International Review of Cytology: A Survey of Cell Biology, Volume 118

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME118

SERIES EDITORS GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER

1949-1988 1949- I 984

19671984-

ADVISORY EDITORS H. W. BEAMS HOWARD A. BERN DEAN BOK GARY G. BORISY BHARAT B. CHATTOO STANLEY COHEN RENE COUTEAUX MARIE A. DiBERARDINO DONALD K. DOUGALL BERNDT EHRNGER CHARLES J. FLICKINGER NICHOLAS GILLHAM M. NELLY GOLARZ DE BOURNE MARK HOGARTH KEITH E. MOSTOV AUDREY MUGGLETON-HARRIS

ANDREAS OKSCHE MURIEL J. ORD VLADIMIR R. PANTIC M. V. PARTHASARATHY LIONEL 1. REBHUN JEAN-PAUL REVEL L. EVANS ROTH JOZEF ST. SCHELL HIROH SHIBAOKA JOAN SMITH-SONNEBORN WILFRED STEIN RALPH M. STEINMAN HEWSON SWIFT MASATOSHI TAKElCHI M. TAZAWA ALEXANDER L. YUDIN

INTERNATIONAL

Review of Cytology A SURVEY OF CELLBIOLOGY

Editor-in-Chief

G. H. BOURNE (Deceased)

Editors

K. W. JEON

M. FRIEDLANDER

Depurtment of Zoology University of Tennessee Knoxville, Tennessee

Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME118

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

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COPYRIGHT 0 1989 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.

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Contents CONTRIBUTORS ............................................................................................ GEOFFREY H. BOURNE ..................................................................................

vii ix

Differentiation of the Bacterial Cell Division Site WILLIAMR. COOK,PIETA. J.

DE

BOER. AND LAWRENCE I. ROTHFIELD

I. Introduction ....................................... I I . Structure o f t n Apparatus ..................... 111. Biogenesis and Localization of the Division Site ..... ........................... IV. Formation of the Septum ......... ............................................ V. Regulation of the Division Process ....................... VI. Conclusions ................... ......................................... ........................................ ........ References .....................

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Properties of the Cell Surfaces of Pathogenic Bacteria R. J. DOYLEAND E. M. SONNENFELD 1. Introduction ....................................................................................... ......... 11. Surface Structures of Bacteria ............................................... 111. Biological Reactions to Peptidoglycans ................................................... IV. Bacterial Cell-Surface Amphiphiles ........................................................ V. Surface Adhesins of Bacteria and Pathogenesis ...................................... VI. Turnover of Cell Wall a thogenesis .................................................. VII. Concluding Remarks ... ......................................... References ........................ .........................................

33 34 43 49 58 14 83 84

Cellular Studies on Marine Algae AHARONGIBOR

.......... .............................. ........................ ................................ ......................................... Eoergesenia ........... .............................. .................. Porphyra ...............

1. Introduction

11. Acetabularia

Ill. IV. V. Concluding Remarks References ...................

V

93 94 102

vi

CONTENTS

The Centrifugal Visual System of Vertebrates: A Century-Old Search Reviewed J . REPERANT,D. MICELI,N . P. VESSELKIN.A N D S. MOLOTCHNIKOFF

I. 11. 111. IV. V. VI. Vll. VIII.

Introduction .... ........ ..................................... ...................................... C yclostomes ............... Fish ....... ....... ............ .......... .... ................... . ................................................................. Amphibians ..... ...

................. ................................................................................... ns ....................................................................................... s ...................................................

1 I5 116

i20 127 130 133 151 160 164

Cell Biology and Kinetics of Kupffer Cells in the Liver K. W A K ~K. . D ~ c K ~A. K ,KIKN.D. L. KNOOK,R. S. M C C U S K ~ Y , L. B O U W ~ N SA.N D E. Wissk

I. 11.

Ill. IV. V. VI.

VII. VIII.

1x.

Introduction ... ................................., .................................................. Morphology of Kupffer Cells ........................................................ Population Dynamics of Kupffer Cells s .............. ............ Isolation, Purification, and Culture of Metabolic Responses of Stimulated Rat Kupffer Cells in Vitro Endocytosis ........ ....................................................................... Kupffer Cells and Endotoxin ................................................................. ......................... Kupffer Cells in Infectious Diseases .... Concluding Remarks .......... References .... .....................................................................................

173 176 181 187 191 198

203 210 220 22 1

Cellular and Molecular Biology of Capacitation and Acrosome Reaction in Mammalian Spermatozoa K. S. SIDHIJ A N D S. S. GUKAYA 1.

Introduction .................,

11. Capacitation ..._............., 111. Acrosome Reaction ............ ............ . ............

1V. Conclusion and Prospects References ....................

INDEX.........................................................................................................

281

Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

L. BOUWENS (173), Laboratorium voor Celbiologie en Histologie (VUB), 1090 Brussel-Jette, Belgium

WILLIAM R. COOK (l), Department of Microbiology, University of Connecticut Health Center, Farrnington, Connecticut 06032 PIET A. J. DE BOER ( l ) , Department of Microbiology, University of Connecticut Health Center, Farrnington, Connee ticut 06032

K. DECKER(173), Biochernisches Institut, Albert-LudwigsUniversitat, Federal Republic of Germany R. J. DOYLE (33), Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292 AHARONGIBOR(93), Department of Biological Sciences, University of California, Santa Barbara, California 93106 S. S. GURAYA (23 l), I.C.M.R. Regional Advanced Research Centre in Reproductive Biology, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, India A. KIRN (173). Laboratoire de Virologie, Faculte' de Mede'cine and INSERM U74, 67000 Strasbourg, France

D. L. KNOOK (173), Instituut voor Experimentele Gerontologie TNO, 2280 HV Rijswijk, The Netherlands vii

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CONTRlBUTORS

R. S. MCCUSKEY(173), Department of Anatomy, School of Medicine, University of Arizona, Tucson, Arizona 85724 D.

MKELI ( 1 15), Laboratoire de Neuropsychologie, UniversitP du Que'bec, Trois-Rivieres, Que'bec, Canada

S. MOLOTCHNIKOFF( 1 15), De'partement de Sciences Biologiques, UniversitP de Montre'al, Montre'al, Que'bec, Canada

J. R E P ~ R A N( 1T1 9 , Laboratoire de Neuromorphologie U106, INSERM, H6pital de la Salpetrie're, Paris, France

LAWRENCE I. ROTHFIELD(l), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 K . S. SIDHU(231), I.C.M.R. Regional Advanced Research Centre, in Reproductive Biology, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, India E. M. SONNENFELD (33), Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292 N . P. VESSELKIN ( 1 15), Institut Sechenov, Leningrad, U.S.S.R.

K . WAKE(173), Department of Anatomy, Tokyo Medical and Dental University, Faculty of Medicine, Yushima, Bunkyoku, Tokyo 113, Japan E. WISSE(173), Laboratorium voor Celbiologie en Histologie (VUB), 1090 Brussel-Jette, Belgium

This Page Intentionally Left Blank

GEOFFREY H. BOURNE (November 17, 1909-July 19,1988)

In Memoriam: Geoffrey H. Bourne On July 19, 1988, the International Review of Cytology-Cell Biology lost its Editor-in-Chief and remaining founder, Professor Geoffrey H. Bourne (co-founder, Professor James F. Danielli, passed away on April 22, 1984). Professor Bourne died rather unexpectedly in a New York hospital while visiting the city to attend a graduation ceremony for the 1988 class of St. George’s University School of Medicine, Grenada, West Indies, of which he was Vice Chancellor and Professor of Nutrition. Among his many prominent achievements, Professor Bourne, along with Professor Danielli, founded the International Review of Cytology in 1950, while he was teaching histology at the University of London. The first volume was published in 1952. Bourne intended to keep the scope of the new international series “as wide as possible to deal with all aspects of cell biology including morphological and chemical studies of both cells and tissues.” Papers presenting new theories of general interest were to be welcomed also. The editors initially published only one volume a year, until 1967,when the number was increased to two. Thereafter, the number of volumes published each year gradually increased to the present rate of five to seven volumes per year, with occasional special volumes introduced during the 1970s. Until 1970, the two founding editors and assistant editor, Kwang W. Jeon, who joined them in 1967, searched the literature to select authors and subjects for review. The task of covering the wide range of rapidly advancing cell biology became too onerous for them alone, so in 1970, they established an advisory board of 22 members drawn from eight different countries. Eight of the original board members still serve. Professor Bourne led a very colorful and successful professional life as a teacher, scientist, administrator, author, editor, and even diplomat. He has many monographs, edited series, and more than 500 scientific articles published in medical and scientificjournals to his credit. Born on November 17, 1909, in Perth, Australia, he received his B.Sc., M.Sc., and D.Sc. degrees in biology from the University of Western Australia. He continued his studies at the University of Oxford, where he received his doctorate in histology in 1943. Between 1943 and 1946, he served in the British

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IN MEMORIAM: GEOFFREY H.BOURNE

Armed Forces as an officer in charge of R&D for special forces and as a nutritional advisor in Southeast Asia. In 1957, he moved from the University of London to the Emory University Medical School in Atlanta to take up chairmanship of the Department of Anatomy. In 1962, he became director of the Yerkes Primate Research Center of Emory University while continuing his active research in histochemistry and ultrastructural studies of various tissues and organs. After retiring from the Yerkes Primate Research Center, Professor Bourne joined the newly established St. George’s University School of Medicine in 1978, as a professor of nutrition. Soon thereafter, he assumed the role of Vice Chancellor. The scientific community at large has greatly benefited from Professor Bourne’s creativity. leadership, vision, and wisdom, and owes him a great debt of gratitude. All who knew and worked with Professor Bourne have suffered a great loss. We shall miss him for his keen insight, charm, and sense of humor. However, we are comforted to know that the fruits of his labor, including the International Review of Cytology-Cell Biology, will remain with us and continue to enrich us for many years. KWANCW. JEON MARTINFRIEDLANDER

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 118

Differentiation of the Bacterial Cell Division Site WILLIAM

R.

J. DE BOER, AND LAWRENCE I. ROTHFIELD

COOK, PIET A.

Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032

I. Introduction

A. BACKGROUND In most bacterial species cell division occurs by the ingrowth of a division septum at the midpoint of the cell. This process is under strict topological and temporal control. We will discuss this subject as a problem in subcellular differentiation, in which a complex structure is constructed at a specific location in the cell in a process that must be coordinated with a variety of other cellular events, most notably with chromosome replication and segregation. In recent years a major change in thinking about bacterial division has come from the demonstration that early events in the differentiation process can be detected long before the initiation of septa1 ingrowth. In addition, more recent evidence suggests that the residual division site continues to carry out specific functions even after cell separation is completed. Thus, a major theme of this review will be the idea that the division site itself has a developmental history in which the stages of genesis, maturation, and localization, and ultimate fate have become accessible to study. In this context we will describe evidence obtained from several disciplines: genetics, microscopy, and, to a lesser extent, biochemistry. We will begin by briefly discussing differences between eukaryotic and prokaryotic cell division, and will describe aspects of bacterial cell envelope organization and chromosomal replication that are relevant to the later discussion of the division process itself. The main body of the review will be divided into three parts concerning (1) the formation and localization of the division apparatus, (2) the formation of the division septum, and (3) the coordination of cell division with other cellular events, such as DNA replication and chromosome segregation. The discussion will be largely limited to information derived from studies of gram-negative bac1 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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teria, most especially of Escherichiu coli, because the combined application of genetics with other techniques to study cell division in this organism has been particularly fruitful in recent years. It should be pointed out that a substantial body of important work has also been done in other species. Although division in both bacteria and eukaryotic cells is accomplished by the circumferential invagination of the cytoplasmic membrane and cell envelope, the details differ considerably, reflecting the different organization of surface structures in the two groups. For example, contractile elements have not been described in most bacterial species. Therefore, it is not surprising that the contractile ring that appears t o play an important role in cytokinesis in animal cells (Beams and Kessel, 1976) is absent from dividing bacteria. On the other hand, the bacterial cell envelope contains a highly crosslinked peptidoglycan structure, the murein (described more fully later), that is not found in eukaryotic cells but plays a key role in the bacterial division process. The murein is the only known rigid structure in bacterial cells and, therefore, although it lies outside of the cytoplasmic membrane, may fulfill some of the roles ascribed t o the intracellular cytoskeleton of higher cells. The driving force in formation of the bacterial division septum may come from the circumferential inward growth of the rigid murein layer. In the simplest model, inner membrane is passively pushed ahead of the ingrowing murein while outer membrane is pulled behind, thereby leading to the coordinate inward movement of the three layers of the septum. This simplistic model provides a convenient starting point for thinking about the septation process.

B. ORGANIZATION OF THE BACTERIAL CELLENVELOPE As indicated in Fig. 1 , the cell envelopes of E. coli and other gramnegative bacteria contain three morphological layers: cytoplasmic (inner) membrane, murein, and outer membrane (Inouye, 1979). The compartment between the inner and outer membranes is termed the periplasmic compartment or periplasmic space. Formation of the division septum requires the ingrowth of this complex structure at the proper site within the cell. The cytoplasmic membrane acts as the major osmotic barrier of the cell, and contains a large number of specific transport proteins as well as proteins involved in energy transduction and other cellular processes. In contrast, the outer membrane contains a more limited spectrum of proteins, including several “porins” that permit the passage of various low molecular weight solutes into the periplasm (Nikaido, 1979). The murein layer is composed of an extensively crosslinked peptidogly-

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FIG. 1. Diagram of the Escherichia coli cell envelope. OM, Outer membrane; OmpA, OmpA protein; Mur, murein layer; PPS, periplasmic space; IM, inner membrane; MLP, bound form of murein-lipoprotein.

can that completely surrounds the cell. This bag-shaped molecule determines the shape of the organism and is responsible for the rigidity and the resistance of the cell to mechanical and osmotic stresses. Cell elongation and septal ingrowth both require that new peptidoglycan units be inserted into the continuous murein structure (Mirelman, 1979). This requires the controlled breaking of covalent bonds within the structure to permit the directed insertion of the new material. There is reason to believe that different biochemical mechanisms are used, at least in part, for insertion of septal murein as opposed to “elongation murein” (i.e., the murein along the length of the cell cylinder) (Mirelman, 1979). When examined by conventional electron microscopy, the murein layer of gram-negative bacteria appears as a thin, dense layer of 2-3 nm thickness that is closely apposed to the outer membrane (Fig. 1). Studies with alternative methods of preparation were interpreted as showing a less densely packed peptidoglycan domain between the dense murein layer and the inner membrane (Hobot et a / . , 1984; Leduc et al., 1985). Biochemical evidence shows that a number of intrinsic outer-membrane proteins (e.g., OmpA and murein-lipoprotein) are involved in attaching the outer membrane to the murein layer (Fig. 1). The outer-membrane proteins that mediate this attachment are sufficiently abundant (-lo5 copies per cell) to provide -400,000 contact points between murein and outer membrane (Park, 1987). The periplasmic compartment contains a number of water-soluble proteins. These proteins include degradative enzymes, proteins that inactivate toxic exogenous substances, and proteins that participate in solute transport and in chemotaxis (Brass, 1986). It is likely that other functional periplasmic proteins remain to be identified, and the possibility must be considered that some of these may play a role in the septation process.

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Proteins can diffuse within the periplasmic space, although the diffusion rate is slower than in free aqueous solution (Brass, 1986; Foley rt ul., 1989). The periplasm can be operationally divided into two regions: the inner periplasm that lies between the inner membrane and the dense murein layer, and the outer periplasm located between murein and outer membrane (Fig. I). Free diffusion within the outer periplasm may be limited by the close apposition of murein and outer membrane and by the very high concentration of fixed proteins (most prominently OmpA and murein-lipoprotein) in this region. Significant movement of proteins within the periplasmic space may therefore be restricted to the inner periplasm.

C. DIFFERENCES BETWEEN CHROMOSOME SEGREGATION IN PROKARYOTIC AND EUKARYOTIC CELLS There are several differences in the process of chromosome segregation between bacteria and eukaryotic cells. Most strikingly, microtubules or analogous structures have not been shown to exist in bacteria, suggesting that the link between the chromosome and the cellular site responsible for directing daughter chromosomes to progeny cells in bacteria is likely to be less complex than in eukaryotes. Consistent with this fact, a mitotic apparatus has not yet been described in bacteria. It is widely believed-though without any direct evidence-that chromosome segregation occurs in bacteria by the attachment of daughter chromosomes to cell envelope sites that direct each of the daughter chromosomes to a different progeny cell. In the model originally proposed by Jacob et ul. (19631, the chromosomal site is located at o r near the origin of replication. In this model, still the most widely accepted one, the processes of chromosome replication and chromosome segregation are coupled and the driving force for chromosome separation is provided by insertion of new cell envelope material between the attachment sites. We will discuss in this review relevant aspects of this problem together with the question of how the cell coordinates the placement and synthesis of the division septum with the processes of DNA replication and chromosome segregation.

XI. Structure of the Division Apparatus A. THE PERISEPTAL ANNULARAPPARATUS Until the past few years, the only visible event in the cell division process was the formation of the division septum. However, earlier stages in the differentiation process have now become accessible to study. This

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was made possible by the discovery of a new organelle, the periseptal annular apparatus, which is present at the future division site before the onset of septal ingrowth (Anba et al., 1984; MacAlister et al., 1983). Studies of the structure and biogenesis of the periseptal annuli have provided a large amount of new information dealing with the process of formation and localization of the division site. The periseptal annuli are two concentric rings that surround the cell at the site of cell division (Fig. 2A). Electron microscopy has revealed that each annulus consists of a narrow zone within the cell envelope, in which the inner membrane, murein, and outer membrane lie in close apposition to each other (Fig. 2B). Three-dimensional reconstructions from serialsection electron micrographs showed that the zones of membrane-murein association are continuous structures that run completely around the cell cylinder. As discussed in more detail later, the annuli appear at the future division site long before the onset of septal invagination, thereby defining the future division site (Cook et al., 1986, 1987). The septum is later formed within the cell envelope domain (the periseptal domain) that lies between the two annuli. In bacterial cells the osmotic pressure resulting from the high intracellular concentration of impermeant solutes pushes the cytoplasmic membrane against the murein layer. Therefore, to visualize structures such as the annular attachments, cells are first plasmolyzed by brief exposure to hypertonic solutions of sucrose or other solutes that can enter the periplasm but do not readily cross the cytoplasmic membrane barrier. This results in a loss of cytoplasmic volume, causing the inner membrane to retract from the rigid murein-outer membrane layer. As shown by Bayer

FIG.2. Diagrammatic representation of periseptal annuli in surface view (A), and cross section (B) at several stages of progression through the cell cycle. PSA, Periseptal annuli; PA, polar annuli: OM, outer membrane; IM, inner membrane; M, murein. From Rothfield er al. (1986).

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et al. in an important series of papers (Bayer, 1979), random electron micrographs of plasmolyzed cells reveal numerous sites where the inner membrane fails to retract from the outer layers of the cell envelope. These attachments were named zones of adhesion by Bayer, and will be referred to as “Bayer bridges” in this review to avoid confusion with the adhesion zones that comprise the periseptal annuli. It is interesting to note that zones of adhesion between inner and outer membranes of isolated chloroplasts and mitochondria can also be visualized following exposure to hypertonic solutions (Cremers et al., 1988; Hackenbrock, 1968). The circumferential membrane-murein attachments that form the periseptal annuli and their biogenetic progenitors resemble the Bayer bridges when viewed in individual thin sections (MacAlister et al., 1983). It is not known whether all of the Bayer bridges that are visible in electron micrographs of random sections are in fact components of continuous structures such as the periseptal annuli, o r whether there are several types of adhesion zones that are structurally and functionally distinct.

B. ROLE OF T H E PERISEFTAL ANNULARAPPARATUS Although there is good evidence that the periseptal annuli are associated with the division process, the physiological role of the annular apparatus has not been established. Two possible roles, which are not mutually exclusive, have been suggested: ( I ) the annuli act as gaskets to segregate the division site from the remainder of the cell envelope, and (2) essential elements of the machinery are components of the annuli themselves. The gasket hypothesis was based on the morphological appearance of the annular attachments. The circumferential membrane-murein adhesion zones that comprise the annuli appear in electron micrographs to separate the periplasmic space into separate compartments, one of which defines the periseptal domain that lies between the paired annuli at midcell. It therefore was hypothesized that the annuli might act as physical barriers to prevent the unrestricted movement of molecules into and out of the periseptal domain. This would permit the cell to accumulate at this site proteins o r other components required for septum formation, ensuring that septation was restricted to the proper location. Studies by Foley et al. (1989) have provided experimental support for the gasket hypothesis. In these studies proteins labeled with fluorescent groups were introduced into the periplasm. The ability of the labeled proteins t o diffuse into different regions of the periplasmic space was followed by measuring the recovery of local fluorescence after the irreversible photobleaching of probe molecules within localized regions. The

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results indicated that certain regions of the periplasm were not in free communication with the remainder of the periplasmic space. These sequestered regions were preferentially located at potential division sites and at cell poles. This supports the view that the periseptal and polar annuli act as functional barriers to the passage of molecules into and out of the periseptal and polar domains that previously had been identified morphologically. It therefore is reasonable to suggest that one role of the annuli is to segregate the periseptal domain from the remainder of the cell envelope. It will be of interest to extend these studies to the inner and outer membranes to determine whether the barrier function is limited to the periplasm or whether membrane components are also sequestered at division sites by the annular gaskets. It is also possible that essential division components may be part of the annular attachments themselves. This question has not been accessible to experimental study because the annuli have not yet been isolated.

c. MOLECULARORGANIZATION OF PERISEPTAL ADHESIONZONES The molecular organization of Bayer bridges and of the periseptal adhesion zones is unknown. It appears likely t o us that the adhesion zones represent sites where inner membrane is attached to murein. This is based on the observation that the inner membrane at these locations resists the strong inward pull resulting from the plasmolysis procedure. This requires that inner membrane be attached to a rigid structure that can itself resist the inward pull. Since the murein is the only rigid structure that could provide an anchor for the inner membrane, we think it likely that protein(s) at these sites interact simultaneously with inner membrane and with the peptidoglycan that comprises the murein sacculus. The following general models are compatible with this formulation (Fig. 3): 1 . The adhesion zones are sites at which proteins directly attach inner membrane to murein without any involvement of outer membrane (Fig. 3A). This would explain the fact that inner membrane remains associated with the murein-outer membrane layer at these sites despite the inward pull of the plasmolysis procedure. 2. The adhesion zones contain protein that attach both the inner and outer membranes to the murein layer (Fig. 3B). This would explain the fact that inner membrane remains associated with the murein-outer membrane layer at these sites despite the inward pull of the plasmolysis procedure and would also be compatible with the evidence that the annular adhesion zones act as diffusional barriers within the periplasm. It should be noted that an outer membrane-murein attachment

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WILLIAM R. COOK

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FIG.3. Hypothetical models of zones of adhesion. For details see text and Fig. 1.

would not be needed to explain the barrier function if lateral diffusion of proteins did not occur at a significant rate in the outer periplasm (i.e., the region between murein and outer membrane), as discussed in Section I,B. Though less likely, in our opinion, than the models just described, it is also possible that the adhesion sites represent regions of fusion between the lipid matrices of inner and outer membranes. Two such models are illustrated in Fig. 3C and D. In Fig. 3C the adhesion zones represent sites where the fusion of inner- and outer-membrane bilayers results in a continuous bilayer that connects the two membranes. This model is untenable in its simplest form, since an aqueous pore would be formed between cytoplasm and the external medium. This problem could be avoided if the sites contained protein(s) that plugged the potential pore. In Fig. 3D the adhesion zones represent sites where the inner- and outer-membrane bilayers have fused to form a new mixed bilayer. This structure would pre-

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

9

C

D FIG.3. (con?.)

sumably be relatively unstable because of the suboptimal packing of phospholipid molecules at the junctions. This problem might be avoided if the sites contained proteins that served both to stabilize the common bilayer phase and to anchor the membrane to murein at these sites.

D. MEMBRANE-PEPTIDOGLYCAN ATTACHMENT AT THE LEADING EDGE OF THE SEPTUM A second differentiated structure based on the attachment of murein to inner and outer membranes has also been identified in high-resolution electron micrographs of the nascent septum (Fig. 4; MacAlister et al., 1987). The structure, called the septal attachment site (SAS) or membrane attachment at the leading edge (MALE), is located at the leading edge of the ingrowing septum throughout the septation process, forming a pursestringlike zone at the leading edge of the ingrowing septum. It consists of a sharply localized zone in which inner membrane, murein, and outer membrane are tightly apposed. The attachment zone at the septal edge

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WILLIAM R. COOK

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FIG 4. Membrane attachment at the leading edge (MALE) of the nascent septum. ( A ) Electron micrograph of septa1 region of a dividing cell from a culture of Esclii~richirrc d i . The cells were plasmolyzed in 20% sucrose prior to fixation. X 88,320. (B) Enlargement of (A) showing the characteristic bulbous enlargement of the murein (M)-outer membrane (OM) layers that lie in close apposition to the inner membrane (IM) at the leading edge. x 320,000. (C) Schematic representation of (B) indicating the relationships of the various cell envelope layers. PPS, Periplasmic space. From MacAlister et al. (1987).

has a characteristic ultrastructural appearance that distinguishes it from the adhesion zones that form the periseptal annuli. After septal closure and cell separation, the attachment site appears to remain at the pole of the newborn cell, where it can be detected as a bacterial birth scar. It has been suggested that MALE may represent the site at which new murein units are inserted into the murein layer of the nascent septum (MacAlister et ul., 1987). If correct, this would explain the vectorial movement of the septum toward the interior of the cell, with the inner membrane being pushed inward by the ingrowing murein. This idea has gained support from the demonstration that incorporation of the murein precursor [3H]diaminopimelic acid ([3H] DAP) occurs predominantly at the leading septal edge, as determined by autoradiography of septating cells that were pulse-labeled with [3H]DAP (Wientjes and Nanninga, 1989). 111. Biogenesis and Localization of the Division Site

A. DIVISIONSITESARELOCALIZED LONGBEFORE THE ONSETOF SEFTATION The association of periseptal annuli with the division septum was originally established by serial-section electron microscopy (MacAlister et uf., 1983). The same experiments showed that structures resembling perisep-

DlFFERENTIATION OF BACTERIAL CELL DIVISION SITES

11

tal annuli were also located at other sites along the length of the cell, where septation was not occurring. Because these structures did not extend completely around the cell cylinder, it was suggested that they might be precursors of the circumferential periseptal annuli that flanked the division septum (Cook et al., 1987; MacAlister et al., 1983). As noted before, the annular attachments form the limiting borders of localized regions of plasmolysis (plasmolysis bays). Thus, although visualization of the attachments themselves requires electron microscopy, the plasmolysis bays that mark their locations can easily be visualized by light microscopy (Cook et al., 1986). The positions of the plasmolysis bays thereby have been used to identify the locations of the annular structures along the length of the cell. This made it possible to study large numbers of cells to establish the pattern of genesis and localization of the annular apparatus during the course of the division cycle. These studies led to the unexpected observation that the future division site was placed at its proper position along the length of the cell during the cell cycle that precedes the cycle in which it participates in septum formation (Cook et al., 1987). In these studies the fact that plasmolysis bays were already localized at midcell in newborn cells originally suggested that new annuli were formed very early in the life of the cell. Evidence that formation and localization of the periseptal annuli in fact occurred during the preceding cell cycle came from the demonstration that predivision cells contained annular structures at one-quarter and three-quarters cell lengths, in addition to the periseptal annuli at midcell. The annuli at the cell quarters were retained during division to become the midcell annuli of the newborn cells. Since the annuli mark the site at which the division septum is formed, the question of how the cell determines the correct location of the division site therefore becomes a question of how the cell localizes the annular apparatus at one-quarter and three-quarters cell lengths.

B. AREDIVISIONSITES GENERATED FROM PREEXISTING SITES BY A REPLICATION-DISPLACEMENT MECHANISM? Studies of annulus distribution at intermediate stages of the division cycle showed that new pairs of annuli were first detected immediately adjacent to the midcell annuli that are present in the youngest cells in the population (Cook et al., 1987). As the cells elongated, the paracentral annuli that were located on both sides of the periseptal annuli appeared to move progressively away from midcell toward the two poles until, in the longest cells, the structures were tightly clustered at one-quarter and three-quarters cell lengths (Fig. 5). These results led to the proposal that new division sites are generated

WILLIAM R. COOK

12

ET A L

I

I

. .

*. 1

.....

i.:. . ,

.

D

FIG 5. Formation and displacement of nascent periseptal annuli during the division cycle. From Cook el ul. (1987).

and localized in a three-step process by a repetitive cycle of (1) replication of the preexisting sites at midcell, generating nascent division sites on each side of the central annuli; (2) displacement of the nascent annuli toward the two poles; and (3) arrest of the lateral displacement when the annuli have arrived at their final positions at one-quarter and three-quarters cell lengths. It is likely that the nascent annuli continue to mature during the displacement stage, since the annuli at intermediate positions appear in electron micrographs not to extend completely around the cell. Although this is difficult to prove, the micrographs suggest that the annuli continue to grow circumferentially around the cylinder during the displacement period with final closure of the ring occurring sometime after their final arrival at one-quarter and three-quarters cell lengths. If, as seems likely, septum formation cannot begin until the periseptal annuli extend completely around the cylinder to seal off the future division site, the coupling of the localization process with the process of annulus maturation would help to ensure that septation is not initiated at ectopic sites. Because the two new sets of annuli are generated on opposite sides of the preexisting central annuli, each of the nascent annular pairs is com-

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

13

mitted to one of the two daughter cells from the moment of its generation at midcell. It has been pointed out that the nascent annuli are attractive candidates to serve as the structures that anchor the daughter chromosomes to the cell envelope during the process of DNA replication and chromosomal segregation (Cook et al., 1987). This would ensure that each daughter cell received one copy of the genome. Although we find this idea attractive, it is at present a hypothesis without supporting evidence. The recent demonstration that the E. coli origin of DNA replication binds specifically to a cell envelope fraction in vitro may facilitate identification of the cell envelope proteins responsible for chromosome binding, and this in turn may make it possible to test the hypothesis that the DNA-binding sites are located within the annuli (Ogden et al., 1988). c . RESIDUAL DIVISIONSITES REMAINAT THE CELL POLES AFTER CELL DIVISIONIs COMPLETED When cells divide, each daughter cell inherits one of the two periseptal annuli that had flanked the septum prior to cell separation. The residual annulus remains at the cell pole, defining a polar domain that lies between the polar annulus and the end of the cell. Because the polar domain is derived from the periseptal domain that delimited the septation site during the preceding division cycle, it is not unreasonable to expect that elements of the division machinery might be retained in this domain after cell separation is completed. Evidence that the residual division site at the new pole has the potential to support another cycle of septum formation has come from studies of minicell mutants of E. coli. These mutants are characterized by the frequent aberrant placement of the division site at the pole of the cell, leading to formation of large numbers of very small spherical cells (“minicells”) that lack chromosomal DNA (Frazer and Curtiss, 1975). It was originally suggested by Teather et al. (1974) that the minicell mutation results in loss of an activity that is needed to inactivate residual polar division sites. Later studies showed that deletion of the minicell genetic locus (minB) results in appearance of the minicell phenotype (de Boer et al. 1989). These studies have identified two gene products of the minB locus, the products of the minC and minD genes, that act together to form an inhibitor of septation that is required to prevent minicell formation. A third gene product, the minE protein, appears to give the minCD division inhibitor specificity for polar sites as opposed to normal sites at midcell. The demonstration that a septation inhibitor is required to prevent formation of polar septa is consistent with the view that the residual sites at

the cell poles retain the capacity to undergo additional cycles of septum formation. The phenotype of minicell-forming strains also includes cells that span a broad range of cell lengths, ranging from normal-sized cells to mediumlength filaments. Analysis of the length distribution of minB cells led Teather et af. ( 1974) t o propose that in minicell-forming strains the number of septation events per unit increase in cell mass remains normal. Thus, only one septum would be formed with each cell doubling, with an equal probability that septation will occur at the “normal” site at midcell or at one of the two residual division sites at the cell poles. If correct, this implies that at least one essential division component is present in limiting amounts, reaching a sufficient level at some point to support one septation event per division cycle. The hypothetical “division potential” would be used up after triggering septation, thereby preventing extra septation events. Alternatively, one could imagine that the initiation of one septation event could provoke a cellular change that excluded subsequent events until cell growth diluted the hypothetical inhibitor. A possible candidate for the hypothetical “division potential” molecule might be the products of the ftsZ gene, since overexpression of ftsZ appears to increase the number of septation events, resulting in formation of minicells and of chromosome-containing cells that are shorter than normal (Lutkenhaus, 1988; Ward and Lutkenhaus, 1985). It has been shown that some septation events in minicell strains result in the formation of rod-shaped cells lacking chromosomes (Jaffe et al., 19881, and that nucleoid distribution patterns are disturbed in the minB filaments (E. Mulder and C. L. Woldringh, personal communication). This suggests that the products of the minB gene may also be involved in maintaining the normal DNA segregation pattern.

IV. Formation of the Septum A. COORDINATION OF INGROWTH OF OUTER MEMBRANE-MUREIN-INNER MEMBRANE Septum formation requires the invagination of the inner-membrane, murein, and outer-membrane layers of the cell envelope. In E. coli the three layers appear to invaginate coordinately, based on thin-section and freeze-etch electron micrographs of septating cells. In a contrary view, Murray and collaborators have suggested that the invagination of inner membrane and murein precedes the invagination of outer membrane during normal septation in E. cofi, based on the use of alternative fixation

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

15

procedures to the usual glutaraldehyde fixation protocol (Burdette and Murray, 1974a,b; Gilleland and Murray, 1975). For a more complete discussion of this question the reader is referred to Rothfield et al. (1986). However, it is clear that the ingrowth of the three layers is not obligatorily coupled, since mutants strains of E. coli and Salmonella typhimurium have been identified in which the ingrowth of the inner membrane and murein is uncoupled from the ingrowth of the outer membrane (Donachie et al., 1984; Donachie and Robinson, 1987; Fung et al., 1978; Weigand et al., 1976). The affected genes are cha and IkyD, respectively. In the uncoupled mutants the inner membrane and murein appear to invaginate normally, resulting in formation of filaments that contain multiple inner membrane-murein crosswalls that divide the cytoplasm into cell-length units. The units are held together by bridges of outer membrane that failed to participate in septal ingrowth. Thus invagination of outer membrane requires one or more proteins that are not required for invagination of inner membrane and murein. These proteins could, for example be involved in attaching outer membrane to the leading edge of the invaginating septum.

B. BIOCHEMISTRY OF SEPTUMFORMATION 1. Role of the ftsl Protein (PBP3) The only division-related protein for which a biochemical function has been identified is the product of theftsl gene (Spratt, 1983). Theftsl gene encodes a 60-kDa inner-membrane protein that has been identified as penicillin-binding protein 3 (PBP3). The specific interaction of the f ts l gene product with p-lactams implies that the protein plays a role in murein biosynthesis or metabolism. It has also been reported that purified preparations of PBP3 show two enzymatic activities presumably involved in murein biosynthesis, a transglycosylase and a transpeptidase, the transpeptidase being the most sensitive to inhibition by p-lactam antibiotics (Ishino and Matsuhashi, 1981). Evidence that PBP3 is required for septum formation has come from studies of thermosensitiveftsl mutants, in which growth at elevated temperature results in formation of nonseptate filaments (Nishimura et al., 1977; Spratt, 1977; Suzuki et al., 1978). Additional support for this view comes from the observation that filament formation also is induced by treatment of normal bacteria with antibiotics, such as cephalexin, that have a high affinity for PBP3 (Botta and Park, 1981; Spratt, 1975). Taken together, the aforementioned evidence strongly suggests that PBP3 plays a direct role in synthesis of septal murein. This implies that PBP3 either

I6

WILLIAM R. COOK

ET Al.

is preferentially located at division sites or is more randomly distributed and can be functionally activated at division sites at the appropriate time in the cell cycle. These possibilities should be accessible to study by immunoelectron microscopy using antibody directed against PBP3 or by autoradiography of cells labeled with isotopically labeled PBP3-specific compounds. 2 . Other Proteins Implicated in Cell Division A number of other gene products have been implicated in the division process based on the isolation of conditional mutants that form nonseptate filaments when grown under nonpermissive conditions. It is not known whether any of the gene products participate directly in the differentiation process. They have been reviewed elsewhere (Donachie and Robinson, 1987; Lutkenhaus. 1988). V. Regulation of the Division Process A. THEE. coli CELLCYCLE 1. Description of the Division Cycle

In E. coli the cell division cycle is a repeating series of events, punctuated by chromosome replication and septum formation (Helmstetter, 1987). For E. coli B/rA grown at a generation time of 60 minutes, chromosome replication (defined as the C phase) requires -40 minutes. Septa1 ingrowth begins 8-10 minutes after completion of replication. Approximately 10-12 minutes are then required for completion of septation and physical separation of daughter cells, although the time required for septum ingrowth increases when doubling times are >60-70 minutes. An additional interdivision period is present before the initiation of the next round of chromosome replication (Helmstetter and Pierucci, 1976; Kubitschek et al., 1967; Skarstad et a / . , 1983, 1985). This may be equivalent to the G , phase of the eukaryotic cell cycle (Cooper, 1984). Similar values have been obtained in both B/r and K-12 strains (Helmstetter, 1987). The components required for formation of the division septum are generated prior to the termination of chromosome replication. This is shown by the observation that in cells that have just completed chromosome replication, septation is not prevented by inhibitors of DNA, RNA, and protein synthesis(BrehmerandChuang, 198lb;Clark, 1968; Dix and Helmstetter, 1973; Helmstetter and Pierucci, 1968; Jones and Donachie, 1973; Kubitshek, 1974; Pierucci and Helmstetter, 1969; Woldringh et al., 1977). The durations of the C phase and septation periods are generally invariant

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

17

when generation times fall between 20 and 60 minutes, but they gradually slow down when generation times are prolonged beyond 60 minutes.

2. Initiation of Chromosomal Replication Slowly growing cells (generation time >50-60 minutes) contain approximately two completely replicated chromosomes per cell at the time of septum formation. On the other hand, the number of partially or completely replicated chromosomes per cell increases by a factor of 2-4 in rapidly growing cells (Helmstetter, 1987). This reflects the fact that initiation of new replication forks at the chromosomal start site (oriC in E. cofi K-12) can begin prior to completion of ongoing rounds of replication. As a consequence, rapidly growing cells contain multiple replication forks at the time of septation. Therefore, replication events can be initiated in one division cycle and completed in the next. This demonstrates that division cycle events can be overlapped. The number of replication forks (and therefore initiation events) per cell (Cooper and Helmstetter, 1968; Helmstetter and Cooper, 1968) and the average mass per cell are both dependent on the growth rate. Donachie (1968) deduced that the ratio of initiation events to cell mass was invariant over a broad range of growth rates, and concluded that initiation began when the ratio of chromosomal origins to cell mass fell below a critical value. This led to suggestions that a positive-acting substance required for initiation of chromosome replication is synthesized at a rate proportional to the overall increase in cell mass (Donachie, 1968; Jacob et al., 1963; Lark, 1979; Margalit et al., 1984; Sompayrac and Maaloe, 1973). Following the initiation event, levels of the initiator would be depleted, thereby preventing further initiations until the critical concentration was reestablished. In the alternative model, initiation of replication would be prevented by an inhibitor molecule that is synthesized once during each replication cycle, after replication of the appropriate gene locus (Pritchard et al., 1969; Pritchard, 1984). The intracellular concentration of the inhibitor would be diluted with increasing cell mass, until the level was too low to prevent initiation. At this point a new round of initiation would take place, followed by a new burst of inhibitor synthesis. 3. Termination of Chromosome Replication It has been suggested that formation of the division septum may be coupled to the termination of chromosome replication. This suggestion was originally based on studies of cells in which DNA replication was permitted to resume after 2 hours of thymine starvation. Both RNA and

18

WILLIAM R. COOK ET

AL

protein synthesis were required to restore septum-forming ability to these cells. By adding rifampicin o r chloramphenicol at various times after thymine supplementation, Jones and Donachie (1973) showed that septation was dependent on a 5- to I0-minute period of RNA and protein synthesis occurring 35-40 minutes after resumption of DNA synthesis. Septation occurred shortly thereafter. The initial round of chromosome replication also terminated at this time, leading to the proposal that “termination protein(s1” formed shortly after termination of chromosome replication are required for the initiation of septation (Jones and Donachie, 1973). The possibility also exists that the period of required protein synthesis reflected the time required for recovery from the SOS response (see later) and that the apparent relationship to termination was coincidental. A correlation between termination of chromosome replication and septum formation was also observed by Grossman et al. (1989). In these experiments, the presence of methionine during amino acid starvation was used to slow chromosome replication, resulting in a delay in the time of termination. There was a corresponding delay in the first burst of division that was seen when protein synthesis was allowed to resume, consistent with the hypothesis that termination is required for septum formation. It is also possible that the delay in division was not causally related to the delay in termination but rather reflected other secondary effects of the rnethionine treatment. It should be noted that septation can occur in cells blocked in DNA elongation and termination (see later), demonstrating that termination of chromosome replication is not absolutely required for septum formation. B.

COUPLING OF SEPTATION TO CHROMOSOME

REPLICATION AND

SEGREGATION Under normal conditions the formation of anucleate cells is extremely rare. This implies that septation is both temporally and topologically coupled to the processes of DNA replication and segregation. Temporal coupling requires that chromosome replication and the decatenation and separation of the daughter chromosomes must be completed prior to completion of the septal crosswall. Topological coupling requires that the new septum must be located between the daughter chromosomes. If temporal and topological coupling did not occur, one can imagine two possible results: ( 1) a mixture of anucleate cells and cells containing more than one chromosome would be produced, and/or (2) septal closure would have a guillotine effect on the unreplicated o r unseparated DNA. Under normal circumstances neither of these effects occurs with significant fre-

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

19

quency, implying that an effective cellular mechanism exists to coordinate septation with DNA replication and/or segregation. Septa1 ingrowth can begin prior to the completion of DNA replication and segregation (Fig. 6; Woldringh et al., 1977). Thus, if direct coupling does exist between DNA segregation and the timing of septum formation it appears unlikely to involve a control on the initiation of septal invagination. On the other hand, a mechanism to prevent closure of the crosswall until DNA replication and separation are completed remains possible. 1. Prevention of Formation of Cells Lacking Chromosomes When DNA

Synthesis Is Blocked

a. Negative Regulation of Septation by the SOS System. The best understood of the pathways that prevent formation of anucleate cells is a part of the SOS response, a complex cascade of reactions in which the presence of damaged DNA or the inhibition of DNA replication results in the induction of a number of genes required for DNA repair and other processes (D’Ari, 1985; Holland, 1987; Little and Mount, 1982; Walker, 1984; Witkin, 1976). One of the consequences of SOS induction is that septation is reversibly inhibited, preventing formation of daughter cells until the defective DNA has been repaired. The mechanism by which septation is halted after induction of the SOS response begins with the activation of the product of the recA gene to an active protease in response to damaged DNA (Little and Mount, 1982;

FIG.6. Thin-section electron micrograph of Escherichiu coli B/rK showing initiation of septal constriction prior to complete separation of nucleoids. X 66,000. From Woldringh et al. (1977).

20

WILLIAM R. COOK Er A L

Walker, 1984). The RecA protease cleaves LexA, a repressor of the SOS regulon (Little and Mount, 1982; Walker, 1984). One of the derepressed genes, the sfiA gene (also known as sufA),codes for an inhibitor of septation (Huisman and D’Ari, 1981; Huisman et a f . , 1984; Mizusawa et a f . , 1983). After removal of the inducing stimulus, LexA is no longer cleaved by RecA protease, and normal division is restored (Maguin et a f . , 1986a; Mizusawa et al., 1983; Mizusawa and Gottesman, 1983; Shoemaker et al., 1984). For further information on the SOS response the reader is referred to Walker (1987). Genetic evidence indicates that the target of SfiA action is the essential cell division gene, f t s Z , or its gene product (Gayda et a f . , 1976; George et a f . , 1975; Huisman et a f . , 1984; Jones and Holland, 1984, 1985; Lutkenhaus, 1983). It has been suggested that inhibition of septation involves a direct interaction between the SfiA and FtsZ proteins. This is based on the observation that the rate of degradation of the SfiA protein by Lon protease is much slower in cells containing wild-type FtsZ protein than in certainftsZ mutants [i.e., sfiB (also known as sufB)mutants] in which the SfiA-mediated division inhibition is lost (Jones and Holland, 1984; Mizusawa and Gottesman, 1983; Mizusawa et al., 1983; Shoemaker et ul., 1984). Consistent with this interpretation is the observation that overproduction of FtsZ can suppress the division-inhibitory effect of SfiA (Lutkenhaus et a f . , 1986; Ward and Lutkenhaus, 1985). Alternative explanations are possible, and a final judgment on the idea that SfiA acts directly on the FtsZ protein must await more direct biochemical experiments. The SOS response appears to be a damage control system rather than a mechanism responsible for coupling DNA replication and septation under normal conditions, since sfiA(sufA)and sfiB(sufB)mutations that inactivate the SOS division inhibition response have no apparent effect on the normal division pattern. A second RecA-dependent coupling between chromosome replication and septation is mediated by the sfiC gene, which is present in some but not all strains (D’Ari and Huisman, 1983). As with SfiA, SfiC is derepressed after RecA activation. However, sfiC derepression is not dependent on LexA cleavage, indicating that an alternative repressor, also subject to RecA cleavage must exist. sfiC has been identified as a component of excisible element €14 (Maguin et a f . , 1986b). Genetic evidence indicates that the target of SfiC action, like SfiA, isftsZ (D’Ari and Huisman, 1983). The action of SfiC is irreversible, and cells do not recover after removing the original inducer (Maguin et a f . , 1986a), making the physiological significance of SfiC action questionable.

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

21

b. Regulation of Septation by SOS-Zndependent Mechanisms. Filamentation also occurs when DNA replication is blocked in strains containing mutations that result in an inoperative SOS pathway. This indicates the presence of a non-SOS mechanism that couples chromosome replication and septum formation (Burton and Holland, 1983, Huisman et al., 1980, 1983; Jaffe et al., 1986; Jaffe and D’Ari, 1985). Such a mechanism could play a role in coordinating chromosome replication and septation in unperturbed cultures (Huisman et al., 1983; Jaffe et al., 1986; Jones and Donachie, 1973), but this will remain conjectural until the responsible gene or genes have been identified and it has been shown that inactivation of these genes perturbs the normal division pattern. Although nonseptate filaments are formed when DNA synthesis is blocked in strains that lack the SOS system, a number of chromosomefree cells are also produced. This indicates that septation inhibition in the non-SOS system is not as effective as that provided by the action of sfiA in the SOS system (Jaffe et al., 1986; Jaffe and D’Ari, 1985). It is possible to envision two types of mechanism by which the SOSindependent coupling of cell division and DNA replication might be mediated. In the first mechanism, coupling could be mediated by a positive control element similar to the “termination protein” proposed by Jones and Donachie (1973), as discussed earlier. In this view the effector would be required for initiation of septation during each division cycle. It has been suggested that the product of thefrsA gene may be the coupling effector (Tormo et al., 1980, 1985a,b, 1986; Tormo and Vicente, 1984). Conversely, the non-SOS coupling could be mediated by a negativecontrol element. In this case the partial inhibition of septum formation that occurs when DNA synthesis is inhibited in SOS-defective cells could reflect the induction of a division inhibitor analogous to the SfiA septation inhibitor of the SOS system.

2. Coordination of Chromosome Replication and Cell Division As noted before, the evidence for direct coupling between chromosome replication and cell division-in the sense that a signal is passed from the replicating or postreplication chromosome to the septation machinery to trigger septation-is not compelling. In an alternative model, DNA replication and septum formation are not directly coupled but both processes are independently triggered by factors related to progression through the cell cycle. This would result in the temporal coupling of the two processes without requiring direct signaling between the chromosome replication process and the septation machinery. This is consistent with the observation that septation occurs in the absence of chromosome replication pro-

22

WILLIAM R. COOK

ET A1

vided that the SOS-induced division inhibition mechanism is not present (see later). The idea that chromosome replication and septation are separate, parallel pathways is also consistent with the fact that the timing of initiation of chromosome replication is much more precisely regulated than the initiation of septation (Brehmer and Chuang, 1981a; Koppes et al., 1978; Kubitschek, 1%2; Newman and Kubitschek, 1978; Schaechter et al., 1962; Skarstad et al., 1986).

c. RELATIONOF CHROMOSOME SEGREGATION TO LOCATIONOF THE DIVISIONSITE 1. Mutations that Affect Nucleoid Segregation

A number of genes have been identified that affect the fidelity of nucleoid segregation into daughter cells. These fall into three general groups: genes coding for products that are known to affect DNA synthesis, genes that affect DNA supercoiling (DNA gyrase mutants), and genes the biochemical function of which is still unknown (par mutants). a. DNA Synthesis Mutants. Inhibition of DNA synthesis induced by temperature shift in thermosensitive mutants blocked in the initiation or elongation steps of DNA replication, or by thymine starvation, results in formation of filaments. Nucleoids are restricted primarily to the center of the filament, presumably representing the original unreplicated chromosome. This was shown most clearly by the studies of Jaffe and collaborators, who examined the effects on the division pattern of blocking DNA synthesis in a number of different ways in strains that d o not express the SOS response (Jaffe et al., 1986; Jaffe and D’Ari, 1985). A two-phase response was observed. During the first few hours, filaments accumulated in the culture, ascribed to the non-SOS-mediated coupling of DNA replication and septum formation. Later, however, substantial numbers of anucleate cells appeared (620% after 4 hours), reflecting the resumption of septation after the initial period of blocked division. Thus the septation block that is mediated by the non-SOS-mediated DNA-division coupling system was transient (Jaffe et al., 1986). The formation of anucleate cells was abolished by cya or crp mutations and therefore is dependent on the presence of of cAMP and the CAMP-binding protein. The mechanism that underlines the cAMP dependence of this septation activity has not yet been defined (D’Ari et ul., 1988; Jaffe et ul., 1986).

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

23

b. DNA Gyrase Mutants. i. gyrA(parD). Hussain et al. (1987a,b) have described a strain containing a mutation (parD) at 88.4 units on the E.coli genetic map, which resulted in the apparent failure to segregate nucleoids, leading to formation of filaments and to the production of anucleate cells. Subsequent experiments showed that the abnormal phenotype was due to an amber mutation in gyrA, coding for the a subunit of DNA gyrase (Hussain et al., 1987b); a second uncharacterized mutation in the same strain also may play a role in the abnormal phenotype. No effects on chromosome replication were noted. The aberrant pattern of septation in this strain has been described in detail, and the results show the presence of chromosome-free cells that span a broad range of cell sizes. The mechanism(s) that couple initiation of septation to cell mass appeared not to be altered, as shown by the fact that the total number of septa formed per mass doubling were similar to the numbers of septa formed in a wild-type strain. ii. gyrB. Mutations in the gene coding for the p subunit of DNA gyrase (gyrB) (Fairweather et al., 1980) fail to decatenate newly replicated chromosomes (Steck and Drlica, 1984). Presumably as a result of this, the cultures contain filaments with centrally localized nucleoids. The cultures also contain small anucleate cells (Orr et a / . , 1979), reflecting the uncoupling of septum formation from chromosome segregation. c. par Mutants. Two other mutations [parA, at 95 units (Hirota et al., 1968a; Norris et al., 1986) and pa&, at 65 units (Kato et a / . , 1988)]affect chromosome partition without any apparent effect on DNA synthesis. In both cases filaments are formed that contain a large centrally located nucleoid. DNA-less cells are also present in the cultures. The fact that chromosome segregation was affected in these mutants whereas DNA synthesis appeared to be unperturbed suggests that the primary defect may be in the process of chromosome segregation. It should be noted that a third par mutant (parB) was originally thought also to fill these criteria (aberrant chromosome segregation in the presence of normal DNA synthesis). The parB mutation was later found to be located in the dnaG gene and was shown to affect the initiation of DNA replication (Filutowicz and Jonczyk, 1983; Norris et al., 1986).

2. Does Nucleoid Position Determine Septa1 Position? a. Nucleoid-Occlusion Model. All of the mutationsjust described result in the formation of filaments with centrally positioned nucleoids, as

24

WlLLlAM R. COOK

ET AL.

well as substantial numbers of smaller, nonfilamentous cells that lack nucleoids. Thus, the cells are capable of septation but appear not to form septa at the normal midcell position. Studies of the pattern of division in these strains have led to the suggestion that the nucleoid(s) play an essential role in establishing the location of the division septum by exerting a local inhibitory effect on septum formation (Donachie et d . , 1984; Woldringh et d . , 1985; Taschner et al., 1987). We will call this the nuclear-occlusion model. We will now examine the implications of this interesting model and the evidence on which it is based. The model states that the location of the nucleoids is the sole determinant of septal positioning (Woldringh et al., 1985; Hussain et al., 1987a,b). In this view the entire cell envelope is competent to initiate septal ingrowth. Following chromosome replication in wild-type cells, the two daughter nucleoids are positioned at regular intervals along the length of the cell by an active nucleoid segregation mechanism, or perhaps by the passive diffusional redistribution of daughter chromosomes. The nucleoids then would act as local inhibitors of septum formation, thereby restricting formation of the septum to a region near the middle of the cell. Three possible mechanisms can be considered for a nucleoid-occlusion effect, if it exists. 1. The nucleoid provides a simple physical block to the inward move-

ment of the septum. 2. The proximity of the nucleoid perturbs the organization of the overlying cell envelope by local physicochemical effects. 3. The nucleoid elaborates a diffusible septation inhibitor that prevents the initiation of septal ingrowth within a limited distance from the nucleoid itself.

b. Evidence for the Model. The idea that nucleoid placement is the sole determinant of septal placement was first suggested by Woldringh et al. (1983, based on studies of dnaZTs cells in which division was permitted to resume after a period of inhibition of DNA synthesis. This resulted in the production of anucleate cells and minicells. It is of interest that rninicell production has not been described when DNA synthesis has been blocked by inactivation of other dna gene products. The authors noted that the coefficient of variation in cell length was much larger for the temperature-shifted culture because both minicells and long chromosome-free cells were formed. On the basis of the increased coefficient of variation, it was suggested that there were no predetermined sites for the execution of constriction in the downshifted cells. E. Mulder and C. L.

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

25

Woldringh (personal communication) have also noted similarly broad coefficients of variation of placement of constrictions in filaments of other DNA mutants in which anucleate cells were produced. The use of coefficients of variation as a indication of the randomness of placement of septa is based on the assumption that the distance from septal constriction to cell pole represents a single unimodal length distribution population. It should be noted that this assumption need not be correct. For example, if preexisting sites were distributed at regular intervals between cell pole and nucleoid, septation at any one of these sites would be possible. Measurement of the distance from constriction to cell pole in a large number of cells would then yield a mixture of discrete size classes, each class representing a multiple of the unit cell length. In this hypothetical case, the coefficient of variation of segment length for the entire data set would be high despite the fact that septal placement was nonrandom. It will be of interest to see whether it is possible to resolve the length distribution pattern into more than one Gaussian population before making a decision on the randomness of septal placement in these experiments. Studies of mutants with DNA gyrase defects have also provided evidence that has been interpreted to support a nucleoid-occlusion model. Growth of gyrA(parD) cells under nonpermissive conditions (see earlier) led to formation of filaments with large centrally located nucleoids, leaving long regions of the filaments devoid of visible nucleoid material. This presumably was due to failure to decatenate daughter chromosomes after completion of replication. Septation within the anucleate regions of the filaments subsequently led to formation of small chromosome-free cells (Hussain et al., 1987a). The chromosome-free cells were said to consist of short rods of all lengths between minicells and normal-length cells, although this is difficult to evaluate based on the resolution of the published results. It was pointed out that these observations are compatible with the idea that the absence of nucleoids near the cell poles led to the random placement of septa in these nucleoid-free regions (Hussain et al., 1987a). It should be noted that inactivation of DNA gyrase changes the expression of a number of chromosomal genes in which the state of chromosomal supercoiling affects promoter activity (Schmid, 1988; Wang, 1985). This raises the possibility that any septal localization defect could be secondary to altered expression of genes that affect septal placement by a mechanism other than nucleoid occlusion. c. Evaluation o f t h e Model. The idea that the entire cell envelope is competent to initiate septation during the latter portion of the division cycle raises certain problems. It is not compatible, for example, with the

26

WILLIAM R. COOK

ET A L .

observation that in slowly growing cells periseptal annuli are localized at the future division site during the previous cell cycle, long before the onset of the C phase of the division cycle in which septation occurs at this site (see Section 11). It is therefore unlikely, in our view, that the potential to initiate septum formation is randomly distributed along the entire length of the cell at the time of formation of the new septum. The most direct way t o test the nucleoid-occlusion model is to determine the size distribution of newborn cells in which nucleoids are absent from the greater part of the cell body secondary to inhibition of DNA replication. This avoids the possible complications of other secondary effects of gyrase defects or of par mutations. If future division sites were localized prior t o chromosome segregation, septa would be expected to form at regular intervals along the filaments, with the exception of the occluded site at midcell. On the other hand, if nucleoid positioning were the sole determinant of septa1 positioning as stated by the nuclear-occlusion model, then septa would be randomly distributed and the anucleate cells that are formed would show a continuum of cell lengths, down to the size of minicells. In the most extensive published study of this type, Jaffe et al. (1986) have studied the division patterns of cultures in which DNA synthesis was blocked in the absence of the SOS system. The inhibition of DNA synthesis was accomplished in a variety of ways, including thymine starvation and temperature shift in dnaA, dnaB, and dnaC mutants. This led to the formation of filaments that contained only one or two nucleoids, primarily located near the midpoint of the filament, plus significant numbers of anucleate cells. The chromosome-free cells were of normal length and were relatively uniform in size, suggesting that the positioning of septation sites was not affected by the absence of nucleoid in the vicinity of the septation event. Similar results were reported by Hirota et a / . (1968b) in studies of a thermosensitive dnaA mutant. In our view, these studies provide the most cogent argument against the proposition that division sites are randomly located along the length of the cylinder and that selection of the site depends only on the absence of nucleoid. One can imagine a more permissive variant of the model in which the nucleoid would affect the placement of the septum by vetoing septation at nearby division sites, while permitting septation to occur at other potential division sites that are located at regular intervals along the length of the cylinder. If this were correct, newly born chromosome-free cells would fall into discrete size classes equivalent to multiples of the presumed unit cell length. The possibility can also be considered that aberrant nucleoid placement affects an earlier stage in differentiation of the

DIFFERENTIATION OF BACTERIAL CELL DIVISION SITES

27

division site, such as the formation or localization of periseptal annuli, rather than affecting the septation event itself. Further studies will be needed to determine whether nucleoid position indeed plays a role in the selection of the septation site. VI. Conclusions

In recent years, significant advances have been made in defining the E. coli cell division process. At the morphological level, the discovery of periseptal annuli has permitted the developmental history of the division site to be followed throughout the cell cycle. At the same time, genetic studies have identified a number of proteins that are implicated in formation and localization of the division apparatus, and in the process of septa1 morphogenesis. Much useful information remains to be obtained in these areas, most notably an understanding of the coordination of division site development and localization with other events of the division cycle. In this regard, the use of immunoelectron microscopy to study the cellular localization of division-related gene products has hardly been exploited. This promises to provide new information that will be essential to any detailed understanding of the mechanism of the division process. Nevertheless, if this rich body of information is to be extended to the molecular level, it will be necessary to develop methods for isolation of the division site itself. Until this is achieved, the picture that emerges will necessarily be incomplete. A second major area of interest concerns the relation between cell division and chromosome replication. It is clear that the two processes are temporally coupled under normal conditions. A considerable body of evidence has permitted the formulation of specific hypotheses regarding the basis of the coupling. However, it is still not known whether or not there is direct coupling between the two processes or, alternatively, whether both respond to a common division clock. Does the initiation or termination of chromosome replication generate a signal that normally regulates the timing of septum formation? Does the differentiating division site send a signal that affects chromosome replication? Do both processes respond to signals generated by other events of the division cycle? It has proved difficult to obtain unequivocal answers to these significant questions, and this general area remains an important field for future study. A third key question is, how does E. coli determine where to place the

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WILLIAM R. COOK

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division septum? There was little experimental information dealing with this question until the past few years, when early stages in the localization process have been described at a morphological level. These studies have suggested that the localization of the division site takes place long before initiation of septum formation. At the same time, studies of cells in which chromosome partition is defective have led to the proposal that the position of the nucleoid plays a role in determining the placement of the division septum. The identification of gene products of the minicell locus that affect the site selection process suggests that considerable additional information about the localization process remains to be obtained from genetic approaches. Exploitation of these relatively recent advances is likely to lead to a more complete understanding of this important but poorly understood aspect of the division process.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 118

Properties of the Cell Surfaces of Pathogenic Bacteria R. J. DOYLEAND E. M. SONNENFELD Department of Microbiology and Immunology, Health Sciences Center, University of Louisville, Louisville, Kentucky 40292

I. Introduction The bacterial cell surface possesses many properties that are important in pathogenesis and immunity. The surface may contain components that stimulate or depress the immune response, depending on concentrations of the components. The surface serves as a site of antigenic recognition. Many bacterial vaccines are composed of surface structure materials. Vaccines against the pneumococcus or Haemophilus influenzae, for example, utilize surface-associated poly saccharides. In many cases, surface structures tend to inhibit phagocytosis of an invading bacterium. Almost any encapsulated bacterium is more resistant to phagocytosis and subsequent intracellular digestion than a nonencapsulated mutant of the same species. The surface also may serve as an anchoring point for a bacterium. It now seems clear that some adherent bacteria have growth advantages over their nonadherent siblings. In both gram-positive and gramnegative bacteria, there are specialized surface structures that enable the bacteria to be tethered to complementary sites on animal cells. In most cases, adhesion of the bacteria to a surface is the initial step in the infectious process. For some bacteria, surface components have direct toxic effects on host cells. Lipopolysaccharides (LPS), surface constituents of many gram-negative pathogens, exhibit several manifestations of toxicity in infected hosts.' Lipoteichoic acids (LTA) from gram-positive bacteria also seem to possess toxic and immunomodulating activities. Finally, the 'Abbreviations used in the text are as follows: Ab, antibodies: Ag, antigens: DAP, diaminopimelic acid: DIC, disseminated intravascular coagulation; EA-IJI, extractable antigens I and 11; FN, fibronectin; Fuc, fucose: GalNAc, N-acetyl-D-galactosamine:GlcNAc, Nacetyl-D-glucosamine: HLA, human lymphocyte antigen (histocompatibility); Ig, immunoglobulins; IL-I , interleukin 1 ; LPS. lipopolysaccharides: LTA, lipoteichoic acids: Man, mannose: MDP, muramyl dipeptide: MS, mannose-sensitive: PG, peptidoglycan. 33 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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surface may serve as a target for antibiotic action. Penicillin and penicillinlike antibiotics and drugs prevent the synthesis of peptidoglycan (PG), resulting in cell death. The purpose of this article is to review a few aspects of bacterial cell surfaces, with an emphasis on the role of surface structures in pathogenesis and in biological responses.

11. Surface Structures of Bacteria

Macromolecular syntheses and nucleic acid replication occur in the bacterial cytoplasm. Most pathogenic bacteria, except for members of the genus Mycoplusma, possess reasonably high internal concentrations of solutes. This requires that the bacteria have extracellular supporting structures to circumvent lysis. Gram-positive bacteria are thought to possess approximately a molar equivalent of internal solute. Such an internal concentration of intracellular material would give rise to a force of -22 atm or about the equivalent of the pressure in an inflated bicycle tire of 70 psi (Koch, 1983). The gram-negative cell is thought to possess an internal solute concentration of -0.2-0.4 M. Solutes are derived from transported materials, precursors, ATP salts, and macromolecules. The extracellular supporting material for bacterial pathogens is PG (also called murein). A simplified structure for a PG is shown in Fig. 1. The PG is composed of p-I ,Clinked repeating units of N-acetylmuramic acid and N-acetyl-D-glucosamine (GlcNAc). Most bacteria do not fully acetylate the amino sugars, whereas others 0-acetylate the C-6 of muramic acid. Resistance to lysozyme is frequently associated with O-acetylation or with free amino groups on the amino sugars. The glycan chains may also be substituted on the lactyl group of the muramic acid by amino acids. Generally, the amino acid directly bonded to the muramic acid is L-alanine. The sequence L-Ala-D-Glu-L-Lys-D-Ala from the muramic acid is common for many bacteria, although diaminopimelic acid (DAP) may replace the L-LYSresidue. The L-LYSor DAP amino acids represent sites for cross-linkages because they possess free amino groups that may form peptide bonds with terminal D-Ala residues of nearby chains. The cross-linkage may be direct, as in an L-Lys-D-Ala bond, or it may involve other amino acids to connect L-LYS(or DAP) to a D-Ala. Staphylococcus izureils, for example, possesses a pentaglycine as a crosslinking unit. The extent of crosslinking varies from bacterium to bacterium, but may be as low as 12% for Bacillirs anthrucis (Zipperle et al., 1984), to as high as 8090% for Legionelh pneurnophila (Amano and Williams, 1983). Prevention of synthesis of PG constitutes the major means for the bac-

BACTERIAL CELL SURFACE

35

O-R' I

L

-Ala

@

I I

D -Glu

I

- Lys ( or DAPI-CROSSLINK D - Ala I L

+o

0

R = H or -C*-CH,

R'= H or LINKAGE to TElCHOlC or TEICHURONIC ACIDS FIG. 1. Peptidoglycan structure showing where bacteriolytic enzymes and autolysins 2, lysozyme; 3, N-acetylmuramyl-L-alanineamidase; may act. 1, N-Acetylglucosaminidase; 4, endopeptidase(s);5, D,L-carboxypeptidase. The extent of amino sugar modification depends on the bacterium. Most pathogens acetylate the amino sugars.

terial activities of cell wall antibiotics such as penicillin. If a bacterium is subjected to a cell wall antibiotic and continues to take up nutrients, then the bacterium will ultimately lyse. This is because no new supporting PG has been assembled to contain the enlarging cytoplasm. In addition to lysozyme (a P-1,4 muramidase), other enzymes, such as autolysins, may degrade preexisting PG. The target sites for autolysins are depicted in Fig. 1. Human serum also contains an N-acetylmuramyl-L-Ala amidase (Mollner and Braun, 1984), but its role as a host defense factor is not well understood. The gram-positive bacterium may contain macromolecules covalently bound to the PG. These include teichoic acids or teichuronic acids (Figs. 2 and 3). The teichoic acid is typically a structure composed of either poly(glycero1 phosphate) or poly(ribito1 phosphate). (It is of course known that some variations in the teichoic acid structure may occur, such as the inclusion of an amino sugar or a mannitol within the chain, but such details are beyond the scope of the present article.) The glycerol or ribitol residues may be substituted by a-or P-D-glucose, D-Ala, or a-or p-GlcNAc or other entities. The nature and extent of substitution de-

36

R.J. DOYLE AND E.M. SONNENFELD

I

( FATTY ACIDS 1 I -

-

-

R = a- or p- D glucose ; D a1anin.e ;OH

FIG.2. Structure of a lipoteichoic acid. (LTA). The fatty-acid end may be firmly bound to the cytoplasmic membrane. whereas the poly(glycero1 phosphate) units may penetrate the PG matrix and extend into the solvent phase. In some bacteria (pyogenic streptococci) the LTA molecules may be released from the cytoplasmic membrane and bind surfaceassociated proteins. Wall teichoic acids are covalently bound to muramic acid residues and are not attached to glycolipid or fatty acid.

pends on the bacterium. A teichuronic acid is typically composed of disaccharide or trisaccharide repeating units containing a uronic acid. The composite cell wall structure of PG and teichoic or teichuronic acid may constitute ~ 4 0 %of the dry weight of a gram-positive cell. The teichoic or teichuronic acids may serve as antigens (Ag), phage receptor sites, proton- or metal-binding sites, wall volume expanders, or autolysin-binding sites, or they may have additional roles in the physiology of the bacterium. Figure 4 shows a thin section of the pathogen B. anthracis. The cell wall is easily recognizable along the cell cylinder and the cell septum. The gram-positive cell surface may also contain LTA. These are

I

01

c=o

0-

H CH

n

FIG 3. Segment of a cell wall-associated teichuronic acid. This teichuronic acid is from Micrococcrrs IvsodeiLricus (Hase and Matsushima, 1972).

