, 434 Picard, D., 335, 363 Picard, M., 421-422, 434 Pickering, A. D., 74, 99 Pickford, G. E., 183, 206,368, 388,390 Pictet, R., 164-165, 177, 189,208, 228, 250,252,265 Pierantoni, R., 34, 58 Pierce, J . G., 136, 157 Pieretti, M., 46, 55 Pillez, A., 82, 99 Pines, J., 424,436 Pintado, J., 247, 269 Pitts, J. E., 259,265 Planas, J. V., 137, 157, 164, 176 Plante, R. K., 337, 359 Plisetskaya, E., 261, 271 Plisetskaya, E. M., 214-216, 220, 222-224,228,231-232,238, 243-244,247-250,256,258, 260-261,262-263,266,268-269 Polenov, A. L., 102, 131, 183, 205 Pollinger, L., 341, 358 Pollock, H. G., 238, 250, 256, 260, 262, 266,269 Pollock, K. M., 197, 207 Poncelet, A.-C., 189, 192,208 Ponglikitmongkol, M., 339, 344, 347, 349-350,363-365 Pongratz, I., 341, 358 Ponta, H., 153, 157 Poonian, M. S., 5-6, 14, 56

Popenoe, E. A., 102,129 Porter, J. C., 68, 98 Posner, B. I., 369, 372,386,390 Posseckert, G., 337, 364 Possilico, J. T., 293, 303 Postel-Vinay, M.-C., 367, 369-371, 389-390 Potter, I. C., 261, 271 Pottinger, T. G., 74,99 Potts, J. T., Jr., 300, 304 Poulos, B., 369, 390 Powell, J. F. F., 47 Powell, D. H., 402, 436 Powell, J., 36 Powell, R. C., 34, 37, 63 Power, D. M., 313,328 Powers, D. A., 164-165, 167,174, 180, 183, 187, 189, 192-193,203-205, 208 Pragliola, A,, 46, 55 Pratt, W. B., 335,358,360,363 Precigoux, G., 39-40, 53 Prehn, S., 229-230, 267, 269 Prendergast, P., 346, 365 Price, D. A., 137, 156 Propato-Mussafiri, R., 18, 63 Prunet, P., 185, 207, 338, 350, 362, 367, 368,373,376-378,381-384,386, 389-391 Pryde, J., 248, 265 Pubols, M. H., 255, 262 Puenta, M., 350, 363 Purrott, R. J., 69, 98 Putnam, C., 369,387 Putnam, C. W., 370,387 Puviani, A. C., 245, 247, 263, 268 Puy, L. A,, 315, 325

Q Quelle, F. W., 371, 391 Querat, B., 140, 150, 155, 157, 332, 359 Quinn, K. A., 217,224 R

Rabin, D. U., 247, 264 Radovick, S., 30, 63, 314, 317, 319, 328 Rahal, J. O., 5, 7, 17, 22, 65

464 Raina, A. K., 71, 94 Raisz, L. G., 274, 303 Rajjo, I., 34, 54 Rall, L. B., 197, 204 Rama Krishna, N. S., 39-40, 45, 65, 107, 131 Ramalho-Ortigao, F. J., 96 Ramirez-Solis, R., 314, 328 Rand-Weaver, M., 29, 57, 160-165, 167-168, 170, 172-173,176-177, 180, 183, 187, 189, 192, 194, 207-208 Rao, N. V., 403,432 Rapoport, T. A,, 228-230, 265,267,269 Ratajczak, T., 344, 363 Raue, F., 300, 305 Rawdon, B. B., 163,176 Rawitch, A. B., 238, 260, 266,269 Rawson, E. J., 170,175, 314, 327 Ray, K., 242, 270 Raynaud, J. P., 344, 362 Raynolds, M. V., 30, 5 7 Read, L. D., 350,363 Reaves, T. A., 104, 107, 129 Reaves, T. A,, Jr., 131 Rechler, M. M., 214, 224 Redenilh, G., 335, 337, 359 Redeuihl, G., 364 Ree, A. H., 349, 363 Reed, D. K., 138,157 Reese, J. C., 344,363 Rehbein, M., 108, 130 Reichlin, S., 5, 10, 62, 63 Reik, A., 336, 363 Reinboth, R., 40, 66 Renard, A., 164-165,177, 189,208 Renfro, J . L., 301, 304 Renkawitz, R., 148, 154, 336-337, 342, 353, 358, 362,364 Renoir, J. M., 335, 358 Rentier-Delrue, F., 183, 185, 189, 192, 207-208,373,377-378,381-383, 386,390-391 Rentouniis, A,, 337, 359 Ressler, C. H., 102, 129 Reynolds, R., 240, 264 Rhodes, S. J., 320, 328 Ribot, G., 150, 154-155, 332, 359 Richard, M., 150, 154 Richards, J. F., 369, 390

AUTHOK INDEX

Richards, R. I., 164-165, 175, 180, 189, 193, 204 Richter, D., 67, 69, 71, 77-78, 80, 85-91, 93, 95-98, 108-112, 114, 117, 129-131 Richter, G., 246, 265 Ricker, A. T., 216, 223 Ridgway, E. C., 319, 326 Rigel, D. F., 261, 262 Riley, D., 336, 361 Rinfret, A. P., 72, 98 Ringold, G. M., 335, 341,359-360 Rissman, E. F., 44, 54 Rivas, R. J., 185, 208 Rivier, C., 68, 70, 72-73, 75, 98-99 Rivier, J., 5-6, 9, 22, 30-31, 49-50, 53, 59-60, 63-65,68, 70-73, 75,91, 94-97,99-100 Rivier, J. E., 7, 9, 12, 18, 30-31, 34-35, 39-40, 50, 57, 59, 61, 63, 65 Roberson, M . S., 153, 157 Roberts, C. T., Jr., 15, 58, 202, 205, 215-218,223,227,266 Roberts, J., 45, 56 Roberts, S., 68, 99 Robertson, E. J., 221,222-223 Robinson, B. G., 89-90, 98 Rochat, S., 232, 269 Roche, P. J., 80, 89-90, 98, 276, 278, 281-283 Rodriguez, J. N., 137, 157 Rodriguez, R., 336, 361 Roeder, R. G., 316,329, 353, 363 Rogers, J., 29, 63 Rogol, A. D., 314, 329 Romano, G., 34,58 Rombout, J.H.W.M., 261,262 Ronald, A. P., 403, 432 Ronner, P., 235, 238, 243-244, 258, 262, 269 Rosenberg, G. B., 247, 266 Rosenblum, I. Y., 167-168, 176, 180, 191-192,206 Rosenfeld, M. G., 5-6, 14, 60, 165, 168-170, 172-173,175-176, 190, 205-206,310-312,314-320, 326-329, 332, 334, 338, 360,365 Rosenthal, E. T., 422, 431,436 Hosenthal, S., 228, 265 Rossant, J., 314, 327

465

AUTHOR INDEX

Rossier, B. C., 372, 388 Roth, J., 71, 97, 213, 216, 224 Rothrock, J. K., 50, 65 Rottman, F. M., 183, 189,209 Rotwein, P., 181, 197, 202, 205-207, 213-215,222-223 Rouille, Y., 102, 128, 131 Rouse, J. B., 238,250,256,260,266,269 Rousseau, P., 30, 65 Roy, A,, 141, 155 Roy, B. B., 69, 72, 98 Roy, R. J., 148, 157 Rozakis-Adcock, M., 371,390 Rubin, D. A., 368, 390 Ruderman, J. V., 422,436 Rui Jin, J., 335, 346, 365 Ruiz-Grande, C., 247, 269 Rumsfeld, H. W., Jr., 68, 98 Rupnow, B. A,, 137,156 Ruppert, S., 108, 114,130-131 Rusconi, S., 338, 362 Russel, D. H., 369, 387, 390 Russel, J., 300, 305 Russell, S. M., 168, 176, 192, 207, 374, 389 Ruth, J. L., 86, 96 Rutter, W. J., 228-229, 250,252, 265-266,268 Ruvkun, G., 169, 175, 316, 326 Ryan, G. B., 276,278,281-283, 303 Ryan, R. J., 138, 154,157 Ryffel, G. U., 150, 156,336, 361

s Sabatier, R., 243, 266 Sabbagh, I., 370,387 Saceda, M., 350,363 Sack, R. A., 320,327 Sadler, S. E., 418, 436 Saffran, M., 68, 98 Sage, M., 69, 98 Sahlin, L., 349, 360 Saito, A,, 137-138, 140, 157 Saito, M., 183, 192, 207 Saitou, N., 115, 131 Sakaguchi, M., 183, 207 Sakai, H., 400, 434 Sakai, M., 320, 326, 337, 361

Sakai, N., 394,402,413-414,416,425, 431-432,435-437 Sakai, S., 372, 390 Sakakibara, S., 31, 60 Sakamoto, T., 194, 207, 220, 224 Sakata, S., 160-163, 167, 176, 187, 189, 192,207 Saki, K., 183, 207 Sala, G. B., 242, 270 Salbert, G., 338, 346, 350,360,362,368, 389 Saligaut, C., 338, 350, 362, 368, 389 Salmain, M., 344, 364 Samadpour, M., 181, 199,204,215-217, 220,223 Sanchez, E. R., 335,358,360,363 Sanchez-Pescador, R., 197,204 Sandra, O., 373 Sano, H., 183,209,376-377,391 Santos, A.J.G., 399, 436 Sara, V. R., 214, 222 Sasaki, A., 10, 60 Sasson, S., 348, 358 Sassone, C. P., 153, 155 Sassone-Corsi, P., 337, 360 Sato, M., 108-109, 114, 118, 125, 131-132,206 Sato, N., 183, 207 Sauer, R., 167-168, 176, 180, 191-192, 206 Saunders, G. F., 232,235-236,267,271 Saunders, T. L., 143,156 Sausville, E., 108-109, 114, 131 Savouret, J. F., 357, 359 Sawangjaroen, K., 19, 63 Sawchenko, P. E., 312,326 Sawyer, W. H., 275,289-290,305 Scanes, C . G., 5, 18, 26, 53, 57, 63, 183, 205 Scanes, N., 183,207 Scarpa, A., 244,258,269 Schafer, H., 40, 63 Schaffner, W., 316,328 Schally, A. V., 5, 30, 60, 63, 68, 71, 91,98 Scharrer, B., 104, 131 Scharrer, E., 104, 131 Schauer, M., 341,358 SchaufeIe, F., 320, 329 Scheer, E., 335,347,364

AUTHOR INDEX

Scheidereit, C., 316,329 Schena, M., 341,363 Scherer, G., 114,131 Schemer, L. C., 335,363 Scheuer, I., 153,155,337,360 Schild, C., 336, 364 Schilling, J. W., 183, 207 Schlesinger, D., 69, 71-72, 96 Schlesinger, D. H., 68, 71, 91, 98 Schmale, H., 108, 130 Schmidt, T. J., 337, 359 Schmidt, W. E., 19, 57, 247, 260, 263, 2 70 Schnee, M. E., 71, 94 Schoderbek, W. E., 153,157 Scholz, K., 39, 56 Schlinrock, C., 67,68-69, 71,77-78,80, 85, 87-89,91,96-97, 108, 111-112, 114, 131 Schooley, D. A., 71,96-97 Schoonen, W.G.E.J., 401,436 Schorpp, M., 336, 361 Schrader, W. T., 336,348,361,364 Schreck, C. B., 77,98-99,400,431 Schreibman, M. P., 38-40, 45, 55-56, 63, 367,390 Schroedter, I. C., 285, 287-288, 300,305 Schule, R., 336-337, 362,364 Schulz, R., 40, 63 Schulz, R. W., 47, 63 Schutz, G., 108, 114,130-131 Schutz, G., 336, 361, 363 Schwaninger, M., 240,270 Schwanzel-Fukuda, M., 46,64 Schwartz, T. W., 261,264 Schworer, H., 19, 57 Scotland, G., 248, 265 Scott, A. P., 400-404,430-431,433,436 Scott, L. A,, 213, 216, 224 Scott, M. P., 317,329 Scully, K. N., 320, 328 Searle, N., 369, 386 Seasholtz, A. F., 80, 89, 98-99 Seavey, B. K., 183,206 Seeberg, P. H., 183,206-207 Seeburg, P. H., 30, 51,53, 57, 60, 64, 192 Segil, N., 39, 56 Seihamer, J. J., 183, 207 Seiler-Tuyns, A., 336, 364

Sekine, S., 137-138, 140, 157, 183, 192, 206-207 Sekkali, B., 189, 192, 208 Selby, M. J., 192, 208 Selman, K., 398,401,436,438 Selye, H., 68, 98 Seppala, M., 45, 64 Sevarino, K. A,, 252, 254-255,270 Shabanowitz, J., 71, 94 Shackleton, C.H.L., 403, 438 Shakur, Y., 248,265 Shamblott, M., 214, 218, 220, 222 Shamblott, M. J., 179, 181, 197, 199-200, 202, 208,215,224 Shamsuzzaman, K. M., 376,389 Shani, J., 372, 386 Shapiro, D. J., 337-338, 365 Sharp, P. A,, 165, 169, 175-176 Shaw, F. D., 106,129 Sheares, B. T., 255, 262 Shen, L., 228, 250,252,265 Sheppard, P. O., 247, 266 Sheridan, M . A,, 244,269 Sherwood, 44 Sherwood, L. M., 300, 305 Sherwood, N., 31, 35, 64 Sherwood, N. M., 3, 7, 9-10, 12, 14, 18-19,30-32,34-36,38-41,46-50, 54, 57, 59, 61-62, 64-66, 108, 110, 117,129, 142,157,232, 260, 268 Shibahara, S., 70. 78-80, 89, 91, 95-96,98 Shibata, N., 418, 439 Shieh, H.-S., 183, 203 Shields, D., 229,253, 257, 263, 270 Shih, S. H., 35, 65 Shiloach, 71, 97 Shimizu, A., 400,437 Shimizu, M., 6, 10, 12, 15, 17, 29, 57, 89 Shimizu, S., 70, 78-80, 91, 98 Shine, J., 164-165, 175 Shinoda, M., 416, 435 Shinoda, N., 416,438 Shirahata, K., 185, 208, 373, 376-378, 390 Shirota, M., 370-371, 386-387, 390 Shiu, R.P.C., 372, 390 Shively, J. D., 253, 259, 262 Shively, J. E., 238, 245, 262 Shuldiner, A. R., 213,216, 224

AUTHOR INDEX

Shupnik, M. A., 136, 142, 149-150, 152, 155,157,350, 364 Siege], E. G., 247, 270 Siegfried, J. M., 217, 224 Siegfried, R., 316, 328 Sigler, P. B., 341, 362 Silbergeld, A., 214, 223 Silkstrom, R. J., 369, 386 Silsby, J. L., 170, 177, 323, 330 Siltala-Roos, H., 341, 359 Silvennoinen, O., 371, 386, 391 Silver, J., 300, 305 Silverman, A. J., 46, 64 Silverman, R. C., 39, 63 Simanis, V., 422,436 Simizu, A., 400, 437 Simmons, D. M., 165, 169-170, 172, 1 7 5 1 7 6 , 3 1 1 , 314-316, 320, 326-327,329 Simmons, J. G., 235, 267 Simons, S. S., 349, 358 Simpson, E. R., 180,208,414,439 Simpson, R. J., 344, 363 Singh, R.N.P., 183, 206 Sirahata, K., 183, 206 Sitton, J., 11, 66 Skibeli, V., 376, 386 Skoblina, M. N., 417,431 Slater, C. H., 34, 53 Slater, E. P., 167, 176, 180, 208, 337, 364 Slusher, M. A., 68, 99 Smeekens, S. P., 230, 257, 270 Smit, A. B., 181,208,213,224 Smith, D. F., 335, 364 Smith, D. N., 360 Smith, G. R., 89, 99 Smith, J. S., 349, 364 Smith, L. D., 400, 438 Smith, R. A,, 247, 266 Smith, W. W., 183, 203 Snell, C . R., 76, 99 Sniffen, C. J., 180, 207 So, S.T.C., 244, 258, 266 So, Y. P., 274-276, 303, 305,401, 438 Sobel, D. O., 74, 99 Soderberg, C., 259-260, 263,266 Sohn, J., 10, 62 Solan, N., 18, 51, 61 Solter, D., 143, 155 Sombre, E. R., 376, 387

467 Somogyvari-Vigh, A., 11, 24, 51, 52, 58, 65-66 Song, S., 185, 208 Sorokin, A. V., 227-228,270 Sower, S. A., 30-31,33-35,38,48, 53-54,58, 64-65, 102, 130 Spagnuolo, A., 34, 58 Spangelo, B. L., 24, 65 Specker, J. L., 183, 185, 187,208-209, 368, 373, 376-378,390-391 Spiess, J., 5-7, 9, 12, 18, 22, 31, 49-50, 63-65, 68, 70, 72, 75, 98-99, 230, 253-255,257,267-268,270 Sprecher, C . A., 247,266 Srivastava, C. H., 50, 65 Stacey, N . E., 399,437 Stachura, M. E., 5, 65 Stack, G., 332, 361 Stannius, H., 273, 275, 305 State, D., 348, 364 Staub, A,, 332, 334, 344, 361 Staudt, L. M., 316, 327 Stein, M., 261, 262 Stein, R., 270 Steiner, C., 148, 154, 353, 358 Steiner, D. F., 181, 204, 213, 214-220, 222-224,227-230,257,263,270 Steiny, S., 376, 388 Steiny, S. S., 183, 207 Stell, W. K., 34, 38-39, 45, 59, 65 Steneveld, A. A., 34, 58 Stenzel, P., 71, 99 Stenzel-Poore, M. P., 71, 99 Sterba, T., 285,287-289,300,305 Stevenson, J. L., 369,388 Stewart, A. F., 336, 363 Stibbs, H. H., 7, 10, 58 Stickney, R. R., 385,391 Stobie, K., 240, 264 Stork, P., 252, 254-255, 270 Strahle, U., 336, 353, 361, 364 Strassburger, W., 259, 265 Strating, M. J., 318, 329 Stricker, C., 335, 358 Stropp, U., 353, 361 Stumpf, W. E., 39-40,61, 66 Sturm, R. A., 169,175,316,318,329 Subhedar, N., 39-40,45,65, 107,131 Sugita, R., 105, 107, 123, 131 Suhr, S. T., 5, 7, 17, 22, 65

468

AUTHOR INDEX

Sullivan, C. V., 243, 248, 269 Sumpter, J. P., 74, 99, 137, 158, 400, 436 Sun, Y. L., 337, 359 Sundararaj, B. I., 395, 403, 432, 437 Sundell, K., 282, 301, 305 Sundler, F., 11, 65 Sundmark, V. C., 315,325 Sussenbach, J. S., 216, 223 Susuki, Y., 213, 223 Suzuki, A., 213,223 Suzuki, K., 135, 136-141, 143-144, 155-158, 399,401, 403,432-433, 435,437 Suzuki, M., 31-32, 35, 39, 45-47, 52, 65, 108, 110, 117, 120,129-130,132 Suzuki, R., 376-377, 391 Suzuki, S., 246-247,268,270 Suzuki, T., 19, 61 Svec, F., 348, 364 Swanson, L., 165, 169,175, 314, 316,327 Swanson, L. W., 169-170, 172-173, 175-1 76,311,314-316,320, 326-327,329 Swanson, P., 31,59, 136-138, 140, 155-158, 164, 172, 176,214, 220, 223-224, 313,328,399,406,433, 437,439 Swarup, K., 294, 302 Sweeney, G., 248, 265 Swennen, D., 189, 192,208,377-378, 390-391 Sydenham, M., 346,360 Szabo, M., 52 Szoke, R . , 68, 71, 91, 98 T

Tada, K., 230, 271 Tagaki, Y., 305 Tager, H., 230, 270 Taillon-Miller, P., 46, 55 Takada, T., 235,265 Takahashi, A., 183, 185, 206, 214, 224, 376,389 Takahashi, H., 70, 78-80, 89, 91, 95-96,98 Takahashi, M., 413,416,436-437 Takahashi, S. Y., 213, 223

