Mammalian Embryo Genomics (Biological Resource Management in Agriculture)

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Biological Resource Management in Agriculture

Mammalian Embryo Genomics

This book contains the proceedings of the first International Meeting on Mammalian Embryo Genomics, held in Quebec City, Canada, on July 20, 2002.

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Mammalian Embryo Genomics

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ISBN 92-64-10426-7 51 2003 11 1 P

Biological Resource Management in Agriculture

For the past decade there has been an intensive international effort to characterise the genomes of several species of plants and animals. As the mapping and sequencing of genes has progressed, scientists have turned their attention to functional genomics, the characterisation of gene expression. Recently a number of laboratories around the world have begun to focus their investigations on the temporal and spatial pattern of gene expression in the early phases of farm animal and human embryo development, an event of utmost importance for the survival and developmental well-being of the embryo. In an agricultural context, this is a critical factor determining reproductive success. This book contains the proceedings of a conference which brought together a group of internationally recognised scientists in the genomics field. The objective was to coordinate activities in the area of embryo genomics and create a number of collaborations dedicated to organising the huge amount of information being generated in this emerging field of research. This information is and will continue to be crucial for most biotechnological applications in the field of animal reproduction.

Biological Resource Management in Agriculture

Mammalian Embryo Genomics

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Biological Resource Management in Agriculture

Mammalian Embryo Genomics

ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT

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ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT

Pursuant to Article 1 of the Convention signed in Paris on 14th December 1960, and which came into force on 30th September 1961, the Organisation for Economic Co-operation and Development (OECD) shall promote policies designed: – to achieve the highest sustainable economic growth and employment and a rising standard of living in member countries, while maintaining financial stability, and thus to contribute to the development of the world economy; – to contribute to sound economic expansion in member as well as non-member countries in the process of economic development; and – to contribute to the expansion of world trade on a multilateral, non-discriminatory basis in accordance with international obligations. The original member countries of the OECD are Austria, Belgium, Canada, Denmark, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland, Turkey, the United Kingdom and the United States. The following countries became members subsequently through accession at the dates indicated hereafter: Japan (28th April 1964), Finland (28th January 1969), Australia (7th June 1971), New Zealand (29th May 1973), Mexico (18th May 1994), the Czech Republic (21st December 1995), Hungary (7th May 1996), Poland (22nd November 1996), Korea (12th December 1996) and the Slovak Republic (14th December 2000). The Commission of the European Communities takes part in the work of the OECD (Article 13 of the OECD Convention).

© OECD 2003 Permission to reproduce a portion of this work for non-commercial purposes or classroom use should be obtained through the Centre français d’exploitation du droit de copie (CFC), 20, rue des Grands-Augustins, 75006 Paris, France, tel. (33-1) 44 07 47 70, fax (33-1) 46 34 67 19, for every country except the United States. In the United States permission should be obtained through the Copyright Clearance Center, Customer Service, (508)750-8400, 222 Rosewood Drive, Danvers, MA 01923 USA, or CCC Online: www.copyright.com. All other applications for permission to reproduce or translate all or part of this book should be made to OECD Publications, 2, rue André-Pascal, 75775 Paris Cedex 16, France.

FOREWORD –

Foreword For the past decade there has been an intensive international effort to characterize the genomes of several species of plants and animals. As the mapping and sequencing of genes has progressed, scientists have turned their attention to functional genomics, the characterization of gene expression. Recently a number of laboratories around the world have begun to focus on investigating the temporal and spatial pattern of gene expression in the early phases of farm animal and human embryo development, an event of utmost importance for the survival and developmental well being of the embryo. In an agricultural context, this is a critical factor determining reproductive success. This book contains the proceedings of a conference which brought together a group of internationally recognised scientists in the genomics field, in an attempt to coordinate activities in the area of embryo genomics and create a number of collaborations dedicated to organizing the huge amount of information being generated in this emerging field of research. This information is and will continue to be crucial for most biotechnological applications in the field of animal reproduction.

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TABLE OF CONTENTS–

Table of Contents Chapter 1. Polyadenylation of Oocyte Transcripts as Markers of Developmental Competence..........................................................

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Chapter 2. Gene Expression Patterns in Bovine In Vitro Produced and Nuclear Transfer Derived Embryos and its Implications for Development............................................................................. 19 Chapter 3. Characterization of cDNA Libraries Developed from Porcine Oocytes and Embryos to Determine Differential Expression Patterns....................................................................... 29 Chapter 4. Systematic Analysis of Mouse Preimplantation Development................................................................................... 37 Chapter 5. Serial Analysis of Gene Expression (SAGE) in Preimplantation Stage Swine Embryos ....................................... 47

Short communications Library Subtraction and Microarray Screening to Isolate Oocyte Specific Transcripts in the Cow.................................................... 57 Characterization of Novel cDNAs that are Stage Specific and Sensitive to Culture Environment During Bovine Early Development................................................................................... 67 Potential Use of cDNA Microarrays to Characterize Gene Expression Patterns of Sex-specific Genes During Early Development................................................................................... 77 A Profile of Gene Expression in Bovine Oocytes Generated by Heterospecific cDNA Array Screening ........................................ 91

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6 – TABLE OF CONTENTS Posters Heteroplasmy Rates Analysis in Single Blastomeres Using Fluorocromes Stained Primers..................................................... 99 Additional Step in Genomics Studies of Bovine Embryos: Proteomics...................................................................................... 101 Quantification of Histone Acetyltransferases Transcripts in the Early Stages of Bovine Embryo Development ...................... 103 Study of Newly Synthesized Proteins During Bovine Oocyte Maturation In Vitro by Two-dimensional Gel Electrophoresis .............................................................................. 104 Study of Genome Activation and Developmental Block Messages Applying Fluorescent DD-RT-PCR ............................................. 106 Identification of First Zygotic Transcripts Using SSH and Validation of RNAi Procedure in the Rabbit Embryo ............... 108 Poly(A) mRNA content and Polyadenylation status of Specific Transcripts During In Vitro Bovine Oocyte Maturation............ 110 Temporal Expression Patterns of Transcription Factors During Cattle Early Embryogenesis ......................................................... 112 Quantitative Real-time PCR Analysis of RNA and DNA in Individual Mouse Embryos and their Blastomeres................ 114 Targeting Gene Expression in the Preimplantation Mouse Embryo Using Morpholino Antisense Oligos.............................. 116 Identification of Novel Products in Maturing Bovine Cumulus-oocyte Complexes.......................................................... 118 Gene Expression and DNA Methylation of an Imprinted Gene Cluster on Ovine Chromosome 18 During Early Embryonic Development................................................................................... 120

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Chapter 1 Polyadenylation of Oocyte Transcripts as Marker of Developmental Competence Tiziana A.L. Brevini, Fabiana Cillo, Chiara Francisci, Fulvio Gandolfi Department of Anatomy of Domestic Animals, Faculty of Veterinary Medicine, University of Milan, 20133 Milano (Italy).

Abstract. RNA and protein molecules synthesized during oocyte growth and maturation contribute to early development prior to zygote genome activation and beyond. RNA storage takes place during oocyte growth and the extent of poly(A) tail at the 3’ end of each transcript has emerged as an important regulatory element for determining its stability. Thus, control of polyadenylation represents a key regulatory step in gene expression and is important for early embryonic development. The developmental competence of an oocyte can be influenced by different factors such as the morphology of the ovary it has been isolated from, the exposure to endocrine disruptors during in vitro maturation or can determine the interval occurring between fertilization and the first cleavage. This makes it possible to divide oocytes in groups with high and low developmental competence and enabling us to study the mechanisms determining the ability of an oocyte to sustain embryonic development. We performed a series of studies investigating the role of maternal mRNA polyadenylation looking at genes that characterize physiological processes involved in early differentiation (oct-4), compaction and cavitation (ß-actin, plakophilin, connexin 43, connexin 32), energy metabolism (glucose transporter type 1, pyruvate dehydrogenase phosphatase), RNA processing (RNA poly(A) polymerase) and stress (heat shock protein 70). Poly(A) tail length of these transcripts was studied with a RT-PCR based method in bovine oocytes whose developmental competence was determined using the parameters mentioned above. All the parameters (ovarian morphology, IVM medium, cleavage kinetic) used for predicting oocyte developmental competence profoundly influenced maternal mRNA polyadenylation, but with patterns specific for each experimental condition, suggesting that a fine tuning of gene expression is responsible for the correct changes and transitions to take place during oocyte maturation and development. These process appear to be largely under the control of post-transcriptional regulatory mechanisms and mostly driven by cytoplasmic components. The latter are synthesized during oogenesis and stored in the oocyte in a dormant state until they are activated at specific stages of development, following a precise temporal and spatial pattern.

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8 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE

Introduction Efficiency of embryo production “in vitro” largely depends on oocyte quality. A series of criteria that allow the identification of oocytes more likely to have the highest developmental competence in vitro have been defined. Some of these result from process occurring during folliculogenesis and include aspects such as morphology of the cumulus oocyte complex or diameter of the follicle [1]. More recently, we demonstrated that also the morphology of the ovary from which the oocyte is isolated could be used as an easy parameter to predict its developmental competence [2]. This indicates that oocyte competence is acquired within the ovary during the developmental stages preceding ovulation or, when the process is carried out in vitro, preceding the isolation of the oocyte from its follicle. This process is defined as “oocyte capacitation” since during this period the oocyte becomes able to sustain embryonic development [3]. However oocyte developmental competence can be deeply influenced also by the environment where in vitro maturation occurs . Although the precise mechanisms involved are still unclear, it can be hypothesized that oocytes become equipped for sustaining embryonic development during capacitation, while during maturation appropriate signals have to be present in order to trigger the developmental program acquired in the previous phase [4]. The study of these aspects may help to gain information on the maturation process and represents a key factor to elucidate the mechanisms controlling oocyte maturation. While a wide body of information on morphological and biochemical parameters is available to date, there is a paucity of data aimed at elucidating the molecular basis and mechanisms involved in these process. Here we present results obtained in our laboratory aimed at gaining a better understanding of these mechanisms. In particular we address our attention on post transcriptional modifications modulating the expression of specific genes along both the capacitation and the maturation process, focusing on the function of the 3’ untranslated region and the possible role

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played by polyadenylation in the control of developmental competence and early embryo development.

The polyadenylation machinery The interval between fertilization and the so-called maternal-embryonic transition (MET), when transcriptional activity is switched on, is supported by maternal RNAs and proteins synthesized during oogenesis. This implies that RNA and protein molecules can be synthesized even several weeks before they are used. A number of strategies to optimize their storage in a quiescent form and to allow their use at the right time during oocyte maturation and early embryonic development have been developed. In particular, it has been shown that oocyte quality and, ultimately, its competence to sustain embryonic development, depends on the efficiency of such storing process as well as on the correct timely reactivation of the stored molecules. Various mechanisms have been described for storing maternal mRNAs and for regulating their expression at the right time. Information available to date indicate that specific sequences regulate mRNA stability, control translational activation and repression and direct mRNA localization (a schematic model of the polyadenylation machinery is presented in Figure 1). These sequences are located in the untranslated region present at the 3’ end of the mRNA molecule (3’ UTR). It has been shown that regulation of maternal mRNA translation is based on changes in the length of the poly(A) tail stretch. Oocyte mRNAs containing short poly(A) tails are translationally inactive. These mRNAs are then activated upon extension of their poly(A) tail during the following stages of development. Similarly, a number of actively translating mRNAs can shorten the poly(A) tail at their 3’ end and, consequently, become deactivated [5]. Two cis-acting sequences are required for a correct regulation of the translation process: the hexanucleotide aauaaa and the cytoplasmic polyadenylation element (CPE), a U rich sequence with general structure UUUUUA4U that is usually located 20 to 30 nucleotides upstream of the hexanucleotide [6, 7]. A number of specific CPE binding proteins (CPEB) were shown to play an important role in the control of polyadenylation of any CPE-containing mRNAs since they bind different mRNAs at specific times of development, promoting the extension of their poly(A) tail and inducing translational activation [8, 9].

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10 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE Figure 1. A schematic model of the components involved in the elongation of the poly(A) tail at the 3’-end of a mRNA

5’ Cap

coding region

MTase

PAP 3’ AAAAAAA

polyA tail shortening

CPSF

CPEB

aauaaa

UAAUUUU

polyA tail elongation

decreased RNA stability

enhanced RNA stability

decreased translational activation

enhanced translational activation

The polyadenylation process requires the presence of poly(A) polymerase (PAP), the hexamere aauaaa and CPE (UAAUUUU), which are bound by cytoplasmic polyadenylation element binding protein (CPEB) and cleavage and polyadenylation specific factor (CPSF). The 5’methylguanosine cap structure (MTase) and the 3’polyA tail of adenosine residues (3’AAAAAAA), also interact in controlling mRNA stability and decay. Shortening of the polyA tail and 5’decapping allow exonucleolitic degradation of the mRNA. PolyA tail elongation, by contrast, prevents enzymatic degradation and increases mRNA stability.

Another group of molecules crucial for polyadenylation control is the Cleavage and Polyadenylation Specific Factor or CPSF, which comprises a complex of three proteins that bind nuclear aauaaa sequence and are involved in the recruiting of poly(A) polymerase. Half life of individual mRNAs may vary also depending on degradation by specific exonucleases. RNA molecules are protected from the attack of these enzymes by a 5’methylguanosine cap structure (MTase) and the 3’polyA tail of adenosine residues (3’AAAAAAA), which together interact in controlling mRNA stability and decay. Shortening of the polyA tail and 5’decapping allows exonucleolitic degradation of the mRNA and is followed by rapid RNA molecule

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decay. PolyA tail elongation, by contrast, prevents enzymatic degradation and increases mRNA stability.

Polyadenylation control in bovine oocyte We have recently shown that bovine oocytes undergoing meiotic maturation display a temporal regulation of maternal mRNA polyadenylation [10, 11]. In our studies we analysed polyadenylation changes of mRNA transcripts stored in bovine oocytes during in vitro maturation, trying to find possible relationships between these changes and oocyte developmental competence. Most of the transcripts that were examined followed a deadenylation pattern known to take place when no specific sequences such as the hexanucleotide or the CPE are present at the 3’UTR (see above). By contrast, when genes such as connexin-32 (Cx 32) and tPA (tissue-type Plasminogen Activator) were analyzed, we could detect an elongation of their poly(A) tail during oocyte maturation (Figure 2). This observation is consistent with data from other species. Both transcripts in fact are known to be present in the primary oocytes but their translation is triggered only on resumption of meiotic maturation [12-14]. Finally, two other genes considered (E-actin and pyruvate dehydrogenase phosphatase), showed no difference in polyadenylation at the end of the maturation process, a pattern not previously described in other species.

Figure 2. Polyadenylation changes of mRNA molecules stored in the bovine oocyte cytoplasm during in vitro maturation

Three examples are given: the mRNA for the transcription factor Oct-4 undergoes a reduction of its poly(A) tail between the germinal vesicle (GV) and the Metaphase II (MII) stage. The mRNA for Connexin-32 (Cx-32) on the contrary, increases the length of its poly(A) tail during the same period, while the transcript for pyruvate dehydrogenase phosphatase (PDP) remains constant during maturation.

MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

12 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE The use of bovine in vitro embryo production as experimental system made it possible to correlate developmental competence and polyadenylation patterns. Our data showed that mRNAs stored in the cytoplasm of oocytes that have not yet reached a full developmental competence have a shorter poly(A) tail than the fully competent ones and this difference is present at the GV stage as well as at the MII phase [11]. These observations indicate a possible relationship between extent of polyadenylation, mRNA stability, and developmental competence during oocyte maturation. The results obtained suggest that an inappropriate length of poly(A) tail at the 3’end may be correlated, at least in the transcript that we examined, with a lower developmental competence. Based on these observations, it is very tempting to speculate that adenylation may represent one of the factors affecting oocyte ability to sustain early embryonic development. This hypothesis finds support in a different set of experiments [10] where we used fully competent bovine oocytes, divided in two groups and matured them using either adequate culture conditions or very poor inadequate ones (i.e., in the presence or absence of gonadotropins). The extent of the poly(A) tail of the two groups was then analyzed at the end of the maturation process. Defective maturation conditions caused incomplete deadenylation of those transcripts that undergo a deadenylation process during maturation. These transcripts had incomplete translational inactivation at the end of the 24-hour maturation period, suggesting that inadequate maturation conditions may adversely affect the control of maternal mRNA translational inactivation in bovine oocytes. However we found that these oocytes reached a polyadenylation extent comparable to those oocytes matured in adequate conditions with a 6 hours delay (after 30 hours of maturation) indicating that the perturbing effect on translational inactivation is mainly of a temporal kind. Furthermore, maturation conditions did not affect the readenylation process in the genes we analyzed. This suggests that no effect on the cis sequence of the 3’UTR involved in the control of translational activation was present in response to the environmental conditions that we tested. A more perturbing effect on polyadenylation was observed when we exposed oocytes to Aroclor-1254 (A-1254), an environmental pollutant, known to affect oocyte maturation, fertilization and developmental competence[15]. In these experiments (see Figure 3) we could demonstrate a more pronounced deadenylation of some of the genes that would deadenylate in control conditions (i.e. GT-Ty1, Cx43, Plako). However, at the same time, a longer poly(A) tail was observed at the 3’-end of Cx32, a gene that normally readenylates during maturation (see above). Finally, the gene for Heat shock protein 70 (HSP70), instead of undergoing a deadenylation process, as in control conditions, showed an extension of the MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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tail at the end of IVM. These findings suggest altogether that A-1254 has the ability to interfere with the polyadenylation machinery. In particular, the

Figure 3. A-1254 effect on polyadenylation changes of mRNA molecules stored in bovine oocytes during in vitro maturation

RNA was extracted at the germinal vesicle stage (GV) and at the end of IVM (MII), which was carried out either in the absence (MII Control) or in the presence (MII + A-1254) of the environmental contaminant. Left hand column MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

14 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE shows the approximate molecular weight of each gene amplification product (bp). Transcripts for Cx32 and HSP70 displayed a longer poly(A) tail when compared with untreated matured oocytes (MII Control). By contrast, a shorter poly(A) stretch was present at the 3’ end of GT-TyI, Cx43 and Plako. A-1254 exerted no effect on polyadenylation of the other mRNA transcripts considered.

contaminant is able to affect not only the mechanisms involved in default deadenylation process but also to interact with the cis sequences located at the 3’ UTR directly controlling the elongation of the polyA tail thus translational activation. However, at the same time, a longer poly(A) tail was observed at the 3’-end of Cx32, a gene that normally readenylates during maturation (see above). Finally, the gene for Heat shock protein 70 (HSP70), instead of undergoing a deadenylation process, as in control conditions, showed an extension of the tail at the end of IVM. These findings suggest altogether that A-1254 has the ability to interfere with the polyadenylation machinery. In particular, the contaminant is able to affect not only the mechanisms involved in default deadenylation process but also to interact with the cis sequences located at the 3’ UTR directly controlling the elongation of the polyA tail thus translational activation.

Polyadenylation and early embryo development Oocyte maternal mRNA has a major role not only during maturation but is also involved in the control of early embryo development[16]. This suggests that polyadenylation may have a regulatory effect on gene expression and translational activation during the early stages of embryogenesis. To explore this possibility we have recently analyzed the pattern of polyadenylation changes that takes place between the resumption of meiosis and the first cleavage of bovine oocytes. Moreover we have investigated whether the delayed occurrence of the first cleavage division, which characterizes embryos of low developmental competence, is accompanied by an altered polyadenylation pattern of individual transcripts[17]. In these experiments RNA was isolated from oocytes or embryos at the germinal vesicle stage, at the end of in vitro maturation, at the end of in vitro fertilization and at the time of the first cleavage. Cleavage was assessed 27, 30, 36, 42 hours post insemination (hpi), and at the latter time the remaining uncleaved oocytes were retained as a group. Between oocyte isolation and first cleavage at 27 hpi (best quality embryos), the poly(A) tail of individual transcripts followed 4 patterns: no changes ((E-actin, PDP); gradual reduction (Cx-43, Oct-4, Plako); gradual elongation (Cx-32, tPA); reduction followed by elongation (PAP, HSP-70, Glut-1). If the interval between insemination and first cleavage was longer than 27 hpi (progressively lower quality embryos) further changes of MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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polyadenylation were observed, which differed for each gene considered (see Figure 4).

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16 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE Differences in the poly(A) tail length of mRNAs isolated from bovine 2-cell embryos cleaved at different times after insemination (hpi) and from oocytes that had not cleaved by 42 hpi (n.c.). The left-hand column shows the appropriate molecular weight marker for each gene (bp).

These data indicate that specific changes in polyadenylation contribute to modulate gene expression in bovine embryos at this stage of development. Defective developmental competence is accompanied by abnormal polyadenylation levels of specific maternal mRNAs. In particular, synchrony between polyadenylation and cleavage emerged as an apparently important factor and temporal uncoupling of the two was evident in embryos with low developmental competence.

Is polyadenylation a good marker for developmental competence? A fine tuning of gene expression is responsible for the correct changes and transitions to occur during oocyte maturation as well as development. These processes appear to be largely under the control of post-transcriptional regulatory mechanisms and mostly driven by cytoplasmic components. The experimental evidences presented in this paper indicate a role of polyadenylation as a post transcriptional regulatory mechanism timely modulating gene expression and translational activation in oocyte maturation and early development of bovine embryos. Indeed we demonstrated that oocytes with different developmental competence always displayed in our experiments different levels of maternal mRNA polyadenylation. Furthermore these differences were present regardless to how developmental competence was predicted and were completely independent on how competence was affected. Altogether these observations point towards polyadenylation as a very fine mechanism controlling gene expression in maturing oocytes and early embryos. Changes in polyadenylation of specific genes at specific times of development induce translational activation/disactivation of the encoded proteins, possibly ensuring the correct concentration of the different molecules to the developing egg. This regulatory system exerts a default tuning of the process and, according to our data, has the ability to respond to experimental challenges. Furthermore, our observations demonstrate a temporal coupling between changes in polyadenylation and the transition of the oocyte along maturation and early development suggesting the use of polyadenylation as a possible promising biomarker for developmental competence of bovine oocyte.

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References

1. Gordon I. Laboratory production of cattle embryos. Wallingford, CAB International; 1994. 2. Gandolfi F, Luciano AM, Modina S, Ponzini A, Pocar P, Armstrong DT, Lauria A. The in vitro developmental competence of bovine oocytes can be related to the morphology of the ovary. Theriogenology 1997; 48: 1153-1160. 3. Hyttel P, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology 1997; 47: 23-32. 4. Moor RM, Gandolfi F. Molecular and cellular changes associated with maturation and early development of sheep eggs. Journal of Reproduction and Fertility 1987; Suppl. 34: 55-69. 5. Richter JD. Dynamics of poly(A) addition and removal during development. In: Hershey JWB, Mathews MB, Sonenberg N (eds.), Translational control. New York, Cold Spring Harbor Laboratoty Press; 1996: 481-503. 6. Hake LE, Richter JD. Translational regulation of maternal mRNA. Biochimica et Biophysica Acta 1997; 1332: M31-38. 7. Paris J, Richter JD. Maturation-specific polyadenylation and translational control, diversity of cytoplasmic polyadenylation elements, influence of poly(A) tail size, and formation of stable polyadenylation complexes. Molecular & Cellular Biology 1990; 10: 5634-5645. 8. Paris J, Swenson K, Piwnica-Worms H, Richter JD. Maturation-specific polyadenylation, in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein. Genes & Development 1991; 5: 1697-1708. 9. Stebbins-Boaz B, Hake LE, Richter JD. CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus. EMBO Journal 1996; 15: 2582-2592. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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18 – POLYADENYLATION OF OOCYTE TRANSCRIPTS AS MARKER OF DEVELOPMENTAL COMPETENCE 10. Brevini-Gandolfi TAL, Favetta LA, Mauri L, Cillo F, Luciano AM, Gandolfi F. Characterisation of mRNA polyadenylation changes during bovine oocyte maturation and effect of IVM conditions. Biology of Reproduction 1999; 60, Supplement 1: 213. 11. Brevini-Gandolfi TAL, Favetta LA, Mauri L, Luciano AM, Cillo F, Gandolfi F. Changes in poly(A) tail length of maternal transcripts during in vitro maturation of bovine oocytes and their relation with developmental competence. Molecular Reproduction & Development 1999; 52: 427-433. 12. Anderson JE, Matteri RL, Abeydeera LR, Day BN, Prather RS. Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos. Biology of Reproduction 1999; 61: 1460-1467. 13. Barnes FL, First NL. Embryonic Transcription in in vitro Cultured Bovine Embryos. Molecular Reproduction and Development 1991; 29: 117-123. 14. Huarte J, Belin D, Vassalli JD. Plasminogen activator in mouse and rat oocytes, induction during meiotic maturation. Cell 1985; 43: 551-558. 15. Pocar P, Brevini TA, Perazzoli F, Cillo F, Modina S, Gandolfi F. Cellular and molecular mechanisms mediating the effects of polychlorinated biphenyls on oocyte developmental competence in cattle. Mol Reprod Dev 2001; 60: 535-541. 16. Brevini-Gandolfi TAL, Favetta LA, Lonergan P, Gandolfi F. The machanism regulating maternal mRNA stability and translation is affected in bovine embryos with low developmental competence. Theriogenology 2000; 53: 268. 17. Brevini TA, Lonergan P, Cillo F, Francisci C, Favetta LA, Fair T, Gandolfi F. Evolution of mRNA polyadenylation between oocyte maturation and first embryonic cleavage in cattle and its relation with developmental competence. Mol Reprod Dev 2002; 63: 510-517.

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Chapter 2 Gene Expression Patterns in Bovine In Vitro Produced and Nuclear Transfer Derived Embryos and its Implications for Development Heiner Neimann & Christine Wrenzycki Dept. Biotechnology, Institute for Animal Science (FAL), Mariensee, Neustadt, 31535 Germany E-mail: [email protected]

Abstract. Bovine in vitro produced (IVP) and nuclear transfer derived (NT) embryos differ from their in vivo developed counterparts in many important characteristics. A frequently observed phenomenon after transfer of IVP or NT-derived embryos is the occurrence of the “large offspring syndrome (LOS)” which is correlated with considerable fetal and postnatal losses, prolonged gestation length, increased birth weights, increased incidence of dystocia and higher perinatal mortality. Although the mechanisms responsible for the developmental dysregulation as a consequence of IVP and NT remain elusive, a current hypothesis is that early and persistent aberrations in embryonic gene expression patterns are critically involved in the occurrence of LOS. Here, we summarize results from studies done over the past years in our laboratory comparing gene expression patterns in IVP- and NT-derived embryos with those from their in vivo developed counterparts, employing a semi-quantitative RT-PCR assay. Numerous aberrations were found in IVP and NT-derived embryos, including a complete lack of expression, an induced expression or a significant up- or down-regulation of a specific gene. In IVP embryos, the relative abundance of several developmentally important genes showed significant alterations related to the basic culture medium, whereas the type of supplement, i.e. serum, BSA, or PVA, had only minor effects. Changes of distinct steps of the nuclear transfer protocol (i.e. time point of activation; cell cycle stage, passage number or origin of donor cells) led to significant aberrations in the mRNA expression patterns of NT-derived embryos. Recently, we have developed cDNA array technology to monitor expression patterns in single embryos. The alterations found in our studies may affect a number of physiological functions in the early embryo and are interpreted as stress response of the embryos to deficient environmental conditions. We hypothesize that the alterations are caused by epigenetic modifications primarily by changes in global methylation patterns. Unraveling these epigenetic modifications is promising to understand the underlying mechanisms of the LOS.

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Introduction Unusually large offspring have been born in ruminants, i.e. cattle and sheep, following transfer of embryos that have either been subjected to somatic nuclear transfer (NT) or exposed to an unusual in vivo or in vitro environment. This phenomenon called “Large Offspring Syndrome (LOS)” is characterized by a great variety of abnormal phenotypes, including significant increases in birth weight and gestation length, the incidence of polyhydramnios, hydrops fetalis, altered organ growth, various placental and skeletal defects, immunological defects and increased perinatal death. This unusual phenotype has also been observed in other species, including humans and mice (for reviews, see [1-6]). We have accumulated evidence that the clue for understanding the underlying mechanism of LOS is alterations in gene expression patterns in the early embryo. Here, we summarize results from recent studies in our laboratory on this subject.

Effect of IVP systems on gene expression patterns Various culture systems have been employed to conduct in-depth and extensive studies to unravel gene expression patterns in bovine pre-implantation embryos, mainly with the aid of sensitive RT-PCR assays. We have accumulated evidence that bovine embryos produced in vitro differ substantially from their in vivo derived counterparts with regard to mRNA expression. Transcripts for the gap junction protein Connexin43 (Cx43) which is necessary for the maintenance of compaction were detected in bovine morulae and blastocysts grown in vivo. In contrast, blastocysts and hatched blastocysts produced in vitro did not express detectable amounts of this gene, independently of whether the culture system was supplemented with serum or BSA [7,8]. Furthermore, the expression pattern of the LIF-LIF-receptor system (Leukemia Inhibitory Factor) was investigated in bovine in vitro produced embryos and was compared with that for in vivo derived embryos [9]. The LIF-LIF-receptor system is thought to be critically involved in early differentiation of the mammalian embryo. No LIF ligand transcripts were found in in vitro produced morulae and blastocysts while transcripts for this gene were detected in their in vitro derived counterparts. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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In contrast to in vivo embryos the LIF-receptor ß (LR-ß) was not detected in in vivo derived morula to blastocyst stages while the gp130 transcript was found from the morula to the hatched blastocyst stage in both in vitro and in vivo produced embryos.

Figure 1. Schematic diagram of the semi-quantitative RT-PCR assay employed in experiments on bovine embryonic mRNA expression

¾ Addition of globin RNA as internal standard ¾ Preparation of RNA ¾ Reverse transcription (RT) of RNA in cDNA ¾ Polymerase chain reaction (PCR) ¾ Agarose gel electrophoresis and ethidium bromide staining ¾ Digitalisation and quantification of the gel photograph employing a computer-assisted image analysis system ¾ Calculation of the relative abundance of each product (ratio between the intensity of the gene specific fragment and that of the globin band) Employing a semi-quantitative RT-PCR assay (Figure 1), the relative abundance of a set of “marker genes” in embryos cultured in media supplemented with either serum or PVA (polyvinyl alcohol, e.g. chemically defined medium) was investigated [10]. Developmentally important gene transcripts that were selected for analysis were involved in compaction and cavitation [(Cx43), desmosomal protein plakophilin (Plako), desmosomal glycoproteins, desmocollin II and III (Dc II and III)], energy metabolism [glucose transporter-1, (Glut-1)], RNA processing [polyA polymerase, (PolyA)], stress-response [heatshock protein 70.1 (Hsp 70.1)] and maternal recognition of pregnancy [interferon tau, (IF W)]. Transcription of the majority of the genes was increased in PVA supplemented/serum-free medium derived embryos compared with their serum-generated counterparts mainly beginning at the 8- to 16-cell stage of development, e.g. maternal-embryonic transition of genomic activity. The exception from that observation was Hsp 70.1 that was significantly increased in embryos derived from serum enriched in vitro culture. These findings indicate a stress response of the early embryo to suboptimal culture conditions. In another MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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22 – GENE EXPRESSION PATTERNS IN BOVINE IN VITRO PRODUCED AND NUCLEAR TRANSFER… study, the effects of two basic culture systems (TCM with 5% CO2 in air vs. SOF with 7% O2, 88% N2, 5% CO2) each supplemented with different proteins (serum, BSA or PVA) on the expression of the above mentioned panel of marker genes was investigated. Results revealed that the basic culture system including medium composition and oxygen tension had profound effects on the level of specific transcripts whereas the protein source was less important [11]. More differences with regard to gene expression were found between TCM-generated embryos and their in vivo counterparts than between SOF and in vivo derived embryos. Interestingly, no differences with respect to transcriptional activities of the above genes in embryos generated under chemically defined conditions in two different laboratories were detected [11]. These findings provide a major step forward towards the standardization of in vitro production systems for the generation of bovine embryos of high quality. We have also shown that in vitro culture affects dosage compensation of X-linked genes, i.e. phosphoglycerate kinase (PGK) and glucose-6-phosphate dehydrogenase (G6PD). Comparing male and female embryos of in vivo and in vitro origin revealed that dosage compensation of PGK was delayed and no dosage compensation has occurred for G6PD in IVP embryos, although Xist transcripts were highly abundant in embryos of in vitro origin. The lack of X-chromosome inactivation inspite of the abundance of Xist transcripts in bovine IVP embryos suggests that timing and magnitude of Xist expression alteration do not necessarily correlate with a tightly controlled inactivation process [11]. Recently, for the first time a relationship between the incidence of altered gene expression patterns in early embryonic development and LOS in IVP calves has been demonstrated [12]. Bovine blastocysts were used to analyze cellular parameters, i.e. the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e. the relative abundance of developmentally important genes, i.e. glucose transporter Glut-1, Glut-2, Glut-3, Glut-4, heat shock protein 70.1 (Hsp), Cu/Zn-superoxide dismutase (SOD), histone H4.1 (H4), basic fibroblast growth factor (bFGF), and receptors of insulin-like growth factor-1 and -2 (IGF1R, IGFIIR). Blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in SOF medium supplemented with BSA or human serum or by in vivo culture in sheep oviducts. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high serum or BSA MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of transcripts for Hsp, SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups (Figure 2).