BACTERIAL CELL SURFACE

37

FIG.4. Thin section of a Bacillus anthrucis cell showing surface array, PG, capsule, and plasma membrane. Courtesy T. J. Beveridge.

poly(glycero1phosphate) molecules that possess a hydrophobic end (Fig. 2). The hydrophobic group is embedded in the plasma membrane, whereas the poly(glycero1 phosphate) may penetrate through the PG to reach the cell periphery. Antibodies (Ab) directed against poly(glycero1 phosphate) frequently agglutinate gram-positive bacteria known to be devoid of wall poly(glycero1 phosphate) (Wicken and Knox, 1975). Other surface components of the gram-positive bacterium may include noncovalently bound polysaccharide (sometimes a group-specific polysaccharide), a surface array, and capsular materials. All of these structures, except for the surface array, have been shown to have a role in pathogenesis (Table I). Flagella may be regarded as virulence determinants because they may cause bacteria to be propelled to nutrient-secreting mucosal sites. Bacillus anthracis is an interesting pathogen from the viewpoint of bacterial cell surfaces (Figs. 4-7). The bacterium contains a PG-polysaccharide wall. There is no teichoic acid, nor does an anti-poly(glycero1 phosphate) Ab agglutinate the bacteria. Bacillus anthracis contains a surface

38

R.J. DOYLE A N D E.M. SONNENFELD

IMPOKTANCE OF

TABLE I BACTERIAL CELL SURFACES

Surface structure Fimbriae (pili) Wall teichoic acid LTA

ffi Protein LPS Secreted polysaccharide and capsular materials Flagella

Surface array Teichuronic acid

IN

PATHOGENICITY

Role in virulence Adhesion and colonization Adhesion, prominent Ag Adhesion, modulates immune response, Ag, pyrogen Modulates immune response, induces inflammation, pyrogen, adjuvant Adhesion, Ag, toxin Toxic, Ag, modulates immune response. pyrogen. adjuvant Adhesion, protection against Ab; sequesters nutrients; retards phagocytosis and subsequent intracellular digestion Propel bacteria t o site of attachment (mucosal surfaces secreting nutrients) None known at present Ag; role in pathogenesis unknown

array (Figs. 4-7) and may secrete a poly(D-glutamic acid) capsule. The capsule seems to be required for virulence. The bacterium also secretes several proteins, some of which may be intercalated within the PG matrix. The protective antigen (PA), for example, can be shown to be surface-bound by use of direct- or indirect-fluorescence methods. The PA is one component of a tripartite toxin essential for the virulence of B. anthrucis. Proteins designated as EA-I and EA-I1 (“extractable antigens”) are closely associated with the PG layer. It is possible that the EA proteins may be subunits of the surface array materials. The gram-positive bacterial surface is relatively thick, resulting in the ability of the bacteria to retain the Gram stain (Beveridge and Davies, 1983). The thickness of a Bacillus subtilis wall is of the order of 25 nm (Beveridge and Murray, 1979). Most cell walls of gram-positive bacteria are solvent-exposed, making them capable of interaction with Ab, enzymes, and environmental molecules (Fig. 8). The wall is reasonably permeable, making it possible for antibiotics, nutrients, and low molecular weight proteins to be taken up or secreted. The gram-negative cell surface is more complex than that of the gram-

FIG.5. Negative stain of capsular material and surface array of Bacillus anthracis. Micrograph courtesy of T. J. Beveridge.

WALL MATRIX

SURFACE ARRAY (EA- I Ag 1 ( E A - I I Ag) LlPOTElCHOlC ACID PA -POLYSACCHARIDE POLY ( D GLUTAMIC ACID)

-

CYTOPLASMIC MEMBRANE FIG.6. Model depicting surface structures of Bacillus anthracis. EA, Extractable antigen; PA, protective antigen. Polysaccharide is covalently attached to PG. Lipoteichoic acid does not penetrate through PG because anti-poly(glycero1 phosphate) Ab will not agglutinate the bacteria. The PG and polysaccharide are turned over during growth to the extent of -50% per generation.

FIG.7. Computer-enhanced image analysis of the surface array of Bacillus anthroris. The apparent symmetry is PI, suggesting that the subunits are assembled free of contact with other subunits. Courtesy of T. J. Beveridge and M. Stewart.

FIMBRIAE (or Fl8RlLLAE 1 TEICHURONIC

-

PEPTIDOGLYCAN LTA PROTEIN CYTOPLASMIC MEMBRANE

41

BACTERIAL CELL SURFACE PROTEIN FlMBRlAE (PILI) LIPOPOLYSACCHIAA:IDE

c

0 and R - A g LIPID A

POPROTE IN EPTIDOGLYCAN

--CYTOPLASMIC MEMBRANE

FIG.9. Cross-sectional representation of the gram-negative cell surface. The PG matrix is relatively thin, of the order of one to three glycan strand equivalents in thickness. The PG may turn over. but in gram-negative rods the turned-over materials are largely reutilized. In many gram-negative bacteria, a lipoprotein is covalently attached to the PG. The fattyacid portion of the lipoprotein is anchored in and stabilizes the inner leaflet of the outer membrane. The outer membrane forms a passive permeability barrier, thereby protecting the PG and cytoplasmic membrane from lysozyme and other potentially deleterious molecules. Small molecules (nutrients) may penetrate the outer membrane through protein channels (porins). The LPS is anchored in the outer leaflet of the outer membrane via hydrophobic interactions. The oligosaccharide structure is completely solvent-exposed and available for interaction with Ab or bacteriophages. Proteins may be embedded in the outer membrane and serve as Ag. Fimbriae may penetrate the PG and outer membrane and extend into the solvent. Lectins at the very tip of the fimbriae may serve to tether the bacteria to complementary surfaces.

positive cell. The gram-negative bacterium possesses a thin layer of PG, possibly equivalent to one to three layers of PG. The wall is crosslinked in a manner similar to that of the gram-positive bacterium and contains the same kinds of amino sugars and amino acids as the gram-positive PG. There is no teichoic acid or teichuronic acid, however. The gram-negative PG is not solvent-exposed, but is surrounded by the outer membrane (Fig. 9). The outer membrane is morphologically similar to the plasma membrane, but it has a much different composition than the inner membrane. The outer membrane contains proteins, phospholipids, a lipoproFIG.8. Representation of a cross section of a gram-positive bacterium. The PG nearest the cytoplasmic membrane is not so stressed as the PG on the surface. When PG turnover occurs, it is usually the wall most stressed and removed from the plasma membrane. In the typical gram-positive cell, there may be 25-35 layer equivalents of glycan to make up the wall thickness. Molecules of LTA may penetrate the PG matrix in some species. Proteins may be embedded in the cell wall or surface-exposed. Teichoic or teichuronic acids are covalently attached- to the muramic acid residues of PG.

K.J.D O Y L E A N D E.M. S O N N E N F E L D

42

0

0 P - A r a N KDO I I KDO

( F A ) GlcN

-

- KDO -

I (FA) GlcN I

@

t

o

r

a p C H O +-

H2 N

D

I

Glc

- Gal

9" "i 9" 'i'

t

C

Gal

-

Re

P -P E T

LIPID A

P-ET I P Hep I I Hcp HepI P

specific pCHO

wo

Qo

-

/ c

P P - ET

NH

(4-Ara

N)

I

0 I

NH 1

FA FA

FA

FA

(Glc N )

(Glc N )

FIG.10. Structures for LPS (A) and lipid A (B). These generic structures show the 0 and R antigens, as well as mutations in the R saccharide. KDO, 2-keto-3-deoxyoctonate; CHO, carbohydrate; ET, ethanolamine; Hep, heptose; Glc N , glucosarnine; FA, fatty acids: Ara N. arabinosamine.

tein (which may be covalently bound to PG, but with the lipid end extending into the outer membrane), trace metals, and an LPS (Fig. 10). The outer membrane may serve to protect a bacterium from environmental molecules or host defense factors. The LPS component of the outer membrane may be the sole virulence determinant for some gram-negative bacteria, as it possesses a spectrum of toxic qualities (Table I). For some gram-negative bacteria, injury to the outer membrane results in increased sensitivity to antibiotics, Ab, or lysozyme. The gram-negative bacterium may secrete capsular materials or protein toxins. In addition, the gramnegative bacterium may possess proteinaceous structures called fimbriae or pili, which may be involved in adhesion of the cells to substrata (Fig. I I). Gram-positive cells may also have surface structures called fimbriae, but these appear to be aggregated proteins rather than the highly ordered structures found in the gram-negative cells. Much of the remainder of this review deals with the biological properties of bacterial cell surface components. In addition, the role of bacterial adhesion and the role of cell wall turnover in bacterial pathogenesis will be discussed.

BACTERIAL CELL SURFACE

43

FIG. 11. Fimbriae associated with Escherichia coli. Arrow points to fimbrial structures possessing MS lectin activity (type 1 fimbriae). Bar = 100 nm. Reprinted by permission of T. I. Beveridge and Academic Press (Krell and Beveridge, 1987).

111. Biological Reactions to Peptidoglycans

In 1957, it was noticed that a prolonged inflammatory reaction occurred in the skin of rabbits after injection of crude streptococcal extracts (Schwab and Cromartie, 1957). Cardiac lesions similar to those seen in rheumatic fever were noted in mice following the intraperitoneal injection of a sterile extract of sonically disrupted group A streptococcal cells (Cromartie and Craddock, 1966). It was thought that the toxic material responsible for the lesions may have been cell wall fragments in both the skin and heart lesions. During this time it was also recognized that bacterial cell walls might contribute to the etiology of rheumatoid arthritis (Hamer-

44

R.J. DOYLE A N D E . M . SONNENFELD

man, 1966) and that similarities between the polysaccharides of human connective tissue and bacterial polysaccharides might be an important factor in the pathogenesis of the disease. Many studies have been completed over the years (Table II), and from these there emerge two important properties associated with a cell wall's capacity to induce a chronic arthritis in laboratory animals: ( 1 ) the molecular weight of the inducing fragments (Chetty et al., 1982) and (2) their ability to persist in host tissue (Schwab and Ohanian, 1967; Lehman et ul., 1985; Stimpson et al., 1986). Cell wall fragments with MW 5.3 x 10' induced only an early acute joint inflammation (Chetty et al., 1982; Fox P t ul., 1982), whereas larger fragments of MW 500 x 10' induced a chronic arthritis of late onset. Intermediate-sized fragments of MW 50 x 10' induced a chronic and acute arthritis. In seeming contradiction to these results, Kohashi et al. (1976) found that arthritogenicity was associated with PG subunits of two or more disaccharides in length and that an attached polysaccharide containing rhamnose had no effect. The vehicle for their cell wall preparations was an oil emulsion as opposed to an aqueous suspension, and it was injected into rats directly in the inguinal lymph nodes as opposed to intraperitoneal injection. Their purpose was to study the nature of adjuvant-induced arthritis (Pearson, 1956). These workers also found that removal of the GlcNAc moiety from a disaccharide-heptapeptide-disaccharide resulted in loss of arthritogenicity. Persistence of cell wall is directly proportional to its resistance to lysozyme (Schwab and Ohanian, 1967). Resistance to lysozyme is enhanced by 0-acetylation TABLE 11 BIOLOGICAL PROPERTIES OF PG A N D DERIVATIVES Property

Reference

Arthritogenicity Pyrogenicity lmmunogenicity Adjuvanticity Stimulation of IL- I production Somnogenic Enhanced resistance to pathogens Stimulation of phagocytic cells Increased production of collagenase and prostaglandin Cytolysis of neoplastic cells Immunosuppression by inhibition of 1L-2 Augmentation of serum interferon production Polyclonal B-cell activator Mitogenic

Fox et ul. (1982) Takada and Kotani (198.5) Heymer et ul. (1976) Ellouz et a / . (1974) Vermeulen and Gray (1984) Krueger ei ul. ( 1982a) Fraser-Smith et ul. (1982) Fraser-Smith et ul. (1982) Wahl ei (11. (1979). Taniyama and Holden (1979) Leclerc and Chedid (1983) lkeda el ul. (198.5) Specter el a/.( 1977) Damais ei ul. (1977)

BACTERIAL CELL SURFACE

4s

of muramic acid residues (Giesbrecht et al., 1982), the presence of a rhamnose-containing polysaccharide attached to the PG (Schwab and Ohanian, 1967), and lack of N-acetylation of glucosamine residues (Hayashi et al., 1973). Resistance of cell wall to degradation by macrophages and monocytes may contribute to its ability to act as a persistent Ag, thereby creating the necessary milieu for the incitement of a chronic arthritis. In fact, it has been shown that streptococcal cell wall will persist in macrophages (Rickles et al., 1969) that demonstrate a cytotoxic effect for mouse L cells (Smialowicz and Schwab, 1977). These authors speculate that the chronic tissue destruction exemplified by streptococcal cell wall in vivo may be the result of this cytotoxic ability of macrophages. The most extensively studied organism with regard to arthritis and cell wall has been Streptococcus pyogenes, which is both resistant to lysozyme and contains a group-specific polysaccharide containing rhamnose. Group A streptococcal cell walls were sensitive to lysozyme when the walls were chemically N-acetylated, but removal of the group-specific carbohydrate before N-acetylation rendered the walls even more susceptible to lysozyme (Gallis et al., 1976). It is thought that the group polysaccharide sterically hinders degradation by lysozyme. In staphylococci, autolytic enzymes appear to be inhibited by anionic polyelectrolytes (Ginsburg et al., 1985) and activated by cationic proteins found in lysosomes. Therefore, the accumulation of sulfated polysaccharides at sites of inflammation may tend to cause persistence of undigested cell wall material within macrophages. It has been noted that a higher relative risk for some forms of arthritis is related to genetic background. It is now known that rheumatoid arthritis is associated with human lymphocyte antigens HLA-DW4 (Rodnan and Schumacher, 1983) and HLA-DR4 (Albert and Scholz, 1987). However, the association with bacterial cell walls, though appealing, is still tentative. It is a tempting premise that similarities exist between cell wallinduced arthritis in laboratory animals and certain forms of arthritis that plague humans (Hadler, 1976). This is partially substantiated by findings demonstrating that rheumatoid factors, developed in animals immunized with streptococci, recognize both the PG of the bacteria and the Fc component of IgG (Bokisch et al., 1973). A variety of tissues, including those from the joint capsule (Rodnan and Schumacher, 1983), appear to be antigenically similar to cellular components of streptococci. This finding is emphasized by the observation that patients with rheumatic fever, juvenile rheumatoid arthritis (Heymer et al., 1976), and rheumatoid arthritis (Braun and Holm, 1970) possess Ab that cross-react with PG. It may be significant to note here that arthritis is a very common symptom often associated with rheumatic fever (Freeman, 1979). A complex interaction

46

R.J. DOYLE AND E.M.SONNENFELD

between Ab to streptococcal group A cell wall components and host tissue seems to be involved in the pathogenesis of this disease (Rotta, 1969). Cell wall material from a wide variety of bacteria is known to induce arthritis in rats and guinea pigs (Hadler and Granovetter, 1978). Some of these organisms would be considered indigenous microbiota. Reiter’s syndrome, a form of reactive arthritis that is HLA-B27-associated (Aho, 1987). has been linked to prior infections caused by Yersinia (Ahvonen et al., 1969), Shigella (Good and Schultz, 1977), Salmonella (Trull et al., 1986), Chlamydia (Keat et al., 1987), Campylobacter (Inman, 1986), and Neisseria gonorrhoeae (Rosenthal et al., 1980). Reactive arthritis involves joints that are anatomically distant from a site of prior infection (Campa, 1983) that is typically enteric or venereal in origin (Aho, 1987). The antecedent infection usually occurs 1-3 weeks before the onset of an arthritis (Aho, 1987) that does not respond to antibiotics. The arthritis is thought to be mediated by persisting microbial Ag (Toivanen et al., 1987) that may involve bacterial cell wall (Bennett, 1978), or perhaps LPS (Yaron et al., 1980). Endotoxins from Escherichia coli, Shigellaflexneri, Salmonella typhosa, and Vibrio cholerae were able to stimulate human synovial and foreskin fibroblasts to secrete prostaglandin E (Yaron et a f . , 1980). Shigella endotoxin was shown to cause secretion of prostaglandin E and hyaluronic acid (Yaron et al., 1980). In addition, LPS is capable of binding to eukaryotic cell membranes (Wicken and Knox, 1977), and this may enhance its ability to persist in host tissues. There may even be an interplay of molecular mimicry, in which HLA Ag crossreact with microbial Ag (Inman, 1986). Another example of a reactivetype arthritis is the arthritis associated with rheumatic fever. It is likely that streptococcal cell wall is involved in its etiology. In the early 1970s, adjuvanticity, which was found originally in oil-water emulsions of killed Mycobacterium tuberculosis organisms (White et al., 1958), was determined t o be a general feature of PG (Nauciel et al., 1973; Nguyen-Dang et al., 1973; Adam et al., 1974), and in 1974 the smallest molecule that still maintained this feature was determined to be a synthetic N-acetylmuramyl dipeptide, or MDP (Ellouz et al., 1974). Figure 12 shows the structure of MDP, which possesses adjuvant properties and will also induce an acute (as opposed to chronic) arthritis in rats treated with repeated subcutaneous injections of MDP in saline (Zidek et al., 1982). Most of the material was rapidly removed from the body, but a small fraction was suspected t o remain present for a longer time. These experiments demonstrated that an oil emulsion (Kohashi et al., 1980) was not a necessary vehicle for MDP in the production of an acute arthritis. Other properties of MDP include its ability to enhance resistance in mice to a wide variety of organisms (Fraser-Smith et al., 1982). Treatment

BACTERIAL CELL SURFACE

47

CH20H

H

I 0 I

CH3- CH

N-$-CH3

H O

- CII - NH - CFH3- C - HN - HCI - CI1 - OH 0

6

CH2O I

C-OH It

0 FIG.12. Structure of MDP (N-acetyl-D-rnurarnyl-L-alanyl-D-isoglutarnine).

of macrophages with MDP produces an entire myriad of effects (FraserSmith et al., 1982), some of which are most likely mediated by interleukin 1 (IL-1). In fact, it is possible that IL-1 is responsible for some of the joint destruction seen in arthritis (Wood et al., 1983a). It is interesting that mouse macrophages will “process” cell wall material from B. subtilis and concomitantly will secrete IL-1 (Vermeulen and Gray, 1984) and glycopeptides that are structurally related to MDP. The ability of MDP to act as adjuvant probably also is a result of IL-1 production by macrophages (Wood et al., 1983b), but this has not been proven conclusively (Fig. 13). In addition to the properties already discussed, MDP also possesses a sleep-inducing effect (Krueger et aL, 1982a). The natural peptide, which is found in urine and cerebrospinal fluid, induces excess slow-wave sleep in rats, rabbits, and cats (Krueger et al., 1982b). The phenomenon of sleep inducement is not species-specific in that the peptide collected from the cerebrospinal fluid of goats could induce sleep in cats that lasted 12-24 hours (Pappenheimer et al., 1967). Factor S, as it was called, was originally thought to contain glutamic acid, alanine, DAP, and muramic acid in molar ratios of 2 : 2 : 1 : 1. Although the composition resembled bacterial PG, the “extra” glutamic acid could not be explained on that basis. In addition, the original collection of cerebrospinal fluid was thought to be free from the possibility of exogenous bacterial contamina-

48

R.J. DOYLE A N D E.M. SONNENFELD

MDP

or

-

PEPTIDOGLYCAN

MACROPHAGE

\ IL- I SENSITIZED CELLS

I

IL-2 EXPANSION of T-HELPER CELL POPULATION

i

ENHANCED HUMORAL and CELL-MEDIATED IMMUNITY Ftc;. 13. Hypothesized action of MDP or PG in the enhancement of humoral and cellmediated immunity.

tion (Pappenheimer rt a/.,1967). Nevertheless, the possibility that the sleep factor was derived from bacterial products absorbed from the gut (Maugh, 1982) could not be refuted because biosynthesis of muramic acid had never been shown in mammalian tissues. In fact, the actual presence of muramic acid tended to support the contention that the substance originated from bacteria (Karnovsky, 1986). Today, there is even some thought that muramyl peptides are similar to vitamins, since they may be required but cannot be synthesized by the host (Krueger, 1986). Some bacterial products can antagonize sleep, supporting the contention that these peptides play an important role in the regulation of sleep (Maugh, 1982). Normal human brain has been shown to contain muramic acid (Zhai and Karnovsky, 1984), while DAP, thought to be of bacterial origin, was found as a normal component of human urine in 1980 (Krysciak, 1980). Also, the L-alanine-D-isoglutamate-containingcompound, as it is found in PG, is biologically active, whereas the L-L or D-D isomers are not (Karnovsky, 1986). All of these data support the assertion that sleep factors are indeed of bacterial origin, and may even be responsible for sleepiness associated with infectious disease (Krueger, 1986). Using fast atom bombardment-mass spectrometry, the structure of the urinary sleep-promoting factor was determined (Fig. 14) (Martin et al., 1984). The major component was found to be a disaccharide containing GlcNAc and N-acetylanhydromuramic acid in linkage with L-alanine, D-

49

BACTERIAL CELL SURFACE CH2 -OH

0

II

C-OH

FIG.14. Structure of the urinary sleep-promoting factor N-acetyl-glucosaminyl-N-acetylanhydromuramyl-L-alanyl-D-glutamyl-diam~nop~melyl-D-alan~ne, as determined by fast atom bombardment-mass spectrometry (Martin et a / ., 1984).

isoglutamine, mesodiaminopimelate (DAP), and D-alanine. The muramyl form of this compound also possesses biological activity (Krueger et al., 1984), but it is less potent than the anhydro form (Krueger et al., 1986). Somnogenic activity is lost when the a-carboxylate of glutamic acid or the ecarboxylate of DAP is amidated (Krueger er al., 1984). Rosenthal and Krueger (1987) found that three types of PG fragments from Neisseria gonorrhoeae were capable of enhancing slow-wave sleep in rabbits. The fragments were N-acetylglucosaminyl- 1,6-anhydro-N-acetylmurarnyl-alanyl-glutamyl-diaminopimelyl-alanine, the corresponding anhydro-N-acetylmuramyl disaccharide with an additional alanine at the C terminus, and anhydro-N-acetylmuramyl tetrapeptide. It is interesting that IL-1 can also induce sleep (Krueger et al., 1984), since as stated earlier, PG can stimulate macrophages to secrete IL-1. This allows one to speculate on the possibility that muramyl peptides may function as somnogens via a mechanism involving IL-1 (Krueger, 1986).

IV. Bacterial Cell-Surface Amphiphiles Amphiphiles may contribute to the pathogenic potential of many bacteria. Both gram-positive and gram-negative bacteria possess surface amphiphiles that have been shown to display a variety of biological properties. In some cases, the amphiphile is toxic to the host, whereas in others, the amphiphile may provoke an immune response. Table I11 lists the most common amphiphiles encountered among bacterial pathogens. The LPS molecules of the Enterobacteriaceae have been one of the most widely studied groups of microbial structures. The LPS molecules

R.J. DOYLE AND E.M. SONNENFELD

50

TABLE 111 1 M W K T A N T BACTERIAL. CELL S U R F A C E

Aphiphile Forssman Ag Enterobacterial common Ag

Lipomannan

LPS

Lipoprotein

LTA

AMPHIPHILES"

Comments A membrane component of the pneumococci. Structure consists of short-chain saccharide attached to lipid. Surface polysdccharide of most members of Enterobacteriaceae. Fatty acids may be linked to polysaccharide consisting of GlcNAc and a uronic acid. From membrane of members of the genus Micwcoccirs. Mannose polymer (partially succinoylated) containing terminal glycolipid. Component of outer membrane of some gram-negative bacteria. Consists of a complex heteropolysaccharide and lipid A (fatty acid, usually P-hydroxymyristic acid, covalently bound t o glucosamine). Covalently bound to PG of some gram-negative bacteria. May confer stability to outer membrane. Membrane component of many gram-positive bacteria. Usually consists of a polymer of glycerol phosphate, with fatty acids esterified a s glycolipids or phosphat idylglycolipids.

"Partially compiled from Wicken and K n o x (1981a.b).

have traditionally been prepared by the Boivin or Westphal procedures (Table IV). The preparations are generally contaminated with other molecules, such as RNA, glycogen, or protein. Regardless of the extent of contamination, the preparations retain their biological properties. The term endotoxin was originally used by Richard Pfeiffer in 1892 while working with cholera vibrios (Westphal et al., 1977). This term seemed appropriate because he observed that this particular toxic material was not excreted from living bacteria, as an exotoxin, but rather was released when the bacteria underwent lysis. Today the term endotoxin is still used, often synonymously with LPS, although this is not exactly correct. Endotoxin is actually LPS with attached protein complexes (Morrison and Ulevitch, 1978). In 1952, Westphal and Luderitz applied the phenol-water extraction method to isolate protein-free LPS (Westphal et ul., 1977). Protein is removed by the phenol, whereas the LPS is recovered from the aqueous phase. Chemically, LPS consists of three sections (Fig. 10): a hydrophilic region, a central acid core, and a lipid-rich region (Osborn et af., 1974). The hydrophilic head contains a heteropolysaccharide chain known as the 0-specific antigenic unit. The 0 Ag refers to the repeating carbohydrate linkages, which impart immunological identity to

BACTERIAL CELL SURFACE

51

TABLE IV LPS PREPARATION^ Preparation Boivin Morrison-Leive Leive-Morrison Hybrid-free Westphal

Freeman Ag

Lipid A

Ethylether

Procedure/properties Supernatants from whole cells extracted with trichloroacetic acid; contaminated with polysaccharide, RNA, acid-soluble proteins Butanol extraction yields a preparation with small amounts of contaminating protein. EDTA releases -50% of the LPS in a high molecular weight form. Hypertonic solution-extracted LPS preparations were more toxic and homogeneous than phenol-water extracts. When whole cells are extracted with 44% phenol at 64°C. a single phase forms. Upon cooling, the aqueous phase containing the LPS may be separated by centrifugation. Preparations are generally contaminated with proteins. When Boivin or Westphal LPS preparations are mildly hydrolyzed with acid, the Freeman Ag (polysaccharide) may be obtained by precipitation with alcohols. It is useful in determining epitopes of LPS preparations. Deep rough mutants may be extracted to yield lipid A (a toxophore), which can then be further purified by hydrophobic chromatography. These preparations are useful in studying the toxicity and biological properties of LPS molecules. Lipopolysaccharide is released in a soluble form from broken cell suspensions by ethylether.

“From Morrison and Leive (1975). Leive and Morrison (1972). Raynaud et d . (1973). Ribi et d.(1959). Jawetz e t a / . (1984). Westphal et a/. (1977). Morrison and Ulevitch (1978). and Rietschel et d. (1982).

the polysaccharide portion of the LPS. These carbohydrates generally occur as tetrasaccharide repeating units, although trisaccharide units are not uncommon. Mutations leading to loss of the repeating 0-Ag sequences result in a structure called the “R” Ag. The R-Ag name is derived from the word rough, which characterizes the colonies of the mutants. The R-Ag polysaccharide core may also be modified by additional mutations, leading to LPS molecules with various core sizes (Fig. 10). The polysaccharide portion of the LPS may be removed from the rest of the molecule by mild acid hydrolysis (Table IV). The released polysaccharide, called the Freeman Ag, is used to study the antigenic specificity of an LPS molecule without the complications arising from its lipid portion. As far as is known, all of the toxic qualities of an LPS molecule can be attributed to its lipid A moiety (Fig. 10). Luderitz ct al. (1966) showed that LPS preparations from various mutants in R- or 0-Ag saccharides were just as toxic as the complex LPS molecule. Some of the reported biological activities of LPS molecules are outlined in Table V. These

52

R.J. DOYLE AND E.M. SONNENFELD TABLE V BIOLOGICAL PROPERTIES OF LIPIDA Property Pyrogenicit y Adjuvanticity Mitogenicity (B-cell polyclonal activator) Elicits Shwartzman reaction Geiation of Limulus lysate Activates alternate pathway for complement Shock Aggregates platelets Antigenic (the lipid A portion is normally immunosilent) induction of hypoglycemia Leukopenia, followed by leukocytosis Release of prostaglandins from macrophages Abortion Activation of Hageman Factor X11 impairment of oxidative phosphorylation Enhancement of lysosomal enzyme activities Necrosis of tumor cells

AND

LPs Reference

Siebert (1952) Freeman (1979) Gery et a / . (1972) Shwartzman (1982) Levin and Bang (1964) Morrison and Kline (1977) Freeman ( 1979) Des Prez et a / . (1961) Westphal (1975) Wolfe et a / . (1977) Athens er a / . (1961) Rietschel et a / . (1982) Freeman (1979) Morrison and Cochrane (1974) Bradley (1979) Martini (1959) Bradley (1979)

range from the well-known pyrogenic property, to B-cell mitogenicity, to the activation of complement, and others. The pyrogenicity of LPS is due to the release of endogenous pyrogens from phagocytic cells following uptake of the LPS. The endogenous pyrogens are low molecular weight proteins that act on the hypothalamus gland. A febrile condition may occur as a result of viral or gram-positive bacterial infections as well, but the pyrogenic response is a characteristic of most gram-negative infections. Pyrogen (LPS) can be detected in very small quantities by use of Limulus amebocyte lysates. The lysates readily gel in the presence of LPS at 0.1 kg/ml. This level of sensitivity is useful not only in detecting LPS in various kinds of solutions and suspensions, but also in the laboratory diagnosis of meningitis. Spinal-tap fluids yielding a rapid gelation of the Limirlus amebocyte lysate suggest a gram-negative infection. The biological effects of LPS are mediated after it adheres to host tissue. Lipopolysaccharide attaches firmly to erythrocyte membranes via a lipoglycoprotein receptor (Springer et al., 1974). This receptor is specific for LPS in that it can reversibly block LPS from binding to red blood cells, (RBC), and it will not bind other bacterial Ag. Additionally, the fixation to RBC was inhibited by the receptor whether the LPS was from smooth or rough cultures. The activity of the receptor was confined to the protein moiety because removal of lipid or carbohydrate did not decrease

BACTERIAL CELL SURFACE

53

activity of the receptor. In addition to RBC, platelets, granulocytes, and mononuclear leukocytes also have high affinity for LPS, but it seems that this binding is due to lipid rather than a lipoglycoprotein (Springer and Adye, 1975). It is probable that hydrophobic interactions are important in the binding of LPS to RBC membranes and platelets and white cells. There is convincing evidence demonstrating that the lipid A part of LPS is responsible for its attachment to host tissue (Luderitz et al., 1973). The phenomenon of lectinophagocytosis (Ofek and Sharon, 1988), whereby lectins on the surfaces of phagocytes recognize specific sugars or saccharides to promote microbial phagocytosis, has not been studied with purified LPS molecules. One of the biological properties of LPS is its ability to activate both the classical and alternate pathways of the complement cascade (Morrison and Kline, 1977). The lipid A region of LPS is responsible for activation of the classical pathway and is antibody-independent, although the extent of the activity is not identical for all species of LPS (Luderitz et al., 1973). The properdin-induced, or alternate pathway, is activated by the polysaccharide portion of LPS. Solubilized lipid A, rendered soluble by alkali treatment or serum albumin, was found to be highly active as an anticomplement agent, in addition to promoting mouse lethality, pyrogenicity, bone marrow necrosis, and Limulus gelation (Luderitz et al., 1973). From these results it was concluded that lipid A exists as the “biologically active center” in LPS. This is further substantiated by results of the effects of synthetic lipid A, which is free of contaminating bacterial components yet retains biological activity (Rietschel et al., 1987). It appears that the polysaccharide section acts to solubilize lipid A in the intact molecule, and polysaccharide can be replaced by serum albumin (Luderitz et al., 1973). Removal of fatty acids (Nowotny, 1987), alkylation (Chedid et af., 1975), or dephosphorylation(monophosphoryllipid A) (Johnson et al., 1987) can lead to detoxification of lipid A, in which the altered molecule retains many of its beneficial aspects (Nowotny, 1987). Lipopolysaccharide can act as an adjuvant, is mitogenic for B cells, and is considered a polyclonal B-cell activator. These characteristics are also attributable to lipid A (Chiller et al., 1973). Another effect of LPS is the Shwartzman phenomenon, which may be considered a type of disseminated intravascular coagulation (DIC) (Jawetz et af., 1984). In this reaction, an animal receives an intradermal injection of endotoxin and the following day receives endotoxin intravenously. Necrosis of the initial skin site occurs in a few hours. When endotoxin is given intravenously on each of 2 successive days, DIC occurs. The initial dose of endotoxin can be replaced by carbon particles or other material that has the effect of obstructing the reticuloendothelial system. The effectiveness of the Shwartzman reaction

54

R.J. DOYLE AND E.M.SONNENFELD

is proportional to the quantity of lipid A in the sensitizing substance (Ohta et al., 1985). Responses of individual types of cells to LPS are varied. The LPS may bind to platelets via its lipid A (Morrison and Oades, 1979) and cause aggregation and release of mediators, such as 5-hydroxytryptamine (Des Prez rt al., 1961). The lytic response appears t o be dependent on the alternate pathway of complement (Momson and Oades, 1979). The mitogenic response of lymphocytes to LPS seems to occur after its administration in vivu or following its mixture with cells in v i m . The B-cell response, limited to mice, results in the synthesis and secretion of immunoglobulins (Ig) from immunocompetent cells. The mitogenic activity of LPS, along with the T-cell mitogenic activities of concanavalin A and other lectins, led t o a much better understanding of the roles of lymphocytes and plasma cells in immunity. Behling et al. (1976) found that when fatty acids were substituted on the amino group of glucosamine, the resulting glycolipids were potent B-cell mitogens, suggesting that the lipid A moiety of LPS is responsible for the mitogenic effects. Treatment with LPS results in alterations in the metabolism of cells, including a decrease in activity of mitochondria1 dehydrogenases and an increase in the incorporation of thymidine, uptake of glucose, formation of lactic acid, and activity of lysosomal hydrolases (Bradley, 1979). Injected into experimental animals, LPS can induce a myriad of nonspecific effects, including fever, hypotension, aberrations in white blood cell counts, DIC, irreversible shock, and death (Rietschel et al., 1982). In contrast, the host may actually require the continual exposure to LPS in the gut for proper maturation of the immune system, while from the bacterium’s point of view, LPS is a protective outer coating, aiding in its ability to resist phagocytosis (Rietschel et al., 1982). Lipopolysaccharide molecules appear t o have several effects on polymorphonuclear leukocytes (PMN). Injection of LPS causes a leukopenia followed by a leukocytosis (Mechanic et al., 1962). The leukopenia may depend on the presence of lipid A (Corrigan and Bell, 1971). The LPS of rhizobia plays a significant role in their ability to interact with legumes during nitrogen fixation. The association is through the 0 polysaccharide and the lectin of the legume. It is selective for each species of nitrogen-fixing rhizobia-legume pair that is found to enter into a symbiotic relationship in nature, and it explains, at least in part, the high degree of specificity exhibited by this relationship (Wolpert and Albersheim, 1976). The LTA molecule (Fig. 2) contains a highly polar poly(glycero1 phosphate) chain and a hydrophobic end, consisting primarily of fatty-acid esters. The fatty-acid composition of an LTA molecule generally reflects

BACTERIAL CELL SURFACE

55

the fatty-acid composition of the bacterial membrane, as shown for analyses of several preparations from different genera. Probably the most useful method for LTA preparation involves extracting a bacterial suspension with 44% phenol at 62"-65"C (Fischer et d . , 1983). A single phase forms at the elevated temperatures. Upon cooling, the aqueous and phenol phases separate and the LTA can be recovered from the aqueous layer. Prepared in this way, LTA is contaminated with nucleic acids, protein, and in certain cases, polysaccharide. Nucleases and proteases can be used to degrade the contaminating materials. Finally, the highly purified LTA is obtained by gel exclusion chromatography of the digest on crosslinked agarose. Silvestri et al. (1978) found that small quantities of LTA could be obtained in highly purified form by extraction with liposomes. An especially promising method for LTA purification has been described by Josephson et al. (1986). After producing LTA from phenolwater extracts of cells, it was purified using a salt gradient elution from DEAE-cellulose. Prepared in this manner, LTA resolved into two main components that differed in fatty-acid content. In order to study the biological properties of LTA, it is necessary to be able to identify and remove potential contaminants. Table VI summarizes some of the reported effects of LTA on biological systems. (Another section details the role of the LTA in adhesion of gram-positive cocci to mucosa.) It is interesting that many of the same effects have been observed for LPS molecules as well. Both types of molecules are amphiphiles. Both have long-chain hydrophilic repeat units, TABLE VI SUMMARY OF SOMEREPORTED BIOLOGICAL PROPERTIES OF BACTERIAL LTA Property

Reference or review paper

Adhere to red cells and other cell types Complex with M protein of streptococci Mitogenic Antigenic Activates macrophages Shwartzman reaction Activates complement Complexes with FN Inhibits streptococcal glucosyltransferase Inhibits autolysins of bacteria Stimulates bone resorption Stimulates nonspecific immunity Solubilizes glucans in alcohol Gelation of Limulus lysate

Hewett et a / . (1970) Ofek et a / . (1982) Mishell et a / . (1981) Wicken and Knox (1975) Wicken and Knox (1980) Wicken and Knox (1980) Fiedel and Jackson (1981) Courtney et a/. (1983) Kuramitsu er a / . (1980) Cleveland et a / . (1975) Wicken and Knox (1980) Wicken and Knox (1980) Cowan et a/. (1988) Kessler (1983)

56

R.J. DOYLE AND E.M. SONNENFELD

and both have hydrophobic ends. Both types of molecules form micellar aggregates and both bind to various kinds of membranes. The reported toxicities of LPS, however, are far greater than those generally acknowledged for LTA. The mitogenic effects of LTA have been studied by several investigators. Mishell et al. (1981) have reported that LTA preparations are B-cell mitogens. Beachey et a / . (1979) previously had reported that LTA was a weak T-cell mitogen. Earlier, Miller et al. (1976) could find no evidence for LTA-induced mitogenic effects. Hamada et al. (1985) have provided strong evidence to show that LTA preparations from several streptococci were good nonspecific B-cell mitogens of murine spleen cell lymphocytes. Mouse thymocytes were not activated by the LTA. In addition, deacylated LTA was a far weaker mitogen than the fully acylated preparations (Hamada et al., 1985). Most of the LTA preparations employed by Hamada et al. were effective mitogens at 0.1-10 pglml. Some of the discrepancies in results for LTA-induced mitogenicity may be due to lack of knowledge of state of purity, extent of acylation, or different concentrations employed by various investigators. Lipoteichoic acid may have a dual effect on immunocompetent cells. Miller and Jackson (1973) found that LTA (and deacylated LTA) from pyogenic streptococci reduced the B-cell response as measured by Ab production to sheep RBC. Miller ef al. (1976) later observed that the LTA could potentiate Ab production against an LPS Ag. Hamada et al. (1985) found that when LTA and sheep RBC were injected simultaneously via the intravenous route, an enhanced immune response occurred. Similarly, both materials added to cell cultures produced an elevated response against the sheep cells. There may be effects of LTA that are as yet poorly understood in terms of immune regulation. A proper balance of immune-cell activation and suppression may be required to mount a normal immune response against bacterial pathogens. It has been known for some time that LTA could initiate the enzymatic events leading to gelation of the Limulus amebocyte lysate. Kessler (1983) studied the structural requirements of LTA for eliciting the gelation reactions. Lipoteichoic acid molecules with shortened poly(glycero1 phosphate) chains were superior to longer chain LTA. In addition, substitution of the glycerol residues by D-Ala or by saccharides resulted in preparations with higher specific activities. The results seem to suggest that hydrophobic LTA are superior to relatively hydrophilic LTA in initiating the gelation reactions. Because the LTA is much less effective, compared to LPS molecules, in promoting the lysate gelation (Kessler, 1983), clinical usage of the reaction has been limited in detecting gram-positive infections or contaminants.

BACTERIAL CELL SURFACE

57

The fatty-acid groups of LTA are also required to sensitize RBC to agglutination by anti-poly(glycero1 phosphate) (Hamada et al., 1985). There has been some degree of controversy surrounding the binding of LTA by RBC. Chorpenning et al. (1979) and Cooper et al. (1978) concluded that lipid-free teichoic acids could readily bind to RBC membranes. Results of Hamada et al. (1979, 1985) and Chiang et al. (1979) support the view that lipid acyl groups are required for the spontaneous association between the RBC membranes and teichoic acid preparations. Chiang et al. (1979), Hewett et al. (1970), and Ofek et al. (1975) have noted that deacylated LTA molecules seem to possess little affinity for animal cell membranes. It is unlikely that the membranes possess specific LTA-binding proteins. Present results suggest that the lipid moiety of the LTA can bind directly to hydrophobic sites in the membranes, causing the poly(glycero1phosphate) chains to protrude outward, where they may interact with Ab or lectins. Lipoteichoic acid has been reported to be a regulator of autolytic activity for several gram-positive bacteria. Cleveland et al. (1975) were the first to show that an LTA preparation could inhibit a bacterial autolytic enzyme. Autolysins from Staphylococcus aureus (Suginaka et al., 1979), Streptococcus pneumoniae (Holtje and Tomasz, 1975), Streptococcus faecalis (Cleveland et al., 1976), and B. subtilis (Rogers et al., 1984) have been reported to be inhibited by LTA preparations. The results of the foregoing researchers were obtained by use of in vitro measurements. How LTA could regulate an autolysin in vivo is unknown. The lipid moiety seems to be a structural requirement for inhibition of autolysins in vitro. Autolysis of whole cells and cell wall turnover seem to be good indicators of autolytic activity in vitro. When Jolliffe et al. (1981) added LTA to growing cultures of B. subtilis, there was no reduction in the rate of cell wall turnover, suggesting that LTA was not interacting with the autolysins. Furthermore, although susceptibility of B. subtilis to nafcillin is proportional to cell wall turnover, there was no effect of LTA on the bacteria when mixed with the antibiotic (Jolliffe et al., 1982). If LTA interacts with autolysins in vivo to regulate autolytic activity somehow, it would be expected that the LTA could dissociate from the enzyme. To date, however, association-dissociation equilibria or kinetics have not been described for LTA-autolysin interactions. Cowan et al. (1988) observed that various LTA preparations could promote the solubilization of D-glucans in concentrated ethanol solutions. A few micrograms of LTA could form alcohol-soluble complexes with nearly 1 mg of glucan. The glucan is normally insoluble in the alcohol, but LTA prevents its precipitation. The only structural requirement for the solubilization reaction is the poly(glycero1phosphate) chain (Doyle et

58

R.J. DOYLE AND E.M.SONNENFELD

al., 1975). Lipid-free teichoic acids also form alcohol-soluble complexes with polysaccharides. Interestingly, Cowan et al. observed that cell-free whole saliva (but not parotid saliva) could also promote the solubilization reaction, using a high molecular weight a4,6 glucan as the polysaccharide. The salivary component capable of complexing with the glucan was microbially derived LTA. When whole saliva was treated with sucrose or penicillin, LTA (or deacylated LTA) was released from the normal microbiota, thereby enhancing the glucan solubilization by the saliva. Furthermore, when saliva was added to a Sephacryl column equilibrated with 80% ethanol, LTA and a 60-kDa protein were retained, only to be eluted by water. It appears that the LTA in saliva not only binds glucans in ethanol, but at least one salivary protein as well. Parrish and Doyle (unpublished observations) have used the alcohol-Sephacryl-water elution system to purify LTA from Streptococcus sobrinus and B . subtilis. The biological significance of LTA-glucan or LTA-protein complexes in alcohols is unknown, but it may reflect interactions between LTA and hydrophilic molecules in hydrophobic milieus. Cowan et al. (1988) suggested that these kinds of interactions may be important in the formation of dental plaques. Macromolecular complex formation, aided by stabilizing hydrophobic interactions, has been considered a driving force in the adhesion of oral streptococci to teeth (Doyle et al., 1982).

V. Surface Adhesins of Bacteria and Pathogenesis Most bacterial diseases require that the bacteria colonize a site in a host so that a sufficiently large number of cells be available to discharge toxic materials or induce inflammatory responses. The colonization must be preceded by adhesive events before bacterial mass can increase significantly. Sometimes the adhesion of bacteria to host cells involves only a few bacteria. Once adherent, the bacteria can then undergo cell division. There are various surfaces in the human body onto which bacteria may adhere. These include keratinized and nonkeratinized epithelial cells, endothelia, bone, and saliva-coated teeth. Many bacteria adhere to all of these surfaces, but may be found only in certain parts of the body. An E. coli for example, would normally be considered as a bacterium residing near or on gut mucosa, but the organism can adhere to many types of cells. An understanding of the apparent tropism of E. coli for gut mucosa must take into account other factors, such as nutrient supply, temperature, and host defense molecules (e.g., lysozyme, Ig). Adhesion of a bacterium to a substratum requires that the bacterium approach a surface closely enough so that complementary molecules can

BACTERIAL CELL SURFACE

59

form a complex (Figs. 15 and 16). Most bacteria are highly negatively charged on the cell surfaces, but they also possess clusters of positive charges on their surfaces. In addition, most bacteria possess hydrophobic molecules (hydrophobins) near or on their cell walls. The presence of oppositely charged groups and hydrophobins make it possible for two highly negative surfaces to form a stable union. Oral streptococci, for example, adhere avidly to negatively charged pellicle proteins even though the bacteria have net negative surface charges. For most pathogenic bacteria, adhesion to some kind of surface is an ecological advantage. Adhesion provides a means for the bacteria to be more effective in obtaining nutrients. Escherichiu coli bound to tissue culture cells is able to divide more rapidly than nonadherent E. coli and exhibits a shorter lag time in division than nonadherent cells (Zafriri et al., 1987). Various surface components of a bacterium may participate in adhesion (Tables I, VII). Fimbriae, for example, possess lectins or lectinlike proWEAK ASSOCIATION (REVERSIBLE 1

u u

%8Q

K,

K-I

COLONIZATION

\ IN VAS I0N

FIG. 15. Steps leading to firm adhesion of a bacterium to a substratum. The bacterium initially approaches the substratum and is bound loosely with only a few reversible interactions ( K ,rate). The bacterial ligands may subsequently combine with complementary receptors, creating an “irreversible” adhesion ( K , rate). The rate constant K.? is very low, showing that dissociation is a critical step in the formation of a stable cell-substratum union. Once firmly adherent, the bacteria may divide (colonize) and may produce symptoms of disease (invasion). (See also Gristina et a / . , 1985; Gristina, 1987).

60

R.J. DOYLE AND E.M. SONNENFELD

111

e-

*

BACTERIA

Ill

BLOOD FLOW CILIARY ACTION COUGHING DES Q UA M AT I 0N EXCRETION PERISTALSIS SECRETIONS SNEEZING

-

Ill AIR,FLUID FLOW

UNCOLONIZED MUCOSA

C0LONIZ E D MUCOSA

FIG. 16. Factors involved in the adhesion of bacteria to mucosal surfaces. Even if the bacterium can interact with complementary receptors on the mucosa, assurance of adhesion may not be realized because of air or fluid flow. Fluids may contain antibodies, complement, lysozyme. lactofenin, and other agents that could interfere with adhesion to epithelial or endothelial surfaces. Addition of sugars or saccharides to the system may also block adhesion. Adapted from Ofek and Beachey (1980b).

teins that enable the bacteria to adhere to carbohydrates on substrata. The specificities of some of the fimbrial and surface lectins are reviewed in Table VIII. Type I fimbriae of the Enterobacteriaceae complex with mannose (Man) or Man derivatives, whereas type P fimbriae bind to Galcrl-4Gal residues. Other fimbriae may interact with GlcNAc, sialic acids, or Gal-containing structures. Nonfimbrial proteins also possess lectin activities. Streptoeoccrrs cricetirs binds a-1.6-linked isomaltooctaosesdecaoses (Drake et al., 1988a,b). In some cases, LTA and surface proteins contribute to adhesion. Secreted polysaccharides may also play a role in the tethering of some pathogens to substrata. The importance of adhesion to infectivity is shown in Table IX. Gramnegative bacteria possessing fimbriae are more infectious than their nonfimbriated variants. In most of these cases, the fimbriae bear lectins specific for sugars or saccharides. In E. coli, the most widely studied fimbriae are specific for Man and Galal-4 Gal. If fimbriae are involved in virulence, then carbohydrates complementary for the lectins may prevent infections. Results from several laboratories are summarized in Table X showing that carbohydrates can reduce experimental infections due to E.

61

BACTERIALCELLSURFACE TABLE VII SURFACE ADHESINS OF SELECTED BACTERIAL PATHOGENS Bacterium Staphylococcus aureus

Streptococcus pyogenes

Adhesin(s) Wall teichoic acid Surface protein Hydrophobins M protein LTA

Streptococcus agalactiae

Streptococcws pneumoniae Streptococcus sanguis

Staphylococcus suprophyricus Staphylococcus epidermidis

Escherichia coli

LTA, with surface proteins LTA Surface protein Lectin Hydrophobins and pol yanions LTA Lectin Extracellular polysaccharide (PCHO) H ydrophobins Type 1 (fimbriae) Type P (fimbriae)

Reference Aly and Levit (1987) Kuusela (1978); Ryden et ul. ( 1983) Mamo er a / . (1987) Tylewska et a / . (1988); Ellen and Gibbons (1972) Beachey and Ofek (1976); Ofek et a/. (1975) Jacques and Costerton (1987) Teti ef a/. (1987); Nealon and Mattingly (1984) Miyazaki et a/. (1988) Anderson et al. (1983; 1986) Nesbitt et a / . (1982); Cowan er a/. (1986a,b); Busscher and Weerkamp (1987) Teti e f a / . (1987) Gunnarson et a / . (1984) Christensen et a / . (1982)

Hogt et a / . (1983) Mirelman and Ofek (1986) Lefler and Svanborg-Eden (1980)

coli, Klebsiella pneumwiae, and Sh. flexneri. The presence of Man or a mannoside prevented the infections associated with E. coli bearing type 1 fimbriae (see also Table XI). The premise that infections may be prevented or inhibited by carbohydrates is attractive (Fig. 17). To date, however, practical considerations have precluded any significant clinical tests. For example, Man added to fluids for intake would be rapidly metabolized or excreted in the human. Some saccharides may be toxic, whereas others are not available in sufficient quantities to institute appropriate human experimentation. Another avenue for prophylaxis may take advantage of the immune response against fimbriae. Several reports have shown that Ab directed against fimbriae afford protection against infection in various experimental animals (Table XII). Possibly, antilectin immune responses are responsible for the observed protection, although this is not known with certainty. The lectin molecule is thought to reside at the very tip of the fimbrium and to comprise only a small percentage of fimbrial protein (DeGraaf and Mooi, 1986). Most antifimbrial Ab would

TABLE VlII LECTINS OF BACTERIAL PATHOGENS"

SPECIFICITIES OF S U R F A C E

Sugar or saccharide

Bacterium

N - Acet yllactosamine

Leptotrichia buccalis. Eikenella corrodens Pasteurella multocida, Escherichia coli, Chlamydia trachomatis Vibrio cholerae E. coli Staphylococcus saprophyticus Actinomyces spp. E. coli Neisseria gonorrhoeae Streptococcus pneumoniae Bartonella bacilliformis Streptococcus cricetrrs E. coli Bacteroides spp., Mvcoplnsma pneumoniae E. coli

N-Acet y Igl ucosamine L-Fucose Gala( 1-4)Gal GalP( 1-4)GlcNAc P-Galactosides Gal, Galp( 1-3)Gal Gal-GalN Ac-Gal GlcNAcP( 1-3)Gal D-Glucose lsomaltooctaose-isomaltodecaose Man and mannosides Sialic acid Sialic acid-Gal

"Derived from Drake er a/. (1988a): Mirelman and Ofek (1986): Wadstrorn and Tmst (1984): Beutheral. (1987j:Gunnarsonef nl. (1984); Uhlenbruck.(l987); Holrngren cr nl. 11983): and Ofek er a/. (1917).

TABLE IX ADHESION OF GRAM-NEGATIVE BACTERIAin Relative adhesion Bucterium

in vitro

ViWO AND

INFECTIVITY"

Relative infectivity

Bacterial variants ~

~~

Poor

High Low

Fimbriate Nonfimbriate

Escherichia coli

Good Poor

High Low

K88 * K88-

E. coli

Good Poor

High Low

CFA' CFA-

Neisseria gonorrhoeae

Good Poor

High Low

Fimbriate Nonfimbriate

Proteus mirabilis

Good Poor

High Low

Fimbriate Nonfimbriate

Salmonella typhimrrrium

Good Poor

High Low

Fimbriate Nonfimbriate

Border ella pertussis

(enterotoxigenic strain)

Good

"Adapted from Beachey cr ol. (1982).

62

TABLE X PREVENTION OF INFECTIONS BY FIMBRIAL LECTININHIBITORS Bacterium Escherichia coli (type P fimbriae)

Animal and site of infection"

Inhibitof

Reference

Mouse, UT

Globotetraose

Leffler and SvanborgEden ( 1980) Roberts et a / . (I 984)

Monkey, UT

Gala4GalpOMe

E. coli (type I fimbriae)

Mouse, UT

MeaMan

Aronson et a / . ( 1979)

E. coli (type I fimbriae)

Mouse, GI

Man

Goldhar et a / . ( 1986)

Shigella jlexneri (type I fimbriae)

Guinea pig, eye

Man

Andrade (1980)

Klebsiella pneumoniae (type I fimbriae)

Rat, UT

MeaMan

Fader and Davis (1980)

"UT. Urinary tract; GI, gastrointestinal tract. *Me. Methyl.

TABLE XI OF INHIBITION OF ADHESIONBY SUGARS OR SACCHARIDESAND MODIFICATION BACTERIALINFECTION'

Tissue

Number of animals

Escherichia coli E. coli

Mouse bladder Mouse bladder

101

E. coli

Mouse bladder

35

Klebsiella pneumoniae K . pneumoniae

Rat bladder Rat bladder

5 5

K . pneumoniae Shigella jlexneri

Rat bladder Guinea pig eye Guinea pig eye

5 20

Bacterium

Sh. j7exneri

99

20

Inhibitor None Methyl-a-w mannoside Methyl-a-Dglucoside None Methyl-a-Dmannoside D-G~UCOSe D-Man (right eye) D-Glucose (left eye)

Percentage colonized or infected

11 22

64 80 20 100

20 70

"Results derived from Andrade (1980); Aronson et a / . , (1979); and Fader and Davis (1980). All the bacteria possessed type 1 fimbriae.

63

64

R.J. DOYLE AND E.M. SONNENFELD

A

Bacterium

FIG. 17. Blocking of bacterial adhesion by adhesin or receptor analogs.

therefore be expected to be directed against nonlectin protein. The use of fimbriae or fimbrial lectin as a vaccine against various infectious agents holds promise. It will probably be necessary t o clone the fimbrial lectin before enough of the protein is available for use as a vaccine. Bacteria, like all life forms, have a tendency to adapt to their environment. There are many examples to show that bacteria must face changing environments, however. A Pseudomonas aeruginosa, normally an inhabitant of plants, may find itself occasionally (in evolutionary history) on an animal cell surface. The Pseudomonas may require different surface structures to adapt from plant to animal. Similarly, an E. coli residing near gut mucosa, rich in Man-containing proteins, characteristically possesses receptors for type 1 fimbriae. On the other hand, bladder and kidney tissue is rich in glycolipids containing the Galcx1-4Gal sequence. It is not surprising therefore that most gut-associated E. coli express type 1 fimbriae and most genitourinary tract E. coli express type P fimbriae. It is as if the E. coli has evolved a means to sense that an environmental change may occur. It now seems clear that the phenomenon of phase

PROTECTION AGAINST Animal

TABLE XI1 Escherichiu co/i PROVIDED

INFECTION WITH

Site“

Fimbriae

Rats Rats Pigs Monkeys

UT GI GI UT

Type Type Type Type

Mice

UT

Type P

Pigs

GI

Type K99

“UT. Urinary tract: GI, gastrointestinal tract

1 1

I

P

BY

ANTIFIMBRIAL AB Reference

Silverblatt and Cohen (1979) Guerina er a / . (1983) Jayappa er a / . (1985) Kaack er a / . (1988); Roberts er a / . (1984) O’Hanley er al. (1985); Schmidt et al. (1988) Isaacson et al. (1980)

65

BACTERIAL CELL SURFACE

transition (Fig. 18) occurs in order for a bacterium to diversify its habitat. Phase transition (Eisenstein, 1981) reflects a change in the expression of a surface component (lectin, etc.) at a predictable rate. Phase transitions seem to occur at the rate of 1 in 102-105,much higher than for a mutation. A phase variant would give rise to a new phase variant at the same rate. Thus, E. coli possessing Man-sensitive (MS) lectins give rise to variants that are devoid of the lectins, but these variants later give rise to MS' phenotypes. Phase transitions occur regularly and often independently of growth conditions. Table VII shows that several bacteria have been reported to adhere to surfaces by multiple mechanisms. Some bacteria, such as Staphylococcus epidermidis, can be found associated with various environments. The presence of S . epidermidis adherent to catheters, implants, and so on, may be a result of the adhesion of a phase variant producing an exopolysaccharide (Christensen et al., 1982, 1987). Most clinical isolates of S . epidermidis do not produce exopolysaccharide, but polysaccharide-producing variants seem to colonize biomaterials selectively (Hogt et al., 1983, 1986).

0 000

0 1

0

GENERATIONS

FIG. 18. Phase transitions in bacteria. In this example, a bacterium possessing an MS lectin undergoes several divisions, and a Man-resistant phenotype ultimately appears. The Man-resistant phenotype may undergo several divisions before it produces a MS phenotype. These kinds of transitions are not mutations, but occur in -1 of 10'-105 cells.

66

R.J. DOYLE AND E.M. SONNENFELD

The view that multiple adhesins may be involved in microbial adhesion is shown in the literature on P . aeruginosa (Table XIII). Various adhesins have been ascribed to the bacterium, including MS type I fimbriae, hydrophobins, exopolysaccharides, sialic acid-specific lectins, GlcNAc-specific lectins, N-acetylmannosamine-bindinglectins, and recently, ganglioside-specific proteins. It is unlikely that phase variation could account for the reported diversity of adhesins for P . aeruginosa. Garber et al. (1985) reported that several isolates containing various surface characteristics (mucoid, rough, etc) were devoid of Man-specific lectin activities. In agglutination reactions using intact P . aeruginosa and papain-treated RBC, no sugars or saccharides were found capable of hemagglutination inhibition. Reagents such as tryptophan and p-nitrophenol were effective inhibitors of hemagglutination, prompting Garber ef al. to conclude that hydrophobic interactions were critical in adhesion. Pseudomonas aeruginosa is known to have the ability to survive under nutrient limitations in a variety of habitats. It may be that the members of the presently defined TABLE XI11 SOMEPROPOSED MECHANISMS FOR THE ADHESIONOF P. ueruginosa ANIMALCELLSO Mechanism or observation Protein-protein complex is enhanced by removal of FN by trypsin. Adhesion of the bacterium may occur more effectively on injured epithelia than on normal epithelia. Adhesin is specific for sialic acid on cellular surfaces or in mucins. Pseudomonas aeruginosa adheres better to injured tracheal cells than to normal tracheal cells. Adhesion to ocular tissue is dependent on pili. Mucins act as receptors for the bacteria. Possible involvement of sialic acid or GlcNAc. An adhesin is specific for Man. Free pili inhibit adhesion to injured tracheal epithelia. Hydrophobic interactions promote hemagglutination Bacterial hydrophobicity appears to be unrelated to adhesion to tracheal epithelial cells of mink Extracellular factors in growth medium enhance adhesion of P. ueruginosa. Mucoid exopolysaccharide is thought to be adhesin for

TO

References Woods e/ a / . (1981) Stern e/ a / . (1982) Ramphal and Pyle (1983a) Ramphal and Pyle (1983b) Uhlenbruck ef a / . (1983) Vishwanath and Ramphal (1985) Speert e/ a / . (1984) Rarnphal el a / . (1984) Garber e / a / . (1985) Elsheikh et a / . (1985) Ogaard et a / . (1985) Ramphal and Pier (1985)

P. aeruginosa.

Multiple adhesins may be involved in attachment of P. aeruginosa to buccal cells. Hydrophobic interactions may mediate nonopsonic phagocytosis.

McEachran and Irvin (1985) Speerl et (it. ( 1986)

67

BACTERIAL CELL SURFACE

TABLE XI11 (conf.) Mechanism or observation Sialic acid-containing receptors may be present in ocular epithelia. Hydrophobic interactions may take place via lipidbinding adhesin. Exopolysaccharide of the bacterium may complex with mucins of host. Adhesins capable of binding to oral bacteria may also bind to animal cell surface. The bacterium appears to bind via its cell pole area to human cilia. Multivalent alginate from the bacterium agglutinated both human buccal cells and tracheal epithelial cells. Sialic acid may be a receptor for P. aeruginosa. Corneal ulceration associated with soft contact lenses may depend on polysaccharide-mediated adhesion of P. aeruginosa to the contact lens. Lectins specific for sialic acids attach the bacteria to various cell types. N-Acetylmannosamine may be an ocular receptor for P .

References Hazlett e t a / . (1986) Ramphal et a/.(1986) Ramphal et a/. (1987) Komiyama et a/. (1987a) Franklin et al. (1987) Doig et a/. (1987) Komiyama et a / . (1987b) Slusher et a/. (1987) KO et a / . ( I 987) Hazlett et a/. (1987)

aeruginosa.

Pseudomonas aeruginosa adhesion to contact lenses was enhanced by mucins and several proteins. The bacterium may bind to gangliotetraosylceramide or gangliotriaosylceramide. _____

~

~~

Miller ef a/. (1988) Krivan et at. (1988) ~~

~

“Stanley (1983) also has provided evidence to suggest that P. aeruginosa adheres to inanimate surfaces by multiple mechanisms.

species of P . aeruginosa possess unique adhesins, making the species more heterogeneous than heretofore thought. In gram-positive bacteria, adhesion has been extensively studied in the genera Streptococcus and Staphylococcus. Lectins are not nearly as prominent in gram-positive bacteria as they are in the gram-negative prokaryotes. Some oral streptococci have been reported to possess glucanbinding lectins (Drake et al., 1988a,b; McCabe et al., 1977; Gibbons and Fitzgerald, 1969). Staphylococcus suprophyticus possesses a surface lectin specific for Ga@(1-4)GlcNAc residues (Gunnarson et al., 1984). Members of the genus Actinomyces possess Gal- and GalNAc-specific surface lectins (Cisar, 1986). Drake et al. (1988a) reported that the glucan-binding lectin of S . cricetus was specific for only glucans containing high contents of a-l,6 anomeric linkages (Table XIV). Furthermore, when inhibition studies were performed, it was observed that the combining site of the lectin optimally accommodated 8-10 hexose residues (Fig. 19 and 20).

68

R.J. DOYLE AND E.M. SONNENFELD TABLE XIV a-1.6 GLUCOSIDIC BONDI N T H E AGGLUTINATIONOF S~reproeorciiscriretits A H T " ROLE OF THE

Glucan

a-I ,6 (%)

B- I208 B- 1225 B- 1255 B- I298 8-742 B- I299 B-I35S(s)

95 90 82

Other linkages (9%)

Decrease in absorbance (95)

5

76 68 71 16

10

64 57

18 36 43

50

so

45

55

0

0 I

"Reprinted courtesy of the authors (Drake P I 01.. 1988a) and the American Society for Microbiology. "Glucans with known molar percent anomeric linkages were used to determine the specificity of the glucan-binding lectin. GBL. Each glucan was prepared in PBS at a final assay concentration of 10 &ml. Glucans such as lichenan ((al,4):, a-1.31. pullulan (a-1.4.a-1.6).amylose (a-1.4).laminarin (p-1.3, p-1.6). rnaltoheptaose (a-1.4). glycogen (a-1.4, a-1.6).along with carbohydrates such as glucose. maltose. isomaltose. isomaltopentaose. isomaltooctaose. nigeran (a1.3. a-I.41.and fructose were incapable of promoting agglutination.

z 0

!c3z

W

U

c3

(3

a

LL

0

z

E! b-

30 20 10

40

0.I

MALTOHEPTAOSE

02

0.3 0.4

c 0.6

0.8

1.0

2.0

!

3.0 4.0

!

:! ! A 1 6.0 8.0 10.0

GLUCAN (rng/rnl) FIG. 19. Inhibition of glucan T2000-mediated aggregation of Streptocorcrrs cricerus by isomaltosaccharides. Suspensions of S. crirerris AHT were incubated with the prospective inhibitors and assayed for glucan T2000-induced aggregation. IM-8, lsomaltooctaose; 1M-6, isomaltohexaose; IM-5, isomaltopentaose. Reprinted courtesy of the authors (Drake ei d., 1988a) and the American Society for Microbiology.

BACTERIAL CELL SURFACE

3.0

-

2.5

-

E 2.0

-

p

69

-0

e .-

-u"\

=r

1.5

0 .c

-

0

E 1.0 L

0 0

5

0 0.5 c

-

\ \

0

0

a

b

\

0 ~ ' " ' ' ' " " ' 0

I

2 3

4 5

\

6 7 8 9 10 I I 12 13 14 15 16 17 18 19 20

55

GLUCOSE RESIDUES FIG. 20. Glucan concentration required for 50% inhibition of glucan T2000-mediated agglutination of Streptococcus cricetus. Inhibition data obtained with glucan TI0 and the isomaltosaccharides were plotted in terms of the concentration of inhibitor needed to achieve a 50% level of inhibition versus the number of glucose residues of each inhibitor. Note that isomaltotriose (date not plotted) essentially represents infinity as no inhibition was observed even at concentrations s 3 3 mg/ml. Reprinted courtesy of the authors (Drake et a / . , 1988a) and the American Society for Microbiology.

This is an unusual combining site as most lectins bind monosaccharides or disaccharides (Goldstein and Hayes, 1978). Saccharides such as maltoheptaose were without effect on lectin activity. Streptococcus cricetus is one of the "mutans" streptococci and may be involved in dental caries. The glucan-binding lectin may enable the bacteria to adhere to tooth surfaces and subsequently initiate dental caries. It is known that sucroseconsuming populations demonstrate higher canes rates than non-sucroseconsuming populations. Sucrose is metabolized directly by extracellular glucosyltransferases to yield a-l,6glucans and fructose. The human diet is usually rich in a-1,4glucans (starches) but poor in a-1,6glucans, so it seems unlikely that most foodstuffs could inhibit colonization of the streptococci. Colonized streptococci could more effectively trap nutrients and could more effectively withstand low concentrations of Ab or lysozyme or other antibacterial agents.