Takayama, Y., 160-165, 167-168, 170-171, 173,176-1 77, 180, 187, 189, 192, 194,207-208,322,328 Takehara, A., 207 Takemura, R., 316,328 Takezawa, T., 127,130 Tamaoki, B., 401,435,437 Tamaoki, B. I., 403, 437 Tan, C. H., 368, 391 Tan, E.S.P., 403, 437 Tan, L., 30, 65 Tanaka, H., 37, 62 Tanaka, M., 138,156, 183,209,393,394, 413-417,435-438 Tanaka, U., 183, 189,208 Tang, B., 371,391 Tang, E.K.Y., 248, 265 Tang, Y., 180, 183, 187, 189, 192-193, 208 Tang, Y.-L., 179, 214, 222 Taniguchi, S., 316, 328 Tanizawa, Y., 247, 264 Tarkey, J. F., 374, 389 Tarpey, J. F., 372, 391 Tashijian, A. H., Jr., 290, 293, 303 Tasi, P. I., 214, 224 Tasset, D., 335, 347, 364-365 Tatemoto, K., 24, 57, 259, 265 Tatsumi, K., 141, 158 Tatsumi, K.-I., 314, 329 Tatsuno, I., 24, 65 Taverne-Thiele, J. J., 261, 262 Tavianini, M. A., 250, 252, 255, 267 Taylor, M. H., 401, 432 Taylor, R. G., 36, 64 Taylor, W. L., 250, 252, 255, 267, 270 Telecky, T. M., 414,416-417,437 Temple, T. E., 99 Tentler, J., 30, 52 Tentler, J. J., 369, 388 Terazono, K., 235, 265 Terenius, L., 24, 57 Terlou, M., 40, 53 Teubner, V., 348,364 Theill, L., 169-170, 173, 175 Theill, L. E., 165, 169, 175, 177, 190, 204,314, 316,318-319,326,329 Theofan, G., 401,438 ThCzC, N., 339, 350, 353,362

469

AUTHOR INDEX

Thim, L., 230,237-238,250,256-257, 260-261,263-264 Thomas, D. Y., 257,263 Thomas, P., 40, 58, 349, 364, 401, 403, 418-419,435-436,438 Thompson, E. B., 338,360 Thompson, R. C., 80,89,98-99 Thoreau, E., 370, 391 Thorens, B., 247, 270 Thorgaard, G. H., 19, 51,216,222 Thorner, J., 5-6, 49, 59 Thorner, M., 22, 49, 63 Thorner, M. O., 5-6, 55 Tickle, I. J., 259, 265 Tierney, M., 123, 129 Tisseran-Jochem, E. M., 278, 290-291, 304 Toft, D. O., 335,364 Tokumoto, T., 425, 432 Tonlorenzi, R., 46, 55 Tonon, M.-C., 7, 10, 18, 54, 73, 76, 99 Tom, L., 44, 58, 152, 155, 335-336, 344, 347, 349, 353-354,358,361,364 Totty, N. F., 71, 96 Trahair, J. F., 276, 278, 281-283, 303 Trakatellis, A. C., 271 Traniu, G., 10, 24, 52, 54, 82, 99 Tran, T. N., 73, 99 Trant, J. K., 418, 438 Trant, J. M., 401, 403, 438 Trautmann, M., 246, 265 Treacy, M. N., 169-170, 172-173,175, 314,316,327,329 Tregear, G. W., 80, 89-90, 98, 276, 278, 281-283,303 Treston, A. M., 217, 224 Trifiro, M. A,, 337, 359 Trinh, K.-Y., 137, 140-141, 150, 158, 185, 208 Tripp, R. A., 77, 99 Trippet, S., 102, 129 Troetschler, R. G., 71, 96 Truong, A. T., 164-165,177, 189,208 Truscott, B., 401,438 Tsai, H. J., 183, 208 Tsai, M. J., 335-336,346,348,357-358, 361,364 Tsai, S. Y., 335-336,346,357-358,361, 364

Tsai-Morris, C. H., 370, 391 Tsamaloukas, A., 229, 269 Tsukahara, T., 33, 38-41, 61-62 Turcotte, B., 353,361 Tyler, C. R., 137, I58

U Uchiyama, M., 160, 175 Uddman, R., 11, 65 Uehara, A,, 7, 9, 18, SO, 60 Uemura, H., 107,132 Ueno, N., 21-22,58 Ulshen, M. H., 235,267 Umesono, K., 364 Underwood, L., 214, 223 Unson, C. G., 241-242,270 Upadhyaya, N., 401, 403,438 Upton, Z., 214, 224 Urano, A., 31-33, 35, 39, 45-47, 52, 54, 65,86, 96,101, 104-105, 107-1 10, 112-115, 117-118, 120-127, 129-132, 139,156 Uyeno, T., 89,99 V

Vaillant, C., 337, 344, 363-364 Vale, W., 5-6, 9, 18, 22, 30-31, 49-50, 53, 59-60, 63-65, 68, 70, 72-73, 75, 94,98-99 Vale, W. W., 7, 9-10, 12, 18, 39-40, 50, 53, 57, 63, 65, 71, 99 Valla, S., 164, 175, 189, 192-193, 205 Valotaire, Y., 331, 337-338, 345-346, 349-350,360,362464,368,389 Valverde, I., 247, 269 Vamvakopoulos, N., 59 van Bohemen, Ch. G., 401, 434 Van den Brande, J. L., 216, 223, 314, 318-319,328 Van Den Hurk, R., 403,434 Van Der Kraak, G., 137, 153, 154, 1.58, 400, 408,431,438 Van Der Kraak, G. J., 408,438 Vanderlaan, W. P., 163, 176 van der Meij, J.C.A., 160, 177

AUTHOR INDEX

470 Van der Nat, H., 314,318-319,328 Van Der Sanden, M.C.A., 47, 63 van der Vliet, P. C., 316, 318, 326 Vandesande, F., 10-11, 24, 40, 52, 60, 62, 82,98, 104-105,129-131,313, 326 Van d e Vliet, P. C., 318, 329 Van Dijk, W., 47, 63-64 van Eys, C.J.J.M., 160, 177 van Goor, F., 153,154 Van Leeuwen, H. C., 318,329 vanSchaik, F.M.A., 216,223 van Weperen, W. W., 318,329 Van Wyk, J . J., 214, 223 Vaudry, H., 7, 10, 18, 30-31, 54, 73, 76,99 Vaughan, J. M., 7, 9, 12, 18, 50, 65 Vegeto, E., 348, 364 Ventimiglia, R., 252, 254-255, 270 Vera, A., 202, 205 Verbost, P. M., 283, 306 Verburg-van Kemande, B.M.L., 73, 99 Verkley, A. J., 47, 66 Verrijzer, C. P., 316, 318, 326, 329 Vessikres, A., 344, 364 Vidal, B., 278, 290-291, 304 Vigh, S., 11, 24, 58, 66 Vigna, S. R., 34, 54 Vijayan, M., 245 Violand, B. N., 183, 203 Voelkel, E. F., 290, 293, 303 von Bartheld, C . S., 38, 60 von Heijne, G., 78,99-100 von Schalburg, 44 von Schalburg, K., 32, 35, 54 Voss, J . W., 170, 172, 176, 310-312, 314-315,317-320,327,329 Vreugdemjo, E., 213,224 Vreugdenhil, E., 181, 208

w Wada, C., 117, 131 Wagner, G., 285, 291, 295,304 Wagner, G. F., 183,208, 273, 274. 276-279,281-285,287-301, 304-306 Wagner, R. W., 71, 94 Wagner, T., 143, 154

Wagner, U., 336, 361 Wahli, W., 336-337, 349, 359,362, 364-365 Wahlstrom, T., 45, 64 Wakamiya, M., 316,328 Walker, K. M., 247, 266 Walker, M. D., 229, 262, 266 Walker, P., 336, 364-365 Walker, S. E., 39, 45, 65 Wall, R., 29, 63 Wallace, R. A., 137, 156, 398, 401-402, 405,410,432,434,436,438 Wallace, R. M., 145, 149, 154 Wallance, J . C., 214, 224 Wallis, A. E., 199, 208, 215-216, 218, 224 Walter, B. T., 183, 207 Walter, P., 337, 360-361 Wang, C., 3 0 , 5 7 Wang, N. C., 137, 141, 150,158 Wang, Y., 256, 271 Wang, Y. C., 140-141,155 Warby, C., 36, 64 Warby, C . M., 35, 61 Warne, J. M., 123, 129 Wasserman, W. J., 400, 438 Watahiki, M., 183, 189, 208-209 Watanabe, K., 183, 207, 209 Watanabe, T., 12, 62 Watanabe, Y. D., 311, 330 Waterman, M. R., 414, 439 Weber, B., 237, 268 Webster, N., 335, 364 Webster, N.J.G., 335, 346-347, 365 Wegner, M., 316,327 Wehrenberg, W., 5-6,21-22, 56, 59 Wehrenberg, W. B., 5-7, 10, 21-22, 52-53,55,58 Weigel, N. L., 335-336, 346, 357, 361, 364-365 Wed, C . , 153, 158, 332, 365, 394, 400, 403,433 Weiler, I. J., 337-338, 365 Weinberger, C., 332, 338,360,365 Weiner, R. I., 143, 158, 187, 204 Weinstein, S. P., 418, 434 Weintraub, B. D., 30, 63, 314, 317, 319, 328 Weir, G. C., 247, 267 Weisbert, M., 418, 434

47 I

AUTHOR INDEX

Weisz, A,, 353, 365 Weith, H. L., 250, 252, 255, 267, 270 Welch, W. J., 335, 358 Weld, M. M., 74-75,100 Wendelaar Bonga, S. E., 160, 177, 274, 276,282-283,289,294,303-304, 306, 377-378,391 Wennen, D., 183, 185,207 Werner, S., 24, 5 7 West, B. L., 320, 329 Wheeler, M. B., 246-247, 264, 271 White, B. A., 372-375,389,391 White, J., 335,364 White, J. W., 232, 235-236, 271 White, R., 335, 337, 344, 346, 348, 350, 359-360,365 White, T. J., 93, 100 Whiting, J., 314, 328 Whitt, G. S., 72, 95 Wierman, M. E., 30, 57, 136, 141-142, 149-150, 152,155 Wilkinson, S. P., 344, 363 Willard, H. F., 46, 55 Willems, P., 283, 306 Williams, E. E., 383, 388 Wilson, A. C., 93, 100 Wilson, C . E., 153, 155 Wilson, D. M., 214, 224 Wilson, J. X., 77, 94 Wilson, L., 317-319, 327, 329 Wilson, S. W., 183, 204 Wilstrom, A. C., 338, 362 Windle, J. J., 143, 149, 155, 158 Winkler, J., 228, 265 Winter, J.S.D., 314, 330 Wirsig, C . R., 46, 66 Wit, J. M., 314, 318-319, 328 Witthuhn, B. A,, 371,386,391 Wolf, U., 92, 97, 108, 114, 117, 131 Wolff, J., 339, 350, 353, 362 Wolffe, A. P., 336, 364 Wolfson, B., 94 Wollmer, A., 259, 265 Wondisford, F. E., 30, 63, 314, 317, 319, 328 Wong, A. O., 153, 154 Wong, E. A., 170,177,323,330 Wvng, K. L., 75, 77,81-82,91,96, 100 Wong, L. Y., 368, 391 Woo. N., 75, 91, 96

Woo, N.Y.S., 77, 88, 100 Wood, J. G., 261,268 Wood, S. P., 259, 265 Wood, W. M., 30, 57,319,326 Woods, D. E., 216,223 Workman, J. L., 336, 357 Woychik, R. P., 183, 189, 192, 209 Wrange, O., 337,359 Wranger, O., 341,359 Wray, S., 40, 46, 66 Wright, A., 339, 341, 359, 365 Wright, D. E., 38-39, 66 Wright, K., 339, 365 Wright, P. E., 318, 325 WU, C.-I., 25, 58 Wu, D., 173, 177, 318, 329 Wu, J., 336, 358 Wu, P., 12, 36, 51, 66 Wu, X. P., 335, 358 X

Xiong, F., 135, 140-142, 145-148, 150-152,158, 173,175, 189, 192, 209, 321, 323-324, 326,330, 354, 365 Xu, W. X., 341,362 Y

Yada, T., 24, 66, 127, 132, 246, 271 Yamada, C., 104, 127,130,132 Yamada, J., 305 Yamada, M., 30, 63 Yamaguchi, A., 419-420,422, 425,439 Yamaguchi, K., 183, 185, 187,206, 208-209,373,376-378,390-391

Yamaji, K., 230, 271 Yamakawa, M., 183,209 Yamamoto, H., 235, 265 Yamamoto, K., 183, 187, 209, 399, 401, 435,439 Yamamoto, K. R., 335, 338-339, 341, 360,362-363 Yamamoto, M., 183, 209 Yamamoto, X. R., 341, 363 Yamanaka, M. K., 183,207 Yamashita, K., 246-247, 268, 270

472 Yamashita, M., 138, 156, 393, 394, 419-420,422,424-427,432-433, 435,439 Yamashita, T., 122-124, 129 Yamauchi, J., 152, 155,354,361 Yamauchi, K., 401,439 Yamazaki, N., 187, 209 Yan, L., 406, 439 Yanagisawa, T., 108, 120, 130 Yanaihara, N., 14, 46, 54, 5 7 Yang, B.-Y., 179, 214, 222 Yang, J., 372, 390 Yang, X., 371, 386 Yanow, C. E., 214,222 Yao, K., 376, 391 Yao, R.-P., 320, 329 Yasuda, A., 29, 57, 168, 175, 180, 183, 185, 187,205-207,209,376-377, 391 Yasuda, H., 420,422,439 Yasunaga, T., 113-114,130, 160-163, 167,176, 187, 189, 192, 207 Yates, N. A., 283, 303 Yi, T., 371, 391 Yi-Qiang, W., 261, 271 Ymer, S. I., 369, 388, 391 Yokoo, Y., 183, 185, 187, 209, 377, 391 Yoo-Warren, D., 247, 264 Yoshikuni, M., 138, 156, 393, 394, 407, 418-420,422,424-426,432,435, 439 Yoshinaga, K., 10, 60 Youdim, M.B.H., 372,386 Young, B. K., 185,208 Young, G., 9,59,214, 220,222-224, 373-376,387,400-401,404-405, 408, 410,417,433,435,439 Young, P. S., 377, 385, 391 Young, W. S., 111, 30, 60

AUTHOR I N D E X

Youngblom, J., 422, 431 Youson, J., 221 Youson, J. H., 238, 246, 256, 261, 262-264, 271, 274,282,304,306 Yu, K. L., 35, 46-47, 65-66, 153, 157 Yu, V. C., 320, 328 Yulis, C . R., 75, 77, 81-82, 85, 88, 91, 96,100, 104, 132 Yun, J., 143, 154 L

Zaidi, M., 302, 303 Zandbergen, M. A., 40, 47, 56, 63, 66 Zandbergen, T., 31, 45, 52 Zanetti, L., 34, 58 Zangger, I., 214, 224 Zanuy, S., 40, 5 7 Zapf, J., 214, 224 Zentel, H. J., 40, 66 Zeytin, F., 5-6, 14, 56 Zeytinoglu, F. N., 290, 293, 303 Zhang, J., 246, 271 Zhang, P., 183, 205 Zhang, R., 370, 391 Zhang, Y.-S., 259, 265 Zhao, Z., 419-420,422, 425,439 Zheng, H., 314,328 Zhu, Y., 419, 439 Zhu, Z., 189, 192-193,209 Zilliacus, J., 339, 365 Zlochevskij, M. L., 227-228,270 Zoeller, R. T., 30, 60 Zohar, Y., 31-32, 56,400, 403, 431 Zuber, M. X., 414,439 Zuffardi, O., 46, 52 Zukoski, C . F., 370, 387 Zwiers, H., 69, 77-78, 90-91, 97

SYSTEMATIC INDEX Names listed are those used by the authors of the various chapters. No attempt has been made to provide the current nomenclature where taxonomic changes have occurred. A

Acanthopagrus schlegeli, 37 Acipenser transmontanus, 9, 10, 14, 19, 20, 32, 34 African cichlid, see Haplochromis burtoni Agnatha, 33-38 Amia calua, 34, 238, 245, 256, 260, 273, 282 Amphioxus, see Branchiostoma californiensis Anglerfish, see Lophius americanus Anguilla, 246 A. anguilla, 10, 34, 48, 82, 139, 140, 239, 258,275,290-291 A . australis, 276 A. japonica, 39, 214 A. rostrata, 10, 39, 238, 239, 241, 260 Arctic char, see Salvelinus alpinus Ascipenser transmontanus, 9, 10, 19, 20, 32,34 Asterina pectinifera, 420, 421, 425, 428, 430 0

Bass sea, see Dicentrarchus labrax; Centropristis striatus striped, see Morone saratilis Blue gill, see Lepomis macrochirus Bonito, see Katsuwonus pelamis

Bowfin, see Amia caloa Branchiostoma californiensis, 181, 219, 227 Bream Black Sea, see Acanthopagrus schlegeli Red Sea, see Pagrus major sea, see Sparus aurata Bufojaponicus, 109, 110, 115, 116 Bullhead, brown, see lctalurus nebulosus L

Caenorhabditis elegans, 316 Calamoichthys calabaricus, 34 Callorhynchus milii, 238 Carassius auratus, 9, 10, 18, 35, 39, 41, 47, 73, 74-75, 77, 81, 89, 104, 119, 123, 137, 153, 259,260, 394-395, 399,408,420-421,423-427 Carp, 7, 9, 23, 70, 78, 89, 92, 93, 141, 187,376-377 Chinese grass, 189, 193 common, see Cyprinus carpio Catfish, 4,15, 22, 29, 31, 33, 42, 47 African, see Clarias gariepinus channel, see Zctalurus punctatus Indian, see Mystus oittatus; Heteropneustes fossilis Malaysian, see Clarias macrocephalus, 10, 19, 20 Tai, see Clarias macrocephalus Ca tostomus commersoni, 69, 70, 74, 8091, 108, 110, 111, 112, 114, 117, 120 473

474

SYSTEMATIC I N D E X

Centropomus undecimalis, 36 Centropristis striatus, 36 Chanos chanos, 35 Chondrichthyes, 38-39 Clarias C . batrachus, 35, 39 C . gariepinus, 35, 44, 45, 47, 139, 403 C . macrocephalus, 9, 14, 17, 32, 35, 50, 403 Clupea harengus pallasi, 35 Codfish, 187, 188 Atlantic, see Gadus morhua Colisu lalia, 285, 373 Coris julis, 37 Cottus scorpius, 237,238, 250,256, 260 Croaker, Atlantic, see Micropogonias undulatus Cynoscion nebulosus, 403-404, 418, 419 Cyprinidae, 92-93 Cyprinus carpio, 10, 12, 14, 69, 137, 139. 163, 183, 193, 194, 228, 229, 230, 399,422

D

Dicentrurchus labrax, 10, 39, 313 Dogfish, 29, 30, 33, 42, 47, 48, 102; see also Squalus acanthias; Poroderma africanum common, see Scyliorhinus canicula Dragonet, see Repomucenus beniteguri Drosophila, 169, 317

E

Eel, 105, 107, 182, 237, 274, 276, 282-283,376-377 American, see Anguilla rostrata Australian, 278-279, 280-282 European silver, see Anguilla anguilla Japanese, see Anguilla japonica moray, see Gymnothorax jimbriatus Elephantfish, see Callorhynchus milii Epinephelus akaara, 37 Eptatretus stouti, 109 Escherichia coli, 343

F

Flounder, 70, 123, 125, 128, 163, 182, 183, 187, 188, 237; see also Platichthys jlesus Japanese, see Paralichthys olicaceus winter, see Pseudopleuronectes americanus Fundulus heteroclitus, 368, 398, 402, 405

G Gadus morhua, 10, 12, 160-163, 164, 187 Gadus morhua morhua, 36 Gar, 237 alligator, see Lepisosteus spatula Geotria australis, 261 Gillichthys, 368 Gobys, 214 Goldfish, see Carassius uuratus Grouper, red spotted, see Epinephelus akauru Guppy, see Poecilia reticulata Peters Gymnothorax jimbriatus, 35 H

Hagfish, see Eptatretus stouti Atlantic, see Myxine gutinosa Hake, see Merluccius capsensis Halibut, 187, 188 Haplochromis burtoni, 31, 36, 45 Hemitripterus americanus, 229 Herring, see Clupea harengus Heteropneustes fossilis, 395 Hydrolagus colliei, 32, 34, 41, 230, 238, 256 I

Ictalurus, 246 1. nebulosus, 241 I . punctutus, 163, 183, 186, 187, 188, 189, 193, 238, 250, 252, 255,258 lctarus mellas, 368

SYSTEMATIC INDEX K

Katsuwonus pelamis, 137, 139, 163 Kisu, see Sillago japonica L

Lamprey, 29, 31, 33, 42, 47, 48, 108, 243; see also Geotria australis; Mordacia mordax sea, see Petromyzon marinus Lepisosteus spatula, 34, 238,260 Lepomis macrochirus, 39 Lillium longiflorum, 420 Locusta migratonia, 71 Lophius americanus, 228-233, 234-236, 238, 245,250-254,257,260,261 Lumpfish, 187, 188 Lungfish, 103 M