Figure 2

1.20

6.00 b

1.00 0.80

5.00

b b

b

4.00

b

0.60

3.00

ab

0.40 0.20

b

a

b b

a

a a

0.00

a

Hsp

a a

SOD

Glut-1

Glut-3

Glut-4

2.00 1.00 0.00

0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10

b a

b b

a a a

a

0.00

IGF-IR

bFGF

H4

IGF-IIR Expression pattern of various developmentally important gene transcripts in bovine blastocysts of different origin (vertical striped bars: in vivo, open bars: ligated sheep oviduct, gray bars: SOF-BSA, dark bars: SOF-serum). Bars with different superscripts differ significantly (a:b p d 0.05). (Data from Lazzari et al., 2002).

Birth weights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI (Table 1).

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24 – GENE EXPRESSION PATTERNS IN BOVINE IN VITRO PRODUCED AND NUCLEAR TRANSFER… Table 1. Gestation length and birth weights of calves derived from the different production systems Group SOF-BSA SOF-serum Sheep oviduct In vivo (SO) In vivo (AI)

Number of calves 23 10 34 63 24

Gestation length [days]

Birth-weights [kg]

Number > 50 Kg [%]

Number > 60 Kg [%]

281.1 ± 7.1a 280.1 ± 5.9a 279.2 ± 5.3a 279.4 ± 5.1a 281.7 ± 4.3a

52.8 ± 56.7 ± 44.1 ± 41.1 ± 43.4 ±

13 (59.1% a) 5 (50.0% a) 4 (11.8%b) 1 (1.6% c) 0

5 (22.7%a) 5 (50.0%a) 1 (2.9% b) 0 0

9.4a 12.1a 5.5b 3.0b 4.3b

Values within columns with different superscripts are significantly different (p < 0.05). Data from Lazzari et al., 2002.

These results support the hypothesis that persistence of early deviations in development may be causally involved in the incidence of LOS, in particular in increased birth weights [12].

Effects of NT procedure on gene expression patterns In an effort to unravel effects of somatic nuclear transfer on transcriptional activities in cloned embryos we have recently determined the relative abundance of eight specific gene transcripts in reconstructed blastocysts derived from various distinct modifications of the nuclear transfer protocol and compared the findings with those for in vitro and in vivo derived embryos [13]. The genes analyzed were Dc II, E-cadherin, Glut-1, Hsp 70.1, interferon tau, Mash2, IGF2r, Dnmt (DNA methyltransferase) employing either fusion before activation (FBA) or activation and fusion simultaneously (AFS). Hsp mRNAs could not be found in NT-embryos from either protocol whereas Hsp transcripts were detected from in vitro produced embryos that were analyzed in parallel. The relative abundance of interferon tau transcripts was significantly increased in embryos derived from AFS and IVP compared to the FBA treatment. The use of either G0 or G1 donor cells significantly reduced the relative amounts of Dnmt transcripts and significantly increased the relative abundance of Mash2 compared to IVP embryos. In addition, the interferon tau transcript levels were significantly elevated in NT-derived blastocysts employing G1 donor cells for NT compared to IVP embryos and those generated from G0 cells. Similar results were obtained when donor cells from passages no. 5/6 vs. passage 8 were compared in nuclear transfer. These data show that modifications of the nuclear transfer protocol are related to distinct alterations in the expression patterns of the resulting embryos. In this study an aberrant expression pattern in NT-derived embryos was found with respect to stress susceptibility, trophoblastic function and DNA methylation during preimplantation development [13]. Nuclear transfer also affects MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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expression of X-chromosome linked genes. While the relative abundances of the genes for the glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK) were similar in in vitro and in vivo produced and NT-derived embryos, the level for the X-inactive specific transcript (Xist) was significantly higher in embryos derived from adult donor cells than in those derived from fetal donor cells [14]. These findings show that the type and treatment of the donor cells affect expression patterns of specific genes in reconstituted embryos. To extend the possibilities of investigating gene expression patterns in embryos derived from livestock species, we have developed a sensitive cDNA technology for single bovine embryos. This technology uses data from a murine cDNA library for which only few (~1000) genes have been mapped and sequenced in cattle. The critical steps involve amplification by T7 and global PCR. A panel of 100 genes was selected that is informative for differentiation, housekeeping, cell cycle, apoptosis, growth and development, etc. Preliminary data show that differences in mRNA expression in embryos derived from various origins can consistently and reliably be determined by this approach [15].

Concluding remarks Collectively, our data show that the in vitro environment has profound effects on gene expression patterns in preimplantation bovine embryos. The marked differences between in vivo and in vitro derived embryos indicate that current in vitro production systems may lead to persistent alterations of gene expression patterns and may thus be a causative mechanism involved in the incidence of LOS. These alterations are thought to be induced by epigenetic modulation of gene expression patterns likely by changes in the methylation pattern [6]. To substantiate this suggestion, we have initiated studies in bovine embryos in which we look at the methylation status of selected genes which are imprinted during murine development as well as at the global methylation pattern employing an antibody against methylated cytosines.

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References

1. Walker SK, Hartwich KM and Seamark RF (1996), The production of unusually large offspring following embryo manipulation, Concepts and challenges. Theriogenology, 45, 111-120. 2. Kruip ThAM and den Daas JHG (1997), In vitro produced and cloned embryos, Effects on pregnancy, parturition and offspring. Theriogenology 47, 43-52. 3. Young LE, Sinclair KD and Wilmut I (1998), Large offspring syndrome in cattle and sheep. Rev. Reprod. 3, 155-163. 4. Renard JP, Chastant S, Chesne P, Richard C, Marchal J, Cordonnier N, Chavatte P and Vignon X (1999), Lymphoid hypoplasia and somatic cloning. Lancet 353, 1489-1491. 5. Sinclair KD, Young LE, Wilmut I and McEvoy TG (2000), In-utero overgrowth in ruminants following embryo culture, lessons from mice and a warning to men. Hum. Reprod. 15, 68-86. 6. Niemann H and Wrenzycki C (2000), Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions, Implications for subsequent development. Theriogenology 53, 21-34. 7. Wrenzycki C, Herrmann D, Carnwath JW and Niemann H (1996), Expression of the gap junction gene connexin43 (Cx43) in pre-implantation bovine embryos derived in vitro or in vivo. J. Reprod. Fertil. 108, 17-24. 8. Wrenzycki C, Herrmann D, Carnwath JW and Niemann H (1998), Expression of RNA from developmentally important genes in preimplantation bovine embryos produced in TCM supplemented with bovine serum albumin (BSA). J. Reprod. Fertil. 112, 387-398. 9. Eckert J and Niemann H (1998), mRNA expression of leukemia inhibitory factor (LIF) and its receptor subunits glycoprotein 130 (gp

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130) and LIF receptor-E (LR-E) in bovine embryos derived in vitro or in vivo. Mol. Human Reprod. 4, 957-965. 10. Wrenzycki C, Herrmann D, Carnwath JW and Niemann H (1999), Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev. 53, 8-18. 11. Wrenzycki C, Herrmann D, Keskintepe L, Martins Jr A, Sirisathien S, Brackett B and Niemann H (2001a), Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum. Reprod. 16, 893-901. 12. Lazzari G, Wrenzycki C, Duchi R, Kruip T, Niemann H and Galli C (2002), Cellular and molecular deviations in bovine in vitro produced embryos are related to the Large Offspring Syndrome. Biol. Reprod. 67, 767-775. 13. Wrenzycki C, Wells D, Herrmann D, Miller A, Oliver J, Tervit R and Niemann H (2001b), Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts. Biol. Reprod. 65, 323-331. 14. Wrenzycki C, Lucas-Hahn A, Herrmann D, Lemme E, Korsawe K and Niemann H (2002), In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK and Xist in preimplantation bovine embryos. Biol. Reprod. 66, 127-134. 15. Brambrink T, Wabnitz P, Halter R, Klocke R, Carnwath J, Kues W, Wrenzycki C, Paul D and Niemann H (2002), Application of cDNA arrays to monitor mRNA profiles in single preimplantation mouse embryos. BioTechniques 33, 3-9.

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Chapter 3 Characterization of cDNA Libraries Developed from Porcine Oocytes and Embryos to Determine Differential Expression Patterns Kristin Whitworth,1 Gordon k. Springer,2 L. Joe Forrester,3 William G. Spollem,2 Jim Ries,2 Clifton N. Murphy,1 Nagappan Mathialagan,4 Brad A. Didion,4 Jonathan A. Green1 and Randall S. Prather1 1) Department of Animal Science, 2) Computer Engineering and Computer Science, 3) University of Missouri DNA Core Facility, University of Missouri-Columbia, Columbia, Missouri 65211. 4) Monsanto Company,Chesterfield, Missouri. E-mail: [email protected]

Abstract. Despite decades of research on oocytes and embryos, there is currently little known about the genetic events that take place during early development in the pig. We have recently initiated a large-scale cDNA sequencing project to provide molecular information regarding the transcripts expressed by porcine oocytes, 4-cell stage embryos (in vivo and in vitro produced) and blastocysts (in vivo and in vitro produced). Plasmid cDNA libraries were generated by using a SMART-PCR based amplification of reverse-transcribed mRNA from each source. Each library was characterized as to the number of clones, average insert size and average poly(A) length. Over two thousand sequencing attempts were performed on each library (10,848 total), resulting in 8,661 ESTs. Analysis of the sequences by clustering algorithms revealed 2,576 unique sequences, of which 1,188 have yet to be identified from other sequencing projects, i.e., they had a Blast score of less than 200 when GenBank and TIGR databases were searched. Sequencing of the libraries identified numerous changes in expression of several of the ESTs at different stages of development and between the in vivo and in vitro produced embryos. The identification and characterization of such differentially expressed genes provides a preliminary view of those genes whose transcription is markedly up- or down-regulated during early development. Such results also define those genes that are aberrantly expressed due to in vitro culture.

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Introduction The goal of the swine genomics consortium at the University of Missouri-Columbia was to determine differential expression patterns of reproductive tissues in the pig at different stages of development. The reproductive tissues were divided into 3 main areas of interest 1) embryo/oocyte; 2) endometrium/oviduct; and 3) follicle/ovary. cDNA libraries from these tissues were created and 51071 expressed sequence tags (ESTs) were sequenced. This sequence analysis was then used to determine a pattern of differential expression. The objective of this paper is to describe the techniques involved in creating cDNA libraries from pig embryos and oocytes and to briefly discuss the results obtained from the sequences. Early embryo development from oocyte to blastocyst is a unique transcriptional process. Porcine oocytes that are matured, fertilized and developed in vitro can be transferred into a recipient pig and produce live offspring [1]. However, these embryos are produced at a lower efficiency and are not as developmentally competent as their in vivo produced counterparts. To determine transcriptional differences between these embryos, cDNA libraries from germinal vesicle oocytes, in vitro produced 4-cell stage embryos and blastocysts and in vivo derived 4-cell stage embryos and blastocysts were sequenced on a large scale (~2000 clones/library). Due the small amount of mRNA in oocytes and embryos, a modified SMART PCR cDNA synthesis (Clontech, Palo Alto,CA) was used [2]. The Switching Mechanism at 5  HQG RI 51$ 7UDQVFLSW 60$57  system (Figure 1) utilizes the terminal transferase (TdT) activity of MMLV reverse transciptase (RT). A modified oligo dT primes first strand synthesis. When the RT reaches the 5  HQGRI WKH P51$ WKH HQ]\PH¶V 7G7 DFWLYLW\ adds additional deoxycytidines (dC) to the 3  HQG RI WKH F'1$ 7KH modified SMART oligo contains a string of deoxyguanidines (dG) at its 3  end. The RT switches templates and continues replicating to the end of the oligonucleotide [3]. This template is then used to amplify the cDNA prior to ligating into a vector and electroporating into bacteria. The PCR-based cDNA library is then sequenced to determine differential expression patterns in oocytes and embryos.

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Materials and methods Tissues and RNA isolation Oocytes were aspirated from slaughterhouse derived ovaries and stripped of their cumulus cells. In vitro produced 4-cell and blastocyst stage embryos were created and cultured 3 to 7 days, respectively [1]. In vivo produced 4-cell and blastocyst stage embryos were collected on days 3 and 6, respectively [5] except that the embryos were flushed with TL Hepes [6].

Poly (A) RNA extraction Poly(A) RNA from 200 oocytes or 50 embryos was extracted by using a micro poly (A) kit (Ambion, Austin, TX) according to the manufacturer’s instructions with minor modifications in the elution buffer which consisted of 10 mM Tris (pH 7.5), 1 mM EDTA, and 0.5% SDS heated to 65qC. Due to the low amount of mRNA, the RNA integrity and purity was not verified.

Reverse transcription and terminal transferase of cDNA. 7ZR /RIP51$IURPRRF\WHVRUHPEU\RVZDVDGGHGWR /RIHDFK ROLJR G7 SULPHU VSHFLILF IRU HDFK OLEUDU\   / RI WKH 60$57   ORQJ SULPHU  J /   $$*-CAG-TGG-TAA-CAA-CGC-AGA-GTA-CGAATT-CGT-CGA-CG-C-GGG 3  /RI51$VH-,QXQLWV /$PELRQ DQG /RI'(3&WUHDWHGZDWHU>@7KLVZDVKHDWHGWRqC for 10 min, and cooled to 37q&IRUPLQ3UHKHDWHG57PL[ / /;VWVWUDQG V\QWKHVLV EXIIHU  /  0 '77  /  P0 G173 DQG  / '(3& ZDWHU  ZDV WKHQ DGGHG $IWHU  PLQ  / 6XSHrScript II MMLV reverse transcriptase was added. The RT was performed by annealing poly(A) RNA with 1 Pg of an oligo(dT) primer (5  *$&-TAG-TTC-TAG-ATC-GCGAGC-GGC-CGC-tag-TTT-TTT-TTT-TTT-TTT-TTT-TTT-TTT 3  ZKHUH tag = library-specific five or six base sequence identifier) at 42qC for 1 hour.

PCR amplification of embryo and oocyte cDNA $ /3&5PL[ZDVDOLTXRWHGRQLFHZLWKWKHIROORZLQJUHDJHQWV /P0G173/LIH7HFKQRORJLHV,QF5RFNYLOOH0'  / 6PDUW 6KRUW SULPHU  QJ /   $$*-CAG-TGG-TAA-CAA-CGC-AGA-GTAC 3   /   3&5 OLEUDU\ SULPHU  QJ /   *$&-TAG-TTC-TAGATC-GCG-AGC-GG 3   / ; FORQHG SIX EXIIHU  /  XQLWV  SIX WXUER SRO\PHUDVH 6WUDWDJHQH /D -ROOD&$  DQG  / '(3& ZDWHU3&5 amplification was performed witK  / RI WKH  / 57 UHDFWLRQ DV template. The PCR conditions consisted of a 95qC initial denaturation for

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32 – CHARACTERIZATION OF CDNA LIBRARIES DEVELOPED FROM PORCINE OOCYTES AND EMBRYOS… 3 minutes and 30 cycles of 95qC denaturation for 30 seconds, 53qC annealing for 30 seconds, and 72qC elongation for 3 minutes.

Smart cDNA Library Production mRNA: Smart primer: SalI -GGG

AAA +

TTT+NotI

:Oligo dT primer

cDNA synthesis (RT) Terminal Transfer of dC GGG

AAA

SalI+CCC

sscDNA

TTT+NotI

PCR with pfu-turbo (Stratagene) SalI +CCC

TTT+NotI

SalI+GGG

AAA+NotI

dscDNA

Not I/SalI Restriction Digest PCR Clean-up (Roche) SIZING Chroma-Spin 400 Chroma-Spin 1000

PCR conditions 95o 3 min (1 cycle) 95o 30 sec 53o 30 sec (30 cycles) 72o 3 min

Ligate into pSPORT vector Transform into DH10E

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Restriction digest and sizing of PCR amplified library The PCR products were restriction digested in NotI and SalI at 37qC for 3 hours. The restriction digested PCR amplified library was cleaned up by using a High Pure PCR product purification kit (Roche, Mannheim, Germany). Following PCR cleanup, reactions were sized using a Chroma Spin-400 column (Clontech) and sized again using a more stringent Chroma Spin-1 000 column.

Ligation of PCR amplified library in pSport and transformation into DH10B. The PCR amplified library was ligated into NotI/SalI digested pSPORT (Life Technologies, Inc.) vector by standard protocol and incubated at 4qC IRU  KRXUV )ROORZLQJ OLJDWLRQ  / RI OLJDWLRQ ZDV HOHFWURSRUDWHG LQWR 25 / RI '+% (OHFWURPD[ FRPSHWHQW Fells (Life Technologies, Inc.). $IWHU UHFRYHU\  DQG  / RI WUDQVIRUPDQWV ZHUH SODWHG RQ /%-agar SODWHV VXSSOHPHQWHG ZLWK  J / FDUEHQLFLOOLQ 6LJPD 6W /RXLV 02  Colonies were counted to determine the number of recombinants for each library.

cDNA sequencing Bacteria clones from each library were randomly picked and grown in 96-ZHOO SODWHV FRQWDLQLQJ  P/ WHUULILF EURWK SOXV  JP/ DPSLFLOOLQ Plasmids were purified by using the QIAprep 96 Turbo Miniprep kit (Qiagen,Valenicia, CA). Sequencing reactions were designed to yield 3  sequence by using Sp6 promoter primer and the ABI Prism BigDye Terminator cycle sequencing chemistry on an ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA).