70

R.J. DOYLE AND E.M. SONNENFELD

The adhesion of pyogenic cocci to mucosa appears to involve multiple mechanisms (Table VII). Streptococcus pyogenes has several components on its cell surface (Fig. 21) that could participate in adhesion reactions. The normal habitat for pyogenic cocci is an animal host. In humans, the bacteria frequently colonize the throat and mucus membranes of the upper respiratory tract and are transmitted by droplets, direct contact, fom-ites, saliva, and so on. The carrier state involves the successful colonization of the bacteria without the appearance of symptoms. Streptococcus pyogenes appears to adhere to mucosa via a bridging mechanism involving fibronectin (FN) (Fig. 22). It is known that LTA molecules can form complexes with M protein via interactions between the poly(glycerol phosphate) and lysine residues on the protein (Ofek et al., 1982).This leaves the hydrophobic end of the LTA exposed to solvent and available for interaction with other proteins or amphiphiles. The F N molecule is thought to be able to bind the hydrophobic residues of the LTA near the amino terminus of the protein. The carboxy terminus, in contrast, anchors the FN molecule to the mucosal surface. The net result is that FN can tether the bacterium to the cell surface utilizing LTA as a ligand, so in reality, both LTA and FN serve as bridging molecules. Table XV reviews some of the evidence for involvement of both LTA and FN in the adhesion reactions. Antibodies against either LTA or FN reduce streptococcal adhesion. Loss of LTA from the bacterial surface also results in reduced adhesion. Tissue culture cells producing relatively low quantities of FN form relatively poor substrata for the streptococcal adhesion. The deacylated form of LTA does not serve as an inhibitor of adhesion, whereas hydrophobin-binding molecules such as serum albumin are good inhibitors. Furthermore, soluble LTA prevents the binding of soluble FN to S. pyogenes. The collective evidence is that both LTA and P E PT ID OG LY C A N HYALURONIC ACID L

M-PROTEIN

-

MEMBRANE

.GROUP SPECIFIC PO LY S A CCH A R IDE

FIG.21. Surface structures of Sfreptococcus pyogenes.

71

BACTERIAL CELL SURFACE

-PLASMA PEPTIDOGLYCAN

LlPOTElCHOlC M-PROTEIN

(m = FATTY ACID 1 EPITHELIAL

~~,

FIG.22. Adhesion of Streptococcus pyogenes depends on an FN bridge. The organism may secrete LTA during growth. The LTA binds to M protein via protein-glycerol phosphate interactions, leaving a solvent-exposed hydrophobic end. This hydrophobic end binds to complementary sites on the FN molecule. The carboxy-terminal amino acids of FN bind strongly to epithelial cells. The net result is that the bacteria can be specifically tethered to the epithelial cells via an FN bridge. The model requires participation of wall matrix, LTA, M protein, and FN. The M protein may contain a segment that interacts with the PG (Pancholi and Fischetti, 1988). The main features of the model were derived from Beachey and Ofek (1976), Beachey et a/. (1983); Courtney e t a / . (1986); and Ofek et a/. (1982).

TABLE XV CONTRIBUTIONS OF LTA AND FN TO THE ADHESIONOF Streptococcus pyogenes TO MUCOSA" LTA

FN

Soluble LTA prevents bacterial adhesion. Deacylated LTA is not an inhibitor of adhesion. Anti-LTA Ab reduces adhesion of S .

Soluble FN inhibits bacterial adhesion. LTA prevents binding of soluble FN to S . pyogenes. Anti-FN Ab reduce adhesion of S.

pyogenes.

Sublethal concentrations of penicillin cause loss of surface LTA from streptococci and result in decrease in adhesion of the bacteria. Molecules that complex with LTA, such as serum albumin, reduce streptococcal adhesion.

pyogenes.

Removal of FN from buccal cells decreases streptococcal adhesion.

Tissue culture cells expressing FN bind streptococci better than cells producing little FN.

"Derived from Beachey (1981): Beachey and Courtney (1987): Beachey et al. (1983): Ofek and Beachey (1980a.b); Ofek et a/. (1982): Simpson et a/. (1980, 1987). and Simpson and Beachey (1983).

72

R.J. DOYLE AND E.M. SONNENFELD

FN contribute to the adhesion of S . pyogenes to mucosa. Other results have suggested that mechanisms in addition to the M protein-LTA-FN interactions may be important in streptococcal adhesion. Tylewska et al. (1988) have suggested that M protein itself may possess lectinlike activity, utilizing fucose or galactose receptors on pharyngeal cells. Streptococcus agalactiae (group B streptococci) is a pathogen for infants and the immunocompromised. There remain several questions about its adhesion to animal cells. Inhibition by anti-LTA, LTA [but not poly(glycero1 phosphate)], and proteases, suggest a surface protein-LTA involvement similar to that of S . pyogenes. The bacterium has a similar surface to S . pyogenes in that it contains solvent-exposed proteins. Mattingly and Johnston (1987), however, showed that LTA is not readily released from the bacterial plasma membrane during growth. Jelinkova et al. (1986) observed that the adhesion of group B streptococci was unrelated to the type-specific protein. Bulgakova et al. (1986) have suggested that capsule may be important in the adhesion of group B streptococci. Some interesting results reported by Cox (1982) provide convincing evidence for a role of LTA in pathogenesis of the group B streptococci. Cox applied LTA to the oral cavity, perineum, and nape of three day-old mice. The mother was then painted with a suspension of S . agalactiae over her nipples. None of the animals receiving the LTA treatment were found to be positive for the streptococci after a 3-day period, whereas 47% of the control, untreated mice were culture-positive. It is difficult to escape the conclusion that LTA is involved in colonization of group B streptococci. Streptococcus pneurnoniae is a normal inhabitant of most humans. The bacterium typically colonizes the upper respiratory tract. The S . pneurnoniae may cause infections of the lungs, throat, ear, and central nervous system. It appears that S . pneurnoniae is one of the few streptococci to possess surface lectins (other than the oral streptococci as discussed earlier). Andersson et al. (1983) found that oligosaccharides bearing Gal-GlcNAc residues were effective inhibitors of adhesion to pharyngeal cells. Beuth et al. (1987) found that GlcNAc-Gal-containing glycolipids would sensitize guinea pig RBC to hemagglutination by S . pneurnoniae. Furthermore, glycolipids in human milk were shown to be effective inhibitors of the bacteria to pharyngeal cells (Andersson et al., 1986), suggesting a role for the glycolipids in natural protection against infection. Like streptococci, staphylococci are normal microbiota of the human. The skin, eyes, intestines, and nasopharyngeal areas provide substrata for the colonization of both S . ai~reiisand S . epidermidis. Staphylococcus aureus is typically a more aggressive pathogen than S . epiderrnidis (Pulverer et cd.. 1987). Most clinical isolates of S . aureus are coagulase-positive, whereas clinical isolates of S . epiderrnidis are coagulase-negative.

BACTERIAL CELL SURFACE

73

Neither S . aureus nor S . epidermidis is known to express surface lectins. Both species of staphylococci have surface proteins and both species possess teichoic acids covalently linked to PG. Staphylococcus aureus clinical isolates frequently express an Ig-binding protein called protein A. Molecules of LTA may also be surface-exposed in S. aureus (Jonsson and Wadstrom, 1984). Staphylococcus aureus has a surface protein that complexes avidly with FN (Froman et al., 1987). Wadstrom et al. (1987) have suggested that the invasiveness of S. aureus clinical isolates is directly proportional to the ability of the bacteria to bind FN. In this regard, Proctor et al. (1983) found that when S . aureus was treated with sublethal concentrations of penicillin, the density of FN-binding sites increased on the bacteria, along with an increased tendency to adhere to FN-coated substrata. Vaudaux et al. (1984) found that anti-FN Ab reduced the adhesion of S . aureus to biomaterial implants. The combined results lead to the conclusion that FN is a receptor for S . aureus. The chemical basis for the recognition of FN by surface protein(s) of S. aureus remains to be worked out. Staphylococcus aureus, similar to other pathogens (Table VII), may also have various means of adhesion. The bacterium is known to bind collagen (Holderbaum et al., 1985), gelatin (Carret et al., 1985), and laminin (Mota et al., 1988). Furthermore, cell wall teichoic acid containing poly(ribito1 phosphate) has been suggested to contribute to the adhesion of S. aureus to nasal epithelial cells (Aly and Levit, 1987). Staphylococcus epidermidis possesses an LTA and a cell wall poly (glycerol phosphate) teichoic acid. The bacterium also expresses surfacebound proteins. The bacterium, unlike S . aureus, does not secrete known toxins. Staphylococcus epidermidis exhibits classical phase variations on its cell surface. Christensen et al. (1987) showed that a single isolate could yield three types of clones with distinct surface characteristics. The wildtype clone adhered well to plastic and synthesized an extracellular polysaccharide. A weakly adherent phenotype was selected from the wildtype clone. From the weakly adherent phenotype, an adherent clone was isolated. The results suggest that S . epidermidis can adhere to surfaces via mechanisms that may be controlled by phase variations. Hogt et al. (1986) believe that hydrophobic phenotypes may selectively adhere to plastics and implants. Once adherent, phenotypes expressing extracellular polysaccharide may become dominant. One member of the genus Staphylococcus, S . saprophyticus, has been shown to express a surface lectin. A common cause of urinary tract infections in females, it is thought to bind to animal cells via a GlcNAc-specific lectin (Gunnarson et al., 1984; Beuth et al., 1987). However, LTA may also contribute to the adhesion of the bacterium because soluble LTA (but not deacylated LTA) inhibited adhesion to uroepithelial cells (Teti et

74

R.J. DOYLE AND E.M. SONNENFELD

al., 1987). In addition, serum albumin was also observed to reduce the binding of S . saprophyticus to urinary cells (Teti et al., 1987). The bacterium may therefore have multiple adhesins on its surface. In fact, multiple adhesins seem to be the rule, rather than the exception, for bacteria that bind to animal cells.

VI. Turnover of Cell Wall and Pathogenesis

Cell wall turnover refers to the excision or shedding of PG and PGassociated polymers during cell metabolism (Doyle et af., 1988). In some cases, the turned-over wall components are not reutilized for new growth. For example, B. subtilis may lose ~ 5 0 % of its wall per generation because of turnover events (Mobley ef al., 1984). There is no evidence that any of the wall turnover products are reutilized or reincorporated into the wall. Other bacteria, such as E. coli or S . typhimurium, exhibit a significant cleavage of preexisting wall during growth, but most of the excised components are reincorporated into new wall (Goodell and Higgins, 1987). Wall turnover occurs as a result of the actions of bacteriolytic enzymes on the PG. These bacteriolytic enzymes may act on the glycan chain (muramidases or glucosaminidases), the linkage between the muramic acid and L-alanine (N-acetylmuramyl-L-alanine amidases, usually referred to simply as amidases), or peptides within the wall structure (peptidases) (see Fig. I). During growth, the autolysins appear to be well regulated. In some species, wall turnover accompanies growth, whereas in others wall turnover may occur in the stationary phase. In B. subtifis, autolysins appear to be regulated by the influence of the energized membrane (Fig. 23) (Jolliffe et al., 1981; Doyle and Koch, 1987). As long as the bacteria are producing protons, the wall is thought to assume a relatively low pH, thereby preventing autolysin activity near the membrane. When the protonic potential approaches zero, the cells begin to lyse. Organisms such as S. aureus have autolysins that seem to be regulated by LTA (Fischer and Koch, 1983; Fischer et al., 1983). Other bacteria, such as the pyogenic streptococci, d o not exhibit wall turnover. The reasons for wall turnover are unclear, but in B. subtilis at least, wall turnover appears to be linked with growth (Koch and Doyle, 1985; Kemper et al., 1988; Doyle et al., 1988). Pooley (1976a,b) found that when the radioactive wall precursor GlcNAc was pulsed into exponential B. subtilis cells, approximately one generation was required before turnover commenced. Furthermore, the rate of turnover was independent of the length of the pulse. Pooley concluded that the newly inserted wall

z

75

BACTERIAL CELL SURFACE

INSIDE

OUTSIDE

ENERGIZED MEMBRANE

0

@%

PROTEASE 831 NACTNATION OF AUTOLYSINS

0

INSIDE

OUTSIDE DEENERGIZED MEMBRANE

0 @% PROTEASE

&OFINACTIVATION AUTOLYSINS 0

Enzyme Inactivated by Energized Membrane

0

Active Enzyme

63 Enzyme Inactivated by Protease

FIG.23. Influence of energized membrane and proteases on autolysins of Bacillus subri/is. Autolytic activity is reduced by soluble proteases, but the proteases do not act on autolysins as they traverse the wall matrix. Wall-bound autolysin is more resistant to proteolysis than soluble autolysin. When protonmotive force is dissipated, autolysins assume an active conformation and cellular autolysis occurs. In B. subrilis, the protonmotive force can be dissipated by uncouplers of oxidative phosphorylation, by ionophores, by starvation for a carbon source, and by anaerobiosis. Restoration of the membrane potential results in the termination of uncontrolled autolysis. From Doyle and Koch (1987). Reprinted by permission of the authors and CRC Press.

76

R.J. DOYLE AND E.M.SONNENFELD

material migrated from the inside of the wall face t o the outside of the wall face, where it became susceptible to turnover. The migration of wall from the inside to the outside took about one generation. The oldest wall contains PG components that are most removed from the energized membrane and are therefore susceptible to surface autolysins. In gram-positive bacilli, this inside-to-outside wall growth causes the cell to elongate. When PG components are added to preexisting wall and crosslinked, the wall then becomes stressed as a result of cellular turgor (Koch and Doyle, 1985). Addition of more wall results in the pushing out and stretching of older wall, thereby causing surface expansion (Fig. 24) (Kemper et a!., 1988).

Figure 25 shows a typical turnover experiment for a gram-positive rod. A culture of B. anthrucis was pulsed with [3H]GlcNAc for either 0.06 or 1.0 generations and then chased with 500 K , values of nonradioactive GlcNAc. Samples were taken during exponential growth and assayed for radioactivity. It is observed that, regardless of pulse time, the rate of loss of label from the cells was -50% per generation. The culture pulsed for

FIG.24. Model depicting the elongation of the side wall of Bucillus subtilis. A cell wall precursor is secreted by the cytoplasmic membrane and then crosslinked into the wall matrix facing the membrane (A). The wall segment is then pushed out by more recently added wall. This causes the wall segment to stretch. By the time it reaches the outer-wall face (about one generation), it has become highly susceptible to the actions of autolysins. An autolysin cleaves one site on the segment, thereby alleviating tension (B), and another autolysin finally cleaves the other bond holding the segment onto the wall matrix (turnover) (C). Addition of new wall with subsequent stretching will result in the elongation of the cell cylinder. Autolysin-deficient cells may have only enough enzyme to clip the most highly stressed bonds. Turnover in these cells will therefore be minimal and their surfaces may be "rough". From Kemper el ul. (1988). Courtesy of the authors and the American Society for Microbiology.

77

BACTERIAL CELL SURFACE

\

:el .-

U K

5'

0

I

I

I

2 GENE RAT I0N S

I

3

I

3.5

FIG.25. Turnover of cell wall of Bacillus anthracis. Cultures were pulsed with ['HIGlcNAc for either 0.06 (0)or 1.0 ( 0 ) generations. See text for details.

0.06 generations exhibited the typical delay before cell wall turnover began. Table XVI lists the pathogens known to turn over their cell walls. The role of wall turnover in pathogenesis is poorly understood. In B. anthrucis, -40% of the cellular dry weight is wall material. Assuming that 50% of the wall is turned over per generation, then growth in vivo would TABLE XVI PATHOGENIC BACTERIA EXHIBITING CELLWALL TURNOVER Bacterium Bacillus anthracis Bacillus cereus Escherichia coli Listeria monocytogenes Neisseria gonorrhoeae Proteus mirabilis Salmonella typhimurium Staphylococcus aureus Streptococcus mutans

References E.M. Sonnenfeld and R.D. Doyle (unpublished 1988) Chung (1971) Chaloupka and Strnadova (1972) Doyle et al. (1982) Hebeler and Young (1976a,b) Grneiner and Kroll (1981a,b) Goodell and Higgins (1987) Rogers (1967) Lesher et al. (1977)

78

R.J. DOYLE AND E.M. SONNENFELD

result in a significant challenge to the immune system by the muramic acid-containing materials. Any Ab directed against wall components, such as teichoic acids, teichuronic acids, or PG, would probably offer little protection to the host, a s long as the wall components are being turned over. Turnover may therefore be one means of helping certain pathogens evade the immune system of the host. Some pathogens d o not turn over their cell walls. In fact, the lack of an active autolytic system may cause the bacteria t o persist in tissues (Fig. 26). Wall remnants of pyogenic streptococci may persist in tissues for months, because of their resistance t o lysosomal enzymes (Ginsburg, 1988). Ginsburg (1988) believes that cationic proteins in neutrophils and macrophages may activate autolysins of certain bacteria, resulting in the elimination of the microbes. On the other hand, proteolytic enzymes (Jolliffe ef a f . , 1982) may reduce the levels of autolysins so as to prevent bacteriolysis in vivo. Work by Wecke er al. (1986) showed that “liquoid,” an anionic polyanethole sulfonic acid, inhibited the autolysins of S. u ~ r e u sreducing , turnover and preventing cell separation. Ginsburg ( I 988) has suggested that polymers, such as heparin or glycosaminoglycans, may inactivate autolysins in vivo and thereby prevent rapid removal of whole or damaged bacteria. Peptidoglycan and muramyl peptides are thought t o play a role in periodontal diseases (Barnard and Holt, 1985). The immunomodulating activity of wall and wall degradation products may play a role in inflammation of gingival tissues leading to periodontal disorders. At this time, it is unknown which oral bacteria exhibit cell wall turnover. As far as is known, turnover in such periodontopathogens as Actinobacillus, Actinomyces, Eikenella, and Baoteroides has not been examined. Mychajlonka el al. (1980) found that in six strains of Streptococcus mutans and eight strains of S . sangrris, there was no evidence for turnover in exponential growth. In addition, wall synthesized during benzylpenicillin o r tetracycline treatment was not susceptible to turnover following removal of the antibiotics. FIG.26. (A) Sfaphylocorrus ~

T C N S incubated

in phosphate-buffered saline (PBS) for

15 hours at 37°C. The cells appear intact. EM x 62.700. (B) Staphylococci incubated for 15

hours at 37°C with leukocyte cationic proteins. Note both the massive destruction of the bacterial cell walls (bacteriolysis) and the dissolution of the inner cytoplasmic structure (plasmolysis). EM ~ 6 2 , 7 0 0 .(C) A macrophage in the synovium of a rat 3 days after the intraarticular injection of viable staphylococci. Both intact and plasmolysed staphylococci are present within the cytoplasm of a macrophage. but no sign ofcell wall breakdown (bacteriolysis) is evident. EM x 15,390. (D) A portion of a macrophage in rat synovium 10 days after the intraarticular injection of viable staphylococci. Note the presence of numerous empty intact shells compatible with plasmolysed cell walls, and the presence of intact cell walls within lysosomelike structures. EM x 15.390. Courtesy of 1. Ginsburg (Ginsburg, 1988) and Blackwell Scientific Publishers.

BACTERIAL CELL SURFACE

79

80

R.J. DOYLE AND E.M. SONNENFELD

FK 26.

(con(.)

BACTERIALCELLSURFACE

FIG.26. (cont.)

81

82

R . J . DOYLE A N D E.M. SONNENFELD

FIG.26.

(cont.)

BACTERIAL CELL SURFACE

83

Lesher et al. (1977) found that subinhibitory concentrations of fluoride anion could induce wall turnover in Streptococcus mutans. The use of fluoride as an additive in drinking water to reduce dental caries is common. It would be interesting to determine whether the decline in dental caries has been marked by an increase in periodontal disease in fluorideconsuming communities.

VII. Concluding Remarks It is now clear, with only a few exceptions, that the bacterial cell surface is essential for pathogenesis. In recent years, the structures of most bacterial surface molecules have become better understood. This has led to a better description of the pathogenic mechanisms of some bacteria. In this article we have tended to emphasize three major features of the bacterial surface: First, the surface is dynamic, resulting in the sloughing off or turnover of wall envelope materials into the surrounding medium. Second, wall components exhibit a remarkable diversity of biological properties, and third, bacterial adhesion to substrata may depend on multiple adhesins. An area related to turnover of walls that has been overlooked is the role of muramic acid-containing materials in the pathogenesis of staphylococci, Neisseria, and B. anthracis. Wall turnover in each of these bacteria (types or species) is greater than that of E. coli or other gram-negative rods. If turnover is a natural consequence of cell division and if turnover products are important in pathogenesis, then a reasonable approach to antibiotic treatment would be to inhibit wall metabolism. It is known that protein synthesis inhibitors such as chloramphenicol cause wall thickening in bacteria. Most protein synthesis inhibitors in bacteria arrest cell division, but their effects on wall degradation in situ are unknown. An important area for future study may be related to the regulation of cell wall-degrading enzymes, such as autolysins. All the major surface structures exhibit multiple effects on biological systems. The recent observations concerning the role of muramic acidcontaining PG components in immune regulation demand that cell wall turnover events be characterized in situ. In addition, the prospect that muramyl peptides may modulate sleep in mammals requires that cell wall turnover be studied using conditions approximating those found in the intestine. It seems strange that a bacterial wall product could be a “vitamin” associated with sleep, but there is now enough literature to convince most that a more than casual relationship exists between normal microbiota and normal sleep. The findings of Lesher et al. on streptococ-

84

R.J. DOYLE AND E.M. SONNENFELD

cal wall turnover in the presence of fluoride anion suggest that certain metabolic challenges may induce wall turnover. This poses interesting questions with regard to rheumatoid arthritis, in which streptococcal wall remnants have been implicated in inducing the symptoms. It is not at all certain whether insoluble wall o r slowly solubilized muramyl peptides are involved in the pathogenesis. Bacterial surface components involved in adhesion to animal cells have been identified in many cases. Furthermore, the molecular bases for adhesive events are known for some bacteria. It is surprising the research on the inhibition of adhesion has not resulted in a new means of preventing infections. A vaccine containing the Man-specific lectin of E. coli may prove to be useful in preventing gastrointestinal infections caused by the bacterium, as preliminary results have suggested. As soon as microbial lectins become available in sufficient quantities (cloning will probably be required for most surface lectins), there will no doubt be a significant effort to develop the proteins as vaccines. A complication in developing vaccines directed against adhesins is the fact that most pathogens can adhere to mucosa by more than one adhesin. It is likely that adhesinbased vaccines will require multiple components in order to provoke an immune response destined t o prevent microbial colonization.

ACKNOWLEDGMENTS Work relating to bacterial cell surfaces has been supported by NIH-NIDR DE-07 199, the Ohio Valley chapter of the March of Dimes, the U.S. Army Research and Development Command. and the N S F (PCM 7808903). The authors thank Prof. T . J. Beveridge for micrographs and discussions, Prof. 1. Ofek for discussions and data, and Prof. Me1 Rosenberg for hospitality and stimulating conversations. Dr. J. Ezzell was instrumental in our understanding of the B. unrhrucis cell surface.

REFERENCES Adam. A.. Amar, C.. Ciorbaru. R . , Lederer. E.. Petit. J . F.. and Vilkas, E. (1974). C. R. Hehd. Siwices Acud. Sci. 278, 799-801. Aho. K. (1987). Clin. Exp. Rheitmatol. 5, Suppl. I . SIS-SI8. Ahvonen, P.. Sievers, K., and Aho. K. (1969). Aclu Rhrrtmutol. Scund. 15, 232-253. Albert. E., and Scholz. S. (1987). Clin. E.rp. Rhectmutol. 5 , Suppl. I . S 2 9 4 3 4 . Aly. R . . and Levit. S. (1987). Rev. Infect. Dis. 9, S341-S350. Amano. K.-I.. and Williams, J. C. (1983). J. Bucteriol. 153, 520-526. Anderson, B.. Dahmen. J.. Frejd, T.. Leffler. H.. Magnusson, G., Noori. G., and Svanborg-Eden, C. (1983). J. Exp. Med. 158, 559-570. Hanson. L. A.. Lagergard. T., and Svanborg-Eden, C. (1986). Anderson. 8 . . Porras. 0.. J. Infect. Dis. 153, 232-237.

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Ofek. I., and Sharon, N. (1988).Infect. Immiin. 56, 539-547. Ofek, I . , Beachey, E. H.. Jefferson. W.. and Campbell. G. L. (1975).J. Exp. Med. 141, 990-1003. Ofek, I.. Mirelman D.. and Sharon. N . (1977).Nuture (London) 265, 623-625. Ofek, I.. Simpson. W. A,, and Beachey, E. H. (1982).J. Bucterid. 149, 426-433. Ogaard. A. R., Bjoro, K., Bukholm. G., and Berdal, B. P. (1985).Acfu Putliol. Microhiol. Immunol. Scund.. Sect. B 93B, 21 1-216. O’Hanley. P.. Lark, D.. Normark, S.. Falkow. S., and Schoolnik. G. K. (1985).J. Exp. Med. 158, 1713-1719. Ohta, M., Rothmann, J.. Kovats, E.. Pham. P. H., and Nowotny. A. (1985). Microhiol. Immunol. 29, 1-12, Osborn, M. J.. Rick, P. D., Lehmann. V.. Rupprecht. E., and Singh, M. (1974).Ann. N . Y. Acud. Sci. 235, 52-65. Pancholi. V., and Fischetti. V. A. (1988).J. Bucteriol. 170, 2618-2624. Pappenheimer, J. R.. Miller, T. B.. and Goodrich, C. A. (1967).Proc. Not/. Acad. Sci. U . S . A . 58, 513-517. Pearson. C. M. (1956).Proc. Soc. Exp. B i d . Med. 91, 95-101. Pooley, H. M. (1976a).J. Bocteriol. 125, 1127-1 138. Pooley, H. M. (1976b). J. Bocreriol. 125, 1139-1147. Proctor. R. A.. Christman. G.. and Mosher, D. F. (1983).J. Lab. Clin. Med. 104,455469. Pulverer, G.. Peters. G., and Schumacher-Perdreau, F. (1987). Zentrulhl. Bukteriol.. Mikrohiol. H y x . . Ser. A 264, 1-28. Ramphal, R.. and Pier. G. B. (1985).Infect. I m m i n . 47, 1-4. Ramphal. R., and Pyle. M. (1983a). Injiect. Immun. 41, 339-344. Ramphal. R.. and Pyle. M.(1983b).Infect. Immun. 41, 345-351. Ramphal, R.. Sadoff, J. C.. Pyle. M., and Silipigni, J . D. (1984). Infect. Itnrnun. 44, 38-40. Ramphal. R., Guay. C., Saunders. J.. and Pier. G . 9. (1986). Clin. Res. 34, A530. Ramphal, R.. Guay, C., and Pier, G. 9. (1987).Infect. Immun. 55, 600-603. Raynaud. M., Kouznetzova, B.. Navarro, M. J.. Chermann, J. C., Digeon, M.. and Petitprez, A. (1973).J. Infect. Dis. 128, S3S-S41. Ribi, E.. Midner. K. C.. and Pemne. T. D. (1959).J . Immimol. 82, 75-84. Rickles. N.. Zilberstein, Z.. Kraus. S., Arad. G., Kaufstein, M., and Ginsburg. 1. (1969). Proc. Soc. E.up. B i d . Med. 131, 525-530. Rietschel, E. T.. Schade, U ..Jensen. M.. Wollenweber, H.-W., Luderitz, 0.and Greisman, S. G. (1982).Scund. J. Infect. Dis..Siippl. 31, 8-21. Rietschel. E. T.. Brade, L.. Brandenburg, K., Flad. H. -D.. de Jong-Leuveninck, J., Kawaham, K., Lindner, B.. Loppnow. H., Luderitz, T., Schade. U., Seydel. U., Sidorczyk, 2.. Tdcken, A.. Zahringer. U.. and Brade, H. (1987).Rev. Infect. Dis. 9, S527-S536. Roberts, J . A., Kaack. B., Kallenius. G., Mollby. R.. Winberg, J.. and Svenson. S. B. (1984).J. Urol. 131, 163-168. Rodndn. G . P.. and Schumacher. H. R. 11983).I n “Primer on :he Rheumatic Diseases” (G. P. Rodnan and H. R. Schumacher. eds.). 8th ed., pp. 30-33 and 94-97. Arthritis Found.. Atlanta, Georgia. Rogers, H. J. (1%7). Foliu Microbid. (Prugue) 12, 191-200. Rogers, H. J., Taylor, C., Rayter. S.. and Ward. J. B. (1984).J. Gen. Microbiol. 130, 23952402. Rosenthal, L.. Olhagen, B., and Ek. S. (1980).Ann. Rheum. Dis. 39, 141-146. Rosenthal. R. S..and Krueger, J. M. (1987).Anfonie van Leeuwenhoek 53, 523-532. Rotta, J. (1969).Curr. Top. Microbiol. Immunol. 48, 63-101. Ryden. C.,Rubin. K., Speziale. P.. Hook. M.,Lindberg, M., and Wadstrom, T. ( 1983).J . Biol. Chem. 258, 3396-3401.

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INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 118

Cellular Studies on Marine Algae AHARONGIBOR Department of Biological Sciences, University of California, Santa Barbara, California 93106

I. Introduction The rapid progress of biology in the past few decades was accomplished by exploiting a few selected experimental organisms, of which Escherichiu coli, yeast, corn, and the fruit fly are outstanding examples. However, progress via the use of a limited number of model systems soon became quantitative in nature, and great qualitative steps forward were accomplished with the introduction of new model organisms. The same experimental organisms can sometimes be exploited for new areas other than those for which it was originally chosen. For example, the fruit fly Drosophila, the salivary glands of which contributed so much to classical genetics, is now being exploited for studies on the role of genes in embryonic development of multicellular animals. In this chapter I describe three of the enormous variety of algae. These three are fascinating and offer a promise for studies on basic biological problems. I would like to present these as challenges to the reader, hoping that ingenious experimenters will be able to exploit these organisms further. The simplest members of the plant kingdom are classified as algae. This subkingdom includes an enormous variety of organisms, from simple single cells to complex giant seaweeds. Among this vast number of plants, some proved to be very useful as model organisms for studies of specific phases in the lives of cells in general and of plant cells in particular. Classical examples are the use of single-cell green algae such as Chlorellu and Scenedesmus in studies on photosynthesis (Bassham, 1962; Lewin, 1962); similarly, Valoniu and Nitella were selected for studies on membrane phenomena and cytoplasmic streaming (Blinks, 1936; Osterhout, 1936). My objectives in writing this chapter are not to review such studies: instead, I describe several algae that are perhaps less famous but that I 93 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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have found to be interesting, because they possess properties that make them suitable for studies of important aspects of biology. One of these, Acetabirlaria, was used for many years for studies on nuclear-cytoplasmic relationships (Hammerling, 1963). These classic studies do not require a rereview, and I prefer to emphasize new or neglected aspects in the life of this alga that possess potential for further fruitful studies. I also chose to consider the green alga Boergesenia and the red alga Porphyra. The choice of the three organisms is purely subjective. With two of them. Acetabularia and Porphyra, I have had a long acquaintance and considerable first-hand experience, whereas with Boergesenia I have had only a short and limited interaction. I like these organisms not only because they have unique biological properties but also they are visibly pleasing and enjoyable to work with.

11. Acetabularia

Acetabularia are green algae, the genus belonging to the family Dasycladaceae of the order Siphonales. The family is characterized by a growth pattern in which the main axis of the plant develops whorls of branches. Some or all of these branches become the reproductive organs. In the genus Acetabularia, branches of the terminal whorl differentiate to become the spore-bearing fruiting body. Different species differ in the degree of association of the branches of the fertile whorl. They form, for example, an “umbrella”-shaped structure in A . mediterranea, while in others such as A . polyphesa they form disconnected individual fruiting branches, similar to a bunch of bananas. Intermediate-type structures are found in other species. Another important attribute of this genus is that their vegetative nucleus is invariably located in the rhizoidal section. Acetabuhria are distinguished by the fact that regular mitosis of the cell nucleus is delayed until the plant completes its vegetative growth and a fully differentiated fruiting whorl has formed. Until then the plant could be regarded as a single cell. However, once nuclear divisions start the plant is to be regarded as a coenocytic organism, similar to all other Siphonales algae. The secondary nuclei divide repeatedly and migrate into the branches of the fruiting body, where they become spaced regularly. Eventually the protoplasm around each nucleus becomes segmented and surrounded by a plasma membrane; a cell wall is then secreted, thus forming the reproductive cysts. Each cyst when formed contains a single nucleus, numerous chloroplasts, and mitochondria. In subsequent development of the cyst the single nucleus undergoes many divisions, eventually to produce many gametes. A gamete is a biflageilated naked cell with

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one nucleus and one chloroplast, resembling a Chlamydomonas cell. After their release, the gametes fuse in pairs to form a zygote, which grows to form the familiar vegetative cell. The gametes from a single cyst do not mate with each other. This suggests that the secondary nuclei are the products of meiosis and thus products of each haploid cyst are of the same mating type. It is the delay in the onset of nuclear divisions in the life of the vegetative cell that made these organisms suitable for the many interesting studies on the role of the nucleus and relationships between the cytoplasm and the nucleus (Hammerling, 1963; Gibor, 1966). Other important properties of Acetabularia are its ability to withstand severe mechanical damage, heal its resulting wounds, and resume growth. Small segments of a cell are capable of regenerating an entire plant as long as they contain the primary nucleus. Enucleated cell segments can continue to grow and even develop for many weeks. Drops of cytoplasm squeezed out of the cell wall can maintain their photosynthetic activity and cytoplasmic streaming for many days in vitro (Gibor, 1966). By centrifugation it is possible to move most of the cell contents to either the rhizoidal or the apical end of the cell. Several hours after the termination of the centrifugation, the cytoplasm moves back and restores the normal green appearance of the cell. The properties of these plants were exploited in classic experiments by Hammerling (1963) to demonstrate the role of the nucleus in determining the phenotypic properties of different species. Transplantation of nuclei by grafting the rhizoids of one species onto the stalk of another was later followed by the transfer of washed nuclei of one species to stalks of another. Such transplantations resulted in the “hybrid,” developing a fruiting body morphologically similar to the nucleus-donor species. In the growing cell, the development of the primary nucleus and the induction of mitosis were found to be controlled by the physiological state of the cytoplasm. Acetabularia could thus serve well for studies on the relationship between the nucleus and the cytoplasm. These biochemical and physiological aspects in the life of Acetabularia were and are still being actively investigated (Berger et al., 1987). There are other aspects of the unique biological properties of Acetabularia that are still underexploited and that deserve further study. An interesting biological problem for which this plant is suitable as an experimental model is aging at the cellular level. Hammerling noted that by repeated amputation of the developing “umbrella,” a cell could be maintained for many years in culture. The primary nucleus of the cell enlarges with the growth of the cell and reaches a maximum size when the fruiting body develops to its maximum size. This occurs just before the onset of mitosis and production of secondary nuclei. The large pri-

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mary nucleus possesses a highly enlarged nucleolus with a characteristic shape. If, however, the bulk of the vegetative cell is amputated prior to the onset of mitosis, the primary nucleus shrinks in size proportionately. The regenerating cell would have a smaller primary nucleus that would commence to enlarge again with the regrowth of the cell. By repeating such amputations every time the cell approaches maturity, it is possible to maintain active growth of the cell for many years. The cell appears to be rejuvenated by the amputations, and theoretically it could be maintained by such manipulations indefinitely. Conversely, a mature fruiting body if grafted onto a young stalk will impose the onset of meiosis and mitosis on the relatively small primary nucleus. The onset of meiosis can be regarded as the end of the juvenile or vegetative phase of the organism and the beginning of a maturation phase. The detailed molecular aspects of the changes in the size and activities of the primary nucleus in response t o the changes in the cytoplasm are yet to be studied. The "physiological age" of the cell can be manipulated by the experimenter. The nature of the signals exchanged between the cytoplasm and nucleus that regulate the activities of the nucleus are not yet known. It was established that primary nuclei can be isolated and maintained alive in vitro. Such nuclei could be returned to enucleated cells, where they functioned normally. With such experimental manipulations it should be possible to expose nuclei to different substances derived from chosen cytoplasms and determine their physiological effects. The physiological activities of the whorls of sterile hair are also of potential interest for research on cellular aging. In laboratory cultures, a growing A . rnedirerruneu cell usually possesses several active whorls. The whorls are symmetrical structures made up, in the case of A. rnediterruneu, of 14 branches. Each branch undergoes further branching. We refer to each level of branching as a tier, numbered by a Roman numeral. Thus there are 14 tier 1 branches, each one of them producing 3 tier 11 branches. Tier I1 branches produce 3 tier 111 branches each, which in turn gives rise to 2 tier IV branches. Tier IV branches produce either 1 o r 2 tier V branches (Gibor, 1973a). Sectors of two whorls are shown in Fig. 1; visible are an upper live whorl with branches full of organelles and a lower, older whorl, appearing to be empty. Simple measurements indicate that the total surface area of all the branches of a single whorl are about equal to the total surface area of the main cell body (Gibor, 1973b). The live whorls are active in the uptake of soluble substances from the environment. This could be readily demonstrated by immersing cells in a solution of a vital dye such as neutral red. The thinnest branches, tier V and IV, appear to be stained in < I minute, followed by tiers 111, 11, and I. The main cell body eventually also accumulates the dye. The uptake

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FIG.1. Sectors of two whorls. The upper whorl is alive, and the branches of tiers I and I1 contain many chloroplasts. The branches of the lower whori appear to be empty. Up to five tiers of branches are clearly seen in this whorl, and the first three are marked by arrows.' x40. From Gibor (1973a,b); Courtesy of Springer Verlag.

of dyes can be inhibited by lowering the temperature, or poisoning the cells with KCN. Older whorls located farther down on the main stem, which appear to be empty, do not accumulate the dyes. Thus the fine branching of the cells is similar in physiological function to the function of root hairs, namely increasing the surface area of the cell in contact with the environment (Gibor, 1973b). Another indication that the whorls function in nutrient uptake is the observation that the length of the branches increased when cultured in a medium deprived of a nitrogen source, while in the presence of nitrate or urea in the culture medium the whorls appeared shorter and more compact (Adamich et al., 1975) (Fig. 2).

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0

1

5

mm

10

2

FIG.2 . Variations in whorl development in cells grown o n different media. Cell I grew for 30 days on 530 pg atom nitrogen per liter. as nitrate. Cell 2 grew for the same length of time in the same concentration of urea. Cell 3 grew in a nitrogen-free medium. From Adamich el d. (1975): Courtesy of the Journal of Phycology.

The cells maintained in nitrogen-free medium also appear to retain a larger number of viable whorls. Up to seven whorls are seen in cell 3 of Fig. 2. Note that in the oldest whorls of this cell only tiers 1 and 11 with perhaps a few tier 111 branches are still viable. In the nitrate-fed cell (cell 1 of Fig. 2 ) . there are only three live whorls. The physiological significance of the sterile whorls to cell elongation was indicated by experiments in which portions of the live whorls, from one side of a cell, were cut off or damaged by U V irradiation. Such cells were found to elongate unequally, causing the cells to bend toward the side of the cell from which the branches were removed or damaged (Fig. 3). The sectors of the cell from which the branches were removed did not elongate as much as the unmanipulated sectors. The unequal elongation of the sides of the cell indicates that the different sectors of the cell periphery are relatively insulated. Apparently there is no free diffusion of substances laterally across the cell. even though no internal barriers (e.g., cell walls) are present in these coenocytic cells.

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FIG.3. Cells 3 days after shaving the branches of whorls from one side of the cell. The line drawn in the background indicates the initial appearance of the cell on February 28, before the operation. Broken lines indicate the shaven side. Shown on the left are two cells with their rhizoids intertwined. From Gibor (1 977a); Courtesy of Springer Verlag.

New whorls form regularly with the elongation of the cell, and only the younger whorls remain alive. The live branches of tiers I and I1 contain many chloroplasts. There are only a few thin, long chloroplasts in tier 111, and chloroplasts are rarely seen in tier IV and above. Examination of the older whorls farther down the main cell axis reveals that the branches appear to become empty of their cytoplasm with aging. This aging process begins with the emptying of tiers V, then IV, and so on until the entire whorl appears empty (Fig. 1). Subsequently, the empty branches are shed and characteristic scars remain on the cell wall marking the earlier location of a whorl. These cellular branches can therefore be thought of as deciduous organelles. The entire complement of whorls of a cell such as cell 3 in Fig. 2 represents a set of cellular structures, or organelles, of progressively increased physiological age. The topmost whorl is young and still growing, with its tiers not having completed their elongation. The seventh whorl of the same cell consists of aged and dying branches; only tier I branches of this whorl appear to be still alive. It appears that the cytoplasm retracts back into the cell body prior to abscission. It is yet to be determined whether components of the cell wall are also recycled back into the cell.

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The movement of the cytoplasm into the branches of a young and growing whorl and the subsequent retraction into the main axis of the cell upon aging are both intriguing phenomena. Such movements are probably related to the striking migration of the cytoplasm and secondary nuclei into the compartments of the mature fruiting body. What role components of the cytoskeleton and plasma membrane play in directing these movements is yet to be determined. We know of several environmental factors that affect the development and aging of the branching whorls of the cell. As mentioned before, the cells retain their whorls in an active state in nitrogen-poor medium, while in nitrate-rich medium the number of active whorls is reduced. Apparently the aging of whorls occurs faster in wellnourished cells. Another important observation is that when cells are incubated in the dark they lose all their whorls in 10 days, and thus aging of the whorls is accelerated in the dark. Cells maintained under red light stop elongating and do not develop new whorls. Exposure to short flashes of blue light immediately induces the resumption of elongation and the growth of whorls (Schmid et ul., 1987) (Fig. 4). An action spectrum for this photomorphogenetic effect indicates that it is similar to other blue-light effects that were described for plant morphogenesis (Schmid e f ul., 1987). The spatial distribution of the blue-light photoreceptors were also investigated by Schmid and co-workers. The apical growing portion of the cell was found to be most sensitive to the bluelight stimulus, but other segments of the cell, including the rhizoid. could also respond to the blue-light stimulus. I t was concluded that the primary photoreceptors for this blue-light effect are probably present throughout the cell length. The blue-light effect on the initiation of elongation and whorl development can be seen in <8 hours after induction. Detailed studies on changes in the architecture of the apical cell membrane after the induction are yet to be performed. Schmid and co-workers (1987) demonstrated that the blue-light stimulation of whorl development occurs in enucleated cells as well as in cells in which transcription is inhibited by actinomycin D. On the other hand, cycloheximide and puromycin, inhibitors of translation, did inhibit the blue-light induction. The fruiting body is a specialized and modified whorl. The molecular changes in the composition of the fertile versus sterile whorls are not known, but the role of nuclear genes in determining the shape of the fertile whorls is well established (Hammerling, 1963). An active role of nuclear genes in the development and function of the sterile whorls is suggested by the fact that whorl development is different in different species of Act.fahirluriu. Thus, A. major elongates rapidly without the regular appearance of whorls. Only upon approach of cellular maturation do cells

-

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FIG.4. Growth and development of the Ace?abu/aria apex after exposure to blue light. The series of photographs were taken at 2-hour intervals. The arrow points to the flattening of the tip, which is the first visible sign of the whorl formation. From Schmid et a / . (1987); Courtesy of Springer Verlag.

begin to develop sterile whorls. In this species the removal of sectors of the whorls on one side of the cell does not cause the cell to bend. It should be possible to demonstrate the nuclear effect by transplanting nuclei between A. mediterranea and A . major. For example, will an A. mediterranea cell into which a nucleus from A. major has been transplanted elongate without the production of whorls? Will such a cell bend in response to cutting of the preexisting sterile whorls from one side of the cell? The apical portion of the cell also possess interesting physiological properties that warrant further study. We demonstrated (Gibor, 1977a) that if the apical segment of a cell is kept in the dark while the rest of the cell is illuminated, the cell does not elongate. The illuminated portions become dark green but they do not elongate. Under similar culture conditions, if the apical portions had been cut off, active regeneration would have taken place. Apparently a new growing apex cannot develop in the presence of an existing apex. Bonnoto and Gelder (1969) observed, how-

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ever, that when Acetablrlaria cells were grown under very crowded conditions a high percentage of the cells developed branched main stems, indicating that new apices could develop in the presence of a cell apex. The latter observation also indicates that the development of cells can be affected by substances that presumably accumulate in the crowded cultures. 111. Boergesenia

Boergeseniu is a coenoecytic green alga belonging to the Valoniaceae family and is usually found in the intertidal zone of tropical seas (Fig. 5). It was first cultured by S. Enomoto (Enomoto and Hirose, 1972). Boergesenia grows readily in enriched seawater at room temperature illuminated for -12 hours daily. Under these conditions plants attain lengths of -2 cm in 2 months. The main body of the plant is made up of a large vesicle up to several centimeters in diameter with a number of smaller vesicles branching from the basal region. It is attached to the substratum by rhizoids. The vesicles show a marked capacity to heal wounds. The healing occurs by the growing together of the exposed edges of the damaged protoplast, thus sealing the open wound. The vesicles are made up of a cellulosic cell wall encompassing a thin layer of protoplasm containing a large number of chloroplasts and nuclei, enclosed between a peripheral plasma membrane and an interior tonoplast. As is the case with all members of the family, the major portion of the volume of the plant is composed of the vacuole. For many physiological studies the voluminous multinucleated vesicle can be regarded as a single cell. Thus Vulonia, a close relative of Boergesenia, served as a model cell for studies of the electrical properties of plant cell membranes for many years (Osterhout, 1936; Blinks, 1936). One of the more interesting properties of Boergeseniu cells is their sensitivity and response to physical and chemical stimuli. Mechanical shocks such as a pinch with forceps, a prick with a needle, or a cut with a blade, immediately initiate a set of responses that result in the fragmentation of the entire protoplast into a large number of rounded cells (Figs. 5 and 6). In the intact cell, chloroplasts appear to be uniformly distributed. Within minutes after an applied shock the distribution of chloroplasts becomes noticeably modified, with a network of aggregated chloroplasts appearing throughout the cell. Subsequently the protoplasm retracts from the cell wall and forms numerous irregularly shaped aggregates interconnected by tine strands. Eventually these irregular masses round up to form spherical protoplasts. These protoplasts soon begin to synthesize new walls, and by -6 hours after the shock the mother cell appears to be a large vesicle

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FIG.5 . Cluster of Boergeseniuforbesii cells. (A) A normally growing cell; (B) a mother cell containing hundreds of rounded protoplasts. (-- ~4.5.)From O’Neil and LaClaire (1984); Reproduced by permission of the AAAS.

composed of a cell wall containing within it many rounded cells referred to as “aplanospores. ” The aplanospores remain quiescent while they are enclosed in the walls of the mother cell. If they are liberated from the mother cell by dissecting the old walls, they germinate and grow rapidly to produce new plants under appropriate culture conditions. The sensitivity of Boergesenia cells to a shock and the rapid and ordered changes in the cytoplasmic organization that follow made this alga an attractive experimental organism for studies of several aspects of cell biology.

FIG.6. Time course of wound healing and protoplast formation in Boergeseniu fiwhesii. ( a k ( e ) I . 15.30.60. and 120 minutes after wounding, respectively. (0 120 hours after wounding. ( - ~ 9 . 5 . From ) LaClaire (1982);Courtesy of the Journal of Phycology.

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Mitosis proceeds in these coenocytic cells without apparent changes in the organization of the cytoplasm. The phase of cytokinesis does not occur regularly in this organism. The stimulus that causes rapid segmentation of the protoplast to aplanospores can be regarded as inducing cytokinesis of the multinucleated protoplast. It thus becomes experimentally possible to initiate at will this important phase of the mitotic cycle and to study it independently of nuclear division. Details of changes in the architecture of the cytoskeleton that accompany cytokinesis were the subject of study in several laboratories. LaClaire (1982, 1984, 1987) studied cytoplasmic movements that are associated with the retraction of the protoplasm from the wounded area and the subsequent rounding up of the protoplasmic aggregates. The movements of the protoplasm require active metabolism, and the presence of calcium ions is essential. The distribution of microtubules (MT) was investigated by electron microscopy (EM) and immunofluorescence techniques. Intact cells were found to have two populations of MT: a highly ordered cortical array consisting of parallel, longitudinal MT and shorter perinuclear MT radiating from the surfaces of the interphase nuclei. The two MT populations differ in some other properties; for example, the perinuclear populations of MT are more sensitive to depolymerization by cold than the cortical MT. The MT cytoskeleton does not appear to be directly involved in the wound-induced motility, since agents that cause the depolymerization of the cortical MT do not inhibit the wound-healing motility. Extensive changes in the plasma membrane and tonoplast occurs soon after wounding of the cell. About 45 minutes after wounding, numerous coated pits and coated vesicles appear near the plasma membrane. These are abundant for the next half hour or so, but thereafter they are infrequently seen. The period of their abundance corresponds to the peak period of protoplasmic contraction that leads to aplanospore production. Measurement of the total plasma membrane surface area in cells before and after wounding indicates that in the transition to aplanospores a decrease of -40% in the total plasma membrane area takes place. The invagination of coated pits apparently represents the process of membrane loss. The new plasma membrane that surrounds the aplanospores originates from both the plasma membrane and the tonoplast of the mother cell. The modification of the tonoplast to become a functional plasma membrane occurs within 2 hours after wounding (O’Neil and LaClaire, 1988). Several investigators took advantage of the induction of the formation of aplanospores in Boergesenia to study mechanisms of cellulose wall biosynthesis (Mizuta and Okuda, 1987; Kudlicka et al., 1987; Itoh and Brown, 1988). The synchronized and rapid rounding up of the protoplasm

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and the immediate initiation of synthesis of new cellulose walls permitted studies on the early steps of assembly of the wall synthesis machinery. Electron-microscopic studies on the appearance of cellulose microfibrils and associated intermembrane particles revealed the development of linear clusters of particles, the so-called “terminal complexes” (TC) prior to the initiation of cellulose microfibril synthesis. These linear TC are thought to be the site of assembly of the cellulose fibrils (Itoh and Brown, 1988). The orientation of the synthesized cellulose fibrils on the cell surface changes temporally with the development of the wall. The first fibrils appear to be randomly directed, but subsequently the fibrillar layers become parallel and oriented. The orientation of these fibrils appears to be independent of the orientation of the cortical MT (Hayano et ul., 1988). Perturbation of the lipid phase of the young plasma membrane of the newly forming aplanospores by the incorporation of cholesteryl-hemisuccinate (Legge and Brown, 1988) caused the appearance of patches that lacked cellulose fibrils on the surface of the cells. Freeze-fracture studies of such membranes revealed the presence of membrane domains that lacked the typical intramembrane particles. Thus the insertion and distribution of the intramembrane particles that presumably are responsible for the synthesis of the microfibrils is affected by the composition of the lipid layer. The organized orientation of the deposited layers of cellulose fibrils necessitates the reorientation of the TC. It is not known how these changes in the orientation of the fibrils in the different lamellae of the synthesized walls are accomplished. An electric potential difference exists across the protoplasm due to differences in ionic composition of the vacuolar fluid and the extracellular medium. The normal physiological activities of the cell are probably affected by this potential. Damage to the cell initiates the retraction of cytoplasm from cell walls, causing the rupturing of the thin layer of protoplasm that separates the vacuolar fluid from the extracellular medium. This undoubtedly changes the ionic composition of the vacuolar compartment. It is likely that the change in the electric potential across the protoplasm plays an important role in initiation of the protoplasmic retraction. The degree of equilibration of the vacuolar fluid with the external medium can be estimated by preloading the cell with a vital dye. The loss of the dye upon wounding and inducing the cells to form aplanospores could be a measure of loss of vacuolar contents. In other studies on the walls of Boergeseniu, it was found that empty walls of the mother cells, after removal of the aplanospores from them, are composed primarily of cellulosic fibrils embedded in a matrix. They were examined by EM and found to be devoid of plasma membranes but to possess appreciable ATPase activity (Mizuta and Suda, 1978). An interesting aspect of the developing

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aplanospores is that they proceed to synthesize a cell wall and then stop further development as long as they remain within the walls of the mother cell. However, if they are removed from the mother cell the aplanospores germinate upon illumination and grow. The germination-inhibiting factors present in the mother cell have not been identified as yet. Since the empty mother cell walls do not possess a membrane, such inhibitors are not likely to be diffusible molecules but are more likely to be polymers or aggregates of macromolecules that are not readily lost through the walls. Polarity can be induced in the germinating aplanospores by the direction of light; the rhizoid emerges at the dark side. Blue light was found to be most effective for the induction of polarity (Ishizawa et al., 1979). The photoreceptors for this induction apparently belong to the “blue-light’’ receptors described for other photomorphogenetic effects in algae including Acetabularia (Schmid et al., 1987). The ready induction of the cytoplasmic movements and cytokinesis make this alga a promising model for further investigations of this phase of the cell cycle of eukaryotic cells.

IV. Porphyra

The genus Porphyra belongs to the family Bangiaceae of the Rhodophyta. The plants are multicellular, forming large, flat thalli 6 1 m long. The large thallus is made up of either one or two cell layers depending on the species. The cells of the thallus are thought to be haploid. This plant can thus be regarded as a two-dimensional multicellular organism. Several differentiated zones are distinguishable in the mature organism (Fig. 7). Attaching the plant to the substratum is a holdfast region composed of uniquely shaped, tailed cells. Above this region is a zone that is obviously polarized, and the cells possess large vacuoles directed toward the holdfast region. Distal to this region is the main body of the thallus. Here the cells are relatively small, highly pigmented, and actively dividing. The peripheral zone of the thallus contains regions with distinct differences in pigmentation. These are the sexually differentiated zones that form either male spermatangia or female carpogonia. The spermatangia subsequently develop and release spermatia while carpogonia develop into carpospores. Fertilized carpospores germinate to produce an alternate generation, the filamentous “conchocelis” generation. In nature the filaments of the cochocelis bore into rocks or shells, hence the name. When mature the conchocelis release spores, the conchospores, which regenerate the familiar Porphyra thallus upon germination. Different species of Porphyra differ with regard to the time of meiosis. In some species meiosis occurs

FIG.7 . Diagrammatic representation of the Porphyru thallus. The appearance of the differentiated cells in the marked zones of the thallus is presented in the photographs on the right side. ( - x 123.)

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early in the germination of carpospores; thus the conchocelis cells are also haploid. In other species the conchocelis cells were reported to be diploid with meiosis occurring prior to the release of concospores (Tseng, 1955; Migita, 1967; Hawkes, 1978). The filamentous conchocelis can be easily propagated vegetatively from detached cells or filaments. Sexuality within the genus Porphyru varies. Thus, some species are monoecious (i.e., the same thallus bears differentiated male and female regions), while other species are dioecious (i.e., individual plants produce either male or female structures) (Hawkes, 1978). Different mature regions of the thallus are readily recognized and it is therefore possible to isolate differentiated tissues mechanically by dissection. Such differentiated tissues can be readily dissociated by enzymatic treatments to yield isolated differentiated cells or protoplasts. Based on morphological differences, it is possible to distinguish the following types of Porphyru cells: (1) rhizoid cells with characteristic “tails,” which are found in the holdfast region, (2) polarized but not tailed cells from the area adjacent to the holdfast, (3) vegetative cells composing the major area of the thallus, (4) differentiated male cells, ( 5 ) differentiated female cells, (6) peripheral cells that do not show overt sexual differentiation but that if isolated and grown in vitro produce filamentous-type growth, (7) vegetative conchocelis cells, (8) sporangial conchocelis cells, and (9) monospores, specialized cells that can directly regenerate the vegetative plant. These morphologically distinguishable cells differ also in their developmental potentials. Differentiated spermatangia could not be induced to divide in vitro (Tseng, 1955; A. Gibor, unpublished data). Isolated rhizoidal, tailed cells dedifferentiated slowly but eventually started to divide and regenerated plantlets. The highest percentage of regenerating cells was obtained from vegetative cells (type 3 in the list). Conchocelis cells only regenerated filamentous-typegrowth (Polne-Fuller and Gibor, 1984). This pattern of differentiation is reminiscent of regeneration of fern gametophytes. The fern gametophyte is also made up of a single cell layer with specific areas of differentiation. Miller (1969), stated in a review of the field: “The regular morphology indicates that the multicellular gametophyte exerts a regulatory control over the growth potential of the cells. A cell which is cut off from communication with its neighbors assumes a sporelike potential. Differencesare found in the ease with which regeneration occurs depending on the original location of the isolated cell.” In the ferns, low molecular weight pheromones were found that induce the differentiation of sexual organs. However, no such inducing substances have been described for Porphyra.

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Little is known concerning the mechanism of differentiation and integration of these assorted cells into a functional organism. In higher plants integration and communication between different tissues and organs is accomplished by chemical signals. These signals move via two channels: ( 1) the simplastic path consisting of the protoplasmic bridges that connect adjacent cells and (2) The apoplasmatic path, the space between the protoplasts. This path requires the secretion of the signaling substances out of the cells that produce them and their absorption by target cells. The apoplasmatic region includes all the space that separates protoplasts from each other, including the cell wall. The directional specificity of movement of different substances within either of these two paths is poorly understood (Gunning and Robards, 1976). An important property of the Porphyru thallus is the absence of plasmodesmata between the cells. Thus the only pathway of communication and integration between the cells must be the apoplasmatic region. Porphyra is therefore a good plant with which to study the apoplasmatic pathway of communication without the added complication of simplastic communication. In the filamentous conchocelis stage, however, cells are interconnected by plasmodesmatalike connections. The biological significance of these differences in communication pathways in the two distinct growth forms of Porphyru-that is, the two-dimensional vegetative thallus and the unidimensional filamentous conchocelis-is not known. The two phases of the life cycle of Porphyru are thus suitable for comparative studies on the translocation of metabolites among cells that communicate only via their apoplasmatic spaces and those that can also communicate by plasmatic bridges. In preliminary experiments we examined the translocation of carbon-labeled substances from a central spot of the thallus. By spot illumination it was possible to label a defined small area of the thallus with radioactive CO,. The translocation of carbon in the thallus was examined after several days. A striking result was the accumulation of labeled substances in sectors of the thallus where active cell division was taking place (healing of wounds), while very little accumulation occurred in quiescent regions of the thallus (Polne-Fuller and Gibor, 1989). Apparently dividing cells function as sinks and somehow cause the attraction of metabolites toward them. The chemical nature of the substances that were translocated is not known. The apoplasmatic region in Porphyru, through which the translocation takes place, is made up of several distinct layers composed of complex polysaccharides and coated by a proteinaceous pellicle. Gunawrdena and Williamson (1974) reported on the chemical heterogeneity of the apoplasmatic material of the Porphyru thallus. They differentiated among the “cell wall,” a “mucilaginous matrix,” and a tough outer “cuticle.” By

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histochemical staining they found that the matrix and outermost layer of the cell wall were rich in sulfated polysaccharides while the inner regions of the cell wall were rich in carboxylated polysaccharides. The cuticle was found to be proteinaceous. Preston (1974) studied the apoplasmatic region of Porphyra by EM. Their work demonstrated the complexity of this region (Fig. 8). Figure 8A is a cross section through the holdfast region of the thallus. The long “tails” of the rhizoid cells grow through the apoplasmatic matrix. The intercellular space is made up of mannan while the “tails” are composed of fibrillar xylan. It appears from the figure that the fibrillar xylan layer is encased in a sheath that is mainly mannan. Figure 8B shows the orientation of the fibrillar xylan over a mannan background. These figures clearly demonstrate the complexity of the apoplasmatic region. Other indicators of the heterogeneity of the apoplasmatic regions of the different sectors of the thallus are the differences in their susceptibility to enzymatic degradation. In our studies on the production of protoplasts from different parts of the Porphyra thallus (Polne-Fuller and Gibor, 1984), it became apparent that while the vegetative central region of the thallus dissociated readily, the spermetangia and the holdfast regions were most resistant to digestion. The ability of the rhizoidal cells to produce and secrete an adhesive substance that anchors the thallus to the substratum is another indicator of the chemical differentiation of the apoplasmatic region of the different zones of the thallus. The physiological significance to the protoplasts of their being embedded in an apoplasmatic material of different composition is obviously important. Little is known concerning the molecular aspects of these interactions, however. The most obvious indicator of this relationship is that removal and isolation of a protoplast from its specific tissue causes an immediate change in its metabolism. The protoplast soon secretes a new apoplasmatic matrix, and then, under proper culture conditions it will dedifferentiate, and start growing and dividing. An interesting example of the spatial uniqueness of the extracellular matrix (ECM) is the demonstration by Quatrano and co-workers (Quatrano et al., 1979) that changes in the composition of the extracellular polysaccharides of the developing Fucus zygote are correlated with the development of new differentiated cells. Fucoidin, a highly sulfated polysaccharide, was found to appear with the differentiation of the rhizoid cell. In the developing embryo the fucoidin remains localized in the rhizoidal region. Similarly, in the red alga Chondrus (Gordon-Mills and McCandless, 1974) two types of carrageenans, a component of the ECM, were found at different phases of the life cycle of the alga. We have characterized a set of glycoproteins that are markers of the

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FIG.8. Electron micrographs of the base of a Porphvru plant (zone I in Fig. 7). (A) A carbon replica of a split face showing a rhizoid (xylan) grown through extracellular material (mannan). (B) A transverse section showing rhizoids with xylan walls in the mannan intercellular substance but surrounded by a distinct layer also made up of mannan. x 20,000. From Preston (1974); Courtesy of Chapman and Hall, Ltd.

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differentiated regions of the Porphyra thallus. These extractable proteins differ in their affinity for different lectins (Kaska et al., 1988). Vreeland and Leatsch (1984) described the use of polyclonal and monoclonal antibodies directed against different algal polysaccharides for studies of the sequential appearance of such polysaccharides in developing algae. From morphological, biochemical, and immunological studies it is becoming evident that the apoplasmatic matrix in which algal protoplasts are embedded is quite complex. The interactions between the protoplasts and the apoplasmatic matrix are obviously crucial to the developing organism. The role of the ECM in animal development is presently a focal area of research. We believe that the Porphyra system can serve as a good model for study of such relationships in a developing multicellular plant. V. Concluding Remarks

Three species of algae, Acetabularia, Boergesenia, and Porphyra, are endowed with unique properties that make them attractive for research on various aspects of development and differentiation. Acetabularia is uniquely suited for investigations of aging at the cellular level. Boergesenia appears to be ideal for studies on cytokinesis and the segmentation of the protoplasts into daughter protoplasts. There are numerous other algae that could be used in studies of different phases of the eukaryotic cell cycle. The Siphonales algae are defined by the lack of the usual septation of the protoplast after nuclear divisions. Different members of this order control or modify different phases of their life cycle. Thus in Acetabularia nuclear divisions are controlled, while in Boergesenia segmentation of the protoplasts is controlled. The two-dimensional multicellular plant, Porphyra, offers opportunities for studies of cellular differentiation and integration. Other algae such as Monostroma, Enteromorpha, and Ulva are also composed of a single or double cell layers, which are, however, interconnected by cytoplasmic bridges. Algologists will find it productive to listen to the problems the cell biologists are tackling. There may be an easy answer to these problems to be found within algal collections. Cell biologists might find it rewarding to hear of the intricate life cycles of some of these organisms. Some of the cell-biologicalproblems might be easier to solve by choosing a new experimental organism from among the algae. REFERENCES Adamich, M., Gibor, A., and Sweeney, B. M. (1975). J . Phycol. 11, 364-367 Bassharn, J. A. (1962). Sci. A m . 206, 88-100.

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Berger. S.. deGroot, E. J., Neuhous. G.. and Schweiger. M. (1987). Eur. J. Cell Biol. 44, 349-370. Blinks. L. R. (1936). CoM Spring Hurbor Symp. Quant. Bid. 4, 24-42. Bonotto, S . . and Gelder. E. B. V. (1969). Plan/ Physiol. 44, 1738-1740. Enomoto. S.. and Hirose, H. (1972). P h y o l o g i a 11, 119-122. Gibor. A. (1966). Sci. Am. 215, 118-124. Gibor. A. (1973a). Protoplasma 78, 195-202. Gibor. A. (1973b). Protoplusma 78, 461-465. Gibor, A. (1977a). Proroplcismu 93, 101-107. Gibor. A. (1977b). I n “Progress in Acetcihitluria Research” (C. L. F. Woodcock, ed.). Academic Press, New York. Gordon-Mills, E.. and McCandless, E. R. (1974). Proc. Int. SeuM,eed Svmp., 8th. 1973 Gunawrdena. P.. and Williamson, F. (1974). Proc. In!. Seaweed Synip.. 8/h, lY73 Gunning, 13. E. S.. and Robards, A. W. (1976). “Intercellular Communication in Plants.” Springer-Verlag. Berlin and New York. Hammerling. J. (1963). Annrt. Rei.. Hunt Ph.vsio/. 14, 65-92. Hawkes. M. W. (1978). Phycologia 17, 329-353. Hayano. S.. Itoh. T.. and Brown, R. M. (1988). Plan/ Ce// Physiol. 29, 785-794. Ishizawa. K., Enomoto, S.. and Wada, S. (1979). Bot. Mag. 92, 173-186. Itoh. T.. and Brown. R. M. (1988). Pro/op/usmu 144, 160-169. Kaska, 0. D.. Polne-Fuller. M.,and Gibor. A. (1988). J. Phycol. 24, 102-107. Kudlicka. K.. Wardrop, A.. Itoh. T.. and Brown, R. M. (1987). Proropplasma 136, 96-103. LaClaire. J . W.. 11 (1982). J. Phwol. 18, 379-384. Laclaire, J . W., 11 (1984). Eitr. J. Cell B i d . 33, 180-189. Laclaire. J . W.. I1 (1987). Planru 171, 30-42. Legge. R. L., and Brown, R. M. (1988). Protopplusmu 143, 38-42. Lewin. R. A,. ed. (1962). “Physiology and Biochemistry of Algae.” Academic Press. New York. Migita. S. (19671. Bull. Fac. Fish.. Nagasaki Unit-. 24, 55-64. Miller. J . H. 11969). Bot. Rev. 34, 361-440. Mizuta. S., and Okuda. K. (1987). Bor. Mar. 30, 205-215. Mizuta. S.. and Sawada, K. 11985). J p n . J. Phycol. 33, 32-44. Protoplasma 136, 96-103. Mizuta, S.. and Suda. S. (1978). Bot. M a g . 91, 57-68. O’Neil. R. M., and Laclaire. J. W., I1 (1984). Science 225, 331-333. O’Neil. R. M.. and Laclaire, J. W.. 11 (1988). Cyrobios 53, 113-125. Osterhout, W. J . V . (1936). Physiol. Rev. 16, 216-237. Polne-Fuller, M.. and Gibor. A. (1984). J . Phvcol. 20, 609-616. Polne-Fuller. M.. and Gibor. A. (1989). In preparation. Preston, R. D. (1974). “The Physical Biology of Plant Cell Walls.” Chapman & Hall, London. Quatrano. R. S.. Brawley. S. H.. and Hagsett. W. E. (1979). I n ”Determinants of Spatial Organization” (S. Subtelny and I. R. Konigsberg. eds.). Academic Press. New York. Schrnid. R.. Idziak. E. M.. and Tunnermann, M. (1987). Plantu 171, 96-103. Tseng. C. K . ( 1955). Sci. Sin. Engl. Ed.) 4, 375-398. Vreeland, V., and Leatsch. M. W. (1984). In “Biotechnology of Marine Algal Polysaccharides” ( R . R. Colwell. E. R. Pariser, and A. J . Sinsley, eds.).