Manducka sexta, 69, 71 Medaka, see Oryzias latipes Merluccius capsensis, 35 Micropogonias undulatus, 403,419 Milkfish, see Chanos chanos Molly, see Poecilia latipinna; P o e c i h formosa Mordacia mordax, 261 Morone saxatilis, 31, 183 Mugil M . cephalus, 36 M . ramada, 11 Mullet, see M u g i l ramada; M . cephalus Mummichog, see Fundulus heteroclitus Myoxocephalus octodecimspinosus, 11 Mystus oittatus, 294, 403 Myxine glutinosa, 181, 219, 228, 229, 230, 256

0 Oncorhy nchus 0. gorbuscha, 107

0. keta, 12, 29, 35, 108, 109, 110, 113, 117, 119, 120, 121, 125, 137, 139, 140, 161-163, 164-165, 166, 169-171, 173, 182, 183, 186, 187, 188, 189, 193,215-218,226-227, 228,229,313,322,376-377,399, 404 0. kitsutch, 77, 137, 140, 163, 164, 172, 181, 183, 197, 199, 214, 215-217, 220, 238, 250,255, 258, 260,261,276-277,278-279, 280-282, 290,399 0. masou, 35, 39, 40, 43, 45, 46, 47, 108, 113, 119, 120, 121, 125 0. mykiss, 9, 10, 18, 35, 43, 74, 104, 105, 113, 119, 120, 121, 123, 125, 127, 128, 140, 141, 145, 146, 150, 164, 167, 171-172, 181, 183, 189, 192, 193, 194, 197, 198, 199, 200-202,215,218-220,241,246, 277, 284, 290, 291, 292, 293, 295, 296, 297, 298, 299, 331, 333, 337, 338, 340, 344, 345, 349, 350, 353, 355, 368, 373, 395,404, 412-418, 429 0. nerka, 9, 10, 14, 17, 19, 20, 32, 35, 43, 44, 108, 113, 117, 119, 120, 121, 286, 287,288, 300,402-403 0. rhodurus, 401, 404,407-410 0. tshawytscha, 11, 35, 43, 139, 140, 141, 142-143, 144, 147, 151, 173, 189, 192, 193, 199, 215-217, 284, 321,323,376-377,404 Oreochromis 0. mossambicus, 373,376, 377-378, 383 0. niloticus, 374, 375, 376, 377-381, 382-384,385,395-396 Oryzias latipes, 123, 398, 402 Osteichthyes, 39-41 P

Paddlefish, see Polyodon spathula Pagrus major, 37, 183, 396-397 Paralichthys olioaceus, 37, 160-163, 418, 419 Perca jlaoescens, 395

476

SYSTEMATIC INDEX

Perch, yellow, see Perca Javescens Periplaneta americana, 71 Petromyzon marinus, 49,221, 256,261 Phoxinus laevis, 104 Plaice, 104 Platichthysflesus, 238, 250, 256 Platyfish, see Xiphophorus maculatus Poecil ia P . formosu, 8 P. latipinna, 36, 39 P. reticulata Peters, 39 Polyodon spathula, 238 Poroderma africanum, 34 Prionace glauca, 183 Pseudopleuronectes americanus, 37

R

Raja R. erinacea, 34 R. rhina, 260 Rana R. ridibunda, 73 R. temporaria, 10 Ratfish, 31, 103, 239 Pacific, see Hydrolagus colliei Hay, electric, see Torpedo marmorata Reedfish, see Calamoichthys calabaricus Repomucenus beniteguri, 419 Rockfish. see Sebastes caurinus

S Sacchuromyces cereoisiae, 257 S a 1in o S . fario, 11 S. gairdneri, 164 S . .salar, 32, 35, 38, 43, 44, 45, 163, 164, 189, 192, 193, 215, 217, 290, 298,376-377 S. trutta, 35, 39, 43, 74 Salmon, 4, 7, 8, 13, 14, 15, 16, 22, 23, 25, 26, 31, 33, 42, 48, 49, 110, 111, 115, 153,273,274,282-283,320-324, 384-385 amago, see Oncorhynchus rhodurus

Atlantic, see Salmo salar chinook, see Onchorhynchus tshawytscha chum, see Oncorhynchus keta coho, see Oncorhynchus kitsutch m a w , see Oncorhynchus masou Pacific, 38 pink, see Oncorhynchus gorbuscha sockeye, see Oncorhynchus nerka Salvelinus S . alpinus, 277, 284 S. fontinalis, 35, 43 Sarotherodon mossambicus, 163 Schizosaccharomyces pombe, 422 Sculpin, see also Myxocephalus octodecimspinosus daddy, see Cottus scorpius Scyliorhinus canicula, 103, 237, 238, 260, 261 Seabass, see Dicentrarchus labrax Sea raven, see Hemitripterus americanus Seatrout, spotted, see Cynoscion nebulosus Sebastes, 246 S . caurinus, 249 Shark, blue, see Prionace glauca Sillago japonica, 419 Skate, see Raja erinacea; Raja rhina Snook, see Centropomus undecimalis Sole, see Solea solea Solea solea, 39 Sparus aurata, 36, 42, 163,214, 261, 313 Syualus acanthias, 32, 34, 103, 261 Starfish, see Asterina pectinifera Sturgeon, 4, 15, 22, 23, 30, 102 white, see Acipenser transmontanus Sucker, 72, 77, 79, 93 white, see Catostomus commersoni

T Thunnus T. obeus, 183,238 T . thynnus, 37 Tilapia, 182, 183, 187, 189, 192, 193, 313, 368, 377; see also Tilapia

477

SYSTEhlATlC INDEX

sparrmanii; Oreochromis mossambicus; Oreochromisniloticus; Sarotherodon mossambicus Tilapia sparrmanii, 36 Tobacco hornworm, see Manducka sexta Torpedo murmorata, 238, 256,259, 260 Trout, 31, 273, 337, 346, 347 brook, see Salvelinus fontinalis brown, see Salmo trutta maw, see Oncorhyncus masou rainbow, see Oncorhyncus mykiss; Salmo gairdneri Tuna, see Thunnus obeus bluefin, see Thunnus thynnus

w Wrasse, see Coris julis X

Xenopus, 150, 337, 338, 349, 354,418, 419,420-425,427,428,430 X . laeuis, 70, 71, 73, 181, 216, 339, 373,420 Xiphophorus maculatus, 39 Y

Yellowfin porgy, 183

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SUBJECT INDEX Acronyms that occur in the text are indexed and identified by a cross reference. A

AII, see Angiotensin I1 Acetylcholine, 107 ACTH, see Adrenocorticotrophin Actinomycin D, translation inhibition, 417 Activation function, 336, 347, 348 Adenohypophysis, see Pituitary, anterior Adenylyl cyclase glucagon stimulation of, 241-243 glucagon-like peptide stimulation of, 235 gonadotropin stimulation of, 408 granulosa cells, 407 inhibition by isotocin, 105 PACAP stimulation of, 18, 20 regulation, 407 SGA stimulation, 407 Adrenal cortex regulation, 311 steroid hormone production, 311 Adrenalectomy, 82, 90, 371, 372 Adrenocorticosteroid hormones, see also individual steroids secretion of urotensin I and 11, 82 Adreriocorticotrophin (ACTH) adrenal cortex regulation, 311 angiotensin 11 stimulated release, 74,75 corticotrophs, 75, 311, 313 isotocin stimulated release, 72-74, 85, 86 PACAP stimulated release, 18 regulation by corticotropin-releasing factor, 68, 74, 85, 86

release from corticotrophs, 73 sea bream, 313 secretion, 68, 69, 72-74, 85 synthesis, 160 urotensin stimulated release, 74 vasopressin stimulated release, 72-74 vasotocin stimulated release, 72-74, 85, 86 Alternative RNA splicing estrogen receptors, 338 GHRH-like/PACAP, 18, 24, 25 gonadotropin I1 p gene, 142 insulin-like growth factor, 197, 199-202,215,217,220 Pit-1, 318, 319, 323, 324 prolactin receptor gene, 368, 370 a-Amanitin, 408 Ames mouse, 314 Aminergic fibers, 311 Ancestral genes corticotropin-releasing factor, 91 GHRH-like/PACAP, 27 glucagon superfamily, 28, 49 gonadotropin-releasing hormone, 42,48 growth hormone, 174, 180, 191-197,203 insulin, 219 insulin-like growth factors, 219 PHI/VIP, 27, 28, 49 PHMNIP, 26-28,49 pancreatic polypeptide family, 259 placental lactogen, 167, 180, 191-197 prolactin, 174, 180, 191-197, 203 PRPIPACAP, 26-28 somatolactin, 174, 180, 191-197, 203 urotensin I, 91 479

SUBJECT I N D E X

Ancestral protein, growth hormone family, 195, 196 Angiotensin I conversion inhibition by captopril, 75 in humans, 75 Angiotensin I1 antagonists, 75 co-localization with vasotocin, 104 human, 74, 75 salmon, 74 receptor, 75 role as adrenocorticotrophin secretagogue, 74 sarcosine analogues, 75 vasotocin immunoreactivity, 104 Antennapedia gene, homeodomain, 317 Antibodies anti-cdc2 C-terminal antibody, 424 anti-cdk2 C-terminal antibody, 425 anti-cdc2 kinase, 424, 425 anti-corticotropin-releasing factor, 82 anti-cyclin B, 424, 425 anti-goldfish cdc2 kinase, 424,425 anti-insulin, 229 anti-Pit-I, 171, 174 anti-phosphotyrosine, 427 anti-PSTAIR, 424,425 anti-somatolactin, 164 anti-urotensin 11, 82 Antidiuresis in rats, 125 plasma osmolarity, 125 Antiestrogen, binding, 344, 345, 347, 348 Antisense cRNA probes, 202 AP-1, see transcription factors Aromatase cytochrome P-450 activity during vitellogenesis, 410, 415 Ad4 binding protein sites, 416 C19 to C18 steroid conversion, 414 cDNA, 412 estrogen responsive element half-sites, 415 Southern analysis, 416 in granulosa cells, 410 medaka, 415 primer extension, 416 S1 nuclease mapping, 416 Southern analysis, 416 Ascorbic acid depletion test, 72 Aspargtocin, 103

Aspartyl proteases in anglerfish, 257 in yeast, 257, 258 Asvatocin, 103 A T splice acceptor, 319 AtT-20 mouse cells, PACAP stimulation, 18 Autocrine hormone, GHRH-like, 51 hormone, PACAP, 51 AVP (arginine vasopressin), see Vasopressin AVT (arginine vasotocin), see Vasotocin

B Bacteriophage repressor, 318 Bladder, urinary, 368,372 Brackish water (BW)-adapted fish, 377, 378, 381-383,385 Brain stem, urotensin I immunoreactivity, 81 Breast cancer cells (T47D & MCF-7), 45, 350 Brockmann bodies effect of epinephrine on, 244 effect of secretagogues on, 244, 258 glucagon-like peptide synthesis, 232, 233 in anglerfish, 229, 232 in sea raven, 229 mRNA of preprosomatostatin I, 11, 252 preproglucagon gene expression, 232

C

C-terminal modifications, glucagon superfamily peptides, 21-23 Calcitonin G-protein coupled receptor, 247 in mammals, 274 inhibition of osteoclastic bone resorption, 274 Calcium gill transport, 274, 275, 283 ionophore A23187,408

SUBJECT INDEX

parathyroid hormone, negative regulation by calcium, 293 regulation by calcitonin, 290 regulation by stanniocalcin, 289-301 regulation of glucagon gene transcription, 240 Calmodulin inhibitor, N-(6-aminohexyl)-lnaphthalenesulfonamide hydrochloride, 408 inhibitor, N-(6-aminohexyl)-5-chloro-lnaphthalenesulfon-amide, 408 inhibitors, 408 CAMP, see Cyclic AMP CAMP response element see response element, CRE CAP site, 89, 145, 174 Captopril, angiotensin I conversion inhibition, 75 Carcinoma (breast) cell lines, GnRH immunoreactivity, 45 a-Casein gene, 371 CAT, see Chloramphenicol acetyl transferase Caudal neurosecretory system, homology between urotensin I and CRF, 69, 72 CCAAT box corticotropin-releasing factor, 89 GHRH gene, human, 14 GHRH gene, rat, 14 human PRP/PACAP, lack of, 14 salmon GHRH-like/PACAP gene, lack of, 14 cdc2 kinase activation by cyclin B, 426, 427 cDNA, 423 in yeast, 422 lambda gtl0 library, 424 Northern blot analysis, 423 poly (A)' tail, 423 protein levels, 425 threonine phosphorylation, 427 cdk2,423 cdk3,423 cDNA aromatase cytochrome P-450, 412 cdc2 kinase, 423 corticotropin-releasing factor, 80, 89, 90 cyclin B, 424

48 1 GHRH, human pancreatic tumor, 5 , 6 GHRH, mouse hypothalamus, 5, 7 CHRH, mouse placenta, 5, 7 GHRH, rat hypothalamus, 5, 6 GHRH, rat placenta, 5, 7 GHRH-like/PACAP, fish, 14, 15 GHRH-like peptide, catfish, 9, 14, 15 GHRH-like peptide, salmon, 7, 9, 14, 15 GHRH-like peptide, sturgeon, 9, 14 gonadotropin I, LY subunit, 138-140 gonadotropin I, Ip subunit, 138, 140 gonadotropin 11, (Y subunit, 138, 140 gonadotropin 11, IIp subunit, 138, 140 gonadotropin-releasing hormone, catfish, 43 gonadotropin-releasing hormone, chicken-11, 43 gonadotropin-releasing hormone, mammalian, 43 gonadotropin-releasing hormone, salmon, 43 growth hormone, 183 Insulin-like growth factor 1, 181, 197, 199, 200,215, 218, 219 Insulin-like growth factor 11, 181, 197 isotocin, 108, 111, 117, 118, 120, 121 mesotocin, 120 PACAP, catfish, 10 PACAP, human, 7, 10, 17 PACAP, rat, 7, 10, 17 PACAP, salmon, 7, 10, 17 PACAP, sheep, 7, 10, 17 PACAP, short precursor, 24 PACAP, sturgeon, 10 pancreatic polypeptide family, 258-260 Pit-1, 170, 171, 174, 321, 322 prolactin, 180, 185 PRP/PACAP, mammalian, 14 somatolactin, 160-164 urotensin I, 89 vasotocin, 108, 110, 111, 117, 120, 121 C DNA library chinook salmon pituitary, 311-313, 32 1 chum salmon pituitary, 170 Corpuscles of Stannius, 276 pig testis 412, 413, 416 rainbow trout liver, 197, 337

482 rainbow trout ovarian follicles, 412, 413 rainbow trout thecal cells, 413 Cell anterior pituitary types, 168, 170, 311 AtT-20, mouse, 18, 255 breast cancer, 45, 350 bronchial epithelium, 217 Chinese hamster ovarian, 354-356 COS-1 monkey kidney tumor, 412, 413 embryonic trout line (STE-137), 348, 356 endocrine types, 311 GH,, rat, 18 GH,, pituitary, 315, 321 glial, 310 gonadotrophs, 41, 139, 140, 143, 145 HeLa, 172-174,315,324 hepatocytes, 333, 350 islet, 230, 231 lactotrophs, 168, 170, 174, 312-315 lymphocyte lines, 369 mouse mammary, 372 Nb2 lymphoma line, 370 neural, 10, 11, 33, 38-41, 310 neuroendocrine, 10, 11, 33, 38-41, 159, 240 pancreatic islet tumors, 5 PC 12, rat, 89 pituitary, fish, 18, 69, 73-75 pituitary gonadotropin, 145 primary, 321 prolactin-secreting rat GH,, 321 salmonid, 321, 324 somatotrophs, 168, 170, 174 cuT3-1, 143 thyrotrophs, 147, 170, 174 volume, 195, 197 Cellular metabolism, 195-197 Central nervous system goldfish, 81-88 gonadotropin regulation, 142, 149 sucker, 81-88 cGMP, see Cyclic C M P Chinese hamster ovarian (CHO) cells, 349, 356, 371 Chloramphenicol acetyl transferase (CAT) gene, 89, 145,348, 349 Chloride levels, plasma, 378 CHO cells, see Chinese hamster ovarian cells

SUBJECT INDEX

Cholesterol side-chain cleavage cytochrome P-450 cholesterol to pregnenolone conversion, 412, 413 radioimmunoassay, 412, 413 rainbow trout ovarian follicle cDNA library, 412, 413 steroidogenesis, 412, 413 transfection of COS-1 cells, 412, 413 Chorionic gonadotropins placental secretion, 136 Chromatin, 336 Chromosomes carp, 92 catostomid, 89, 92 condensation induced by lily pachytene microsporocytes, 420 Cyprinidae, 92 goldfish, 92 Cleavage sites, see Processing sites Clustered mutations, 316 CNS, see Central nervous system Co-evolution, growth hormone and insulin family, 203 Co-expression AVP and OXT mRNA in rats, 86 AVT and IST mRNA in rainbow trout, 86 CRF and AVT mRNA in goldfish, 86, 88 CHF and IST mRNA in goldfish, 86, 88 Co-localization GHRH and NPY, 24 PHI, CRF and enkephalin, 24 CHRH and vasotocin, 24 Concanavalin A, 282 Conservation LRF see GnRH GHRH family, 8, 20, 49 GHRH-like molecule, salmon, 15, 19-21 GHRH, mammalian, 8, 12, 20, 22, 49 GnRH, 29, 32, 33, 43, 49, 71, 90 GnRH-associated peptide, 43 growth hormone, 182-185 insulin-like growth factor, 197-200 PACAP, 8, 12, 19, 20 PHI and PHM family, 49 Pit-1. 173

SUBJECT INDEX

prolactin, 185-187, 191-194 somatolactin, 162, 166-168, 187-189 Conserved domains growth hormone (A, B, C, D), 182-185, 187, 191, 192 insulin-like growth factor, 181, 198-202,215,216 placental lactogen, 191 prolactin (A, B, C, D), 185-187, 191, 192 somatolactin (A, B, C, D), 162, 166-168, 187-189, 191, 192 Conserved motifs, corticotropinreleasing factor, 90 Constructs, PLRICAT, 321, 324 Co-ordinated release, model (GH, ACTH and prolactin), 24 Copeptin, in mammals, 109-111, 114, 115 Cordycepin, 408 Corpuscles of Stannius bowfin, 272 cDNA library, 276 coho salmon, 276,277,290 electrophoretic pattern comparisons, 276 kidney tubule cells, 272 nuclear hypertrophy, 289,294 primary cell culture model, 294 rainbow trout, 276, 290 regulation of calcium homeostasis, 274 secretion of stanniocalcin, 274, 290 secretory granules, 287 stanniocalcin gene expression, 299 Cortical alveoli, 420 Corticosteroids. 82, 87, 127 Corticotrophs adrenocorticotrophin, 73, 75, 311 CRF receptor I, goldfish, 76 CRF receptor 11, mammals, 76 CRF receptor 111, amphibian, 76 goldfish, 73, 75, 76 interaction with CRF nerve terminals, 77 niouse AtT20 cells, 255 sea bass, 313 Corticotropin-releasing factor ACTH releasing activity, 68, 73, 75, 86 affinity column, 81

483 ancestral gene, 91 antiserum, sucker, 82 CAAT box, 89 CAMP responsive element, 89 CAP site, 89 caudal neurosecretory system, fish, 69, 72,81 cDNA, 80, 89, 90 co-expression with isotocin mRNA, 86, 88 co-expression with vasotocin mRNA, 86,88 co-localization with vasotocin, 82, 104 dendrogram, 70 dexamethasone effect on mRNA levels, 90 duplication by tetraploidization, 72, 92 eel, Anguilla anguilla, 82 effect of urophysectomy on synthesis, 87 endorphin release in Xenopus, 73 evolution, 90, 92, 93 function, 77 gene, 89, 90 gene family, see Corticotropinreleasing factor gene family gene organization, 89 gene transcription, 89, 90 glucocorticoid responsive elements, 90 human, 71,89,92 hypothalamus, goldfish, 69 immunoreactivity, 81-84 in dexamethasone-blocked goldfish, 69 in situ hybridization, 85 a-melanocyte-stimulating hormone release, 73 metopyrone effect on synthesis, 87, 90 mRNA, 80, 85, 86, 88, 90, 91 nerve terminals, 77 Northern blot analysis, 80, 85 ovine, 68, 69, 75, 89, 90 ovine, immunoreactive fibers, 81 pituitary cells, goldfish, 83 precursor, see Corticotropin-releasing factor precursor rat, 89 receptor I in goldfish, 76 receptor I1 in mammals, 76 receptor 111 in amphibians, 76 receptor ligand-binding portion, 76