Results and discussion Quality assessment of cDNA libraries The quality of each cDNA library was first assessed by restriction digestion of ninety-six randomly picked clones to determine the average insert size and the percentage of clones without inserts. Acceptable libraries were assessed by sequencing 288 randomly picked clones to determine the percentage of clones with long poly(A) tails [> 40 adenines]. The goal for the cDNA libraries to qualify for large-scale sequencing was the following criteria: 1) more than 95% of the clones contained inserts; 2) average insert size was greater than 1 kb; 3) less than 10% of the clones contained long MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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34 – CHARACTERIZATION OF CDNA LIBRARIES DEVELOPED FROM PORCINE OOCYTES AND EMBRYOS… poly(A) tails; and 4) the complexity of the library was at least 106 colony forming units.

Library quality The library quality is presented in Table 1. The quality of these libraries was lower, i.e. short inserts were obtained, than we achieved without amplification of the cDNAs [7, 8]. Nevertheless a large number of ESTs were generated that provided information about stage specific expression. These individual ESTs were clustered into a unigene set of 2,559 members. The number of unigene ESTs that do not have any matches in the genome databases (unique ESTs) was 1,173 (45.8%). The individual novelty rate between libraries ranged from 45% to 70.8%.

Table 1. Quality of oocyte/embryo libraries

Total sequenced Total recombinants Empty vector (%) Average insert size (bp) Long Poly(A) Tails(%) Novelty Rate

pgvo 2 304 2.8 X 106

p4civp 2 016 3.3 X 106

Library namea p4civv pblivp 2 016 2 304 2.1 X 106 2.0 X 106

pblivv 2 208 3.7 X 105

119 (5.2)

341 (16.9)

14 (0.7)

8 (0.4)

42 (1.9)

617

821

356

512

610

167 (7.2)

95 (4.7)

59(2.9)

31(1.3)

465(21.1)

70.8%

45.0%

52.6%

51.1%

57.1%

a) pgvo- germinal vesicle stage oocyte. p4civp- in vitro produced 4-cell stage embryo. p4civv- in vivo produced 4-cell stage embryo. pblivp- in vitro produced blastocyst stage embryo. pblivv- in vivo produced blastocyst stage embryo.

Differential expression Interesting differential expression patterns were observed between porcine oocytes, 4-cell stage embryos and blastocysts. Differential expression patterns were also observed between in vitro produced and in vivo derived embryos of the same stage. These details of these interesting differential expression patterns will be published in an upcoming manuscript [9]. The differential expression patterns observed in the endometrium/oviduct libraries and the follicle/ovary libraries will also be MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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published in upcoming manuscripts [7, 8, respectively]. After publication of this data, the clone names, sequence information and annotations will be submitted to GenBank for public access.

Conclusions High quality cDNA libraries can be made from porcine embryos and oocytes with a small amount of mRNA by using a modified SMART PCR protocol. Microarray analysis and real time PCR will be performed to confirm the differential expression patterns suggested by these libraries. Additionally, the importance of specific differentially expressed transcripts will be further investigated individually. A detailed description of the project and the analysis pipeline is presented at our website (porcine.rnet.missouri.edu/). A similar USDA-funded project on bovine reproductive tissues is underway and all data will be posted on a weekly basis during the sequencing phase of the project (bovine.rnet.missouri.edu/).

Acknowledgements Project was supported by Monsanto Company.

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References

1. Abeydeera LR. (2000), Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 54: 787-97. 2. Adjeye J., Bolton V, and Monk M. (1999), Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos. Gene 237: 373-383. 3. Chenchik A, Zhu YY, Diatchenko L, Li R, Hill J, and Siebert PD (1998), Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR. BioTechnique Books, MA 305-319. 4. Abeydeera, LR, Wang W, Prather RS, and Day, BN (1998): Maturation in vitro of pig oocytes in protein-free media: Fertilization and subsequent embryo development in vitro. Biol Reprod 58:1316-1320. 5. Anderson JE, Matteri RL, Abeydeera LR, Day BN, and Prather RS (1999), Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo-and in vitro-derived 4-cell porcine embryos. Biol Reprod 61: 1460-1467. 6. Hagen DR, Prather RS, Sims MM, and First NL (1991), Development of one-cell porcine embryos to the blastocyst stage in simple media. J Anim Sci 69: 1147-1150. 7. Green JA, et al., (in preparation). 8. Jaing et al., (in preparation). 9. Whitworth KM, Springer,GK, Forrester LJ, Spollen WG, Ries J, Lamberson WR, Murphy CN, Mathialigan N, Didion BA, Green JA, and Prather RS. Developmental expression of over 2,500 genes during pig embryogenesis: An EST project (in preparation).

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Chapter 4 Systematic Analysis of Mouse Preimplantation Development Wency l. Kimber, Yulan Piao, Tetsuya s. Tanaka, Toshiyuki Yoshikawa, Toshio Hamatani, Mark G. Carter, and Minoru S.H. Ko Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging (NIA), National Institutes of Health (NIH), Baltimore, MD 21224, USA. E-mail: [email protected]

Abstract. Many important and complex events occur during the preimplantation period of mouse development as the embryo transitions from a single totipotent cell to the differentiated structure of the blastocyst. Using an embryogenomics approach, we aim to identify and study genes with key roles in these processes. Our study utilizes the unique resource of the NIA mouse cDNA clone set, which contains many novel, full-length cDNAs representing all mouse preimplantation stages. This clone set provides the basis for microarray experiments enabling us to study global gene expression and assemble gene expression profiles. In addition, we are using EST frequency in individual libraries to identify genes with restricted expression profiles, implying a role at specific stages of preimplantation development. This work is complemented with functional assays including gene “knock-down” experiments using antisense oligo technology. These studies lay the foundation for a systematic evaluation of the role of large numbers of genes in preimplantation development.

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Introduction Mouse preimplantation development encompasses embryonic development from fertilization up until implantation of the blastocyst into the uterine wall. Many complex developmental events take place during this period, including the transition from maternal to zygotic transcription, accompanied by massive degradation of maternal proteins and transcripts (reviewed in [1-5]). The embryonic axes become established during this time, and complex changes at the genomic level also occur, including the loss and then selective reapplication of DNA methylation. Preimplantation development culminates in the formation of the blastocyst, representing the first mammalian differentiation event. During this developmental phase, the embryo has become transformed from a single totipotent egg to a multipotent trophectoderm and a pluripotent Inner Cell Mass (ICM). We are interested in this progressive loss of totipotency, the transition from an immortal to mortal cellular state, and the developmental processes and genes that govern this.

Embryogenomics approaches The complexity and scale of events during embryonic development mean that the traditional “one gene one effect” analytical approaches are of limited value, and therefore gene interactions must be studied on a larger, more global scale. The advent of genomics has raised this possibility, with an approach called “embryogenomics” which allows the simultaneous analysis of large numbers of developmentally expressed genes using large-scale genomics methodologies [6]. The core of this approach is expression profiling by cDNA microarrays. This technology relies heavily on the availability of appropriate Expressed Sequence Tags (ESTs), and therefore the first step towards this goal is the collection of all expressed genes from the appropriate tissues and embryonic stages (project [7]), to produce complementary DNA (cDNA) libraries.

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Mouse cDNA project Our laboratory’s particular emphasis is on early mouse embryonic development, utilizing preimplantation embryos and stem cells. This stage is of special scientific importance, as it is under-represented in both mouse and human transcript databases, and therefore appropriate microarray platforms are unavailable. The study of preimplantation development in human embryos is difficult due to the unavailability of early stage embryos for ethical and technical reasons. The mouse does not suffer from these limitations, and the close parallels between mouse and human preimplantation development enables us to use the mouse as a model organism. The collection of sufficient material is challenging however even in the mouse, due to the small size of preimplantation embryos. To overcome this, we developed a PCR-amplification method to construct cDNA libraries from very small amounts of embryonic RNA [8-11]. Clones from these cDNA libraries have inserts ranging from 0.5 to 3.0 Kb, with an average insert size of 1.5 Kb. In addition to sample size, another challenge faced, was the incomplete coverage of open reading frames due to relatively small insert size. Recently we have overcome this by employing a novel linker-primer design that allows differential amplification from a complex mixture (average 3.0 Kb with size ranges of 1 – 7 Kb)[12]. This method facilitates the generation of cDNA libraries enriched for long transcripts without size selection of insert cDNA. All of our recent cDNA libraries have been made by this new method, and thus, a significant fraction of these cDNA clones contain complete open reading frames, and can be considered “full length” clones. To date, the NIA cDNA project has generated 224,511 ESTs, from nearly 50 individual libraries. Individual NIA cDNA clones are currently available from the American Type Culture Collection (ATCC) and information on cDNA clones, including DNA sequences, can be found on our web site (http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html), as well as in public databases such as GenBank.

Nia 7.4k and 15k mouse cDNA clone set In order to utilize these cDNA clones for cDNA microarray analysis, a condensed set of unique cDNA clones is desirable. The generation of such a cDNA clone set containing all preimplantation transcripts/genes has been one of the main goals of our cDNA project. Although the original strategy was the collection of unique cDNA clones by the cDNA normalization procedure [8, 9], the sequence information (ESTs) from many cDNA clones makes it possible to select unique clones based on their sequence information. The first condensed, non-redundant clone set assembled from our collection was the “NIA 15K Mouse cDNA Clone Set” containing MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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40 – SYSTEMATIC ANALYSIS OF MOUSE PREIMPLANTATION DEVELOPMENT 15,247 cDNA clones [13]. These unique clones were selected from approximately 53,000 3’-ESTs derived from preimplantation stages (unfertilized eggs, 1-cell, 2-cell, 4-cell and 8-cell embryos, morulae and blastocysts [11], micro-dissected tissues of embryonic and extra-embryonic parts of E7.5 embryos [10], female gonad/mesonephros from E12.5 embryos, and ovary from newborn fetus. Approximately half of the clones from this collection represent unique transcripts with unknown functions [14]. Recently, we completed assembly of the “NIA 7.4K Mouse cDNA Clone Set [15]”, a non-redundant collection of cDNAs non-overlapping with the 15K clone set. These cDNA clones were derived from embryonic tissues (E0.5 to E12.5), as well as the following stem cell lines: embryonic stem (ES) cells, trophoblast stem (TS) cells, mesenchymal stem (MS) cells, neural stem (NS) cells, hematopoietic stem (HS) cells, and embryonic germ (EG) cells. Both the NIA mouse 15K and 7.4K clone sets are available without restriction, and have been distributed to 10 academic centers, for further distribution to over 200 research centers world-wide (see our web site for details, http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html).

Preimplantation ESTs: stage-specific genes In addition to their use in microarrays, EST collections can give information on the expression levels of each gene by the frequency in which they appear in individual libraries. Using the PCR-based cDNA library construction method described above, we generated cDNA libraries from preimplantation embryos, at various stages from unfertilized eggs to blastocysts, and obtained around 3,000 ESTs from each library [11]. These ESTs were grouped into clusters based on their sequence similarity, and the frequency for each cluster (which represents individual genes) was calculated. The EST frequency in each cDNA library roughly corresponds to the expression level, and so the gene expression patterns during preimplantation development can be determined. In addition to a collection of genes whose expression levels are relatively constant, we found four interesting patterns of gene expressions (Fig. 1). First, there is massive degradation of the large number of RNAs stored during oogenesis. Second, there are genes that initiate their transcription at a certain stage and then maintain their expression. Third, there are genes following both first and second patterns. These three expression patterns have been previously described, and our work has confirmed this with numerous examples. However, we were surprised to see a fourth pattern, that of a spike-like transient expression at a specific stage of development. For example, we observed genes whose expression was low in the beginning, but increased dramatically at 4-cell stage, and then declined sharply afterwards. Based on this new finding, we speculated that these stage-specifically expressed genes MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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play important roles and may indeed drive preimplantation development. In addition, we have localized 798 genes from this collection on the mouse genetic map and observed interesting trends in the map location of genes, i.e., the clustering of co-expressed genes [11], which we have also shown previously in the gene collection from extraembryonic tissues [10]. A large number of genes from these collections have been also been mapped to the Radiation Hybrid map of the mouse genome by the MRC UK Mouse Genome Center [16].

Figure 1. Schematic representation of gene expression patterns during mouse preimplantation development

Microarray analysis of morula vx blastocyst Since the EST frequency is only an approximate measure of gene expression levels, the stage specific nature of genes has to be further MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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42 – SYSTEMATIC ANALYSIS OF MOUSE PREIMPLANTATION DEVELOPMENT validated by microarray analysis. We have recently performed the global gene-expression profiling of morula versus blastocyst gene expression using the NIA 15K mouse cDNA microarray [17]. The gene expression levels were measured four times for blastocyst and five times for morula stage embryos. The student’s t-test at a 5% confidence level identified 428 genes up-regulated in the blastocyst, and 748 which were down-regulated. Up-regulated genes included Mist1, Id2, Hd1, and Requiem, whereas down-regulated genes included the CREB-binding protein, Per3, the zinc finger protein Zf217, Krox-25, and Miwi1. Among these differentially expressed genes, ten genes were subjected to semi-quantative RT-PCR analysis, seven of which showed consistent expression levels with those obtained from the microarray analysis. Thus, the microarray analysis further supports the notion of stage-specific genes, by clearly identifying genes that are down-regulated from morula to blastocyst. To further validate these results, we have collected approximately 2000 embryos from each stage of preimplantation development and are currently examining the expression profiles of ~22,000 genes by combining the NIA 15K and 7.4K clone sets.

Functional assays: antisense RNA experiments Follow up analysis of stage specific genes typically includes loss of function studies, which traditionally has been done on a gene by gene basis, using knockout techniques. This approach however, is time-consuming, and can only handle a few genes at a time, and, for preimplantation studies, also has the disadvantage that genes expressed at these stages typically exhibit an embryonic lethal phenotype, thus severely limiting the scope of the study. Therefore, in order to facilitate large scale gene analysis, a simple, high throughput in vitro assay must be developed, where embryonic development can be closely monitored and samples easily retrieved. We have been evaluating the use of antisense oligonucleotides to “knockdown” gene expression in vitro, as this technology should be applicable on a large scale. Previously, antisense oligos have been hampered by poor specificity and stability; however there have been a number of advances in oligo chemistry in recent years which have overcome these problems. We have evaluated two such oligo modifications, to identify one applicable for use in large scale gene suppression studies in mouse preimplantation embryos in vitro. Towards this end, we tested two modified antisense oligonucleotides [18], one, a 2’-O-methoxyethoxy (MOE) modification at the 2’-sugar position (2’-MOE), [19], and the other a Morpholino oligo, in which the riboside moiety of each subunit has been converted to a morpholine moiety with phosphorodiamidate linkages between subunits [20]. We chose E-cadherin as our test gene due to its well-characterized role in mouse preimplantation development [21-23], and MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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designed antisense oligos of both types with this sequence. These oligos were microinjected in fertilized eggs in vitro, and their development followed for 5 days. Over 1500 embryos were scored, and we found that injection of 2’-methoxyethoxy (2’-MOE)-modified oligonucleotides blocked development to the blastocyst stage (indicative of loss of E-cadherin function) in two-thirds of embryos. In contrast, embryos injected with 2’-MOE missense control oligonucleotides, or oligonucleotides with a Morpholino modification developed normally. Thus, antisense technology utilizing 2’-MOE-modified antisense oligonucleotides is a candidate approach for effective examination of the role of large numbers of genes during early embryological development. Further functional information can be deduced by examining the spatial location of gene expression profiles using RNA in situ analysis. This technique has been extensively applied to tissue sections and late stage embryos, but there are significant challenges in applying this technique to extremely small preimplantation embryos. In our lab, we have developed a high throughput in situ version of this methodology, which can successfully visualize gene expression patterns in preimplantation embryos. Using this technique, we have been able to distinguish between the two lineages of the blastocyst, and identify genes whose expression is restricted to each.

Prospect Embryogenomics approaches include the expression profiling by EST projects and cDNA microarrays, the analysis of spatial gene expression patterns by a large-scale whole-mount and tissue-section in situ hybridization, and functional analysis by gene knockdown and knockout technologies. Taken together, these resources should provide us with a better understanding of the global changes of gene expression levels during preimplantation development. Genes identified in these studies will provide insights into mammalian preimplantation development, and such information may allow us to evaluate the well-being of preimplantation embryos for both human and farm animals. Thus, these approaches are important, not only for fundamental basic research, but also for the applied reproductive sciences.