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 118

The Centrifugal Visual System of Vertebrates: A Century-Old Search Reviewed J. REPERANT," D. MICELI,'~N. P. VESSELKIN,$ AND s. MOLOTCHNIKOFF§

*Laboratoire de Neuromorphologie U106, INSERM, H6pital de la SalpetriPre, Laboratoire de Neurochimie-Anatomie, Institut des Neurosciences CNRS, and Laboratoire d'Anatomie Compare'e M.N.H.N., Paris, France TLaboratoire de Neuropsychologie, Universite' du Quebec, Trois-RiviPres, Quebec, Canada $Laboratory of Evolution of Neuronal Interactions, Institut Sechenov, Leningrad, U.S.S.R. $De'partement Sciences Biologiques, Universite de MontrPal, Que'bec, Canada

I. Introduction

For over a century, the question of the existence of a centrifugal visual system in vertebrates has been the subject of extensive discussion and controversy. Indeed, although its presence in birds was demonstrated toward the end of the last century (Ramon Y Cajal, 1888; Perlia, 1889; Dogiel, 1895; Jelgersma, 1896; Wallenberg, 1898), it was not clearly demonstrated in other groups of vertebrates until several decades later, in spite of repeated attempts. It was only in the 1970s that this question was reconsidered with some degree of success and mainly as a result of newly developed morphological techniques involving the orthograde and retrograde axonal transport of tracers. Although numerous points regarding its anatomical organization still remain controversial, it is now generally accepted that a centrifugal visual system exists in the different vertebrate radiations. In parallel to the anatomical studies, numerous electrophysiological investigations have been performed, particularly in mammals and birds. Despite the amount of experimental data accumulated, the functional role of the centrifugal system is to this day still poorly understood and the source of diversified hypotheses. The present review has been undertaken with a comparative perspective and we shall approach the subject from both anatomical and functional points of view, group by group, from the most primitive vertebrates (lamprey) to the most evolved (mammals). Particular emphasis will be placed on the anatomy in view of the fact that, contrary to the functional 115 Copyright 0 1989 by Academic Press. Inc.

All rights of reproduction in any form reserved.

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aspects, there is a considerable volume of significant morphological data available. Furthermore, we shall give special attention to the centrifugal system of birds because of the large volume and diversity of work done in this vertebrate class.

11. Cyclostomes

The myxiniforms and the petromyzontiforms (cyclostomes), which are parasitic forms, today constitute the sole representatives of a vast body of agnath vertebrates of the Paleozoic period that gave rise to gnathostome vertebrates. Most of the data related to the centrifugal visual system of cyclostomes has essentially been obtained in Lampetra jluviatilis, a species belonging to the petromyzontiform group. Tretjakoff (1916) was the first investigator to demonstrate the existence of efferent fibers to the retina of L.fluviatilis by emphying the Golgi method and axonal labeling using methylene blue. He described two terminal zones, one situated in the external part of the inner plexiform layer (IPL), the other within the outer plexiform layer (OPL). His work went unnoticed for some time, and it is only recently that the subject of the centrifugal visual pathway was taken up again and examined in greater detail by using histophysiological and electrophysiologicaltechniques (Kosareva et al., 1977; Kosareva, 1980; Reperant et a / . , l980c,d, 1981, 1982, 1985; Vesselkin et al., 1980, 1984, 1988; Vesselkin and Reperant, 1985, 1987). Following intraocular injections of different tracers (horseradish peroxidase or HRP, [3H]adenosine. Evans blue) or the iontophoric deposit of HRP into the proximal stump of the cut optic nerve, retrogradely labeled neurons were identified essentially in the mesencephalic tegmentum, bilaterally within the nucleus M5 of Schober and contralaterally in the reticular mesencephalic area or RMA (Kosareva et al., 1977; Kosareva, 1980; Reperant er al., 1981, 1982; Vesselkin et al., 1980, 1984). Comparing the various orthograde and retrograde labeling results indicated that neurons of M5 and RMA were labeled via the retrograde axonal transport of the different tracers in the retinopetal system and not by orthograde transneuronal processes or from extraretinal pathways (Vesselkin at af., 1984). In addition, part of the anatomical data regarding RMA as a site of origin of the centrifugal visual system was confirmed using electrophysiological techniques. These involved evoked potentials and unit recordings in the RMA following electrical stimulation of the optic nerve. The experiments were performed in curarized animals under conditions of normal blood circulation, or with perfusions of adapted physiological saline or of a solu-

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tion known to block chemical transmission. Various electrophysiological criteria, including the results obtained under the conditions of reversible and chemically induced synaptic blockage, indicated that the responses in the RMA reflected antidromic processes (Vesselkin et af., 1984). The anatomical experiments demonstrated that the M5 complex comprises -560 centrifugal neurons, 85% of which project to the contralateral retina. On the other hand, the RMA contains only 190 centrifugal cells that project almost entirely to the contralateral retina. The neuronal morphology of these two cell types seems to be very different. In fact, the M5 cell bodies are pyramidal in shape and larger than those observed in the RMA, which generally appeared to be fusiform and multipolar. Dendrites of the latter neurons are flexuous and free of spines. While those of M5 are oriented lateroventrally, with some of them penetrating within the tegmentomesencephalic optic area, the majority of the dendrites of RMA neurons are oriented dorsally toward the tectum opticum (TO). In some cases, the latter penetrate the deep and intermediate tectal layers and occasionally extrude as far as the superficial layers, where retinal fibers terminate (Vesselkin et al., 1984). By using a double-labeling technique at the electron-microscopic (EM) level (Reperant et al., 1988), it has been shown that the dendrites of M5 and RMA cells receive optic endings. Thus, in L. Jluviatilis, two visual-feedback systems would appear to exist: retina + tegmental mesencephalic optic area + M5 + retina, and retina + superficial layers of TO + RMA + retina. After producing the degeneration of ganglion cells and their axons with intraocular injections of a calcium chloride solution, Vesselkin and Reperant (1985) succeeded in selectively labeling centrifugal neurons by iontophoretically depositing HRP into the optic nerve. It was shown that the centrifugal axons regrouped in the anterior mesencephalic tegmentum in a region ventral to M5 and RMA, where they gave rise to the axial optic tract. Approximately 750 centrifugal axons were counted, representing -2% of the total population of fibers contained in the optic nerve. Vesselkin et af. (1988, 1989) have examined the mode of termination of centrifugal fibers at the retinal level following the iontophoretic deposit of HRP into in vitro preparations of the nerve. On flat-mounted retinas, extremely thin (0.2 and 0.3 pm), nonmyelinated fibers were localized within the same fasciculi as the ganglion cell axons. Near the periphery of the retina these fibers often followed one or two large ganglion cell axons. Then they left the larger axons, became even thinner, and exhibited some bifurcations. The examination of transverse sections revealed that such fibers terminated within the most external zone of the IPL bordering the inner nuclear layer. The centrifugal endings in the OPL de-

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scribed by Tretjakoff (1916) could not be confirmed in this material. The EM observations showed that the centrifugal optic terminals had diameters of 0.5-1.2 pm, contained ovoid vesicles, and were most often situated in the external part of the IPL. In most cases they establish type 1 synaptic contacts upon either unlabeled somata or labeled dendrites (belonging to ganglion cells), or unlabeled profiles containing synaptic vesicles (PCSV). Unlabeled cell bodies were usually found in the internal part of the inner nuclear layer. In such cells, dense-core vesicles were observed comparable to those described in amacrine cells of the carp by Witkovsky (1971). Moreover, the unlabeled PCSV, located postsynaptically to the centrifugal elements, could not be identified as to their nature but could be subdivided into two classes: those containing pleomorphic synaptic vesicles and microtubules and those displaying flattened synaptic vesicles (Fig. I ) . In a comparative autoradiographic study of the bidirectional axonal and transcellular transport of different amino acids and sugars from the eye in L.fluviariiis, it was shown that only glycine and proline could be transported retrogradely (Reperant er al., 1980c,d, 1985). It also appeared that the M5 and RMA somata labeled after intraocular injection of [3H]glycine and ['Hlproline probably belong to two different neuronal populations. Selective retrograde transport has been documented notably for amino acids (for reviews, see Streit ef al., 1979). According to these authors, selective transport would result from a high-affinity uptake of the injected marker by the axon terminal due to the structural similarity between the marker and the axon terminal transmitter. There are numerous indications that glycine and proline might both be inhibitory transmitters. For example. high-affinity uptake has been observed for glycine and proline in synaptosomes. Also, iontophoretic application of these substances induces an inhibition of neuronal discharge in the vertebrate brain (for review, see Felix and Kunzle, 1974, 1976). In view of this, the selective retrograde transport of glycine and proline in the lamprey centrifugal visual system may be related to the fact that they are indeed the neurotransmitters of this system. However, there is as yet no direct evidence indicating that the centrifugal pathway in Lampetra is indeed prolinergic and glycinergic. There is very little information available in cyclostomes regarding the physiological properties of their centrifugal visual system. The sole experiment involving the recording of antidromic and synaptic potentials obtained upon optic nerve stimulation in the lamprey indicated activation of amacrine cells, which i n turn influenced ganglion cells, as well as direct influences of centrifugal fibers on ganglion cells (Vesselkin et al., 1989).

FIG. 1. Electron micrograph of the lamprey retina showing (A) HRP-labeled centrifugal boutons (LCB) making contacts with nonlabeled profiles containing synaptic vesicles ( X 31,320), and (B) a synapse between the LCB and a nonlabeled somatic profile ( x 32,480).

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111. Fish

A. ANATOMY 1. Teleosts

For a long time the existence of a retinopetal system has been postulated in teleosts. Indeed, from the end of the nineteenth century until the 1930s, anatomists continued providing fragmentary, though at times contradictory, information regarding the presence of centrifugal optic fibers in these fish. In 1893, Ramon y Cajal observed the presence of axons in the superior region of the IPL of the retina, which terminated in free endings in the vicinity of amacrine cells. In 191 1, this same investigator believed that some neurons of the TO sent their axons to the retina. In enucleated fish, Krause (1898) observed a nondegenerated optic fascicle emanating from the TO (fasciculus fibrae tectalis nervus optici) that terminated within the retina. However, Jansen (1929) contested this interpretation and described this fascicle as a component of the postoptic commissure. Catois (1902) noted the existence of centrifugal optic fibers stemming from the corpus geniculatum thalamicus in different fish. A retinopetal fascicle (tractus isthmoopticus or TIO) emanating from the isthmic nucleus was also described by Franz (1912). Subsequently, however, these results were contested by Kudo (1923), Jansen (1929), and AriensKappers et al. (1936). According to Holmgren (1918, 1920), four retinopetal contingents are present in teleosts: the tractus recessoopticus stemming from the nucleus preopticus recessi, the tractus opticus posterior from the nucleus preopticus pars magnocellularis, and finally, emanating from the olfactory region, the tractus olfactoris lateralis optici and the tractus olfactorius opticus. The existence of the tractus preopticus opticus was confirmed by Jansen (1929), whereas Kudo (1923) observed the tractus olfactorius opticus in his preparations. In this early period, evidence for centrifugal innervation of the teleost retina was also derived from studies showing that retinomotor responses depended on the integrity of the optic nerve (Arey, 1916). The question of centrifugal fibers in teleosts was reexamined in the 1970s. at first mainly using electrophysiological techniques, and then later with histophysiological and immunohistochemical met hods. Indirect proof of the existence of a retinopetal pathway was provided by Sandeman and Rosenthal (1974), who showed that retinal ganglion cells are excited by a variety of somesthetic stimuli. Other studies have found myelinated fibers synapsing on the goldfish retina (Witkovsky, 1971), and there is electrophysiological evidence that the TO contains neurons that project to the retina (Vanegas et al., 1973; Schmidt, 1979).

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Subsequent studies have attempted to identify the central cells that projpect to the teleost retina by retrogradely labeling them with different tracers (HRP, cobaltous lysine, fluorochromes) administered by either intraocular injection or iontophoretic deposit in the optic nerve. Labeling obtained via the latter route has generally been interpreted as demonstrating the sites of origin of centrifugal visual pathways. However, it is important to note that the topographical localization of these different structures of origin varies greatly from one species to another, and also within the same family. In addition, contradictory results have sometimes been obtained in the same species. In teleosts, 30 species belonging to 15 different families have been investigated using these methods, and the results obtained were extremely varied. In the Ameridae and Notopteridae, no retrogradely labeled structures were observed (Munz et al., 1982), whereas in the Cyprinidae, the results were contradictory. In Zdus idus (Munz et a/., 1982) and Rutilus rutilus (Peyrichoux et al., 1977), no labeled neurons were found after the intraocular injection of HRP. In Carassius auratus the data are again even more contradictory. Schmidt (1979) described the retinopetal cells in this species and found them to be distributed bilaterally in the TO. There were more cells in the contralateral than in the ipsilateral TO, and these were distributed in the stratum fibrosum and griseum superficiale. However, no labeled cells were found in the TO by administering the same tracer through intraocular injection (Uchiyama et al., 1981; Demski and Northcutt, 1983; Munz et al., 1982), or by applying cobaltous lysine to the optic nerve (Springer and Gaffney, 1981; Springer, 1983). However, labeled neurons have been observed either bilaterally interspersed between the olfactory nerve fibers or rostrally along the ventromedial aspect of the olfactory bulb (Demski and Northcutt, 1983; Springer, 1983; Stell et al., 1984). These cells as a whole have been reported to correspond to the ganglion cells of the nervus terminalis. In other families investigated, the number of contralaterally labeled structures has differed between one (Pontodontidae: Gerwerzhagen et al., 1982; Centrarchidae: Bazer and Ebbesson, 1985; Belontiidae: Oka et al., 1986; Anguillidae: Grober et al., 1987; Percichthyidae: Zucker and Dowling, 1987), two (Balistidae: Uchiyama et al., 1981; Matsutani et al., 1986; Anabandidae: Munz et al., 1982; other Centrarchidae: Munz et al., 1982; Chamnidae: von Bartheld and Meyer, 1988; some Ciclidae: Springer and Mednick, 1985; Fritzsch et a / . , 1987), three (Mockolidae: Ebbesson and Meyer, 1981; Meyer and Ebbesson, 1981; Poeciliidae: Munz and Claas, 1981; Munz et al., 1982), four (Tetraodontidae: Ebbesson and Meyer, 1981; Meyer et al., 1981; most Ciclidae: Munz et al.,

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1982), and five (in other Ciclidae: Ebbesson and Meyer, 1981). The latter are situated within the TO (stratum griseum and fibrosum superficiale and stratum griseum centrale, Tetraodontidae: Ebbesson and Meyer, 1981 ; Meyer et al., 1981; Mockolidae: Ebbesson and Meyer, 1981, Meyer and Ebbesson, 1981; Ciclidae: Ebbesson and Meyer, 19811, in the pretectum (subnucleus of the pretectal complex, Ebbesson and Meyer, 1981; or pretectal nucleus, Munz at al., 1982; Tetraodontidae: Ebbesson and Meyer, 1981; Meyer et a [ . , 1981; Mockolidae: Ebbesson and Meyer, 1981; Meyer and Ebbesson, 1981; Ciclidae: Ebbesson and Meyer, 1981; Munz et al., 1982; Anabandidae: Munz et al., 1982; Centrarchidae: Munz et al., 1982; Poeciliidae: Munz and Claas, 1981; Munz et al., 1982), in the medial thalamus (dorsomedial optic nucleus: Ebbesson and Meyer, 1981; or nucleus thalamoretinalis: Springer and Mednick, 1985; Tetraodontidae: Ebbesson and Meyer, 1981: Meyer et al., 1981; Mockolidae: Ebbesson and Meyer, 1981; Meyer and Ebbesson, 1981; Ciclidae: Ebbesson and Meyer, 1981; Springer and Mednick, 1985; Fritzsch et al., 1987; Chamnidae: von Bartheld and Meyer, 1988), in the lateral thalamus (corpus geniculatus lateralis ipsum, Tetraodontidae: Ebbesson and Meyer, 1981; Meyer et al., 1981; Ciclidae: Ebbesson and Meyer, 1981), in the ventral hypothalamus (Poeciliidae: Munz and Claas, 1981; Munz et al., 1982), in the preoptic region (preoptic area, Balistidae: Uchiyama et al., 1981; Uchiyama and Ito, 1984; Matsutani et al., 1986; nucleus intrachiasmaticus, Pontodontidae: Gerwerzhagen ef a / . , 1982). Other similarly labeled structures have been found in the ventromedial anterior telencephalon (nucleus olfactoretinalis: Munz et al., 1981, 1982; or ganglion cells of the nervus terminalis: Springer, 1983; or the telencephalic optic nucleus: Ebbesson and Meyer, 1981; Ciclidae: Ebbesson and Meyer, 1981; Munz et al., 1982; Springer and Mednick, 1985; Crapon de Caprona and Fritzch, 1983; Munz at al., 1982; Centrarchidae: Munz el al., 1982; Anabandidae: Munz et al., 1982; Balistidae: Matsutani et ul., 1986; Belontiidae: Oka et al., 1986; Anguillidae: Grober et al., 1987; Percichthyidae: Zucker and Dowling, 1987; Chamnidae: von Bartheld and Meyer, 1988). Ipsilateral labeling has rarely been reported (in Centrarchidae: Munz et a / . , 1982; Anguillidae: Grober et al., 1987; Balistidae: Matsutani er al., 1986; Ciclidae: Capron de Caprona and Fritsch, 1983). Because of insufficient controls in several experiments, the positive findings just described may reflect the identification of some cell groups that actually project to the retina but also may represent cells that have been labeled either transneuronally or from extraretinal axon terminals that are intraocular or extraocular. However, taking into account the validity of the different controls performed, it would seem that at least two neuronal

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structures indeed do project onto the retina: the preoptic retinopetal nucleus (PRN) and the nucleus olfactoretinalis. a. Preoptic Retinopetal Nucleus (PRN). This visual centrifugal core has been mainly identified in Navodon modestus (Uchiyama et al., 1981, 1985, 1986; Uchiyama and Ito, 1984) and contains 8000-10,000 ovalshaped cells of between 7 and 10 pm diameter, regrouped in a peripheral ring around a central neuropilary zone. The centrifugal axons stemming from the core represent -45% of the total number of fibers in the optic nerve. These axons have been traced between the IPL and the inner nuclear layer of the retina. It has also been demonstrated that the PRN receives a discrete projection from the contralateral retina and a strong projection from the TO (mostly ipsilateral) and more particularly from large pyriform neurons in the stratum album centrale. The latter are themselves the targets of retinal terminals and nonoptic afferents. Therefore, in this species, a reciprocal connection between the retina and the PRN (retina --f PRN + retina) exists, together with a more complex circuit involving the TO (retina + TO --., PRN retina). On connectional grounds, this feedback loop is, in some respects, comparable to the retino + tecto + isthmo --f retinal system of birds (see Section VI on birds). --f

b. Nucleus Olfactoretinalis. This comprises all of the ganglion cells of the nervus terminalis (GCNT), which project onto the retina. In species with a sensile olfactory bulb, the GCNT are located ventrally at the border behind the olfactory lobe and the forebrain (notably in poecilid and ciclid fish). In contrast, in teleosts with an olfactory bulb connected to the forebrain by a rather long olfactory bulb tract, the GCNT are located both rostra1 and ventral to the olfactory bulb (especially in goldfish). The number of GCNT varies according to the species (from -20 to 500). It has been shown that the GCNT increase throughout life (Crapon de Caprona and Fritzsch, 1983). Only some of these cells project onto the retina (50% in Astronotus: Springer and Mednick, 1985; 6040% in different ciclid, centrarchid, and poecilid fish, Munz et al., 1982). Two types of GCNT project onto the retina: (1) large multipolar neurons (225 pm diameter) and (2) pear-shaped medium-sized neurons (12-15 Krn diameter) (Munz et al., 1982; Crapon de Caprona and Fritzsch, 1983; Matsutani et al., 1986). The second type are five to eight times more numerous than the first. The GCNT receive afferents from different areas of the telencephalon (Matsutani et al., 1986), but also from the isthmic region (von Bartheld et al., 1986). On several occasions it has been shown that the dendritic processes of type 2 cells penetrate the olfactory bulb and proba-

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bly enter into contact with mitral cells of the olfactory glomeruli (Springer, 1983; Crapon de Caprona and Fritzsch, 1983; Oka et a / . , 1986; Matsutani et a / . , 1986). von Bartheld and Meyer (19861, in work carried out on the goldfish, showed that GCNT axons could be classified into three categories according to their trajectory to the retina. All fibers course within the lateral part of the medial optic tract. Cells of the first type cross at the level of the anterior commissure and recross in the optic chiasma before terminating in the ipsilateral retina. Axons of the second type course across the telencephalon, reach the medial optic tract, cross over at the level of the chiasma, and arborize in the contralateral retina. In both cases the axons exhibit little collateralization. Axons of the third type show different characteristics. Indeed, these fibers are of large diameter and display a high degree of collateralization at the level of the rostra1 diencephalon. Some of the collaterals cross through the horizontal commissure and several continue toward the contralateral retina, whereas others course through the posterior commissure. Thus, a single GCNT fiber, besides innervating the retina, projects to numerous ipsilateral and contralateral structures of the diencephalon and mesencephalon. Elsewhere, Stell et a / . (1984) have demonstrated the existence of some GCNT projecting bilaterally upon the retina using the double-labeling technique in this same species. Upon reaching the retina, the centrifugal axons pass through the IPL and course along the limit between the inner plexiform and inner nuclear layers. A number of studies in a variety of teleost species have shown that the olfactoretinalis system contains substances immunochemically related to luteinizing hormone-releasing hormones (LHRH) and molluscan cardioexcitatory tetrapeptide (FMRF amide) and substance P (Munz et a / ., 1981, 1982; Stell et al., 1984, 1988; Zucker and Dowling, 1987; Kah et a / . , 1986; Muske er al., 1987; Grober et al., 1987). In the goldfish, Stell et a/. (1984) have shown that the three peptides coexist in the GCNT efferent to the retina. In the white perch, Zucker and Dowling (1987) showed that LHRH and FMRF amides are colocalized within the large vesicles present in the olfactoretinal terminals (Fig. 2). According to Stell et af. (1984, 1988), in the goldfish these boutons terminate upon two types of amacrine cells, and possibly bipolar and ganglion cells. On the other hand, Zucker and Dowling (1987) found that in the white perch the olfactoretinal fibers terminate mainly on the dopaminergic interplexiform cells (IPC). 2. Other Fish Species Very little information is available concerning the existence of a centrifugal visual system in fish apart from teleosts. No data are available on chondrosteans, holosteans, and dipnoans. In brachiopterygians (polypteri-

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OPllC 1.Cl"rn

FIG.2. Summary diagrams of the origins and synaptic connections of the olfactoretinalis-dopaminergic interplexiform cell (DA-IPC) system in the white perch. In the retina, the centrifugal fibers (solid black) ramify in the most distal inner plexiform layer (IPL) and make extensive synaptic contact onto the perikarya and proximal dendrites of DA-IPC (stippled). Dopaminergic interplexiform cells, in turn, provide extensive synaptic output to cone horizontal cells (HLH3) in the outer plexiform layer (OPL) and some synaptic input to bipolar (B)-cell dendrites in the OPL and to amacrine (A) cells in the IPL. The IPC themselves also receive input from amacrine cells, and some amacrine cells are probably contacted by centrifugal fibers. C, Cones; H4, rod horizontal cell; G, ganglion cell. From Zucker and Dowling (1987).

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forms), a single study by Meyer et a f . (1983) refers to a centrifugal optic system (described as an isthmooptic system) that has its origin in the mesencephalic tegmentum. Projections toward the retina are bilateral, but with a contralateral predominance. The retinopetal neurons form two rather close neuronal groups, one of which is located near to the ventricle (nucleus opticus pars superficialis), and the other more laterally and ventrally (nucleus opticus profundus). As far as the selacians are concerned, the data are equally scarce. Only Luiten (1981), in a study of the shark Gingfymostoma cirratum, noted the presence of labeled neurons within the TO (zona externum of the stratum cellulare externum) following the contralateral intraocular injection of HRP. Moreover, Witkovsky (1971), using EM, described centrifugal terminals that synapse upon amacrine cells in the retina of sharks. B. FUNCTIONAL CONSIDERATIONS The findings in the white perch-that olfactoretinal fibers synapse upon dopaminergic IPC (Zucker and Dowling, 1987), which in turn synapse upon horizontal and bipolar cells-suggest that the routing of centrifugal information is from the IPL to the OPL. Thus, centrifugal influence can reach bipolar and horizontal cells via dopaminergic IPC, and even the photoreceptors via feedback from cone horizontal cells (Baylor et a f . , 1971). It follows, then, that in the teleost, every retinal neuron is potentially susceptible to central influence. Umino and Dowling (1988) have suggested that LHRH permits dopamine release from IPC, whereas FMRF amide produces an opposite effect. The centrifugal control of photoreceptors by way of the IPC and the presence of two peptides with antagonistic effects further suggests that retinopetal fibers may facilitate as well as reduce photoreceptor signals. Working on the isolated superfused goldfish retina, Stell et af. (1984) have studied the effect produced by FRMF amide and LHRH present in the olfactoretinal endings on the activity of ganglion cells. “These two substances caused increased spontaneous activity in the dark loss of lightinduced inhibition, and increased incidence of light-retrained pulsative response. The authors conclude that the olfactoretinal system can modulate the output of ganglion cells responsive to color contrast. However, according to Davis et a f .(1988), the olfactoretinal system in goldfish neither enhances nor inhibits scotopic photosensibility for large unfocused stimuli. Several lines of research have suggested that the GCNT in teleosts may respond to sex pheromones and thereby regulate sexual and reproductive behavior (Demski and Northcutt, 1983; Stacey and Kyle, 1983). The GCNT “may respond to olfactory or chemical cues present in ”

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the water or pheromone present during sexual behavior, and in turn may have either a general or a specific excitatory effect on retinal transmission. The ultimate consequence may be the initiation of a variety of visually-mediated behaviors. ”

IV. Amphibians A.

ANATOMY

Although some investigators have provided physiological evidence for the existence of a centrifugal system in amphibians (Borchers and Ewert, 1978; Byzov and Utina, 1971; Branston and Fleming, 1968; Tasaki et al., 1978), anatomists have had far greater difficulty in revealing its presence, and consequently the overall data is very contradictory. We shall consider the case of anurans, where the search for a retinopetal pathway has been the object of numerous studies. Larsell (1924) was the first investigator to signal the existence of a centrifugal visual system in the frog. In Golgi preparations he described an isthmothalamic tract where some of the fibers reached the retina. Some years later, Rozemeyer and Stolte (193I ) observed several centrifugal fibers arborizing within the IPL in retinas of Rana esculenta prepared according to the Golgi Cox method. Maturana (1958) also noted the presence of -40 myelinated and nondegenerated fibers in the central end of the nerve 200 days after sectioning the optic nerve in Bufo bufo. He interpreted these fibers to be of nonretinal origin and efferent in nature. In two studies on tectal projections employing the degeneration techniques, Rubinson (1968) and Lazar (1969) described degenerating fibers in the optic nerve on the side contralateral to the lesioned tectum, in Rana pipiens and R . esculenta, respectively. These investigators thus assumed the existence of a tectoretinal pathway in anurans. From 1975 onward, the repeated use of tracing techniques in many species ( R . esculenta, R. pipiens, Rana catesbiana,Bufo marinus, Bufo americanus, Xenopus laevis) following either the intraocular injection or the iontophoretic deposit of tracers such as HRP, did not lead to the confirmation of the earlier data. Some authors (Scalia and Teitelbaum 1978) go as far as to refute the existence of a retinopetal pathway in anurans. In contrast, Fuller and Prior (1975) suspected the existence of a hypothalamoretinal pathway after noting some labeled neurons in the anterior hypothalamus following the deposit of cobalt salts into the optic nerve. But numerous other investigators using HRP and short survival periods (intraocular injection or iontophoretic deposit into the nerve) have reported

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a total absence of somatic labeling in the brain (Scalia and Teitelbaum, 1978; Vesselkin et al., 1979; Wilczynski and Zakon, 1982; Ermakova et al., 1981; Fritzsch and Himstedt, 1981; Wirsig-Wiechmann and Basinger, 1988). On the other hand, after long survival periods, very intense somatic labeling has been reported within the TO and attributed to the transcellular transfer of HRP from the optic endings (Ermakova et al., 1981; Wilczynski and Zakon, 1982). This question has reemerged in light of new immunohistochemical findings in R. pipiens (Wirsig-Wiechmann and Basinger, 1988), demonstrating the existence of a centrifugal visual system that is peptidergic, FMRF amidelike, and stemming from the rostral telencephalon. Within the optic nerve, the FMRF amide immunoreactive (ir) centrifugal fibers, numbering eight on the average, are confined to one small area. The fibers, which have diameters ranging between 0.5 and 1 pm, are nonmyelinated and display varicosities every 20 pm. As they enter the retina, the centrifugal fibers usually display frequent branching before reaching the peripheral retina, and in the vicinity of the optic disk pass mainly within the nerve fiber layer. After they leave the optic nerve head, frequent bifurcations occur and collaterals are seen coursing through the IPL, reaching its sublayer 1. They cross the IPL in a quasivertical direction, o r pass through it over long distances (200-400 pm) until they course horizontally along the proximal boundary of the amacrine cell layer, where they appear to synapse. A distribution analysis revealed that twice as many fibers project upon the dorsal half of the retina as the ventral half, and that the right retina receives more centrifugal fibers than the left. Very few centrifugal fibers, if any, were noted to cross over at the optic chiasm to project to the contralateral retina. Wirsig-Wiechmann and Basinger concluded, however, that there were at least two origins of FMRF amide-ir retinopetal fibers: a rostral cell group possibly corresponding to the nervus terminalis, and a caudal population of cells situated somewhere between the lamina terminalis and the optic chiasma. Comparable results have been obtained in two other anuran species: R. catesbiuna and X . luevis (Uchiyama et al., 1988). The immunohistochemical data revealed the presence of -30 fibers in the optic tract that were either FMRF amide- or N-terminal substance P-immunoreactive. Their mode of arborization within the retina resembles that observed in R. pipiens (Wirsig-Wiechmann and Basinger, 1988). However, compared to this latter species, the branching of the fibers were here described to occur less frequently. Furthermore, Uchiyama et al. (1988) reported that their number is very high in the central region of the retina compared to the periphery. Within the most proximal portion of the optic nerve, the fibers constitute a tight and superficial bundle that penetrates the ipsilateral

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preoptic area, then continues to the septopreoptic functional area (lamina terminalis), a region observed to contain numerous FMRF amide- and N-terminal substance P-immunoreactive terminals. Following the intraocular injection of HRP, some labeled neurons were observed within this same area. Reciprocally, the injection of HRP into the lamina terminalis resulted in the labeling of a few fibers within the optic nerve. Uchiyama et al. concluded that the FMRF amide and/or N-terminal substance P efferent fibers to the retina originate essentially in the lamina terminalis. In contrast to the results of Wirsig-Wiechmann and Basinger, Uchiyama et al. (1988) considered that the GCNT, which are essentially LHRH-immunoreactive, do not project directly to the retina in anurans, as is the case in teleosts. Instead, they would appear to innervate the centrifugal optic neurons of the lamina terminalis, which in turn would relay to the retina. In the other amphibian forms, data concerning the retinopetal pathway are few and fragmentary. In 1933, Herrick described on Golgi preparations in Ambystoma a tract of fibers entering the optic nerve of the same and opposite side and arising in the posterior part of the nucleus preopticus. However, Herrick was unable to reconfirm this observation in a later study performed in 1948. In axolotl, Weber (1945) noted the existence of nondegenerating fibers in the central stump of the optic nerve several months after it was sectioned. He considered these fibers to be centrifugal, and situated their origin within the hypothalamus. Using the HRP method, Fritzsch and Himstedt (1981) have traced retinopetal fibers to their cell bodies in the pretectal region in several salamander species (Salamandra salamandra, Triturus vulgaris, Triturus cristatus). The labeled cell bodies, which are fusiform in shape, have been found bilaterally at the diencephalic-mesencephalic border, caudolateral to the commissura posterior. Their numbers (26 or 28) are distributed almost equally on both sides of the brain.

B. FUNCTIONAL CONSIDERATIONS Following optic nerve stimulation, Byzov and Utina (1971) recorded excitatory postsynaptic potentials (EPSP) associated with a series of high-frequency spikes in the frog retina and considered these to be amacrine cell responses to synaptic activation by retinopetal terminals. In addition to an antidromic component, some ganglion cells displayed a late postsynaptic potential, which was believed to correspond to synaptic potentials from amacrine cell contacts, the latter being directly stimulated by retinopetal fibers. By using extracellular recordings obtained from the frog retina and optic nerve, Branston and Fleming (1968) showed that

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associating auditory or cutaneous stimuli with light pulses presented in the receptive fields of retinal cells led to a decreased light-evoked discharge in OFF and ON-OFF retinal neurons. The spontaneous activity in the retina was also altered by association with clicks and somatosensory stimulation. A shift in the peaks of the interval histogram of the responses in an ON-OFF unit was also observed, and the authors concluded that the efferent fibers influence the inhibitory cellular elements of the retina.

V. Reptiles

A. ANATOMY Although some electrophysiological studies had provided indirect evidence of a centrifugal visual system in reptiles (Marchiafava, 1976; Cervetto et al., 1976), the anatomists must take credit for its definitive identification. The question was first raised in various experiments examining axonal degeneration in the optic nerve following retinal ablation (Armstrong, 1951; Kruger and Maxwell, 1969; Reperant el al., 1981). The survival of optic nerve fibers observed several months after production of the lesion was generally considered as proof of the presence of a retinopetal pathway in reptiles. In the grass snake enucleated unilaterally at 19 weeks, Armstrong (1951) noted perfectly normal fibers in the central portion of the optic nerve, the origin of which could be situated within the supraoptic and suprachiasmatic nuclei. In the optic nerve of Alligator mississipiensis and Cuirnan crocodilus, 20 months following enucleation, Kruger and Maxwell (1969) found -4000 myelinated fibers of relatively normal appearance, representing -5% of the total population of myelinated optic axons. Considering the slow process of axonal degeneration in these forms, they concluded that these fibers corresponded either to a predominant efferent population or to a slowly degenerating population of afferent fibers. Finally, in the optic nerve of Viperu aspis 4 months after retinal ablation, Reperant et ul. (1981) observed -650-700 normal myelinated fibers of large diameter, situated at the nerve periphery. The latter were found to degenerate only 11-12 months after the lesion and were considered to be centrifugal, forming 1% of the total axon population of the optic nerve. Further proof of the existence of these fibers in various species, by demonstrating their source neurons, was later provided through the use of hodological techniques based on the retrograde axonal transport of tracers from the retina (HRP, [-'H]adenosine, nuclear yellow, granular blue). In most species, the labeling of the centrifugal neurons is bilateral

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but exhibits a contralateral bias. The location of labeled neurons also varies considerably. For example, they may be situated either in the caudal mesencephalic tegmentum (in the crocodilian C. crocodilus: Ferguson et al., 1978; in the turtle Pseudemys scripta elegans: Cervetto, 1976; Schnyder and Kunzle, 1983; Weiler, 1985; in the lizards Gerrhonotus coeruleus: Halpern et al., 1976; Varanus exanthematicus: Hoogland and Welker, 1981; Ophisaurus apodus: Kenigfest et al., 1986, in the ventral thalamus (in the snakes Crotalus viridis: Schroeder, 1981; Thamnophis sirtalis and Thamnophis radix: Halpern et al., 1976; V . aspis: RepCrant et al., 1980a,b, 1981; in the lizard Cordylus cordylus: Halpern et al., 1976), and in the basal areas of the telencephalon (in the snake Python reticulatus: Hoogland and Welker, 1981). 1 . Caudal Mesencephalic Tegmentum

According to work by Ferguson et al. (1978) in C . crocodilus, many retrogradely labeled cells are to be found bilaterally but predominantly contralaterally in one large oblong cell field at the isthmic level, bounded medially by the trochlear nucleus and laterally by the isthmic nucleus. Those in the lateral portion of the isthmic cell field are smaller (10-19 pm), more compactly organized than those found medially (15-20 pm), and they seem to be multipolar and spindle-shaped neurons. The isthmic cell field receives a strong projection from the TO. On the basis of its location, the number of its neurons, and its relations with the TO, the isthmic cell field of Caiman appears very similar to the avian nucleus isthmoopticus (NIO; and its ectopic satellite cells). In the turtle Ps. scripta elegans, the authors (Schnyder and Kunzle, 1983; Weiler, 1985) agree that the location of the centrifugal neurons is a rather large and poorly defined region of the caudal mesencephalic reticular formation, which is situated approximately between the trochlear and the isthmic nuclei. These neurons have a diameter of 10-20 pm. They are generally bipolar and are far fewer than in Caiman. Between 4 and 12 labeled neurons were found on the side contralateral to the injection of the tracer, whereas only 1-3 such cells were observed ipsilaterally. Very little information is available in lizards regarding the characteristics of the centrifugal optic neurons in the mesencephalic tegmentum. 2. Thalamus In all of the coenophidian snakes examined (C. veridis, Th. sirtalis, Th. radix, V . aspis), the centrifugal visual neurons are found within a ventrolateral thalamic nucleus described as the nucleus of the ventral commissure (Halpern et al., 1976; Schroeder, 1981), or the centrifugal optic thalamic nucleus (Reperant et al., 1980b, 1981). Situated rostrally

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to the n. ovalis, it continues caudally up as far as the boundary of the posterior thalamus beneath the nucleus geniculatus lateralis pars ventralis. In Thamnophis, the HRP labeling of centrifugal neurons was essentially contralateral, whereas it is bilateral with a strong contralateral bias in Crotalus and Viperu (estimated at 82.5% in the latter species). In contrast to that found in Viperu, this nucleus comprises two portions in Crotalus: a rostral portion with large neurons and a caudal portion containing smaller cells. Furthermore, the former projects bilaterally, whereas the latter’s connections are essentially distributed to the contralateral retina. In V. uspis, a total of 660 centrifugal neurons have been identified (a number comparable to that of nondegenerdting fibers of the optic nerve 4 months after retinal ablation). Such neurons are bipolar, exhibit diameters of 15-22 pm, and show clear signs of degeneration 6 months after lesions of the retina (Reperant et ul., 1981). 3. Tek?ncepha/on

In the hoenophidian snake P. reticulutis, the centrifugal optic fibers arise within the telencephalon. Following intraocular injections of HRP, granular blue, or nuclear yellow, Hoogland and Welker (1981) observed numerous retrogradely labeled multipolar neurons of medium size in a basal area of the telencephalon. These cells were distributed more or less equally on both sides of the brain. They were not restricted to a particular cell group, but rather were scattered throughout a large region of the basal telencephalon. They were mostly located rostral to the anterior thalamus, although some were found in the lateral preoptic area. The labeled neurons formed a continuous horizontal band immediately in front of the anterior commissure. Even more rostrally, this band of cells could be divided into a medial and lateral group. When nuclear yellow was injected into the left eye and granular blue into the right eye, no double labeling of neurons was detected. In a n immunohistochemical study performed in the turtle Ps. scriptu eleguns, Weiler ( 1985) demonstrated the peptidergic nature of certain cells of the centrifugal optic pathway, while also describing their mode of innervation in the retina. He showed that one-third of the centrifugal neurons of the mesencephalic tegmentum exhibit mer-enkephalinlike immunoreactivity (ELI). In the nerve, the ELI axons number three to six, have a diameter of 1.5 pm, and are beaded and unmyelinated. They penetrate the retina and then course toward the visual streak, a fovealike structure in the turtle retina, entering the inner retina at this level. These axons collateralize at the level of the ganglion cell layer, and the collaterals terminate within the IPL. Their terminal arborization extends 100 pm horizontally and branches predominantly within layers 3 and 4 of the IPL.

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Furthermore, in colocalization studies of retrograde-transported nuclear yellow and serotonin immunoreactivity performed in this same species, Schutte and Weiler (1988) showed a single neuron of the mesencephalic tegmentum to be serotoninergic. The centrifugal axon stemming from its soma arborized exclusively in the temporal hemiretina, exhibiting very numerous ramifications that covered about one-third of the total surface of the retina.

B. FUNCTIONAL CONSIDERATIONS Intracellular recordings of amacrine cells of the turtle retina to optic nerve stimulation have demonstrated centrifugal excitatory effects on amacrine cells as witnessed by large depolarizations with properties of EPSP (Marchiafava, 1976). Also in turtles, Cervetto et al. (1976) studied the interaction between visual and centrifugal inputs to ganglion cells. It was found that when the photoresponse in the ganglion cell coincides with the gradual synaptic response obtained via optic nerve stimulation, there is increased sensitivity to illumination of the central with respect to peripheral areas. This redistribution of excitability over the ganglion cell receptive field suggested a specialized role of the centrifugal system for the detection of light stimuli of low intensity. The excitation appears to be transmitted to ganglion cells by way of the extensive connections stemming from amacrine cells. Consequently, ganglion cells are influenced both by visual input derived via the photoreceptors and by centrifugal input possibly mediated by amacrine cells.

VI. Birds A. ANATOMY Ramon y Cajal was the first investigator to become aware of the possible existence of centrifugal projections to the avian retina. From 1888 onward, working on retinas of sparrows processed according to the Golgi method, the Spanish author noted the presence of axons that emerged from the fiber-optic layer, crossed the IPL, and appeared to terminate among the amacrine cells along the internal or vitreal aspect of the inner nuclear layer. Considering that such axons did not stem from any intraretinal cell body (recurrent collaterals of ganglion cells have never been demonstrated in birds), he concluded that they originated in the brain and thus represented centrifugal visual fibers. In 1895 the Russian Dogie1 (followed by his fellow countryman Ressnikoff, 1897) confirmed the exis-

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tence of such fibers in chicken and pigeon retinas by using either the Golgi or the methylene blue technique. Ramon y Cajal’s hypothesis regarding the centrifugal nature of such fibers received further support after Dogie1 was able to trace the fibers back into the optic nerve head. Unfortunately, these different authors were probably not aware that relevant observations had been made at about the same time by Perlia (1889), who provided the first experimental evidence regarding the central origin and course of the centrifugal fibers. Several months after performing a monoenucleation in the newborn chicken, he observed through retrograde degeneration the atrophy of a nucleus in the dorsal mesencephalic tegmentum (which he named nucleus medialis opticus) and its ascending tract (designated medialis opticus bundel) situated on the side contralatera1 to the operation. Comparable results were obtained some years afterward by Jelgersma (1896), and subsequently by Kosaka and Hiraiwa (1915), Huber and Crosby (1929), and Cowan el al. (1961). But it was in 1898 that the German investigator Wallenberg, after performing an extremely refined experiment, provided unquestionable proof that this nucleus indeed did project to the retina. Wallenberg showed that after lesions of the nucleus (which he called nucleus isthmi) in the chicken, degenerating fibers stained with the Marchi method could be traced rostrally in the “medialis opticus bundel,” across the optic chiasma, and contralaterally through the medial part of the optic nerve into the nerve fiber layer of the retina, where they spread out to all regions of the retina. As far as it was possible to determine, they seemed to terminate close to the ganglion cells. These important findings went unnoticed for the next 50 years. As a matter of fact, it was only in 1961, at a time when anatomical work on the visual system of birds was undertaken again in Great Britain (Cowan. et NI.,1961; Cowan and Powell, 1963), that attention was focused on the earlier observations of Wallenberg. Important progress in our understanding of the centrifugal visual system of birds was accomplished from 1964 onward with the remarkable work of McGill (1964) and McGill ef al. (1966a,b) in the Department of Human Anatomy at the University of Oxford. By using tracing techniques in the pigeon based on the demonstration of axonal degeneration after creating lesions, these investigators showed that the centrifugal optic nucleus (or nucleus isthmoopticus, NIO) constituted the key structure in a feedback loop (retino + tecto -+ isthmo + retina) in which retinotopy was preserved. It was also shown that the different zones of the N10 project upon the corresponding retinal zones from which they receive their input relayed via the tectum. In other words, each part of the retina is connected through the TO with the corresponding region of the N 1 0 from which it receives its centrifugal input. These results were subse-

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quently confirmed in the chicken by using other anatomical tracing methods (Crossland et a/., 1974; Catsicas et al., 1987). Numerous studies in the last two decades have concentrated on the centrifugal visual system. Furthermore, the use of intraocular injections of tracers transported by the retrograde axonal flow [HRP, Wheat germ agglutinin (WGA)-HRP, fluorescent tracers] enabled investigators not only to label the neuronal somata of the NIO (La Vail and La Vail, 1972), but also to demonstrate that other cells surrounding the NIO and referred to as “ectopic” project to the retina (Clarke and Cowan, 1976; Hayes and Webster, 1981; O’Leary and Cowan, 1982; Weidner et a/., 1987; WolfOberhollenzer, 1987). Various other experiments were aimed at the study of the development of the centrifugal system, most notably those by Cowan and his students and co-workers (Cowan and Wenger, 1968; Cowan and Clarke, 1976; Clarke and Cowan, 1976; Clarke et a/., 1976; Clarke, 1982; Clarke and Caranzano, 1985; O’Leary and Cowan, 1982). Finally, many authors attempted to determine the functional role of the system (Holden, 1968a,b; Holden and Powell, 1972; Miles, 1970, 1971, 1972a-d; Galifret et al., 1971; Pearlman and Hughes, 1976a,b; Rogers and Miles, 1972; Shortess and Klose, 1977; Knipling, 1978; Uchiyama er al., 1987; see Section V1,B). The following sections contain a review of the different data concerning the NIO, the ectopic centrifugal optic neurons, the TIO, and the mode of termination and distribution of the centrifugal fibers in the retina. 1 . The Isthmo-optic Nucleus (NIO}

In birds, the majority of neurons projecting to the retina are aggregated within a nucleus (nucleus isthmoopticus NIO), situated in the dorsal aspect of the avian midbrain adjacent to the pia and medial to the caudodorsomedial edge of the TO at the level of the trochlear nucleus. The nucleus, though variable in size depending on the species, has been observed in all birds examined-with the exception of the kiwi (Craigie, 1930), ibis (Showers and Lyons, 1968), and ostrich (Verhaart, 1971). The nucleus has been described under different names. Jelgersma (1896) termed it the ganglion opticum dorsale; Perlia (1889) called it the medial optic nucleus; Wallenberg (1898), Edinger and Wallenberg (1899) named it ganglion isthmi; Craigie (1928) named this cell mass the nucleus tractus isthmo-optici. Finally, Huber and Crosby (1929), and Ariens-Kappers, et al. (1936) designated it as the nucleus isthrno-opticus (NIO) and this term was subsequently adopted by the majority of authors. The NIO receives a heavy projection from the TO by way of the tectoisthmic tract, while its efferents constitute the TIO.

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a. Development of the NIO. The ontogenetic studies of the NIO and its associated afferent and efferent pathways were performed in Anus platyrhynchos (Sohal and Narayanan, 1974,1975; Sohal, 1976)and pigeon (Bagnoli et al., 1987), but most particularly in Gallus domesticus (Cowan and Wenger, 1968; Clarke and Cowan, 1976; Vaage, 1973; Clarke et al., 1976; Cowan and Clarke, 1976; Clarke, 1982; O’Leary and Cowan, 1982; Angaut and Rafin, 1981; Clarke and Caranzano, 1985; Catsicas et ul., 1987). In Gallus, the cells of the N10 are generated sometime between the end of day 5 and day 7 of incubation (Vaage, 1973; Cowan and Wenger, 1968; Clarke e f al., 1976; Clarke, 1982). They arise within the ventricular area of the caudal mesencephalic alar plate, then migrate ventrolaterally over a distance of -200 pm before aggregating to form the anlage of the nucleus. The first NIO cells generated occupy the ventrolatera1 portion of the nucleus, whereas the last neurons are localized along its dorsomedial edge (Clarke, 1982). The nucleus clearly is differentiated by day 8 of incubation, but is not numerically complete until day 11 (Cowan and Wenger, 1968). At this time it contains -22,000 cells. Between day 13 and day 17 of incubation, -60% of the NIO cells degenerate, and thereafter the population remains stable at -9500 cells. This process of N10 cell death occurs later in A. platyrhynchos (between day 16 and day 21 of incubation), and in this species affects only 45% of the neurons. After day 21 the neuronal population is stabilized at -3600 cells (Sohal and Narayanan, 1974). In G. domesticus, Clarke and Caranzano (1985) showed that the NIO neurons are already polarized by day 12 of incubation, and exhibit a morphology resembling that found in older embryos and hatched chicks (see Fig. 3). In their ultramicroscopic study, Angaut and Raffin (1981) reported that the NIO neuropil in 10-day chick embryos was similar to that of the mature state. However, the dorsomedial NIO suffers from a particular delay in maturation. It thus appears that synaptogenesis and neuropil maturation continue for some time after hatching. Various observations made in the chicken after the intraocular injection of different tracers that are transported retrogradely (HRP, WGA-HRP, fluorochromes) indicated that the earliest time at which it is possible to detect retrograde labeling of NIO neurons is on day 9 of incubation (Cowan and Clarke, 1976; Clarke and Cowan, 1976; Clarke et al., 1976; O’Leary and Cowan, 1982). At this point, the labeled cells are situated in the ventrolateral portion of the contralateral NIO. During the next 48 hours, an increasing number of cells can be labeled in a distinct ventrolateral-to-dorsomedial progression within the nucleus (O’Leary and Cowan, 1982). Thus, the sequence of labeling parallels the time course of generation of the NIO

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5Oum

FIG.3. Three neurons traced with a camera lucida from rapid Golgi preparations of newly hatched chicks. Axons are marked by arrows. Note in (A) that the dendrites tend to branch within a central area. Also observe that branchpoints frequently are somewhat widened and flattened as well as fringed by numerous appendages. From Crossland (1979).

neurons. There would therefore seem to be a close relation between the time that neurons withdraw from the cell cycle and that when their axons attain the target area (O’Leary and Cowan, 1982). Moreover, during the course of these experiments, neurons were found to be labeled on the side ipsilateral to the injection. Between day 10 and day 13 of incubation their numbers are the most elevated (-60), but represent < I % of the total number of NIO neurons (O’Leary and Cowan, 1982). Their number diminishes progressively until birth, and stabilizes at 10 neurons. O’Leary and Cowan (1982) demonstrated transitory collaterals in some of these neurons.

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b. Structural Organization of the NIO in Adult Birds. i. General Anatomy. The NIO has occasionally been described in classical neuroarchitectonic studies performed in many avian groups. It has been reported in numerous passeriforms (Hirschberger, 1971; Huber and Crosby, 1929; Showers and Lyons, 1968), galliforms (Hirschberger, 1971; Showers and Lyons, 1968; Reperant, 1978), anseriforms (Showers and Lyons, 1968; Sohal and Narayanan, 1974; Reperant, 1978), psittaciforms (Hirschberger, 1971; Verhaart, 1971 ; Reperant, 1978), strigiforms (Hirschberger, 1971; Reperant, 1978; Weidner et al., 1987), and falconiforms (Shortess and Klose, 1975; Weidner et al., 1987), but not in struthioniforms (Verhaart, 1971), apterygiforms (Craigie, 1930), and ardeiforms (Showers and

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Lyons, 1968). In his comparative study of 23 species of birds, Reperant (1978) described three levels of organization of the NIO. In galliforms, passeriforms, and columbiforms, which are mostly seed or fruit-eating, the NiO is always very well differentiated and laminated, and contains 7000-12,OOO neurons. In contrast, in birds with a well-developed trigeminal system that generally search for food by poking or burrowing, as is particularly the case in anseriforms, the NIO is clearly less well differentiated and contains fewer neurons (on the average 3000-4000 neurons). Finally, in all raptors examined (see also Shortess and Klose, 1975;Weidner et al., 1987), the NIO was found to be poorly differentiated and reticular in appearance, and contained between 900 and 1400 neurons depending on the species. The anatomical organization of the NIO has been most extensively studied in the pigeon and chicken. The structure is well developed, exhibiting a convoluted laminar arrangement and somewhat folded appearance. In the pigeon, the dimensions of the nucleus are 1000 p m in the mediolateral, 700 p m in the dorsoventral, and 735 pm in the rostrocaudal direction (Wolf-Oberhollenzer, 1987). Precise counts performed in the pigeon and chicken have shown that the NIO contains between 8000 and I 1,000 neurons (Cowan, 1970; Cowan and Powell, 1963; Hayes and Webster, 1981; O’Leary and Cowan, 1982; Wolf-Oberhollenzer, 1987). The convoluted laminae of neuronal somata consist of two layers separated by a clear neuropilar layer. The two-layered lamina is continuous throughout the nucleus, which can be considered as a flattened and folded hollow spherical body. The flattened sheet is oriented mainly in the vertical dimension and, approximately in the coronal plane, it is folded basically in the shape of an S. The Golgi impregnations revealed that the cells of the NIO (15 p m average diameter in their longest dimension) are typically flask shaped and characterized by an irregular and spined cellular contour. They possess one to four main dendrites extending in a single direction, which rapidly give off branches that are oriented toward the neuropilar zone (Cowan, 1970; Angaut and RepCrant, 1978; Crossland, 1979; Angaut and Raffin, 1981; Clarke and Caranzano, 1985). The axons of the isthmooptic cells emerge from the side opposite to the dendritic trunk and gather within the dorsal aspect of the nucleus, forming a compact bundle. They then extend dorsolaterally toward the medial edge of the TO, where they regroup to constitute the T I 0 (Fig. 3). Based on differences in evoked discharges, Holden (1968b) proposed the existence of a system of recurrent collaterals of such axons upon the NIO. To date, none of the anatomical work has confirmed this hypothesis. Similarly, Holden’s idea (1968b) of the existence of interneurons in the NIO has not been confirmed by anatomists. All the NIO neurons ap-

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pear clearly to be Golgi types. The intraocular injections of retrogradely transported tracers (HRP, WGA-HRP, fluorochromes) performed after hatching in different avian species have shown that almost all the NIO neurons project to the contralateral retina. However, in the chicken (O’Leary and Cowan, 1982; Weidner et al., 1987) and in the nocturnal rapter Tyto alba, it has been shown that some rare isthmooptic neurons project to the ipsilateral retina. Acetylcholinesterase has been found in the NIO (Hayes, 1982; D. Miceli, J. RepCrant, L. Marchand unpublished observations), and centrifugal fibers may contribute to one of the stained bands in the IPL (Hayes, 1982). ii. Electron Microscopy. Two studies, in the pigeon (Angaut and RepCrant, 1978) and the chicken (Crossland, 1979), have examined the ultrastructural properties of the NIO. The somata contain an excentric nucleus with a prominent nucleolus. The cytoplasm contains stacks of rough endoplasmic reticulum (RER), many scattered polyribosomes, numerous mitochondria, occasional microtubules, and a prominent Golgi apparatus. The cell membrane is smooth but irregular for long distances, and occasionally displays spinous extensions linked to the presence of attachment plaques. In the pigeon (Angaut and Reperant, 1978), the attachment plaques are encountered between the apposed membranes of neighboring cells. More often, however, smooth regions of the two membranes joined each other and became closely entwined. Conspicuous attachment plaques were found along each straight portion of the folded membranes. In some instances, two regions of apposed membranes coated with attachment plaques flanked small areas where the membrane came in close apposition, making gap junctions. These cytological features have not been observed in the chicken (Crossland, 1979). In G. domesticus, a large part of the cell perimeter remains free of synapses (Crossland, 1979). On the other hand, in Columbia liviu, close to 50% of the smooth membrane of NIO cells are apposed to axon terminals often represented by alternating boutons possessing either rounded or pleiomorphic synaptic vesicles (Angaut and RepCrant, 1978). The neuropil comprises dendrites of various caliber. The largest correspond to stem dendrites containing abundant RER and the spines are particularly frequent in the fine dendritic profiles. Bundles of myelinated axons are scattered throughout the neuropil and thin glial processes are numerous. A variety of shapes, elements, and vesicular synaptic contents are observed among the terminals. However, three main types of terminal boutons were described based on whether they contained rounded (R type), flattened (F type), or pleiomorphic vesicles (P type). The R types are the most numerous, and in the chicken make up 64% of all the synapses found in the NIO (Crossland,

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1979). They may belong mainly to tectal afferents and make asymmetric contacts, particularly upon small dendrites (Crossland, 1979). The F types, with flattened synaptic vesicles, establish symmetric synapses mostly upon large dendrites. In the chicken, they make up -20% of the total population of NIO boutons (Crossland, 1979), but their site of origin has not been determined. The third type of boutons containing pleiomorphic vesicles are the least numerous and make asymmetric contacts mainly upon the base of large dendritic trunks. Their origin has also not been determined. It is interesting to note that glomerular structures characterized by the presence of glial wrappings around small assemblies of synaptic contacts are frequently observed within the neuropil.

iii. Afferents to the NIO. Optic Tecturn (TO). It is worth mentioning that the large majority of NIO afferents stem from the TO, and that the tectoisthmal tract approaches the nucleus ventrally (Cowan and Powell, 1963; McGill et al., 1966a,b). The first anatomical studies using degeneration techniques suggested that this projection originates in the superficial layers of the TO (Cowan and Powell, 1963; McGill et al., 1966a). On the other hand, based on electrophysiological data, Holden (1968b) suggested that some fibers could emanate from the deep layers of the TO. The use of the HRP technique following injections of the enzyme into the NIO of the chicken (Crossland and Hughes, 1978) and quail (Uchiyama and Watanabe, 1985) made it possible to establish the precise location of the tectal neurons projecting upon this structure. In both species, the somata of the tectal-NIO neurons are situated in layer h of the stratum griseum et fibrosum superficiale. No labeled neurons have been observed in any other tectal layer, and particularly in the deep layers. In the quail, Uchiyama and Watanabe (1985)showed that most of the numerous dendrites of such neurons that extend toward the deep part of the tectum give off branches in lamina h and in the functional zone between laminae i and j. Few branches, however, attain the superficial layers or come into contact with optic fibers. Other Afferents to the NIO. We have seen previously, while reviewing the EM studies, that endings of unknown origin coexist next to tectoisthmic terminals. Consequently, other a e r e n t s to the NIO must be present besides the tecto-projection. The literature pertaining to this question provides some fragmentary information that is sometimes contradictory. Using conventional preparations in the pigeon, Huber and Crosby (1929) claimed the existence of interconnections between the NIO and the nuclei responsible for extrinsic ocular muscle command. Angaut and

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RepCrant (1978) confirmed the presence of a moderate projection from both the abducens and trochlear nerve using the degeneration technique. However, Crossland and Hughes (1978) and Uchiyama and Watanabe (1985) were unable to reproduce these results using the HRP method. Moreover, Crossland and Hughes (1978) described other possible sources of afferents to the NIO in the chicken: the nuclei intercollicularis et mesencephalicus lateralis dorsalis and the locus coeruleus, results that have not been confirmed by Uchiyama and Watanabe (1985) in the quail. However, the latter authors are in agreement with Crossland and Hughes, and describe a possible projection to the NIO from the medial and ventromedial tegmentum. Finally, it should be noted that the NIO can be activated through its contralateral homolog by way of the intertectal commissures (Galifret et al., 1971) and receives information from the visual Wulst via a polysynaptic pathway involving the TO (hyperstriato + tecto + NIO) (Uchiyama et al., 1987). 2. Centrifugal Optic Neurons Referred to as “Ectopic” The existence of centrifugal optic neurons situated outside the NIO was first demonstrated by Cowan and Clarke (1976) following the intraocular injection of HRP in the chicken embryo. The latter authors referred to such cells as “ectopic” to the NIO. Subsequently, such neurons were described in posthatched animals in different avian species: C . livia (Streit and Reubi, 1977; Hayes and Webster, 1981; Wolf-Oberhollenzer, 1987), G. domesticus (Cowan and Clarke, 1976; Clarke and Cowan, 1976; O’Leary and Cowan, 1982), Coturnix coturnix japonica (Weidner et al., 1987) and T . alba (Weidner et al., 1987). These cells have been most extensively studied in the chicken. Most of the ectopic centrifugal neurons (ECN) are clustered around the NIO, where they form a diffuse halo of cells. They are more evident in the central gray and in the tegmentoreticular fields of the caudal mesencephalon. Here they are dispersed among the tectoisthmo fibers and the occipitomesencephalic tracts. Some are situated in the T I 0 and in the dorsal division of the lateral lemniscus. More than 80% of the ECN are multipolar, with three or more primary dendrites exhibiting long secondary and tertiary dendritic branches. The remainder are bipolar and spindleshaped, displaying two primary dendrites (Hayes and Webster, 1981; O’Leary and Cowan, 1982; Wolf-Oberhollenzer, 1987; Weidner et al., 1987). The axons seemed mostly to originate from cell bodies. However, in some cases they stemmed from one of the primary dendrites (O’Leary and Cowan, 1982). The ECN are larger than the NIO cells (average diameter 18 Fm), and their axons seem to follow the same centrifugal course as the NIO fibers. In fact, O’Leary and Cowan (1982) were able to trace the ECN axons into the tract where

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it lies dorsolateral to the NIO in the chicken. They also showed that such neurons receive afferents from the TO by way of the tectoisthmic tract. The proportion of ectopic cells in relation to NIO neurons on the contralateral side is 15% in the chicken (O’Leary and Cowan, 1982),22% in the pigeon (Hayes and Webster, 1981; Wolf-Oberhollenzer, 1987),and 27.5% in T. alba (Weidner et al., 1987). The use of intraocular injections of different tracers (HRP, WGA-HRP, fluorochromes) revealed that some of the ECN (from 30 to 100, depending on the species) project upon the ipsilateral retina. The double-labeling fluorescence method used in birds after hatching indicated the absence of collateralization of ECN neurons (O’Leary and Cowan, 1982; Wolf-Oberhollenzer, 1987; Weidner et al., 1987). In the chicken, O’Leary and Cowan (1982) showed that the ECN axons penetrate the contralateral retina as of day 9 of incubation. At day I 1 of incubation, the number of ECN projecting to the contralateral retina is -3500, and 140 ECN project to the ipsilateral eye. By the end of incubation day 17, the number of contralateral ECN drops by -53% to a mean figure of 1500. Approximately the same number of ECN neurons survive in the posthatched and adult animal. In the case of ECN displaying ipsilateral projections, their number stabilizes at -30 by day 17 of incubation. It has been suggested by Cowan and collaborators (Clarke and Cowan, 1976; Cowan and Clarke, 1976; O’Leary and Cowan, 1982) that the ECN represent misplaced NIO neurons that are misdirected during their migration from the neuroepithelium in which they arose. This suggestion arose mainly from the observation that ECN are generated during the same period as the NIO neurons themselves. It is also possible that ECN constitute a mesencephalic neuronal population that is very distinct from that of the NIO. Several arguments based on the morphological properties of these two cell categories support this notion. It should be borne in mind that, in the adult, the somata of the ECN are larger than the cells of the NIO, which are generally flask shaped with one or two large stem dendrites extending from the perikaryon toward the center of the nucleus. Most of the ECN are multipolar with three or more primary dendrites oriented in different directions. Furthermore, it has long been assumed that during the early stages of development, the cells of the NIO display features similar to those of ECN (Clarke and Cowan, 1976; Angaut and Raffin, 1981; O’Leary and Cowan, 1982). These have been described as multipolar with dendrites growing in several directions. In fact, studies in the chicken (Clarke and Caranzano, 1985) have shown that by day 1 1 of incubation, the NIO neurons are already polarized and exhibit morphological features similar to those found in older embryos and hatched chicks.