SUBJECT INDEX receptor subtypes, 76 regulation of gene transcription, 90 relation to PACAP, 18 role in osmoregulation, 77 splice junctions, 90 start codon, see CAP site stress-induced release, 73, 74 sucker, 90-92 synthesis in placental tissue, 90 TATA box, 89 tissue specific expression, 89, 90 transfection assay, 89 untranslated region, 3', 89 untranslated region, 5', 89, 90 Xenopus, 71 Corticotropin-releasing factor gene family CRF-1, 69 CRF-2, 69 CRF major, 91 CRF minor, 91 diuretic peptide (DPI), 69, 71 diuretic peptide I1 (DPII), 71 gene organization, 89, 350 a-helical conformation, 76 hydrophilic domains, 76 hydrophobic domains, 76 urotensin I, 69, 71 Corticotropin-releasing factor precurso1 amidating signal, 78 cryptic peptide, 78, 91 exon, 89, 350 hormone moiety, 78,80, 91 human, 78-80 intron, 89, 90, 350 monobasic cleavage, 78 mutations, 91 ovine, 78-80 polyadenylation signals, 80 proteolytic site, 78 rat, 78-80 sCRF1, sucker CRF1, 80, 87, 89-93 sCRF2, sucker CRF2, 80, 87, 92, 93 sequence identities, 80, 89 signal cleavage sites, 78, 79 signal peptide, 78, 91 structure, 78, 79, 338, 339, 341, 342 sucker, 78, 80 Corticotropin-releasing hormone, see Corticotropin-releasing factor

Cortisol negative regulation of vasotocin, 82 role in hyperosmotic environmental adaptation, 383 Cortisone, 372 COS-1 monkey kidney tumor cells, 412, 413 Cosecretion GHRH and PACAP, 24 model (PHI, CRF and enkephalin), 24 Coumestrol, 347 CRE, see CAMP response element Cretinism, 314 CRF, see Corticotropin-releasing factor CRF-related peptide, see Corticotropinreleasing factor gene family CRH, see Corticotropin-releasing factor 434 Cro, 318 Cryptic peptide hydrophobicity, GHRH precursor, 26 in CRF/UI precursor, 78 in GHRH(PRP)/PACAP precursor, 14, 15, 25, 26 in GHRH-like/PACAP cDNA, 14, 15, 24-26 CS, see Corpuscles of Stannius Cyanoketone, 400 Cyclic AMP activation of glucagon gene, 240 dibutyryl, 407 effect of forskolin, 407, 408 in granulosa cells, 408 inhibition, 407 protein kinase type 11, 79 release by PACAP, 9 response element (CRE), 15, 89, 144, 240, 242 response element binding protein, 240 response element in CRF, 89, 90 Cyclic GMP, dibutyryl, 408 Cyclin B, cDNA, 424 cyclin box, 424 from goldfish, 424 germinal vesicle breakdown, 426 oocyte maturation, 425, 426 protein levels, 425, 427 serine phosphorylation, 427 Xenopus A, 424 Xenopus B1,424 Xenopus B2,424

SUBJECT INDEX

485

Cycloheximide, protein synthesis inhibition, 422 Cyclostomes agnatha insulin superfamily 219-221 hagfish and lamprey VT precursors, 108, 109 hagfish insulin, 228-230 hagfish insulin-like growth factor I, 181, 219, 221 hagfish neurophysis, 109 hypothalamic fibers, 311 lamprey GnRH, 29, 31-34, 37,42,48 lamprey GnRH neurons, 33,38 lamprey insulin-like growth factor, 221 lamprey pancreatic polypeptide family, 261 lamprey somatostatin, 256 neural regulatory factors, 311 pituitary organization, 311, 367 Cytokine receptor family, 371 Cytoplasmic domain, 370 D

D-Box, steroid receptors, 340-342 Dendrogram of CRF/UI peptide sequences, 70 Deuteros tomes as ancestors, 194, 195, 197 growth hormone, 194, 203 prolactin, 194, 203 somatolactin, 194, 203 Development anterior pituitary cells, 150, 170, 172, 310-313 anterior pituitary hormone, 310-313 embryogenesis, see Embryogenesis GHRH gene transcripts, 17 GnRH neurons, 45, 46 gonadal differentiation, 143 IGF-I tissue expression, 200-202, 221 IGF-I1 tissue expression, 200-202, 221 of lactotrophs with pit-1, 170-172, 174 of somatotrophs with pit-I, 170-172, 174 of thyrotrophs with pit-1, 170-172, 174 paracrine factors, 312 Rathke’s pouch, 310

Dexamethasone effect on glucocorticoid levels, 90 effect on urotensin I and I1 immunoreactivity, 82 effect on vasotocin immunostaining, 85 Diencephalon, in pituitary development 310 Diethylstilbestrol, 346, 347 Differential splicing, see Alternative RNA splicing 17a,20P-Dihydroxy-4-pregnene-3-one (17a,20P-DP) African catfish, 403 aniago salmon, 401, 406 binding to oocyte cortices, 418 binding to zona radiata-oocyte membrane, 418 conversion from precursor, 402, 404, 405,411 cyclin B synthesis, 430 forskolin-induced production, 407, 417 germinal vesicle breakdown, 401-403 gonadotropin I1 levels, correlated with, 401 gonadotropin I1 regulation of, 137, 138,400,401,404 in postvitellogenic oocytes, 137, 405, 410,411 Indian catfish, 403 Malaysian catfish, 403 maturation-inducing hormone, 137, 401, 402 medaka, 402 murnmichog, Fundulus heteroclitus, 402,405 oocyte maturation, 400, 402, 410, 411 protein kinase C activation, 408 sockeye salmon, 403 Dinierization, 335, 340, 344 Direct repeat sequences growth hormone, rat, 191, 192 placental lactogen, human, 191, 192 prolactin, human, 191, 192 prolactin, rat, 191, 192 Diuretic peptide (DP) cockroach, Periplaneta americana, 71 locust, Locusta rnigratoria, 71 moth, Manduca serta, 69

SUBJECT INDEX

Diuretic peptide I1 (DPII) moth, Manduca sexta, 71 Divergence time, vasotocin and mesotocin precursors, 114, 115 DNA, see also cDNA binding domains in transcription factors, 316-318, 339 binding protein (GSEB), 143, 149 binding protein, see also transcription factors content, carp, 92 content, goldfish, 94 gonadotroph-specific element (GSE), 143, 149 major groove, 317, 318 palindromic, 340 Pit-1 binding region, 316-318 recognition, 317, 341, 342 response elements, see Response elements transposition, in growth hormone gene, I92 DNase 1 footprints, 316 Domains estrogen receptor, 344-348 gonadotropin I (a, Ip), 138 gonadotropin I1 (a, IIp), 138 growth hormone (A, B ,C, D), 182-185, 187, 191, 192 homeodomains, see Homeodomains insulin (A, B, C), 181 insulin-like growth factor (A, B, C, D, E), 181, 198-202 placental lactogen, 181 POU homeodomain, see POU, homeodomain POU-specific domain, see POU, specific domain prolactin (A, B, C, D), 185-187, 191, 192 somatolactin (A, B, C, D), 162, 166-168, 187-189, 191, 192 Dopamine, 107 Dotislot blot analysis of isotocin, 118 of vasotocin, 118 DP, see Diuretic peptide DPII, see Diuretic peptide I1 Dual control, of ACTH synthesis and release, 85 Duplication, see Gene duplication

Dwarfism in mice, 314 phenotype, 314 Dyad symmetry, 342

E

Early-vitellogenic oocytes, 396, 397, 418 Ectoderm cells, 310, 367 origin of anterior pituitary, 168, 310 Rathke’s pouch, 168, 170, 310 Egg yolk protein, synthesis, 331, 333 EGTA, 420 Embryogenesis, 220, 221, 310-313 Embryonic trout cell line (STE-137), 348, 349, 356 Endorphin release stimulation by ovine CRF, 73 release stimulation by sauvagine, 73 release stimulation by urotensin I, 73 Endosomes, 369 Engrailed gene, homeodomain, 317 Enhancers, 232, 353, 354 Enkephalin, isotocin immunoreactivity, 104 EPO, see Erythropoietin Equol, 347 ERE, see Estrogen response element Erythropoietin hormone, 371 receptor, 371 Estradiol (E,) binding, 344-347 E,, 331, 346, 349 gonadotropin I1 stimulation, 410 in follicle cells, 410 levels during vitellogenesis, 410 production in granulosa cells, 410, 411 production stimulation by prolactin, 368 steroid precursor, 404, 405 Estriol, 347 Estrogen gonadotropin I1 p gene expression, 150-152 nuclear receptor, 332-336 receptor, 320, 332-336, 344-349 receptor, gene, 349, 350, 351

SUBJECT INDEX

receptor, rainbow trout, 337, 338, 344-349 response element (ERE), see Estrogen response element responsiveness, 334 Estrogen response element (ERE) binding specificity, 341-343 distal (sGTHIIp), 150 estrogen receptor binding to, 151, 348, 349 gene transfer experiments, 151 gonadal steroid regulation, 149 in aromatase cytochrome P-450 gene, 4 15 in gonadotropin releasing hormone gene, 44 in prolactin gene, 320 in sGTHIIP promoter, 150 palindromic structure, 340 proximal (sGTHIIp), 150 transient expression studies, 150, 348, 349 trout pituitary cells, 145 Estrone, 347 Euryhaline fishes, 313 Evolution corticotropin-releasing factor, 90, 92,93 crossopterygian fish, 92 from fish to amphibia, 103 gene duplication, 91, 93, 338 genome duplication, 91, 92, 338 GHRH gene, mammals, 20, 26, 27, 49 GHRH/PACAP gene, 19,20, 26,27 glucagon, 231, 237 glucagon superfamily, 26-29, 49 GnRH-associated peptide, 43, 44 GnRH midbrain neurons, 40 GnRH peptides, 3, 41, 42, 47 growth hormone gene family, 167, 168, 174, 180, 181, 192,203 higher vertebrates, 92-94 insulin gene family, 181 insulin-like growth factor, 181, 197, 203 mutational events, 92 oxytocin hormone family, 110, 111, 113, 115, 116 PACAP gene, 19,49 PHM/VIP gene, 20, 26, 27,49 placental lactogen, 179, 180, 191-194

487 polyploidization in vertebrates, 91, 93 posterior pituitary hormones in gnathostomes, 102-103, 108 prolactin, 179, 180, 187, 191-197, 203 PRP/PACAP gene, 19, 20,26,27,49 somatolactin, 167, 168, 174, 180, 187, 191-197, 203 urotensin I, 92-94 vasopressin hormone family, 110, 111, 113, 115, 116 vertebrate HPA(1) axis, 75 Evolutionary distance, 114, 119 Evolutionary model growth hormone family, 168, 192, 203 insulin family, 203, 219 Exon-intron organization, see also Gene organization corticotropin-releasing factor gene, 89,90 estrogen receptor gene, 350, 351 gonadotropin gene, 135, 141 growth hormone gene, 180, 189-191 glucagon gene, 232-235 prolactin gene, 180, 189-191 somatolactin gene, 165, 167, 168, 180, 189-191 splice junctions, 16, 19, 22, 25, 27, 90, 320 splice site, see splice junctions Extracellular domain, 370

F

FABMS analysis, of prosomatostatin I, 254 Feedback regulation, 127, 332, 333 Fibroblasts, 324 First meiotic metaphase maturation-promoting factor activity, 420 Flanking sequences, 5’ estrogen receptor gene, 353 gonadotropin gene, 143, 145, 146, 148 gonadotropin-releasing hormone, 44 growth hormone gene, 168, 190-193 insulin gene 228, 229 prolactin gene, 168, 169, 190-193 somatolactin gene, 191-193

SUBJECT INDEX

Follicle 17a,20fi-dihydroxy-4-pregnene-3-one, 395,404 granulosa layer, 395, 396, 404 maturation-inducing hormone, 406 special thecal cells, 395, 406 structure, 395, 396 thecal layer, 395, 396, 404, 406 Follicle-stimulating hormone anterior pituitary secretion, 139 p gene promoter, 144 gonadotrophs, 311 human p gene, 138, 144 ovarian regulation, 311 PACAP effect on release, 18, 50 testicular regulation, 311 tetrapod, 138 Follicular stellate cells in pituitary, 24 interleukin-6 release, 24 PACAP stimulation, 24 Foot printing analysis, in sGTHIIp, 151 Forskolin, 416 Fresh water-adapted fish, 105, 123, 125, 373, 377, 382, 383, 385 FSH, see Follicle-stimulating hormone Functional model, growth hormone family evolution, 194-197, 203 Fusion protein, 171

G G-protein, see Guanine nucleotidebinding regulatory protein Gametogenesis gonadotropin regulation, 135 receptors in ovaries, 137 receptors in testes, 137 GAP, see GnRH-associated peptide Gastric inhibitory peptide, 23, 231 GC, see Glucocorticoids Gel mobility shift, 319 Gel-shift assays, 343 Cenbank, 278 Gene, see also specific genes bacterial chloramphenicol acetyl transferase, 89, 145, 348, 354, 355 conversion, 114 CRF, 89, 90 duplication, see Gene duplication

estrogen receptor, 350, 351 estrogen receptor, rainbow trout (rtER), 353, 354 GHRH, human, 5, 6. 14, 16, 19, 20, 26,27 GHRH, rat, 5, 6, 14, 17 GHRH-likeiPACAP, salmon, 10, 14, 16, 19, 20, 25-27 GLP-1 and GLP-2, 232-235 glucagon, 232-235 gonadotropin I, 11, 140-142 gonadotropin-releasing hormone, 30-32,38,44 growth hormones, 165-168, 172-174, 180, 189-194 insulin, 181,226, 227 insulin-like growth factor, 181, 200 KALIG-1, neural cell adhesion molecule, 46 ovalbumin, chicken, 152 PACAP, human, 7, 10, 14-16, 19, 20,26 Pit-1, 190, 313-315 placental lactogen, 180, 191 prolactin, 164-168, 170-174, 180, 189-191, 193, 194 promoters, 143, 144, 320, 321 PRP/PACAP, human, 6, 7, 10, 14-16, 19, 20,26 pseudo, 93 rainbow trout estrogen receptor (rtER), 353,354 silent, 93 somatolactin, 165-168, 173, 174, 180, 189, 191, 193 thyroid-stimulating hormone, 148 urotensin I, 93 vasoactive intestinal peptide (VIP), 16, 26-28 vitellogenin, 150 Gene duplication CRF gene by tetraploidization, 92, 93 crossing over between two homologous chromosomes, 91 exchange between sister chromatids of one chromosome, 91 GH/PRL/PL/SL genes, 167, 168, 180 GHRH, 26,27,49 GHRHIPACAP, 19, 26, 27, 49 glucagon superfamily, 28, 29, 49

SUBJECT INDEX

gonadotropin-releasing hormone, 41, 44,48 isotocin, 93, 114, 115 of a genome, 92, 338 oxytocin hormone family, 116 PACAP and VIP genes, 26-29,49 polyploidization, 91, 93 regional, 91, 93 vasoactive intestinal peptide, 26-28,49 vasotocin precursors, 93, 114, 115 vasopressin and oxytocin ancestral gene, 114 VT-1, VT-l’, 93 VT-1, VT-2, 93 Gene organization, see also Gene corticotropin-releasing factor, 89, 90 estrogen receptor, 350, 351 GHRH, 14, 16 GHRH-like/PACAP, 14, 16 GLP-1 and GLP-2,232-235 glucagon, 232-235 gonadotropin, 140-143, 147 gonadotropin-releasing hormone, 44,45 growth hormone, 180, 189-194 insulin 226, 227 PACAP, 16, 19, 20, 25, 27 placental lactogen, 180 prolactin, 180, 189-194 somatolactin, 189-194 TSH, 140, 141 Gene transfer see also Transgene and Transgenic mouse follicle stimulating hormone, 144 estrogen response elements (ERE), 150, 151, 348, 349, 356 a-T-antigen oncogene, 143 GENETYX sequence analysis software, 111 Germinal vesicle effect of 17a,20pDP, 400 migration, 420, 421 Germinal vesicle breakdown in medaka, 402 indicator of hormone action, 394 induced by cyclin B, 426 induced by lily pachytene microsporocytes, 420 GH, see Growth hormone GH response element (see Pit-1 binding element)

489 GH3 rat cells, stimulation by PACAP, 18 GH4 cells, see Pituitary, anterior GHF-1, (GH factor-1 protein) see Pit-1 CHRH, see Growth hormone-releasing hormone GHRH-like molecules (see Growth hormone-releasing hormone-like molecules) GHRH-like/PACAP (see Growth hormone-releasing hormone-like/ PACAP precursor) Gill calcium transport, 274-276,283 effect of stanniocalcin, 274 membranes, 373,374,378,379,383 physiology, 383 GIP, see Gastric inhibitory peptide Glial cells, 310 GLP, see Glucagon-like peptide 1 or 11 GLUC, see Glucagon Glucagon I, 232, 233 11, 232, 233 adenyl cyclase activation 241-243 anglerfish, 232, 233 dogfish, gut tissue, 237 eel, 239 effect on plasma glycerol levels, 245 effect on plasma unesterfied fatty acid levels, 245 expression, 237 6-protein coupled receptor, 247 gene, 232-235 gene promoter, 237 glucagon superfamily member, 23, 49, 23 1 gluconeogenesis activation, 242-244 glycogenolysis activation, 241-244 homology with prealbumin, 232 hormone family, 231 human, 238,239 rat, 232, 239, 243 induced hyperglycemia, 244 lipolysis activation, 244, 245 preproglucagon expression in Brockmann bodies, 232 primary structures, 238 proglucagon, 232,233, 237 promoter, 237 receptor, 235, 240, 244, 247 regulation of DNA synthesis, 235

490 release inhibition by glucose, 244 synthetic analogues, 242 ureagenesis activation, 245 Glucagon-like peptide I expression in Brockmann body, 232,233 function in mammals, 247 gene, 232-235 glucagon superfamily member, 23 gluconeogenesis activation in fish, 247 glycogenolysis activation in fish, 246, 247 anglerfish, 236, 245 increase in cyclic AMP levels, 246 lipolysis activation in fish, 247 metabolic role in fish, 247 pancreatic secretion in fish, 237 primary structures, 239 receptor, 235, 247, 248 stimulation of insulin synthesis, 247 suppression of glucagon transcription, 247 synthesis in intestinal L-cells, 237 Glucagon-like peptide I1 anglerfish, 233, 236, 245 expression in Brockmann body, 232 function in mammals, 247 gene, 232-235 gluconeogenesis activation in fish, 247 glycogenolysis activation in fish, 247 lipolysis activation in fish, 247 mammals, 233 metabolic role in fish, 247 primary structures, 239 receptor, 247, 248 stimulation of adenylyl cyclase, 235 stimulation of insulin synthesis, 247 suppression of glucagon transcription, 247 synthesis in intestinal L-cells, 237 Glucagon superfamily ancestral gene, 28, 29, 49 C-terminal modifications, 23 evolution, 28, 49 exon duplication, 28, 29 GHRH, GIP and secretin precursors, 28 K-terminal residues, 23 PACAP family, 19, 49 PACAP, VIP and glucagon precursors, 24,28 precursors, 28

SUBJECT INDEX

Glucocorticoids hormone, 190, 191, 350, 371, 372 receptor, 338, 353 release of growth hormone, 9, 190, 191 response element (GRE), 90, 341, 354 Gluconeogenesis, glucagon activation, 242-244 Glucose, inhibition of glucagon release, 244 Glutamic acid, 107 Glutathione S-transferase, 171 Glycentin, synthesis in intestinal L-cells, 237 Glycentin-related polypeptide, 232, 237 Glycogenoly sis activation of adenylyl cyclase, 244 activation of protein kinase A, 244 glucagon activation of, 241-243 Glycoprotein hormone a subunit gene hormone basal element, 153 phorbol myristate acetate treatment, 153 GnRH response element, 143 Glycoprotein hormone family a subunit, 136 /3 subunit, 136 chorionic gonadotropins, 136 thyroid-stimulating hormone, 136 Glycoprotein hormones follicle-stimulating hormone, 31 1 luteinizing hormone, 311 thyroid-stimulating hormone, 311 GnKH, see Gonadotropin-releasing hormone GnRH-associated peptide (GAP) conservation, 43 evolution, 43, 44 in GnRH precursor molecule, 43 inhibition of prolactin, 43, 44 Gonadal differentiation, 143 Gonadal steroids development of pituitary gonadotropin cells, 150 gonadotropin release, 142 gonadotropin synthesis, 140 negative feedback mechanisms, 149, 150 Gonadectomy estrogen treatment, 150 gonadal steroid replacement, 149 plasma gonadotropin levels, 140, 149 testosterone treatment, 150