Acknowledgments We would like to thank present and past members of the Ko laboratory for invaluable contributions to the research programs described above. In particular, we would like to thank Dawood Dudekula, Yong Qian, Vincent VanBuren, and Alexei Sharov for their contributions to the computational analyses of DNA sequence, and microarray data analyses, and Patrick Martin, Uwem Bassey, and Carole Stagg for their contributions in the cDNA project. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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References

1. Hogan, B, Beddington, R, Costantini, F, Lacy, E, Manipulating the Mouse Embryo: A Laboratory Manual. Second ed. 1994, New York: Cold Spring Harbor Laboratory Press. 2. Watson, AJ, Kidder, GM, Schultz, GA: How to make a blastocyst. Biochem Cell Biol 1992, 70:849-855. 3. Latham, KE, Schultz, RM: Embryonic genome activation. Front Biosci 2001, 6:D748-59. 4. Solter, D, de Vries, WN, Evsikov, AV, Peaston, AE, Chen, FH, Knowles, BB, Fertilization and activation of the embryonic genome, in Mouse Development. Patterning, Morphogenesis, and Organogenesis, J. Rossant and P.P.L. Tam, Editors. 2002, Academic Press: London. p. 5-19. 5. Zernicka-Goetz, M: Patterning of the embryo: the first spatial decisions in the life of a mouse. Development 2002, 129:815-29. 6. Ko, MSH: Embryogenomics: developmental biology meets genomics. Trends Biotechnol. 2001, 19:511-518. 7. Adams, MD, Kelley, JM, Gocayne, JD, Dubnick, M, Polymeropoulos, MH, Xiao, H, Merril, CR, Wu, A, Olde, B, Moreno, RF, Kerlavage, AR, McCombie, WR, Venter, JC: Complementary DNA sequencing: expressed seqeunce tags and human genome project. Science 1991, 252:1651-1656. 8. Ko, MSH: An ’equalized cDNA library’ by the reassociation of short double-stranded cDNAs. Nucleic Acids Res 1990, 18:5705-11. 9. Takahashi, N, Ko, MSH: Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos. Genomics 1994, 23:202-10. 10. Ko, MSH, Threat, TA, Wang, X, Horton, JH, Cui, Y, Pryor, E, Paris, J, Wells-Smith, J, Kitchen, JR, Rowe, LB, Eppig, J, Satoh, T, Brant, L, Fujiwara, H, Yotsumoto, S, Nakashima, H: Genome-wide mapping of unselected transcripts from extraembryonic tissue of 7.5-day mouse MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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embryos reveals enrichment in the t-complex and under-representation on the X chromosome. Hum Mol Genet 1998, 7:1967-78. 11. Ko, MSH, Kitchen, JR, Wang, X, Threat, TA, Hasegawa, A, Sun, T, Grahovac, MJ, Kargul, GJ, Lim, MK, Cui, Y, Sano, Y, Tanaka, T, Liang, Y, Mason, S, Paonessa, PD, Sauls, AD, DePalma, GE, Sharara, R, Rowe, LB, Eppig, J, Morrell, C, Doi, H: Large-scale cDNA analysis reveals phased gene expression patterns during preimplantation mouse development. Development 2000, 127:1737-49. 12. Piao, Y, Ko, NT, Lim, MK, Ko, MSH: Construction of Long-Transcript Enriched cDNA Libraries from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method. Genome Res 2001, 11:1553-8. 13. Tanaka, TS, Jaradat, SA, Lim, MK, Kargul, GJ, Wang, X, Grahovac, MJ, Pantano, S, Sano, Y, Piao, Y, Nagaraja, R, Doi, H, Wood, WH, 3rd, Becker, KG, Ko, MSH: Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc Natl Acad Sci U S A 2000, 97:9127-32. 14. Kargul, GJ, Dudekula, DB, Qian, Y, Lim, MK, Jaradat, SA, Tanaka, TS, Carter, MG, Ko, MSH: Verification and initial annotation of the NIA mouse 15K cDNA clone set. Nat Genet 2001, 28:17-8. 15. VanBuren, V, Piao, Y, Dudekula, DB, Qian, Y, Carter, MG, Martin, PR, Stagg, CA, Bassey, UC, Aiba, K, Hamatani, T, Kargul, GJ, Luo, AG, Kelso, J, Hide, W, Ko, MS: Assembly, Verification, and Initial Annotation of the NIA Mouse 7.4K cDNA Clone Set. Genome Res 2002, 12:1999-2003. 16. Hudson, TJ, Church, DM, Greenaway, S, Nguyen, H, Cook, A, Steen, RG, Van Etten, WJ, Strivens, MA, Trickett, P, Heuston, C, Davison, C, Southwell, A, Hardisty, R, Varela-Carver, A, Haynes, AR, Rodriguez-Tome, P, Doi, H, Ko, MSH, Pontius, J, Schriml, L, Wagner, L, Maglott, D, Brown, SDM, Lander, ES, Schuler, G, Denny, P: A Radiation Hybrid Map of Mouse Genes. Nature Genet. in press. 2001. 17. Tanaka, TS, Ko, MSH: A global view of gene expression in the preimplantation mouse embryo. Submitted. 18. Kimber, WL, Nitin Puri, N, Borgmeyer, C, Ritter, D, Seidman, M, Ko, MSH: Efficacy of 2-methoxyethoxy (2’-MOE)-modified antisense oligonucleotide for the study of mouse preimplantation development. Reprod Biomed Online in press.

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46 – SYSTEMATIC ANALYSIS OF MOUSE PREIMPLANTATION DEVELOPMENT 19. Altmann, KH, Fabbro, D, Dean, NM, Geiger, T, Monia, BP, Muller, M, Nicklin, P: Second-generation antisense oligonucleotides: structure-activity relationships and the design of improved signal-transduction inhibitors. Biochem Soc Trans 1996, 24:630-7. 20. Summerton, J, Weller, D: Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997, 7:187-95. 21. Takeichi, M: Cadherin cell adhesion receptors as a morphogenetic regulator. Science 1991, 251:1451-5. 22. Kemler, R: From cadherins to catenins: cytoplasmic protein interactions and regulation of cell adhesion. Trends Genet 1993, 9:317-21. 23. Pey, R, Vial, C, Schatten, G, Hafner, M: Increase of intracellular Ca2+ and relocation of E-cadherin during experimental decompaction of mouse embryos. Proc Natl Acad Sci U S A 1998, 95:12977-82.

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Chapter 5 Serial Analysis of Gene Expression (SAGE) in Preimplantation Stage Swine Embryos K.A. Zuelke,1,3 L. Blomberg,1E.L. Long,1 J.R. Dobrinsky,1 C.P. Van Tassell,2 T.S. Sonstegard2 USDA Agricultural Research Service, Biotechnology and Germplasm Laboratory,1 and Bovine Functional Genomics Laboratory,2 Beltsville, MD 20705 3

Correspondence: Dr. Kurt A. Zuelke USDA ARS ANRI BGL Bldg 200, Rm 124A, BARC-East Beltsville, MD 20705 Ph: +1 301 504 8545 Fax: +1 301 504 5123 Email: [email protected]

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Introduction Mammalian embryo development represents a continuum of molecular and cellular interactions whereby early events dictate or influence subsequent developmental outcomes. For example, conditions under which oocytes are matured can dramatically impact the rates of pronuclear and blastocyst development after in vitro fertilization and embryo culture [1]. Swine often exhibit high rates (> 30%) of early embryonic mortality following either artificial insemination or natural mating [2]. The efficiencies of producing swine embryos in vitro are also poor compared to other livestock species. Typically, approximately 20% of transferred in vitro produced swine embryos develop into live offspring after embryo transfer [3]. The long-term objective of the present study is to establish comprehensive gene expression profiles from the 4 critical stages of porcine embryo development depicted in Figure 1. Development from the 2- to 4-cell stages (day 2 (D2) after insemination) coincides the transition from maternal to embryonic genome expression within the embryo [4]. The blastocyst stage (D6) represents the earliest stage at which cellular polarity and morphologic differentiation occur within the embryo. Blastocyst stage embryos are also the stage most commonly transferred and cryopreserved in swine [5, 6]. The maternal recognition of pregnancy combined with the dramatic embryo elongation that occurs between day 11 (D11) and day 12 (D12) of gestation denote critical stages in porcine development that immediately precede tight adherence of the embryo to the uterine endometrial epithelium [7, 8]. Serial analysis of gene expression (SAGE) enables both qualitative and quantitative analysis of gene expression on a whole-transcriptome level [9]. The SAGE process consists of extracting mRNA template from cells of interest and performing a series of manipulations to isolate and sequence a population of 10-12 base-pair nucleotide “tags” (i.e. a SAGE library) wherein each tag is uniquely representative of an individual transcript represented in the original mRNA population. Bioinformatic analysis of SAGE tag sequence data identifies and quantitates the occurrence of each tag; the tag frequency is correlated to the frequency at which its respective transcript occurs in the mRNA population. We performed SAGE on in MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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vivo-derived D11 and D12 porcine embryos to identify and characterize critical gene expression events that occur during and between these key stages of early porcine embryo development.

Figure 1. Critical stages of porcine preimplantation embryo development

Filamentous (D12)

4-Cell (D2)

Expanded Blastocyst

Elliptical (D11)

(D6)

Materials and Methods Construction of SAGE Libraries Cycling gilts were synchronized, superovulated and artificially inseminated as previously described [6]. Total RNA was extracted from D11 and D12 embryos collected immediately after slaughter from 2 to 3 gilts per treatment group. The SAGE libraries were constructed from 5 Pg aliquots of total RNA extracted from pooled embryos (between 5 and 10 embryos per each treatment group) using the MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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50 – SERIAL ANALYSIS OF GENE EXPRESSION (SAGE) IN PREIMPLANTATION STAGE SWINE EMBRYOS I-SAGE® kit (Invitrogen, Carlsbad, CA). The protocol of this kit is based on the original SAGE methodology [9; see also (http://www.sagenet.org)]. The anchoring and tagging restriction enzymes used were NlaIII and BsmFI, respectively. Following PCR-amplification, di-tags (26mers) were released using NlaIII and separated via (12% w/v) polyacrylamide gel electrophoresis (PAGE). The 26mer di-tags were purified from the polyacrylamide gel, ligated to form concatemers, and size fractionated via (8% w/v) PAGE. Three size ranges of di-tag concatemers (300 – 500 bp, 500 – 800 bp, and >800 bp) were isolated. The 500 – 800 bp fraction was ligated into Sph I-linearized pZErO®-1 vector (Invitrogen) to construct the present libraries. Ligation products were transformed into One Shot®TOP10 ElectrocompTM cells (Invitrogen) by electroporation, and cultured overnight at 37°C on low salt LB agar plates containing 50 µg/ml of zeocin. SAGE tag inserts were amplified by inoculating individual colonies into separate wells of a 96-well PCR plate containing 25 µl of a PCR reaction mix consisting of 2.5 µl 10X PCR Buffer (200 mM Tris-HCL (pH 8.4), 500 mM KCL), 1.25 µl DMSO, 500 µM dNTP, 1.7 PM MgCl2, 0.2 µM M13 forward (5’-CCC-AGT-CAC-GAC-GTT-GTA-AAA-CG-3”) and reverse (5’-AGC-GGA-TAA-CAA-TTT-CAC-ACA-GG-3”) primers, and 1 Unit of PLATINUM®Taq DNA polymerase (Invitrogen). The thermocycling profile was denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 70°C for 90 sec for 27 cycles. PCR products were purified with a 96-well Montage PCR cleanup kit (Millipore, Bedford, MA) and recovered in 50 µl of nuclease-free water. For sequence analysis, 2 µl of the purified PCR product template was transferred to a 384-well plate containing 0.5 µl of Big-Dye v. 2.0 (Applied Biosystems, Foster City, CA), 1.5 µl of Big Dye extender (Sigma, St. Louis, MO), and 3.2 pmole of SP6 primer (5’-ATT-TAG-GTG-ACA-CTA-TAG-3”), and were then amplified using standard thermocycling conditions recommended by manufacturer of Big Dye. Reactions products were precipitated with four volumes of 70% (v/v) isopropanol and washed with four volumes of 70% (v/v) ethanol. After drying, reaction products were resuspended in 25 µl of Hi-Di Formamide (Applied Biosystems), denatured at 95°C for 5 min, and analyzed on an ABI-3700 automated DNA analyzer (Applied Biosystems, Foster City, CA).

Processing and Analysis of SAGE tag sequences Sequence quality assessment and trimming were performed using phred v0.980904.e. Vector sequence was identified and trimmed using “cross_match” with the “-minscore 18” and “-minmatch 12” options. Sequence information in the processed trace files was converted into text files, and the SAGE tags were extracted and quantified using SAGE 2000 software version 4.12 [http://www.sagenet.org/Software/software2000.htm]. Tag nucleotide sequences and frequency data were then output to MS MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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Access database files for subsequent analyses. For determining differential expression, tag frequencies between both SAGE libraries were analyzed for significance (P<0.05) using tools in the SAGE 2000 software based on a Chi-square analysis combined with Monte-Carlo simulations [10].

Annotation of SAGE tags Since the NlaIII recognition sequence “CATG” lies immediately five-prime of the SAGE tag sequence, “CATG” was appended five-prime to each SAGE tag to yield 14 bp tags for use in database comparisons [9]. Two primary sequence databases were used to ascribe tentative annotation to the porcine-derived SAGE tags. First, tag sequences were compared with a database of human SAGE tags (National Center for Biotechnology Information (NCBI), Bethesda, MD, human “SAGEmap”; ftp://ftp.ncbi.nih.gov/pub/sage/map) [11]. Porcine-derived SAGE tags were then compared against cDNA sequence information in the porcine gene index (SsGI, Version 4) available from The Institute of Genome Research (TIGR, Rockville, MD; http://www.tigr.org/tdb/tgi/ssgi) [12]. In select cases where the porcine tags annotated to SAGEMap and TIGR cDNA assemblies, the tag sequences were also compared with the Genbank “nr” database (NCBI; http://www.ncbi.nlm.gov/). All database comparisons were performed using BLAST algorithms [13] (http://www.ncbi.nlm.nih.gov/BLAST/) and required a perfect 14-base match for inclusion in the final dataset. To increase the likelihood that these tentative SAGE tag annotations were ascribable to a defined functional category (e.g. metabolism, cytoskeletal protein, etc), initial database matches were filtered to the following criteria: 1) those that aligned with differentially expressed (P < 0.05 or P < 0.001) embryonic SAGE tags; 2) were represented in a library 5 or more times; 3) linked to either a Unigene or TIGR Tentative Consensus (TC) Id (i.e. tentative contiguous EST assemblies representing a predicted gene annotation); and 4) annotated with a gene designation rather than simply an EST clone (i.e. “non-EST” annotations). Functional protein categories were assigned to SAGE tag annotations with the aid of the SWISS-PROT knowledge database (http://us.expasy.org/sprot/). These search criteria have yielded the most consistent gene annotations in analyses of turkey sperm storage tubule and swine embryo SAGE library datasets in our laboratory [14].

Results and Discussion A total of 42,389 and 42,392 SAGE tags representing 14,464 and 13,098 unique putative genes were sequenced from the D11 and D12 libraries, respectively. Comparative statistical analysis of the SAGE tag frequencies MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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52 – SERIAL ANALYSIS OF GENE EXPRESSION (SAGE) IN PREIMPLANTATION STAGE SWINE EMBRYOS indicated that at P < 0.05 and P < 0.001 significance, 434 tags and 87 tags, respectively, were differentially expressed between D11 and D12. Thus, approximately 1.6% of the 27,562 putative genes identified by SAGE within the D11 and D12 embryos were differentially expressed. SAGE tags annotated following blasts of NCBI and TIGR databases could be clustered into the functional groups depicted in Table 1. This assignment of differentially expressed genes to putative functional pathways further enhances the utility of SAGE in facilitating the discovery of new genes in poorly characterized systems such as porcine embryos. Real-time PCR has confirmed differential expression between D11 and D12 of several of these transcripts (data not shown). The maternal recognition of pregnancy combined with the dramatic embryo elongation that occurs between D11 and D12 of gestation denote critical stages in porcine development. Despite the paucity of publicly available porcine embryo DNA sequence data, SAGE enabled the identification and relative quantification of genes associated with porcine embryo development from D11 to D12. Identifying differentially expressed genes during this interval represents an important first step in developing new tools and strategies for decreasing embryonic mortality. These results provide the foundation for future elucidation of the most critical differentially expressed genes during mammalian embryo development.

Table 1.

Putative Differentially Regulated Proteins Comparing D11 and D12 Porcine Embryos

Protein Categorization* Membrane/Membrane Associated Proteins Cytoskeleton Proteins Neuropeptide/Steroidogenic Proteins Ribosomal Proteins Signal Transduction/RNA Processing General Metabolism Protein Processing Cytokines Cell Cycle proteins Antioxidant Proteins Heat Shock Proteins Misc. Proteins

Higher in D11** X X X X X X X

X

Higher in D12** X X X X X X X X X X

* Functional protein categories were assigned to SAGE tag annotations with the aid of the SWISS-PROT knowledge database. ** P < 0.001.

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Acknowledgements Technical assistance of Lori Schreier, Delbert Parsons and Jim Piot in recovering the porcine embryos is gratefully acknowledged. This work was supported by USDA ARS CRIS Project No. 1265-31000-082.