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3. Isthmo-Optic Tract The intracerebral trajectory of the tractus isthmoopticus (TIO), sometimes termed the medial optic tract or axillary optic tract, has been described in detail in the pigeon (Cowan and Powell, 1963; Galifret et al., 1971) and the chicken (Wallenberg, 1898; Crossland and Hughes, 1978). The latter data have been obtained either by using the degeneration techniques after lesioning the NIO or sectioning the TIO, or with autoradiography following injections of tritiated amino acids into the NIO. The centrifugal fibers regroup dorsolaterally to the NIO to form a compact elliptical bundle. They progress caudally and medially within the stratum griseum et fibrosum superficiale of the TO, and begin to course more ventrally upon reaching the level of the diencephalon. Even more rostraIly, the centrifugal fibers continue to course ventrally, and form an elongated tract that is bordered laterally and ventrally by the optic tract. The T I 0 continues medially along the optic tract and penetrates the marginal optic tract rostra1 to the chiasma, where it becomes less compactly organized. In the chiasma, the decussation of the centrifugal fibers occurs gradually at different levels, so that they are found in every interdigitation, before finally entering the contralateral optic nerve. The extraretinal projections of the NIO in the chicken (Wallenberg, 1898) and pigeon (Cowan and Powell, 1963; Galifret et al., 1971) have been described using the degeneration techniques after creating lesions of the nucleus. The NIO has been found to project upon the nucleus pretectalis lateralis and nucleus superficialis synencephali (Galifret et al., 1971), nucleus externus (Cowan and Powell, 1963), nucleus geniculatis lateralis ventralis (Wallenberg, 1898; Cowan and Powell, 1963; Galifret et al., 1971), and the nucleus ventrolateralis thalami (Wallenberg, 1898). Using the autoradiographic technique after the injection of a tritiated amino acid into the NIO of the chicken, Crossland and Hughes (1978) failed to observe such projections and suggested the possible existence of a projection from the NIO to the nucleus lentiformis mesencephali pars magnocellularis. Although the extraretinal projections of the NIO have not been demonstrated conclusively, the existence of collateralization of centrifugal optic axons upon different cerebral structures might explain why some NIO neurons do not degenerate after retinal ablation (McGill et al., 1966a,b; Raffin and Reperant, 1975). Cowan’s ultrastructural study of the T I 0 in the pigeon (1970) revealed that virtually all the centrifugal fibers are myelinated and are among the largest axons in the visual system. Axon diameter measurements performed in this species indicated that the tract is made up of a unimodal population of fibers with a diameter ranging between 0.5 and 2.5 Fm, with

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the mode at - I .5 pm. The T I 0 in the pigeon and chicken contains between 10,OOO and 12,000 fibers. Since in these species the optic nerve contains -2.5 million axons (Binggeli and Paule, 1969; Duff et ul., 1981; Rager and Rager, 1978), the centrifugal fibers comprise -0.4% of the total number of optic nerve fibers. In birds of prey, where the centrifugal visual system is poorly developed, it has been estimated that in one species (Buteo buteo) the proportion of centrifugal to retinofugal axons was only 0.4% (Weidner el al., 1987). 4. Termination of Centrificgal Fibers in the Retina

The mode of distribution and termination of centrifugal fibers at the retinal level has been the subject of repeated studies since the end of the last century. most often in the chicken and pigeon. Initially, the Golgi method and, to a lesser degree, that using methylene blue, were extensively used (Ramon y Cajal, 1888, 1889, 1893, 191I ; Dogiel, 1895; Ressnikoff, 1897; Maturana and Frenk, 1965). The degeneration methods, whether used in light- or electron-microscopic studies, were less commonly employed (Cowan and Powell, 1963; Dowling and Cowan, 1966). This problem has since been investigated using various histophysiological methods based upon the axonal transport of different tracers (Crossland and Hughes, 1978; Hayes, 1982; Hayes and Holden, 1983; Catsicas et al., 1987). a. Features and Mode of Arborization of Centrifitgal Endings. It has been shown throughout these studies that after leaving the optic fiber layer, the centrifugal fibers course through the IPL and reach the zone of the inner granular layer. They break up into arborizations with short, thick, and very varicose branchlets. The collateralization of such fibers either prior to their entry into the eye Gust beyond the lamina cribrosa) or as they course through the IPL, has been suggested on occasion (Maturana and Frenk, 1965; Cowan, 1970). However, numerous other authors (Ramon y Cajal, 1889; Dogiel, 1895; Hayes and Webster, 1981) have never observed such collaterals. The mode of termination and localization of centrifugal fibers has sometimes received rather different descriptions. Ramon y Cajal(1888, 1889, 1893, 1911) described three types of centrifugal endings in the most internal part of the inner nuclear layer (see Fig. 4). He showed that numerous centrifugal fibers in the pigeon form a delicate pericellular nest around the somata of amacrine cells that he called “associative.” This type of arborization would subsequently be described by Dogiel in 1895. Ramon y Cajal also showed two other types of endings in the pigeon: ascending and descending (inferior or basilar). The

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FIG.4. Schematic representation of a transverse section through the retina of the bird Passer domesticus, stained according to the Golgi method and showing the different types of arborization of centrifugal fibers (a-f). (A) Displaced ganglion cell; (B, C) bistratified and tristratified ganglion cells. From Ram6n y Cajal (1911).

ascending arborizations were relatively thin and terminated deeper within the inner nuclear layer, whereas the descending endings were horizontal and meandered at the internal border of this same layer. Both types of endings made contact upon ordinary amacrine cells. In 1889, Ramon y Cajal considered the third type of arborizations to be the most numerous. However, in 1911 he reported that the first type were more widespread. Furthermore, he noted that the different types of endings varied in their degree of development from one species to the next. For example, in passeriforms and galliforms, arborizations of the first type are sometimes reduced to a simple mass bristled with varicosities and immediately adjacent to associative amacrine cells. Dogiel (1895), who generally recognized Ramon y Cajal’s three types, described another category of centrifugal endings in the pigeon (which the Spanish author was unable to identify). According to Dogiel, these fibers crossed the IPL and branched frequently above this layer, forming vast delicately flattened and complex arborizations. The centrifugal fibers of the pigeon retina were investigated by Maturana and Frenk (1965) using the methylene blue-staining procedure. The situation they described was rather different. Two types of centrifugal terminals were reported-convergent and divergent-the convergent type apparently corresponding to Ramon y Cajal’s pericellular nest. The branches follow different paths but converge onto a single cell body in the amacrine cell layer, where they may give off small branches, which make additional loops that terminate either on the same nest or on neighboring cells. The convergent fibers synapse with no more than three or four cells, and frequently with only one. The divergent type was charac-

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terized by the fact that the ramifications successively make synaptic contacts with different cells, and sometimes at different levels of the same cells. They penetrate the inner nuclear layer to a lesser extent than the convergent type of endings, and are generally situated at the border between this layer and the IPL. Maturana and Frenk reported that these fibers terminate on three types of cells: “flat” amacrines, small amacrines of the parasol type, and displaced ganglion cells. The flat arnacrines apparently consist of the small amacrines of the first level described by Ramon y Cajal. while the small amacrines of the parasol type might be the equivalent of small stratified amacrines according to Ramon y Cajal’s nomenclature. The displaced ganglion cells receive boutons belonging to the three types of endings. The terminals occur not in a nest, but near the axon hillock. The flat amacrines are solely innervated by the diverging type of centrifugal fiber, whereas the small parasol type receives the pericellular nest of the convergent type. Cowan and Powell (1963),after destroying the NIO of the pigeon, used the Nauta silver stain method to trace the degenerating fibers, and found that these fibers terminate in the innermost inner nuclear layer. The majority were seen to end in two or more fingerlike processes embracing amacrine cell bodies. Centrifugal fiber endings have been investigated by Dowling and Cowan (1966) using EM following lesioning of the N10 in the pigeon. It was found that the centrifugal synaptic endings were larger than most other terminals in the plexiform layer, in general, frequently displaying diameters of s8 pm. Most were identified along the internal aspect of the inner nuclear layer, in contact with the proximal portion of the principal process of amacrine cells. Some boutons were occasionally found in contact with the amacrine cell body, and some rare terminals were detected deep within the amacrine cells. Thus, the majority of terminals observed by Dowling and Cowan resembled, in their arrangement and localization, the three cell types defined by Ramon y Cajal (see Fig. 5). However, these authors noted that the arborizations of the third type are far more numerous than those of the first or second types. Dowling and Cowan, contrary to Maturana and Frenk, did not observe terminals upon the displaced ganglion cells. Hayes (1982), in his architectural study of the pigeon retina, was able to identify centrifugal endings with HRP labeling after injecting the enzyme into the NIO. Although he confirmed the overall results of Dowling and Cowan, he also noted that the centrifugal boutons contain spherical synaptic vesicles of 30-50 nm diameter and densecore vesicles. These boutons establish symmetric-type synapses upon the cell bodies of amacrines or their “processes.” Elsewhere. Crossland and Hughes (1978) investigated the intraretinal endings of centrifugal fibers in the chicken by employing the autoradio-

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*-',.'fb

FIG.5. Mode of arborization of centrifugal optic fibers in the retina of the pigeon. A, Centrifugal axon; a, b, c, ramifications of the centrifugal axon; B, amacrine cell. From Ram6n y Cajhl (I91 1).

graphic technique, after injecting [3H]proline into the NIO. They observed that the highest concentration of labeling was situated along the junction between the inner nuclear and inner plexiform layers. Nevertheless, occasional clusters of silver grains were present within the inner nuclear layer. Finally, in their study of centrifugal fiber terminals stained by the anterograde axonal transport of HRP from the NIO in the pigeon, Hayes and Webster (1981) were able to identify and examine the distribution of the two types of endings described by Maturana and Frenk (1965). The majority of terminal arborizations (68%) were convergent, with up to six branches converging to form a cluster of a few large terminals ( ~km 4 diameter) around a single unlabeled cell body in the amacrine cell sublayer of the inner nuclear layer, and often penetrating this layer by as much as 10 bm. As many as 10 widely spaced branches were found in the remaining divergent arborizations, apparently contacting a number of unlabeled cells in the amacrine sublayer with s 2 0 small terminals (- 1 k m diameter) that seldom penetrated the inner nuclear layer. b. Distribution of Centrifugal Endings in the Retina. After lesioning the NIO in the pigeon, Cowan and Powell (1963) observed centrifugal fiber degeneration to be mainly concentrated in the temporal retina. But in their study using the retrograde degeneration technique following ablations of different retinal fields, McGill et al. (1966b) considered that the

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NIO, in this species, projects upon the whole extent of the retina. Maturana and Frenk (1965) also concluded that centrifugal fibers were distributed throughout the full surface of the pigeon retina. Later studies using various techniques based on the axonal transport of tracers have provided very different results. After the injection of HRP into the region of the NIO in the pigeon, Hayes and Holden (1983) showed that the labeled centrifugal endings were grouped in a 3- to 5-mm-wide and IZmm-long band exhibiting a density >50 endingdmm’. This band was near to the projection area of the horizontal meridian of the retina, and two areas of high densities were observed within it: one in the temporal yellow field -3.5 mm from the area centralis and the other in the central yellow field just overlying the area centralis. The maximum densities observed in the temporal yellow field and near the area centralis were 610 and 570 endingdmm’, respectively, and the concentrations declined rapidly toward the superior retina, being < 10 endingdmm’ in the red field. Only rarely were terminals found in the superior third of the retina. The densities were observed to decline more gradually laterally and ventrally. Centrifugal endings were present in most of the inferior retina, but were absent from a circular area of -500 pm diameter centered on the area centralis (see Fig. 6). Hayes and Holden estimated the total number of terminals in the pi-

I

.

FIG. 6 . Map of a flat-mounted retina showing the density of HRP-labeled centrifugal terminal arborizations (tetramethyl benzidene: TMB incubation). Numbers should be multiplied by 10 to give number of arborizations per square millimeter. The band of arborizations is at -7“ to the horizontal and contains two areas of high density (0).Dashed line delineates the red field; the rest of the retina is the yellow field. ( 0 ) Area centralis. Left retina: N , nasal; T , temporal; S, superior; 1, inferior retina. From Hayes and Holden (1983).

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geon retina to be -7000. This is considerably lower than the estimate of 100,OOO made by Maturana and Frenk (1965) in this same species. Somewhat different data were obtained by Crossland and Hughes (1978) in the chicken by using the anterograde axonal transport of radioactive proteins from the NIO. According to the latter authors, the centrifugal endings are most dense near the central portion of the nasal half of the retina and least concentrated in the temporal half. Moreover, they noted that centrifugal terminals were absent throughout the margin of the retina. Catsicas et al. (1987) have shown, by depositing a retrograde tracer into discrete parts of the chick retina, that the NIO neurons project only to the ventral two-thirds of the retina, with the exception of the extreme periphery. They confirmed the projection to be topographic, with the medial part of the NIO projecting temporally and the ventral part dorsally in the retinal projection field. They also noted that the ectopic centrifugal neurons exhibit a different projection pattern in the retina, where their overall projection field is more ventrally placed. Furthermore, the projection field of the ectopic cells seems to be larger and clearly lacking the topographic precision of NIO neurons. Medially placed ecto,pic cells tended to project temporally in the retina, whereas the dorsal, ventral, and laterally situated cells tended to project nasally. B. FUNCTIONAL CONSIDERATIONS The centrifugal visual system in the pigeon and chicken has been investigated extensively during the last decade using both electrophysiological and behavioral approaches. However, the data obtained from these studies, though abundant, have not provided any satisfactory information regarding the function of this system. In a study by Miles (1972a-d), a few ganglion cells that increased their responses to an ON stimulus after the recent passage of a dark moving edge moving forward through the receptive field, failed to show this enhancement after depression of the retinopetal path. A similar action of centrifugal fibers was found, though more widespread, in another study by Pearlman and Hughes (1976a,b), where 75% of ganglion cells in the pigeon were found to be influenced by cooling of the NIO. The major finding was that the cryoblockade of the NIO produced a decrease in the responsiveness of most retinal ganglion cells without altering the trigger features specific to receptive fields. The suggestion made was that the efferent fibers excite amacrine cells, which, in turn, would be inhibitory to ganglion cells. Moreover, when the N10 is directly stimulated, the retinal ganglion cell response to stationary stimuli is enhanced but no specific modulatury effects on retinal responsiveness have been reported (Miles, 1972a-d). In comparison to retinal cells,

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most neurons of the NIO have ON-OFF field centers and purely inhibitory surrounds. The receptive fields are more than five times larger and the units tend to diminish their responses to repetitive inputs because they habituate. Among the stimulus characteristics, forward target movements are particularly suitable in eliciting a response, for most NIO neurons and cells preferred dark edges to light edges. Thus, this result seems to indicate that isthmooptic neurons deal most efficiently with transient and dynamic features of the visual environment. The units detect what abruptly changes in the visual field (novelty detectors). The behavioral data appear somewhat contradictory. According to Hodos and Karten ( 1974), coarse visual intensity and pattern discrimination performance in the pigeon are unimpaired following creation of bilateral NIO lesions. Chickens (Rogers and Miles, 1972) and pigeons (Shortess and Klose, 1977) with similar lesions have been observed to peck more slowly at grains scattered on the floor. Furthermore, the birds displayed deficits in the detection of low-luminance targets (Rogers and Miles, 1972). Rogers and Miles have suggested that this system might play a role in enhancing contrast under certain dim-light conditions and that the efferents may assist in the detection of stimuli that are novel or difficult to discriminate through a mechanism of “dynamic adaptation.” I n view of the mild deficits observed, Shortess and Klose (1977) have suggested that the centrifugal visual system plays a minor supplemental role rather than a central role in the visual process. In contrast to the latter data, Knipling (1978), who performed the same experiments in the pigeon, observed no deficits and concluded that the system is probably not involved in the processing of spatially detailed visual information. Instead, and because of its direct connections with amacrine cells, the system would be more likely involved in the modulation of processing temporal properties of stimuli (Knipling, 1978). Several other functions of the centrifugal visual system have been proposed. One is that it mediates visual guidance of motor behavior (Rogers and Miles, 1972), whereas another is that it prevents confusion between self-produced movements and motion exterior to the birds (Miles. 1972a-d). Galifret er al. (1971) have postulated that such a system may provide a sort of scanning mechanism without any eye movement. On the basis of comparative neuroanatomical data obtained in 23 bird species, Reperant (1978) and Weidner er al. (1987) suggested that the centrifugal system was more particularly involved in ground-feeding, pecking, and food selection among static stimuli at a short viewing distance.

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VII. Mammals

A. ANATOMY Since the end of the nineteenth century, many studies have been carried out to demonstrate the existence of a retinopetal system in mammals. Despite these efforts, much controversy still surrounds this question today, and the data available seem generally nonconclusive and contradictory. Although the most significant results were obtained using anatomical techniques, a large body of electrophysiological data exists that supports claims for the existence of retinal efferent fibers (Dodt, 1956; Palestini et al., 1959; Abe, 1962; Jacobson and Gestring, 1958a,b; Hernandez-Peon, 1961; Jacobson and Suzuki, 1962; Nagaya e? al., 1962; Tasaki et al., 1962; Ogden and Brown, 1964; Ogden, 1966; Spinelli et al., 1965; Weingarten and Spinelli, 1966; Gills, 1966; Spinelli and Weingarten, 1966; Haft and Harman, 1967; Vatter, 1967; Haft, 1968; Van Hasselt, 1969, 1972a,b, 1972-1973; Molotchnikoff and Tremblay, 1983a,b, 1986; Molotchnikoff et al., 1984, 1988; see also Granit, 1962; Brindley, 1960; Ogden, 1968; Rodieck, 1973; see also Section VI1,B). For more than a century, many anatomists have tried to obtain morphological proof of the existence of a centrifugal visual system in mammals. These studies have been undertaken using many different techniques, and focused on three levels: retinal, optic nerve, and central. 1 . Retina The Golgi method and other related techniques have often been used on the retinas of mammals, but they have not been as successful as they were in birds in revealing the existence of centrifugal fibers (see Fig. 7). Only four studies (Ramon y Cajal, 1893; Marenghi, 1900; Polyak, 1957; Schonik-Jarros, 1965) have identified a few rare axons, usually poorly stained, which originate in the fiber-optic layer and cross the IPL before terminating in the amacrine cell region. According to Ramon y Cajal, Polyak, and Schonik-Jarros, these axons have a central origin, whereas Marrenghi describes them as ganglion cell collaterals. This latter point of view is shared by Dacey (1985), who bases his opinion on results obtained by the use of the HRP technique after iontophoretic enzyme injections within the retina. Gros-Bielchowsky’s reduced silver-staining technique, modified by Gallego (1953) for in rot0 staining of the retina, has emerged as the most suitable method for the study of intraretinal centrifugal fibers. As well as staining the neuronal bodies, ganglion cells, amacrines, and horizontal cells (Gallego, 1953, 1954, 1965, 1967, 1971; Gallego and Cruz, 1965; Hon-

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FIG.7. Schematic representation of a transverse section of the dog retina stained according to the Golgi method, showing the centrifugal fibers 6)arborizing at the level of the external limit of the inner plexiform layer (B). (A) Outer plexiform layer. a, Cone; b, rod; c, d, e, f, bipolar cells; g, cells with ascending axons; h, amacrine cell; m, nerve fiber penetrating IPL; n, ganglion cell. From Ramon y Cajal (I91 1).

rubia, 1%6), this technique also provides for excellent staining of the retinal layer of optic fibers (Gallego, 1953; 1954; Honrubia, 1966). Thus it has been possible to describe accurately the trajectory of the ganglion cell axons that are afferent or associative on flat-mounted retinas (Gallego and Cruz, 1965; Honrubia, 19661, and also to identify intraretinal fibers of central origin in various mammals, including humans (Gallego and Ventura, 1953; Ventura and Mathieu, 1959; Honrubia, 1966; Honrubia and Grijalbo, 1968; Honrubia and Elliott, 1968, 1970; Goldberg and Galin, 1973; Reperant and Gallego, 1976; Reperant et al., 1981; Stone, 1981). Stemming from the papilla, they appear among the centripetal optic fibers as large, very argyrophilic axons. These axons, the diameter of which diminishes progressively, course for several millimeters through the optic fiber layer before giving off a large number of branches, which can occupy up to a complete retinal quadrant (Reperant et a / . , 1981). As they move away from the papilla, the centrifugal fibers and their branches gradually leave the optic fiber layer, cross the ganglion cell layer, and seem to end in the IPL. Such recurring fibers, stemming from the papilla, have been observed in various species (cat, mouse, rat, dog, rabbit, monkey, human). In humans it is rare to observe >I0 retinopetal fibers per retina (Reperant et al., 1981). (These results are fundamentally different

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from those obtained by Wolter, who detected 100,OOO centrifugal optic fibers in the human by using degeneration techniques at the optic nerve level.) The abundance of collateral branches given off by each fiber compensates for the low number observed by Reperant and Gallego (see Fig. 8). This method has also revealed the existence of axons that penetrate the retinal arteries and branch out along their inner wall, forming fine strands that sometimes have flattened, very argyrophilic, and enlarged expansions at their endings (RepCrant and Gallkgo, 1976). Using EM following optic nerve section in the cat and the monkey, Brooke et al. (1965) and Wakakura and Ishikawa (1982) noted the presence of degenerated profiles in the outer area of the IPL, which they attributed to centrifugal optic terminals. From our point of view this evi-

P

FIG.8. Drawing of a centrifugal fiber (CF) derived from a reconstruction of 90 photomicrographs taken from the human retina stained in toto according to the Gallego method. P, Papilla. From Rep6rant and Gallego (1976).

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dence is not conclusive, since the observed degeneration is too advanced to allow for accurate identification of these profiles. In the rabbit, Reale et a / . (1971) and Lucian0 et a / . (1971) have described myelinated fibers that exhibit acetylcholinesterase activity and are situated in the IPL adjacent to the boundary with the inner nuclear layer. The fact that the intrinsic axons of the retina are always nonmyelinated led these authors to assume that these fibers are centrifugal. However, although Witkovsky ( 1971) has demonstrated the existence of myelinated centrifugal optic fibers in fish, there is no proof that the same will apply to the rabbit. In fact, these authors have been unable to confirm that the fibers actually terminate in the retina. It is therefore possible that the myelinated axons originate in displaced ganglion cells. Using the HRP method after depositing the enzyme in the optic nerve of the Mongolian gerbil, Larsen and M@ller(1986) described some labeled axons in the IPL, the inner nuclear layer, and the OPL. In a comparable experiment performed by Perry et al. (1984) in the macaque monkey, a few labeled fibers were observed to emanate from the papilla and showed a very high degree of collateralization. The terminals of these fibers appeared to be close to the inner nuclear layer. The various authors concluded that the observed labeling resulted from the orthograde transport of the enzyme by the retinopetal axons. Foilowing the iontophoretic injection of Phasedus vulgaris leukoagglutinin in the nucleus oculomotorius of the rat, Hoogland et a / . (1985) have described labeled fibers and preterminal arborizations in the inner nuclear layer of the retina. These fibers would be centrifugal and originate from this nucleus. Using the immunohistochemical technique on the retina of the mouse, Drager et d.(1984) described centrifugal axons. They were thicker than most of the retinofugal axons and tended to run superficially from the optic disk in their course over the retina. Their number is weak and many terminate high in the IPL or at the inside edge of the inner nuclear layer. Some can be followed to the OPL and even to the photoreceptor outer processes. 2. Degeneration Studies of Centrifuga/Elements in the Optic Nerve and Centers Numerous studies have approached the question of the existence of a centrifugal visual system in mammals by carrying out an analysis of the degeneration of the optic pathway at the optic nerve and central levels after lesioning the retina, the optic nerve, or the centers thought to be the source of the centrifugal pathway. The guiding principle is that orthograde degeneration should take place before retrograde degeneration. It is possible to detect the former by using special stains for degeneration

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fibers-for example, the Nauta and Fink-Heimer method. If centrifugal fibers exist in the optic nerve and retina, then they should become visible in a shorter time after lesion than would be the case for the degeneration of optic centripetal axons. On the other hand, following enucleation or optic nerve section, the retrograde degeneration of centrifugal fibers is very slow and they would persist in the optic nerve for a long time after the ganglion cell axons have disappeared. Thus, by this method, the existence of efferent optic fibers would appear to have been demonstrated in various species (mouse: Honjin el al., 1964; rat: Tsang, 1937; Hayhow, 1959; Schober, 1974; Fulcrand and Privat, 1977; Bons, 1987; rabbit: von Monakow, 1889; Cragg, 1962; dog: Elinson, 1896; Holmes, 1901; Schonik-Jarros, 1958; Okun and Collins, 1962; Haschke, 1963; Gorikov, 1969; cat: Edinger, 1911; Fillenz and Glees, 1961; Scully and Giolli, 1966; Gorikov, 1969; Lin, 1969; monkey: Noback and Mettler, 1973; human: Wolter, 1956, 1957, 1960, 1961, 1965a,b, 1968, 1979; Wolter and Liss, 1956; Liss and Wolter, 1956; Pfister and Wolter, 1963; Wolter and Knoblich, 1965; Wolter and Lund, 1968; Matsuyama, 1968; Sacks and Lindenberg, 1969; Novokhatsky, 1987). In addition, various sites have been proposed as being the origin of the centrifugal visual pathway: nucleus suprachiasmaticus (Bons, 1987), hypothalamus (Wolter, 1965b; Wolter and Knoblich, 1965; Wolter and Lund, 1968; Sacks and Lindenberg, 1969), hypophysis (Wolter, 1965b; Wolter and Knoblich, 1965; Wolter and Lund, 1968), lateral geniculate body (Holmes, 1901; Wolter, 1965b; Wolter and Knoblich, 1965; Wolter and Lund, 1968), superior colliculus (von Monakow, 1889; Edinger, 1911; Noback and Mettler, 1973), nucleus of the basal optic root (Tsang, 1937; Hayhow, 1959),and occipital lobe (Schkolnik-Jarros, 1958; Haschke, 1963). However, the same methods applied to the same material have sometimes produced negative results, and this has led certain authors to refute the existence of centrifugal fibers in mammals (Munzer and Wiener, 1902; Bodian, 1937; Packer, 1941; Mantz, 1954; Brindley and Hamasaki, 1961, 1962, 1966; Hess, 1958; Lin, 1972, 1973; Lin and Ingram, 1972a,b, 1973). 3. Retrograde Axonal Transport Methods in Determining the Origin of Centrijiuga1 Optic Fibers Researchers have mainly sought to identify the origin of centrifugal fibers in mammals by using intraocular injection of axonal tracers (HRP, WGA-HRP, fluorochromes), which are likely to be retrogradely transported. Such studies have been carried out in the dog (Terubayashi et al., 1983), the prosimian Microcebus murinus (Bons and Petter, 1986), the Mongolian gerbil (Larsen and Mgller, 1985), and especially the rat (Scalia and Colman, 1974; Reperant, 1975; Reperant et al., 1981; Colman et al.,

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1976; Itaya. 1980; Itaya and Itaya, 1985; Davis and McKinnon, 1982; Hoogland et a / . , 1985; Schnyder and Kunzle. 1984; Weidner e t a / . , 1983; Villar et a / . , 1987). The results obtained were once more highly contradictory. In the dog, after intraocular injection of HRP or propidium iodide, Terubayashi er al. (1983) mainly observed a single somatic labeling zone in the ventral part of the hypothalamus. Between 176 and 260 labeled neurons were counted. -80% of them on the contralateral side to the eye injection. This labeling became visible 2 days after the injection. Various controls enabled the authors to observe that the labeling could not result from extracellular diffusion of HRP along the optic nerve, nor from systemic transport through vessels or transneuronal labeling from the labeled optic endings. They conclude that the labeled neurons in the ventral hypothalamus of the dog are the only source of centrifugal fibers to the retina . In the gerbil. 12 hours after intraocular injection of either HRP or WGA-HRP, Larsen and M#ller ( 1985) observed the presence of contralaterally labeled neurons in the nucleus geniculatus lateralis pars dorsalis (GLd), the nucleus pretectalis, and the stratum griseum intermedium of the superior colliculus. Although the precise number was not specified, labeled neurons were noted to be particularly numerous in the rostra1 part of the GLd. Given the very short survival time. the authors excluded the possibility of labeling by synaptic transport, and considered these neurons to be centrifugal. In the prosimian M. murinus, Bons and Petter (1986) studied the origin of centrifugal fibers using intraocular injection of different fluorescent tracers (fast blue, true blue, rhodamine f3-isothiocyanate or RITC). Regardless of which tracer was used, the authors observed considerable somatic bilateral labeling, mainly in two areas: the ventromedioposterior region of the suprachiasmatic nuclei, and the anteromedial region of the arcuate nuclei. Since it was visible 2 days after injection, they did not consider labeling via the transsynaptic pathway to be possible, concluding instead that the labeled neurons projected to the retina. It should be noted that the hypothalamic labeling zones in this study are considerably different from those identified by Terubayashi et a / . (1983) in the dog. In the rat, work carried out using these methods to identify the existence of tile centrifugal visual system has provided contradictory results. Following intraocular injection of several different retrograde tracers (HRP, WGA-HRP, fast blue. nuclear yellow) in albino rats, Itaya (1980) and Itaya and Itaya (1985) described bilaterally retrogradely labeled neurons in the medial pretectal area (MPA). The neurons varied between 10 and 14 in number and were situated mainly on the contralateral side. The

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various controls carried out enabled the authors to conclude that these were in fact centrifugal neurons. Comparable results were subsequently obtained in the developing rat (Bunt et al., 1983) and the adult rat (Villar et al., 1987). On the other hand, following intraocular injection of various tracers (HRP, WGA-HRP, fast blue, true blue, Evans blue, nuclear yellow) in the same material, other investigators (Scalia and Colman, 1974; Reperant, 1975; Reperant et a / . , 1981; Davis and McKinnon, 1982; Weidner et al., 1983; Schnyder and Kiinzle, 1984; Hoogland et al., 1985) never observed labeled neurons in the MPA resulting from axonal tracer transport from the retina. For example, Schnyder and Kiinzle (1984) describe mainly some labeled glial cells in this area. According to Weidner et al. (1983), the numerous fluorescent somata found in the MPA resulted from transsynaptic pathway labeling of tracers from optic terminals. After intraocular injection of different tracers, Itaya and Itaya (1985) also observed a constant somatic labeling in the contralateral periaqueductal gray matter (PAG). Varying between 12 and 15 in number, these neurons are scattered within the PAG between the dorsal raphe and the mesencephalic trigeminal nucleus. According to these authors, these labeled cells could belong to the rostra1 coeruleus, the caudal dorsal raphe nucleus, and/or the dorsal tegmental nucleus. They therefore represent a second centrifugal visual contingent. More or less comparable results were obtained by Villar et al. (1987). In fact, after intraocular injection of HRP, the latter authors observed some labeled neurons (between 1 and 10) predominantly contralaterally in the pars lateralis of the dorsal raphe nucleus (DRL), which they reported to be serotoninergic. After creating lesions of the DRL and measuring 5-hydroxytryptamine in the retina using high-performance liquid chromatography with electrochemical detection, a 60% decrease of serotonin in the retina was detected compared to sham-operated and control animals. Other studies (Reperant, 1975;Reperant et al., 1981; Hoogland et al., 1985) make no mention of labeled neurons in these areas. However, within 2 days of injection of WGA-HRP, Schnyder and Kiinzle (1984) described labeled cells in the ventrolateral mesencephalic tegmentum (bilaterally), in the medial and lateral parts of the ventral PAG corresponding to the dorsal raphe, and in the laterodorsal tegmental nucleus (bilaterally). These authors indicated that labeling arose not from retrograde axonal transport of the tracer from the retina, but rather from somatic labeling by way of transsynaptic pathways from the optic terminals, since in these areas the number of labeled elements increased with the survival period. Schnyder and Kiinzle (1984) concluded that there is no centrifugal visual system in the rat. Using intraocular injection of HRP or nuclear yellow in six strains of white rat, Hoogland et al. (1985) essentially detected labeling of 20-50

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neurons in the contralateral dorsolateral part of the nucleus oculomotorius (NOC). Various control experiments enabled them to conclude that the labeling of cells in the NOC is not the result of leakage of the tracer out of the eyeball into the muscles, and that the cells were indeed the site of origin of centrifugal fibers. In parallel experiments using iontophoresis, they injected a tracer into the NOC and were able to observe labeled axons of medium size from the injected area penetrating the optic tract and then the contralateral nerve to terminate in the inner nuclear layer of the retina. Taken as a whole, these data could therefore indicate that in the rat, centrifugal fibers originate in the NOC. Using the same methods, Jaeger and Benevento (1980) also described neurons of the oculomotor complex that project to the eye in the macaque monkey and the rabbit. However, these authors put forth the hypothesis that these cells were connected to the intraocular muscles. It is worth noting that after intraocular injection of tracers, several authors (Itaya and Itaya, 1985; Schnyder and Kunzle, 1984; Weidner et al., 1983; Villar el a / . , 1987) have described labeling of cells in the NOC and trochlear nucleus. The latter has been interpreted as resulting from extraocular leakage of the tracer and subsequent uptake by oculomotor nerve terminals or fibers innervating extraocular muscles. Following the intraocular injection of different tracers (RITC, HRP, cholera toxin-conjugated HRP) in the rat and adult rabbit, Muller and Hollander ( 1988) described the presence of 100 labeled ganglion cells in the eye contralateral to the injection. They were able to demonstrate the existence of a retinoretinal projection in both species. Such a system had previously been suggested by Parsons (1902) in the rabbit and subsequently described in the rat during the course of development (Bunt and Lund, 1981; Bunt et al., 1983). In conclusion, the overall data seem to indicate the existence of a centrifugal system in mammals, but its exact origin has yet to be specified. The difficulties encountered are probably due to the paucity of centrifugal visual neurons in this class of vertebrates.

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B. FUNCTIONAL CONSIDERATIONS Rather curiously, there is a large body of indirect physiological data that provide evidence of the existence of a retinopetal system in mammals, in contrast to the relatively more controversial anatomical data. Dodt ( 1956) recorded action potentials from ganglion cells to electrical shock applied to the contralateral optic tract in the rabbit. This elicited a typical antidromic action potential superseded by a second delayed spike (delay of 7-25 milliseconds), which was attributable to the activation of

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the centrifugal fibers. Molotchnikoff and Tremblay (1983a,b 1986) recorded retinal ganglion cell activity at the optic chiasm following the cryogenic blockade of the visual cortex and/or the pretectum in the rat, and have suggested that the cortex controls retinal output. Cooling the visual cortex produced both a shortening of the latencies of the poststimulus time histogram and the appearance of postexcitatory inhibitory pauses. Measurements of interspike intervals have shown that the depression of the cortex results in a more oscillatory pattern of the cells’ firing rate, and the effects are more pronounced when the light stimuli are of stronger intensity. Similar effects on ganglion cell activity have been obtained after blocking the pretectum that, together with the cortex, appear to act synergistically on the retina. Finally, the ganglion cells that appeared to be most affected by centrifugal fibers were those that responded strongly to the dimming (OFF) of the light and lacked a concentrically organized receptive field. Spinelli and Weingarten (1966) combined auditory and somatic stimuli with light to study the influence of nonvisual stimuli on light-evoked discharges of retinal ganglion cells of the cat. Among 300 fibers, 29 were identified as efferent because they were selectively activated by clicks, shocks, or both. The coupling of nonvisual stimuli with flashes produced early (shortened latencies) responses. In monkeys, Ogden and Brown (1964) employed intraretinal recording techniques to study responses evoked by electrical stimulation of the optic nerve. The stimulation evoked a single antidromic action potential and, in addition, a long-latency slow positive wave (P wave) believed to be initiated by the excitation of efferent fibers running through the optic nerve. Several investigators (Jacobson and Gestring, 1958a,b; Jacobson and Suzuki, 1962; Nagaya et al., 1962; Abe, 1962; Van Hasselt, 1972a,b) have observed an increase in the cat or rabbit electroretinogram (ERG) following either optic nerve section or chronic manipulations of the eye. These results, however, could not be repeated by others (Arden et al., 1960; Brindley and Hamasaki, 1962). Jacobson and Gestring (1958a) indicated that in cats and monkeys, the injection of pentylenetetrazol (Metrazol) and hexamethonium during electrical stimulation of the reticular formation produces a depressed ERG, which is then eliminated by sectioning the optic nerve. Interestingly, double-flash experiments in cats and rabbits showed that retinal excitability following a conditioning flash is abolished or reduced (Van Hasselt, 1969; Molotchnikoff et al., 1988). Also accompanying these changes, the oscillatory potentials of the ERG have been shown to increase significantly in magnitude both in the rabbit (Molotchnikoff et al., 1988) and following lesioning of the pretectal area in the rat (Takahashi et al., 1987). A Fourier analysis of the ERG compo-

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nents in the rabbit indicated that the highest power increases following the treatment occurred at -200 Hz. It is worth mentioning that in the rabbit retina, the narrow-field bistratified amacrine cells fire at this frequency (Dacheux and Raviola, 1986). In humans, the existence of a centrifugal system stems from ERG and behavioral studies. In 1966, Gills reported that two patients who underwent a surgical section of one optic nerve near the chiasm showed an increase in ERG relative to the untreated eye. Eason et al., (1983) studied central influences on the human retina by using a selective-attention paradigm. Their results indicated that the amplitude of the ERG obtained to peripherally shone flashes were influenced by experimental manipulation of the subject’s attention. Retinal responses to stimuli presented at attended locations were larger than those at unattended sites. The authors claimed that the larger responses are due to a centrifugal path that controls retinal transmission. However, since the eye movements were not monitored in these experiments, no indication regarding actual eye positions were provided, a factor that in itself could alter subject responsiveness.

VIII. Conclusions Extrinsic axons to the retina are found in most of the large taxonomic groups of vertebrates, from the lamprey to the human. These fibers always terminate upon amacrine cells, more rarely upon ganglion cells (lamprey, birds) and upon IPC (white perch). In numerous species (the majority of teleosts, amphibians, turtles, mammals), the number of centrifugal fibers is low (6-50). However, upon entering the retina they show a considerable degree of collateralization and thus may be considered to be divergent. These fibers are generally of small diameter and unmyelinated (except in the human). In other groups (lampreys, snakes, crocodiles, birds), the number of centrifugal fibers is much higher (750-10,OOO). These axons are usually of large diameter and myelinated (except in the lamprey). It has also been demonstrated that the retinopetal fibers are predominantly convergent in birds. In teleosts, several neuropeptides have been colocalized within retinopetal fibers, including LHRH-like, FMRF amide-like, and substance Plike peptides. In the frog, the retinopetal fibers are amide- and substance P-immunoreactive. In the turtle, the retinopetal fibers contain a met-enkephalinlike peptide, and in the same reptiles as is the case in the rat, some centrifugal axons have been shown to be serotoninergic. The sites of origin of centrifugal fibers appear to vary considerably

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from one vertebrate group to the next and have been identified within the mesencephalon, diencephalon, or telencephalon. Consequently, it is very difficult to establish adequate homologies between these structures. At least three hypotheses that attempt to explain either the high degree of variability or the a priori contradictory results can be advanced. 1. The centrifugal visual system of vertebrates is polyphyletic in origin, having appeared on several occasions and independently in different vertebrate lineages and arising from rather diverse structures. As such, the wide range of centrifugal visual structures identified may be functionally different and cannot be considered as being truly homologous. Instead, they may be referred to as being homoplasic (Campbell and Hodos, 1970). It should be noted, however, that a centrifugal visual system is present in one of the most primitive living vertebrates, the lamprey, and consequently represents a very primitive anatomical arrangement that probably arose at the agnathan stage. Elsewhere, if one assumes that gnathostome vertebrates stemmed from the main lineage of jawless vertebrates (Romer, 1973), the phylogenetic hypothesis implies that this specific anatomical arrangement was lost at the gnathostome stage, but was then reestablished during the course of evolution in the different lineages of gnathostome vertebrates. 2. According to Ebbesson (1980), neuronal systems evolve by a process involving the loss of connections instead of the creation of new connections with hitherto-unrelated targets (parcellation theory). For example, the extensive retinopetal system seen in teleosts may reflect a primitive organizational arrangement, and the reduction in such connections that has been described in more advanced vertebrates (reptiles, birds, mammals) may be the result of the neuronal system evolving by a loss of pathways (Ebbesson and Meyer, 1981). This evolutionary hypothesis is questionable because in lampreys, which constitute a more primitive vertebrate form than the teleost fish, the number of structures of origin of centrifugal visual pathways appear more reduced (two) than those found in fish (as many as five), which represent the most evolved forms of the actinopterygian lineage (Romer, 1973). 3. The neuronal labeling that has been observed within various structures following the intraocular injection of different tracers may indeed be the result of retrograde axonal transport after being taken up by centrifugal fibers. However, because of insufficient controls in several experiments previously reported, labeling may be due to either the transsynaptic transport of tracer substances from the optic terminals, or the retrograde transport of tracers from extraretinal axon terminals that

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are intraocular or extraocular. Therefore, such structures cannot be considered to represent the actual sites of origin of centrifugal visual pathways. Consequently, it would be difficult, given our present state of knowledge, to explain clearly the existing interspecies differences in the topographic location of the structures believed to be retinopetal. The questions must be reconsidered using new experimental approaches, particularly immunohistochemical ones. In addition, it would be interesting to investigate the centrifugal visual system in other primitive forms, such as the chondrosteans and holosteans of the actinopterygian lineage and the dipnoans of the sarcopterygian lineage. This would provide additional information for testing any of the aforementioned hypotheses. Furthermore, a comparative analysis of these structures at both the hodological (intracerebal connections, precise localization of their retinal projection zones) and functional levels should offer a better understanding of the basis for such marked interspecies variation and its phylogenetic significance.

FUNCTIONAL ROLESOF THE RETINOPETAL SYSTEM Although there appears to be an abundant body of anatomical and physiological data accumulated, the role of the retinopetal system is far from being elucidated. It is conceivable that a given functional role may apply to all species or, conversely, it may be species-specific. A centrifugal control of peripheral sensory organs is common in other systems. For instance, one of the best-known systems is the control of muscle spindles by y-efferent fibers. In a sense, the y loop “recalibrates” the sensitivity of the spindles and thereby plays a role in controlling the degree of muscular oscillation. In the auditory system and the lateral line of fish, there is evidence of a similar central control of their response spectrum. In these systems, the centrifugal fibers allow rapid adjustments of sensitivities, which are mostly necessary after sudden stimulation. Similar roles that seem to accomplish important functions in other sensory and motor domains might also be attributable to the centrifugal visual system. The functions associated with the retinopetal system may be divided into two broad classes, which are not necessarily exclusive and may even be complementary to each other. The first function links the retinopetal system to processes associated with eye movements. A second function links the efferent visual system to the control of retinal sensitivity. The first hypothesis was supported by Miles ( 1972a-d), who suggested that the functions of the retinopetal system may be considered as a feed-for-

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ward mechanism related to motor programming processes. An efferent neuronal signal such as a “corollary discharge” (Trevarthen, 1968)would act to compensate or cancel out the effects produced by the animal’s own movement. Hence, any voluntary or involuntary movement would be accompanied by centrifugal inhibition. For instance, the absence of a blur during saccades would be due to a transient elevation of the visual threshold-thus masking the slippage of the image on the retina that occurs during eye displacement. In order to be effective, a corollary discharge must contain sensory as well as motor components. There is, however, a difficulty in associating the retinopetal system with the corollary discharge. The major drawback is the often small number of retinopetal axons counted in the optic nerve. Hence, it is somewhat difficult to reconcile the necessity of an elaborate neuronal network that is capable of handling the visual sensorimotor coordination with such a rather small number of retinopetal cells. In birds, however, the NIO receives a robust input from the TO, and in both structures, cells are particularly responsive to moving stimuli. Thus, a full corollary signal may be elaborated at the collicular level and converge upon the NIO, to be then selectively directed to the retina. The second role attributed to the retinopetal fibers is to provide a dynamic local adaptation or to modulate retinal sensitivity. Light and dark adaptations have relatively long time courses, as they depend on the kinetics of pigment turnover. Furthermore, these adaptations are homogeneous across the retina. However, in everyday life the eyes constantly scan the visual environment; hence the amount of light that strikes the retina varies quasi-instantly in time and space. Since the biochemical mechanisms are relatively slow, one needs a rapid and local process of adaptation of retinal sensitivity, which might more adequately be accomplished by neuronal mechanisms of the centrifugal system. The notion of a link between the retinopetal system and sensitivity offers the advantage that it does not require a large number of cells, since few synaptic contacts located at strategic sites such as axonal hillocks or somata may suffice to influence considerably the unit firing rate. Since centrifugal axons terminate preferentially upon amacrine cells, which are interneurons between bipolar and ganglion cells, they can effectively influence the output stage of the retina. Indeed, in all experiments where the presumed centrifugal system has been manipulated either through cryoblockade or with the addition of nonvisual stimuli, the ganglion cell responses were altered but the trigger features were not. It is the strength or the pattern of the responses that is influenced, while the visual properties remain unaffected. Finally, the centrifugal system may provide a path for cross-influences

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between various sensory modalities at the peripheral level. The discovery that centrifugal fibers originate in the olfactory system in fish and frogs lends support to an anatomical basis for an olfactovisual loop. It is thus conceivable that olfactory cues such as specific molecules dissolved in water have a bearing on retinal sensitivity.

ACKNOWLEDGMENTS This work was supported by grants from INSERM. Convention d'Echange Franco-Soviof Sciences of the U.S.S.R.).. etique (C.N.R.S./Affaires Etrangeres-Academy C.R.S.N.G., C.R.M.C.. Cooperation France-Quebec MA1 and A.U.C.C.-CanaddMINVUZ-U .S.S.R. scientific exchange grants.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 118

Cell Biology and Kinetics of Kupffer Cells in the Liver K. WAKE,* K. DECKER,?A. KIRN,$ D. L. KNOOK,§R. S. MCCUSKEY,11 L. BOUWENS,**AND E. WISSE** *Department of Anatomy, Tokyo Medical and Dental University, Faculty of Medicine, Yushima, Bunkyo-ku, Tokyo, 113 Japan iBiochemisches Institut, Albert-Ludwigs-Universitat,0-7800 Freiburg i . Br., Federal Republic of Germany SLaboratoire de Virologie, FacultP de Medicine and INSERM U 74, 67000 Strasbourg, France Unstituut voor Experimentele Gerontologie TNO, 2280 HV Rijswijk, The Netherlands 11 Department of Anatomy, School of Medicine, University of Arizona, Tucson, Arizona 85724, **Laboratorium voor Celbiologie en Histologie (VUB), 1090 Brussel-Jette, Belgium

I. Introduction

The history of the Kupffer cell is a long and tortuous one. The current concept has evolved over more than a century. The first concept was established with the discovery of the Sternzeflen by von Kupffer in 1876. Von Kupffer (1876) and his pupil Rothe (1882) extensively described the Sternzelfen in the livers of various mammals by using a unique gold chloride-staining method (Fig. 1). These authors were of the opinion that the Sternzeffenwere localized outside the hepatic sinusoids and belonged to the “perivascular connective tissue cells” described by Waldeyer. Twenty-two years after his first report, however, von Kupffer changed his opinion (von Kupffer, 1898, 1899). He observed that rabbit sinusoidal endothelial cells took up india ink after intravenous injection (Fig. 2), and equated them with the Sternzelfen in his gold chloride preparations of human livers. This misunderstanding (see Wake, 1980) led him to conclude that the gold-reactive Sternzeffen were phagocytic in nature and were integral elements of the syncytial endothelium of the hepatic capillaries. His theory that particles were directly taken up from the bloodstream by the endothelial lining cells in the hepatic capillaries was widely accepted among contemporary researchers. Vital staining was introduced by Ribbert in 1904 for the purpose of identification of phagocytic cells. Kiyono (1914) used vital staining extensively, testing >1000 dyes and systematically examining phagocytic cells in various tissues and organs. According to Kiyono, histiocytes and the 173 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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cells of the reticuloendothelium had the same ability of taking up dyes, and it was supposed that they had a common origin. He equated the Kupffer cells with the liver histiocytes. Taking this concept into consideration, Aschoff (1924) proposed the “Reticulo-Endotheliales System,” or RES. This system consisted of (1) reticular cells in the lymphoid organs, (2) endothelial cells of the hepatic lobules (Kupffer cells), splenic sinus, and lymph nodes, (3) histiocytes in the connective tissue, (4) splenocytes, and (5) monocytes (endothelial leukocytes or blood histiocytes). At that time, Zimmermann (1928) demonstrated three kinds of cells in and around the sinusoids of the liver: (1) endothelial cells, (2) “endocytes” (Fig. 2), and (3) “pericytes” (Fig. 3). He denied the syncytial nature of the endothelial cells and concluded that only the endocytes were Kupffer cells. The endocytes had been described by Browicz in 1898 (cited from Browicz, 1900). Browicz described pear-shaped cells hanging in the lumen of the sinusoid with their processes attached on the inner surface of the sinusoidal walls. The concepts as conceived by Browicz and Zimmermann were proven by modern techniques and methods of investigation to be fundamentally correct. However, their ideas encountered strong criticism from devotees of the RES, who proposed that the three profiles of cells corresponded to various functional stages of one cell type. Under the fluorescence microscope, quick-fading vitamin A fluorescence is emitted from cells scattered in the hepatic lobules. These fluorescent cells were thought to be reticuloendothelial Kupffer cells. Investigations by Wake (1964, 1971), using Kupffer’s gold chloride method, fluorescence microscopy, and electron microscopy (EM) provided new evidence indicating that Sternzellen, stained by the original gold chloride method, were persinusoidally located and stored vitamin A. Refined techniques such as the application of bone marrow chimeras, parabiosis, and autoradiography extended our knowledge on macroFIG. 1 . Light micrograph of perisinusoidal cells in rat liver, stained with the classical gold chloride method, developed by Kupffer in 1876. Kupffer referred to these cells as Sternzellen (stellate cells). X 240. FIG. 2. Light micrograph of endothelial cells and Kupffer cells, labeled with intravenously injected india ink particles. Kupffer cells can be recognized by large aggregates of dark material; endothelial cells contain smaller amounts of the same material. x 500. FIG.3. Light micrograph of a Golgi-stained rat liver preparation. Silver precipitates are present on a cell, called a pericyte by Zimmermann in 1923, and today mostly designated as a fat-storing, or stellate cell. X 2000. FIG. 4. Autofluorescence of vitamin A as demonstrated by fluorescence microscopy, which inspired Kudo in 1938 to recognize these cells as reticuloendothelial cells. At present we know that this reaction is also typical of fat-storing cells. X 500.

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phages considerably after 1960. Van Furth et al. (1972) proposed the concept of the mononuclear phagocyte system (MPS). According to this system, Kupffer cells originate from blood monocytes. Concerning the origin of macrophages and Kupffer cells, however, van Furth’s concept has not been fully accepted. Wisse (1970, 1972, 1974a,b). demonstrated, using perfusion fixation of rat livers, that endothelial and Kupffer cells are different types of cells without transitional forms, even under conditions reported to enhance the frequency of such transitions, such as partial hepatectomy, blockade of the RES, and RES stimulation by zymosan or splenectomy. Both cells also differed in cytochemical properties and endocytosed particles of different sizes by different mechanisms. With these refinements, the study of Kupffer cells has developed rapidly and has been extended to problems concerning the origin, structure, and function of these cells in normal and pathological conditions. In this review we present an overview of the current morphological and biochemical data. Many of the major advances in our knowledge about Kupffer cells have resulted from the development of new methods, such as cell isolation, purification, and culture.

11. Morphology of Kupffer Cells

A. FIXATION AND MICROSCIPIC RECOGNITION OF KUPFFERCELLS

Studying the morphology of the different types of sinusoidal cells and distinguishing among them has been made possible by the combination of perfusion fixation (Fahimi, 1967; Wisse, 1970), transmission electron microscopy (TEM) (Wisse, 1970, 1972), enzyme cytochemistry (Widmann et al., 1972; Wisse, 1974a,b), and particle (mainly 0.8-pm latex) injections (Widmann et al., 1972). By using a proper combination of these methods, it is possible to characterize four different cell types as integral inhabitants of the sinusoidal wall: endothelial cells (Wisse, 1970, 1972), Kupffer cells (Fahimi, 1970; Wisse, 1974a,b), pit cells (Wisse et al., 1976), and fat-storing cells or perisinusoidal stellate cells (Ito and Shibasaki, 1968; Wake, 1971, 1980). It is important to note that perfusion fixation not only causes better fine-structural preservation of the cells but also preserves the histological arrangement, position, and shape of the sinusoidal cells. We might conclude that perfusion fixation has enabled us to disprove the theory that various sinusoidal cells are different functional stages of a single cell type. By using the specific staining techniques for peroxidase (Fahimi, 1970; Wisse, 1974a,b), in combination with a small dose of 0.8-pm latex parti-

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cles, it is possible to visualize rat liver Kupffer cells unequivocally in light-microscopic (LM) sections, in ultrathin sections, in frozen sections, and in freshly isolated sinusoidal cell suspensions. After culturing, however, Kupffer cells lose their peroxidatic activity (Emeis and Planque, 1976). Other criteria for the characterization of Kupffer cells, such as (a) cell shape or the position in the sinusoid, (b) the uptake of small particulate colloids such as carbon, colloidal gold, and thorotrast, or (c) esterase or acid phosphatase activity or the presence of lysosomal dense bodies or other dense inclusions, are not conclusive evidence to characterize Kupffer cells in situ (Emeis and PlanquC, 1976).These considerations are valid for rat liver; the characterization of Kupffer cells in mouse liver is further complicated by the fact that some sinusoidal endothelial cells are also positive for peroxidase (Stohr et af., 1978). The situation in human liver is at present unclear. The recognition of Kupffer cells at low magnification in the light microscope facilitates the study of lobular distribution of Kupffer cells and allows the quantitation of Kupffer cells in different experimental situations by counting the number of cells within a given microscopic field (Bouwens et al., 1984; Bouwens and Wisse, 1985). Promising new possibilities for recognizing sinusoidal cells may be found in the development of antibodies, which has been made possible by isolation and purification methods for different types of sinusoidal cells. B. KUPFFER CELLSIN TEM Kupffer cells and pit cells have variable shapes and positions in the sinusoids, in contrast to endothelial and fat-storing cells. This aspect suggests that the latter cell types are true sessile components of the sinusoidal wall, whereas the former types might be more plastic and mobile. This is supported by the fact that Kupffer cells and pit cells move in experimental circumstances to different positions, such as the space of Disse or the space between parenchymal cells, or even take part in the formation of liver granuloma (Wisse, 1974a,b; Wisse et al., 1976). Nevertheless, both Kupffer cells and pit cells can be regarded as true inhabitants of the sinusoidal wall, because they anchor with microvilli in the space of Disse and they show local proliferation after partial hepatectomy ,just as endothelial and fat-storing cells do. The shape and the surface of Kupffer cells is irregular; the cells have elongated cytoplasmic processes that can stretch along or underneath the endothelium (Fig. 5). Kupffer cells lie upon, or are embedded in or covered by the endothelium. In some cases, they may traverse the sinusoidal lumen with a thick cytoplasmic process. The presence of fenestrae easily

I78

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distinguishes endothelial processes from those of Kupffer cells. No special cell contacts exist between Kupffer cells and endothelial cells; they simply touch each other and lack gaps or specialized junctions of any kind. Kupffer cells also contact other sinusoidal cells, like fat-storing cells, pit cells, or collagen bundles in the space of Disse, but no special morphological structures result from these contacts. Kupffer cells do not contact other Kupffer cells, except during granuloma formation, such as is seen after zymosan injections (Wisse, 1974b). With special precautions or experimental approaches, a fuzzy coat can be visualized covering the Kupffer cell surface. Perfusion fixation with glutaraldehyde does not preserve this fuzzy coat. However, it can be found (a) 3 minutes after intravenous injection of colloidal particles or erythrocyte ghosts, which probably stabilizes the coat by attachment (Heifer, 1970; Wisse, 1974a);(b) in osmium-fixed frozen sections; and (c) in different intracytoplasmic structures such as wormlike structures (Toro et af., 1962) and fuzzy-coat vacuoles (Wisse, 1974a). The nature of this fuzzy coat is unknown and forms one of the intriguing problems of Kupffer cell biology in that phagocytosis starts with the attachment of particles during which the presence of receptors and opsonic proteins might play a decisive role. The further analysis of molecules in the fuzzy coat seems possible by using available visualization techniques such as immunogold staining of ultrathin frozen sections. Kupffer cells play a major role in the uptake of endotoxin, altered blood cells, or foreign particles. To accomplish endocytosis, Kupffer cells possess several morphologically recognizable mechanisms that have been shown to contain or to transport material. The mechanisms involved are bristle-coated micropinocytosis, fuzzy-coat vacuoles (a kind of macropinocytosis), wormlike structures, and phagocytosis by engulfment and further internalization. Interestingly, these structures (with the exception of phagocytosis) can be seen in normal Kupffer cells of animals that have received no injection or treatment, implying that these endocytotic mechFIG. 5 . Transmission electron micrograph of a Kupffer cell in rat liver. The cell can be specifically recognized by peroxidase staining, resulting in electron-dense reaction product in the RER and the nuclear envelope. Further details in the picture represent dense bodies or lysosomes and ruffles at the cell surface. L, Sinusoidal lumen; Pc, parenchymal cell; bc, bile capillary; SD, space of Disse. X 4526. FIG. 6. Scanning electron micrograph of a Kuppfer cell in siru. The surface of the cells shows numerous folds and plicae. The cell body bulges into the lumen, and interaction with blood cells is to be expected. The sinusoidal wall contains endothelial fenestrae (arrows). The space of Disse is deprived of endothelial lining, and shows the microvillous sinusoidal surface of the parenchymal cells (*). C, A small bundle of collagen is situated at the parenchymal cell surface. X 15,425.

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anisms may be constantly operational and active (Wisse, 1977). Apart from vacuoles of different sizes, a heterogeneous population of dense bodies (lysosomes) can be found in the cytoplasm. The size, density, and shape of these bodies are highly variable, but their abundance and relative volume (Blouin el a / . , 1977) clearly indicate a considerable capacity of Kupffer cells in vivo for endocytosis and digestion. The absence of recognizable structures in these lysosomes under normal circumstances suggests a rapid digestion and/or the uptake of molecules rather than particles or (parts of) cells. Fat droplets, autophagic vacuoles, or multivesicular bodies have not been described for Kupffer cells in siru. Kupffer cells possess a normal set of cellular organelles. The nuclear envelope, together with the rough endoplasmic reticulum (RER) and the mysterious annulate lamellae, is the site where the peroxidatic activity is located. The shape of the nucleus is different from other sinusoidal cells; flat or blunt endings can give the nucleus an almost rectangular appearance in some sections. The presence of a number of RER cisternae and Golgi apparatuses suggest the involvement of Kupffer cells in protein secretion. For further morphological descriptions of Kupffer cells, the reader is referred to Fahimi (1970, 1982), Wisse (1974a,b, 1977), and Wisse and Knook (1979).

c. KUPFFERCELLSIN SCANNING ELECTRON MICROSOPY(SEM) Most Kupffer cells are easily recognizable in SEM specimens taken from perfusion-fixed livers (Fig. 6). In these preparations, natural surfaces of cells and sinusoids become available for SEM observation by applying freeze-fracture, critical-point drying, and gold sputtering. The intactness of the sinusoidal wall, mainly the fenestrated endothelium, critically depends on the application of a “physiological,” low-pressure perfusion fixation (De Zanger and Wisse, 1982), which results in the sinusoids being cleared of cells and plasma. Kupffer cells are infrequently present and take positions at bifurcations or larger periportal sinusoids. Unfortunately, not all Kupffer cells can be recognized with 100% certainty in SEM, because the two reliable TEM criteria (i.e., peroxidatic activity and ingestion of latex particles) have limited value in SEM. In SEM, Kupffer cells have variable surface irregularity (Motta, 1977). In some cases, the cell surface bears some small and slender microvilli, in other cases microvilli are accompanied by different pseudopodia and larnellipodia. Sometimes, regions with a surface irregularity corresponding to wormlike structures can be observed. The characteristic irregularity of the Kupffer cell surface differs drastically from the smooth surface of endothelial cells, or the branched, flat luminal surfaces of fat-storing

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cells, occasionally seen through large gaps in the endothelial lining. Kupffer cells branch, and this phenomenon can give the cells a stellate appearance. In SEM, one has a better three-dimensional impression of the shape and magnitude of Kupffer cells whereas LM or TEM sections represent only a finite two-dimensional sample. Thinner projections might radiate from the Kupffer cell surface to the endothelial wall. These thin processes resemble the guy ropes of a tent. In Motta’s opinion (1977), such processes attach the Kupffer cell to the endothelial lining and might cause the appearance of gaps (openings larger than fenestrae). At higher magnification small pits can be observed that correspond to the presence of coated pits. At these places, no attachment of material can be observed. In the case of particle injections, SEM preparations vividly illustrate some of the events taking place at the surface of Kupffer cells. The surface can be seen to be covered with small colloidal particles or can be seen engulfing larger particles, such as latex. In all cases, the presence of a number of platelets surrounding the Kupffer cell during this phagocytic activity becomes apparent. The platelets seem to be attracted by an unknown factor, but they are not phagocytosed. Their presence can be confirmed by in vivo microscopy and sectioned material studied in LM or EM. Many hematologists have been questioned about the possible meaning of this phenomenon, but at present no obvious reason for this platelet clustering seems to exist.

111. Population Dynamics of Kupffer Cells

A. DISTRIBUTION AND POPULATION

SIZE

Kupffer cells are the most important category of fixed reticuloendothelial cells (Jones and Summerfield, 1982; Singer et al., 1969). They are thought to constitute the largest population of fixed macrophages in humans and various vertebrate species. In many diseases, an increase and sometimes a depletion of liver macrophages is known to occur. It is therefore of interest to investigate the change in numbers and distribution of Kupffer cells in normal and experimental situations, together with the mechanisms of their generation. A prerequisite to allow easy and reliable quantitation of Kupffer cells is an unequivocal identification of these cells at the LM level. Uptake of latex beads >O. 1 pm allows distinction between Kupffer cells and sinusoidal endothelial cells (see also Section II,A), but it has been demonstrated that not all Kupffer cells can be labeled by intravenous injection of

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such particles. Especially cells located in central parts of the liver lobule seemed less active in phagocytosis, even when these particles were perfused retrogradely through the hepatic vein (Sleyster and Knook, 1982). We found that after latex particle (0.8-km) administration in doses approaching overload condition, a maximum of only 65% of Kupffer cells, as recognized by peroxidase staining, were labeled in liver sections (Bouwens et al., 1989). The best method currently to stain rat Kupffer cells specifically is endogenous peroxidase staining. At the EM level, all cells that can be recognized as Kupffer cells by position and ultrastructural characteristics (Wisse, 1974a,b) contain peroxidase activity in the RER, nuclear envelope, and annulate lamellae. Moreover, no other sinusoidal cell type exhibits this peroxidase pattern (Wisse and Knook, 1979). The merit of this technique is that it also can be used at the LM level. Furthermore, the peroxidase marker is found in all cell intersections and it seems to be independent of the functional state or position of the Kupffer cells in the liver lobule. In this way, small Kupffer cells in the central region are not overlooked or confused with other sinusoidal cell types. Using peroxidase staining and morphometric methods, the total number of Kupffer cells was estimated to be -200 x lo6 for a rat weighing 250 g and a liver weighing 9 g (Bouwens ef a/., 1986a). After cell isolation, however, a recovery of 4-14 X 10" Kupffer cells per gram liver has been reported, representing -22% of all nonparenchymal liver cells (Knook et al., 1977). In situ, the distribution of Kupffer cells over the liver lobule is not homogeneous. When the liver lobule is divided in three regions from portal to central, the Kupffer cells have been reported to be distributed in an approximate ratio of 4 : 3 : 2, by means of morphological and endocytotic characterization (Kaneda and Wake, 1983; Sleyster and Knook, 1982). There is an obvious enrichment of Kupffer cells around portal veins, where these cells are larger and more active than those in the central region of the lobule. After cell isolation, two Kupffer cell subpopulations can be discerned that differ in size and several activities (Sleyster and Knook, 1982). This means that the Kupffer cell population is probably functionally heterogeneous. When the Kupffer cell population is stimulated to expand by a single intravenous injection of zymosan, its cell number was seen to increase 4fold within a period of 5 days (Bouwens et a!., 1984). This dense Kupffer cell population was distributed relatively homogeneously throughout the liver lobule, although there again existed a slightly higher density around portal veins, together with focal accumulations of macrophages in granulomas. A comparable increase of liver macrophages after stimulation by

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a variety of inflammatory agents has been reported (Diesselhoff-den Dulk et al., 1979; Kelly et al., 1960, 1962; Kelly and Dobson, 1971; North, 1969, 1970; Warr and Sljivic, 1974). It has been demonstrated that the glucan-induced numerical increase of liver macrophages correlated directly with an increase in reticuloendothelial clearance of particles from the blood (Di Luzio et al., 1970). Such a Kupffer cell increase undoubtedly represents an important mechanism for host defense but can also contribute to the damage of parenchymal cell structure and function. The understanding of the mechanisms generating this population expansion is therefore of considerable interest for the understanding of the pathogenesis and therapeutic intervention of chronic inflammatory diseases. It is a commonly held belief that increased tissue macrophage numbers are the direct consequence of an enhanced influx of monocytes, the putative macrophage precursors, to the site of inflammation. Moreover, according to the current concept of the MPS, all tissue macrophages or resident macrophages, including Kupffer cells, should be considered as nondividing end cells derived from blood monocytes (Van Furth et al., 1972; Van Furth, 1982). Therefore, little attention has been paid to the possible role of an enhanced local replication of tissue macrophages after stimulation. Nevertheless, there is an increasing amount of evidence indicating that macrophages in several tissues retain their mitotic potential. The quantitative significance of such local proliferation of tissue macrophages as compared to the derivation from blood monocytes is the subject of considerable controversy. B. ONTOGENY

A replicating population of sinusoidal macrophages is established from day 11 of gestation in fetal rat liver (Deimann and Fahimi, 1978; Naito and Wisse, 1977; Pino and Bankston, 1979). This is well before bone marrow is formed (from day 19) and before monocytes have appeared in the circulation (from day 17). These fetal Kupffer cells are already phagocytic and have the characteristic peroxidase localization in RER and nuclear envelope, which differs from the granular peroxidase pattern encountered in monocytic cells. These observations exclude the monocytic origin of Kupffer cells at that stage of development and suggest that they have an early embryonic or extraembryonic origin, probably to be found in the yolk sac.

c. BONEMARROWDERIVATION VERSUS LOCALPROLIFERATION In the liver of sublethally irradiated recipients that have received bone marrow grafts, macrophages of the donor type were identifiable by means

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of sex chromosome staining in humans (Gale et a f . , 1978) or by immunological markers within rat-mouse chimeras (Shand and Bell, 1972). After human liver transplantation, recipient-type macrophages recognizable by karyotyping invaded the donor liver (Portmann et al., 1976) or even seemed to replace the original Kupffer cell population (Porter, 1969). This experimental evidence in favor of a bone marrow derivation of Kupffer cells, however, also demonstrated their mitotic potential in that karyotypic characterization required the occurrence of Kupffer cell metaphases. The origin of liver macrophages accumulating after RES stimulation by inflammatory agents such as zymosan, Corynebacteriurn parvurn, Listeria monocytogenes, bacterial endotoxin, and estrogens has been investigated by different experimental approaches. When tritiated thymidine was administered to animals before the application of the inflammatory stimulus, an influx of extrahepatically labeled macrophages was demonstrated to occur into the liver. Because free tritiated thymidine was no longer available at the time of stimulation, the observed increase in labeled liver macrophages could not be attributed only to local proliferation (Diesselhoff-den Dulk et a f . ,1979; North, 1970). However, local proliferation of resident macrophages that were shown to be already present in the liver before the time of stimulation also was reported under these experimental circumstances (Kelly and Dobson, 1971; North, 1969). The existence of local and extrahepatic Kupffer cell derivation has been observed after partial irradiation of animals with either the liver o r the bone marrow shielded (North, 1970; Warr and Sljivic, 1974). This dual origin of Kupffer cells also can be concluded from reports of apparently contradictory results with parabiotic experimental animals (Kinsky e f a f . , 1969; Volkman, 1976). Because of this dual origin, one may have to discriminate between two different Kupffer cell populations: those proliferating in situ and those recently recruited from bone marrow. Mitotic activity of Kupffer cells in siru has been reported in normal liver (Bouwens et al., 1989; Kelly et a f . , 1962; Widmann and Fahimi, 1975; Wisse, 1974b), in regenerating liver (Bouwens et a f . , 1984; Widmann and Fahimi, 1975; Wisse, 1974b), after liver stimulation with varying agents such as zymosan (Bouwens et al., 1984; Warr and Sljivic, 1974; Wisse, 1974b) estradiol (Kelly et a / . , 1960), L . monocytogenes (North, 1969), glucan (Deimann and Fahimi, 1979), and in fetal liver (Deimann and Fahimi, 1978; Naito and Wisse, 1977). These data demonstrate that Kupffer cells in different animal species have the potential of self-replication. Although this activity is low in steady-state conditions, cell division can be enhanced by different stimuli. In an attempt to estimate the relative importance of local production

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and extrahepatic recruitment of Kupffer cells during liver regeneration after two-thirds partial hepatectomy and after zymosan stimulation, the metaphase-arrest method was used to determine the potential doubling time of the expanding Kupffer cell population (Bouwens et al., 1984). These experiments revealed that during liver regeneration, the Kupffer cell population grew with a mean doubling time of 3.6 days and that local replication ensured a potential doubling time of 3.7 days. From these data it can be concluded that the total production of new Kupffer cells could be provided by local proliferation. In the case of zymosan-induced exponential growth, these data were 2.5 and 3.5 days, respectively, which means that -70% of the population expansion was due to local proliferation (Bouwens et al., 1984). In the latter experimental model, >90% of Kupffer cells could be labeled by repeated administration of tritiated thymidine over a period of 48 hours (Bouwens e? al., 1986a). This means that nearly all Kupffer cells were in the cell cycle. Furthermore, selective Xirradiation of the liver with a sublethal dose of 850 rad significantly inhibited Kupffer cell growth, whereas irradiation of the whole body with the liver shielded did not have this effect (Bouwens e? al., 1986b). In both experimental models-partial hepatectomy and zymosan stimulation-it could be demonstrated that the dividing Kupffer cells were mature macrophages that were already residing in the liver before the time of stimulation, and thus, were not recruited by the stimulus. This was supported by labeling the cells with latex particles several days before the stimulus (Bouwens et al., 1984). This leads us to conclude that Kupffer cells have a dual origin (i.e., local proliferation and extrahepatic recruitment), but the predominant mechanism underlying their population growth consisted of local proliferation of mature Kupffer cells. In other experimental situations, however, the relative importance of both mechanisms may vary depending on the conditions. D. THE KUPFFERCELLPRECURSOR It is established that Kupffer cells can originate from tissues outside the liver, and because the Kupffer cell precursor must be present in peripheral blood, the most likely candidate for this is the monocyte. Monocytes are the only leukocytes that can develop macrophage characteristics, both in vivo and in vitro, yet there is as yet no incontrovertible proof for the existence of differentiation from monocytes into Kupffer cells. In spite of extensive ultrastructural investigations on this problem (Bouwens et al., 1984; Bouwens and Wisse, 1985; Wisse, 1974a,b), there exists only one report claiming the existence of so-called transitional forms between monocytes and Kupffer cells (Deimann and Fahimi, 1979). Such transi-

tional forms occurring in low numbers after glucan stimulation in rats, were described as cells with the typical peroxidase pattern of Kupffer cells, with the additional presence of monocyte-specific granules. However, our observations on the ultrastructure of sinusoidal cells have revealed the following.