SUBJECT INDEX

Gonadotrophs a subunit gene expression, 143, 144 aT3-1 precursor lineage, 143 expression of Diphtheria Toxin-A chain, 143 follicle-stimulating hormone, 311 human follicle-stimulating hormone p gene expression, 144 rainbow trout, 140, 145, 150 salmon gonadotropin 110 gene, 141, 143, 150, 165 termination of GnRH axons, 41 Gonadotropin central nervous system regulation of, 142 characterization, 145 cloning, 140, 141 feed back controls, 142, 149 follicle-stimulating hormone, 136, 138, 144, 149, 152 gametogenesis, 137, 398 gene expression, transcriptional control, 143-145, 149 gonad regulation, 142 gonadal function, 137 luteinizing hormone, 136, 138, 145, 149, 152 mammals, 136, 149, 152, 153 maturation of oocytes, 137, 394, 398-400,406 ovulation, 136, 138, 399, 400 phosphoinositide turnover, 152 pituitary regulation, 142 protein kinase C activation, 153 receptor binding domain, 138 receptors in ovaries, 137 receptors in testes, 137 receptors, type I, 11, 400 reproductive process, 140 secretion, 140, 142, 152 sexual maturation, 138 spermiation, 132, 138 steroid regulation, 149-152 steroidogenesis, 136, 137, 398, 404, 405 subunit interaction domain, 138 synthesis, 139, 140 teleosts, 135-140 vitellogenesis, 136 Gonadotropin a subunit gene bovine, 141, 143

49 I bovine a subunit promoter, 143 carp, 137, 139-141 chinook, 141 DNA-binding protein (gonadotrophspecific element binding protein, GSEB), 143, 149 flanking sequence, 5’,143 genomic organization, 140 gonadotroph-specific element (GSE), DNA, 143, 149 gonadotroph-specific expression, 143 human, 141 mouse, 141 organization, 141 production by aT3-1, 143 rat, 141 salmon, 141 size, 141 thyrotroph-specific expression, 147 upstream regulatory sequences (URSj, see Flanking sequences, 5’ Gonadotropin p subunit gene alternative RNA splicing, 142 expression, 144 genomic organization, 142, 147 genomic sequence, 141 CAP site, 145 carp, 140, 141 cDNA, 140, 141 chinook salmon, 140, 141 common carp, 140, 141 E2 induced transcription, 151 estrogen control, 152 estrogen response element, 150 exodintron junctions, see Splice junctions flanking sequences, 5’, 145 GSE homologous sequence, 149 minimal promoter (sGTHIIP), 145, 146, 148 multiplicity of mRNAs, 142 organization, 142, 147 phorbolester induced transcription, 153 pituitary primary cell cultures, 145, 146, 151 polyadenylation, 141 promoter, 145 rainbow trout, 140, 145, 150 RNA blotting analysis, 151 silencer sequence (pSilj, 148

492

SUBJECT INDEX

splice junctions, 141 immunohistochemical analysis, 140 TATA box, 145, 147, 148 in spawning trout, 140 testosterone control, 151 induction of maturational competence, upstream regulatory sequences, see 419 Flanking sequences, 5’ induction of meiotic maturation, 400 Gonadotropin I 11-ketotestosterone production, 137 bonito, 137 maturation-inducing factor, 137, 404, carp, 137 405 cDNA, subunits a and Ip, 140, 141 maturation of gametes, 137, 399 chum salmon, 137, 312, 322, 399 ovulation, 136, 138, 400 coho salmon, 137,399 puromycin inhibition, 407 common carp, 137, 139 rainbow trout, 140, 400 estradiol-17p production, 137 receptor, type 11, 406 European eel, 139 release by CnRH, 42 gametogenesis, 137 sexual maturation, 138 gametogenic period, 137 similarity to luteinizing hormone, 138 immunohistochemical analysis, 140 subunit a, 138 11-ketotestosterone production, 137 subunit IIp, 138 receptor, type I, 406 testes, early spermatogenic, 137 sea bream, 313 testes, late spermatogenic, 137 similarity to follicle-stimulating vitellogenin incorporation, 137 hormone, 138 Gonadotropin-releasing hormone, 332 subunit a,139, 140 action with PACAP, 50 subunit Ip, 140 amino acids, critical, 41 testes, early spermatogenic, 137 ancestral gene, 42, 48 trout, juvenile, 140 axons, termination at base of trout, prespermatogenic, 140 telencephalon, 39 trout, previtellogenic, 140 axons, termination in anterior vitellogenin incorporation, 137, 410 pituitary, 41 Gonadotropin I1 axons, termination near gonadotrophs, amago salmon, 137,406,408 41 bonito, 137 axons, termination in posterior calcium regulation, 408 pituitary, 33, 38, 40, 41 carp, 137, 399 axons, termination on portal blood cDNA, subunits a and IIp, 140 system, 40, 41 chinook salmon, 137 carcinoma cell lines, breast, 45 chum salmon, 137, 399 catfish form, 29,31-33,35,42-45, coho salmon, 137,399 48,50 common carp, 137, 139 cDNA, 30, 31,43-45, 117 cycloheximide inhibition, 407 cells, midbrain, 39, 40, 47 17a,20P-dihydroxy-4-pregnene-3-one, cells, spinal cord, 39, 40 137, 138, 400-403 chicken form, 29-37, 42-48 enhancement of RNA and protein conservation, 29, 32, 33, 43, 49 synthesis, 408 developmental expression, 45, 46 European eel, 139 distribution, 33-42, 45-48 estradiol-17p production, 137, 404, 410 dogfish form, 29,31-34,42,48,50 goldfish, 399 evolution, 3, 41, 42, 47 17a-hydroxyprogesterone, 137 exons, 44 20~-hydroxysteroiddehydrogenase, family, 32, 47, 49 137 flanking region 5’, in salmon gene, 44

SUBJECT INDEX

gene, Atlantic salmon, 31,32,38,44 gene, human, 30,38,44 gene, Pacific salmon, 31, 32,38, 44 gene, rat and mouse, 30,38,44 gene duplication, 41,44,48 gene number, 44,45 gene structure, 43,44 genes, multiple, 44,45 GH release, 50 GnRH-I, chicken, 29,31-33,36,37,

42,48 GnRH-I, lamprey, 29,31-34,37,42, 48 GnRH-11, chicken, 29-37,42-48 GnRH-111, lamprey, 29,31-34,42 goldfish, 153 gonadotropin secretion and synthesis,

33,42,142 HPLC analysis, 32,41 hypophyseal portal vessels, 40,41, 142 hypothalamic neuron release, 142 immunoreactivity, brain and spinal cord, 33,38-41 immunoreactivity, non-neural, 33 immunoreactivity, retina, 45 in situ hybridization, salmon mRNA,

46,47 introns, 44 Kallmann’s Syndrome, 46 lamprey form, 29,31-34,37,42,48 location, 33,38-41,45,46 mammalian form, 29,30,32-35,37, 42,46,48,SO, 152 midbrain neurons, evolutionary trend,

39,40,47 niRNA, 45,46 multiple forms, 41,47,50 neurons, agnatha, 33,38 neurons, bony fish, 39-41 neurons, cartilaginous fish, 38,39 neurons, immunoreactivity, 33,38-41 neurons, migration pathway, 33,39,

40,45,46 neurons, teleosts, 39-41 Northern blot analysis, 45 novel forms, 34-36,41 olfactory placode, 33,39,45 PCR analysis, 45 peptide sequences, 32,41 phylogenetic map, 34-37,41,42,

45,48

pituitary, GnRH fibers, 47 pituitary-gonadal system control, 142 polymorphism, 41 precursor, see Gonadotropin-releasing hormone precursor primary structure, 30-32,41 protochordates, 49 pulsatile release, 152 radioimmunoassay, 32,41 receptor recognition, 41 receptor signalling, 152 release of gonadotropin 11, 42 release of growth hormone, 50 release of LH and FSH, 41 responsive elements for API, AP2, 143 salmon form, 29, 31-37,41-48,50, 117 secretion, 142,152 Southern blot analysis, 44,45 spinal cord, 39,40 structural analysis, 41-45 sturgeon, 32 terminal nerve, 38-40 tissue expression, 45 tumor immunoreactivity, mammary gland, 45 yeast a mating factor, sequence similarity, 49 Gonadotropin-releasing hormone precursor catfish, 43 GnRH-associated peptide, 43,44 GnRH hormone, 43,44 length of GAPS, 43 mammalian, 43 processing site, 44 salmon, 43,44,117 signal peptide, 43,44 untranslated region, 3’, 44 untranslated region, S ’ , 44 Granulosa cells adenylyl cyclase, 407 amago salmon, 407-409 aromatase activity, 410 calmodulin inhibitors, 408 17a,20p-dihydroxy-4-pregnene-3-one synthesis, 404,406,407,411 endoplasmic reticulum, 398 estradiol-17p synthesis, 411 GnRH action, 50,51

494 Golgi apparatus, 398 gonadotropin activity, 408 gonadotropin receptor, 406 guanine nucleotide-binding regulatory protein, 407, 408 20P-hydroxysteroid dehydrogenase, 404, 408, 409,411 20P-hydroxysteroid dehydrogenase, de novo synthesis, 404, 408, 409 medaka, 398 maturation-inducing hormone, 404 mummichog, Fundulus heteroclitus, 398 oocyte maturation, 398 receptor, type I1 gonadotropin, 406 GRE, see Glucocorticoids, response element Growth hormone (GH) cy helical structures, 184, 185 adaptive osmoregulation, 164, 220 amphibians, 180, 183 ancestral gene, 167, 168, 174, 180, 191-197, 203 Atlantic salmon, 164, 189, 192, 193 birds, 180, 181, 183 blue shark, 183 bony fish, 183 bovine, 180, 182, 187, 189, 190 carp, 164, 183, 189, 190, 193, 194 catfish, 182-185, 189, 190, 193 cDNA, 120, 183 chicken, 182, 189, 190, 193 chinook salmon, 189, 192, 193 chordates, 194 chum salmon, 120, 169, 172, 183, 190 cod, 160, 162, 163 coho salmon, 120, 183 comparison to somatolactin, 162,165-168 conservation, 182-185 conserved domains (A,,, BGH, CGH, DGH),182-185, 187, 191, 192 direct repeat sequences in gene, 166-168 disulfide bonds, 183, 185, 187 DNA transposition, 192 eel, 182 evolutionary model, 168, 192, 203 family, ancestral gene, 167, 168, 174, 180, 191-197, 203

SUBJECT INDEX

family evolution, 167, 168, 179, 180, 187, 191-194 flanking region, 5’, 168, 190-193 flounder, 160, 162, 163, 182, 183 functional model, 194-197, 203 functions, 180, 194-197 gene, 164-168, 172-174, 180, 189-194, 314 gene family, 165-168, 180, 189, 191, 194,202 gene organization, 180, 189-194 gene transcription, 169, 173 glucocorticoids regulate, 9, 190, 191 growth specific domain CGH,185, 187 human, 165-167,182-184, 189-191, 193 hypothalamic extracts, 5 hypothalamic lesions, 5 insulin-like growth factor induction, 9, 180,220 internal repeat sequences, 166-168, 191, 192 invertebrates, 194-197, 203 lactogenic action, 180 mammals, 164-167, 173, 174, 180, 189, 190, 192, 193 osmoregulatory responses, 164, 180, 194-197 PACAP stimulated release, 24 Pit-1 regulation, 165, 168-174, 190-193 primary structure, 166, 180, 183, 192 primitive bony fish, 183, 193 primordial domain, 191 promoter function, 169, 172-174 rainbow trout, 120, 127, 164-167, 172, 182-185, 189, 190, 192, 193 rat, 165-167, 169, 172, 173, 189-191, 193, 311 receptor, 203, 371 receptor binding, 184, 185, 371 regulation of expression, 168-174, 180, 190-194 release by cfCnRH, 50 release by dfGnRH, 50 release by mCnRH, 50 release by sCnRH, 50 reptiles, 180, 183 salmon (see Atlantic, chinook, coho, chum) sea bream, 163, 183

SUBJECT INDEX

sea turtle, 182, 183 sequence alignments, 166, 167, 182, 183, 192 silencer sequences (pRE, Sil-l), 147, 148 somatic growth, 180, 196 striped bass, 183 structural model, 203 surface probability statistics, 184, 185 three-dimensional structure, 183,184 thyroid hormone regulates, 9, 190, 191 tilapia, 182-185, 189, 190, 192, 193 tissue-specific expression, 190, 192 tuna, 182-185 yellowfin porgy, 183 Growth hormone-releasing factor, see Growth hormone-releasing hormone Growth hormone-releasing hormone alternative RNA splicing, 16 amino acids, critical, 21 ancestral gene, 26, 49 as a local hormone in gonads, 5 0 , 5 1 CCAAT box, human and rat, 14, 15 cDNA, human pancreatic tumor, 5, 6 cDNA, mouse hypothalamus, 5, 7 cDNA, mouse placenta, 5, 7 cDNA, rat hypothalamus, 5,6 cDNA, rat placenta 5, 7 cleavage site, see Processing sites co-localization with NPY, 24 co-localization with vasotocin, 24, 104 conservation, 8, 20, 22, 49 cryptic peptide, 26 cryptic peptide, hydrophobicity, 26 evolution, 3, 19, 26, 27, 49, 50 exon loss, GHRH/PACAP gene, 26,27 exonlintron splice site, see Splice junctions expression, rat and mouse ovaries, 17, 50, 51 expression, rat and mouse placenta, 17 expression, rat and mouse testes, 17, 50, 51 family, 20, 49, 50 follicular maturation, 18 functional role, 18, 50, 51 gene, human, 5, 6, 14, 16, 19, 20, 26, 27 gene, mammals, 26, 27, 49 gene, rat, 5, 6, 14, 17

495 gene duplication, 26, 27, 49 GHRH1-,gNH2, activity, 22 GHRHl-2gNH2,activity, 22, 49 GHRHd2,mouse, 8, 23 GHRH43, rat, 6, 8, 23 GHRH44, COW,7 , 8 , 2 3 GHRH,, goat, 7, 8 , 2 3 GHRHM,human, 6, 8, 9, 13,23 GHRH4, pig, 7, 8, 23 GHRHu, sheep, 7, 8, 23 GHRH-like4s, carp, 7-9, 18 GHRH-like4s, catfish, 8 GHRH-like45, salmon, 7, 8 GHRH-like45, sturgeon 8 GHRH-like peptides, see Growth hormone-releasing hormone-like molecules growth hormone release in fish, 18 growth hormone release in tetrapods, 18,24 hGHRH1_370H, 5, 6 hGHRHlAONH2,role in golfish GH release, 9, 18 hGHRH1_MOH,5 , 6 , 11,21 hCHRHlAdNH2, 5, 6, 11, 13,21-23, 49 hCHRH1-&NH2,action on chicken pituitary, 5, 18 hGHRH1-&NH2,action on dwarf chicken pituitary, 5, 18 hGHRH1-MNH2,action on frog pituitary, 5, 18 hGHRH1-&NH2,action on turtle pituitary, 5, 18 homology to PACAP-related peptide, 26 immunoreactivity in arcuate/ ventromedial nuclei, human, 10 immunoreactivity in arcuate/ ventromedial nuclei, rat, 10 imrnunoreactivity in cells, frog, 10 immunoreactivity in non-neural tissues, 10 immunoreactivity in paraventricular nucleus, guinea pig, 10 immunoreactivity in gut, gonads and placenta, 10, 18, 50 interaction with GnRH, 50, 51 intron addition, GHRH gene, 27 mammals, 4, 10, 18, 50 mRNA, 16, 17,50

496 h’-terminal residues, deleted CHRHl-44, 21 N-terminal residues, substituted GHRHl_,o, 21 negative feedback of growth hormone, 17 pancreatic islet cell tumors, 5 peptide, cow, 8, 12, 23 peptide, goat, 8, 12, 23 peptide, human, 8, 12, 23 peptide, mouse, 8, 12, 23 peptide, pig, 8, 12, 23 peptide, rat, 8, 12, 23 peptide, sheep, 8, 12,23 peptide, structure, 8, 12, 23 precursor, 13 primary structure, 8 processing site, 13, 20 receptor, GHRHIVIP, 50 reproductive function, 18, 50, 51 splice junctions, 22 superfamily, 23, 49 TATA box, human and rat, 14, 15 transcript, human, 16, 17 transcript, rat, 17 tumors, pancreatic islet cell, 5 Growth hormone-releasing hormone-like molecule (GHRH-like) alternative RNA splicing, 18, 24, 25 amino acids, critical, 21, 22 autocrine role in the gonads, 51 carp, 7, 9-12 catfish, 4 cDNA, catfish, 9, 14 cDNA, salmon, 7, 9, 14 cDNA, sturgeon, 9, 14 chromatography, 12 cod, 10, 12 conservation, 19, 20, 49 GHRH1-2gNH2, carp, 9 GHRH1-450H,carp,9, 23 GHRH-liked5, fish, 7-9, 18 immunoreactivity, fish, 10, 11 paracrine role in the gonads, 50, 51 peptide, carp, 12, 23 peptides, 5, 7, 8, 9, 12, 13, 18, 20-24 position 1 amino acid, 22, 23 precursor cleavage, 20,22 precursor, 14, 15, 22, 24 processing site, 15, 20, 22, 24

SUBJECT I N D E X

rainbow trout, 10 receptor binding, 50 release of GH, fish, 18, 50 salmon, 4, 11, 12, 14, 15, 20, 22, 23, 25 sturgeon, 4, 14, 15, 22, 23 teleosts, 10 Growth hormone-releasing hormonelike/PACAP precursor (GHRH-like/ PACAP) ancestral gene, 26-28 cDNA, catfish, 14, 15 cDNA, salmon, 14, 15 cDNA, sturgeon, 14, 15 cryptic peptide, 14, 15, 24-26 gene, GHRH/PACAP, salmon, 10, 14, 16, 19, 20, 24, 25 gene, salmon, 10, 14, 24, 25 gene duplication, GHRH-like/PACAP, 19, 26, 27 gene organization, 19, 25 GHRH-like region, 14, 15, 24, 25 mRNA transcript, 14, 17, 18, 25 PACAP region, 14, 15, 19, 20, 24, 25 precursor, fish, 14, 15, 20, 24, 25 preprohormone, short, 18, 24, 25 processing site 18, 20, 24, 25 signal peptide, 14, 15, 25 transcript, salmon, 14, 25 Growth regulation, 181, 196, 202, 203 GTH, see Gonadotropin Guanine nucleotide-binding regulatory protein (G protein) granulosa cells, 407 inhibitory G-protein, 105, 407 stimulatory G-protein, 407 GV, see Germinal vesicle GVBD. see Germinal vesicle breakdown H

H1, see Histone H1 Heat shock protein hsp 56, 335 hsp 70, 335 hsp 90, 335 HeLa cells, 172-174,315, 324 Helix-turn-helix, 229, 318 Helodermin, 231 Helospectin, 231

SUBJECT INDEX Hematopoieticicytokine receptor superfamily, 370 Hepatic hypertrophy, 369 nuclear factor HNF,, 353 Hepatocyte cell culture, 350 rainbow trout, 333, 349, 350 HepG2, 324 Heterodimer, 319-320 hGH, see Growth hormone, human High performance liquid chromatography GHRH-like molecule analysis, 12 GnRH analysis, 32, 41 hGHRH,-,,NH2, retention time comparison, 12 somatostatin analysis, 254 Histone H I as an exogenous substrate, 422 kinase activity, 421, 422 to assess presence of maturationpromoting factor, 422 Homeobox genes, 169,317 Homeodomain, 169, 170, 173, 316-318 Homodimer, 335, 340,341 HOMOGAPN program, calculation of homology scores, 111 Hormone-binding domain, 344, 345, see also Receptor, steroids Hormone-dependent transactivation, (AF.), 348 Hormone response element (HRE), 336 HPA( I), see Hypothalamic-pituitaryadrenal (interrenal) system HPLC, see High performance liquid chromatography BOP-HSD, see 206-Hydroxysteroid de hydrogenase hsp, see Heat shock proteins Hydrocortisone, 372 Hydromineral imbalance, 382 Hydrophilicity, growth hormone, 184 Hydrophobicity, cryptic peptides, GHRH precursor, 26 17a-Hydroxylase/17,20-lyase cytochrome P-450 17a-hydroxylase activity, 411, 414 17,20-lyase cytochrome P-450 activity, 411, 414

pregnenolone to progesterone, 414 rainbow trout thecal cell cDNA library, 413 17a-hydroxyprogesterone to testosterone, 411 11,BHydroxylase metopyrone inhibition of activity, 85 3P-Hydroxysteroid dehydrogenase dehydroepiandrosterone substrate, 414 Aj-3P-hydroxysteroids to A4-3ketosteroids, 413 in mammals, 413 pregnenolone to progesterone, 414 pyrimidine-nucleotide binding domain, 414 rainbow trout thecal cell cDNA library, 413 Southern hybridization analysis, 414 17P-Hydroxysteroid dehydrogenase, 411 BOP-Hydroxysteroid dehydrogenase, 395, 404,405,408,409,411,412, 416 activity enhanced by CTHII, 137 homology with human carbonyl reductase, 413 pig testis cDNA library, 412, 413, 416 rainbow trout ovary cDNA library, 412, 416 Hypercalcemia, in the eel, 275 Hyperglycemia, induced by glucagon, 244 Hyperosmotic environment, 124, 125, 377 exposure, 195, 196 Hypoosmotic exposure, 195, 196 Hypophysectomy, 368, 378, 384 Hypophysis, see Pituitary, anterior Hypoplastic pituitaries, 314 H ypothalamic-pituitary-adrenal (interrenal) system control mechanisms, 72, 86 evolution, 72, 75 in coho salmon, 77 interaction with the immune system, 77 H ypothalamo-hypophysial axis, 103, 332, 333 portal plexus, 311 Hypothalamus aminergic fibers, 311