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Brackett BG, and Zuelke KA. (1993), Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology 39:43-64. Pope WF, and First NL. (1985), Factors affecting survival of pig embryos. Theriogenology 23:91-105. Abeydeera LR. (2002), In vitro production of embryos in swine. Theriogenology 57:257-273. Telford NA, Watson AJ, and Schultz GA. (1990), Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 26:90-100. Dobrinsky JR. (1997), Cryopreservation of pig embryos. J Reprod Fertil Suppl 52:301-312. Dobrinsky JR, Pursel VG, Long CR, and Johnson LA. (2000), Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 62:564-570. Geisert RD, and Yelich JV. (1997), Regulation of conceptus development and attachment in pigs. J Reprod Fertil Suppl 52:133-149. Yelich JV, Pomp D, and Geisert RD. (1997), Ontogeny of elongation and gene expression in the early developing porcine conceptus. Biol Reprod 57:1256-1265. Velculescu VE, Zhang L, Vogelstein B, and Kinzler KW. (1995), Serial analysis of gene expression. Science 270:484-487. Audic S, and Claverie JM. (1997), The significance of digital gene expression profiles. Genome Res 7: 986-995. Lash AE, Tolstoshev CM, Wagner L, Schuler GD, Strausberg RL, Riggins GJ, and Altschul GD. (2000), SAGEmap: A public gene expression resource. Genome Res 10:1051-1060. Quackenbush J, Cho J, Lee D, Liang F, Holt I, Karamycheva S, Parvizi B, Pertea G, Sultana R, and White J. (2001), The TIGR MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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gene indices: analysis of gene transcript sequences in highly sampled eukaryotic species. Nucleic Acid Res 29:159-164. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. (1990), Basic local alignment search tool. J Mol Biol 215:403-410. Long EL, Sonstegard, TS, Long, JA, Van Tassell CP, and Zuelke KA. (2003), Serial Analysis of Gene Expression (SAGE) in Turkey Sperm Storage Tubules in the Presence and Absence of Resident Sperm. Biol Reprod (In press).

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Library Subtraction and Microarray Screening to Isolate Oocyte Specific Transcripts in the Cow C. Robert,1 S. McGraw,1 D. Gagné1 and M.A. Sirard1 1. Centre de Recherche en Biologie de la Reproduction, Département des Sciences Animales, Université Laval, Sainte-Foy, QC, Canada G1K 7P4 e-mail:[email protected]

Abstract. This study was conducted to isolate stored maternal mRNA specific to the oocyte in order to better understand the molecular mechanisms, that confer the unique features of the bovine oocyte. Suppressive subtractive hybridization was performed to compare the RNA content of germinal vesicle oocytes to a total RNA pool extracted from several sources of somatic cells. The end product of this subtraction as well as two other library subtraction reactions were used to fabricate the construction of a microarray containing 2 347 bovine oocyte expressed sequence tags. Since the amount of starting material is limited when using oocytes or pre-implantation embryos, a comparative study was done to evaluate 3 protocols of probe preparation. Higher signals were obtained from the probes prepared from the amplified cDNAs resulting of the library subtractions. By considering a 5-fold intensity ratio to be significantly different, 31 clones were isolated and 16 different genes were identified. Even with such a severe cut off ratio their identification show they are mostly not exclusive to the oocytes but evidence suggest that they must be present in a very high level in the oocytes relatively to the somatic cells. However, Growth Differentiation Factor-9 and zona pellucida protein A, two well known oocyte specific genes were isolated. SSH can be an efficient method to isolate a large amount of candidates and when combined to a high throughput screening method to sort out the valuable candidates, it is an appropriate approach to study the oocyte’s maternal mRNA pool.

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Introduction The oocyte is a very peculiar cell that bears unique features. Amongst these, the oocyte cytoplasm has the unique ability to reprogram and rejuvenate a differentiated nucleus >1,2,3,4@ and it has also the capacity to remain in a dormant state for years, in large mammals, without compromising its ability to resume meiosis and grow. We hypothesized that these oocyte specific characteristics must require some oocyte specific genes and to understand the molecular mechanisms responsible for them, comparisons, or more precisely, subtractive analysis between RNA pools of somatic cells and oocytes were constructed to isolate oocyte specific transcripts. Oocyte specific gene expression has not been widely investigated and to date, only a few genes have been associated specifically to the oocyte. Among these, genes encoding the zona pellicida >5@ proteins and the growth differentiation factor 9 (GDF-9) >6@ are the most well known. The main reason for the lack of knowledge regarding the study of gene expression in the mammalian oocytes and early embryos is due to the limited amount of starting material available which is insufficient for the application of standard techniques. The development of new molecular based technologies has provided an avenue to study gene expression in oocytes and early embryos. The PCR based Suppressive Subtractive Hybridization (SSH) method results in the isolation of a large number of candidate genes must be coupled to a screening process to isolate true positives. The use of the microarray technology as a screening procedure is an interesting approach because of its ability to evaluate thousands of candidates simultaneously. However, the same limitations regarding the amount of starting material applies to the probe preparation as the standard procedures requires levels of RNA in excess of what is conventionally collected from oocyte and preimplantation embryo pools. In general, standard probe preparation procedures require about 20 µg of total RNA. Since the total RNA content of a bovine oocyte has been calculated to be 2.4 ng >7@, this would necessitate the use of large pools of oocytes which would most certainly reduce the specificity of the analysis by increasing the heterogeneity of the samples. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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Materials and methods Microarray fabrication Briefly, a microarray containing 2 347 uncharacterized bovine oocyte expressed sequence tags (ESTs) was fabricated from the end product of three cDNA library subtractions. The subtractions were performed using suppressive subtractive hybridization (SSH) procedures as described by Diatchenko and collaborators >8@. Since the amount of starting material was insufficient to directly perform the cDNA libraries constructions and subtractions, a global pre-amplification step was applied to each total RNA samples using the SMART procedures (Clontech Laboratories, Palo Alto, Ca, USA). All SSH experiments were performed using the PCR Select Kit (Clontech Laboratories) and the protocols were followed as indicated by the manufacturer. The first library subtraction compared oocytes collected from < 2 mm follicles considered as developmentally incompetent to the cDNAs of oocytes from the 3-5 mm follicles which were considered to have greater competence. This subtraction method has previously been described in Robert et al. (2000) >9@. The second subtraction compared high developmental competence oocytes to developmentally impaired oocytes in order to find stored maternal mRNA associated with developmental competence. The highly competent germinal vesicle (GV) oocytes were collected by transvaginal aspiration of follicles pre-matured in vivo according to the ovarian stimulation protocol described previously >10@. This ovarian stimulation protocol supports the production of oocytes that reach the blastocyst stage in vitro with a frequency as high as 80.4% >10@. The incompetent fully grown oocytes were collected from slaughter house ovaries by aspiration of follicles found deeper in the ovarian cortex. A portion of each oocyte group was cultured in vitro under standard in vitro production conditions >11@ to confirm their difference in terms of developmental capability. The third subtraction compared the cDNA library of a pool of 700 bovine GV oocytes collected from slaughter house ovaries by aspirating 3-5 mm follicles to a cDNA library fabricated by combining the total RNA of a pool of several somatic cell sources (liver, kidney, theca cells and corpus luteum). On average, 800 positive PCR products were selected for each subtracted library. The selected ESTs were purified up using 384 wells DNA binding filter plates (Millipore, Nepean, ON, Canada). and printed on CMT-GAPS coated glass slides (Corning, Corning, NY, USA) using a ChipWriter robot (Virtek, Waterloo, ON, Canada) at the Ontario Cancer Institute (Toronto, ON, Canada).

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60 – SHORT COMMUNICATIONS Oocyte handling and total RNA extraction. The oocytes were dissociated from the surrounding cumulus cells mechanically and washed 3 times in PBS buffer to remove the contaminating cumulus cells. The denuded oocytes were snap frozen in liquid nitrogen and stored at -80qC until RNA extraction. Total RNA extraction and DNase I treatment was done simultaneously using StrataPrep columns (Stratagene, La Jolla, CA, USA). For each oocyte sample, the total RNA was co-precipitated with 1 µg of glycogen (Roche Molecular Biochemicals, Laval, QC, Canada) as carrier by the addition of 1/10 volume of sodium acetate 3M pH 5.2 and 1 volume of isopropanol (Sigma, St Louis, MO, USA). The RNA was pelleted by centrifugation and washed using 400 µl of 70% ethanol. The RNA/glycogen pellet was dried quickly at 65qC using a thermal cycler (MJ Research inc., Watertown, MA, USA) and was resuspended in 10 µl of nuclease free water. The total RNA of the somatic cells (liver, kidney, theca cells and corpus luteum) was extracted using Trizol Reagent (Life Technologies). Total RNA from each tissue was pooled and the sample was treated with DNase I/RNAse free (Ambion inc, Austin, TX, USA) to prevent contamination of the sample with genomic DNA. The total RNA was re-extracted using StrataPrep columns (Stratagene), the RNA was precipitated and the pellet was processed as described above. The RNA concentration was evaluated by measuring the absorbency at 260 nm using a spectrophotometer (Amersham-Pharmacia, Baie D’Urfé, QC, Canada) and by colorimetric staining using QuickSticks (Clontech Laboratories) about 10 µg of total RNA from the pooled tissues was used in the subtraction.

Subtracted probe preparation A set of probes were prepared from the end products of the subtracted cDNA libraries by standard random labeling. Briefly, for each probe, 10 µg of cDNA was labeled using 1 µl of Klenow enzyme (Roche Diagnostics, Laval, QC, Canada) to incorporate the Cy3 or Cy5 labeled dCTP (NEN, Boston, MA, USA). The reaction buffer contained unlabeled dATP, dGTP and dTTP at 10 mM as well as 2 µl of labeled dCTP. The reaction was primed using 1 µg of random hexamers (Roche Diagnostics) and was place in a thermal cycler (MJ Research Inc.) for 30 minutes at 37qC. After the completion of the reaction, the unincorporated nucleotides and primers were removed by Qiaquick column purification (Qiagen inc., Mississauga, ON, Canada). The probes were combined and co-precipitated with 1 µg of glycogen (Gibco BRL) using 1/10 volume of sodium acetate 3M pH 5.2 and 1 volume of isopropanol (Sigma).

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Direct probe preparation Two direct probe preparation protocols were tested. The oocyte specific probes were prepared from total RNA extracted with the Strataprep columns (Stratagene) as described above from a single pool of 600 GV oocytes. The total RNA from the pool of tissues was prepared as described above. Both total RNA samples were divided in half to test the different labeling procedures. The first approach uses an oligo dT18 in combination with random hexamers to prime the reverse transcription reaction while the other reaction was reverse transcribed using the Genisphere specific primers (Genisphere inc., Montvale, NJ, USA). Overall, both reverse transcription reactions are similar. The RNA samples were denatured along with the primers in a 11 µl volume at 80qC for 10 min and quickly quenched on ice. After, 4 µl of 5X buffer, 2 µl of DTT at 100 mM, 1 µl of each dNTP at 10 mM, 1 µl of RNAsin (Promega, Madisson, WI, USA) and 1 µl of Superscript II were added to the mix. The reactions were done in a thermal cycler (MJ Research Inc.) for 1h at 42qC. Following the incubation, the cDNA samples primed with the oligo dT18 and the random hexamers were purified by Qiaquick column purification (Qiagen) and precipitated as described above. The labeling of the other probes were done following exactly the manufacturer’s recommendations (Genisphere inc.). Briefly, for each sample the reverse transcription was done using an oligo dT primer bearing an upstream sequence complementary to the DNA sequence on the dendrimers that carries the dyes that will be subsequently added. Following the reverse transcription, the reaction was stopped using 3.5 µl of 0.5 M NaOH/50 mM EDTA, incubated at 65qC for 10 min and neutralized using 5 µl of 1 M Tris-HCl pH 7.5. The samples were pooled and the DNA was co-precipitated with 15 µg of linear acrylamide using 4 µl of 5M NaCl. The pellet was washed with 70% ethanol, dried and ressuspended in 5 µl of sterile water. The DNA was denatured by heating the sample at 80qC for 10 min and labeled by adding both types of capture reagents which contains the dye carrying dendrimers (Cy3 or Cy5) coupled with the specific DNA sequence complementary to the reverse transcription primer sequence and incubating the sample at 55qC for 30 min.

Hybridization of the microarrays The random labeled probes were resuspended in 5 µl of sterile water, 2 µl of human Cot-1 DNA (Life Technologies) was added as competitor and the mix was denatured by incubating the samples at 95qC for 5 min in a thermal cycler (MJ Research Inc.). The probes were added to 30 µl of the pre-warmed ExpressHyb hybridization buffer (Clontech Laboratories) and MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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62 – SHORT COMMUNICATIONS quickly added to the microarray under a clean LifterSlip cover slip (Erie Scientific, Portsmouth, NH, USA). The probes labeled with the Genisphere dendrimeres were added to the warmed-up hybridization buffer included in the kit with 2 µl of oligo dT blocker, 1 µl of Cot-1 DNA and 1 µl of differential expander reagent. The Genisphere probes where also added to the microarray under a clean LifterSlip cover slip (Erie Scientific). The hybridization were performed in CMT-GAPS chambers (Corning) in a water bath at 55qC for a day away from any light source. The slides were washed in a Coplin jar with 2X SSC, 0.2% SDS at 55qC for 10 min followed by 2X SSC at room temperature for 10 min and a final wash was performed in 0.2X SSC at room temperature for 10 min. The slides where dried by centrifugation at 1000 RPM for 5 min and they were scanned using a microarray scanner (GSI Lumonics, Kanata, ON, Canada) at the microarray center in the “Institut de Recherche en Biotechnologie” (Montreal, Canada).

Results and discussion The probes prepared by direct labeling using the incorporation of the dyes by random priming only produced strong signals for a small portion of the clones. However, the overall background was relatively low considering the low intensity signals. The other direct probe preparation protocol employed the Genisphere technology that labels the probes by using a complementary DNA sequence to the reverse transcription primer to anneal the dendrimers filled with the respective dye. This method resulted in higher background and the signals were weak. However, the oocyte sample hybridization shows stronger signals than with the direct random labeling. By contrast, the pooled tissue sample showed very weak signals that were merely above the background. The high background was addressed by Genisphere and according to the manufacturer, the dendrimers are sticky and bind non specifically to the coating of the first generation of CMT-GAPS microarray slides (Corning). Since these hybridization were done, Corning modified the CMT-GAPS slides and now offers CMT-GAPS II slides. We have not tested the binding of the dendrimers to this second generation of microarray slides which could resolve the high background issue. The subtracted probes resulted in strong signals. In order to find genes that are exclusively expressed in the oocyte, only greater than 5-fold differences were considered. Generally, in microarray analysis, when a greater than 2-fold difference in the intensity between both signals is observed, the clones are considered to be different >12@. Overall, 31 clones were selected to be associated with the oocyte from which 14 were MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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identified. A total of 9 different clones could not be matched to any sequence in Genbank. Further characterization of these unidentified candidates is needed as they could be involved in novel molecular pathways associated with the unique features of the oocyte. Among the identified candidates, the only true positive is GDF-9 which is exclusively expressed in the oocyte >6,13,14@. The zona pellucida protein A was also isolated and although it is not strictly oocyte specific because the granulosa cells also express the gene in the early stages of folliculogenesis in large mammals >15,16@, it is also a good positive clone since the sample from the pooled tissues did not contain granulosa cell RNA. Since the subtracted probes are enriched in differentially expressed gene products, the use of such probes could be productive for identifying low expressed genes. In conclusion, library subtractions results in a high number of candidates to evaluate. When this method is combined with a high throughput screening approach such as the microarray technology it allows for the efficient isolation of true positives. These methods will enable a comparative analysis to study precise physiological characteristics using restricted amounts of starting material and this will be of benefit to investigators working with oocytes or pre-implantation embryos.

Acknowledgments This work was supported by the Natural Sciences and Engineering Research Council of Canada and Semex Canada.

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References

1.Campbell KH, McWhir J, Ritchie WA, and Wilmut I. (1996), Sheep cloned by nuclear transfer from a cultured cell line. Nature 380:64-66. 2.Fulka J Jr, Loi P, Ledda S, Moor RM, and Fulka J. (2001), Nucleus transfer in mammals: how the oocyte cytoplasm modifies the transferred nucleus. Theriogenology 55:1373-1380. 3.Wakayama T, and Yanagimachi R. (2001), Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 58:376-383. 4.Betts D, Bordignon V, Hill J, Winger Q, Westhusin M, Smith L, and King W. (2001), Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proc Natl Acad Sci USA 98:1077-1082. 5.Philpott CC, Ringuette MJ, and Dean J. (1987), Oocyte-specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida. Dev Biol 121:568-575. 6.McGrath SA, Esquela AF, and Lee SJ. (1995), Oocyte-specific expression of growth/differentiation factor-9. Mol Endocrinol 9:131-136. 7.Bilodeau-Goeseels S, and Schultz GA. (1997), Changes in the relative abundance of various housekeeping gene transcripts in in vitro-produced early bovine embryos. Mol Reprod Dev 47:413-420. 8.Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, and Siebert PD. (1996), Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A 11:6025-6030. 9.Robert C, Barnes FL, Hue I, and Sirard MA. (2000), Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes. Mol Reprod Dev 57:167-175.