I . Kupffer cells can exhibit a large amount of different lysosomal and other granular structures with varying morphology and density. 2. Kupffer cells often contain erythrocyte debris that also reacts with the peroxidase substrate, and these phagosomes can be confused with peroxidase-positive monocyte granules. 3. Kupffer cells have catalase activity in microperoxisomes that may be confused with peroxidase-containing lysosomes. 4. Even in material that was not incubated for peroxidase demonstration, dense granular structures can occur in Kupffer cells. The occurrence of Kupffer cells containing some dense granules, or “monocyte-type” lysosomes with a halo, is therefore not reliable evidence for the existence of transitional forms between monocytes and Kupffer cells. Moreover, monocytes can be readily seen in sinusoids of normal or stimulated animals, and these cells were never observed developing ER peroxidase activity or other typical Kupffer cell features. On the contrary, after zymosan stimulation, large numbers of monocytes were seen in liver sinusoids developing into large, activated, inflammatory macrophages that remained distinct from the resident Kupffer cells (Bouwens and Wisse, 1985). Thus, it can be suggested that two phenotypically different macrophage types occur during liver inflammation: an “exudate” or monocytic type, and a “resident” type, like the Kupffer cell. This situation is comparable with the situation described in the peritoneal cavity (Daems ct al., 1976; Volkman, 1976; Volkman et al., 1983). In contradistinction to Kupffer cells, the rnonocyternacrophages, attracted by inflammatory stimuli, remain in the liver for a limited period and thus belong to a transient population. The direct Kupffer cell precursor has been described as a peroxidasenegative phagocytic cell that looks like an immature cell, containing few organelles and, in particular, few lysosomes and vacuoles (Bouwens and Wisse, 1985). Whether these latter cells belong to a monocyte subset that have lost their granular peroxidase activity, as is known to occur in monocytes in vitro (Breton-Gorius ct al., 1980), or whether these cells form a distinct macrophage sublineage is not yet established. However, colonies of macrophages, clonigenically derived from individual bone marrow precursors, have been reported to express different phenotypes, suggesting the existence of distinct macrophage lineages (Bursuker and Goldman,

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1983). The existence of separate, stable macrophage subsets differing in functions, phenotype, and origin, however, is still a controversial but fascinating subject. IV. Isolation, Purification, and Culture of Kupffer Cells

Several procedures are presently available for the preparation of isolated and purified Kupffer cells. Each procedure has its own effect on the final yield, the purity, and the functional characteristics of the isolated Kupffer cells. Generally, these procedures involve perfusion and incubation of the liver with dissociating enzymes. The use of collagenase results in a suspension that contains all types of liver cells (Brouwer et al., 1982a,b; Knook e f al., 1982; Nagelkerke et al., 1982).The use of pronase, which selectively destroys parenchymal cells, results in a suspension that consists almost exclusively of sinusoidal cells (Brouwer et al., 1982b). The advantages and disadvantages of these methods have been discussed elsewhere (Brouwer et al., 1982b). The various steps in the purification of Kupffer cells include the following: 1. Perfusion of the liver with dissociating agents. With small experimental animals like the rat and the mouse, perfusion of the total liver is performed. For larger animals and humans, generally only a part of the liver is perfused. 2. Incubation of liver tissue for further dissociation or destruction of parenchymal cells. 3. Further purification of Kupffer cells by centrifugation procedures or by selective attachment. The following is a summary of our present knowledge with regard to the various steps of isolation and the behavior of Kupffer cells during culture. If not stated otherwise, the description refers to the isolation, purification, and cultivation of Kupffer cells removed from the livers of rats. A. ISOLATION OF KUPFFERCELLS

Isolation starts with perfusion of the liver with a fluid containing dissociating agents such as collagenase, pronase, or a combination of both enzyme preparations (Table I). Only collagenase perfusion can be employed for the isolation of both parenchymal and sinusoidal cells from the same liver (Nagelkerke et al., 1982; van Berkel, 1982). The perfusion is generally performed at 37°C for optimal activity of

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TABLE I CHARACTERISTICS OF

KUPFFEKCELLS

ISOLATED B Y VARIOUS

Sinusoidal cell preparation s/r of

Isolation method Pronase 37°C

10°C Collagenase Pronasecollagenase

Kupffer cells

21 17 14 19

Yieldh of Kupffer cells (X

10")

34 35 39 46

METHODS"

Kupffer cell fraction' Compositiond

L

E

K

3 6 15

24 18 7

74 76 78 76

24

Protein content (&lo6 cells)

87.4 113.9 n.d. 114.2'

Viability' of Kupffer cells

90 90 85 90

"Average of last four experiments. Cells were isolated with pronase at 37°C (Knook and Sleyster. 1976). with pronase at 10°C (Praaning-Van Dalen and Knook. 1982). or with pronase and collagenase (Knook C f ul., 1982). 'Yield represents the number of cells per liver (mean weight 4.8 g ) of 3- to 4-month-old female Brown Nonvay/Billingham Rijswijk rats with an average body weight of 150 g. 'Standard procedures were applied for purification of Kupffer cells by centrifugal elutriation. The standard separation chamber was replaced by the Sandreson chamber for the purification of the Kupffer cells obtained by the pronase 10°C method. dLymphocytes ( L ) . endothelial (EJ. and Kupffer ( K ) cells are expressed as percentages of total cell number. 'Percentage of cells that exclude 0 . 2 5 8 trypan blue. Results were confirmed by EM studies. 'Not determined.

the dissociating enzymes. However, methods are now available that are performed at d 10°C. using either pronase (Praaning-Van Dalen and Knook, 1982) or collagenase (Nagelkerke et al., 1982). After perfusion, the hepatic tissue is incubated for further dissociation by mechanical or enzymatic means. In addition, selective destruction of parenchymal cells can be accomplished by pronase treatment (Knook et a/., 1982), extended collagenase treatment (Brouwer et al., 1982a), or incubation with enterotoxin (Berg et al., 1979). The incubation step can be omitted if one employs the "two-step perfusion" method, that is, an initial calcium-free perfusion, followed by perfusion with collagenase and calcium (Nagelkerke et ul., 1982), or the coldpronase method employing pronase at 10°C (Praaning-van Dalen and Knook, 1982). €3. PURIFICATION OF KUPFFER CELLS

The yield of Kupffer cells in sinusoidal cell preparations varies with the isolation method used (Table I). For further purification, most labora-

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tories employ the method of Knook and co-workers, that is, the application of a discontinuous metrizamide gradient to eliminate red blood cells and parenchymal cell debris, followed by centrifugal elutriation to obtain highly purified Kupffer cells (Knook and Sleyster, 1976, 1980; Knook et al., 1977; Brouwer et al., 1984). Centrifugal elutriation has no detrimental effects on the Kupffer cells and results in relatively pure cell preparations (Table I). As an alternative method, selective adherence of Kupffer cells to culture dishes during overnight culture of sinusoidal cells has been employed (Munthe-Kaas et al., 1975). This method has several disadvantages, including the phagocytosis of cell debris by Kupffer cells and contamination with endothelial cells, which sometimes attach even faster than Kupffer cells (Brouwer et al., 1980, 1982a; De Leeuw et al., 1982). Some characteristics of isolated Kupffer cells are given in Table I. The yield of Kupffer cells in the various isolation methods does not differ much; the largest number, 10 X lo6 cells per gram liver, is obtained with the pronase-collagenase method. Kupffer cell viability usually exceeds 90%. Sinusoidal cells prepared by the collagenase method are contaminated with -46% parenchymal cells in spite of attempts to remove these cells by differential centrifugation. Prolonged differential centrifugation results in a considerable loss of Kupffer cells (van Berkel, 1982; Brouwer et al., 1982a). After separation of sinusoidal cells by centrifugal elutriation, the relative distribution of the cell types in the “Kupffer cell fraction” varies with the isolation method (Table I).

c. EVALUATION OF THE ISOLATION METHODS Endocytosis can be considered as one of the most important functions of Kupffer cells, and thus the quality and usefulness of a particular isolation method should be judged by the retention of endocytic properties in the isolated cells. However, each isolation method has its own advantages, depending on the cellular characteristics demanded and the specific aim of a study. If a high yield of pure Kupffer cells is needed, the method of preference is the pronase-collagenase method (Table I). When the capacity of Kupffer cells to endocytose in vitro is to be determined, the cold-pronase isolation method (Praaning-Van Dalen and Knook, 1982) or the cold-collagenase method (Nagelkerke et al., 1982) appears to be suitable. Advantages of these low-temperature (10°C) methods include (1) prevention of degradation of substances ingested in situ, which allows the study of in vivo endocytosis; (2) prevention of the ingestion of cellular debris during the isolation procedure, which may lead to an incorrect re-

190

K. WAKE

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distribution of cellular components (e.g., enzymes); and (3) a substantial retention of membrane receptors, which allows in vitro studies on endocytosis, directly after Kupffer cell isolation. Several membrane receptors will be damaged by the use of pronase during the isolation procedure. Kupffer cells, freshly isolated by the coldpronase method, immediately bind and endocytose various substances. Receptors for mannose-terminated glycoproteins and for colloidal albumin are preserved to a large extent after cold-pronase treatment (Praaning-Van Dalen and Knook, 1982). Thus, Kupffer cells isolated by the cold-pronase method do not need a recovery period during short-term culture. Some lipoprotein receptors are destroyed by pronase probably even at low temperature. For studies on these receptors, the collagenase isolation method is to be preferred (Nagelkerke er al., 1982).

D. CULTURE OF KUPFFEK CELLS During a short-term culture (8-24 hours), the Kupffer cells show recovery from trauma inflicted during isolation (Brouwer et al., 1982a). Longterm maintenance cultures (2-10 days) of isolated Kupffer cells may be used for studying several of their functions, such as ( I ) interaction with other cells, microorganisms, and molecules; (2) synthesis and secretion of effector substances; (3) involvement in the immune processes; and (4) the tumoricidal and natural killer (NK) cell activity of cultured Kupffer cells. Kupffer cells in maintenance culture largely retain their differentiated characteristics with regard to morphology, enzyme cytochemistry, membrane receptors, endocytosis, and lysosomal functions for periods of 510 days (Brouwer et al., 1982a,b; De Leeuw et al., 1983). Cultures of Kupffer cells can be very useful in studying functional aspects that cannot be investigated in vivo or in situ. Although several investigators have used maintenance cultures of Kupffer cells for these purposes, a comparison of various functional capacities of cultured cells with those of cells in intact livers has not been sufficiently performed. It is encouraging that one of the few studies reporting such a comparison demonstrated that the maximal rate of uptake of colloidal albumin by Kupffer cells in maintenance culture was in the same order of magnitude as the rate of uptake by Kupffer cells in the intact liver (Brouwer et al., 1985). However, one should consider the possibility that both qualitative and quantitative changes in Kupffer cell function may arise as a consequence of culture conditions.

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191

V. Metabolic Responses of Stimulated Rat Kupffer Cells in Vitro Kupffer cells provide a first line of defense against noxious material (bacteria, viruses, toxins, etc.), approaching the liver through the portal vein. As a result, the cells are expected to be in a more-or-less activated state, and this raises the problem of defining the resting versus the stimulated state of the Kupffer cells. In particular, the procedures of their isolation from livers with the help of hydrolytic enzymes, such as collagenase and pronase or mixtures thereof, will always bring them in contact with cell debris and might stimulate the cells to some extent. Using Kupffer cells, kept in primary culture for 2 days, one can observe strong reactions to various stimuli, such as opsonized erythrocytes, zymosan particles, immunocomplexes, endotoxin, or Ca2+ionophores. The intensity and the velocity of the responses to these stimuli are not uniform; the most conspicuous reactions are seen after the phagocytosis of different particles or exposure to endotoxin. A. METABOLICEFFECTS OF ENDOCYTOSIS Phagocytosis of particulate matter appears to involve several receptors of variable specificity. Binding sites for Fc, C3b, mannose (N-acetylglucosamine), galactose (N-acetylgalactosamine), and apolipoprotein B are well known (for a review, see Knook and Wisse, 1982). Receptors for fibronectin-containing particles (Rieder et al., 1982) and sites for the uptake of insulin and glucagon (Kreusch, 1980) have also been observed. Cell organelles like parenchymal cell mitochondria are endocytosed rapidly by cultured Kupffer cells; the typical enzymatic activities of these organelles can be detected in cell extracts for several hours after ingestion (Rieder and Decker, 1984). This finding supports the morphological observation that intact material is present for some time after internalization. One of the early events observed after stimulation by particles is the so-called oxygen burst that has been described for many phagocytosing cell types (for a review, see Klebanoff, 1980). In Kupffer cells, most of the extra 0, taken up can be accounted for as 0 ; in the extracellular fluid (Bhatnagar et al., 1981). The superoxide anion radical is formed by NADPH reduction of 0,, a reaction that is catalyzed by a membraneassociated NADPH oxidase: 20,

+ NADPH > 20; + NADP' + H'

The large amount of NADPH required during phagocytosis is provided

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K. WAKE

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by a strongly enhanced flux of glucose through the pentose phosphate pathway (Bhatnagar et a!., 1981). Kupffer cells neither contain nor synthesize glycogen, in contrast to alveolar macrophages (Hoffmann et al., 1978). Therefore, the instant requirement for glucose cannot be met by an intracellular store but must be satisfied by an enhanced uptake. The observation (Bhatnagar et al., 1981)that cytochalasin B prevents 0; formation is in line with this interpretation. This compound was shown to inhibit carrier-mediated hexose transport (O’Flaherty et al., 1984). The crucial role of the pentose phosphate pathway in this process may explain the high content of glucose-6-phosphate dehydrogenase (G6PD) in Kupffer cells, and this, in fact, accounts for most of the enzyme activity present in the liver (Knook et al., 1980). The suppression of 0;formation by SH-blocking agents, such as iodoacetamide, has been traced to the inhibition of G6PD (Bhatnagar et al., 1981). Superoxide is considered as an activated oxygen species participating in the oxidative destruction of invading particles, especially of the cell membrane of bacteria. In the presence of superoxide dismutase, superoxide is converted to H,O, and in the presence of the myeloperoxidase of leukocytes, the powerful -OH radical is formed. It also should be mentioned that the NADPH oxidase reaction produces, rather than consumes, intracellular protons. It is not known what role is played by the accumulation of protons near the site of the interaction between the oxidase and NADPH; countertransport with metal ions such as Na’ or Ca” is a possibility. The 0; production by phagocytosing Kupffer cells was found to depend on the presence of Ca” in the medium (Birmelin and Decker, 1983). B. THEROLE OF CALCIUM A rapid influx of Ca” into Kupffer cells occurs after the onset of particle phagocytosis (Birmelin and Decker, 1983) (Fig. 7). The uptake of 4SCaZ+ by rat Kupffer cells in primary culture is stimulated 7-fold over the basal exchange rate and lasts 15minutes. It is immediately followed by an enhanced active release of Ca” from the cells. It appears that the Ca” fluxes, rather than the concentration in the intracellular compartments, are responsible for several secondary effects. They include, in addition to the 0; production just mentioned, an activation of phospholipase A?, the synthesis and release of various arachidonic acid (AA) derivatives, and a transient rise of the intracellular levels of cyclic nucleotides. It is obvious, however, that the superoxide production and the synthesis of eicosanoids are not necessarily correlated (Dieter et al., 1986) (Fig. 8). These processes are provoked simultaneously by the phagocytosis of particles and by phorbol 12-myristate acetate (PMA). However,

-

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

193

30-

m22-

D

QX

Ep0

min

FIG.7. Effect of zymosan stimulation on the uptake of calcium ions by Kupffer cells cultivated for 72 hours. Calcium uptake was evaluated by adding a small quantity of radioactive calcium to the culture dishes. After washing, the uptake of the isotope by the cells was measured in a liquid scintillation spectrometer. Uptake in zymosan-treated cells ( ); untreated cells, (0),

+

prostaglandins (PG) are also released after the exposure of Kupffer cells to lipopolysaccharides (LPS) or to the calcium ionophore A 23187, but little 0 ; is released under those conditions. Furthermore, the formation of superoxide is not affected by pretreatment of the cells with dexamethasone, while the arachidonate cascade is strongly suppressed by glucocorticoids. In addition, the calmodulin antagonist calmidazolium (R 2457 1) also does not affect the zymosan-triggered formation of superoxide but inhibits the liberation of arachidonate (Birmelin el al., 1984).

C. ARACHIDONATE METABOLISM The intracellular availability of free arachidonate appears to be the limiting factor for eicosanoid synthesis by rat Kupffer cells. The phospholipase A, reaction serves both the cyclooxygenase and the lipoxygenase pathway by providing tetraenoic acid. Kupffer cells kept in primary culture for 48 hours respond to a phagocytic stimulus by enhancing the activ-

I94

K. WAKE

E'I'AL

A

1

bl, .:.. .:

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

T

.:....... .......

T

T h PAF

T

LPS

AA

FIG.8. Effect of various stimuli on PGE, release and superoxide production by Kupffer cells treated with dexamethasone. Dotted bars represent cells treated with dexamethasone ( 1 p M ) : open bars show the effect of 0.5 mg/ml zymosan (Zy). I p M phorbol myristate acetate (PMA). 20 p M A 23187. 5 nM platelet-activating factor (PAF) 30 pg LPS, or 30 pM AA. The release of PGE, was determined by radioimmunoassay. and the released superoxide was measured by cytochrome C reduction.

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

195

ity of phospholipase A,, which is specific for the addition of an arachidonoyl group in the 2-position of the phosphatidylcholine at a pH optimum of 8.1 (Birmelin et al., 1984). Calmodulin inhibitors prevent this rise of phospholipase A, activity if given to the cells prior to or together with zymosan. Calmodulin is present in cultured Kupffer cells in amounts (3.2 pg per lo6 cells as determined by CAMP phosphodiesterase assay) exceeding those of parenchymal cells and various other cells (Birmelin et al., 1984). Calmodulin inhibitors are without effect if added to the cellfree extract of stimulated cells. Furthermore, calmodulin added to the extract of unstimulated Kupffer cells does not increase phospholipase A, activity. It appears that the zymosan-triggered stimulation either is mediated by lipocortin or is brought about by a direct calmodulin-dependent interconversion of phospholipase A,. Kupffer cells are the most potent producers of prostanoids among the different liver cell types. Two kinds of stimulators are known to elicit eicosanoid release: (1) contact with phagocytosable material (e.g., zymosan, opsonized erythrocytes, mitochondria, heat-inactivated bacteria), and (2) inflammatory and immunomodulating agents (e.g., LPS, PMA) or calcium ionophores. While the arachidonate cascade (Fig. 9) starts immediately after contact of the Kupffer cells with the phagocytotic stimulus, reverting to a quiescent state after 1 hour, this response is much slower but lasts longer (324 hours) after exposure to LPS (Birmelin et al., 1986).

-

PraUcyclm

Prata$andim

Iffib1

(0.E.F SmOSI

1hmmbOl.M Ap

W E E

LNlIOh”9

FIG.9. Schematic survey of the formation of the difficult kinds of eicosanoids from AA.

196

K . WAKE ET AL

The major AA metabolite released after stimulation is PGD2, but PGE,, PGFZct, a PGA,-like compound, thromboxane (measured as TXB,) accompanied by 15-hydroxy-5,8,iO-heptatrienoicacid (HHT), small amounts of prostacylin (measured as 6-keto-PGF,, or 6-KPGF,, and HETEs are also released (Birmelin and Decker, 1984; Decker et ul., 1986) (Table 11). N o PGE, and very little peptidoleukotrienes are synthesized by stimulated rat Kupffer cells. Quantitative, perhaps even qualitative differences may exist in the eicosanoid patterns of Kupffer cells from different species. Interestingly, rat Kupffer cells only slightly inactivate prostanoids and leukotrienes (LT); they are able, however, to convert LTC, to LTD,, LTE,, and N-acetyl LTE,. Hepatocytes, on the other hand, do not produce but quite actively degrade added eicosanoids (Fig. 10) (Tran-Thi et d . , 1986). Neither phagocytosis nor synthesis of PGE, by Kupffer cells is influenced by added PGE, or PGE,, in contrast to what is observed with other mononuclear phagocytes, such as periotoneal macrophages (Oropeza-Rendon er d . , 1980). The amount of PGEz released by zymosan is the same as that elicited by treatment of Kupffer cells with Ca" ionophore. The effects of A 23187 and zymosan, however, are not additive. Ca'+ channel blockers like verapamil do not inhibit PGEz release. Ca" entry into Kupffer cells seems to use mechanisms different from those operating in heart muscle and other verapamil-sensitive tissues (Fleckenstein, 1980). A highly efficient inhibitor of PGEz synthesis that may also be of physiological significance is LTB, (Decker and Birmelin, 1984). This LT suppresses

TABLE I1 CAPACII Y O F LIVER CELLSTO SYNTHESIZE PROSTANWDS

Maximal rates of synthesis" (ng/ hour) by Cells

PGD,

PGE,

Hepatocyteb Endothelial cells Kupffer cell\

<0.5 23

<0.1

<0.1

I2

n.d.

76

52

220

by I g of liver (wet wt)

lohcells TXB,

PGD,

n.d.' 4.5

<0.003 28

(50



n.d.

805

n.d.

158

9X(

6

110

5500

1900

1300

I50

275(

PGF?, 6-KPGFl,

W E Z PGF,,,

6-KPGF1,, TXB


arachidonate to cells in primary culture. One gram liver wet weight is taken tq "Determined after addition of 30 consist of 100 x 10" hepatwytes. 35 x 10" endothelial cells. and 25 x 10" Kupffer cell\. "Not determined.

I

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

197

16

the zymosan-provoked PGE, release by >70% at I nM concentration in the medium. Neither LTA, nor LTE, shows any effect. It is tempting to speculate on the role of PGE, and LTB, in liver physiology and pathology. Noxious substances that interact primarily with Kupffer cells (such as bacteria or endotoxins) elicit the production of WE,, which can be considered as a parenchymal cell-protectingagent. Prostaglandin E, activates adenylate cyclase and stimulates the energy production of these cells. On the other hand, damaged parenchymal cells may produce LTB, (Perez el al., 1984) or signals that elicit LTB, release in adjacent cells. This LT, by virtue of its inhibitory capacity for PGE, synthesis in Kupffer cells, could potentiate the injury developed in parenchymal cells and enhance their destruction (Fig. 1 I). The surprising effects of LTB, focuses interest on the role of lipoxygenase products during Kupffer cell activation. Compounds known to inhibit 5-lipoxygenase, not only suppress LT but also PG formation, if added to Kupffer cells in primary culture (Table 111). This unexpected finding does not appear to be due to an aspecific reaction of cyclooxygenase. An an-

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ET A L .

KUPFFER CELL

HEPATOCYTE

stimulus

phosphatidyl choline

PGD2 PGE 2

arachidonic acid

-

-

*

2

TXA2 HHT

PGD2

dinor - and

PGEZ 6 - K PGF,,,,

tetranor derivatives

TXBz

polar and

HHT

products

?

HETE LTC4 LTC4-LTE4

low m.w.

LTD4'

LTE4eN-acetyl-LTE

N-acetyl-LTE4

LTC,

blood

bile

I urine

feces

I

FIG. I I . Scheme showing the contribution of Kupffer cells and parenchymal cells to the synthesis and degradation of AA metabolites.

tagonist of the SRS-A receptor, FPL 55712 (Sheard, 1981), also inhibits the zymosan-provoked PGEz release (Decker and Birmelin, 1984). Thus, it appears that the majority of PG formation by Kupffer cells requires the presence of lipoxygenase products.

VI. Endocytosis Kupffer cells and endothelial cells represent a major part of the RES and are responsible for the clearance of a large variety of substances from the circulation. Endogenous and exogenous substances that can be endocytosed by Kupffer cells include various colloids, and particles such as latex beads and liposomes, cells like erythrocytes and bacteria, denatured proteins, toxins like endotoxin, viruses, hormones like insulin, antigens, and glycoproteins. The endocytosed substances will be transported mainly to the lysosoma1 compartment where the endocytic vesicles generally fuse with primary lysosomes. Morphological investigations of Kupffer cells revealed that the cells contain a considerable number of lysosomes that are structurally heterogeneous in format and contents. Endocytosed substances will be de-

199

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

INHIBITION OF

TABLE 111 PGEz FORMATION BY SUBSTANCES RELATED TO THE LIPOXYCENASE PATHWAY^

~

~~

Concentration (P M )

PGE, release (ng/1o6cells/30 minutes)

Uninhibited cells (7%)

None

-

17.3 2 1.7

100

LTB,

0.001 0.002 0.010

4.8 5 0.4 3.5 2 1.3 2.3 2 1 . 1

28 20 13

1.2

Inhibitor

Nordihydroguiaretic acid

Esculetin Diethylcarbamazine FPL 55712

I 5 10 20

17.1 2 11.0 1.7 2 1.5 2

0.5

99 64 10 8

1 2

<1 2.5

<5 14

100 200

6.0 6.8

35 39

4.4 f 1 . 1 4.7 f 1.6 6.9 f 1.9 5.1 1.7

25 27 40 29

0.5 1 .o 2.0 5.0

* 0.9

*

0.6

“ProstaglandinE2was measured in the medium of 72-hour-culturedrat Kupffer cells with 500 pg zymosan added at zero time together with the inhibitors. Basal release (in the absence of zymosan and inhibitors) was 1.5 ? 0.4 ng PGEl per lo6 cells per 30 minutes.

graded in the lysosomes. Some degradation products can be exocytosed; others will be used by the cell itself. Endocytosed substances that are not degradable can remain in the lysosome, which will finally form a residual body. Although in some cases nondegradable material may be exocytosed, it will generally remain accumulated within the cells for a considerable time. A. ADSORPTION OF LIGANDS TO THE KUPFFERCELL MEMBRANE The process of endocytosis consists of three major steps: (1) the events that occur at the cell membrane (i.e., binding of substances or attachment of particles), (2) the mechanism of internalization (i.e., pinocytosis or phagocytosis), and (3) the intracellular transport of endocytosed material. A wide variety of exogenous and endogenous substances can be internalized by Kupffer cells via adsorptive endocytosis. These ligands are bound to receptors. Biochemical and ultrastructural studies have demon-

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strated the presence of several receptors on Kupffer cells. Some of these receptors perform a highly specific interaction with certain ligands; others interact less specifically and can be regarded as general binding sites. The various types of receptors and binding sites on Kupffer cells as well as their specificities have been reviewed (cf. Praaning-Van Dalen ef al., 1982). The following receptors or binding sites are known to be present on Kupffer cells: I . Fc receptors 2. C3 receptors 3. N-Acetyl-D-galactosamine receptors, which mediate the endocytosis of particulate asialoglycoproteins in contrast to the comparable receptor on parenchymal cells, which has a specificity for soluble glycoproteins 4. N-Acetylglucosamine or mannose receptors, recognizing glycoproteins terminated by N-acetylglucosamine or mannose 5. Binding sites for hormones such as insulin and glucagon 6. Receptors or binding sites for bacteria, yeast, viruses, apolipoprotein B-containing lipoproteins 7. The so-called foreign body receptors

Substances that enter Kupffer cells without prior adsorption to the cell membrane are endocytosed by the mechanism of fluid-phase pinocytosis. Generally, fluid-phase pinocytosis may occur through either small bristlecoated micropinocytotic vesicles or fusion of membrane folds that engulf the fluid and give rise to a larger smooth-walled macropinocytic vesicle. Macropinocytotic vesicles of 0.7 p m have been observed in endothelial cells, but not in Kupffer cells; bristle-coated micropinocytotic vesicles are present in both cell types (Wisse and Knook, 1979). Although polyvinylpyrrolidone (PVP), which is a marker for fluidphase endocytosis, is known to enter Kupffer cells (Praaning-Van Dalen er af., 1981), the rate of uptake is very low compared to substances taken up by adsorptive endocytosis. However, a certain amount of PVP will always enter the Kupffer cell in the fluid phase, since some fluid will always be trapped in the bristle-coated micropinocytotic vesicles. In comparison to parenchymal liver cells, Kupffer and endothelial cells possess a relatively high activity for fluid-phase endocytosis (Praaning-Van Dalen et al., 1981).

B. INTERNALIZATION Ligands bound by receptors on rat liver Kupffer cells can be internalized by two different mechanisms, bristle-coated micropinocytosis and

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

20 I

phagocytosis. For particles bound by the galactose receptor, endocytosis via coated vesicles is restricted to particles <35 nm. Larger particles are endocytosed via smooth membrane-bound phagocytic vacuoles (KolbBachofen et al., 1983). Latex beads 6 2 9 nm are taken up by micropinocytosis as already described. Larger beads are endocytosed by phagocytosis (Praaning-Van Dalen et al., 1982).

c. INTRACELLULAR TRANSPORT OF ENDOCYTOSED MATERIAL After formation of the endocytic vesicles, intracellular transport of the vesicles forms the last part in the endocytic process. In case of adsorptive endocytosis, the bristle-coated vesicles fuse very rapidly after being pinched off from the cell membrane (Praaning-VanDalen et d.,1982). The larger vesicles no longer possess a coated membrane and may together form the so-called macropinocytotic vesicles. Apart from the large smooth-walled vesicles, tubular structures filled with endocytosed ligand have been described (Praaning-VanDalen e f al., 1982). Whether these structures represent a special uptake mechanism or have a function in the dissociation and transport of receptors and ligands is still obscure. As described before, Kupffer cells can take up particles larger than a single bristle-coated pit by phagocytosis or by cooperation of several coated pits. In both cases, the intracellular route of the endocytotic vesicles will probably be comparable to that for coated pits. This implies that the ligands will eventually be found in large (>0.7 pm) vesicles, while the ultimate fate will be accumulation and digestion in the lysosomal system. It is generally assumed that most, if not all, endocytotic vesicles in Kupffer cells will fuse with lysosomes. Calmodulin plays an essential role in this fusion process (van Berkel et al., 1982), which further depends on the acidification of the endocytic vesicle (Tolleshaug and Berg, 1982). It is noteworthy that even easily degradable substances such as serum albumin will not be completely digested by Kupffer cell lysosomes, even after several days (Brouwer and Knook, 1977; Garvey and Caperna, 1982). Thus, undegraded material may contain antigenic determinants essential for the function of antigen presentation, as has been shown to reside also in Kupffer cells (Garvey and Caperna, 1982). D. LYSOSOMES Kupffer cells contain well-developed lysosomes. Morphometric studies revealed that 64% of the volume of Kupffer cells is made up of lysosomal

dense bodies and that these cells contain up to 17% of the total volume of lysosomes in the rat liver (Blouin et al., 1977). In accordance with this important volumetric contribution are the high lysosomal enzyme activities of isolated Kupffer cells, which, depending on the enzyme, are 2-20 times higher (on a milligrams protein basis) than similar enzyme activities in isolated parenchymal cells (Knook and Sleyster, 1980). Table IV presents several lysosomal enzyme activities determined in purified Kupffer cells. The enzymes were selected for their role in the hydrolysis of a variety of substrates. The presence of acid phosphatase in Kupffer cells in situ was also demonstrated by cytochemical staining (Wisse, 1974a; Wilson er al., 1982). Several forms of this enzyme could be demonstrated in isolated Kupffer cells (Sleyster and Knook, 1980). Periportal Kupffer cells have larger and more heterogeneous lysosomes, and higher lysosomal enzyme activities on a per-cell basis, when compared with that of midzonal and pericentral Kupffer cells (Sleyster and Knook, 1982). Degradation of heat-aggregated '251-labeled bovine serum albumin (BSA) and excretion of breakdown products by Kupffer cells in maintenance culture were inhibited by chloroquine, a specific inhibitor of lyso-

TABLE IV LYSOSOMAL ENZYME ACTIVITIES I N ISOLATED KUPFFEKCELLS Enzyme"

Activity per lo6cells

Activity per mg protein

Acid phosphatase Acid lipase Acid DNase Cathepsin D Aminopeptiddse B p-Galactosidase p-Glucuronidase P-Acetylglucosaminidase Arylsulfatase B

11.8 2 1.2 6.2 t 0.4 26.1 2 5.1 2.0 2 0.2 1.4 t 0.2 2.4 2 0.2 3.2 2 0.2 16.4 2 0.8 4.8 2 0.7

104.6 2 53.1 2 235.6 2 17.4 2 12.5 2 21.0 2 28.4 2 144.3 2 42.7 2

10.3 5.3 43.4 1.8 1.4 1.6 2.1 6.6 7.0

"Enzyme activities are expressed as nanomoles 4-methylumbelliferone (acid phosphatase, acid lipase, p-galactosidase, p-acetylgluosaminidase). nanomoles nucleotide equivalents (acid DNase), nanomoles tryptophan (cathepsin Dt. nanomoles P-naphthylamine (aminopeptidase B), nanomoles phenolphthalein (glucuronidase), or nanomoles nitrocatechol (arylsulfatase B) reIeased per minute at 37°C. Values are the means 2 SEM of eight experiments. Cells were isolated by pronase treatment at 37°C and purified by centrifugal elutriation (Knook er 01.. 1977). Lysosomal enzyme activities were determined as described previously (Knook and Sleyster, 19801.

BIOLOGY AND KINETICS OF KUPFFER CELLS IN LIVER

203

somal degradation (Brouwer and Knook, 1982). This indicates the degradation of ingested colloidal albumin in Kupffer cell lysosomes. The high specific activities and the multitude of different lysosomal enzymes in Kupffer cells reflect the great capacity of these cells for degradation of a large variety of endocytosed extracellular materials. The relationship between endocytosis of certain substances and the activity of lysosomal enzymes involved in their degradation was studied in vivo by determining the effect of phagocytosis of latex or of IgG-coated sheep red blood cells (IgG-SRBC) on lysosomal enzyme activity. Kupffer cells were isolated and purified 24 hours after the rats had received an injection of IgG-SRBC, and 24 hours or 7 days after latex administration (Brouwer et al., 1981; Sleyster and Knook, 1982). Phagocytosis of IgG-SRBC did not change the activity of cathepsin D, acid phosphatase, and N-acetyl-P-Dglucosaminidase in Kupffer cells (Brouwer et al., 1981). Also, phagocytosis of latex particles did not result in significant changes in most lysosomal enzyme activities with the exception of cathepsin D activity, which increased (Sleyster and Knook, 1982). These results indicate that the response of Kupffer cells to a phagocytic stimulus causes neither a generalized nor a substrate-specific induction of lysosomal enzyme synthesis. Rat Kupffer cells upon stimulation do not seem to secrete substantial amounts of lysosomal enzymes. Activites of p-glucuronidase, P-N-acetylglucosaminidase, and cathepsin D, found in the medium after contact with zymosan and a variety of other elicitors, are always accompanied by strictly correlated activities of typical intracellular enzymes such as lactate dehydrogenase (LDH) and G6PD genase (P. Dieter, unpublished), indicating cell injury rather than specific secretion of lysosomal activities.

VII. Kupffer Cells and Endotoxin

A.

UPTAKE, STORAGE, AND

DETOXIFICATION

The liver takes up circulating infectious agents and ensures the clearance of bacterial endotoxin. Rutenberg et al. (1967) found that mortality in rabbits was reduced from 85% to 11% when a certain dose of bacterial endotoxin was administered via a mesenteric vein instead of a systemic vein. There is some evidence that parenchymal cells play a role in the uptake and detoxification of endotoxin (Zlydaszik and Moon, 1976; Ramadori et al., 1980; Maitra et al., 1981), but the Kupffer cells seem to be the principal site for the removal of circulating endotoxin (Mathison and Ulevitch, 1979; Ruiter et al., 1981; Maier and Ulevitch, 1981a; PraaningVan Dalen et al., 1981). According to Ulevitch et al. (1979), the interac-

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tion of LPS with recipient cells is improved by the presence of highdensity lipoproteins. Hepatic macrophages, obtained by the magnetic isolation of iron-loaded cells, displayed a marked endotoxin-detoxifying capacity in comparison with blood monocytes (Filkins. 1971). Trejo and Di Luzio (1973) found that, within different populations of rat macrophages, Kupffer cells manifested the greatest endotoxin-detoxifying capacity. Yamaguchi et a f . (1982) demonstrated that endotoxin interaction with Kupffer cells conforms to classical saturation kinetics. Partial degradation (Freudenberg et af., 1982)of endotoxin and the transfer of myristic acid residues from LPS to specific macrophage proteins (Aderem et af., 1986) have been reported. B. EFFECTSON METABOLISMA N D FUNCTION Endotoxin from Safmonefla minnesota in concentrations 630 pg/ml has no effect on the viability and phagocytotic capacity of Kupffer cells over a period of 48 hours (Birmelin et a f . ,1986). Exposure of rat Kupffer cells in culture to endotoxin leads to an enhanced activity of phospholipase A2 (Birmelin et al., 1984), followed by a triggering of the arachidonate cascade (Fig. 9). Kupffer cells not only release PG after endotoxin treatment; they also react metabolically to these mediators in a process that might be called autostimulation. Prostaglandin E, elicits an activation of adenylate cyclase (Bhatnagar et a f . , 1982) and an increased CAMPlevel for a period of >24 hours (Decker and Birmelin, 1984). These responses and the LPS-provoked collagenase synthesis and release are suppressed by R 24571 when given simultaneously with LPS (Bhatnagar et a f . , 1982; Decker et al., 1982). The collagenase response can be inhibited by cycloheximide, indicating de novo synthesis rather than the release of stored enzyme. The indomethacin inhibition of this process is overcome by addition of PGE, or dibutyryl-CAMP (Bhatnagar et a f . , 1982). Lipopolysaccharides added to cultured hepatic macrophages stimulate a selective increase in the activity of several cellular enzymes, such as LDH, lysozyme, and plasminogen activator, a procoagulant factor (Maier and Ulevitch. 1981b). In vitro activation of rat Kupffer cells with Escherichia coli endotoxin renders them capable of phagocytosing C3-opsonized SRBC (Steffan, 1983). Keller et a f . (1984b)have shown that the extrinsic antiviral properties of isolated rat Kupffer cells are considerably enhanced by prior treatment with LPS. Further evidence of a correlation between Kupffer cell function and endotoxin levels in the afferent hepatic vessels comes from experiments with portocaval anastomosis (PCA). The paucity of phagocytic Kupffer

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cells observed in animals having PCA provides an explanation for the reduced clearance of colloidal material reported in PCA animals (Grun et al., 1980). -It is suggested that this reduction may be due to a reduction of a gut-derived stimulus, such as endotoxin, which is normally present in the portal blood. Following end-to-side PCA, such blood is diverted into the systemic circulation and reaches the liver secondarily. Since some portal blood may reach the liver in animals with side-to-side PCA, the finding of slightly more phagocytotic Kupffer cells in these animals is not surprising.

c. MEDIATORSRELEASEDBY LPS-STIMULATED KUPFFERCELLS A variety of mediators are released from Kupffer cells following uptake of endotoxin; they include interleukin 1 or IL-1 (Bauer et al., 1984, 1986), parenchymal cell-stimulating factor (Wolosky and Fuller, 1985), interferon or IFN (Neumann and Sorg, 1978), tumor necrosis factor or TNF (Decker et al., 1987), and eicosanoids (Decker, 1985). These substances are thought to participate in the host’s responses to endotoxin and may modulate the resistance of the host to infection and disease (Berry, 1977; Morrison and Ulevitch, 1978; Rosenstreich and Vogel, 1980; McCabe, 1980; Leser et al., 1982; Urbaschek and Urbaschek, 1982; Decker, 1985). Interleukin 1 (Dinarello, 1984) and parenchymal cell-stimulating factor (Wolosky and Fuller, 1985) are instrumental in inducing the acute-phase response following an inflammatory insult. Both factors stimulate, in the presence of permissive doses of glucocorticoids, parenchymal cells to synthesize and secrete acute-phase proteins (Bauer et al., 1984). These acute-phase glycoproteins consist mainly of proteinase inhibitors of different specificity. Interferons produced by macrophages are thought to inhibit mitogen-induced T-cell proliferation (Mjorner et al., 1978), besides their general antiviral and antitumor (Kirchner, 1984) effects. The latter properties are shared by the TNF (Beutler et al., 1985; Kirchner 1984). Interferon also stimulates IL-1 and PGE, production in resting macrophages (Bachwich et al., 1986), together with IL-1 release by vascular endothelial cells (Nawroth et al., 1986) and acute-phase protein synthesis (Kampschmidt, 1984; Darlington et al., 1986). The toxic part of endotoxin (LPS, or even the LPS-derived lipid A portion) induces rat Kupffer cells in culture to synthesize and release eicosanoids (Bhatnagar et al., 1981). The pattern of the AA derivatives is quite similar to that seen in phagocytosing Kupffer cells, but the rate of synthesis is much slower, and the PGD,/PGE, ratio is different (Birmelin et al., 1986). The latter effect, however, may be due to the time span of stimulation rather than to the action of LPS itself, since repeated exposures of

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Kupffer cells to phagocytotic stimuli also lead to a reverted PGD2/PGE, ratio. D. MORPHOLOGICAL OBSERVATIONS During the first 5 minutes following injection of nonlethal doses of endotoxin, the rate of phagocytosis of latex particles by Kupffer cells is accelerated as observed by in vivo microscopy (Fig. 12). At 15 minutes, phagocytosis is inhibited (McCuskey et af., 1982a,c, 1983). Concomitantly, leukocytes and platelets adhere transiently to the sinusoidal lining, and lymphocytes frequently are associated with Kupffer cells. These responses are more pronounced at higher doses of endotoxin. By 2 hours, at the peak of mediator release from Kupffer cells, the velocity of blood flow is reduced and patterns of flow are altered by platelet aggregates and leukocytes that plug sinusoids. The sinusoids are narrowed by swollen endothelial and Kupffer cells.

FIG. 12. In vivo photomicrograph of a video image of the liver using a 59 X water-immersion objective. S, Sinusoid; KC, Kupffer cell; E, endothelial cell; N. nucleus of parenchymal cell. Arrow indicates bile canaliculus.

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In vivo microscopic examination (McCuskey et al., 1984d) revealed that Kupffer cell phagocytotic function relative to sinusoids that contained blood flow, had increased between 50 and 75% depending on the lobular region. This peak of Kupffer cell activity was followed by a progressive decrease to below that of controls by 12 hours. Pretreatment with minute doses of endotoxin minimized changes in Kupffer cell function (McCuskey et al., 1984d) and protected the mice from death caused by massive doses of endotoxin introduced by cecal ligation and puncture (Urbaschek and Urbaschek, 1984). At the ultrastructural level (Boler and Bibighaus, 1967; Frenzel et al., 1977; McCuskey et a / . , 1982a,c; McCuskey, 1983b),the surfaces of Kupffer cells are smoothed by the presence of endotoxin, due to a loss of microplicae and filopodia. The cells appear inactive within 2 hours after administering endotoxin. The endothelial lining is disrupted by endotoxin, allowing blood cells and platelets to penetrate the space of Disse through sites of desquamation and enlarged intracellular and intercellular gaps in the sinusoid lining. Enlargement of the endothelial fenestrae, mitochondrial swelling, and dilatation of their cristae are consistent findings (Boler and Bibighaus, 1967; Frenzel et al., 1977; McCuskey el al., 1982a, 1983). E. ENDOTOXIN TOLERANCE Endotoxin exerts a special effect on Kupffer cells according to the dose, the method of administration, and the length of the pretreatment. Thus, the injection of low doses of endotoxin into rats induces a protection against a lethal dose (Arrendondo and Kampschmidt, 1963) as well as an enhanced resistance against infections (Cluff, 1970). This resistance seems to be principally mediated by Kupffer cells (Ruggiero et al., 1980). One may thus postulate that in normal conditions a continuous stimulation of Kupffer cells by circulating endotoxin increases their antiinfectious and antitumor capacities. Kupffer cell phagocytosis and microvascular hemodynamics were studied in mice after small tolerance-producing and LD,, doses of endotoxin, alone and in combination with bacillus Calmette-Gdrin (BCG) (McCuskey et al., 1982a,c, 1983). In the endotoxin-induced hyperreactive state, the tolerance-inducing dose of endotoxin produces no change in the rate of phagocytosis, but the LD,, dose reduces the rate almost totally. If tolerance is induced, there is no reduction in the rate of phagocytosis. The rate of phagocytosis is accelerated slightly 24 hours after the induction of tolerance. This suggests that the Kupffer cells had been activated and probably are more effective in clearing subsequent endotoxin from

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the blood (Urbaschek and Urbaschek, 1982), but without sufficient release of toxic substances to be lethal. That some mediators were released, however, is suggested by the microvascular alterations that accompanied the phagocytic responses; elevated levels of mediators also have been found in the posthepatic blood (Parant et al., 1980; Urbaschek and Urbaschek, 1982, 1983). Swiss Webster mice were rendered tolerant to endotoxins by pretreatment with detoxified endotoxin (Urbaschek and Nowatney, 1968). These animals demonstrate no suppression of phagocytosis compared to nonpretreated mice, when normal endotoxin was administered. Rather, the rate of Kupffer cell phagocytosis is only slightly modified (McCuskey et al., 1982a). Electron-microscopicexamination also suggests that the Kupffer cells had been activated by detoxified endotoxin. This suggests that they are more effective in clearing subsequent endotoxin from the blood but apparently without much release of toxic and vasoactive mediators, since hepatic microcirculatory dynamics were not altered in these mice. These results further support the concept that not only do endotoxins and Kupffer cells play a significant role in the pathophysiology of septic shock, but minute concentrations of endotoxin in the portal blood also modulate Kupffer cell function and are beneficial in contributing to nonspecific resistance of the host to infective processes.

F. SPECIES SENSITIVITIES Different species exhibit a wide range of sensitivities to endotoxin, when studied by in vivo microscopy (McCuskey et al., 1984b). The species sensitivity to endotoxin (LD,,) is guinea pig > hamster > mouse > rat, correlating the rate of latex phagocytosis and the number and lobular distribution of Kupffer cells. Guinea pigs exhibit the fastest rate of phagocytosis and the highest density of phagocytic Kupffer cells. In all species, there is a portal to centrolobular decrease in the density of Kupffer cells that phagocytose latex. In addition, a low level of Kupffer cell activation was found in C3H/HeJ endotoxin low-responder mice (McCuskey et al., 1982a,c, 1983, 1984a), whereas increased endotoxin sensitivity was observed in mice whose Kupffer cells had been activated by BCG (McCuskey et al., 1982a,c, 1983).These results suggest that the intrahepatic density and level of activation of Kupffer cells affect the animal’s sensitivity to circulating endotoxins. After BCG activation of the RES, minute amounts of endotoxin are lethal (McCuskey et al., 1982a,b,c, 1983; McCuskey, 1983a). Electronmicroscopic examination reveals extensive damage to the Kupffer cells,

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the endothelial lining, and adjacent parenchymal cells. Such damage is not seen when endotoxin is given to animals not treated with BCG. The responses are not seen in endotoxin low-responder, C3H/HeJ mice which have a paucity of phagocytic Kupffer cells and a deficiency in several lysosomal enzymes (McCuskey et al., 1982c, 1984a). However, the lysosoma1 and phagocytic deficiencies, aqd the sensitivity to endotoxin, can be restored to near-normal levels within 14 days following infection of C3H/HeJ mice with BCG (Vogel et al., 1980; McCuskey et al., 1982~).

G. ROLE OF ENDOTOXIN IN LIVERINJURY There is evidence that endotoxin plays a role in liver injury (Munford, 1978; Nolan, 1981; for a review, see Nolan and Camara, 1982). However, it is not clear whether direct or indirect effects are responsible for hepatocellular damage. Parenchymal cells in culture are not damaged by LPS S50 p,g/ml medium (Tran-Thi et al., 1985). Endotoxemia affects many tissues and organs besides the liver, and death of an animal due to endotoxin is not the result of hepatic failure. Nevertheless, there is no doubt that Kupffer cells play a major role in protecting the organism, and its liver from endotoxins. When Kupffer cells are selectively destroyed by frog virus 3 (FV3), they are no longer capable of clearing the circulating endotoxin of gut origin, and severe hepatocellular damage and death of the animals ensues. If, in this case, the source of endotoxin is suppressed by colectomy (Gut et al., 1982a,b) or if the biological activity of endotoxin is impaired, hepatic damage and death do not occur (Gut et al., 1984). In several other cases, impairment of RES activity is seen to sensitize the host to endotoxin. The administration of lead acetate (Seyberth et al., 1972), beryllium phosphate (Vacher et al., 1977), and streptococcal and staphylococcal exotoxins (Schlievert et al., 1980; Schlievert, 1982), leads to a decrease in the phagocytic function of Kupffer cells. This may result in a diminution in the clearance of endotoxin and thus in a heightened susceptibility to lethal endotoxic shock (Seyberth et al., 1972). These observations are at present difficult to reconcile with the finding that reticuloendothelial blockade in normal animals significantly reduces or eliminates the toxic response to injected endotoxin (Altura, 1982). Increased sensitivity to endotoxin is seen in animals treated with Dgalactosamine (Galanos et al., 1979; Freudenberg et al., 1986). The high sensitivity to the lethal effect of LPS could be transferred to LPS-insensitive C3H/HeJ mice by injection of macrophages cultured from bone marrow of sensitive C3H/HeN mice.

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VIII. Kupffer Cells in Infectious Diseases A. UPTAKEOF INFECTIOUSAGENTSBY THE LIVER Viruses whose intravenous-clearance curves have been studied in mice include Rift Valley fever (Mims, 1956), Newcastle disease virus, vesicular stomatitis virus (Brunner et ul., 1960), and FV3 (Gut et al., 1981). Although the scrapie agent disappears from the blood very rapidly, its clearance seems not to be related to the activity of the RES (Hotchin er al., 1983). Blood clearance studies of bacteria and parasites have been carried out in intact animals, or in the isolated perfused liver. Maximal trapping of Salmonella typhimurium occurs when the Kupffer cells of mice are intact and viable (Friedman and Moon, 1980). Hepatic lectins seem to be involved in the nonimmune clearance of E. coli (Perry and Ofek, 1984). Treatment with silica results in a significant decline in infectious sporozoites of Plasmodium yoelii in the isolated perfused liver and in a parallel decrease in the infection rate of the host in vivo (Sinden and Smith, 1982). Immunological factors may play a decisive role in the blood clearance of infectious agents. In guinea pigs, anticapsular antibodies increase the clearance rate of Streptococcus pneumoniae via the activation of the classical complement pathway (Brown et al., 1983). The clearance of virulent E. coli in turkeys is markedly enhanced by antibody-mediated liver phagocytosis (Arp, 1982). which is likewise largely responsible for the clearance of Trypanosornu hrucei in immune mice, both being dependent on opsonization involving C3 (Macaskill et a f . , 1980). Finally, the trapping of a lafge number of larvae in the livers of mice, following reinfection with Toxocaru canis, could well be a T-cell-dependent reaction (Sugane and Oshima, 1983).

B. I n Vivo INTERACTIONOF VIRUSESWITH KUPFFER CELLS Since clearance curves do not provide information on the type of cells involved, immunofluorescence and ultrastructural studies have been carried out to determine the way in which virus particles infect the liver. Numerous viruses have been revealed inside the Kupffer cell of infected animals or patients; in most experimental infections, the damage to the Kupffer cell preceded that of the parenchymal cells (for a review, see Kirn et al., 1982a). In human hepatitis, the data are somewhat controversial. Hepatitis A antigen was detected by immunofluorescence in the cytoplasm of Kupffer cell in experimentally infected chimpanzees (Mathiesen et al., 1977) and marmosets (Mathiesen el al., 1978). In patients, during the acute phase

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of illness, typical particles sharing specific hepatitis A antigen were found in the cytoplasm of parenchymal cells and sinusoidal cells (Shimizu et al., 1978; Tanikawa et al., 1986). These particles were similar to those detected in marmosets after an experimental infection (Huang er al., 1979). Kupffer cells of chronic carriers of hepatitis B show orceine-positive material presumably associated with the HBs antigen (Karasawa and Shikata, 1977). In fulminant hepatitis B, HBs antigen was revealed in viable and necrotic parenchymal cells and Kupffer cells (Abe et al., 1982). Crystalline structures with particles have been found by McCaul et al. (1982a) inside human endothelial cells from patients with nonA-nonB hepatitis, similar to those described by Bradley et al. (1980) in the cytoplasm of the Kupffer cells and endothelial liver cells of experimentally infected chimpanzees. These particles are not viral; they are a reflection of the pathological response of the host to infection in nonA-nonB hepatitis (McCaul et al., 1982b). Electron-microscopic studies of liver biopsy specimens of two homosexual patients with AIDS, one without hepatitis and one with chronic active hepatitis in remission, revealed the cytoplasmic tubular structures in Kupffer cells that are characteristic of chimpanzee nonA-nonB hepatitis (Watanabe er al., 1984). Nuclear particles, comparable to those found in parenchymal cells, were also demonstrated in different types of sinusoidal cells in the livers of patients with nonAnonB hepatitis as well as in healthy chimpanzees (De Vos et al., 1984). In the case of a chimpanzee, inoculated with human serum infectious for the 6 agent, nuclear alterations similar to those found in nonA-nonB hepatitis were observed occasionally in sinusoidal liver cells (Canese et al., 1984). OF FV3 VIRUS INFECTION C. In Vivo MICROSCOPY

In vivo and electron microscopy also have been used to elucidate the sequelae of events occurring in the sinusoids of NMRI mice, following infection with lethal doses of (FV3), (McCuskey et al., 1984~).Frog virus 3 destroys Kupffer cells and produces acute degenerative hepatitis of toxic origin (Kirn er al., 1982a, 1983). Within the first hour after FV3 infection, Kupffer cells became swollen and impeded blood flow in many sinusoids. Blood flow was further hindered by the adhesion of leukocytes to the sinusoidal walls. However, limited phagocytosis of latex particles by Kupffer cells was still possible in spite of extensive nuclear and cytoplasmic injury. By 3 hours these responses were exacerbated by the fragmentation of Kupffer cells, which further embolized the sinusoids. Following fragmentation, the remainder of such Kupffer cells became rounded but were still capable of removing

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some latex particles from the blood by phagocytosis as well as by surface attachment. Latex particles also adhered to and were phagocytosed by the endothelium, which was swollen and, in some sites, absent. Aggregates of platelets were adherent to sinusoidal walls, particularly to the injured Kupffer cells. As a result, many sinusoids were congested and blood flow through the lobule was tortuous as a result of flow being shunted around these areas. Vacuolization of parenchymal cells, observed as early as I hour, was more evident by 4 hours, and became quite pronounced by 10 hours when dilated bile canaliculi and swollen mitochondria were evident. At this time, blood flow was limited and intermittent, and many leukocytes were adherent to the walls of portal and central venules. In the scattered sinusoids where flow was maintained, only an occasional Kupffer cell was found; these had little or no capacity to endocytose latex particles. These cellular responses were more pronounced in periportal regions (Kirn et al., 1982a, 1983), probably reflecting the high density of phagocytic Kupffer cells in this region of the hepatic lobule (McCuskey et al., 1984b). This suggests that the initial injury to endothelial and parenchymal cells may result from the variety of mediators and toxic substances released from injured Kupffer cells (Howard et al., 1959; Cook et al., 1982; Brouwer ef a/., 1982a; Cohen et al., 1982; Decker et al., 1982; Kirn et ul., 1982b; Leser et al., 1982). The subsequent loss of Kupffer cells is thought to provoke further injury to surrounding endothelial cells and parenchymal cells, which are now exposed to infectious particles (Steffan and Kirn, 1982) or toxic substances in the blood, such as gut-derived endotoxins (Kirn et d., 1982a, 1983). Endotoxin may play a significant cytotoxic role during FV3 infection, as is suggested by decreased sensitivity to FV3 in colectomized animals and in endotoxin-tolerant animals (Kirn el al., 1982b, 1983; Gut at ul., 1982a). In addition, many of the microvascular and hepatocellular responses elicited by FV3 infection mimicked the responses produced by bolus injections of endotoxin. These include the adhesion of leukocytes and platelets to the swollen sinusoidal lining and the deranged flow pattern produced by these blood cells, as well as subsequent hepatocellular vacuolization, and bile canalicular dilation (McCuskey, 1983a; McCuskey rf ul., 1982a). Further support is provided by the slight but significant elevation of endotoxin in the systemic circulation 30 minutes to 4 hours following FV3 infection (McCuskey et al., 1984~).This suggests hepatic spillover of gut-derived endotoxin due to injured Kupffer cells, which are the principal site for removal of these toxins (Mathison and Ulevitch, 1979; Ruiter et ai., 1981; Maier and Ulevitch, 1981a). In addition to gutderived endotoxins, hypoxia, and the lack of nutritional blood flow, sinus-

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oidal embolization may contribute to the alterations resulting from FV3induced Kupffer cell necrosis. These results support the concept that Kupffer cells and gut-derived endotoxins play a significant role in the hepatocellular injury produced by FV3. D. FATEOF INFECTIOUS AGENTS As a result of phagocytosis, the ingested particles are enclosed in phagosomes, which will fuse with lysosomes. For some viruses, however, there may be a fusion of the particle with the plasma membrane of the Kupffer cell, allowing the viral material to penetrate directly into the cytoplasm (Gendrault et al., 1981) (Fig. 13). The fate of the internalized particles will depend on one hand, on their nature, and on the other hand, on the physiology and immunological status of the host. In the case of neutrophils, killing depends on superoxide-independent bacteriocidal proteins that are highly specific for certain microbial species and on superoxide-requiring systems that nonspecifically attack all cells (for a review, see Elsbach and Weiss, 1983). Although macrophages lack myeloperoxidase, which amplifies the cytotoxicity of superoxide radicals, they seem to have the same oxygen reduction system as neutrophils (Johnston,

FIG. 13. Infection of a cultured Kupffer cell by FV3 particles. Virus particles fuse with the plasma membrane of the Kupffer cell. Two particles show the liberation of their mucleoprotein content into the cytoplasm. x 83,000. Courtesy of Dr. Jean Louis Gendrault.

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1978). A significant correlation between the augmentation in the production of superoxide radicals in Kupffer cells and the inhibition in the growth of L. monocytogenes has been reported (Hashimoto et al., 1984). It may therefore be stated that superoxide-dependent mechanisms represent a significant antimicrobial weapon in Kupffer cells as is the case in peritoneal macrophages. Until now, Kupffer cells have not been explored for superoxide-independent mechanisms. Killing of bacteria in Kupffer cells also may require humoral factors. The rat liver, washed free of blood and perfused with Salmonella typhi, avidly traps the bacteria but does not significantly destroy them (Moon et al., 1975). However, when blood or plasma was added to the perfusion medium, >50% of the trapped bacteria were killed within 30 minutes. Complement is also required for the bacteriocidal activity of Kupffer cells; the specific antibodies merely enhance bacterial trapping (Friedman and Moon, 1980). Finally, T-cell mechanisms contribute to the resistance of mice to L. monocytogenes infection (Takeya et al., 1977). In some cases, parasites are not killed within the macrophages; moreover, they survive because they have developed mechanisms that allow them to resist host microbicidal capacities. For example, Mycobacterium tuberculosis (Armstrong and Hart, 19711, Toxoplasma gondii (Jones and Hirsch, 1972), and Chlamydia psittacci (Friis, 1972) inhibit phagosome-lysosome fusion. However, when fusion is restored by coating the parasites with specific antibodies, M . tuberculosis still survives and multiplies within the macrophages (Armstrong and Hart, 1975). Phagosome escape is another survival strategy developed by certain intracellular parasites, as illustrated by the behavior of Trypanosoma cruzi in mouse peritoneal macrophages (Nogueira and Cohn, 1976) and Rickettsia prowazeki in cultured human monocytes. Little is known about this phenomenon in Kupffer cells. The nucleoprotein of FV3 may escape from Kupffer cell phagosornes by fusion of the viral shell with the surrounding membrane (Gendrault et d., 1981). Sporozoites of Plasmodium herghei can possibly escape completely from the Kupffer cell and reach the space of Disse as shown by Meis et al. (1983), who suggested that Kupffer cells are implicated in the transport of sporozoites toward adjacent parenchymal cells.

E.

VARIATIONS IN SUSCEPTIBILITYTO

INFECTION

1. Chemically Induced Variations

Macrophage blockade produced by the injection of thorotrast or silica has been used in the study of interactions between Kupffer cells and vi-

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ruses. The clearance of Newcastle disease virus was greatly reduced at 5 hours after thorotrast treatment (Brunner et af., 1960). In mice, liver macrophage functions impaired by silica facilitated the early invasion of the brain by yellow fever (Zisman et af., 1971). Diminished Kupffer cell functions also augmented the susceptibility of adult mice to coxsackie virus B3 infection (Rager-Zisman and Allison, 1973). Pretreatment with silica increased the severity of hepatic lesions produced by herpesvirus in mice (Mogensen and Andersen, 1977) and diminished resistance to a highly virulent strain of MHV 4 induced by preinfection with a low-virulence strain of MHV 4 (Taguchi et al., 1980). Treatment of hamsters with antimacrophage serum, both before and after administration of ameba, not only significantly increased the size of the abscesses in the liver, but also allowed the metastatic foci to disseminate to other organs (Ghadirian et al., 1983). These experiments demonstrate that macrophage blockade increases susceptibility to different infections. However, such an increase in sensitivity is difficult to correlate with the sole diminution in Kupffer cell functions, since blockade-producing agents have not been shown to affect only Kupffer cells and since it has not been determined whether the target cells for the viral infections studied were indeed Kupffer cells and/or other macrophages. In mice inoculated intraperitoneally with FV3, the blockade of macrophages with thorotrast decreased virus penetration into the liver and allowed the animals to survive (Kim et al., 1973-1974). Diets also influence the susceptibility of mice to bacterial and viral infections. Thus, it has been found that MHV-resistant mice fed on hypercholesterolemic diet became fully susceptible to this virus (Pereira et al., 1986). This resistance may be related to a defect in Kupffer cells, which could no longer be activated in vitro with bacterial endotoxin. Galante et af. (1982) showed that in livers isolated from ethanol-fed rats, phagocytosis of bacteria was significantly depressed and killing did not occur. The serum of these rats perfused into control livers had no effect on the phagocytosis of E. cofi,but greatly reduced bacterial destruction. Biliary obstruction provoked by ligation of the common bile duct in the rat decreased hepatic phagocytosis of E. coli, a phenomenon that could explain the increased susceptibility to infections noted during obstructive jaundice (Katz et al., 1984). The effect of macrophage activation by specific or nonspecific mechanisms has been extensively studied (North, 1978). Most data, however, come from peritoneal macrophage experiments and only a few concern Kupffer cells. Mice pretreated with bacterial phospholipids, which were claimed to activate macrophages (Fauve and Hevin, 1974), survive a lethal challenge of FV3 whose target cells are Kupffer cells (Anton and Kirn, 1979). Di Luzio and colleagues (1982) and Williams et af. (1983)

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have closely studied the effect of glucan on E. coli sepsis and MHV hepatitis in mice. They found that the administration of glucan rendered the animals resistant to either the bacteria or the virus and at the same time enhanced the phagocytic functions of the Kupffer cell. When the glucaninduced Kupffer cell hyperphagocytosis was reversed to normal by methylpalmitate, there was a complete loss of protection. Administration of kinins into mice also rendered them resistant to a lethal challenge of L. monocytogenes, increased the blood clearance of the bacteria and decreased their multiplication in the liver (Fauve and Hevin, 1979). Wing et (11. (1983) found that starvation led to a decrease in L. monocytogenes multiplication in the livers of mice and to an enhancement of macrophage functions. Vaccination of mice with P. yoelii induces an activation of the Kupffer cell, allowing the animals to clear the otherwise lethal infection within 7 days (Dockrell er al., 1980). 2 . Host-Determined Variations

The genetic basis of susceptibility to virus infections involving macrophages was first studied with peritoneal macrophages. More viral antigen has been revealed in the sinusoidal lining cells of mice sensitive to MHV than in resistant mice (Taguchi et al., 1976). Furthermore, a direct relationship has been demonstrated between the resistance of A/J mice and the ability of their peritoneal macrophages to resist partially MHV 3 replication (Virelizier and Allison, 1976). In Kupffer cells, the situation seems to be different. Both MHV 3 and JHM, a strain of lower virulence, multiply in comparable amounts in cultured Kupffer cells and endothelial cells from susceptible and from resistant mice. However, in the cells isolated from the livers of resistant animals there was a delay in multiplication of 24-36 hours, which might allow the local and systemic responses to clear the infective particles (Pereira, 1984a). In piglets, <2 weeks old infected with porcine cytomegalovirus, the mortality rate was high with a strong tendency for the virus to multiply within the RES, including Kupffer cells. By contrast, in animals >2 weeks old, the infection was usually inapparent and the Kupffer cell rarely involved (Edington et al., 1976). Swanson and O’Brien (1983) have claimed that the innate resistance of mice to Salmonella typhimurium is controlled by mouse chromosome 1 locus Icy. This gene regulates the level of survival of intracellular bacteria that accumulate within Kupffer cells and resistant macrophages of the spleen. On the other hand, congenitally athymic mice exhibited an abnormally high resistance to L. rnonocytogenes and other bacteria, and this could be in correlation with a more pronounced clearance and an augmentation in bacterial destruction, on account of the presence of naturally occurring activated macrophages in the RES (Nickol and Bonventre,

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1977). The pathological background of the host may also influence the susceptibility to infections or affect the function of the RES, as is the case for diabetic mice, which were found to be more susceptible to streptococcal infections than control mice; this enhanced virulence was associated with prolonged bacteremia (Edwards and Fuselier, 1983).

F. STIMULATION AND IMPAIRMENT OF KUPFFERCELL FUNCTIONS Viral infections may stimulate Kupffer cell functions. Susceptible strains of mice infected with leukemogenic oncornaviruses remove carbon particles from the blood more rapidly (Gledhill et d . , 1965). An increase in the activity of Kupffer cell lysosomes is another consequence of the infection of mice with Friend leukemia virus (Drozhennikow et al., 1979). Yet another example of virus macrophage activation is the infection of mice with lymphocytic choriomeningitis or ectromelia virus where the Kupffer cells show an increased ability to kill bacteria within 8 days after the start of viral infection (Blanden and Mims, 1973). Kupffer cells transformed with Simian virus 40 (SV40) exhibit properties associated with neoplastic transformation in culture (Clark and Pateman, 1978). Stimulation of the hepatic RES leading to increased phagocytic activities was also reported after infection of mice with C. parvum (Toki et al., 1981), BCG (Ghadirian and Meerovitch, 1982), and Trypanosoma lewisi (Ferrante et al., 1978). In mice, injected intravenously with Lactobacillus casei, there was an increase in the production of oxygen radicals, together with an inhibition in the growth of L. monocytogenes (Hashimoto et al., 1984). Kirn et al. (1982~)reported the synthesis of IFN by cultured Kupffer cells and endothelial cells, infected with Sindbis virus or treated with bacterial endotoxin. Since this IFN may diffuse into the space of Disse toward the parenchymal cells, its synthesis by the infected sinusoidal cells may play an important role in the defense of the parenchymal cells against viruses. Conversely, the properties of the RES may also be impaired by the infection. Depression of the Kupffer cell function was thus observed after the onset of virus infection in mice (Mahy, 1964) and in a coxsackie virus B2 infection in a child (Hinkle et al., 1983). Mice pretreated with C. parvum showed an increased susceptibility to E. coli that may correlate with the high in vitro sensitivity of their macrophages to the toxic effect of LPS (Yoshikai et al., 1983). The chronicity of infection in CBA mice after the injection of Brucella abortus is related to a number of factors, one of them being the incapacity of the bacteria to activate the macrophages in which they persist (Cheers and Pagram, 1979). In Kupffer cells infected in vitro with FV3 there is an inhibition in the phagocytosis of IgG-SRBC,

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whereas the uptake of colloidal carbon by bristle-coated vesicles remains unaffected (Kirn et d . , 1980). A drastic impairment of Kupffer cells and endothelial cell functions occurs in murine FV3 hepatitis. Kupffer cells are destroyed and endothelial cells heavily injured by the virus, thus leading to the loss of parenchymal cell protection. Four hours after the administration of a sublethal dose of FV3 to mice, the parenchymal cells have acquired endocytic capacities for colloidal carbon or latex particles (Kirn Pt nl., 1978). Likewise, such FV3-preinfected mice become susceptible to different virus infections to which they are normally resistant (Steffan and Kirn, 1979; Pereira, 1984b).