498

SUBJECT INDEX

anterior pituitary regulation, 311 corticotropin-releasing hormone production, 87 extracts, 5 fibers, 311 lesions, 5 neural regulatory factors, 311 neurosecretory fibers, 311 nucleus lateralis tuberis, 69, 81 nucleus recessus lateralis pars lateralis, 81 nucleus recessus lateralis pars medialis, 81 oxytocin synthesis, 159 peptidergic fibers, 311 role in growth hormone secretion, fish, 8, 9 tetrapods, 311 urotensin I immunoreactivity, 81 vasopressin synthesis, 159 I

ICI 164, 347, 384 IGF, see Insulin-like growth factor I and 11 Immune system interaction with the HPA(1) axis, 77 involvement of corticotropin-releasing factor, 77 Imniunohistochemistry gonadotropin I and 11, 140 Pit-1 in pituitary, 171, 172, 174 Immunoreactivity GHRH, 10, 50 GHRH-like peptide, 10, 11 gonadotropin-releasing hormone, 33, 38-41,45 isotocin, 104, 127 PACAP, 11,51 Pit-1, 171, 174, 314 stanniocalcin, 283, 285 vasotocin, 85, 104, 123, 124, 127 urotensin I, 81 Immunoregulation, 367 Inimunostimulatory factors, 369 I n situ hybridization corticotropin-releasing factor mRNA, 85-88

GnRH mRNA, 46,47 isotocin and vasotocin mRNA, 86, 104, 118 oxytocinivasopressin probe, 118 prolactin receptor localization, 371 somatostatin I and I1 rnRNA, 254 stanniocalcin mRNA, 274, 285-289 stanniocalcin digoxigenin-labelled probe, 287,289 synthetic oligonucleotide probe, 118 vasotocin 104, 118, 126, 127 Intestinal L-cells glicentin synthesis, 237 glucagon-like peptide I synthesis, 237 glucagon-like peptide I1 synthesis, 237 oxyntomodulin synthesis, 237 Insulin amphioxus, 227 ancestral gene, 219 anglerfish, 228-230 C-peptide, 226-228 carp, 228-230 chum salmon, 226-229 domains (A, B, C), 181 endoproteolysis of C-peptide, 230 enhancer elements, 229 evolution, 203 Far box, 229 family members, 203 gene, 226,227 gene family, 181 gene transcription, 228, 229 hagfish, 228-230 human, 228 IEB2, 229 IEG1, 229 Nir box, 229 preproinsulin, 228 primary structure, 228 processing site, 230 proinsulin, 229,230 promoters in mammals, 228 protein translation, 229 ratfish, 230 rodent, 226 sea raven, 229 secretion, 230, 231, 247 signal peptide, 228 signal recognition particle, 229 suppression of gluconeogenesis, 231

SUBJECT INDEX

suppression of glycogenolysis, 231 TATA box, 228 untranslated (UT) region, 226, 227 Insulin-like growth factor I alternative RNA splicing, 197, 199-202,215,217,220 amphioxus, 181 ancestral gene, 219 cDNA, 181, 197, 199,200, 215,218, 219 cRNA, 202 chicken, 181,202, 218 chinook salmon, 199,200, 21.5-218 chum salmon, 215-218 coho salmon, 181, 197, 199,200, 214-217,220 developmental role, 202, 221 domains (A, B, C, D, E), 181, 198-200, 202, 215, 216 evolution, 181, 197, 203 expression, 197, 199-202,214-221 expression during embryogenesis, 220, 22 1 function, 181, 202 gene, 181, 200,213-221 growth hormone mediation, 180, 214, 220 hagfish, 181,219,221 human, 197-199,216,218, 219 Ia, Ib, 200 in liver, 197, 200-202, 215,220 lamprey, 221 mammals, 181, 197,200,202,213-215 members of insulin family, 181 mRNA (Ea-1, Ea-2, Ea-3, Ea-4) 199-202 multiple E peptides, 199-202, 217, 218 non-allelic genes, 216 nonmammals, 181, 197,214 Northern blot analysis, 220-221 polymerase chain reaction analysis, 197, 199, 200,215, 216 postnatal liver synthesis, 220 preproIGF-I, 200, 215, 218 proIGF-IA-1, 215-217 proIGF-IA-2, 215 proIGF-IA-3, 215, 216 proIGF-IB, 21.5-217 primary structures, 198

499 rainbow trout, 181, 197-202, 21.5, 216, 218 release of growth hormone in fish, 9 RNase protection assay, 200, 202, 220 rodents, 202, 215, 218, 221 sea bream, 214 sequence alignment, 197-199 signal peptides, 181, 197, 216, 219 teleost, 213-216, 219-221 tissue-specific expression, 200-202 Xenopus, 181,216 Insulin-like growth factor I1 ancestral gene, 219 cDNA, 181, 197 cRNA, 202 developmental role, 202, 221 domains (A,B,C,D,E,), 181, 198-201 evolution, 181, 197, 203 expression during embryogenesis, 221 function, 181, 202 human, 197-199,218 in liver, 197, 200-202 member of insulin family, 181 mRNA, 199-202 Northern blot analysis, 220, 221 polymerase chain reaction analysis, 199 primary structure, 198 rainbow trout, 181, 197-199, 201, 202, 2 18 RNase protection assay, 200, 202 signal peptide, 181, 198 teleost, 214, 219, 221 tissue-specific expression, 200-202 Insulin-related peptide, molluscan, 181 Insulin superfamily agnatha, 219-221 amphioxus, 219 hagfish, 219, 220 hybrid insulin/IGF gene, 219 insulin, 213 insulin-like growth factor I, 213, 216 insulin-like growth factor 11, 213 Intermediate lobe, see Pars intermedia or Pituitary, intermediate Internal repeat sequences in growth hormone gene, 166-168, 191, 192 Internalization, 372 Interrenal cells, 71

500 Intestinal L-cells glicentin synthesis, 237 glucagon-like peptide I synthesis, 237 glucagon-like peptide I1 synthesis, 237 oxyntomodulin synthesis, 237 Intracellular metabolites, 195-197 osmolytes, 195 Intronlexon organization, see Exonl intron organization Ion balance, 320 pumps, 368 regulation, 164, 174 Ion-retaining effect, 377 Islet cells, see also Brockmann body effect of secretagogues on, 258 fish, 229, 230, 261 mammals, 230, 231, 248

3-Isobutyl-1-methylxanthine(IBMX), phophodiesterase inhibition, 407, 408, 417 Isoforms, multiple, 318, 319 Isotocin cDNA, salmon, 108, 111, 117, 118, 120 cDNA, white sucker, 108, 111 cortisol secretion in dexamethasoneblocked goldfish, 72 expression, 107, 117, 119-121, 124 gene group I, 11, 108, 110-113, 116, 118 immunoreactivity, 104, 127 in parturition, 105 i n situ hybridization, 104, 118, 126, 127 in spawning salmon, 105 inhibition of adenylate cyclase, 105 niRNA, 86, 119, 121, 124, 125, 127 protein sequence, 103 stimulation of adrenocorticotrophin release, 72-74, 86 sucker, 89 Isotocin precursor chum salmon, 108, 109, 117-120 effect of hyperlhypo-osmotic stimuli, 125 gene duplication, 93, 115, 120 isotocin 1, 108, 118, 120, 125 isotocin 2, 108, 118, 120, 125 niasu salmon, 117, 120

SUBJECT INDEX neurophysin, 109, 110 signal peptide, 109 structural organization, 109 sucker, 93, 108, 111, 120 IST, see Isotocin IT, see Isotocin J Jackson, 314 JAK family kinases, 371

JAK2,371 JAK2 tyrosine kinase, 371 K

Kallmann’s Syndrome GnRH neurons, 46

KALIG-1,46 Ketanonestrol aziridine, 344 Knock-out mice, 221

L Lactation growth hormone role, 180 prolactin role, 180, 311, 367, 379 a-Lactoglobulin, 371 growth hormone role, 180 prolactin role, 180, 311, 367, 379 Lactotrophs Pit-1, 168-172, 174, 311-335 prolactin synthesis, 163, 179, 311-315 LexA, 318 LexA binding sites, 318 LH, see Luteinizing hormone LHKH, see gonadotropin-releasing hormone (GnRH) Lindane, 347 Lineage growth hormone family, 167, 168, 174,

179, 180, 187, 191-197,203 prolactin, 180, 191-197, 203 somatolactin, 167, 168, 174, 179, 187,

191-194 Lipolysis glucagon activation, 244, 245

SUBJECT INDEX

501

Maturation-promoting factor activity, assessed by H1 kinase activity, 420 catalytic subunit, 422 homology to serinelthreonine protein kinase, 422 induced maturation of starfish oocytes, 420, 421 pre-MPF, cdc2 kinaselcyclin B complex, 425 regulatory subunit, cyclin, 422 similarity among vertebrates and invertebrates, 420 transfers between goldfish and Xenopus, 419 Maturational competence gonadotropin induction, 419 relationship with 20p-S concentrations, 419 Melanin-concentrating hormone, in chum salmon, 117 a-Melanocyte-stimulating hormone release stimulation by CRF-like peptides, 73 release stimulation by ovine CRF, 73 release stimulation by sauvagine, 73 release stimulation by urotensin I, 73 Melanotrophs amphibian, 73 M brown trout, 74 CRF immunoreactivity in Magnocellular neurons, CRF perikarya, 81 mammals, 73 imniunoreactivity, 81, 86 Mammary gland, 369-372 rainbow trout, 74 Maturation-inducing hormone Rana ridibunda, 73 release of a-melanocyte-stimulating amago salmon, 404 17a,20fl-dihydroxy-4-pregnene-3-one, hormone, 73 400, 404,405 Mesotocin cDNA in toad, 120 dragonet, Repomucenus beniteguri, 419 evolution, 115 kisu, Sillago japonica, 419 gene duplication, 115 neurophysin, 109, 110 mummichog, Fundulus heteroclitus, precursor signal peptide, 109 405 oocyte insensitive phase, 419 sequence, 103 structural organization, 109 oocyte maturation, 394, 398 oocyte sensitive stage, 419 Metabolic activation, 203 5a-pregnan-3P,17a,20P-triol, 402 Metabolism, 195-197, 309, 367, receptors, 417 368 17a,ZOp,21-trihydroxy-4-pregnene-3- Metopyrone one, 400, 419 effect on CRF synthesis, 87

Lipotropin, 160 LRF, see Gonadotropin-releasing hormone (GnRH) Ltk, 324 Luciferase expression, 169, 172 Luteinizing hormone anterior pituitary secretion, 136 /3 gene, 141 bovine fl gene promoter, 145 CAP site, 145 equine p gene, 145 gonadotrophs, 311-313 GnRH-stimulated release, 41 GSE homologous sequence, p subunit, 149 ovarian regulation, 311 PACAP-stimulated release, 18, 19, 50 rat /3 gene, 145 rat p promoter, 169 TATA box, 145 testicular regulation, 311 tetrapod, 138 Luteinizing hormone-releasing hormone (LHRH), see Gonadotropin-releasing hormone (GnRH) 17,20-Lyase, 411-414 Lysosomes, 369

SUBJECT INDEX

effect on urotensin I and I1 immunoreactivity, 82 inhibition of 11-p hydroxylase activity, 85 Microiontophoresis, 105, 123 Mid-vitellogenic oocytes estradiol-17p production, 137 steroidogenic enzyme levels, 416, 417 Migratory pathway GnRH neurons, 33,39,40,45,46 MIH, see Maturation-inducing hormone Milk production prolactin control of, 311 Mitogenic stimulation human proIGF-IB, 217 of IGFs in general, 203 Mouse mammary cell cultures, 372 MPF, see Maturation-promoting factor mRNA corticotropin-releasing factor, 85, 86,90 GHRH, 16, 17, 50 GHRH-like/PACAP, 14, 17-19, 25, 50 GnRH, 45,46 gonadotropin p subunit, 142, 144, 151 insulin-like growth factor I, 199-202 insulin-like growth factor 11, 199-202 isotocin, 86, 119, 121, 124, 125, 127 oxytocin, 121, 127 PACAP, 9, 17, 19, 50 Pit-1, 314, 315 prolactin, 127 somatostatin, 160-163, 254 stanniocalcin, 277, 279, 296 vasopressin, 86, 121, 127 vasotocin, 86, 88, 119, 121, 124, 125, 127, 128 a-MSH, see a-Melanocyte-stimulating hormone MT, see Mesotocin Mutant, 316 Mutation rate, 114

N

N-terminal sequence amino acids (residues), glucagon superfamily peptides, 21-23 amino acids, pit-1, 318, 319, 322, 324 transactivation region, 318, 319 Na', see Sodium

Nb2 lymphoma cell line, 370 Neural cell adhesion molecule, 46 Neural cells, 310 Neural regulatory factors, 311 Neural tube, 314 Neurohypophysis, see Pituitary, posterior Neurointermediate lobe GHRH immunoreactive fibers, fish, 11 Pit-I, 171, 174 somatolactin, 160, 161 Neuropeptide Y appetite stimulation, 261 effect on blood pressure and heart rate, 261 co-localization with GHRH, 24 Neurophysin hagfish, 109 mammals, 116 teleost, 109-111, 116 toad, 116 Neurosecretory axon terminals, 103 Neurosecretory cells (magnocellular) effect of hyperosmotic stimuli, 124-126 hormone production in preoptic nucleus, 103 secretion, 106, 117, 121, 128 stimulation from sodium loading, 121, 125, 126 stimulation from water deprivation, 121, 125, 126 teleost, 72, 105, 126, 128 Neurosecretory fibers, 311 Neurosecretory system caudal, U I immunoreactivity, 77, 81 homeostatic osmoregulation, 124 responses to environmental salinity, 125 NLT, see Nucleus lateral tuberis Noradrenaline, 107 Northern blot analysis corticotropin-releasing factor, 80, 85, 88 estrogen receptor, rainbow trout, 337 gonadotropin-releasing hormone, 45 human follicle-stimulating hormone p gene, 144 insulin, 228 insulin-like growth factor I, 220, 221 insulin-like growth factor 11, 220, 221

503

SUBJECT INDEX

isotocin and vasotocin precursors, 119, preprosomatostatin I, 11, 25 somatolactin, 162 stanniocalcin, 277,295 NPO, see Preoptic nucleus NPY, see Neuropeptide Y NRLl, see Nucleus recessus lateralis pars lateralis NRLm, see Nucleus recessus lateralis pars medialis NSCs, see Neurosecretory cells (magnocellular) Nuclear estrogen receptor, 332 Nuclear localization, 335 Nuclear magnetic resonance, 318 Nuclear receptor superfamily, 332,333 Nucleosome, 334,336,337 Nucleus lateral tuberis control of adrenocorticotrophin biosynthesis, 69,85 corticotropin-releasing factor production, 69 CRF genes, sucker, 85,86 destruction, lesion, 85 UI-like CRF peptide, 85 urotensin I immunoreactivity in perikarya, 81 urotensin I transcripts, 88 Nucleus recessus lateralis pars lateralis CRF immunoreactivity in perikarya, 81 Nucleus recessus lateralis pars medialis CRF immunoreactivity in perikarya, 81

0 Oct-1 transcription factors, 169,316,320 Oct-2 transcription factors, 169,316 Olfactory chemosensory system effect of diluted seawater, 123 suppression of electrical activity, 123 Olfactory placode GnRH neuron development, 33,39,

45,46 Oncogenes simian virus 40 T-antigen oncogene,

143

Oocyte maturation, induced by cyclin B,

426,427

120

Oocytes Atlantic croaker, 403 first meiotic division, 394 first meiotic prophase, 393 follicle layer, 395 G2lM border, 393 germinal vesicle breakdown, 394 goldfish, 394 Indian catfish, 395 maturation, 394 micropyle, 395 postvitellogenic, 400 second meiotic metaphase, 394 teleost, 394 yellow perch, 395 zona radiata, 395 Oogenesis, 406 oPRL, see Prolactin, ovine Ornithine decarboxylase, 369 Orphan receptors, 332 Osmoconformers, 195 Osmolarity, intracellular, 195 Osmoregulation bioassay, 373 calcium, 180 effects, 180 evolution of hormone control,

194-197,203 growth hormone, 194-197 in neurosecretory system, 124-126 insulin-like growth factor I, 220 organs, 381,382,384,385 osmoconformers, 195 prolactin, 180,194-197,311-325 response, 194-197,203 role, 124,125 role of corticotropin-releasing factor, 77 somatolactin, 180,194-197 Osmoregulatory surfaces, 368 Osmotic pressure, 195 Osmotic stress, 203 OT, see Oxytocin hormone family Ovary 17~~,20p-dihydroxy-4-pregnene-3-one production, 406,408 follicles, 395 oocyte maturation, 394 teleost, 393,394

504

SUBJECT INDEX

Oviparous species, 331 OXT (oxytocin), see Oxytocin Oxpntomodulin synthesis in intestinal L-cells, 237 Oxytocin antisera, 127 aspargtocin in Squalus acanthias, 103 asvatocin in Scyliorhinus caniculus, 103 co-expression with vasopressin in rats, 86 effect of hyperosmotic stimulation, 125 evolutionary pathway, 110, 111, 113, 115, 116 gene duplication, 116 isotocin, 102, 120 mammals, 109, 110, 117, 120 mRNA, 121, 127 neutral peptides, 103 osmotic regulation in mammals, 125 phasvatocin in Scyliorhinus caniculus, 103 ratfish, 103 valitocin in Squalus acanthias, 103 P

P-450arom, see Aromatase cytochrome P-450 P-450~17,see 17a-Hydroxylasell7,20lyase cytochrome P-450 see Cholesterol side-chain P-~SOSCC, cleavage cytochrome P-450 P-box steroid receptors, 340-342 PSSIhsp 56 proteins, 335 PACAP, see Pituitary adenylate cyclase activating peptide PACAPIPHP, see PRPIPACAP PACAP-related peptide, see pituitary adenylate cyclase activating polypeptide-related peptide Pachytene microsporocytes of lily induce CVBD in Xenopus, 420 Palindromic DNA, 336, 340 Pancreatic polypeptide family amino acid sequence, 260 ancestral peptide, 259 anglerfish, 259, 260 cDNA, 259

coho salmon, 261 dogfish, 260, 261 goldfish, 259, 260 lampreys, 261 mammals, 259, 261 neuropeptide Y, 259 pancreatic polypeptide, 258, 259 peptide Y, 259 tertiary structure, 258 Torpedo marmorata, 259, 260 Pancreatic tissue, see also Brockmann bodies A cells, 261 islet cell tumors, GHRH, 5 islet cells, see Islet cells production of proglucagon, 233, 237 regulation of glucagon secretion, 244 Paracrine, 369 factors, 312 hormone, GHRH-like, 51 hormone, PACAP, 51 Parathyroid hormone G-protein coupled receptor, 247 negative regulation by calcium, 293 Paraventricular neurons oxytocin mRNA levels, 125 vasopressin mRNA levels, 125, 127 Pars distalis, see also Pituitary, anterior adrenocorticotrophin-producing cells, 311 CHF in rostra1 pars distalis, 81 gonadotrophs, 145 prolactin-producing cells, 313 rainbow trout, 145 Par5 intermedia (see also Pituitary, intermediate) CRF action on aMSH, 73 CRF-U1 action on POMC, 74 melanocyte-stimulating hormone, 73, 160 origin, 310 proopiomelanocortin (POMC), 160 rat, 73 somatolactin, 160, 163, 170, 180 Pars tuheralis, 310 Parvocellular neurons, CRF immunoreactivity, 86 PAS reagent, see Periodic acid-Shiff reagent PCH, see Polymerase chain reaction