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10.Blondin P, Bousquet D, Twagiramungu H, Barnes F, and Sirard MA. (2002), Manipulation of follicular development to produce developmentally competent bovine oocytes. Biol Reprod 66:38-43. 11.Blondin P, and Sirard MA. (1995), Oocyte and follicular morphology as determining characteristics for developmental competence in bovine oocytes. Mol Reprod Dev 41:54-62. 12.Bassett DE Jr, Eisen MB, and Boguski MS. (1999), Gene expression informatics – it’s all in your mine. Nat Genet 21:51-55. 13.Jaatinen R, Laitinen MP, Vuojolainen K, Aaltonen J, Louhio H, Heikinheimo K, Lehtonen E, and Ritvos O. (1999), Localization of growth differentiation factor-9 (GDF-9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B. Mol Cell Endocrinol 156:189-193. 14.Aaltonen J, Laitinen MP, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, Hovata O, Dale L, and Ritvos O. (1999), Human growth differentiation factor 9 (GDF-9) and its novel homolog GDF-9B are expressed in oocytes during early folliculogenesis. J Clin Endocrinol Metab 84:2744-2750. 15.Lee VH. (2000), Expression of rabbit zona pellucida-1 messenger ribonucleic acid during early follicular development. Biol Reprod 63:401-408. 16.Sinowatz F, Kolle S, and Topfer-Petersen E. (2001), Biosynthesis and expression of zona pellucida glycoproteins in mammals. Cells Tissues Organs 168:24-35.

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Characterization of Novel cDNAs that are Stage Specific and Sensitive to Culture Environment During Bovine Early Development David R. Natale1,2 and Andrew J. Watson2,1 Departments of Physiology and Pharmacology1 and Obstetrics and Gynecology,2 The University of Western Ontario, London, ON, Canada. email: [email protected]

Abstract. The pre-implantation or pre-attachment period of development is characterized by a high frequency of embryo-loss. In species in which assisted reproductive technologies (ART) are routinely applied, less than 60% of oocytes that are fertilized and subsequently cultured in vitro, will develop to the blastocyst stage. In addition, recent studies have indicated that the pre-implantation embryo is susceptible to disruptions in the developmental program, (for example, gene expression or metabolism), that stem from exposure to sub-optimal culture environments. Research in our laboratory is focused on defining the basic program that controls pre-implantation development and thus, in revealing how gene expression directs development to the blastocyst stage. In addition, we are interested in understanding how the embryo interacts with its environment to modulate gene expression to ultimately control development. To address these objectives, we have applied the technique of differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) to in vitro produced (IVP) bovine embryos collected under different experimental protocols to investigate: 1) changes in patterns of gene expression as the embryo progresses through the different stages of pre-attachment development; and 2) the influence of culture environment on patterns of gene expression during this period. The application of this method has also resulted in the identification of a number of mRNA transcripts previously uncharacterized in the bovine pre-attachment embryo.

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Introduction Pre-implantation development begins with the fertilization of a mature oocyte and concludes with the implantation or attachment of a fluid-filled embryonic structure called the blastocyst to the uterus. To reach this point, the inseminated oocyte proceeds through a series of important morphogenetic events including; zygotic genome activation (ZGA), compaction (trophectoderm differentiation), and cavitation (blastocyst formation) (reviewed in [1-4]). The blastocyst consists of two distinct cell-types, the inner cell mass (ICM) cells and the trophectoderm, which develops as a consequence of the first differentiative events of mammalian development (reviewed in [3, 5]). From the beginnings of conception the embryonic developmental program is susceptible to influences from the external environment. This dependency on the external environment can result in negative consequences to development that impact on the pre-implantation period and can also be propagated throughout the developmental program to negatively influence fetal development, postnatal development and increase susceptibility to disease in adult years [6-8]. Recent studies in several species including the human have indicated that developmental abnormalities in offspring at the time of birth and diagnosed up to one year later, occur at a greater frequency following conception through assisted reproductive technologies (ART) versus natural conception [9-14]. In many species including the human, in vitro embryo culture systems are not optimized and this is revealed in lower than desired frequencies of embryo development to the blastocyst stage [15]. The results from studies that have been applied to pre-implantation embryos from a variety of species have demonstrated that gene expression is dramatically influenced by embryo culture environment [16-19]. Studies investigating changes in patterns of DNA methylation have implied that embryo culture can be associated with altered patterns of genomic imprinting and gene expression [20, 21]. These data support the hypothesis that ART-associated developmental abnormalities could originate, in part, in culture medium induced insults to embryonic gene expression patterns. However the precise molecular nature of these disruptions on overall embryonic gene expression MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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patterns have not been fully characterized. To gain a full appreciation of the impact of culture on development and to identify ways to assess the effects of culture medium on embryo development and enable the formulation of improved culture systems, we applied a gene discovery approach to assess gene expression in bovine pre-attachment stage embryos.

Materials and methods Until recently, the predominant approach to characterizing gene expression in early embryos involved the application of a “candidate gene” approach, whereby a gene or small grouping of related genes were investigated following the presentation of a specific hypothesis. This hypothesis would be based upon preliminary data or deductive reasoning that supported a role for the gene or gene family in regulating preimplantation development. For example, expression of members of the Na+/K+ ATPase gene family have been characterized in the developing trophectoderm based on their contribution to maintaining trans-epithelial ion gradients in epithelial layers [22, 23]. However, the introduction of genomics techniques that are capable of investigating the expression of large numbers of genes in a single assay has enabled researchers to formulate a hypothesis and then analyze the outcomes in terms of effects to large pools of gene products. The principal advantage of this approach is that one can now consider effects to gene networks and develop a greater appreciation for the global impact of gene expression on the developmental period under investigation. We have applied this approach to investigating gene expression by applying the technique of differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) to bovine pre-attachment stage embryos to investigate stage-specific patterns of gene expression and to characterize the influence of culture environment on embryonic gene expression. DD-RT-PCR is a polymerase chain reaction-based technique that is an unbiased method of contrasting pools of mRNA from two or more samples. First introduced in 1992 [26], it has now been widely applied to search for new genes and investigate gene expression in the pre-implantation embryo [27-30]. It offers the advantages of being inexpensive, applicable to small amounts of starting mRNA and able to compare many different samples simultaneously. In addition, differentially expressed cDNA bands are easily extracted from the gel for identification by nucleotide sequencing. We applied DD-RT-PCR in two experimental protocols. In the first, we screened mRNA pools isolated from a developmental series of bovine in vitro-produced embryos that included mature oocytes, 2-5 cell, 6-8 cell, 8-16 cell, morula and blastocyst stage embryos [31]. In order to identify MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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70 – SHORT COMMUNICATIONS genes that were being expressed from the embryonic genome, we included a treatment with the RNA polymerase II-specific transcriptional inhibitor, alpha-amanitin [31]. In these experiments zygotes from all developmental stage excluding oocytes were exposed to treatment with alpha-amanitin for both four and twelve hours. Embryos from each treatment group and developmental stage were then assessed for changes in gene expression patterns by DDTRTTPCR using two different primer combinations. In the second experiment, mRNA pools isolated from bovine blastocysts produced under non-defined (TCM-199 +serum + coculture) and defined (cSOFMaa and KSOMaa) conditions were screened by DD-RT-PCR using seven different primer combinations [19].

Results and discussion The results from the DD-RT-PCR screens in experiment one illustrated the overall variations in gene expression that arise as the embryo progresses from the 2-cell to blastocyst stages of development in a very unique way [31]. In this experiment, the mature oocyte samples served as a control for maternal mRNA products and we observed that this oogenetic or maternal pattern of transcript bands remained constant through the oocyte and 2-5 cell stages. At the 6-8 cell bovine embryo stage, there was a pronounced change in mRNA banding pattern that continued until the 8-16 cell stage. From the 8-16 cell to blastocyst stages, however, we observed a reduction in the variation of these mRNA banding patterns suggesting that expression of new gene products required to support blastocyst formation are in fact “turned on” comparatively early by the 8-16 cell stage. The transitions in mRNA expression that we observed support many other investigations of the events of early development, whereby, following fertilization of the oocyte, maternal stores of mRNA begin to degrade and by the 8-cell stage in bovine development, zygotic genome activation occurs (reviewed in [32]). In addition, we observed a small number of mRNA transcripts that were sensitive to alpha-amanitin at the 2T5 cell stage and that alpha-amanitinG sensitivity was greatest at the 6T8 cell stage of development [31]. These results also support findings that ZGA in the bovine occurs in at least two stages as has been found to occur in the early mouse embryo. These stages consist of a “minor” transcriptional activation that is detectable early, (ie at the 2-4 cell stage in the bovine) followed by the “major” activation of embryonic transcriptional activity that occurs during the bovine 8-16 cell stage transition [33-38]. However the most important outcome from these experiments is that they have directly contributed to the isolation and initial characterization of a large pool of new embryonic MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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cDNAs that have already stimulated new avenues of research and have fostered a greater understanding of the genetic program that controls pre-implantation development. From the first set of experiments (alphaamanitin experiments) we have isolated and identified 34 differentially expressed cDNA bands of which 13 displayed significant sequence identity to nucleotide sequences published in GenBank non redundant databases. Of these 13, 5 were found in embryo stages following activation of the full embryonic genome (post 8-16 cell stage) while the remaining 8 were considered to be oogenetic or maternal transcripts. The majority of these clones did not match any known or published sequences in the GenBank nucleotide sequence databases. However, of the clones that displayed an identity to known GenBank sequences, one in particular, IQGAP-2, was identified in our screen as sensitive to alpha-amanitin beginning at the 8T16 cell stage [31]. We have subsequently characterized the expression of IQGAP-2 and related proteins in murine pre-implantation development and we are investigating the hypothesis that this group of gene products is involved in regulating the onset of E-cadherin-mediated cell-to-cell adhesion at the time of compaction [39]. The results from the second set of experiments that examined culture effects on embryonic gene expression patterns demonstrated with each primer set that culture environment influences blastocyst mRNA expression [19]. When directly contrasting patterns of mRNA expression from blastocysts cultured in the two serum-free defined media (citrate supplemented synthetic oviduct fluid medium + amino acids; cSOFMaa vs. potassium simplex optimized medium + amino acids; KSOMaa), we did not observed a single difference in banding pattern between these two groups. This was an interesting result as in our hands bovine embryos cultured in cSOFMaa have a much higher frequency of blastocyst development compared to bovine embryos cultured in KSOMaa (unpublished data). However, when we contrasted banding patterns in blastocysts produced in defined media to those produced in the presence of oviduct cell co-culture and serum-supplemented TCM-199 medium, we observed a series of differences in the banding patterns of these two groups. Overall, we determined that 3.8% of all blastocyst cDNA bands were differentially expressed between these two groups [19]. From these screens, 41 additional differentially expressed products were cloned and sequenced. The majority displayed little sequence similarity to known sequences in the GenBank non-redundant database. At this time, however, seven clones have shown similarity to entries in the GenBank nucleotide sequence databases and are reported in Natale et al.([19].

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72 – SHORT COMMUNICATIONS Conclusions Our experiments utilizing DD-RT-PCR have provided a wider view of the overall variations in gene expression patterns that arise during bovine pre-attachment development and have revealed new insights into the timing of zygotic genome activation in the bovine embryo and its effect on global patterns of mRNA expression [31]. We have also shown that bovine embryos cultured in defined versus co-culture/serum-supplemented culture systems display differences in patterns of gene expression that directly stem from exposure to those particular culture environments [19]. Finally and most importantly, these studies have resulted in the identification of approximately 70 differentially expressed gene products by utilizing the DD-RT-PCR approach. Some of these cDNAs have already become the principal subject of new studies aimed at revealing their function during the first week of development. These include IQGAP [39], and more recently, p38 related/activated kinase, PRAK (Natale et al., 2003, Submitted). In conclusion, our results provide additional insight into the way in which gene expression supports progression through the pre-attachment interval of bovine development. We have shown that DD-RT-PCR is a versatile and useful tool for investigating patterns of gene expression in early development and we have used this tool to identify gene products that are novel in the context of pre-attachment/pre-implantation development.

Acknowledgments The authors wish to thank Dr(s) Paul De Sousa, Mark Westhusin, Michele Calder, Lisa Barcroft, Quinton Winger, and Gerald Kidder for assistance with embryo collections and technical details regarding the application of DD-RT-PCR methods. We also acknowledge operating grant support from NSERC and CIHR Canada to AJ Watson and the National Cooperative Program on Non Human in vitro Fertilization and Preimplantation Development supported by NIH Cooperative Agreement UO1HD34580 to both M E Westhusin and AJ Watson.

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References

1. Kidder, G.M., The genetic program for preimplantation development. Dev Genet, 1992. 13(5): p. 319-25. 2. Watson, A.J., G.M. Kidder, and G.A. Schultz, How to make a blastocyst. Biochem Cell Biol, 1992. 70(10-11): p. 849-55. 3. Fleming, T.P., L. Butler, X. Lei, J. Collins, Q. Javed, B. Sheth, N. Stoddart, A. Wild, and M. Hay, Molecular maturation of cell adhesion systems during mouse early development. Histochemistry, 1994. 101(1): p. 1-7. 4. Wiley, L.M., G.M. Kidder, and A.J. Watson, Cell polarity and development of the first epithelium. Bioessays, 1990. 12(2): p. 67-73. 5. Watson, A.J., The cell biology of blastocyst development. Mol Reprod Dev, 1992. 33(4): p. 492-504. 6. Barker, D.J., In utero programming of cardiovascular disease. Theriogenology, 2000. 53(2): p. 555-74. 7. Young, L.E., Imprinting of genes and the Barker hypothesis. Twin Res, 2001. 4(5): p. 307-17. 8. Hales, C.N. and D.J. Barker, The thrifty phenotype hypothesis. Br Med Bull, 2001. 60: p. 5-20. 9. Van Steirteghem, A., M. Bonduelle, I. Liebaers, and P. Devroey, Children born after assisted reproductive technology. Am J Perinatol, 2002. 19(2): p. 59-65. 10. McEvoy, T.G., K.D. Sinclair, L.E. Young, I. Wilmut, and J.J. Robinson, Large offspring syndrome and other consequences of ruminant embryo culture in vitro: relevance to blastocyst culture in human ART. Hum Fertil, 2000. 3(4): p. 238-246. 11. Stromberg, B., G. Dahlquist, A. Ericson, O. Finnstrom, M. Koster, and K. Stjernqvist, Neurological sequelae in children born after in-vitro fertilisation: a population-based study. Lancet, 2002. 359(9305): p. 461-5. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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74 – SHORT COMMUNICATIONS 12. Leese, H.J., I. Donnay, and J.G. Thompson, Human assisted conception: a cautionary tale. Lessons from domestic animals. Hum Reprod, 1998. 13 Suppl 4: p. 184-202. 13. Hansen, M., J.J. Kurinczuk, C. Bower, and S. Webb, The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med, 2002. 346(10): p. 725-30. 14. Ceelen, M. and J.P. Vermeiden, Health of human and livestock conceived by assisted reproduction. Twin Res, 2001. 4(5): p. 412-6. 15. Gardner, D.K., Development of serum-free media for the culture and transfer of human blastocysts. Hum Reprod, 1998. 13 Suppl 4: p. 218-25. 16. Niemann, H. and C. Wrenzycki, Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology, 2000. 53(1): p. 21-34. 17. Ho, Y., K. Wigglesworth, J.J. Eppig, and R.M. Schultz, Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev, 1995. 41(2): p. 232-8. 18. Watson, A.J., P. De Sousa, A. Caveney, L.C. Barcroft, D. Natale, J. Urquhart, and M.E. Westhusin, Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol Reprod, 2000. 62(2): p. 355-64. 19. Natale, D.R., P.A. De Sousa, M.E. Westhusin, and A.J. Watson, Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR. Reproduction, 2001. 122(5): p. 687-93. 20. Young, L.E. and H.R. Fairburn, Improving the safety of embryo technologies: possible role of genomic imprinting. Theriogenology, 2000. 53(2): p. 627-48. 21. Doherty, A.S., M.R. Mann, K.D. Tremblay, M.S. Bartolomei, and R.M. Schultz, Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod, 2000. 62(6): p. 1526-35. 22. Barcroft, L.C., S.E. Gill, and A.J. Watson, The gamma-subunit of the Na-K-ATPase as a potential regulator of apical and basolateral Na+-pump isozymes during development of bovine pre- attachment embryos. Reproduction, 2002. 124(3): p. 387-97. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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23. Betts, D.H., L.C. Barcroft, and A.J. Watson, Na/K-ATPase-mediated 86Rb+ uptake and asymmetrical trophectoderm localization of alpha1 and alpha3 Na/K-ATPase isoforms during bovine preattachment development. Dev Biol, 1998. 197(1): p. 77-92. 24. Watson, A.J., C. Pape, J.R. Emanuel, R. Levenson, and G.M. Kidder, Expression of Na,K-ATPase alpha and beta subunit genes during preimplantation development of the mouse. Dev Genet, 1990. 11(1): p. 41-8. 25. Watson, A.J. and G.M. Kidder, Immunofluorescence assessment of the timing of appearance and cellular distribution of Na/K-ATPase during mouse embryogenesis. Dev Biol, 1988. 126(1): p. 80-90. 26. Liang, P. and A.B. Pardee, Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 1992. 257(5072): p. 967-71. 27. Lee, K.F., J.F. Chow, J.S. Xu, S.T. Chan, S.M. Ip, and W.S. Yeung, A comparative study of gene expression in murine embryos developed in vivo, cultured in vitro, and cocultured with human oviductal cells using messenger ribonucleic acid differential display. Biol Reprod, 2001. 64(3): p. 910-7. 28. Ma, J., P. Svoboda, R.M. Schultz, and P. Stein, Regulation of zygotic gene activation in the preimplantation mouse embryo: global activation and repression of gene expression. Biol Reprod, 2001. 64(6): p. 1713-21. 29. Minami, N., K. Sasaki, A. Aizawa, M. Miyamoto, and H. Imai, Analysis of gene expression in mouse 2-cell embryos using fluorescein differential display: comparison of culture environments. Biol Reprod, 2001. 64(1): p. 30-5. 30. De Sousa, P.A., Q. Winger, J. Hill, K. Jones, A.J. Watson, and M.E. Westhusin, Reprogramming of fibroblast mRNA expression following nuclear transfer in bovine embryos. Cloning, 1999. 1: p. 63-69. 31. Natale, D.R., G.M. Kidder, M.E. Westhusin, and A.J. Watson, Assessment by differential display-RT-PCR of mRNA transcript transitions and alpha-amanitin sensitivity during bovine preattachment development. Mol Reprod Dev, 2000. 55(2): p. 152-63. 32. Memili, E. and N.L. First, Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote, 2000. 8(1): p. 87-96.