G . KUPFFER CELLSI N OTHERPATHOLOGICAL STATES Macrophages in general, and Kupffer cells in particular, are involved in several pathological processes. In this review, only a few examples of pathological situations in which Kupffer cells seem to play a major role will be given. Roos and Dingemans (1977) observed Kupffer cells encircling apparently intact tumor cells shortly after they penetrated into liver sinusoids. At later stages, the tumor cells were completely encircled and degenerated. However, there was a rapid blockage in the encirclement process possibly caused by tumor cell products. Tumor cells bind to Kupffer cells in vivo (Burkart and Friedrich, 1982) and in vitro (Schlepper-Schafer et nl., 1981). The binding seems not to be mediated by a galactose-specific lectin as is the case with parenchymal cells. A correlation was also found between the size of experimentally injected tumors in rats and the activity of the hepatic RES; the animals with the smallest tumors showed an increased activity in their RES (Ryden et al., 1983).The pronounced depletion of the Kupffer cells in the liver lobules in chronic hepatitis, biliary cirrhosis, and other types of cirrhosis led Manifold et a / . (1983) to postulate that this RES impairment might play a role in hepatic carcinogenesis. In addition, stimulation of Kupffer cell function with giucan significantly enhances survival and inhibits hepatic metastases in a syngeneic model of hepatic metastatic disease in mice. Keller et nl. (1984a) found that cultured rat Kupffer cells demonstrated a strong cytostatic effect on a human tumor cell line as well as on virus-transformed or normal target cells. Unlike peritoneal macrophages, Kupffer cells inhibit target cell proliferation with great efficiency without any prior activation. The in vitro cytostatic effect requires the binding of the macrophage to the target cells. Kupffer cells may play a role in the prevention of immune complexdetermined nephritis. Accordingly, mice treated with C. parvurn cleared and degraded BSA immune complexes more rapidly than control mice.

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Furthermore, in stimulated animals the amount of immune complexes deposited in the kidneys was reduced (Barcelli et af., 1981). Specific IgA receptors seem to be involved in the removal of IgA immune complexes by mouse Kupffer cells (Rifai and Mannik, 1984). On the other hand, Nishi et af. (1981) showed that circulating immune complexes may produce loss of Fc and C3 receptor function of Kupffer cells in rats and provide evidence that this might result in diminished clearance of complexes, particularly in patients with systemic lupus erythematosus, who have high levels of circulating complexes. A preexisting defect in the RES function has also been suspected to be the cause of other immune complex-mediated diseases such as rheumatoid arthritis and lupus erythematosus. A depression in the phagocytic function of the RES occurs in cirrhosis (Wardle et af., 1980; Lahnborg et af., 1981), cholestasis (Drivas et af., 1976), and biliary obstruction (Katz et af., 1984). This may explain the enhanced frequency of infections in these patients. Kuratsune et af. (1983) stated that the endotoxemia in liver cirrhosis may result in part from a spillover of endotoxin from the portal flow due to diminished Kupffer cell functions. During the fatty-liver stage in rats fed a cirrhotogenic diet, the Kupffer cell functions are significantly depressed and this may contribute to the increase in sensitivity to endotoxin; the resulting hepatic necrosis may set the stage for fibrosis and the ultimate cirrhosis (Ah and Nolan, 1969). Finally, a possible relation between damage of Kupffer cell functions and fulminant hepatic encephalopathy has been suggested (Canalese et d., 1982). Accumulation of iron in Kupffer cells follows an excessive breakdown of erythrocytes in patients with transfusional siderosis. Hepatocellular siderosis is due either to excessive intestinal absorption or to enhanced redistribution of iron recycled from the macrophages-a genetically determined defect called idiopathic hemochromatosis (IH). In transfusional siderosis, the iron content of Kupffer cells reflects the accumulation of iron in the other macrophages. In IH, macrophages exhibit an impaired ability to store iron and can thus promote parenchymal cell iron overload (Dullmann et al., 1982). A syndrome of iron overload, which shares most of the histopathological findings with human IH, has been discovered in mynah birds (Gosselin and Kramer, 1983). Hultcrantz et af. (1983) have shown that iron overload does not affect lysosomal enzymes and that the buildup of iron in both parenchymal and sinusoidal cells can be mobilized in response to an increased demand. Kupffer cells seem to be involved in several pathological syndromes related to coagulation. They may thus initiate microvascular thrombosis as in endotoxemia (Maier and Hahnel, 1984) or, when their capacity for

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clearing microthrombi is impaired, they may be responsible for an increase in the severity of disseminated intravascular coagulation (Oka et a ] . , 1983). Kupffer cells may also play a role in inflammatory responses, since they secrete a substance that increases the synthesis of fibrinogen in cultured parenchymal cells (Sanders and Fuller, 1981). Depression in Kupffer cell phagocytotic function has been reported after trauma (Kaplan and Saba, 1979), thermal injury, or burn toxoid administration in rats (Nedoshivina et al., 1981). Hypoglycemia induced in rats after insulin administration directly impaired RES functions (Kober and Filkins, 1981), whereas reticuloendothelial hyperphagocytosis occurred in streptozotocin-diabetic rats; this enhanced phagocytosis could be prevented by chronic insulin replacement therapy (Cornell, 1982).

IX. Concluding Remarks A controversy concerning the identity of different types of sinusoidal lining cells has been resolved by modern histological methods. The current concept of four different types of sinusoidal lining cells-endothelial, Kupffer, fat-storing (stellate), and pit cells-has now been firmly established. Throughout this review, evidence has been presented from light and electron microscopy, cytochemistry, and in vivo microscopy, and from methods such as cell isolation, purification, and culture, including biochemical and immunological characterization of normal and activated Kupffer cells. Although substantial information about Kupffer cells in human liver diseases is somewhat lacking, we have presented aspects of Kupffer cell function in various experimental pathological conditions. Evidence is accumulating quickly that Kupffer cells are the resident macrophages of the hepatic sinusoid, demonstrating local proliferation in addition to the recruitment from another source like the spieen or the bone marrow. However, we still lack knowledge about the differentiation of Kupffer cells in early embryonic life. It has been proposed that Kupffer cells originate from cells in the yolk sac, but further studies concerning this topic are needed. Since the descriptions of the isolation, purification, and culture of Kupffer cells from rat liver, the study of the function of these cells in vitro has made considerable progress. Elaborate biochemical analysis has clarified the mechanism of oxygen burst followed by the release of a number of mediators. Kupffer cells have been found to play a major role in the clearance

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of endotoxin from the blood, which induces the release of a number of mediators. Experimental virus infections have provided us with indications of how viral hepatitis may develop in human liver. We have learned that the susceptibility of Kupffer cells to bacterial and viral infections is influenced by hormonal, immunological, nutritional, and genetic factors. The regulatory role of Kupffer cells in various hepatic disorders as well as in liver transplantation, however, still awaits further investigation. Recent evidence suggests the involvement of Kupffer cells and NK cells in the defense against tumor development. We therefore need to know how Kupffer cells coordinate their activities with liver-associated NK cells (pit cells) in tumor cell cytotoxicity.

ACKNOWLEDGMENTS Ronald De Zanger and Marleen De Pauw are thanked for their efforts to process the manuscript through different stages of preparation.

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INTERNATIONAL REVIEW OF CYTOLOGY. VOL. I18

Cellular and Molecular Biology of Capacitation and Acrosome Reaction in Mammalian Spermatozoa K. S. SIDHU AND S. S. GURAYA I.C.M.R. Regional Advanced Research Centre in Reproductive Biology, Department of Zoology, College of Basic Sciences and Humanities, Punjab Agricultural University, Ludhiana, India

I. Introduction The concept of capacitation proposed originally by Austin (195 1) and Chang (1951) independently is found to be universal in all the eutherian mammals and has been discussed in several reviews and books (Bedford, 1970; Bedford and Cooper, 1978; Shapiro et al., 1981; Yanagimachi, 1981; Clegg, 1983; Bedford, 1983; Chang, 1984; Hinrichsen-Kohane et al., 1984; Meizel, 1984, 1985; Shapiro, 1984; Langlais and Roberts, 1985; Metz and Monroy, 1985; Austin, 1985; Flechon, 1985; Hedrick, 1986; Guraya, 1987; Sidhu, 1988; O’Rand, 1988; Schatten and Schatten, 1989). These reviews and books deal with different and isolated aspects of capacitation and acrosome reaction (AR). However, no single compilation in literature brings out clearly the various concepts involved in the complex phenomena of capacitation and AR in totality. Therefore, the objective of the present review is to integrate the information available on different aspects of capacitation and AR and to propose a tentative but unified view of the mechanisms involved in these phenomena. Although capacitation is achieved synergistically and efficiently in the female reproductive tract, it has also been accomplished outside the female reproductive tract in numerous in vitro studies using well-defined media in several mammalian species. These in vitro studies have greatly enhanced our understanding of the biochemical or (molecular) aspects of capacitation and AR. There is reasonable consensus that capacitation per se involves no morphological change in spermatozoa that can be discerned, even under the electron microscope; it does facilitate the occurrence of a distant morphological event in spermatozoa known as AR. The AR appears to involve point fusions between the outer acrosomal membrane and overlying plasmalemma leading to the release of acrosomal 23 1 Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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contents or enzymes required at the site of fertilization, and its onset does not appear to require a specific stimulus, but only an adequate level of Ca” in a physiological milieu once capacitation has been completed (Bedford, 1983; Guraya, 1987; Sidhu, 1988). Most of the current studies have attempted t o elucidate various biochemical or molecular changes occurring during capacitation that prepare the spermatozoa for AR. However, the present state of knowledge still does not allow us t o distinguish clearly the events occurring during capacitation and AR, but the former is considered to be the prerequisite for the latter. N o unified view of the biochemical o r molecular mechanism of capacitation has emerged from the results of numerous in vitro studies, but most of the observations point to the conclusion that capacitation involves two important changes at the molecular level in spermatozoa: ( 1 ) sperm surface alteration and/ or intramembranal molecular mobility to facilitate Ca” influx, and (2) changes in sperm energetics via alterations in oxygen uptake and glucose utilization manifested in the form of changes in pattern of flagellar beat (hyperactivated motility). These molecular changes during capacitation ensure the timely occurrence of AR. The release of hydrolytic enzymes from the acrosome during AR and the generation of greater thrust in sperm showing hyperactivity during capacitation greatly facilitate the entry of spermatozoa through egg investments for fertilization. Since the mid- l970s, considerable information has been generated, but there are still lacunae in our knowledge about the regulatory mechanisms involved in the process of fertilization (Metz and Monroy, 1985). There are also species variations in regard to the time, site, and stimulus of capacitation and AR in different eutherian mammals. Several hypotheses have been put forward to explain capacitation and AR, but none of these hypotheses in isolation can explain these phenomena. In the present review, we have made an attempt to compile and analyze critically the results of recent studies carried out on capacitation and AR to elucidate the complexities involved and the progress made in the better understanding of capacitation and AR.

11. Capacitation

A. BIOCHEMICAL, MOLECULAR, A N D PHYSIOLOGICAL ASPECTS

Capacitation is considered to involve a series of incompletely understood subcellular and molecular changes that prepare the spermatozoa for AR (for reviews, see Rogers and Bentwood, 1982; Clegg, 1983; Meizel, 1985; Metz and Monluy, 1985; Guraya, 1987; Sidhu, 1988). Our knowl-

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edge about capacitation has increased considerably during the last few years because of the development of in vitro fertilization systems in several mammalian species and because of advancements of technologies involved in the study of molecular events, but it is not a well-understood phenomenon. No criteria for analyzing capacitation or AR independent of each other have been developed. The biochemical and physiological changes occurring during capacitation are not well defined and these changes may not be directly correlated with the actual acquisition of fertilizing ability by spermatozoa. Most of the changes occurring during capacitation are studied in relation to the following parameters: (1) Sperm surface alterations, (2) sperm intramembrane molecular alterations, and (3) metabolic alterations. I . Sperm Surface Alterations During maturation in the epididymis and also during ejaculation, spermatozoa are coated by epididymal and accessory gland secretions. This coating phenomenon inhibits or masks the ability of spermatozoa to fertilize the egg (decapacitation) and thus may protect the premature occurrence of capacitation (Sidhu, 1988). Decapacitation factors are present in epididymal fluid (Weinman and Williams, 1964) and seminal plasma and may have either low (Pinsker and Williams, 1967) or high molecular weight (Reyes et al., 1975; Reddy et al., 1979, 1982; Audhya et al., 1987). Mclaughlin and Shur (1987) have shown that mouse caput epididymal fluid contains galactosyltransferase and galactosyltransferase modifiers that inhibit binding of mature cauda epididymal sperm to the egg zona pellucida. Sperm galactosyltransferase plays a significant role in fertilization (Lopez et al., 1985; Lopez and Shur, 1987; Macek and Shur, 1988; Shur, 1989). Eng and Oliphant (1978) had earlier isolated a glycoprotein from rabbit seminal plasma that has a reversible decapacitation activity. This glycoprotein, the acrosome-stabilizing factor (ASF), decapacitates sperm apparently by preventing the AR. This factor is synthesized in the corpus epididymis (Thomas et al., 1984a,b) and can be rapidly purified from seminal plasma utilizing monoclonal antibodies (mAb) via affinity chromatography (Reynolds and Oliphant, 1984). Extensive chemical characterization of ASF has been carried out (Thomas et al., 1986; Wilson and Oliphant, 1987). A direct correlation between the structure of the ASF molecule and its biological activity has been demonstrated (Wilson et al., 1986). Koehler et al. (1980) used fluorescence to indicate the presence of a molecule on the ejaculated sperm surface in rabbit having antigenic determinants similar or identical to those of fibronectin. The activity is localized to the surface over the acrosomal region, can be removed by trypsin, and is rapidly reestablished by incubation of trypsinized

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sperm in seminal fluid. Its molecular weight of 40,000 is very similar to the active subunit of ASF (38,000). Preliminary data indicate that it is lost during the capacitation process. Sidhu and Guraya (1988) have isolated a calmodulinlike protein from buffalo seminal plasma that inhibits the accumulation of Ca’ + in ejaculated sperm by stimulating a calcium-extruding phenomenon, and thus inhibits the onset of capacitation and AR in ejaculated spermatozoa. During capacitation, this protein is probably removed from the sperm surface, which enables the sperm to accumulate the Ca’+ required for capacitation and AR. It has been shown that decapacitation could be induced in capacitated spermatozoa by pronase-denuded vesicles that hydrolyze glycoproteins quantitatively, and also by cholesterolbearing liposomes, indicating the roles of glycoprotein and cholesterol in sperm decapacitation (Davis and Hungund, 1976; Davis, 1976, 1980; Davis and Davis, 1983). These decapacitation factors are present on the surface of ejaculated sperm and possibly are removed along with other peripheral membrane components during their stay in the female tract (Oliphant and Brackett, 1973; Koehler, 1976; Sidhu et al., 1984; Sidhu, 1988). There is considerable evidence that the surface of mammalian spermatozoa is most dynamic and is in a state of continuous modification from the time of spermatogenesis to capacitation (Moore, 1985). The hydrophobic regions of the molecules in plasma membrane have their hydrophilic regions exposed at the membrane surface, which includes carbohydrate, sialic acids (glycoproteins), glycolipids, and gangliosides. The presence of sialic acid and sulfated carbohydrate is probably responsible for the net negative charge on the sperm surface (Sidhu and Guraya, 1980, 1985; Guraya. 1987). These polar molecules on the surface of sperm can bind other proteins and ions from the male and female reproductive tracts during spermiation, maturation in the epididymis, ejaculation, and capacitation, and other processes. Thus there could be a continuous change in molecules at the sperm surface (Guraya, 1987; Sidhu, 1988). Several studies of sperm topography point to the conclusion that the sperm surface is stabilized during maturation in the epididymis and during ejaculation by selective coating with secretions from male accessory glands (Guraya, 1987; Sidhu, 1988), and the reverse occurs in the female reproductive tract, where the sperm surface is modified to destabilize the membranes for occurrence of AR. Sperm surface antigenic determinants are restricted to distinct domains (Villarroya and Scholler, 1986; Huneau et al., 1988; Hardy et al., 1988; Esaguy et af., 1988). Monoclonal antibodies have been used to study modifications of the sperm surface that occur during capacitation, AR, and fertilization (Myles and Primakoff, 1984; Wolf et al., 1985; Hinrichsen-Kohane et af., 1985; Saxena et af.. 1986~;

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Masaro et al., 1986; Kallajoki et al., 1986; Lakoski et al., 1988). Sperm surface components that are involved in specific events of the fertilization process have been identified and purified. These surface components include mouse sperm autoantigens in the anterior acrosomal region that are identified by mAb and are involved in sperm AR, sperm-zona interaction, and sperm-egg fusion (Saling and Lakoski, 1985; Saling et al., 1985; Saling, 1986; Primakoff et al., 1987; Lakoski et af.,1988); guinea pig acrosoma1 antigens (AAI) involved in AR and binding of spermatozoa to the zona pellucida (Hardy et al., 1985, 1988; Guienne and DeAlmeida, 1986); human sperm acrosomal antigen (aHS1A. 1) identified by mAb and immunofluorescence undergoing important modifications during in vitro capacitation and AR (Villarroya and Scholler, 1986, 1987; Huneau et al., 1988); and rabbit sperm plasma membrane glycoprotein autoantigen (RSA- l ) , most concentrated over the postacrosomal-equatorialregion probably involved in zona binding (O’Rand et al., 1984a, 1985; Fisher et af., 1987; Esaguy et al., 1988). However, it is not always possible to take full advantage of these tools when the subcellular localization of the recognized determinants remains equivocal. The immunofluorescence techniques that are generally used fail to identify the exact labeled organelles in spermatozoa. Moreover, mAb directed to surface components are liable to bind to internal sites through unsuspected membrane breaks, and hence immunoelectron microscopy can be an alternative approach as used by Huneau et al. (1988). Several studies using lectins and antibodies against sperm surface antigens indicate that the capacitation involves decoating of the sperm surface of the epididymal and seminal plasma proteins and also possibly an alteration in the glycoproteins of the sperm surface (Clegg, 1983; Andre, 1983; Ahuja, 1984; Flechon, 1985; Oliphant et al., 1985; Singer et af., 1985; Peitz and KO, 1987). Lectin binding to spermatozoa progressively disappears during capacitation (Talbot and Franklin, 1978; Koehler, 1978; Lewin et al., 1979; Byrd, 1981; Singer et af., 1985; Cross and Overstreet, 1987). We have also shown that treatment of buffalo bull spermatozoa with hypertonic salt solution, which liberates peripheral proteins (Oliphant and Brackett, 1973; Singer, 1974), greatly enhances the onset of capacitation and AR (Sidhu et al., 1984), as also reported in several other studies (Johnson, 1975; Aonuma et af., 1982; O’Rand, 1982; Hyne and Garbers, 1982; Reddy et al., 1982; Fraser, 1984). Follicular fluid or serum factors (Barros and Garavagno, 1970; Lui et af., 1977) evidently also modify surface components, since they are known to capacitate spermatozoa. Enzymes (Gwatkin et al., 1974; Shur and Hall, 1982; McNutt and Killian, 1986; Guerette et al., 1988) also may contribute to the sperm surface alterations. The source of these enzymes and their regulation in the female reproductive tract need to be determined in future

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studies. Studies using lactoperoxidase-catalyzed iodination of sperm surface proteins demonstrate substantial alteration of surface proteins (Oliphant and Singhas, 1979; Esbenshade and Clegg, 1980). Shur and Hall (1982), using genetic and biochemical probes, have suggested that in mouse spermatozoa, capacitation involves selective removal of a surface glycoside (i.e., poly-N-acetyllactosamine),which masks the activity of sperm-specific galactosyltransferase involved in binding N-acetylglucosamine residues in the egg zona pellucida. Shinohara et a / . (1984) suggested that stimulation of AR by purified trypsin, chymotrypsin, and kallikrein in hamster spermatozoa is due to the hydrolysis of the sperm surface gl ycoprotein coat. Other capacitationassociated changes reported are activation of ATPase (Gordon and Dandekar, 1977) and decrease in net negative charge (Vaidya et a/., 1971; Rosado et a / . , 1973; Courtens and Fournier-Delpech, 1979). Interestingly, the net negative charge on the sperm surface increases during maturation in the epididymis and is implicated in the enhanced Ca” binding required for motility. The electronegativity of the sperm surface is due to the presence of sialic acid residues (Sidhu and Guraya, 1985; Guraya, 1987). Decrease in electronegativity of the sperm surface occurs during treatment with follicular fluid or neuraminidase. Uterine fluid and follicular fluid contain neuraminidase and proteases, and these enzymes are related to the induction of capacitation (Gordon et a / . , 1986; Guerette er al., 1988). However, the major unresolved question is, what is the significance of decrease in sperm surface electronegativity. One possibility is that because egg vitelline membrane also has a negative charge, the decrease in sperm surface negative charge will facilitate binding (Bedford, 1983). A decrease in free surface-active -SH and -NH, groups during capacitation has also been reported (Rosado et al., 1973; Yanagimachi et al., 1983; Thomas et a / . , 1984a,b; Fleming er a / . , 1986); this may be due to conformational changes secondarily induced by changes in surface polarity. Such changes are important for membrane breakdown during AR. Changes in the surface proteins as studied by radioiodination and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Oliphant and Singhas, 1979; Esbenshade and Clegg, 1980; Oliphant et al., 1985), and modification of lectin binding (Gordon et al., 1975; Talbot and Franklin, 1978) also occur during capacitation. These observations suggest that the peripheral proteins and associated components are either removed or altered from the sperm surface (Fraser, 1984). Such alterations may facilitate membrane fusions during AR, possibly by reducing the net negative charges or by increasing membrane fluidity. Another possibility is that such changes may have something to do with gamete interactions (Ahuja, 1982, 1985; Webb, 1983; Shapiro, 1984; Lambert,

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1984; Lamber and Van Le, 1984; Lambert et al., 1985; O’Rand et af., 1984a,b, 1985; O’Rand, 1986, 1988; Shalgi et al., 1986; Macek and Shur, 1988). Although the study of sperm glycocalyx (sperm surface) has been the subject of several recent investigations-particularly in regard to capacitation-we do not understand fully the interrelationships between these molecules and their mode of anchoring to membrane continuum (Guraya, 1987). According to Clegg (1983), very little information is available about the internal surface of the plasma membrane and the external surface of the acrosome membrane, which become more closely oriented during AR. Microtubules and microfilaments in the acrosomal region possibly play some role in AR (Stambaugh and Smith, 1978; Peterson et af., 1978; Campanella et a / . , 1979; Tamblyn, 1980; Clarke et a / . , 1982; Welch and O’Rand, 1985). Actin has been demonstrated in the acrosomal and postacrosomal regions, where it coexists with calmodulin (Camatini and Casale, 1987; Casale et al., 1988). Uncapacitated human sperm contain abundant G-actin, but F-actin could not be detected in these cells (Virtanen et al., 1984). Saxena et a/. (1986b) have demonstrated the appearance of F-actin in most regions of boar spermatozoa during capacitation. In the same species, it has been demonstrated that proteins are cleared from the plasma membrane overlying the head of ejaculated spermatozoa during capacitation in v i m (Saxena et al., 1986a). The use of mAb has indicated that two plasma membrane proteins migrate from the head plasma membrane into the flagellar plasma membrane by a process that is inhibited by cytochalasin D. These proteins move back into the head plasma membrane when fresh seminal plasma is added to capacitated spermatozoa. This movement is also blocked by cytochalasin D. These observations suggest that actin is involved in the translocation of these membrane proteins. Saxena et al. (1986b) have proposed that actin polymerization is directly involved in capacitation. O’Rand (1979) summarized all the surface changes in sperm and proposed a molecular model of sperm capacitation. In this model the plasma membrane prior to capacitation (Fig. 1A) has four classes of molecules involved in the surface change: (1) glycoproteins that are mobile within the plane of the membrane, (2) nonmobile glycoproteins, (3) glycolipids, and (4) peripheral membrane components (see also Guraya, 1987). This precapacitation model is supported by several observations. Antibodies to seminal plasma (class 4 in the foregoing list) will bind to the sperm surface (Oliphant and Brackett, 1973). Antibodies to whole semen (all classes) will not show patching because of the involvement of both mobile and nonmobile classes (Romrell and O’Rand, 1978). Lectin binding of class 1, 3, or 4 to class 2 molecules will show some degree of patching

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P

P

FIG. 1. A model for the surface changes in sperm membranes associated with capacitation. (A) Prior to capacitation; (B) after capacitation. Four classes of molecules are shown: 1, mobile glycoproteins; 2, nonmobile glycoproteins; 3, glycolipids; 4, peripheral components. The lower sketches show the surface pattern expected for intrinsic mobile glycoproteins (class I ) in association with peripheral components (class 4) before capacitation (A) and without peripheral components after capacitation (B). From O’Rand (1979).

(O’Rand, 1977, 1978). Complement-dependent antibody immobilization of ejaculated spermatozoa depends on the relationship between the intrinsic surface antigen and class 4 (O’Rand and Metz, 1976). After capacitation the relationship among the four classes of molecules changes (Fig. 1B). Peripheral components have been modified or removed, and mobile and nonmobile classes (1 and 2) have reassociated. This is consistent with several observations. The model shows that there should be a region of membrane glycoprotein and separate domains relatively free of glycoproteins. Thus, a protein-poor, high-fluidity area ready for membrane fusion (Friend et al.. 1977) may coexist with protein-rich areas of decreased fluidity (O’Rand, 1977) in a patchwork-quiltlike topography. This would be consistent with the pattern of membrane fusion seen during the AR (Bedford and Cooper, 1978). 2. Sperm Intramembrane Molecular Alterations Sperm membrane changes that occur during capacitation include changes in the lateral mobility of proteins (O’Rand, 1977, 1979) and

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changes in intramembranous particles (Koehler and Gaddum-Rosse, 1975; Friend et al., 1977; Kinsey and Koehler, 1978; Friend, 1980; Bearer and Friend, 1982; Flechon, 1985). These intramembranous changes facilitate the formation of areas depleted of membrane proteins, which might be the site for occurrence of AR in spermatozoa. The transitional mobility of these elements depends on many factors such as fluidity of membrane lipids as determined by their phase-transition temperature, the length and degree of saturation of fatty-acid (FA) chains, the amount of cholesterol, the mobility of attached surface receptors, and the extent of their association with cytoplasmic proteins (Friend et al., 1977). The presence of intramembranous particles in mammalian spermatozoa was first studied under various physiological conditions (Koehler and Gaddum-Rosse, 1975; Friend et al., 1977; Friend, 1980; Friend and Heuser, 1981; Bearer and Friend, 1982; Enders and Friend, 1985; Guraya, 1987). Deriving inferences from earlier studies (see references in Lucy, 1978, 19841, a number of investigations have been made in mammalian spermatozoa correlating redistribution of intramembranous particles to the onset of capacitation and AR. The two most common features observed during capacitation and AR are the following: 1. The well-organized strands of small particles enveloping the middle piece become disorganized during incubation of spermatozoa in capacitation medium (Fig. 2), and this structural change correlates with the modification of flagellar pattern (hyperactivity) during capacitation (Koehler and Gaddum-Rosse, 1975). 2. Deletion of fibrillar intramembranous particles from the E-fracture faces of both acrosomal and plasma membrane and the clearing of globular particles from the P face of the plasma membrane occur (Fig. 3) immediately preceding AR (Friend et al., 1977; Bearer and Friend, 1982).

Davis et al. (1979) proposed that the decrease in the cholesterol/phospholipid ratio in the sperm plasma membrane facilitates the occurrence of capacitation culminating in AR. According to the model, destabilization of this membrane is associated with increased permeability to Ca” , which chelates anionic phospholipid molecules in the adjacent membrane lipid bilayer to promote fusion (Papahadjopoulos et al., 1974; Lucy, 1984) between the plasma membranes and outer acrosomal membrane (Davis et al., 1974; Davis, 1978). The relationship between anionic lipid concentration and the functional properties of plasma membrane domains has been explored using the antibiotic polymyxin B (PXB) as a probe for anionic lipids and filipin as a probe for j3-OH sterols such as cholesterol (Bearer and Friend, 1982; Enders and Friend, 1985). According to these

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FIG. 2. (A) A portion of the middle piece. The original face fracture (A) exhibits the particle strands. The true surface (s) reflects the presence of the underlying strands. x 50,000. (B) A portion of the middle piece from a spermatozoa incubated for 16 hours in Hanks’ balanced salt solution. The membrane-associated strands are still present, but the quantity and organization of the strands are reduced. X 56,000. From Koehler and GaddumRosse (1975).

workers, areas of plasmalemma specialized for fusion during the AR had a higher affinity for the probe than adjacent nonfusigenic regions. During capacitation, enlargement of the area susceptible to PXB binding occurs over the acrosomal cap. These studies indicate that varying concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane. The enhanced PXB binding after capacitation suggests that more anionic lipid becomes accessible to the probe, either by flip-flop of anionic lipids from the cytoplasmic to the external leaflet or by neosynthesis of these lipids during capacitation, as already reported by some workers (Evans et al., 1980, 1987; Bearer and Friend, 1982; Sidhu er al., 1989). Many studies support Davis’s model. For example, modification of albumin-bound lipid alters its sperm capacitation ability (Davis, 1976; Lui and Meizel, 1977);synthetic phospholipid vesicles containing cholesterol and sterol sulfate, which stabilize the plasma membrane, reversibly inhibit sperm fertilizing capacity (Davis, 1976, 1980; Davis and Byrne, 1980; Fayrer-Hosken et al., 1986, 1987);detergents and lysophosphatidyl-

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FIG.3. (A) Circular clearing of intramembranal particles (IMP) (arrow) in capacitated sperm treated with polymyxin B (PXB). x 54,000. (B) A capacitated sperm, with macular clearing (arrows) in the postacrosomal segment, remains unaffected by PXB, which induced perturbations of the adjacent proximal cap. x 36,000. (C) Filipin attacks the proximal cap of a capacitated sperm heavily, and also minimally affects the postacrosomal segment. Particle-free clearings are clearly visible. ~45,000.From Bearer and Friend (1982).

choline induce the AR, and incubation with albumin lowers the cholesterol/phospholipid ratio in the rat sperm plasma membrane (Davis et al., 1979).

Albumin, a multiple ligand carrier, has been shown to mediate sperm cholesterol efflux during in vitro capacitation of rat (Davis et al., 1980), mouse (Go and Wolf, 1985), human (Moubasher and Wolf, 1986; Langlais et al., 1988), and bull (Ehrenwald el al., 1988). In human serum, verylow-density lipoprotein (VLDL), low-density lipoprotein (LDL), highdensity lipoprotein (HDL), and albumin-and in human follicular fluid, HDL and albumin-induce efflux of cholesterol and cholesterol sulfate from spermatozoa during capacitation (Langlais et al., 1988). Evans et al. (1980) have shown that during in utero incubation the phosphatidylcholine contents of porcine sperm increased-not necessarily as a result of phospholipid synthesis but more likely adsorption from female reproductive tract secretions. The result of these studies would be a decreased cholesteroYphospholipid ratio, which is compatible with the hypothesis of Davis. Davis (1982) has identified albumin as a uterine sterol acceptor that removes cholesterol molecules from sperm membranes during in utero incubation, thereby reducing the cholesterol/phospholipid ratio. This in turn leads to membrane destabilization during capacitation, thus facilitating AR. Menezo et al. (1984) have also shown that a high choles-

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terol content in the follicular fluid inhibits fertilization. The hypothesis proposed by Davis for the mechanism of capacitation has been supported by several observations, showing an exchange of lipids between sperm and albumin and particularly the efflux of sterol from sperm during capacitation in albumin-containing media (Go and Wolf, 1983, 1985; FayrerHosken ef al., 1986; Ehrenwald ef al., 1987). Cholesterol is the major sterol in most mammalian cells, but desmosterol, a precursor of cholesterol and sulfo-conjugated sterols has also been detected in mammalian spermatozoa (Go and Wolf, 1983), Sulfated sterols have been implicated in the stabilization of sperm membranes during transit in the epididymis (Legault ef ul., 1979). Sterol sulfates play a role in capacitation (Langlais er al., 1981, 1984; Tesank and Flechon, 1986). The sterol sulfatase activity in the female reproductive tract could hydrolyze sterol sulfates of spermatozoa, leaving free sterol in the membrane. Cleavage of membrane phospholipid by phospholipase A2 or 1ecithin:cholesterol acyltransferase could allow a free FA from the phospholipid to esterify cholesterol, thus producing lysophospholipid and cholesteryl acyl ester. This leads to the accumulation of lysophospholipids and an altered cholesteroVphospholipid ratio in the sperm membrane causing membrane perturbation culminating in AR. The cholesteryl ester could be sequestered by albumin in the medium. It is also possible that acyltransferases responsible for esterification of cholesterol are activated by albumin. Whereas the attention of researchers has been focused primarily on cellular sterols in most of the studies mentioned, a pivotal role in capacitation for the other major membrane lipids (i.e., phospholipids) has been suggested (Clegg, 1983; Takei ef al., 1984). The phospholipids as substrate of sperm-associated phospholipase A? could give rise to lysophospholipids, which facilitate membrane fusion during AR particularly in hamster. human, and mouse spermatozoa (e.g., Section III,C,2). As already discussed, capacitation involves modifications in sperm lipid composition such as hydrolysis of sterol sulfates to free sterol, cholesterol efflux, and formation of lysophosphatides by endogenous phospholipase A2. These changes are generally considered to have a destabilizing effect on the membrane bilayer and thus to promote fusion. Much has been published concerning the total lipids of sperm from a variety of species (Sidhu and Guraya, 1985; Guraya, 1987). However, capacitation envisages changes in the plasma membrane and outer acrosomal membrane, which are directly involved in AR. Although procedures have been described for the isolation and purification of the sperm membranes (Lunstra ef ul., 1974: Gillis ef al., 1978; Peterson ef al., 1980; Holt, 1983; Soucek and Vary, 1984; Holt and North, 1985; Sidhu and Guraya, 1989c),

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information on the lipid composition of the sperm membranes is limited (Wolf and Voglmayr, 1984; Parks and Hammerstedt, 1985; Nikolopoulou et al., 1985; Wolf et al., 1986a,b,c, 1988; Parks et al., 1987), and a few reports implicate sperm membrane lipid in capacitation and AR (Vijayasarathy and Balaram, 1982; Hinkovska et al., 1986; Wolf et al., 1986a, 1988; Lee et al., 1986a,b;Sidhu and Guraya, 1989c,d).Although phosphatidylinositol represents only a small percentage of total phospholipids in bovine and other sperm membranes, the importance of phosphoinositol metabolism in intracellular signaling has been demonstrated (Berridge, 1984) and appears to be related to events associated with the AR. Nikolopoulou et al. (1985) reported that hybrid vesicles resulting from the AR of boar sperm in vitro incorporated 32Piinto polyphosphoinositides at a rate of 5- to 15-fold that of plasma membrane alone. Furthermore, boar sperm membranes phosphorylated proteins when incubated in the presence of r3’P]ATP, calcium, and diacylglycerol (Nikolopoulou et al., 1986). Diacylglycerol is an important component of intracellular signaling by phosphoinositides, stimulating the activity of protein kinase C. Phorbol esters, which mimic the effect of diacylglycerol on protein kinase C, accelerate the AR of mouse sperm once bound to the zona pellucida (Lee et al., 1986a,b). Phosphoinositides are reported in the plasma membrane and outer acrosomal membrane in bull and boar spermatozoa (Berruti and Franchi, 1986; Parks et al., 1987). The precise role of phosphoinositide metabolism and its effects on calcium mobilization and protein phosphorylation during capacitation and AR are yet to be determined. 3 . Changes in Sperm Physiology Physiological changes accompanying sperm capacitation are expressed in sperm motility, respiration, and substrate utilization (Fraser and Lane, 1987). Modulation of sperm physiology by metal ions and cyclic nucleotides occurs during capacitation (Sidhu and Guraya, 1985;Guraya, 1987). Both in vitro and in vivo studies have shown an increase in respiration during capacitation. Rabbit sperm in utero takes up four times more oxygen than ejaculated sperm, and in vitro uterine, oviductal, and follicular fluids also stimulate oxygen consumption in spermatozoa (Boel, 1985). Rogers et al. (1977) have shown that fertilization ability of hamster sperm is reduced by inhibitors of oxidative phosphorylation such as oligomycin, rotenone, and actinomycin A. All these studies indicate the importance of respiration in capacitation and fertilization. However, it is not clear whether a change in the metabolism is directly attributed to capacitation or merely coincidental with it (Mann and Lutwak-Mann, 1981). Boel (1985) has shown that increased respiration of sperm in a capacitating

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medium is due to the presence of oxidizable substrates and, as such, is a component of the process of capacitation rather than a cause of its occurrence. Fraser and Quinn (1981) have shown that there is no need for oxidative metabolism, and that successful fertilization in the mouse can be achieved with glucose as substrate under anaerobic conditions in the presence of oligomycin. Most of the in vitro cultures employed for the study of sperm capacitation, AR, and fertilization include three potential exogenous substrates that are required to support fertilization: pyruvate, lactate, and glucose (Rogers, 1978; Fraser and Quinn, 1981; Sidhu et al., 1984). The presence and relative concentration of certain energy sources in a capacitating medium have been related to the timing and efficiency of capacitation. In mouse and guinea pig, the presence of pyruvate and lactate in culture increases the percentage of fertilization (Miyamoto and Chang, 1973; Toyoda and Chang, 1974; Rogers and Yanagimachi, 1975). However, Hoppe (1976) has concluded from studies with mice that the glucose is the major source of energy for capacitation. This concept has been confirmed by Boel (1985). In contrast, Rogers and Yanagimachi (1975) showed in guinea pig that glucose actually retards AR. Mujica and Valdesruiz ( 1983) also demonstrated that in guinea pig spermatozoa, capacitation could occur within a short time with glucose as the only exogenous substrate, but the AR was arrested-probably by a glucose metabolite. The intracellular levels of glucose 6-phosphate may play a key role in the expression of AR. Similarly, Hyne and Edwards (1985) have shown that 2-deoxy-~-glucoseand 2-amino-2-deoxy-D-g~ucosestrongly inhibit the AR without affecting sperm motility in the guinea pig, probably by causing the depletion of the sperm ATP concentration. The optimal energy source for capacitation may vary among species, but various energy sources used affect metabolism, which in turn affects capacitation (Rogers and Bentwood. 1982). Takei et al. (1984) examined the phospholipids in guinea pig spermatozoa before and after capacitation in vitro using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC). They observed no change in total phospholipid content of spermatozoa during capacitation. However, the phospholipid content of the medium was reduced by 20% during incubation, suggesting that sperm may utilize extracellular phospholipids as an energy source during capacitation. A distinct change in the patterns of sperm motility undergoing capacitation have been shown commonly known as activation or hyperactivity (Yanagirnachi, 1970, 1981). The hyperactivated sperm mobility during capacitation of spermatozoa was first observed in hamster (Yanagimachi,

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1969), and later in the guinea pig (Yanagimachi, 1972), mouse (Fraser, 1977), dog (Mahi and Yanagimachi, 1976), rabbit (Cooper et al., 1979), bat (Lambert, 1981), dolphin (Fleming et al., 1982), sheep (Cummins, 1982), and buffalo (Sidhu et af., 1984). The beating of the flagella of activated hamster sperm is considerably greater than that of uncapacitated spermatozoa. Hyperactivated motility in spermatozoa is also seen in vivo in the ampullary portion of the oviduct, indicating that activation reflects an important change occurring during capacitation (Suarez, 1987). This hyperactivity of sperm motility during capacitation, described as more energetic, may be the outcome of increased metabolism. It can be hypothesized that all the surface changes on spermatozoa occurring during capacitation might influence the permeability of membranes to the substrates and then in turn favor activation of spermatozoa. The significance of the hyperactivated motility of sperm during capacitation is not clear. It might increase the chances for contact of the few ampullary spermatozoa with the egg mass (Katz et al., 1978) or help promote sperm transport from the isthmus to the ampulla of the oviduct (Cummins, 1982). In rabbit spermatozoa there occurs a physiological interaction between the spermatozoa and the isthmic environment leading to modulation of sperm motility. A relatively vigorous motility displayed by spermatozoa in the rabbit uterus is largely inhibited after residence for several hours in the lower isthmus (Overstreet et al., 1980; Johnson et al., 1981). Active swimming, however, can be readily induced by dilution of the isthmus contents with ampullary fluid or artificial media; pyruvate in the medium stimulates while potassium inhibits hyperactive sperm motility (Burkman et al., 1984). According to Johnson et af. (1981), hyperactivated motility increases 20-fold the hydrodynamic-power output of rabbit spermatozoa and thus might augment the directed sperm thrust necessary for penetration of the egg investments (Bedford, 1983). Suarez et al. (1984), using high-speed videomicrography , have shown that unreacted hyperactivated sperm have a different movement potential than acrosome-reacted sperm. Ca2+may affect motility twice during capacitation: during the initiation of hyperactivation and again during the initiation of the AR (Suarez et al., 1987). The change in motility accompanying the AR could serve to optimize two different functions that have been suggested for hyperactivated motility by Katz et al. (1978), namely providing a search pattern for finding the cumulus mass and providing increased capability for generating thrust against the resistance provided by the cumulus matrix, corona radiata, and zona pellucida. Prior to AR, hamster sperm probably cover space more rapidly than after the reaction by virtue of having higher beat frequencies. More rapid coverage of space offers an advan-

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tage for locating the cumulus. After the AR, the flagellar bending accompanying the AR would provide increased thrust, generating potential for penetration of the remaining oocyte investments. Alterations in sperm metabolism, respiration, and motility during capacitation are affected by various agents that increase the level of cyclic nucleotides. In various mammalian species a relationship has been shown between the levels of cAMP and capacitation (Morton and Albagli, 1973; Peterson et al., 1978; Hyne and Garbers, 1979; Cornett et al., 1979; Mrsny and Meizel, 1980; Garbers and Kopf, 1980; Berger and Clegg, 1983; Rufo et al., 1984). Consequently, the effect of exogenous cAMP has been shown t o augment capacitation in rat and rabbit spermatozoa. Because of the limited permeability of intact sperm cells to CAMP, other means of elevating the levels of CAMP (e.g., the use of phosphodiesterase inhibitors such as papaverine, theophylline, o r caffeine) have been shown to activate motility and AR in spermatozoa (Rogers and Bentwood, 1982). Monks and Franser (1987) have shown that in mouse spermatozoa, adenylate cyclase increases and phosphodiesterase decreases, thereby increasing cAMP availability during capacitation. Delgado et al. (1976) postulated that the increase in metabolism and motility produced in human spermatozoa by cAMP is accompanied by a labilization of the membrane structure (less stable conformation of membrane proteins, that is, a. helix, and/or random coil from stable configuration, that is, antiparallel p conformation) that can be considered a prerequisite to the AR. In addition to CAMP, 8-bromo-cGMP has also been shown to stimulate AR in guinea pig spermatozoa (Santos-Sacchi et al., 1980; Santos-Sacchi and Gordon, 1980). These investigators showed that an elevated cGMP/cAMP ratio was important for induction of AR. Garbers and Kopf (1980) indicated that a 50-fold increase in cGMP occurred in pig spermatozoa incubated in oviductal fluid without affecting cAMP level. The presence of calmodulin has also been shown in spermatozoa (Jones et al., 1978, 1980; Feinberg et a / . , 1981; Tash and Means, 1982; Yamamoto, 1985) and probably plays a role in organizing events associated with calcium uptake and function (Rufo ez al., 1984; Sidhu and Guraya, 1989a,b). But the major question remains of the interplay between CAMP,calcium, and calmodulin in regulating either sperm motility o r metabolism during capacitation. Peterson et al. (1983) showed that calmodulin plays a role in regulating calcium uptake in boar spermatozoa but does not appear to be involved in ATPdriven Ca” efflux. Ca” extrusion in boar sperm may depend on regulators other than calmodulin. Such calmodulin-independent regulation has also been suggested for guinea pig sperm adenylate cyclase by Garbers c’t al. (1982). However, the involvement of calmodulin in AR in guinea

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pig, and buffalo bull spermatozoa have been shown (Lenz and Cormier, 1982; Nagae and Srivastava, 1985; Sidhu and Guraya, 1989a,b). B. FACTORS INDUCING CAPACITATION The site of capacitation in the female reproductive tract is still open to question. Earlier observations (Chang, 1951, 1955; Austin, 195 I) showed the involvement of the female reproductive tract in capacitation of rabbit spermatozoa. The uterus and oviduct work synergistically in this regard, since the time required for capacitation is 5-6 hours when spermatozoa are exposed sequentially to both organs, but 10-1 1 hours when incubated in either organ independently. This implies involvement of at least two factors, one contributed by the uterus and the other by the oviduct (Bedford, 1970). It has been shown that the oviduct is the site for capacitation of spermatozoa and that sperm capacitation results from their intimate association with the cumulus cells and depends on one or more heat-stable low molecular weight substances provided by the intracellular matrix. During the association with the cumulus cells, sperm are deeply embedded in the cell and their membranes are altered by glycosidases of cumulus cells (Gwatkin, 1977). Partial capacitation can also be induced in sperm by P-amylase and 6-glucuronidase, which are known to remove seminal plasma proteins from sperm. Following these observations, several workers have shown in vitro capacitation of spermatozoa in various fluids such as follicular fluid (Meizel, 1978; Lenz et al., 1982; Mollova et a/., 1983; Suarez et al., 1986; Siiteri et a/., 1988a; Yudin et al., 1988), fallopian tube fluid (Barros et al., 1972), uterine fluid (Lewis and Ketchel, 1972; Davis, 1982; Sidhu et al., 1986), and homologous or heterologous sera (Yanagimachi, 1970). The chemical similarities between the blood serum and secretions from the female reproductive tract prompted the suggestion that the agents responsible for capacitation are synthesized at a place other than the female genital tract and are subsequently distributed via the bloodstream to the site of capacitation (Barros and Garavagno, 1970). The capacitation-inducing ability of serum is influenced by the hormonal status of the female; a progesterone-primed uterus inhibits sperm capacitation. Capacitation with blood sera is dependent on macromolecules, which are probably associated with the albumin fraction. Sidhu et al. (1986) have shown that the capacitation-inducing ability of estrous uterine fluid is present in an albuminlike fraction that is nondialyzable and heat-labile. Yanagimachi (1969) showed that in bovine follicular fluid there are two fractions involved in capacitation; one is responsible for activation of sperm and is dialyzable and heat-stable, whereas the

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other is nondialyzable, is heat-labile, and is involved in the induction of AR (Section 111,A). Lui et al. (1977) showed that albumin is the bovine follicular fluid protein involved in in vitro induction of the hamster sperm AR. Menezo et al. (1984) have shown that lipids in the human preovulatory follicular fluid are important for capacitation. Davis (1982) demonstrated the presence of a uterine protein that binds cholesterol from rabbit spermatozoa during capacitation and induces membrane destabilization leading to AR. Glycosaminoglycans (GAG) present in bovine and mouse cumulus matrix, hamster zona pellucida, and bovine follicular fluid (Eppig, 1979; Ball et a / . , 1982; Lenz et al., 1982; Talbot, 1984b) also have roles in capacitation and/or AR (Meizel, 1985; Lee et al., 1986a,b; Cherr et al., 1986; Didion and Graves, 1986; Handrow et al., 1986). The work of Wassarman and colleagues (1985) has shown that three glycoproteins designated as ZP,, ZP,, and ZP, are present in mouse zona pellucida, and that ZP, stimulates AR (for details see Section 111,A).

c. EFFECTSOF CHEMICALS ON CAPACITATION In Virro Various substances such as detergents, drugs, hormones, catecholamines, taurine, hypotaurine, enzymes, and lipids affect capacitation and AR in mammalian spermatozoa (Yanagimachi, 1975). Although the conditions employed by these in vitro studies may be different from those encountered in vivo, these studies contribute to an understanding of the basic mechanism of capacitation and AR. The molecular mechanism(s) by which detergents and several other reagents induce activation or AR is not known. These reagents might modify the surface components or structural components of the sperm plasma membrane, making them more permeable to CaZ+as is required for AR. Of the several enzymes tested by Yanagimachi (1979, only chitinase and P-glucosidase were capable of inducing the AR and activation of guinea pig spermatozoa. All the reagents, which stimulate adenylate cyclase and inhibit phosphodiesterase, also stimulate capacitation and AR (Tash and Means, 1983). Epinephrine and norepinephrine and a-and P-adrenergic agonists stimulate, while a and P antagonists inhibit hamster sperm capacitation and/ or AR; moreover, a agonists are more potent than P agonists (Cornett and Meizel, 1978; Cornett et al., 1979; Meizel and Working, 1980; Meizel er al., 1985). These results show that both a-and P-adrenergic receptor mechanisms might be involved in the stimulation of hamster sperm capacitation andlor AR. The data of Meizel et al. (1985) show that an a-adrenergic receptor rather than a 0-adrenergic receptor is involved in the stimulation of capacitation by catecholamines. The P-adrenergic agonist

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isoproterenol is not as effective as epinephrine and norepinephrine in stimulating capacitation. Incidently, a P-adrenergic receptor on hamster sperm could not be detected with a fluorescent receptor probe (Cornett and Meizel, 1980). a-Adrenergic stimulation of hamster spermatozoa leads to increased Ca” in hamster spermatozoa. Ca2+ might stimulate glycolysis via phosphofructokinase, and glycolysis is important for sperm capacitation and AR. a-Adrenergic receptor stimulation might increase CAMP, which in turn might be involved in an increase in CaZ+(Exton, 1982). The sulfonated amino acid taurine, and its precursor hypotaurine and other structurally related compounds, are known to be essential for sustaining sperm motility during in vitro capacitation and AR of hamster spermatozoa (Meizel, 1981;Liebfried and Bavister, 1981; Gwatkin, 1983). Taurine is also required for capacitation of human spermatozoa (Chen, 1985). Mrsny and Meizel (1985) showed that taurine and hypotaurine stimulate hamster sperm motility by their ability to inhibit Na’ ,K+-ATPase. Inhibition of Na’ ,K’-ATPase might support motility by increasing the ATP available for contractile proteins or by increasing the intracellular Na’, which can be exchanged for extracellular Ca”.

111. Acrosome Reaction

The acrosome is a baglike structure consisting of an inner and an outer acrosomal membrane present at the anterior side of the head covered by plasma membrane (see Fig. 4A). The acrosome contains several hydrolytic enzymes (i.e, proacrosin/acrosin, hyaluronidase, neuraminidase, acid phosphatases, esterases, etc.) (Morton, 1976; Guraya, 1987). The role of only few of these hydrolases such as acrosin, hyaluronidase, and neuraminidase has been investigated during fertilization. Several ultrastructural and biochemical studies have shown that these enzymes are released by an exocytotic event involving point fusions between the outer acrosome membrane and plasma membrane known as AR (Bedford, 1983; Yanagimachi, 1981; Clegg, 1983; Meizel, 1984, 1985; Langlais and Roberts, 1985; Guraya, 1987; Sidhu, 1988). The mammalian AR is essential for fertilization and occurs in sperm that have already undergone capacitation. Three distinct morphological stages of AR-acrosome swelling, vesiculation, and shedding (Fig. 5)-have been identified in buffalo bull spermatozoa (Sidhu et al., 1984, 1986). The AR allows release of the hydrolytic enzymes required for penetration of sperm through egg investments and related steps, and appears to modify the equatorial and/

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K . S. SlDHU AND S. S. GURAYA I

A

-.,

B

C

B

A

C

FIG.4. Fusion events in speratozoa during AR. The lower sketches show series of point fusion between the contiguous plasma and outer acrosomal membranes. From Barros el ( I / . (l%7).

A

B

C

D

E

FIG. 5 . Diagrammatic representation of the acrosomal stages during AR in buffalo bull spermatozoa. (A) Sperm head showing acrosome (black): (B) acrosome swelling: (C) vesiculation; ( D ) shedding; ( E) sperm head denuded of acrosome. From Sidhu et (I/. (1986).

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25 1

or postnuclear cap for attachment with the egg membrane (Bedford, 1983). The AR and exocytosis of somatic exocrine cells are similar in regard to the requirement of Ca2+influx and clearing of intramembrane particles at the exocytotic sites. However, AR differs from somatic cell exocytosis in that (1) it includes loss of the membranes unlike in somatic cell exocytosis and (2) in AR the exocytotic sites become cholesterol-poor (Friend, 1980) whereas Orci et al. (1981) showed in pancreatic acinar cells that exocytotic sites contain cholesterol. Mammalian sperm AR also differs somewhat from that in invertebrates; for example, it includes many more loci of point fusion between the outer acrosome membrane and plasma membrane (Barros et al., 1967), and these loci create a fenestrated shroud around the apex of the reacted sperm head that persists for some time. This process allows the release of acrosomal contents while still preserving much of the sperm surface containing receptors for zona pellucida (Bedford, 1983). Mammalian AR is distinguished by two additional characteristics: (1) it does not appear to require some specific stimulus, but an adequate level of Ca2' in a physiological milieu once capacitation is completed, and (2) there is little synchrony of the reaction among the spermatozoa in a medium. However, in invertebrates a specific substance from the egg jelly coat is required, which induces synchronous occurrence of the AR in the entire spermatozoa population. The observation that capacitation is not necessary if Ca2+ is driven by other means (Yanagimachi, 1975) indicates that the primary role of capacitation is to regulate the timing of Ca2+ influx. In invertebrates Decker et al. (1976) showed that ionophore can substitute for egg jelly in inducing A R . Bedford (1983) proposed a mechanism to equate the action of egg jelly with the end result of capacitation in the functional sense as shown here: Echinoid

Mammals

5. Egg jelly factor

+

5.

Sperm membrane change

Sperm membrane change

5. Ionophore

+

Capacitation factor(s)

5.

CaZ+influx

5.

Ca2+influx

Ionophore

5.

ACROSOME REACTION

A.

SITEAND FACTORS CAUSING ACROSOME REACTION

There is reasonable consensus that AR involves vesiculation of membranes and release of acrosomal hydrolases (Fig. 4B,C) required for penetration through the egg investments. Thus it would not be reasonable to

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consider any site in the female reproductive tract other than the immediate vicinity of the egg for induction of AR. The occurrence of AR in the uterine fluid of bovine species (Wooding, 1975; Sidhu el al., 1986) does not preclude the contribution from the oviductal fluids. Considering the ampulla of the oviduct as the site for fertilization, the uterus is thus too early a site for the occurrence of AR, which might lead to premature release from sperm acrosome of enzymes required at the site of fertilization. The identification of a nondialyzable, heat-labile factor from the follicular fluid inducing AR in hamster spermatozoa (Yanagimachi, 1970; Lui et al., 1977) supports the contention that the environment near the egg in the oviduct is the probable site for AR. Ovarian follicular fluid from several species has been shown to increase the number of sperm undergoing AR in vitrn (Meizel, 1985; Suarez et al., 1985, 1986; Sidhu, 1988; Siiteri et al., 1988a; Yudin et ul., 1988). Suarez et a / . (1986) showed that the AR-inducing ability of human follicular fluid resides in a fraction of M,45,000 and is stable to heating at 58°C. The component(s) in the follicular fluid inducing AR is(are) not derived from the blood serum, as serum alone failed to induce significant levels of AR. Siiteri et al. (1988a,b) further characterized the human follicular fluid inducing AR in human spermatozoa to have the following properties: ( I ) M , -50,000, (2) lack of extraction by treatment with chloroform-methanol, (3) resistance to protease, (4) partial stability to boiling, (5)a decrease in the apparent molecular weight of at least part of the activity following protease and chondroitinase ABC treatment, (6) resistance to various GAG hydrolases, (7) partial loss of activity upon dialysis, (8) a decrease in the size of at least part of the active component following treatment with the peptide N-glycosidase F, and (9) ability to initiate AR rapidly within a5 minutes. Cumulus fragments and human mural granulosa cells are the sources of the AR-initiating activity of human follicular fluid (Siiteri et al., 3988b). Although capacitation may be favored in the oviduct, its environment is not critical for the induction of AR. Studies employing retrieval of oviductal spermatozoa several hours after insemination could detect AR in only a small percentage of spermatozoa (Suarez et af., 1983; Yanagimachi and Phillips, 1984). However, others have shown visible signs of AR in the oviductal spermatozoa after several hours of incubation (Cummins, 1982; Cummins and Yanagimachi, 1982). Cumulus oophorus and zona pellucida are involved in the induction of AR (Gwatkin, 1977; Meizel. 1984, 1985; Lee and Storey, 1985; Tesarik, 1985; Cross et nl., 1988; Siiteri et ul., 1988a,b). Bedford (1983)quoted references supporting the idea that cumulus cell-free eggs are fertilized readily in the oviduct in the absence of follicular fluid, and in vitro when exposed to samples of capacitated spermatozoa without cumulus oophorus, corona radiata, or

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follicular fluids in the medium; this is supported by other findings refuting the role of cumulus in AR (Suarez et al., 1983; Talbot, 1984a; Cherr et al., 1984; Jacobs et al., 1984). However, others have shown the involvement of cumulus in AR and have also found that cumulus-intact rather than cumulus-free eggs are fertilized in v i m (Yanagimachi et al., 1983; Yanagimachi and Phillips, 1984). As already indicated, GAG present in cumulus stimulate AR in bovine spermatozoa (Lenz et al., 1982, 1983a,b; Handrow et al., 1984; Perrish et al., 1985; Ax and Lenz, 1985; Meizel and Turner, 1984, 1986; Cherr et al., 1986; Didion and Graves, 1986). Heparin and chondroitin sulfate among GAG were most effective in inducing AR (Thompson and Cummins, 1986). Delgado et al. (1976) showed that 3H-labeled heparin binds to specific receptors on sperm membrane and that binding is dependent on Ca”, pH, and temperature. Brown and Jones (1987) have identified a zona pellucida protein that binds a boar sperm polypeptide of MW 53,000. Similarly, murine sperm plasma membrane has been shown to have a binding site for zona pellucida (Poirier el al., 1986; Fournier-Delpech and Courot, 1987). Hydrolysis of chondroitin sulfate by sulfatase reduces its ability to stimulate the AR, indicating that AR-stimulating ability resides in the amino sugar in the sulfate residue (Handrow et al., 1982). But the question arises, how do GAG stimulate AR? Stimulation of conversion of proacrosin to acrosin by GAG has been shown (Parrish et al., 1979; Lenz et af., 1983b), but GAG cannot have access to proacrosin through the sperm membranes. Meizel (1985) hypothesized that GAG can stimulate sperm surface enzymes required for AR. Some observations suggest that interaction with the zona surface may specifically induce the AR in the fertilizing spermatozoon (Saling and Storey, 1980; Florman and Storey, 1982; Lee and Storey, 198% since acrosome-intact spermatozoa were observed attached to the zona pellucida (Storey et af., 1984; Myles et al., 1987). However, reacted hamster spermatozoa move freely within the cumulus oophorus (Cummins and Yanagimachi, 1982), and it is reported that guinea pig spermatozoa cannot even adhere to zona before they react (Huang et af., 1981). Also, AR occurs readily in spermatozoa around washed zona-free oocytes, leading to the incorporation of the reacted sperm by the oocyte (Toyoda and Chang, 1968). However, some observations have indicated the role of zona pellucida in AR. Monoclonal antibodies have been raised against murine and porcine zona pellucida antigens having sperm receptor activity (East et al., 1985; Yurewicz et al., 1987). Purified ZP, with M , -80,000, stimulates AR (Bleil and Wassarman, 1983; Wassarman et af., 1985). Similarly, heatsolubilized hamster zona can stimulate AR in hamster, human, and rabbit sperm (Cherr et al., 1984, 1986; O’Rand and Fisher, 1986; Cross et al.,

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1988). Florman et al. (1984) showed that pronase can destroy the ability of mouse ZP, to stimulate AR, indicating that the protein component of the glycoprotein is important in the AR whereas the oligosaccharide portion of ZP, might serve as the ligand to which sperm bind (Wassarman er al., 1985). The AR may be initiated in the cumulus oophorus matrix (Cummins and Yanagimachi, 1986), and it is completed while the spermatozoa bind ZP,. Binding of ZP, probably induces a fusogenic cascade involving guanine nucleotide-binding proteins (Endo et al., 1987). Wassarman (1987a,b) has shown that the AR-inducing ability of ZP, depends on both the polypeptide chain and oligosaccharides; neither component alone can induce the AR. Autoradiographic visualization of iodinated ZP, reveals that ZP, binds preferentially to acrosome-intact sperm, while iodinated ZP2 binds to acrosome-reacted sperm (Bleil and Wassarman, 1986). Therefore, the spermatozoa switch their glycoprotein binding specifically following the AR. Fraser (1982) suggested that mouse vitellus lacking zona can initiate the AR in vitro. However, acrosome-reacted spermatozoa have more tendency to attach to zona-free oocytes (Talbot and Chacon, 1982). The possibility that the substance released from cortical granules of the oocyte might provide stimulus for AR (Nicosia et a / . , 1977) is negated by the fact that the fertilizing spermatozoon penetrates the oocyte before exocytosis of the cortical granulosa has occurred. According to Bedford (1983), AR is an endogenous phenomenon and its onset is coordinated by complex events during capacitation.

B. EFFECTSOF EXOGENOUS FACTORS ON ACROSOME REACTIONIn Vitro In several in vitro studies various exogenous factors o r substances have been shown to influence the AR. However, an important question remains as to whether the exogenous factors o r substances influence the capacitation o r AR. Another important question is whether any of these effects observed in vitro can play a role in AR in vivo. Nevertheless, these in vitro studies are important in understanding the basic mechanism involved in AR. 1. Serum Albumin

Serum albumin is the most important component of the media used in all the in vitro studies on fertilization. Similarly, the most important-protein in the uterine fluid, oviductal fluid, follicular fluid, and serum responsible for inducing AR is the albumin fraction (see Meizel, 1978, 1985; Sidhu et al., 1986). Bavister (1981) reported that although motility in the epididymal hamster spermatozoa could be maintained in vitro in the presence of an artificial polymer, polyvinyl alcohol, capacitation and AR did

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not occur. When bovine serum albumin (BSA) was added, 3 hours elapsed before it had an effect on AR, indicating the importance of serum albumin in both capacitation and AR. The biochemical mechanism of albumin's effect on AR is much debated and needs to be understood more precisely. The major emphasis is whether the primary structure of albumin or its ligands are involved in AR, and if ligands are involved, then what is their chemical identity. Meizel's group has shown that the FA content of albumin affects its ability to induce AR. Removal of the FA from albumin greatly enhances its AR-inducing ability (Lui and Meizel, 1977; Lui et al., 1977). Fleming and Yanagimachi (1984) demonstrated the involvement of FA and FA moieties of sperm membrane phospholipids in the AR of guinea pig spermatozoa. Dilauroylphosphatidylcholine liposomes have also been used to induce the AR rapidly in bull, ram, and stallion spermatozoa with subsequent penetration of zona-free hamster eggs (Graham and Foote, 1984, 1985; Graham et af., 1986, 1987). Meizel and Turner (1983) showed that exogenous cis-unsaturated FA (oleic, arachidonic, and cis-vaccenic) stimulated the hamster -sperm AR, while trans-isomers did not have any affect in vitro. While arachidonic acid (AA) apparently initiates the AR through metabolites, oleic acid must initiate the AR through some other mechanism (Meizel and Turner, 1984). It is important to note that the cis-unsaturated FA content of follicular fluid is very high (Yao et al., 1980), and that most of these FA are probably bound to the considerable amount of serum albumin present in the follicular fluid. Cis-unsaturated FA cause perturbation in membranes leading to increased membrane fluidity (Karnovsky et al., 1982), which is important for AR (Fraser, 1982). Serum albumin might also bind FA released by the action of sperm phospholipase A, on phospholipids and thus prevent end-product inhibition of the enzyme. Lysophospholipids produced by phospholipase A, are important for AR (see Meizel, 1984, 1985). Serum albumin also chelates heavy-metal ions such as Zn" (Kragh-Hansen, 1981), and Zn2+ can inhibit hamster sperm capacitation and/or AR in vitro (Meizel, 1978). Jacobs et af. (1984) showed in a protein-free defined medium that the heavy-metal chelator penicillamine can replace albumin in stimulating fertilization. According to Meizel and Working (1980), several chelators including penicillamine stimulate capacitation and/or AR in hamster sperm in vitro. Also, Aonuma et al. (1982) showed that inhibition of mouse fertilization by Zn2+is relieved by serum albumin. Davis's group showed that high cholesterol levels in serum inhibit sperm capacitation and AR. Davis et af. (1979) incubated rat sperm in medium containing BSA and analyzed the cholesterol and phospholipid contents of both albumin and the sperm. Cholesterol content decreased in sperm while phospholipid content in-

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creased, and the reverse occurred in albumin. According to Davis, serum albumin removes sperm membrane cholesterol, thereby destabilizing the membranes leading to AR. It has been proposed that the serum proteins present in the fluid of the female reproductive tract sequester cholesterol from the sperm and/or donate additional phospholipid to the sperm (e.g., Section H,A,2). In either case, the net outcome is lowering the cholesteroUphospholipid ratio of the sperm during capacitation, facilitating membrane destabilization and leading to AR (Davis e f al., 1980; Davis, 1981). Llanos et al. (1982) showed that phospholipase A2 activity associated with commercial preparations of serum albumin does induce AR in hamster spermatozoa. Similarly, protease activity in serum albumin might influence the AR (Meizel, 1984). Serum albumin has a hydrophobic nature, and Lucy (1984) explained how the hydrophobic sequence cleaved from cellular polypeptide may induce membrane fusion. Garcia e f al. (1984) showed that fusion in unilamellar vesicles can be induced by a low molecular weight fragment of serum albumin. 2. Metal Ions There is a general consensus that Ca” is essential for the occurrence of AR in invertebrate and vertebrate mammalian spermatozoa. The development of a synchronous system for the induction of AR in guinea pig by Yanagimachi and Usui (1974), which involves incubating sperm in Ca2+free medium for 10 hours and then adding Ca” when 40430% of sperm show AR, gives support to the role of Ca” in AR. Similarly, several studies utilizing the calcium ionophore A 23 187 have demonstrated the role of Ca” in AR (see Yanagimachi, 1981; Hyne er al., 1984). Singh et al. (1980) suggested that although uptake of Ca2+ by sperm is required for the AR, a high concentration of Ca2+ exerts an adverse effect on the survival of spermatozoa that have undergone AR. They proposed that some additional mechanism may operate to protect the sperm by limiting the entrance of Ca” during capacitation in vivo. The likely candidate for such a control would be the Ca”-ATPase pump (e.g., Section III,C,3). It has been shown that the uptake of 45Caoccurs slightly before AR and that the Ca” uptake is a voltage-dependent and pH-sensitive mechanism (Babcock and Pfeiffer, 1987). S i + but not Mgz+o r Zn2+ can replace Ca2’ in inducing AR (Yanagimachi and Usui, 1974; Meizel and Lui, 1976; Mortimer, 1986; Mortimer ef al., 1986). Mortimer e f al. (1988) demonstrated that while Sr” ions can substitute fully for Ca” in the capacitation and AR of human spermatozoa, sperm-zona and sperm-oolemma seem to involve some more Ca’+-specific processes. Similar observations were made in mouse sperm by Fraser (1987). The mechanism by which Ca2+ induces AR is not clear. Yanagimachi and Usui (1974) have indicated that

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Ca2+ neutralizes negative charges on the outer surface of the outer acrosoma1 membrane and the inner surface of the overlying plasma membrane, allowing the membrane to come closer for fusion. Second, Ca" might bind to membrane acidic phospholipid-producing crystalline domains facilitating membrane fusion. Alternatively, Ca" might inhibit Mg2'-dependent ATPase activity involved in pumping water out of the acrosome. An influx of water into the acrosome would bring the outer acrosomal membrane and the plasma membrane into close approximation, facilitating fusion. Ca2' might be required for acrosin activity, and the latter is involved in membrane vesiculation or acrosome matrix loss. Acrosin might activate inactivated phospholipase A,, which induces membrane vesiculation. A high K'/Na' ratio in the medium increases fertilization frequency in rat spermatozoa (Toyoda and Chang, 1974). Roblero et al. (1988) have also shown that a high K' concentration improves the rate of AR in human spermatozoa. According to Mrsny and Meizel (1982), K' influx seems to be important for hamster sperm AR. However, Fraser (1983) showed that high levels of K' inhibit mouse sperm motility and decrease AR in vitro. Bize and Santander (1985) showed that epinephrine decreased the K' requirement of hamster sperm capacitation. Hyne et al. (1984) showed the Na' requirement for capacitation and membrane fusion during the guinea pig sperm AR. Zn2+can also inhibit golden hamster sperm capacitation and/or AR in vitro (Meizel, 1978).