SUBJECT INDEX

PCs, see Prohormone convertases Peptide 7B2, release by PACAP, 18 Peptide family, definition, 49 Peptidergic neurohormones, intracellular communication, 102 PEPTIDESTRUCTURE program growth hormone, 184 computer program, 184 surface probability statistic, 184, 185 Wisconsin GCG program, 184 Periodic acid-Schiff reagent, 160 Phasvatocin, 103 PHI and PHM family conservation, 26, 27, 49, 50 glucagon superfamily member, 23,49,50 PHM/VIP ancestral gene, 26, 27, 49 evolution, 26-28 gene, human, 26-28 precursor, 24 Phosphatase inhibitors, 420 Phosphodiesterase inhibitors IBMX, 407,408,417 theophylline, 407, 408, 417 to measure cyclic AMP concentrations, 24 1, 242 Phosphorylation, of pit-1, 316 in pathway activated by GH binding, 371 protein kinase A-mediated, 245 Phylogenetic analysis Boot Strap probability, 116 distance matrix, 115 maximum likelihood, 11.5, 116 PROTMEL method, 115 star decomposition, 115 Phylogenetic divergence, 115, 116, 320-324 Phylogenetic map, GnRH, 34-37, 41, 42, 45,48 Phytoestrogens, 347 Pit POUHD, see Pit-1, POU homeodomain Pit-1, also culled GH factor-1 antibodies, 171, 174 binding regions, 16.5, 169, 172-174, 190-192,315-318,320 bovine, 169 cDNA, 170, 171, 174, 321, 322 cDNA expression vector, 322

505 chinook salmon, 173 chum salmon, 170-174 combined pituitary hormone deficiency, mutation, 317 conservation, 173 cotransfection, 172, 174, 319-321 deficient animals, 314 dependent cells, 315 development, 314 dimerization, 319, 320 distal binding regions, 320, 321 D N A interactions, 316, 317 domain, 319 expression, 314, 315 function, 169, 170, 172, 174, 316-318 gene, 190, 313-315 growth hormone gene, 165, 168-174, 190-192 human, 15, 315 immunoreactivity, 171, 174 in anterior pituitary, 171, 172, 174 in lactotrophs, 314, 315 in neurointermediate pituitary, 171, 172, 174 in somatolactin-producing cells, 171, 172, 174 in somatotrophs, 314, 315 in thyrotrophs, 315 independent cells, 31.5 isoform, 170, 318, 319, 323, 324 isoform Pit-la, 319, 323 isoform Pit-lp, 319, 323, 324 isoform Pit-ly, 319, 323 lactotroph development, 170-172, 174 mammals, 170-174, 313-315 mouse, 170 mRNA, 170, 171, 314, 315 mutant form, 173, 314 pituitary cell differentiation, 170, 172, 310-313 POU domain, 169,170, 173,316-318, 322,323 POU family, 169, 171 POU homeobox, 169, 170, 173 POU homeodomain, 316-318 prolactin gene, 165, 170, 172-174, 190-192 protein, 171, 172, 174 proximal binding regions, 321 PRP/PACAP, human, 15

506 rainbow trout, 171, 172, 174 rat, 169-174, 317, 322 response element, 165, 169, 172-174, 190-192,315-318,320 salmon, 170-174,320-324 sequences, 322 somatolactin gene, 165, 170-174, 191, 192 somatotroph development, 170-172, 174 stimulation of GH/PRL/SL promoters, 172-174, 190, 191 thyrotroph development, 170-172, 174 tissue-specific expression, 168, 172, 174, 190 transcriptional activation, 173 turkey, 170, 323 Pituitary adenylate cyclase activating polypeptide (PACAP) action with GnRH, 19, 50 alternative RNA splicing, 24, 25 as a local hormone in gonads, 50, 51 autocrine role in the gonads, 51 catfish 4, 51 cDNA, catfish, 10 cDNA, human, 7, 10, 17 cDNA, rat, 7, 10, 17 cDNA, salmon, 7, 10, 17 cDNA, sheep, 7, 10, 17 cDNA, short precursor, 24 cDNA, sturgeon, 10 cleavage sites, see processing sites cosecretion with GHRH, 24 evolution, 19, 20, 26, 27, 49 exodintron splice site, see splice junctions exons, 19,20,24-27 follicular stellate cells, interleukin-6 release, 24 frog, 7 FSH stimulated release, lack of, 18 functional role, 10, 18, 24, 50, 51 gene, human, 7, 10, 14-16, 19, 20, 26 gene, salmon, 10, 14, 16, 19, 20, 24-26 gene duplication, GHRH/PACAP, 19, 26, 27, 49 glucagon hormone family, 19, 23, 49, 231 gonads, 51 immunoreactivity, ferret, 11

SUBJECT INDEX

immunoreactivity, lack of in fish, 11 immunoreactivity, guinea pig, 11 immunoreactivity, human, 11 immunoreactivity, monkey, 11 immunoreactivity, pig, 11 immunoreactivity, rat, 11, 51 immunoreactivity, sheep, 11 in mammals, 4, 51 mRNA tissue expression, 9, 17, 19, 50 PACAP,-27, human, 13 PACAP1-27,sheep, 9 PACAP,_27NH2,catfish, 20 PACAPI_,,NH2, conservation, 20 PACAP,-,7NH,, salmon, 13, 20 PACAP,_27NH2,sturgeon, 20 PACAP,_,, functions, 19 PACAP1-3B,catfish, 8 PACAP,-3H,frog, 8, 10 PACAP,-,, human, 8, 13,23 PACAP,_,,, salmon, 8, 13 PACAP,_,,, sheep, 9, 23 PACAP1-,NH2, conservation, 20 paracrine role in the gonads, 50, 51 peptide family, 19 peptides, frog, 8, 12 peptides, sheep, 9, 12 23 peptides, structure, 8, 23 precursor, cryptic peptide, 24-26 precursor, GHRH-like region, 13, 19, 20, 24, 25 precursor, PACAP region, 9, 13, 19, 20, 24, 25 precursor, PACAP1_27,13 precursor, PACAP,_3B,13 precursor, short, 24, 25 precursor, signal peptide, 13, 25 processing sites, 13, 20, 25 receptors, ovarian, 51 receptors, spermatogonia, 51 receptors, spermatozoa, 51 role in reproduction, 18, 19, 50, 51 salmon, 4 sheep, 7 , 9 splice junctions, 22-24 stimulation of ACTH release, 18 stimulation of adenylate cyclase, 18, 20 stimulation of cyclic AMP, 9 stimulation of GH release, 9, 18 stimulation of LH release, 18, 19

SUBJECT’INDEX

stimulation of peptide 7B2, 18 stimulation of prolactin release, 18 sturgeon, 4 superfamily, 23, 49 thyroid-stimulating hormone release, lack of, 18 Pituitary adenylate cyclase activating polypeptide-related peptide (PACAP-related peptide, PRP) amino acid in position-1, 23 brain, rat and sheep, 6 cleavage site, see Processing site function, 23, 24 GH release, lack of, 9, 24 glucagon hormone family, 23,231 homology to GHRH, 9, 14 mammals, 4, 14, 23 precursor, 9, 13, 15, 23, 24 processing site, 13 testes, human, 6 Pituitary adenylate cyclase activating polypeptide-related peptide/PACAP (PRP/PACAP) ancestral gene, 26, 27 cDNA, mammalian, 14 evolution, 26, 27 flanking sequence, 5’, 15 gene, human, 6, 7, 10, 14-16, 19, 20, 26 precursors, 15, 24 transcription factors, 15 Pituitary, anterior adenomas, 315 adrenocorticotropic hormone, 74, 160 cell lineages, 310-313 cells, 311-313 chum salmon, 312 coho salmon, 312 cultured cells, 315 differentiation, 310-3 13 development, see Pituitary, anterior, development endocrine cell types, 311 endocrine regulation, 311 follicle-stimulating hormone, 144 GH, cell line, 315 gonadotrophs, 145 gonadotropin release, 142, 153, 160 gonadotropins, 160 growth hormone, 160

hypophysis, 310-313 hypoplastic, 314 lactotrophs, 163, 168, 170-172, 174, 179 lipotropin, 160 lobe, 310 luteinizing hormone, 145 mammalian development, 310-311 ontogeny, 310-313 prolactin, 160, 168, 170, 174 rat, 73, 311, 312 somatotrophs, 168, 170-172, 174, 179 termination of GnRH axons, 41 thyroid-stimulating hormone, 160, 170 thyrotrophs, 147, 170-172, 174 urotensin I potency in goldfish, 73 Pituitary, anterior, development cell lineages, 311-313, 314 cells, 170, 311-313 corticotrophs, 311-313 diencephalon role, 310 ectodermal origin, 168, 310 endocrine factors, 310 glycoprotein hormone a-subunit, 313 hormones, 311 oral cavity, 168, 310 paracrine factors, 311, 312 Rathke’s pouch, 168, 170, 310 salmonid cells, 321, 324 Pituitary, intermediate proopiomelanocortin (POMC) derived peptides, 160 melanocyte-stimulating hormone, 160 Pit-1, 171, 172, 174 somatolactin, 160, 163, 170, 180 Pituitary, posterior diencephalon, outpouching of, 310 glial cells, 310 hormones, see Pituitary, posterior, hormones neural cells, 310 neuromodulators, 105 neurotransmitters, 105 termination of GnRH axons, 33, 38, 40, 41 Pituitary, posterior, hormones activation of second messenger system, 106, 107 catostomids, 117, 127 circannual control of expression, 120

SUBTECT INDEX

diurnalicircadian control of expression, 120 electrical activity, 105-107 gene expression, 107, 124 goldfish, 86 isotocin, 72, 85, 105 lunar control of' expression, 120 mesotocin in lungfish, 103 mRNA poly(A) tail size, 121, 124 oxytocin hormone family, 102, 103, 111, 113, 159 phylogenetic tree, 114, 116 precursors, 109, 116 salmonids, 114, 115, 117, 127 seasonal control of expression, 120, 127 stimulus transcription coupling, 106 vasopressin hormone family, 102, 103, 111, 113, 159 vasotocin, 72, 85, 105, 123, 124 Pituitary-adrenalcortical axis relationship to caudal neurosecretory system, 82 Pituitary cells goldfish, 18, 69, 73-75 rainbow trout, 18 PL, see Placental lactogen Placenta CRF mRNA synthesis, 90 placental lactogen synthesis, 180 Placental lactogen ancestral gene, 180, 191 conserved domains, 191 expression, 180 family evolution, 179, 180, 191-194 fimctions, 180 gene, 180, 191 gene sequence, 180, 191 precursor, 167 primary structure, 180 svncytiotrophoblasts, 180 Plasma chloride levels, 378 la',2~a-dihydroxy-4-pregnene-3-one, 401 gonadotropin, 401 sodium levels, 378 somatolactin, 164 thyroxine, 373 vasotocin, 123, 125, 126 PhlA, a phorbol ester, 153, 408

Point transversions, 316 Polydipsia, in rats, 125 Polymerase chain reaction (PCR) gonadotropin-releasing factor, 45 insulin-like growth factor 197, 199, 200, 215, 216 primers, 323 Polyploidization, 91, 93 Polyurea, in rats, 125 PONmg, see Preoptic nucleus Postvitellogenic follicles, 405 Postvitellogenic oocytes, 400 17a,20P-dihydroxy-4-pregnene-3-one production, 137, 404 20P-hydroxysteroid dehydrogenase activity, 137, 404, 409 granulosa layer, 137, 404 maturation-inducing factor, 137, 401, 402 POU domain, in rat Pit-1, 169, 170, 173 domain, in salmon Pit-1, 170, 173, 321, 322 domain, structure and function, 316-3 18 domain proteins, 316-318, 320 homeodomain (POUHD), 169, 170, 317, 318 specific domain (POUS),316-318, 321-323 transcription factors, 169, 170, 173, 317, 324 POU,,, see POU, homeodomain POUS, see POU, specific domain PPSS (preprosomatostatin), see Somatostatin Precursor corticotropin-releasing factor, 78-80, 87, 89-93, 338,339, 341, 342, 350, GHRH-like peptide, 14, 15, 22, 24 GHRH(PRP)/PACAP, 14, 15, 18, 20, 24, 25 glncagon/GLP, 28 gonadotropin-releasing hormone, 43,44 insulin, 181 insulin-like growth factors, 181 PACAP long precursor, 9, 13, 19, 20, 24,25 PACAP short precursor, 18,24,25 PHMNIP, 24

SUBJECT INDEX

placental lactogen, 167 somatolactin, 166, 167, 187 somatostatin, 250, 252-255, 257, 258 vasoactive intestinal peptide, 24 vasotocin 93, 108-111, 115, 117-120, 125 Pregnenolone, 402, 403 Preinitiation complex, 334, 335 Preoptic-neurohypophyseal tract CRF immunoreactivity in perikarya, 81 Preoptic nucleus acetylcholine excitation, 107 control of adrenocorticotrophin release, 69 corticotropin-releasing factor production, 69 cortisol pellet implants, 82 CNF immunoreactivity in perikarya, 81 CRF-like peptide, 80, 85 destruction, lesion, 85 effects of angiotensin 11, 107 effects of dopamine, 107 effects of isotocin, 107 effects of nionoaniines, 107 effects of neuropeptides, 107 effects of noradrenaline, 107 effects of vasotocin, 107 electrical response to osmotic stimuli, 124 glutaniic acid excitation, 107 hormone synthesis, 107 magnocellular neurons, 85 magnocellular region, 81, 82 rnuscarinic receptors, 107 parvocellular region, 82, 85, 86 sucker CRF genes, 80 urotensin I transcripts, 88 Preprohormone, see also Precursor glucagon, 232-236 insulin, 181 insulin-like growth factors, 181 somatolactin, 187 somatostatin, 251 Pretranslational regulatory model, 315 Primary structures glucagon, 238 glucagon-like peptides, 239 gonadotropin-releasing hormone, 30-32,41 growth hormones, 166, 180, 183, I92

509 insulin, 228 insulin-like growth factors, 198 prolactins, 166, 167, 180, 185, 186 somatolactins, 180, 187, 188 stanniocalcin, 278-279 Primer extension experiments, 353 PRL, see Prolactin Processing sites CRF, signal peptide, 78, 79 dibasic enzyme, 20 GHRH 13, 20 GHRH-like molecule, 13, 20, 22, 25 CHRH precursor, 13, 20 CHRH(PRP), 13, 20 GnRH, 44 insulin, 229, 230, 257 PACAP, 13,20 PACAP-related peptide, 13, 20 somatostatin, 251 Progesterone hormone, 373,400 receptor, 353 Prohormone convertases (PC) aspartyl protease homologue in yeast, 257,258 aspartyl protease inhibition of PC2, 257 calcium activation, 230 conversion of proinsulin to insulin, 229, 230,257 conversion of prosomatostatin to somatostatin, 255, 257 in anglerfish, 229, 230 in mammals, 230 PC2, 230,257 PC3,230 Prolactin action, 220, 367 amphibians, 180 ancestral gene, 180, 191-197, 203 binding, 370, 378 binding sites, 369, 372-376 birds, 180 bovine, 163, 189 bull frog, 186, 187 carp, 186, 187, 189, 190 catfish, 186, 187, 189, 190 cDNA, 180, 185 cDNA, receptor, 370, 377 cells (lactotrophs), 168, 170, 174, 311-315

510 chicken, 186, 187 chinook salmon, 189, 193, 194 chum salmon, 186, 187,312 coho salmon, 312 conservation, 185-187, 191-194 conserved domains(A,,l, B,,I, C,,,, Dprl), 185-187, 191, 192 construct with CAT, 321, 324 disulfide bonds, 185, 187 euryhaline fishes, 164, 313 family evolution, 179, 187, 191-197, 203 flanking sequence, S’, 168, 169, 190, 191, 193, 321 function, 164, 180, 194-197, 311 gene, 164-174, 180, 189-191, 193, 194, 315, 316,320, 321 gene duplication, 180 gene expression, 168, 173, 174, 180, 320,321 gene promoter, 169, 315, 316, 320, 321 genomic organization, 180, 189, 190, 320, 321 GH, cells, 321 human, 163, 166, 167, 186, 187, 189, 190 inhibition by GAP, 43, 44 invertebrates, 194 lactation, 180 lactotrophs, 168, 170, 174, 311-315 mammals, 163, 165-167, 169, 173, 174, 180, 185, 189, 190,311, 312, 314 milk production, 311 osmolarity intracellular, 180 osmoregulatory response, 194 ovine, 145, 163, 167, 186, 187, 372-376,379 Pit-1 regulation, 168-174, 190-193 primary structure, 166, 167, 180, 185, 186 promoter, 169, 173, 174, 315, 316, 320, 32 1 rainbow trout, 127 rat, 18, 165-167, 169, 173, 186, 187, 189-191 receptor, 367-391 receptor binding, 368, 370 receptor binding proteins, 370 receptor cDNAs, 370, 371 receptor mRNAs, 369-371

SUBJECT INDEX

receptor, ti PRL-I, 374, 375, 377-383 receptor, ti PRL-11, 374, 375, 377-383 reptiles, 180 reproduction, 196 response to PACAP, 18 salmon (sPRL), 189, 190, 193, 377,384 sea bream, 313 sea turtle, 186, 187 sequence alignment, 185, 186 target genes, 315, 316 teleost, 185 tetrapod, 161, 185 ti PRL, 374, 375,382, 384, 385 tilapia, 186, 187, 189, 190, 193, 368, 373-378 Prolactin/CAT constructs, 321, 324 Prolactin-secreting rat GH4 cells, 315, 321 Proliferin, 376 Promoters, see also Flanking sequences, 5’ bovine gonadotropin a subunit, 143 bovine luteinizing hormone, 145 follicle-stimulating p subunit, 144 glycoprotein hormone a subunit, 143 gonadotropin p subunit, 144, 145, 147 growth hormone, 172, 173 prolactin, 147, 173 somatolactin, 173 Proopiomelanocortin-derived peptides from teleost melanotrophs, 74 release stimulation by CRF, 74 release stimulation by urotensin I, 74 Protein, see also specific protein or peptide interactions, 319, 320 kinase A or C, 240, 242, 316 protein-protein interaction, 334, 337, 342 Prothoracicotrophic hormone, insect, 181 PROTMEL method analysis of distant phylogenetic relation, 115 distance matrix, 115 star decomposition algorithm, 115 PRP, see Pituitary adenylate cyclase activating polypeptide-related peptide PRPIPACAP, see Pituitary adenylate cyclase activating polypeptiderelated peptide/PACAP

511

SUBJECT INDEX

Pseudogene urotensin I sequence, 93 PSS (prosomatostatin), see Somatostatin PTH, see Parathyroid hormone PTTH, see Prothoracicotrophic hormone Puromycin, translational inhibition, 417 R

Radioimmunoassay cholesterol side-chain cleavage cytochrome P-450,412, 413 gonadotropin, 412, 413 gonadotropin-releasing hormone, 32,41 human follicle-stimulating hormone fl gene, 144 insulin-like growth factor I, 214 insulin-like growth factor 11, 214 somatolactin, 164 vasotocin plasma levels, 123 Radioreceptor assay, 374-376, 378,379 RAR, see Retinoic acid receptor Rathke’s pouch, 168, 310, 325 Reabsorption, 368 Receptor Arg-vasopressin, 104 binding, growth hormone, 184, 185 binding, prolactin, 368, 370 binding proteins, prolactin, 370 cDNA, prolactin, 370, 371 CRF-RI in goldfish, 76 CRF-RII in mammals, 76 CRF-RIII in amphibians, 76 D-box, 340-342 dimers, 335, 340, 344 erythropoietin, 371 estrogen, 320, 332,334-336, 344-349 estrogen, rainbow trout, 337, 338, 344-349 glucagon, 240 glucocorticoid, 338, 353 gonadotropin, 137 gonadotropin-releasing hormone, 153 growth hormone, 203, 371, 373 heat shock proteins, 335 heterodimers, 320 homodimers, 335,340,344 hormone-binding domain, 344-347

hormone-dependent transactivation (AFZ), 347,348 insulin-like growth factor, 214 muscarinic, 107 nuclear translocation, 335 P-box, 340-342 prolactin, 368-372, 382-384 prolactin, mRNA, 370, 371 protein-protein interactions, 334, 337, 342 retinoic acid, 332, 338 steroid receptor, 151-153 thyroid receptor, 332, 338 vasotocin 104, 105 vitamin D receptor, 332 Receptor binding assay insulin-like growth factor I, 213, 214 insulin-like growth factor 11, 213, 214 Recognition helix, 317 Relaxin, 181 Reproduction, 18, 19, 29-51, 107, 140, 164, 311, 367 Response element AP-1 and AP-2 response elements, 143 CRE, cyclic AMP response element, 15, 89, 144, 240, 242 ERE, estrogen response element, 44, 144, 145, 149-151, 320, 341,342, 348,349 ERE, half site (half palindrome), 144, 4 15 Far box, in insulin gene, 229 GnRH-RE, gonadotropin-releasing hormone response element 144, 153 GRE, glucocorticoid response element, 90, 341, 354 CSE, gonadotroph-specific element, 143, 144, 149 HRE, hormone response element, 340-341 Nir box, in insulin gene, 229 PGRE, glycoprotein hormone (basal) response element, 153 Pit-1-RE, Pit-1 response (binding) element, 165, 168, 173, 174, 191-193, 315, 322, 324 TPA response element, 15, 353 TRE, thyroid hormone response element, 144

512

SUBJECT INDEX

TRH, TSH-releasing hormone, 144 TSE, trophoblast specific element, 144 Retina GnRH immunoreactivity, fish, 45 Retinoic acid receptor (RAR), 332 Reverse transcriptase/PCR, insulin-like growth factor, hagfish, 219 insulin-like growth factor, lamprey, 22 1 method, 199 RNA splicing alternative, see Alternative RNA splicing RNA synthesis actinomycin D, 408 a-amanitin, 408 cordycepin, 408 inhibitors, 408 RNase protection assay, 200, 202, 220 RPA, see RNase protection assay rtER, see Estrogen receptor, rainbow trout RTH, see Hepatocytes, rainbow trout S 2Op-S, see 17a,20P,21-Trihydroxy-4pregnen-3-one Salinity, 367, 368, 382, 383, 385 Sarcosine, analogs of angiotensin 11, 75 Sauvagine amphibian skin CRF-like peptide, 72 endorphin release in Xenopus, 73 homology to urotensin I, 72, 89 a-melanocyte-stimulating hormone release, 73 Scatchard analysis, 373, 379, 380, 382, 418 Sea water-adapted fish, 105, 123, 125, 128 Secretagogues, 230, 231, 244, 258, 290 SECR, see Secretin Secretin, 23, 231, 247 Sex steroids, see individual steroids SGA, 407 Signal peptides cleavage sites, CRF, 78, 79 cleavage sites, UI, 78, 79 GHRH precursor 13, 27

GHRH-IikeiPACAP precursor, 13-15, 25, 27 GnRH precursor, 43, 44 insulin-like growth factor I precursor, 215 insulin precursor, 228 isotocin precursor, 109 mesotocin precursor, 109 PRP/PACAP precursor, 13-15, 27 somatostatin precursor, 252, 253 urotensin I precursor, 78, 89, 91 vasotocin precursor, 109, 111 Signal transduction, 382 Signal transduction pathways G-protein coupling, 76 GnRH receptor, 153 cyclic AMP, 76 prolactin, 371 protein kinase C , 76 second messenger systems, 76, 106 Silencer, 353, 354 Silencer sequences, 148, 353, 354 Silent gene urotensin I sequence, 93, 94 Site-directed mutagenesis, 341, 344 SL, see Somatolactin Snell mouse, 314 mutants, 314 Sodium effect of sodium loading, 121, 125 retaining effect, 368 transport, 368 Somatolactin alternative polyadenylation, 161 amino acids, Cys, 161, 166, 167, 187 ancestral gene, 167, 168, 174, 19-194 antibodies, 164 Atlantic cod, 160-164, 187, 188 Atlantic salmon, 163 bonito tuna, 163 bull frog, 163 catfish, 163, 187-189 cDNA, 160-164, 180, 187 chum salmon, 161-165, 172, 173, 187-189, 193 cod, 161, 187-189 coho salmon, 163, 164 common carp, 163 concentration in plasma, 164 conservation, 162, 166-168, 187-189

SUBJECT INDEX

conserved domains (As,, B,,, C,,, Dsl), 162, 166-168, 187-189, 191, 192 distribution, 163 effect on hepatic IGF-I, 220 evolution, 167, 168, 187, 191-197, 203 family, 179, 187, 191-197 flounder, 160-163, 187-189 function, 160, 164, 174, 194-197 gene, 162, 164, 165, 167, 168, 173, 174, 180, 189, 191, 193 gene duplication, 167, 168 gene family, 167, 168, 376 GHiPRL family, 160, 164, 167, 168, 173, 174 gilthead sea bream, 163 halibut, 187-189 human, 163 immunoreactivity, 163 in PAS positive cells, 160, 164 in PRL-producing tumor cells, 163 invertebrates, 194 ion-regulation, 164, 167, 174 location, 162, 180 lumpfish, 187-189 maturation in fish, 164 mouse, 163 mRNA, 160-163 K-glycosylation sites, 162 Northern blot analysis, 162 osmoregulation, 180, 194-197 osmoregulation of calcium, 180 Pit-1 binding elements, 165, 168, 173, 174, 191-193 Pit-1 stimulation of gene, 169, 172, 173 precursor, 166, 167, 187 primary structure, 180, 187, 188 promoter, 172, 173 radioimmunoassay, 164 rat, 163 reproduction role, 164 sequence alignment 188, 189 steroidogenesis, 164 TATA box, 165 tetrapods, 163, 174 tilapia, 163 Somatomedin hypothesis growth hormone, 214 insulin-like growth factor I mediation, 214 Somatostatin I, 250

513 11, 250 anglerfish, 250-256 Atlantic hagfish, 257 bowfin, 256 channel catfish, 250,252, 255 coho salmon, 250, 255 daddy sculpin, 250, 256 decrease of glucagon levels, 258 decrease of glucagon-like peptide levels, 258 decrease of plasma insulin, 258 flounder, 250, 256 HPLC analysis, 254 immunohistochemistry analysis, 254, 256 immunoprecipitation, 253 in situ hybridization, 254 induced hyperglycemia, 258 lampreys, 256 lysine hydroxylation, 253 mammals, 255 Northern blot analysis, 252 Pacific ratfish, 256 preprosomatostatin I, 11, 250, 252, 254 prosomatostatin I, 11, 253-255, 258 signal peptides, 251-253 Southern blot analysis, 250 SST-14 I, 11,250-257 SST-22,252, 255 SST-25 11, 255, 256, 258 SST-26,256 SST-28 11,250-258 SST-34,256, 257 SST-37, 256 Torpedo marmorata, 256 Somatotroph growth hormone synthesis, 168, 179 lineage, 312 Pit-1, 170-172, 174 sea bream, 313 stem cell, 312 Southern blot analysis aromatase cytochrome P-450, 415, 416 estrogen receptor gene, rainbow trout,

338 gonadotropin a , p subunits, 140, 141 gonadotropin-releasing hormone, 44,45 3P-hydroxysteroid dehydrogenaseisomerase, 414

514

SUBJECT INDEX

isotocin precursor, 112,113,117, 120 Pit-1, 323 somatostatin, 250 stanniocalcin, 283,284 C R F l cDNA fragment, sucker, 89 vasotocin precursor, 112, 113,117, 120 Spawning, in pink salmon, 105 Species-specific expression, 321 Spemi cells, GHRH immunoreactivity,50 Splice junctions, 16,19,22,25,27,90,

141,319,320 SH, see individual steroids, receptor or Receptor

SST,see Somatostatin Stanniocalcin Australian eel, 276,277,279 bowfin, 282 ectopic production in fish, 283 European eel, 275 glycosylation sequence, 281 homology between salmon and eel,

276,277, 281,282 immunoreactivity in fish brain and pituitary, 283 lowering of gill calcium transport, 274,

283,292 phenylalanine N-terminal, 279,281 prevention of hypercalcemia, 275,290 primary structure, 278-280 rainbow trout, 276 salmon, 276,277,279 STC, see Stanniocalcin Steroid, see individual steroids Cl9 steroids, germinal vesicle breakdown, 401

phophodiesterase inhibitors, 417 puromycin inhibition, 417 Steroidogenesis gonadotropin stimulation, 404,405,

410 in ovaries, 137 in testes, 137 somatolactin stimulation, 164 Steroidogenic activity, of the adrenals, 71,76 cells, 395 Steroidogenic enzymes aromatase cytochrome P-450, 411 cholesterol side-chain cleavage 411 cytochrome P-450, 17a-hydroxylasel17,20-lyase cytochrome P-450, 411 SOP-hydroxysteroid dehydrogenase,

404,411 3P-hydroxysteroid dehydrogenaseisomerase, 395,398,400,411 Stress adrenocortical enlargement, 68 gut ulcer bleeding, 69 induced release of corticotropinreleasing factor, 68 thymolymphatic complex involution,

68,69

Structural model, growth hormone family, 203 Structure, see Primary structure Superfamily nuclear receptors, 332,see also receptors Supraoptic neurons oxytocin mRNA levels, 125 C21 steroids vasopressin mRNA levels, 125,127 cortisol, 401 Surface probability statistic, growth deoxycorticosterone, 401 hormone, 184,185 1701,20p-dihydroxy-4-pregnene-3-one, SVG (CRF-like peptide sauvagine), see 137,138,400,411 Sauvagine 17a-hydroxyprogesterone, 137,400, Syncytiotrophoblasts, placental lactogen 411 synthesis, 180 initiation of germinal vesicle Synonymous nucleotide substitution rate breakdown, 400 in neurophysin, 114 progesterone, 400 17a,20P,21-trihydroxy-4-pregnene-3T one, 400,401 Steroid-induced oocyte maturation CAMP inhibition, 417 aT3-1cell cholera toxin inhibition, 417 human gonadotropin I1 01 subunit, 143 forskolin inhibition, 417 human a-T-antigen oncogene, 143

515

SUBJECT INDEX murine FSH a subunit, 149 T3, see Thyroid hormone T4, see Thyroid hormone Tamoxifen, 347, 348 Tamoxifen aziridine, 344 Target gene, 315, 316 Target-gene specificity, 315, 316 Targeted ablation (knock-out), 314 TATA box corticotropin-releasing factor gene, 89 estrogen receptor gene, 353 GHRH gene, human, 14 GHRH gene, rat, 14 GHRH-like/PACAP gene, salmon, lack of, 14 GTHa subunit gene, salmon, 143 GTHIIP gene, salmon, 145 human PRP/PACAP gene, lack of, 14 insulin gene, 228 luteinizing hormone gene, 145 prolactin gene, 165, 321 somatolactin gene, 165 thyroid-stimulating hormone gene, 147, 148 TATNCAT box, 316 Telencephalon urotensin I immunoreactivity, 81 ventral, 81, 82, 84, 86 Terminal nerve agnatha, absence of GnRH neurons, 33, 38 bony fish, GnRH neurons, 39,40 cartilaginous fish, GnRH neurons, 38 Testosterone aromatase conversion, 410 formation by 17a-hydroxylase/l7,20lyase cytochrome P-450, 412 germinal vesicle breakdown, 401 gonadotropin I1 p gene expression, 151 prolactin receptor regulation, 371 Tetraploidization CRF gene duplication, 72, 92 growth hormone family, 193 in catostomids, 89, 92, 93, 108, 117 in Cyprinidae, 92, 93, 108 of ancestral vertebrates, 92 of salmonid fish, 108, 117 of vasotocin and isotocin precursors, 114 Thecal cell layer

amago salmon, 404, 405 CAMP formation, 407

17a,20P-dihydroxy-4-pregnene-3-one, 404 gonadotropin activity, 407 17a-hydroxylase/ 17,20-1yase cytochrome P-450 activity, 414 1701-hydroxyprogesteronesynthesis, 404 oocyte maturation, 407 receptors, type I, I1 gonadotropin, 406, side-chain cleavage enzyme systems, 411,412 steroid precursor, 405, 406 testosterone production, 410 tilapia, 397, 398 Thecal cells, special amago salmon, 404, 405 endoplasmic reticulum, 395 17a,20P-dihydroxy-4-pregnene-3-one, 395 gonadotropin activity, 407 17a-hydroxyprogesterone synthesis, 406,407 3P-hydroxysteroid dehydrogenase, 395, 398 17P-hydroxysteroid dehydrogenase, 395 steroid precursor, 406 tilapia, 395, 407 type I, I1 gonadotropin receptors, 406 Theophylline, phophodiesterase inhibition, 407 Thin layer chromatography, 401 Thymidine kinase (TK) promoter, 148, 348,349,354,355 Thyroid, 311, 332 Thyroid hormone GH gene regulation, 190, 191 negative regulation, 147, 148 receptor, 320, 322, 338 release of growth hormone in fish, 9 T3, 190, 311 T4, 190,311 transcription regulation 190, 191 Thyroid-stimulating hormone /3 gene, 141 glycoprotein hormone family member, 136 human a subunit gene, 148 negative regulation, 147

516 negative TREs, 148 Pit-1, 170 promoter, 147, 319 rat a subunit gene, 147 synthesis, 170 TATA box, 148 thyrotrophs, 147, 312, 319 TRE hexamer half-site [AGGT(C/A)A], 148 Th yrotrophs Pit-1, 170, 172 thyroid-stimulating hormone expression, 148, 160, 170 tiPRL, see Prolactin, tilapia Tissue-specific expression corticotropin-releasing factor, 89, 90 estrogen receptor, 337, 338 glucagon, GLP-1, GLP-2, 237 gonadotropin p subunit gene, 144, 145 growth hormone gene, 190, 192 insulin-like growth factor I gene, 200-202,220 insulin-like growth factor I1 gene, 200-202,220 Pit-1, 168, 172, 174, 190, 321 prolactin gene, 190 TLC, see Thin layer chromatography TPA response element AP-1, 15 AP-1 consensus sequences, 353 TR, see Thyroid hormone, receptor or Receptor Transactivation AF1, AF,, 335, 347, 348 domains, 173, 317, 344, 347 function, 317, 335, 347 Transcription regulation of gonadotropin IIp gene, 144-147, 150-152 repression, 337 start site, see CAP site Transcription factors antennapedia, 317 AP-1, 15, 143, 153, 353 434 Cro, 318 CTF, 347 cyclic AMP binding protein, 15, 242 engrailed, 317 for PRP/PACAP gene, 15 GHF-1, see Pit-1

SUBJECT INDEX

GSEB, gonadotroph-specific element binding protein, 143, 149 helix loop helix family 229 HNF4, hepatic nuclear factor, 353 Oct-1, 169, 316 Oct-2, 169, 316 Pit-1, see Pit-I POW domain, 169,316-324 POW homeobox, 169 Spl, 347 ultrabithorax (homeodomain) Unc-86, 169, 326 yeast transcription factor Gal 4, 347 Transgene, 312 bacterial chloramphenicol acetyl transferase gene, 145 human follicle-stimulating hormone p gene, 144 human a-T-antigen oncogene, 143 Transgenic mouse chloramphenicol acetyl transferase gene, bacterial, 145 Diphtheria Toxin-A chain expression, 143 follicle-stimulating hormone p gene, human 144 gonadotropin a subunit gene, human, 144 gonadotropin a subunit promoter, bovine, 143 insulin-like growth factor 1, 11, 221 luteinizing hormone p subunit promoter, bovine 145 simian virus 40 T-antigen oncogene, 144 Translational start sites, dual, 319 Transmembrane domain, 370, 371 17cu,20P,21-Trihydroxy-4-pregnene-3-one Atlantic croaker, 403 concentrations, 419 gonadotropin effect on receptor concentrations, 419 maturational competence, 419 oocyte maturation, 400, 401, 403, 419 receptor in oocyte, 419 spotted seatrout, 403,419 TSH, see Thyroid-stimulating hormone Tumors in mammary glands, 45 Tyrosine kinase, 371

517

SUBJECT INDEX

U

UI, see Urotensin I UII, see Urotensin I1 Ultrabithorax, 317 Unc-86, see Transcription factors Upstream regulatory sequence, see Flanking sequences, 5’ U rophysectomy effect on CRF synthesis, 87 effect on urotensin I synthesis, 88 Uroph ysis urotensin I immunoreactivty, 81 Urotensin I ancestral gene, 91 ancient form, 92 binding sites in goldfish, 77 carp, 69, 78, 89, 92, 93 cDNA, 89 effect of urophysectomy on synthesis, 88 endorphin release in Xenopus, 73 evolution, 92-94 fragment U-4-28 activity, 75 gene, 93 homology to CRF, 69, 72, 90, 91, 93 homology to sauvagine, 89 immunoreactivity, 81 in urophysis of carp, 81 in urophysis of sucker, 81 metopyrone effect on synthesis, 82 neuron secretory activity, 82 potency, 72 release of adrenocorticotrophin, 73-75 release of a-melanocyte-stimulating hormone, 73 silent gene, 93, 94 sucker, 69, 81 vasodilatory activity, 75 Urotensin I precursor carp, 78, 79 cryptic peptide, 78, 89, 91 hormone moiety, 7 8 , 8 9 , 9 l sequence identities, 80 signal cleavage sites, 78, 79 signal peptide, 78, 89, 91 Urotensin I1 neuron secretory activity, 82 sucker antiserum, 82

V

Valitocin, 103 Vanadate, protein phosphatase inhibition, 427 Vasoactive intestinal peptide (VIP) evolution, 26-28, 49 gene, human, 16,26-28 gene duplication, 26-28, 49 GHRHNIP receptor, 50 glucagon hormone family, 23, 24, 26-28,49 precursor, 24 Vasopressin basic peptides, 103 co-expression with oxytocin in rats, 86 cortisol secretion in dexamethasoneblocked goldfish, 72, 73 evolutionary pathway, 110, 111, 113, 115, 116 osmotic regulation in mammals, 125 mRNA, 86, 121, 127 stimulation of adrenocorticotrophin release, 72 vasotocin, 102 vasotocin and isotocin homology, 72, 103, 125 Vasotocin cDNA, salmon, 108, 110, 111, 118 cDNA, white sucker, 108 cortisol negative regulation, 82 cortisol secretion in dexamethasoneblocked goldfish, 72 divergence, 111, 114, 115 dogfish, 102 effect of dexamethasone on immunostaining, 85 expression, 86, 107, 117, 119-121, 123, 124 gene group I, 11, 108, 110-113, 116, 118 hypo-osmotic adaptation in euryhaline species, 128 immunoreactivity, 85, 104, 123, 124, 127 in parturition, 105 in preoptic neurons, 85, 107, 123, 125 in sitti hybridization, 104, 118, 126, 127 in spawning salmon, 105 inhibition of adenylate cyclase, 105

518 mRNA, 86,88, 119, 121, 124, 125, 127, 128 plasma levels in euryhaline species, 123, 125, 126 plasma levels in stenohaline species, 123, 125, 126 rainbow trout, 104, 105, 117, 120 receptor, Arg-vasopressin binding site, 104 receptors in gill epithelium, 105 sequence, 103 stimulation of adrenocorticotrophin release, 72, 73, 86 synthesis, 85 Vasotocin precursor chum salmon, 108,109, 117-120 cyclostomes, 108, 109 effect of hyper/hypo-osmotic stimuli, 125 gene duplication, VT-1, VT-l', 93, 115, 120 gene duplication, VT-1, VT-2, 93, 115, 120 m a w salmon, 117, 120 neurophysin, 109, 110 post-translational cleavage of salmon neurophysin, 110 signal peptide, 109, 111 structural organization, 109 sucker, 93, 108, 111 toad, 108, 115, 118 VT-1, 93, 108, 110, 118, 120, 125 VT-l', 93 VT-2, 93, 108, 110, 118, 120, 125 VDR, see Vitamin D, receptor or receptor Vertebrate growth, 309 VIP, see Vasoactive intestinal peptide VIPIPHM, see PHM/VIP Vitamin D, receptor, 332 Vitellogenesis, 331, 333, 394, 410

SUBJECT INDEX

Vitellogenin carp, 137 goldfish, 137 gonadotropin I, 137 gonadotropin 11, 137 induced by E2. 332,333 salmon, 137 Xenopus Al, A2 genes, 150 Viviparous species, 331 VP, see Vasopressin VT, see Vasotocin

w Western blot analysis, 319

X

Xenobiotics (lindane), 347

Y

Yeast a mating factor, sequence similarity with GnRH, 49

Z

Z-box, 320 2-box sequence, 320 Zinc fingers amino acid sequence, 340 D-box, 340-342 P-box, 340-342 Zn13, 320 Zona radiata-oocyte membrane complex, 395,418

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