3. Effect of Biogenic Amines, Detergents, and Other Reagents Capacitation and AR of hamster spermatozoa in vitro are stimulated by epinephrine, norepinephrine, and a-and P-adrenergic antagonists (Meizel et al., 1985). Serotonin or its agonist 5-methyltryptamine, but not other catecholamines, stimulated the hamster AR within 15 minutes (Meizel and Turner, 1983). Thus it appears that catecholamines d o not stimulate the AR directly but rather increase capacitation, the prerequisite for AR, while serotonin stimulates the latter step in capacitation and/or AR. Adrenergic stimulation in hamster sperm does lead to increased cytosolic Ca" (Meizel et al., 1985). Various detergents induce AR by perturbing the membrane structure and chemistry. Lysophosphatides, which can be produced in vivo in spermatozoa by the action of sperm phospholipase A, on phospholipids, induce AR in mammalian spermatozoa (On0 et al., 1982; Ohzu and Yanagimachi, 1982; Llanos and Meizel, 1983; Koichi et al., 1984; Kyono et al., 1984, 1985). Meizel and Turner (1983) showed that cis-unsaturated FA (oleic, arachidonic, and cis-vaccenic), the end products of sperm phospholipase A,, stimulate AR in hamster spermatozoa by possibly local in-

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crease in membrane disorder. In other cells, metabolism of AA via the cyclooxygenase pathway leads to the formation of prostaglandins, prostacyclins, and thromboxanes (Hall and Behrman. 1982), whereas metabolism involving the lipoxygenase pathways leads to production of hydroperoxy- and hydroxyeicosatetraenoic acids and leukotrienes. These products of the oxidative metabolism of AA appear to be important for exocytosis in somatic cells (Laychock and Putney, 1982). Phenidone or nordihydroguaiaretic acid, inhibitors of both the cyclooxygenase and lipoxygenase pathways of AA metabolism, and docosahexaenoic acid, a cyclooxygenase pathway inhibitor, inhibit the AR of hamster sperm induced by AA (Meizel and Turner, 1984). Prostaglandin E, (PGE,), a product of the cyclooxygenase pathway of AA metabolism and 5- or I2-hydroxyeicosatetraenoic acid, products of the lipoxygenase pathway, stimulate the AR when added to sperm capacitated by incubation for 4.5 hours. Prostaglandins not derived from AA, such as PGF, and PGA,, do not stimulate AR. It has been suggested that the AA metabolites produced by sperm and the female reproductive tract are important for mammalian sperm AR (Meizel and Turner, 1984; Meizel, 1985). Drummond and OldsClarke (19861, using specific inhibitors for phospholipase A, and cyclooxygenase, showed that in mouse sperm, capacitation involves phospholipase A, but not cyclooxygenase activity. Inhibition of hamster AR by transmethylation inhibitors indicates the involvement of increased transmethylation of proteins or phospholipids during exocytotic events (Meizel, 1981). Meizel and Turner (1984) also showed that PGE, and PGEI, the products of AA metabolism, stimulate AR in hamster spermatozoa possibly by inhibiting phosphodiesterase and elevating cAMP levels. The involvement of cAMP in AR of mammalian spermatozoa in in ritru studies was shown (Tash and Means, 1983). Sperm motility factor (SMF), a low molecular weight, heat-stable substance originally derived from hamster adrenal gland (Bavister el al., 1976) and also found in a variety of body tissues (Bavister and Yanagimachi, 1977; Bavister et al., 1978), stimulates capacitation and/or AR in hamster and buffalo bull spermatozoa (Bavister et al., 1976; Sidhu et al., 1984).

c. ROLE OF HYDROLYTIC ENZYMES The role of acrosomal hydrolases in fertilization processes has not yet been eludicated. Considerable interest has resurfaced in the study of the role of acrosomal hydrolases in AR. Various enzymic mechanisms for membrane vesiculation during AR have been proposed. However, no single mechanism can fully explain the mechanism of AR. Various recent

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observations made in this regard point to the conclusion that these hydrolases play a significant role in AR. 1. ProacrosinlAcrosin

Acrosin, a sperm serine proteinase, has been shown to be involved in fertilization by facilitating sperm penetration through egg investments particularly the zona pellucida (Morton, 1976; Urch et al., 1985a,b). In the mammalian spermatozoa studied so far, acrosin is always present as a zymogen called proacrosin. In boar spermatozoa, the activation of proacrosin under appropriate conditions to acrosin is inhibited by proacrosin activation-inhibitory factor, and the acrosin itself is also inhibited by low molecular weight sperm acrosin inhibitors (Parrish and Polakoski, 1979; Guraya, 1987). If acrosin is necessary for zona pellucida penetration or AR, then proacrosin must be activated under appropriate conditions. Activation of proacrosin to acrosin does not occur during capacitation and it does occur during AR (Goodpasture et al., 1981; Mack et al., 1983). The role of acrosin in AR is much debated. The involvement of trypsin in exocytosis in various somatic cells has been shown by the use of trypsin inhibitors and stimulators (Ahkong et al., 1978; Lui and Meizel, 1979; Shinohara et al., 1984; Van der Ven et al., 1985). There are two schools of thought in regard to the role of acrosin in AR. Meizel’s group has shown that acrosin is directly involved in membrane vesiculation during AR (Lui and Meizel, 1979; Dravland et al., 1984), whereas others disagree with the Meizel group and instead propose that it is the subsequent loss of acrosomal contents that is dependent on acrosin (Green, 1978, 1982; Shams-Borhan and Harrison, 1981; Perreault et al., 1982; Fraser, 1982; Thomas et al., 1986). If we assume that membrane vesiculation and loss of acrosomal matrix are the events of AR, then how is acrosin bringing about these events during AR? Meizel(l984) proposed four mechanisms of acrosin’s action in inducing AR:

1 . Acrosin might activate the putative zymogen form of phospolipase A, and the phospholipase A, is involved in AR (Llanos et al., 1982).. Acrosin and kallikrein are involved in zymogen activation of phospholipase A, in human spermatozoa (Guerette et al., 1986, 1988). 2. Acrosin might hydrolyze sperm membrane proteins, thereby promoting membrane fusion. 3. Acrosin might stimulate sperm adenylate cyclase as in other cells (Anderson and Jaworski, 1981), thus increasing intracellular CAMP,which may be important for capacitation and AR (see Tash and Means, 1983). 4. Acrosin might also modify the sperm surface, thus increasing Ca’+ influx leading to membrane vesiculation (Sidhu and Guraya, 1989a).

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Sperm proacrosin and acrosin have also been implicated in zona binding (Thomas et al., 1986; Benau and Storey, 1987; Robinson et al., 1987; Jones and Brown, 1987). Previously doubts were raised about the role of sperm acrosin as the zona penetrant (Bedford and Cross, 1978; Saling, 1981). However, a limited but specific proteolysis of the boar zona pellucida is carried out by the acrosin from boar spermatozoa (Urch et al., 1985a,b; Urch, 1986). It has also been demonstrated that the acrosin that binds zona pellucida becomes membrane-bound in the zona-binding regions during the AR. This would facilitate the binding-and-release cycle postulated as the mechanisms for sperm penetration of zona (O’Rand et id., 1986; Esaguy et al., 1988). Other enzymes like neuraminidase have also been implicated in sperm capacitation and AR (Srivastava and Farooqui, 1980; Srivastava et al., 1988). 2 . Phospholipase A , Activity Phospholipase A, activity appears during AR in sea urchin spermatozoa (Conway and Metz, 1976; SeGall and Lennarz, 1981). Involvement of phospholipase A2 during exocytotic events in somatic cells has been shown (Papahadjopoulos, 1978). In mammalian spermatozoa the involvement of phospholipase A2 during AR has been shown by several observalions: 1. Phospholipase A, activity is present in spermatozoa, and it increases several fold during AR in man, ram, rat, and hamster (Soupart et al.. 1979; Wang et al., 1981; Llanos et al., 1982; Langlais et al., 1982; Ono et al., 1982; Takkar et al., 1983, 1984; Lindahl et al., 1987; Hinkovska et al., 1987). 2. Inhibitors of phospolipase A, such as p-bromophenacyl bromide, mepacrine. and Upjohn compound 1002 inhibit AR but not fertilization in hamster spermatozoa (Yanagimachi, 1981 ; Dravland and Meizel, 1982; Llanos et al., 1982). 3. Exogenous phospholipase A, stimulates AR in hamster and guinea pig spermatozoa (Singleton and Killian, 1981, 1983; Llanos et al., 1982). 4. The products of phospholipase A2 action on phospholipids (i.e., lysophospholipids) stimulate AR in human, guinea pig, rabbit, and bull spermatozoa (Fleming and Yanagimachi, 1981, 1984; Ohzu and Yanagimachi, 1982: Llanos and Meizel, 1983; Kyono et al., 1984, 1985; Yanagimachi and Suzuki, 1985; Byrd and Wolf, 1986; Wheeler and Seidel, 1988; Chen et al., 1988). Antaki et al. (1988), using a radiation inactivation technique, determined that the molecular weight of phospholipase A, was 12,000 when radioactive phosphatidylcholine was used as substrate and 8000 when

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radioactive phosphatidylethanolamine was used as substrate. Based on thermosensitivity, two forms of phospholipase A,, one temperatureresistant and the other temperature-sensitive (60°C, 10 minutes), have been demonstrated in human seminal plasma and spermatozoa. The lysophosphatides produced by the action of phospholipase A, on phospholipids are the important fusogens inducing membrane vesiculation. If sperm phospholipase A, is involved in the AR, the enzyme must be released by that event, and the localization of the enzyme is also very important. Allison and Hartree (1970) showed phospholipase A, activity associated with ram sperm acrosomal preparations. Fleming and Yanagimachi (1981) suggested the localization of phospholipase A, on the outer acrosomal and plasma membrane of spermatozoa. Phospholipase A, activity and phospholipid composition of isolated sperm head membrane have been measured in guinea pig and ram (Dunlap and Killian, 1986; Hinkovska et d . , 1986, 1987). Llanos et al. (1982) demonstrated that phospholipase A, activity is released during AR in hamster spermatozoa. Conway and Metz (1976) also observed the release of phospholipase A, activity during AR of sea urchin spermatozoa. They also suggested another role of phospholipase A, in sperm-egg fusion. However, Yanagimachi (1981) found that nontoxic levels of mepacrine did not inhibit sperm-egg fusion in vitro in the hamster. Phospholipase A, activity has also been demonstrated in human seminal plasma (Kunze et al., 1974; Wurl and Kunze, 1985). Phospholipid methylation involved in the formation of phosphatidylcholine, the substrate for phospholipase A,, might be involved, since the inhibitor of phospholipid methylation inhibits AR in hamster spermatozoa (Meizel, 1981; Llanos and Meizel, 1983). Sidhu et al. (1989) have shown the increased incorporation of [l - I4C]acetate and [U - ''C]glucose into the phospholipids during capacitation of buffalo bull spermatozoa. Evans et al. (1980) also showed the increase of phosphatidylcholine during uterine capacitation of porcine spermatozoa. The accumulation of phospholipids in the postnuclear cap of ejaculated buffalo bull spermatozoa (see Sidhu and Guraya, 1985) might be of some relevance here. A relationship exists between phospholipid methylation and somatic cell membrane fluidity (Hirata and Axelrod, 1980), and the increased membrane fluidity is important for AR (Friend, 1982). Meizel and Turner (1983) showed that several cis-unsaturated FA, including oleic and arachidonic acids, stimulate hamster sperm AR. These cis-unsaturated FA may perturb membrane fluidity, which is important for AR (Karnovsky et al., 1982). The question remains whether the release of FA by the action of phospholipase A, include cis-unsaturated FA. Lysophosphatidylcholine produced by hydrolysis of phosphatidylcholine by phospholipase A, is more soluble in water and is considered to be involved in phase transition in mem-

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branes resulting in cell fusion (Lucy, 1984). Lysophosphatidylcholine, an important fusigenic agent, once formed, will thus cause lysis of the membranes. Although the involvement of lysophosphatidylcholine in AR has been shown, the major question remains of the regulation of lysophosphatidylcholine levels. Its continuous formation catalyzed by phospholipase A? may damage sperm and egg membranes nonspecifically during fertilization. Sperm lysophospholipase has been speculated to play a role in this regard in hamster (Meizel, 1984). Alternatively, Langlais and Roberts ( 1985) have suggested that since lysophosphatidylcholine and cholesterol combine stoichiometrically to form a stable complex (Rand et ul., 1975), their interaction could reduce both the lytic action of lysophosphatidylcholine on the membrane and its induction of cell fusion. Thus membrane cholesterol levels affect the fusigenic properties of lysophosphatidylcholine; fusion in membranes will occur with decreased sterol content. Interestingly, one of the mechanisms proposed for sperm capacitation is a decrease in the sperm cholesterol/phospholipid ratio that may consequently play a regulatory role in the fusigenic effect of lysophosphatidylcholine during AR. Srivastava et al. (1982) proposed that phospholipase C is involved in the AR. Phospholipase C eventually produces phosphatidic acid, which is a calcium ionophore and thus facilitates the influx of Ca2' required for AR. Phospholipase C has the advantage that it does not form more damaging lysophosphatides as does phospholipase A,. Ribbes et al. ( 1987) have isolated a phospholipase that is predominantly located in the human sperm head. The enzyme is activated by calcium and inhibited by EGTA with optimum pH 6.0. Bennet et al. (1987) demonstrated that in human spermatozoa the Ca" entry activates both a phospholipase A, and a phospholipase C, leading to the formation of lysophospholipid, diacylglycerol, or phosphatidic acid, which might be involved in AR. Serum albumin is the most important ingredient of the media used for capacitation AR, and fertilization in v i m . It is also the major component of follicular and oviductal fluids. Saturation of the albumin with FA or cholesterol decreases its ability to induce AR. Meizel (1984) suggested that albumin may remove from the sperm membrane FA or lysophosphatides produced by phospholipase A2, and thus may prevent end-product inhibition of the enzyme. Davis ef al. (1979) showed that albumin removes cholesterol from the sperm membrane and thus destabilizes sperm, leading to their vesiculation. The sperm phospholipase A2 has been considered to be present in the form of the inactive precursor prophospholipase A? and is activated during AR (Langlais and Roberts, 1985). Guerette er ul. (1988) have demonstrated the presence of a prophospholipase A2 in human spermatozoa that could be activated by exogenous proteases. Among the proteases tested (trypsin, kallikrein, and

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plasmin), trypsin is the most potent activator of prophospholipase A, in intact spermatozoa. Although the endogenous protease (i.e., acrosin formed from proacrosin) could activate prophospholipase A,, this activation is more efficiently achieved by exogenous proteases. The phospholipase A, present in the endometrium exhibits a peak of activity at 2-4 days postovulation (Bonney, 1985) that could accelerate the onset of the AR. Similarly, the presence of plasmin and kallikrein in human follicular fluid (Gordon et al., 1986; Guerette et al., 1988) would facilitate the occurrence of AR by activating prophospholipase A, to phospholipase A, in spermatozoa. 3 . ATPases

Several ATPases are present in mammalian spermatozoa (see Guraya, 1987). The relative significance of these ATPases in the AR of mammalian spermatozoa was reviewed by Meizel (1984). Gordon et al. (1978) showed the presence of a Ca2'-dependent ATPase

on the outer acrosomal membrane of mammalian spermatozoa that pumps Ca2+ into the acrosome, thus stimulating AR. However, an inward-directed Ca2+pump is present only in sarcoplasmic reticulum and not in any other somatic cells (Stekhoven and Bonting (1981). Ashraf et al. (1982, 1984) and Breitbart et al. (1984) demonstrated the presence of an outward-directed Ca2+pump Ca2'-ATPase in the head and flagellar membranes of boar spermatozoa. These studies suggest that inhibition of Ca2'-ATPase should facilitate Ca" influx. In fact, Santos-Sacchi and Gordon (1982) showed that depletion of ATP inhibits Ca2'-ATPase in guinea pig spermatozoa, which facilitates the Caz+influx required for AR. The regulation of calcium transport in spermatozoa is poorly understood. The kinetics of Ca2+transport in spermatozoa changes during their maturation in the epididymis; the ejaculated spermatozoa are relatively impermeable to Ca" (Babcock et al., 1979; Rufo et al., 1984), and the permeability to CaZc increases during their sojourn in the female reproductive tract (Meizel, 1985). Various components are believed to be involved in calcium transport: Ca2+,Mg2+-ATPase,Na+,K'-ATPase, Na'/ Ca" antiporter, and calmodulin have been demonstrated in spermatozoa (Bradley and Forrester, 1980; Ashraf et al., 1982; Mrsny et al., 1984; Nelson, 1985; Weinman et al., 1986), but very divergent views exist about their localization. Sidhu and Guraya (1989a,b) have demonstrated the presence of a calmodulinlike protein in buffalo seminal plasma that stimulates Ca2+,Mg2'-ATPase in erythrocyte ghosts and also in buffalo bull spermatozoa. Caz+,Mg2+-ATPaseis predominantly localized in the purified plasma membrane from tail fraction. Interestingly, calmodulin, which regulates events associated with Ca2+ transport, is localized predomi-

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nantly in the sperm head in bull (Jones er al., 1980). The calmodulinlike protein in buffalo seminal plasma did stimulate Ca",Mg"-ATPase of plasma membrane from sperm head and tail. In boar sperm plasma membranes, Ca" binds with high affinity, especially in the presence of sodium and potassium, and the CaZ+uptake is regulated by calmodulin (Peterson er al., 1983). We propose that in buffalo bull spermatozoa the calmodulinlike protein in seminal plasma binds to the spermatozoa during ejaculation and stimulates the Ca" extrusion phenomenon by activating the Ca2' pump. During capacitation some sperm surface proteins are dislodged (Sidhu ef a / . , 1984, 1986), and likewise, the calmodulinlike protein is also probably removed, thus reducing the activity of the Ca" pump and leading to the accumulation of Ca" in capacitating spermatozoa. This elevated intracellular Ca" is required for hyperactive motility and also, consequently, for AR. Ouabain-sensitive Na' ,K'-ATPase is present in rat, rabbit, and boar spermatozoa (Gordon, 1973; Ashraf er al., 1982; Mrsny and Meizel, 1982). Inhibition of capacitation and AR of hamster spermatozoa by ouabain indicates the role of Na+,K'-ATPase in these events (Meizel, 1984). Mrsny er al. (1984) showed that N a + , K -ATPase activity increases during capacitation and/or AR of hamster spermatozoa, and that the increase in Na'-ATPase activity occurring during capacitation is probably mediated by intracellular cGMP but not CAMP, although both cyclic nucleotides stimulate the hamster sperm AR. It is proposed that K + influx is important during capacitation and AR in hamster spermatozoa (Mrsny and Meizel, 1982). However, in rat spermatozoa higher levels of K' ions decrease AR in mouse and guinea pig spermatozoa (Rogers er al., 1981; Fraser, 1983). In sea urchin spermatozoa K' efflux and Na' influx are important for AR (Schackman and Shapiro, 1981). How Na+,K'-ATPase is involved in AR is not clear. According to Meizel, K + influx may be coupled to H' efflux, resulting in intraacrosomal increase in pH, which brings about activation of proacrosin required for AR. Influx or efflux of either Na+ o r K' may also change the membrane potential and thus facilitate AR (Section III,B,2). Mrsny and Meizel (1985) showed that the sulfur-containing @-amino acids taurine and hypotaurine can inhibit Na+,K ' -ATPase in hamster spermatozoa and thus play a role in regulating sperm motility and fertility. Inhibition of Na' ,K'-ATPase by these amino acids stimulates N a + in spermatozoa, which can be exchanged with extracellular Ca" as Ca*+/Na+antiport, has been demonstrated in sperm (Bradley and Forrester, 1980), and Ca" is necessary for sperm motility and AR (Nelson, 1985; Sidhu, 1988). Mg" -dependent proton pump (Mg"-ATPase) is present in exocytosing somatic cells responsible for the acidic internal pH of these cells. +

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Working and Meizel(l983) showed the existence of a similar proton pump (Mg2+-ATPase)in hamster sperm acrosome that is inhibited by low levels of Ca2+.Inhibitors of sperm Mg2+-ATPase,4,4’-dicyclohexyl-carbodiimide or 4-chloro-7-nitrobenzofurazan(DCCD or NBD-CL) or proton ionophores such as carbonyl cyanide p-trifiuorometho-xyphenyl hydrazone (FCCP) stimulate AR in capacitated hamster spermatozoa (Working and Meizel, 1983), possibly by increasing the acrosomal pH. Meizel showed that the intraacrosome pH of hamster spermatozoa is -5, and it increases during the late stage of capacitation. Increase in acrosomal pH may stimulate activation of proacrosin to acrosin, which might be involved in membrane vesiculation. Phospholipase A,, which is involved in AR, is also active at alkaline pH. Increased acrosomal pH may also stimulate Ca2+ influx (Working and Meizel, 1983). Usui and Yanagimachi (1986) described the cytochemical localization of Mg2+-dependentATPase in guinea pig sperm head before and during AR. In uncapacitated spermatozoa Mg2+-dependentATPase is localized on both the inner surface of the plasma membrane and the outer surface of the outer acrosomal membrane. The activity is MgZ+-dependentand inhibited by both Ca2+ and SH-blocking agents. The activity disappears in acrosome-reacting spermatozoa, probably because of its inhibition by influx of Ca”. It is proposed that this enzyme may play a role in pumping water out of the spermatozoa, and that its inactivation by Ca2+would allow an influx of water, resulting in a swelling of the acrosomal cap and close approximation of the outer acrosomal membrane with the overlying plasma membrane and thus leading to their vesiculation. IV. Conclusions and Prospects

The probable relationships among events occurring during capacitation and AR in mammalian spermatozoa are depicted in Fig. 6. The concepts described in this figure are based on the available data and might need to be revised as further advances reveal the complexities involved in capacitation and AR. Spermatozoa of all eutherian mammals must undergo capacitation before they undergo AR. But the phenomenon of capacitation is still far from clear. Apparently capacitation does not involve any discernible morphological changes and the changes observed during capacitation may not be correlated with actual acquisition of fertilizing ability by spermatozoa. Exogenous and endogenous stimuli (Fig. 6) are thought to bring about surface alterations and intramembrane molecular alterations in spermatozoa during capacitation that are manifested in altered metabolism and mo-

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FIG. 6. Model showing various changes observed in the acrosome during capacitation and AR in mammalian spermatozoa. Several exogenous and endogenous stimuli bring about membrane alterations (both on the surface and intramembranal) during capacitation and probably facilitate Ca” influx. The increased intraacrosomal Ca” might bring about ( I ) membrane vesiculation of opposing membranes (outer acrosomal and plasma membranes); (2) inhibition of Mg”-ATPase thus conserving intraacrosomal HZOrequired for acrosomal swelling; (3) binding to acidic phospholipids (PL), forming crystalline domains in the membrane at the sites of fusion: (4) activating a putative zymogen protease (I) (inhibited) to protease (A) (activated) required for activation of proacrosin to acrosin; ( S ) stabilizing acrosin activity, which might be involved directly for membrane vesiculation or might activate prophospholipase A2 (ProPLipase A) producing fusogen lysophosphatides (LysoPL) plus fatty acids (FA). The role of serum albumin might be to remove these FA thus preventing end-product inhibition of PLipase A, or it might be involved in chelating cholesterol, thus lowering the cholesterol (CH0L)IPL rat0 (i.e., increasing the PLKHOL ratio), leading to membrane destabilization. The increased intraacrosomal pH possibly brought about by the proton pump is necessary for activation of proacrosin to acrosin and also for PLipase activity. PM, Plasma membrane; OAM. outer acrosomal membrane; IAM, inner acrosomal membrane; NM. nuclear membrane; ES, equatorial segment.

tility. Some of these molecular events have been defined using extrinsic and intrinsic membrane probes. Remodeling of sperm surface architecture and intramembrane molecular alterations occur during capacitation in mammalian spermatozoa. The predominant change in spermatozoa involves surface alterations in glycoproteins and glycolipids. The significance of other changes observed during capacitation is not clear. Recent

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studies of intramembranous particles in spermatozoa during capacitation and AR observed two salient features: (1) well-organized strands of small particles enveloping the middle piece disorganized during capacitation (Fig. 2), and (2) deletion of fibrillar intramembranous particles from the E-fracture faces of both acrosomal and plasma membrane and the clearing of globular particles from the P faces of the plasma membrane (Fig. 3). Thus a protein-poor, high-fluidity area ready for membrane fusion coexists with a protein-rich area of decreased fluidity in spermatozoa immediately before AR. Recent evidence demonstrates lipid exchange between albumin and sperm cells; consequently a decrease in the cholesteroVphospholipid ratio occurs during capacitation, leading to vesiculation of membranes during AR. The areas of plasmalemma specialized for fusion during AR have a higher affinity for anionic lipid probes than do adjacent nonfusigenic regions. Similarly, crystalline domains of anionic phospholipids are formed by binding with Ca” drawn in immediately before AR. The consequence of molecular changes observed during capacitation (Section I,A,1-3) is the increased influx of Ca2+ and increase in sperm metabolism. Although changes in sperm motility, respiration, and substrate utilization accompanying capacitation have been observed, it is not clear whether these changes bring about capacitation or simply accompany capacitation. The mechanism for capacitation in vivo is not clear. The lack of data on in vivo studies is probably due to difficulties involved in retrieving a sufficient number of spermatozoa from the female reproductive tract. Culturing of female tract in various media with optimal numbers of spermatozoa could be a logical method for overcoming this difficulty. But the major question is on the site within the female tract where capacitation occurs. The specific composition of the milieu required for capacitation in the female tract is a matter of speculation. Analysis of factors causing capacitation has great future prospects in understanding this phenomenon at the molecular level and exploiting it for contraception. The AR seems to perform two important functions during fertilization: (1) the release of hydrolytic enzymes, which facilitate sperm penetration through egg investment, and (2) modifications of the membranes in the postacrosornal region, where possibly interaction between egg and sperm takes place. The AR in mammalian spermatozoa does not require any specific stimulus, but it does require an adequate level of Ca2+once capacitation has been achieved. Thus the primary role of capacitation is to schedule the influx of Ca2+.Although our knowledge about AR is more precise than it is about capacitation, most of our inferences are drawn from in vitro studies. Controversies exist about the site and the molecules in female reproductive tract responsible for AR. It is unreasonable to think of any

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other site than the vicinity of egg for induction of AR. Considering AR as a rapid phenomenon, the oviduct would be a too early a site for this process. Similarly, a role for cumulus oophorus, corona radiata, or follicular fluid in AR has been refuted by several studies. A requirement for GAG in AR has been demonstrated, but the question remains how these molecules bring about AR. We feel that the milieu of the female reproductive tract is important, and each component of the female tract seems to have priming effects on sperm till the final vesiculation is achieved. Serum albumin is important for in v i m and perhaps also for in uiuo AR. Controversies remain about whether the primary structure of albumin o r its ligants are involved in AR. Meizel’s group has proposed that FA contents, particularly the unesterified cis-unsaturated FA of albumin, are important for AR. These unsaturated FA cause local perturbation in membranes, leading to disorder and increased membrane fluidity important for AR. Davis’s group has proposed that albumin removes cholesterol from sperm membranes during capacitation, thus lowering the cholesterol/phospholipid ratio and facilitating membrane destabilization leading to AR. The other roles proposed for albumins such as chelating heavy metals (e.g., Zn”), which inhibits capacitation and AR, and binding of FA released by phospholipase A, to prevent end-product inhibition of this enzyme, are interesting and need to be analyzed more intensively to arrive at a definite conclusion. Recently a role for hydrolytic enzymes such as acrosin (protease) and phospholipase A, has been proposed in membrane vesiculation events during AR (Section III,C,I-3). But the controversies remain as to how these enzymes bring about membrane vesiculation. Two schools of thought mentioned earlier differ in regard to the role of acrosin. According to Meizel’s group, acrosin is directly involved in membrane vesiculation, while others consider that acrosin is responsible in the subsequent loss of acrosomal matrix. Presumably both vesiculation and the loss of acrosomal matrix are stages of AR. The pertinent question here is whether the protease involved in these stages of AR is the acrosin or some other serine protease not yet characterized. If the protease involved is acrosin, as hypothesized by several workers, then how does the acrosin in the acrosome accomplish both membrane vesiculation and zona penetration? Whether there are two populations of acrosin with specific distributions, each with a specific function, needs to be explored in future studies. Of the several roles of acrosin hypothesized by Meizel (1984), the involvement of acrosin in activation of the zymogen form of phospholipase A, is interesting and has been supported by Guerette rt al. (1986). Phospholipase A? is emerging as an important candidate for AR in mammalian spermatozoa (Section III,C,2). The fusogen lysophospha-

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tides, the product of phospholipase A, activity, could play a role in the AR. The precise mechanism of its activation has not been worked out although protease enzyme is implicated in activation. The endogenous substrate for phospholipase A, appears to be phosphatidylcholine. The ready availability of this substrate during AR either by phospholipid methylation or by neosynthesis is a matter of speculation and needs to be studied in more detail. Caz+influx is considered to be essential for exocytosis during AR and there has been a great interest in understanding Ca2+-gatingmechanisms, but how Ca” assists in vesiculation is not clear. Various hypotheses have been proposed for the role of Ca”. Caz+-dependentATPase of sperm membranes is implicated in regulating Ca2+levels in the acrosome. But controversies exist whether the CaZ+-ATPasepump is involved in influx or efflux of Ca”. Purified acrosomal and plasma membrane preparations need to be employed to study the Ca2’-gating mechanism in spermatozoa. The involvement of ATPase in regulating intraacrosomal levels of Na’ and/or K’ and intraacrosomal pH needs to be studied. It can now be envisaged that phenomena of sperm capacitation and AR are endogenous molecular events at the membrane level that can be modulated by the external environment. The application of various molecular probes and other biotechnologies will resolve controversies involved in understanding the mechanism of capacitation and AR at the subcellular and molecular levels.

ACKNOWLEDGMENTS The work from our laboratory discussed in the text was supported by the Indian Council of Medical Research. The authors thank Ms. Indejit Kaur for typing the manuscript.

REFERENCES Ahkong, Q. F., Blow, A. M. J., Botham, G. M., Launder, J. M., Qurik, S. J., and Lucy, J. A. (1978). FEBS Lett. 95, 147-152. Ahuja, K. K. (1982). Exp. Cell Res. 140, 353-362. Ahuja, K. K. (1984). Dev. Biol. 104, 131-142. Ahuja, K. K. (1985). Am. J . Anat. 174, 207-223. Allison, A. C., and Hartree, E. F. (1970). J. Reprod. Fertil. 21, 501-515. Anderson, W.B., and Jaworski, C. J. (1981). Arch. Biochem. Biophys. 207, 465468. Andre, J. (1983). “The Sperm Cell: Fertilizing Power, Surface Properties, Motility, Nucleus

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Index

Amino sugars, bacterial cell surface and. 34, 35, 41 Amphibians, centrifugal visual system of vertebrates and, 160 anatomy, 127-129 function, 129, 130 Antibiotics bacterial cell division site and, 15 bacterial cell surface and, 34, 83 amphiphiles, 57 cell wall turnover, 79 peptidoglycan. 46 surface structures, 35. 38, 42 Antibody bacterial cell surface and amphiphiles. 53, 56, 57 cell wall turnover, 79 peptidoglycan, 45, 46 surface adhesions, 64. 69, 73 surface structures, 37, 38, 42 Kupffer cells and, 177, 210, 214 marine algae and, I13 spermatozoa and, 235, 238 Antigen bacterial cell surface and, 33 amphiphiles, 50-52 peptidoglycan, 45, 46 surface structures, 36, 38 Kupffer cells and, 198. 201, 210, 211 spermatozoa and, 233, 234, 248, 253 Aplanospores, marine algae and. 103, 105I07 Arachidonate, Kupffer cells and, 193, 195199, 204 Arachidonic acid Kupffer cells and, 192, 196, 205 spermatozoa and, 255, 258, 261

A

Acetabdur.iu, 94-102, 107, 113 N-Acetylmuramyl dipeptide. bacterial cell surface and, 46,47 Acrosin, spermatozoa and, 257, 259-261, 265, 268 Acrosome reaction in spermatozoa, see Spermatozoa Acrosome-stabilizing factor, spermatozoa and, 233, 234 Actin, spermatozoa and, 237 Actinomycin D, marine algae and, LOO Adhesion bacterial cell division site and, 6-9 bacterial cell surface and, 58-74, 83, 84 amphiphiles, 55 surface structures, 42 Adsorption, Kupffer cells and, 19920 I AIDS, Kupffer cells and, 21 I Albumin Kupffer cells and, 190, 218 spermatozoa and. 267, 268 acrosome reaction, 254-256, 262 capacitation, 240-242, 247, 248 Algae, marine, see Marine algae Amacrine cells, centrifugal visual system of vertebrates and, 160, 163 birds, 145-147, 149, 150 mammals, 151, 160 Amino acids bacterial cell division site and, 18 bacterial cell surface and, 34, 41 centrifugal visual system of vertebrates and, 118, 143 spermatozoa and, 249, 264

28 1

282

INDEX

Arthritis. bacterial cell surface and, 4 3 4 7 . 84

ArP bacterial cell surface and. 34 spermatozoa and. 244. 263 ATPase marine algae and. I06 spermatozoa and, 236. 256. 257, 263-265. 269 Autolysins. bacterial cell surface and, 83 amphiphiles. 57 cell wall turnover. 74. 76. 79 peptidoglycan. 45 surface structures. 35. 36

n B cells, bacterial cell surface and. 53. 54. 56 Bacdlrts crnihrrrci~.bacterial cell surface and. 83 cell wall turnover. 76. 77 surface structures, 37. 38 Bacillus Calmette-Guerin. Kupffer cells and, 207-209. 217 Buri/lrr.s srrhtiiis. bacterial cell surface and amphiphiles. 57. 58 cell wall turnover, 74 peptidoglycan. 47 surface structures, 38 Bacterial cell division site, see Differentiation of bacterial cell division site Bacterial cell surface. 33. 34. 83, 84 amphiphiles. 49-58 cell wall turnover, 74-83 peptidoglycan. 4 3 4 9 surface adhesions. 58-74 surface structures. 34-43 Bayer bridges. bacterial cell division site and. 6, 7 Birds. centrifugal visual system of vertebrates and. 115. 116. 151 anatomy. 133-149 function. 149, 150 Hoergesenia. 94. 102-107. I13 Hone marrow. Kupffer cells and, 220 endotoxin. 200 population dynamics. 183-186 Bovine serum albumin Kupffer cells and, 218 spermatozoa and. 255

C

Calcium Kupffer cells and, 188. 191-193, 195. 196 spermatozoa and. 232, 267, 269 acrosome reaction. 251. 253, 256, 257. 262-265 capacitation. 234. 236, 239. 243. 245, 246. 248, 249 Calmidazolium. Kupffer cells and, 193 Calmodulin Kupffer cells and, 193, 195. 201 spermatozoa and, 237, 246, 263, 264 Capacitation, spermatozoa and. 231-233. 265-267 chemicals. 248. 249 factors. 247. 248 molecular alteration. 238-243 physiology. 243-247 surface alteration, 233-238 Carbohydrate. bacterial cell surface and amphiphiles. 50-52 peptidoglycan. 45 surface adhesions. 60.61 Carbon. marine algae and. 110 Catecholamines, spermatozoa and. 248. 257 Cathespin D. Kupffer cells and. 203 Caudal mesencephalic tegmentum. centrifugal visual system of vertebrates and. 131 Cell envelope. bacterial cell division site and. 1-4. 13 chromosome replication. 24, 25 division apparatus. 5 septum formation. 14 Cell wall turnover, bacterial cell surface and. 74-83 amphiphiles, 57 surface structures. 42 Cellulose. marine algae and, 105. 106 Centrifugal visual system, 115, 116. 160, 162 amphibians anatomy. 127-129 function. 129. 130 birds anatomy. 133-149 function. 149. 150 cyclostomes. 116-1 I9 fish anatomy. 120-126 function. 126, 127

INDEX mammals anatomy, 151-158 function, 158-160 reptiles anatomy, 130-133 function, 133 retinopetal system, 162-164 Cerebrospinal fluid, bacterial cell surface and, 47 Chemotaxis, bacterial cell division site and, 3 Cklumydomonos, marine algae and. 95 Ciilorella, marine algae and, 93 Chloroplasts bacterial cell division site and, 6 marine algae and, 94, 99, 102 Chloroquine, Kupffer cells and, 202 Cholesterol, spermatozoa and, 267, 268 acrosome reaction, 251, 255, 256. 262 capacitation. 234, 239, 242, 248 Cliondrus, marine algae and, 1I I Chromosome replication, bacterial cell division site and, I , 4, 18, 19, 27 coordination, 21, 22 DNA synthesis, 19-21 Escherichia coli. 16-18 mutation, 22, 23 nucleoids, 23-27 Chromosome segregation, bacterial cell division site and, I, 4, 13, 18, 19 coordination, 21, 22 DNA synthesis, 19-21 mutation. 22, 23 nucleoids, 23-27 Chromosomes bacterial cell division site and, 13, 14 Kupffer cells and, 184, 216 Cirrhosis, Kupffer cells and, 218, 219 Clones, bacterial cell surface and, 73 Coagulase, bacterial cell surface and, 72 Collagen, bacterial cell surface and. 73 Collagenase, Kupffer cells and, 187-191. 204 Concanavalin A, bacterial cell surface and, 54 Contractile ring, bacterial cell division site and, 2 Cyclic AMP bacterial cell division site and, 22 Kupffer cells and, 204 spermatozoa and, 246, 249, 258, 259

283

C ycloheximide Kupffer cells and, 204 marine algae and. 100 C ycloox ygenase Kupffer cells and, 193, 197 spermatozoa and, 258 Cyclostomes, centrifugal visual system of vertebrates and, 116-1 19 Cytochalasin D. spermatozoa and, 237 Cytoplasm bacterial cell division site and, 2, 5, 8 bacterial cell surface and. 34, 35 marine algae and, 94, I13 Acetabuluriu. 95, 96, 99, 100 Boorgeseniu, 103, 105-107 Cytoskeleton bacterial cell division site and, 2 marine algae and, 100, 105

D Dendrites, centrifugal visual system of vertebrates and birds, 138-142 cyclostomes, 118 DiacyIglycerol, spermatozoa and, 243 Diaminopimelic acid, bacterial cell surface and, 34. 47-49 Differentiation, marine algae and, 109-1 1 I. 113 Differentiation of bacterial cell division site, I, 2. 27, 28 bacterial cell envelope, 2-4 biogenesis, 11-13 chromosome replication, 18, 19 coordination, 21, 22 DNA synthesis, 19-21 mutations, 22. 23 nucleoid position, 23-27 chromosome segregation, 4 Eseherichicr cofi. 16- I8 localization, 10. I1 membrane-peptidoglycan attachment, 9, 10 molecular organization, 7-9 periseptal annular apparatus, 4 7 residual sites, 13, 14 septum formation, 14-16 Displacement, bacterial cell division site and, 11-13

INDEX Disseminated intravascular coagulation, bacterial cell surface and. 53. 54 Division potential. bacterial cell division site and. 14 DNA. bacterial cell division site and, I . 13, 14 chromosome replication, 18-26 chromosome segregation. 4 Eschcvicliirr cmli. I 6, 18 DNA gyrase. bacterial cell division site and. 23, 25 Dopamine. centrifugal visual system of vertebrates and. I26 Dorsal raphe nucleus. centrifugal visual system of vertebrates and. 157 L)rosopMci. marine algae and. 93

E Ectopic centrifugal neurons, centrifugal visual system of vertebrates and, 141. 142 Electron microscopy bacterial cell division site and, I I . 12 bacterial cell envelope. 3 division apparatus. 6. 10 septum formation, 14 centrifugal visual system of vertebrates and birds, 139. 140, 144, 146 cycloslomes. 117. I18 mammals, 153 Kupffer cells and. 175. 181, 220 endotoxin. 208 infectious diseases, 2 I I population dynamics, 182 marine algae and. 105. 106. I 1 I spermatozoa and, 23 I Electroretinogram. centrifugal visual system of vertebrates and. 159. 164 Endocytosis, Kupffer cells and. 198-7-03 isolation. 189, IW morphology. 180 population dynamics. 182 Endoplasmic reticulum. Kupffer cells and. I86 Endot helium bacterial cell surface and, 58 Kupffer cells and, 173. 175. 176. 220 endocytosis, 198

endotoxin, 205, 207, 209 infectious diseases. 211, 212. 216218 morphology. 176. 177. 179-181 population dynamics. 181 Endotoxin bacterial cell surface and. 50. 53 Kupffer cells and. 203-209, 220 endocytosis, 198 infectious diseases. 212, 213. 215. 217. 219 metabolic responses, 191 morphology, 179 population dynamics, 184 Enkephalinlike immunoreactivity centrifugal visual system of vertebrates and. I32 Enterobacterial common antigen. bacterial cell surface and, 50 Enterotoxin, Kupffer cells and. 188 Epididymis, spermatozoa and, 233. 234. 236. 242. 263 Epithelium, bacterial cell surface and, 58.73 Erythrocytes Kupffer cells and. 179, 191. 198 spermatozoa and. 263 Escherichici coli bacterial cell division site and, 2 . 13. 27 cell cycle. 16-18 chromosome replication, 23 septum formation, 14. 15 bacterial cell surface and, 83, 84 cell wall turnover. 74, 77 peptidoglycan. 46 surface adhesions. 58-65 Kupffer cells and. 204. 210. 215, 217 marine algae and. 93 Eu karyotes bacterial cell division site and. 2. I6 bacterial cell surface and. 46 marine algae and. 107, 113 Excitatory postsynaptic potentials. centrifugal visual system of vertebrates and. 129. 133 Exocytosis Kupffer cells and. 199 spermatozoa and, 251. 254. 258. 260, 264. 269 Exotoxin bacterial cell surface and. 50 Kupffer cells and, 209

.

INDEX Extracellular matrix, marine algae and, I 1 I , I I3

F

Fatty acid bacterial cell surface and. 53, 55, 57 spermatozoa and. 239. 255, 268 Fibroblasts, bacterial cell surface and, 46 Fibronectin, bacterial cell surface and, 7073 Filaments bacterial cell division site and, 14 chromosome replication, 22, 23, 25, 26 septum formation, IS, 16 marine algae and. 109, I10 Fimbriae, bacterial cell surface and surface adhesions, 59-61, 63, 64,66 surface structures, 42 Fish, centrifugal visual system of vertebrates and, 161, 164 anatomy, 120-126 function, 126. 127 Fluid-phase pinocytosis, Kupffer cells and. 200 Fluorescence bacterial cell division site and, 6 bacterial cell surface and, 38 centrifugal visual system of vertebrates and. 142, 156, 157 Kupffer cells and, 175 spermatozoa and, 249 Fluoride. bacterial cell surface and, 83, 84 FMRF amide, centrifugal visual system of vertebrates and, 124, 128, 129. 150 Forssman antigen, bacterial cell surface and. 50 Freeman antigen, bacterial cell surface and, 51 Frog virus 3, Kupffer cells and, 209-215, 218 Fircrrs, marine algae and, 1 1 I Fusion, bacterial cell division site and. 8

G G d u s dornesticirs, centrifugal visual system of vertebrates and, '136, 139

285

Ganglion cells. centrifugal visual system of vertebrates and, 160, 163 amphibians, 129 birds. 133, 134, 146, 149 cyclostomes, 118 fish. 120, 124, 126 mammals, 151. 152, 154, 155, 158 nervus terminalis, 123, 124, 126, 129 reptiles, 132, 133 Genetics bacterial cell division site and, I . 2, 28 marine algae and, 93 Geniculatus lateralis pars dorsalis, centrifugal visual system of vertebrates and, 156 Glucose bacterial cell surface and, 54 Kupffer cells and, 192 spermatozoa and, 244 Glucose-&phosphate dehydrogenase, Kupffer cells and, 192, 203 Glutaraldehyde. bacterial cell division site and, I5 Glycerol, bacterial cell surface and. 35, 56 Glycine, centrifugal visual system of vertebrates and. I18 Glycogen, bacterial cell surface and, 50 GI ycoprotein Kupffer cells and, 190, 198, 200, 205 marine algae and, 11 1 spermatozoa and. 266 acrosome reaction. 254 capacitation. 233-238, 248 Glycosaminoglycans, spermatozoa and, 248. 252. 253. 268 Golgi method, centrifugal visual system of vertebrates and amphibians, 127, 129 birds, 133, 134, 138, 139, 144 cyclostomes, 116 Gram-negative bacteria bacterial cell division site and, 1-3 bacterial cell surface and, 33, 83 amphiphiles, 49. 52 surface adhesions, 60. 62, 67 surface structures, 41, 42 Gram-positive bacteria, bacterial cell surface and, 33 amphiphiles. 49. 52. 56, 57 cell wall turnover, 76

286

INDEX

Gram-positive bacteria (continrred surface adhesions. 67 surface structures. 34. 36-38. 42

H Hemagglutination. bacterlal cell surface and. 66. 72 Histiocyte\. Kupffer cells and. 173. 175 Hormones Kupffer cells and. 198, 200 spermatozoa and. 247. 248 Horseradish peroxidase. centrifugal visual system of vertebrates and amphibians. 127-1 29 birds, 135. 136, 139-142, 146-148 cyclostomes. 116. I17 fish, 121, 126 mammals, 151. 154-158 reptiles, 132 Human lymphocyte antigen. bacterial cell surface and, 45,46 Hyaluronic acid. bacterial cell surface and.

46 Hybrids. marine algae and, 95 Hydrolytic enzymes, spermatozoa and. 249. 258-265 Hydrophobins. bacterial cell surface and, 59. 70

I

Idiopathic hemochromatosis, Kupffer cells and, 219 lmmunoelectron microscopy. bacterial cell division site and. 16. 27 Immunoglobulins bacterial cell surface and. 45. 54. 73 Kupffer cells and. 203, 217. 119 Infectious diseases. Kupffer cells and, 210220 Inflammation bacterial cell surface and cell wall turnover, 79 peptidoglycan. 43-45 surface adhesions. 58 Kupffer cells and endotoxin, 205 infectious diseases, 220

metabolic responses, 195 population dynamics, 183, 184. 186 Inhibition bacterial cell division site and, 13. 14, I922. 24. 26 Esclierichiu coli, 16 bacterial cell surface and, 33, 52. 63. 69. 83, 84 amphiphiles. 57 surface adhesions. 61, 67, 69. 70, 72 surface structures, 45 centrifugal visual system of vertebrates and. 163 amphibians, 130 birds. 149 cyclostomes. I18 fish. 126 mammals. 159 Kupffer cells and endocytosis, 202. 204. 205 infectious diseases, 214, 217. 218 metabolic responses. 195- I98 population dynamics, 185 marine algae and. 97. 100, 105, 107 spermafozoa and. 268 acrosome reaction, 255, 258-265 capacitation, 234. 240, 242. 243, 249 Inner membrane bacterial cell division site and, 2 division apparatus, 5-10 septum formation, 14, 15 bacterial cell surface and, 41 Inner plexiform layer, centrifugal visual system of vertebrates and amphibians. 127. 128 birds. 133, 139, 144-146 cyclostomes, 116-1 18 fish. 120. 123, 124, 126 mammals, 151-154 reptiles. 132 Interferon, Kupffer cells and. 205, 217 Interleukin- I bacterial cell surface and, 47. 49 Kupffer cells and. 205 Internalization. Kupffer cells and, 200. 201, 213 lnterplexiform cells. centrifugal visual system of vertebrates and. 124, 126, 160 Iron, Kupffer cells and, 219

INDEX K Kallikrein, spermatozoa and, 236, 259, 263 Kupffer cells, 173-176, 220, 221 culture, 190 endocytosis, 198, 199 adsorption of ligands, 199. 200 internalization. 200, 201 intracellular transport, 201 lysosomes, 201-203 endotoxin, 203, 204 liver injury, 209 mediators, 205, 206 metabolism, 204, 205 morphology, 206, 207 species sensitivities, 208, 209 tolerance, 207, 208 infectious diseases, 210 fate of agents, 213, 214 functions, 217, 218 pathology, 218-220 susceptibility, 214-217 viruses, 210-213 isolation, 187-190 metabolic responses, 191 arachidonate, 193, 195- I99 calcium, 192-194 endocytosis. 191, 192 morphology fixation, 176, 177 scanning electron microscopy, 180, 181 transmission electron microscopy, 177180

population dynamics, 181 distribution, 181-183 ontogeny, 183-185 precursor, 185-187 purification, 188, 189

L

Lactate dehydrogenase, Kupffer cells and, 203, 204 Laminin, bacterial cell surface and. 73 Lampetru fltrviurilis, centrifugal visual system of vertebrates and, 116-1 18 Lamprey, centrifugal visual system of vertebrates and, 118, 160, 161

287

Lectin bacterial cell surface and, 84 amphiphiles, 54, 57 surface adhesions, 59-67, 73 marine algae and, 113 spermatozoa and, 235-237 Leukocytes bacterial cell surface and, 54 Kupffer cells and, 185,206, 21 I , 212 Leukotrienes Kupffer cells and, 196, 197 spermatozoa and, 258 Ligands Kupffer cells and, 199-201 spermatozoa and, 241, 254 Light microscopy bacterial cell division site and. 10 centrifugal visual system of vertebrates and, 144 Kupffer cells and, 177, 181, 182, 220 L i m t h s , bacterial cell surface and, 52, 53. 56 Lipid bacterial cell division site and. 8 bacterial cell surface and, 50-53. 57, 58 marine algae and, 106 spermatozoa and, 239, 240, 243, 248, 267 Lipid A, bacterial cell surface and, 51, 53, 54 Lipomannan, bacterial cell surface and. 50 Lipopol ysaccharide bacterial cell surface and, 33 amphiphiles, 49-56, 58 peptidoglycan, 46 surface structures, 38, 42 Kupffer cells and endotoxin, 204-206, 209 infectious diseases, 217 metabolic responses, 193, 195 Lipoprotein bacterial cell surface and, 41, 42. 50 Kupffer cells and, 190, 204 spermatozoa and. 241 Lipoteichoic acid, bacterial cell surface and, 33 amphiphiles. 50. 54-57 cell wall turnover, 74 surface adhesions, 60,70-73 surface structures, 36, 38

288

INDEX

Lipoxygenase Kupffer cells and, 193. 197-199 spermatozoa and, 258 Liquoid. bacterial cell surface and, 79 Liver. Kupffer cells in, see Kupffer cells Localization bacterial cell division site and, 1. 1 6 1 2 . 27, 28 chromosome replication. 23. 25. 27 division apparatus. 5, 6, 9 marine algae and, I I I Luteinizing hormone-releasing hormone. centrifugal visual system of vertebrates and. 124, 126, 129. 160 Lymph nodes. bacterial cell surface and. 44 Lymphocytes bacterial cell surface and. 54, 56 Kupffer cells and. 206 Lysosomes bacterial cell surface and, 45. 79 Kupffer cells and culture, 190 endocytosis. 198. 199. 201-203 endotoxin. 209 infectious diseases, 213, 217 morphology, 177, 180 population dynamics, 186 spermatozoa and. 234 Lysozyme bacterial cell surface and surface adhesions, 69 surface structures. 34, 42, 44. 45 Kupffer cells and, 204

M Macrophages bacterial cell surface and, 45. 47. 48 Kupffer cells and. 175. 176, 220 endotoxin. 204, 205, 209 infectious diseases, 213-219 metabolic responses. 192, I% population dynamics. 181-187 Mammals, centrifugal visual system of vertebrates and anatomy. 151-158 function. 158-160 Mannan. marine algae and. 1 I 1 Marine algae, 93. 94. 113

Acerubdaria. 94-102 Boergeseniri, 102-107 Porphym, 107- I I3 MDP. see N-Acetylmuramyl dipeptide Medial pretectal area, centrifugal visual system of vertebrates and, 156, 157 Meiosis. marine algae and. 95. 107, 109 Membrane attachment at the leading edge, bacterial cell division site and. 9 Methionine, bacterial cell division site and. 18

MHV. Kupffer cells and, 215, 216 Microfibrils. marine algae and, 106 Microfilaments. spermatozoa and, 237 Microtubules bacterial cell division site and, 4 centrifugal visual system of vertebrates and, 118. 139 marine algae and, 105. 106 spermatozoa and, 237 Microvilli. Kupffer cells and. 177, 180 Minicell. bacterial cell division site and. 13, 14. 24-26 Mitochondria bacterial cell division site and. 6 bacterial cell surface and, 54 centrifugal visual system of vertebrates and, 139 Kupffer cells and, 191, 212 marine algae and, 94 Mitogen bacterial cell surface and. 53, 54. 56 Kupffer cells and, 205 marine algae and, 97. 98, 100 Mitosis bacterial cell division site and. 4 Kupffer cells and. 183. 184 marine algae and, 93. 96, 105 Monoclonal antibodies marine algae and, 1 13 spermatozoa and, 233. 235, 237. 253 Monocytes. Kupffer cells and, 175, 176 endotoxin, 204 infectious diseases, 214 population dynamics, 183, 185, 186 Mononuclear phagocyte system, Kupffer cells and, 176, 183 Morphology bacterial cell division site and, 2, 6, 7. 27.28 bacterial cell surface and, 41

INDEX

289

centrifugal visual system of vertebrates 0 and birds, 142 Optic nerve, centrifugal visual system of cyclostomes, 117 vertebrates and, 163 mammals, 151 amphibians, 127- 129 Kupffer cells and, 176-181 birds, 134, 141, 144 culture, 190 cyclostomes. 116, I17 endocytosis, 198 fish, 120, 121, 123 endotoxin, 206. 207 mammals, 151, 152, 154, 155, 159. 160 metabolic responses, 191 reptiles, 130. 133 population dynamics, 182, 186 Osmotic stress, bacterial cell division site marine algae and, 95, 109, 113 and, 2, 3, 5 spermatozoa and, 23 I , 249, 265 Outer membrane Muramic acid, bacterial cell surface and, 83 bacterial cell division site and. 2. 3 cell wall turnover, 74, 79 division apparatus, 5-9 peptidoglycan, 45. 47, 48 septum formation, 14, 15 surface structures, 34 bacterial cell surface and, 41. 42 Murein, see ulso Peptidoglycan Oxygen bacterial cell division site, 2 Kupffer cells and, 192, 217, 220 bacterial cell envelope, 2-4 spermatozoa and, 232, 243 division apparatus, 5-9 septum formation, 14, 15 Mutation P bacterial cell division site and, 13 chromosome replication, 22, 23, 26 Parenchymal cells, Kupffer cells and septum formation, 15, 16 endocytosis, 200, 202 bacterial cell surface and, 33, 51, 65, 69 endotoxin, 203, 205, 209 infectious diseases, 210, 212, 214-220 metabolic responses, 191, 197 N morphology, 177 population dynamics, 183 NADPH, Kupffer cells and, 191, 192 purification, 189 Natural killer cells, Kupffer cells and, 190, Penicillin, bacterial cell surface and, 33, 34 22 1 amphiphiles, 58 Neuraminidase. spermatozoa and, 236 surface adhesions, 73 Neutrophils, Kupffer cells and, 213 surface structures, 35 Nirella, marine algae and, 93 Penicillin-binding protein 3, bacterial cell diNitrogen, bacterial cell surface and, 54 vision site and, 15, 16 Nucleic acids. bacterial cell surface and, 34, Peptides 55 bacterial cell surface and, 84 Nucleoids, bacterial cell division site and, cell wall turnover, 74, 79 14, 22-28 peptidoglycan, 4 7 4 9 Nucleus isthmoopticus, centrifugal visual centrifugal visual system of vertebrates system of vertebrates and, 163 and, 124, 126, 160 birds, 134-143, 146-150 Peptidoglycan reptiles, 131 bacterial cell division site and, 2 Nucleus oculomotorius, centrifugal visual bacterial cell envelope, 2, 3 system of vertebrates and. 157, 158 division apparatus, 7, 9, 10 Nucleus olfactoretinalis, centrifugal visual bacterial cell surface and, 34, 4 3 4 9 , 83 system of vertebrates and, 123, 124 cell wall turnover, 74, 76, 79

290

INDEX

Peptidoglycan ( c . o r i r i n i t 4 surface adhesions. 73 surface structures. 34-38. 41 Periaqueductal gray matter. centrifugal visual system of vertebrates and. 157 Periseptal adhesion zones. bacterial cell division site and. 7-9 Periseptal annular apparatus, bacterial cell division site and. 4-7. 10. 1 I , 27 chromosome replication. 26. 27 Peroxidase. Kupffer cells and morphology, 176. 177, 180 population dynamics, 182, 183. 186

PH bacterial cell surface and, 74 269 spermatozoa and. 253. 262. 264. 265. Phagocytosis bacterial cell surface and, 33. 52-54 Kupffer cells and. 173 endocytosis. 201. 203 endotoxin. 204-208 infectious diseases. 21&213. 215-217. 219. 220 metabolic responses. I9 1 - 193. I 95. 196 morphology, 179. 181 population dynamics. 182. 183, 186 purification, I89 Phenotype bacterial cell division site and. 13. 14. 23 bacterial cell surface and. 65, 73 Kupffer cells and, 186. 187 marine algae and. 95 Pheromones. marine algae and. I 0 9 Phospholipase A? Kupffer cells and. 192. 195. 204 spermatozoa and. 268, 269 acrosome reaction. 255-264 capacitation. 242 Phospholipid bacterial cell division site and. 9 bacterial cell surface and. 41 spermatozoa and. 267. 269 acrosome reaction. 255-257, 260-262 capacitation. 239-244 Photobleaching. bacterial cell division site and, 6 Photosynthesis. marine algae and, 93, 95

Plasma Kupffer cells and. 180 spermatozoa and acrosome reaction, 261. 264 capacitation. 233-235. 237. 247 Plasma membrane bacterial cell surface and, 34, 41. 72 Kupffer cells and, 213 marine algae and, 94, 100. 102. 105 spermatozoa and, 267, 269 acrosome reaction. 249, 253. 257. 261. 263-265 capacitation. 234. 236. 237. 239-243. 248 Plasminogen activator, Kupffer cells and, 204 Plasmolysis. bacterial cell division site and. 7. 1 1 Polymers. marine algae and. 107 Polymorphonuclear leukocytes, bacterial cell surface and, 54 Polymyxin B. spermatozoa and. 239. 240 Polypeptides. spermatozoa and, 253. 254, 256 Pol ysacc harides bacterial cell surface and, 33 amphiphiles. 51. 53. 55. 58 peptidoglycan. 44. 45 surface adhesions. 60. 65. 73 surface structures. 37, 38 marine algae and. 110. 1 I I , I13 Porins. bacterial cell division site and, 2 P<WP/I!IY~. 94. 107-1 13 Portocaval anastomosic. Kupffer cells and. 204. 20s Preoptic retinopetal nucleus, centrifugal visual system of vertebrates and, 123 Proacrosin. spermatozoa and. 253, 259-261. 265 Prokaryotes. bacterial cell division site and, 4 Proline. centrifugal visual system of vertebrates and. I18 Pronase. Kupffer cells and. 187-191 Prostaglandins bacterial cell surface and, 46 spermatozoa and, 258 Prostanoids, Kupffer cells and. 195. 196 Proteases. bacterial cell division site and. 19. 20

29 I

INDEX Protective antigen, bacterial cell surface and, 38 Protein bacterial cell division site and, 13, 27 bacterial cell envelope, 2-4 chromosome replication, 20-22 division apparatus, 6-9 Escherichia coli, 16, 18 septum formation, 15, 16 bacterial cell surface and, 83 amphiphiles, SO, 52, 55, 57, 58 peptidoglycan, 45 surface adhesions, 59-61,64,66.70,72, 73,79 surface structures, 38, 41 centrifugal visual system of vertebrates and, 149 Kupffer cells and, 179. 198, 204, 205 marine algae and, 110, I 1 1 spermatozoa and, 267 acrosome reaction, 253, 254, 256, 258, 263. 264 capacitation, 234-239, 243, 247, 248 Protein A. bacterial cell surface and, 73 Protein kinase C. spermatozoa and, 243 Protoplasts, marine algae and, I13 Boergeseniu, 102, 105 Porphyru, 10%I 11, 113 Pseudornonas, bacterial cell surface and, 64,66, 67 Puromycin, marine algae and, 100 Pyrogen, bacterial cell surface and, 52,53, 56

R R antigen, bacterial cell surface and, 51 Ranu pipiens, centrifugal visual system of vertebrates and, 127, 128 Red blood cells, bacterial cell surface and amphiphiles, 52, 53, 67 surface adhesions, 68, 72 Replication bacterial cell division site and, 11-13 bacterial cell surface and, 34 Reticular mesncephalic area, centrifugal visual system of vertebrates and, 116118

Reticulo-Endotheliales System, Kupffer cells and, 175, 176 endocytosis, 198 endotoxin, 208, 209 infectious diseases, 210, 216-220 population dynamics, 184 Reticuloendothelial cells, Kupffer cells and, 175, 181. 183. 209, 220 Retina, centrifugal visual system of vertebrates and, 160, 162-164 amphibians, 127-130 birds, 133-135, 139, 142-149 cyclostomes, 117 fish, 120, 121, 123, 124, 126, 127 mammals, 151-154, 156160 reptiles, 130. 132, 133 Retinopetal system, centrifugal visual system of vertebrates and, 160, 162-164 amphibians, 129 fish, 120, 121, 126 reptiles, 130 Rheumatoid arthritis bacterial cell surface and, 43,45, 84 Kupffer cells and, 219 RNA bacterial cell division site and, 16-18 bacterial cell surface and, 50 Rough endoplasmic reticulum centrifugal visual system of vertebrates and, 139 Kupffer cells and, 180, 182, 183

S Scanning electron microscopy, Kupffer cells and, 180, 181 Scenedesmus, marine algae and, 93 Septa1 attachment site, bacterial cell division site and, 9 Septum, bacterial cell division site and, I , 2. 10, 13, 14 bacterial cell envelope, 2, 3 chromosome replication, 18-27 chromosome segregation, 4 division apparatus, 4, 5, 9, 10 Escherichiu coli. 16-18 formation, 14-16 Serotonin, centrifugal visual system of vertebrates and, 133, 157, 160

292

INDEX

Serum albumin. hpermatozoa and. 254-256 Simian virus 40. Kupffer cells and. 217 Sinusoids. Kupffer cells and. 173, 220 endotoxin. 206. 207 infectious diseases. 21 I . 212. 216-219 isolation. 1x7 morphology. 176. 177. 179. 180 population dynamics, 181-183. 186 purification. 188. I89 Sleep, bacterial cell surface and. 47-49. 83 SOS system. bacterial cell division site and. 18-22. 26 Sperm motility factor, 258 Spermatozoa, 231. 132. 365-269 acrosome reaction, 249-25 I exogenous factors, 254-258 hydrolytic enzymes. 258-265 site. 251-254 capacitation. 232. 233 chemicals. 14X. 249 factors. 747. 248 intramembrane molecular alterations. 238-243 physiology. 243-247 surface alteration. 233-238 Spleen bacterial cell surface and. 56 Kupffer cells and. 175. 220 .Sfcrpliy/oe~~c.c.rt.s. bacterial cell surface and. 67. 73 S/aphy/oc~oc~crr.s ortre~rts.bacterial cell surface and, 72-74. 77. 79 .S/Nplt?.ktc.oc.c.rrsc~pidcrmis.bacterial cell surface and. 65. 72, 73 Sternzellen. Kupffer cells and, 173. I75 Streptococcal cells. bacterial cell surface and. 83. 84 amphiphiles, 56. 58 cell wall turnover, 74, 79 peptidoglycan. 43. 45. 46 surface adhesions. 59. 70. 72 .Streptoeoccrr.s. bacterial cell surface a m . 67 Strrploc.occrts c.ricerrrs. bacterial cell surface and. 40.67-69 Strrpfoc~oc.crr.s: t i i t t a r i s , bacterial cell surface and, 77. 79. 80 S/rqmcoccws pnerrmonicre. bacterial cell surface and. 72

Sfrepfococcrrs pncrc:nonicw. bacterial cell

surface and. 72 bacterial cell surface and, 70-72 Substance P. centrifugal visual system of vertebrates and. 124 Sugar. centrifugal visual system of vertebrates and. 118 Synaptosomes, centrifugal visual system of vertebrates and. 118 Systemic lupus erythematosus. Kupffer cells and, 219 Slrcymtc'ocwrs pyogmrs.

T T cells bacterial cell surface and. 54. 56 Kupffer cells and. 205. 210. 214 Tectumum. centrifugal visual system of vertebrates and. I63 amphibians. 128 birds. 134. 135. 138. 140-143 cyclostomes, I17 fish. 12OL123. 126 reptiles. 130 Teichoic acids. bacterial cell surface and amphiphiles. 57, 58 cell wall turnover, 79 surfact- adhesions. 73 surface structures. 35-38. 41 Teichuronic acids, bacterial cell surface and cell wall turnover. 79 surface structures, 35, 36, 38, 41 Telencephalon, centrifugal visual system of vertebrates and, 132, 133 Teleosts. centrifugal visual system of vertebrates and. 161 amphibians, 129 fish. 12@--124, 126 Terminal complexes. marine.algae and. 106

Termination protein. bacterial cell division site and. 21 Thalamus. centrifugal visual system of vertebrates and, 122. 131. 132 Thallus, marine algae and. 107. 109-1 I I. I13 Thymidine bacterial cell surface and, 54 Kupffer cells and. 184, 185

INDEX Thymine, bacterial cell division site and, 17, 18. 22. 26 Thymocytes. bacterial cell surface and, 56 Tractus isthmoopticus, centrifugal visual system of vertebrates and, 120, 135, 138. 143 Transcription, marine algae and, 100 Transmission electron microscopy, Kupffer cells and. 176-181 Trypsin, spermatozoa and, 233, 236, 259, 263 Tumor, Kupffer cells and, 218, 221 Tumor necrosis factor, Kupffer cells and, 205 Turnover, cell wall, see Cell wall turnover

U

293

Valonia, marine algae and, 93. 102 Vesicles centrifugal visual system of vertebrates and birds, 139, 140, 146 cyclostomes, 118 fish, 124 Kupffer cells and, 201, 217 marine algae and. 102, 105 spermatozoa and, 240 Virus, Kupffer cells and, 210-213, 216, 217, 22 1

X Xenopus laevis, centrifugal visual system of vertebrates and, 127, 128 Xylan, marine algae and, I I I

Urine, bacterial cell surface and, 47, 48

V

Vaccine, bacterial cell surface and, 33, 64, 84

Vacuoles Kupffer cells and, 179, 180, 201, 212 marine algae and, 107

Zymogen, spermatozoa and, 259, 268 Zymosan, Kupffer cells and, 176 endocytosis. 203 metabolic responses, 193, 195-197 morphology, 179 population dynamics, 184-186

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