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76 – SHORT COMMUNICATIONS 33. King, W.A., A. Niar, I. Chartrain, K.J. Betteridge, and P. Guay, Nucleolus organizer regions and nucleoli in preattachment bovine embryos. J Reprod Fertil, 1988. 82(1): p. 87-95. 34. Kopecny, V., J.E. Flechon, S. Camous, and J. Fulka, Jr., Nucleologenesis and the onset of transcription in the eight-cell bovine embryo: fine-structural autoradiographic study. Mol Reprod Dev, 1989. 1(2): p. 79-90. 35. Viuff, D., P. Hyttel, B. Avery, G. Vajta, T. Greve, H. Callesen, and P.D. Thomsen, Ribosomal ribonucleic acid is transcribed at the 4-cell stage in in vitro-produced bovine embryos. Biol Reprod, 1998. 59(3): p. 626-31. 36. Viuff, D., B. Avery, T. Greve, W.A. King, and P. Hyttel, Transcriptional activity in in vitro produced bovine two- and four-cell embryos. Mol Reprod Dev, 1996. 43(2): p. 171-9. 37. Plante, L., C. Plante, D.L. Shepherd, and W.A. King, Cleavage and 3H-uridine incorporation in bovine embryos of high in vitro developmental potential. Mol Reprod Dev, 1994. 39(4): p. 375-83. 38. Memili, E. and N.L. First, Developmental changes in RNA polymerase II in bovine oocytes, early embryos, and effect of alpha-amanitin on embryo development. Mol Reprod Dev, 1998. 51(4): p. 381-9. 39. Natale, D.R. and A.J. Watson, Rac-1 and IQGAP are potential regulators of E-cadherin-catenin interactions during murine preimplantation development. Gene Expression Patterns, 2002. 2(1-2): p. 17-22.

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Potential Use of cDNA Microarrays to Characterize Gene Expression Patterns of Sex-specific Genes During Early Development Maria Nino and W. Allan King Dept. of Biomedical Sciences, University of Guelph, Guelph, Ontario N1G 2W1

Abstract. X-chromosome inactivation is the mechanism by which mammals equalize X-linked gene products between females and males. The choice of X chromosome to be inactivated is random, occurs early in embryonic development and results in stable silencing of genes on the inactive X chromosome. However, some genes are known to escape the inactivation process and while X-inactivation is a universal phenomenon among mammals, species-specific differences dictate which ones will be expressed or silenced. With the X chromosome representing approximately 5% of the human genome, its anomalies lead to severe phenotypical and physiological alterations. On the other hand, X-inactivation is a major epigenetic event and therefore can provide invaluable insight into processes such as nuclear reprogramming during nuclear transfer or the impact of the environment on gene expression. Starting with total RNA extracted from muscle samples obtained from male and female bovine fetuses we hybridized human 19K cDNA microarray slides (Ontario Cancer Institute). The hybridization of human chips with bovine samples was successful with consistent high hybridization spots and low background. A total of 188 ESTs were consistently up regulated in female versus male samples. Of these, 36 (19.1%) were unknown, 147 (78.2%) were autosomal and 5 (2.7%) were X-linked. The expression differences of these candidate genes will be confirmed using real-time PCR. These results suggest that the use of heterologous hybridization is a useful tool in the screening of bovine genes. Based on this observation we are currently working with RNA amplification methods to increase the amount of starting material to study differential X-linked transcription profiling of bovine female and male embryos.

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Introduction X-chromosome inactivation is the mechanism by which mammals equalize X-linked gene products between females and males. The choice of X chromosome to be inactivated is random, occurs early in embryonic development and results in stable silencing of genes on the inactive X chromosome. However, some genes are known to escape the inactivation process and while X-inactivation is a universal phenomenon among mammals, species-specific differences dictate which ones will be expressed or silenced. With the X chromosome representing approximately 5% of the human genome, anomalies in their number (i.e. Turner syndrome) or deviations in the inactivation process (i.e. Rett syndrome) lead to severe phenotypical and physiological alterations. On the other hand, X-inactivation is a major epigenetic event and therefore can provide invaluable insight into processes such as nuclear reprogramming during nuclear transfer or the impact of the environment on gene expression. Even though there is a great deal of research in this area, many questions pertaining to the expression or silencing of X-linked genes and the consequences of the disruption of their normal patterns, particularly among domestic animals, remain to be answered. Powerful gene expression profiling techniques such as cDNA microarray and real-time quantitative RT-PCR are used to study differential gene expression and have great potential to expand our knowledge of the X-inactivation process in a variety of species [7,8,11,14,15,19,45,49].

The x inactivation mechanism X-inactivation is the dosage compensation mechanism whereby all X chromosomes except one are inactivated in each somatic female cell in a developmentally regulated manner that coincides with cellular differentiation in early embryogenesis. This process solves the problem of differential X-linked gene copy number and expression imbalances between the sexes. X inactivation can be achieved in one of two ways: imprinted, which leads to preferential silencing of the paternal chromosome and random, where both chromosomes have equal probability of remaining MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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active. In marsupials and extra-embryonic tissues of mice the X-inactivation process is imprinted and recently, an antisense gene to Xist called Tsix has been characterized in mouse and has been implicated in imprinted X-inactivation, by regulating XIST expression. In the embryo proper, the active X chromosome is normally chosen at random although in mice, the X-controlling element (Xce) locus influences the choice of the X chromosome to be inactivated [1,20,21,23,25,32]. The process is directed by the X inactivation Centre (XIC) of which at least two copies must be present for X inactivation to occur. Inactivation comprises several steps: counting, selection of a single X chromosome to remain active, initiation of inactivation from the XIC coinciding with transcription of X inactivation specific transcript (XIST), spreading of inactivity in cis along the chromosome and finally, stabilization and maintenance through future cell divisions by epigenetic modifications. [2,27,32,34,35]. The inactive X chromosome is recognized by its late replication and condensed heterochromatic structure. At the molecular level it shows marked differences in DNA methylation and histone acetylation with respect to its active counterpart, making X-chromosome inactivation an epigenetically regulated process. DNA hypermethylation and histone hypoacetylation work synergistically to accomplish high fidelity inheritance of the inactive state of a whole chromosome and at the same time, making the process a target for gene expression disruption due to environmental conditions like in vitro culture systems [30, 37,40]. Although X-inactivation represents a wide repression of a complete chromosome, there are genes that escape the silencing. In humans, it is considered that approximately 15% of the X-linked genes escape inactivation, most of which are expected to be located in the pseudoautosomal region and to have a functional Y homologue. For genes located near the X pseudoautosomal region, the lack of inactivation was first attributed to a position effect, but even if some of them have a Y homolog, these appear to be mostly nonfunctional pseudogenes. Finally, some genes escaping inactivation in humans are located at considerable distances from the pseudoautosomal region [9,14,23,46]. There are wide differences in the X-inactivation process between mouse and humans. The X chromosome of the mouse appears to be more completely inactivated, which may explain the phenotypic differences between humans and mice X-chromosome aneuploids. XO female mice (Turner syndrome) have a near-normal phenotype and are capable of reproducing whereas XO women present a characteristic phenotype with short stature, ovarian failure and severe fetal loss. Haploinsuficiency of MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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80 – SHORT COMMUNICATIONS genes escaping X-inactivation in humans might explain the abnormal phenotype of individuals with Turner syndrome. The large number of genes escaping X-inactivation in humans may be important in a yet unidentified female-specific manner in processes like ovarian development [14,23]. XO aneuploids have been described in several domestic species associated with reproductive failure but even though X chromosome trisomies have been identified in cattle there are no reports of XO cases in this species. This suggests that the phenotype associated with the defect may be severe enough to cause elimination during pregnancy or through the stringent reproductive selection process of the cattle industry or that it may not have any effect at all [5,6,33,39]. Very little is known about X-inactivation in domestic animals. A comparative study of the XIC region between human, mice and bovine revealed many conserved genes, CpG islands and even pseudogenes but it also showed marked species differences such as the lack of evidence for the existence of TSIX in human or cattle. Previous studies in cattle have shown that Xist transcription starts at the 2-cell stage in the pre-implantation embryo with a late replicating X chromosome evident at the early blastocyst stage. It has also been shown that Xist is expressed in adult bovine testis as well as in fetal, newborn, and prepubertal testes long before spermatogenesis is established and that it is probably not involved in gene silencing during spermatogenesis in this species [10,13,16,17]. There is considerable evidence that suggests that there are clear differences between male and female embryos that might be related to sex chromosome gene expression imbalances during the period when dosage compensation is incomplete. It is also becoming apparent that embryo production techniques such as in vitro fertilization (IVF) and nuclear transfer (NT) are associated with gene expression changes that can be related to the disruption of epigenetic events. Therefore, profiling the expression of X-chromosome linked genes has the potential of providing insight into both these aspects of development [26,36,38,42,47,49].

The cDNA microarray approach The relatively recent development of high throughput techniques for the analysis of gene expression patterns has provided researchers with the power to screen the expression profile of thousands of genes in a single experiment. Among them, DNA microarray analysis has features that make it the most attractive method for profiling mRNA expression [24]. DNA microarrays studies consists of an orderly, high-density arrangement of nucleic acid spots or “probes” immobilized on a suitable MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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substrate, which are then exposed to a “target”, consisting of a labeled free nucleic acid from the sample under analysis. This procedure provides a way to analyse thousands of genes at the same time and to establish general patterns of expression under specific conditions [24]. The probes most commonly used are cDNAs obtained by PCR. For the analysis of gene expression in most eukaryotes, the expressed sequence tag (EST) data represent the most extensive database for gene identification and has been widely adopted. The substrates used include glass, silicon, nylon and nitrocellulose membranes. Glass is particularly well suited, because it is durable and non-porous, allows the covalent attachment of probes, and has low background fluorescence. The slides are prepared by printing the PCR amplified products suspended in either high-salt or other denaturing buffer to glass microscope slides using a high-speed robotic system. Factors such as the slide surface and the spotting buffer are critical components for reproducible, high fidelity microarray analysis [24,31]. The target probe is generated by reverse transcribing the mRNA isolated from the specimens under investigation. Currently, fluorescence is the detection system of choice, although radioactivity may be used. Fluorescence analysis usually requires the use of two target samples that differ in a pre-defined parameter (i.e. normal versus diseased, female versus male) to enable a comparison of the gene expression of one to the other, thus identifying up or down regulation. Each of the targets is labeled with a different fluorochrome and both samples are simultaneously hybridized to the slide. Hybridization and washing are critical to the generation of high-quality data. The purpose of hybridization is to enable complementary target and probe sequences to specifically bind while the washing removes non-hybridized probes, reduces non-specific hybridization and minimizes background [18, 31]. Microarray image analysis is required to assess the relative amounts of fluorochromes (relative response ratios) that relate to the targets hybridized to each probe. The first steps are background subtraction and normalization. Background subtraction pulls the non-specific background noise out of the signal detected for each spot and allows comparison of specific signals, whereas normalization is the process of accounting for differences in different areas of the array as well as between separate arrays, allowing consistent comparisons. These analyses generate a massive number of individual points that must then be analyzed using a data-mining tool that is sufficiently sophisticated not only to correlate all of these data, but also to group them together in a meaningful manner [18,31]. DNA microarrays are considered by many as “fishing trips” that allow the large-scale analysis of mRNA abundance as an ultimate indicator of MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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82 – SHORT COMMUNICATIONS gene expression and one of the most common criticisms of this technique is that when hundreds or thousands of genes are examined at once, some will appear changed by random chance. This is a statistical reality and highlights the requirement for post-hoc confirmation of the changes seen with the arrays in individual samples either at the mRNA, protein or activity levels or a combination of methods, since it is a known fact that increased levels of transcription do not always translate into increased levels of proteins. Also, due to the several factors that influence the outcome of cDNA microarray screening, the data obtained must be confirmed using techniques such as quantitative real-time RT-PCR, which provides a way to quantify relative changes in gene expression for a large number of genes with limited RNA in a rapid and precise manner and has been widely and successfully used in conjunction with array technology [18,22,31,41,43]. A limiting factor at the moment is the large amount of RNA required per hybridization. One way to bypass this problem is the use of multiple rounds of linear amplification based on cDNA synthesis and a template-directed in vitro transcription reaction which generates sufficient quantities of labeled material starting from very little amounts (1 – 50 ng total RNA), is highly reproducible and introduces much less quantitative bias than PCR-based amplification, being perfectly suited to work with embryos in the early developmental stages [4,29,31]. Another concern in domestic animal research is the lack of species-specific arrays. Most of the published studies using microarray technology have used homologous probing, where cDNA probes and targets originate from the same species. For mammals, commercial microarray availability is mostly restricted to human, rat and mouse, while studies in species like cattle depend on the production of custom-made arrays that are in most cases restricted to a very specific area of research such as the immune system or embryo development. It is clear that the production of homologous arrays for every species or area of interest would be a daunting task and with this in mind several groups have chosen heterologous hybridization as an alternative. The success of heterologous hybridization will depend basically on a high percent of cDNA homology between the probes and the targets and with more than 80% sequence identity between most gene homologues in placental mammals there is a high probability of successful cross-reaction. Based on this, heterologous probing of cDNA arrays can provide an important first step in gene discovery [12,44]. One excellent example is found in studies on hibernation and gene expression using bat and ground squirrel tissues on rat and human arrays. Cross-reactivity was found in the range of 73 – 93% when using rat microarrays but when the same tissues were used on human microarrays the figure was reduced to 15 – 20%. Nonetheless, cross-reactivity on the human MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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arrays was increased by up to 85 – 90% using a reduction in the post-hybridization washing temperature from 50qC to room temperature (~21qC). However, it has to be kept in mind that this gain in cross-reactivity is obtained at the expense of the specificity of the reaction, probably leading to a large number of false positives that will then have to be carefully screened [44]. Considering the almost complete conservation of synteny between the human and bovine X chromosomes we have chosen this approach to address the issue of X inactivation in the bovine [3].

Material and methods – experimental design The experiments were design to detect X-linked differential gene expression between male (XY) and female (XX) samples that could be due to escape from X inactivation. Reciprocally labeled hybridizations were performed three times for a total of 6 hybridizations. The slides used were the commercially available human 19K3 cDNA microarray from the Ontario Cancer Institute (OCI), printed on sets of two glass CMT-GAPS slides (Corning Inc). The complete list of ESTs and their sequences are available at http://www.microarrays.ca/support/glists.html.

Samples : extractions and labeling protocol Samples of male and female bovine fetal muscle were obtained at the abattoir and placed immediately on dry ice, transported to the laboratory and stored at -80qC until further processing. Total RNA was extracted using TRIzol“ Reagent (Invitrogen Life Technologies Corp.) according to the manufacturer instructions. The total RNA samples obtained were treated with Dnase I using DNA-free (Ambion Inc.) and afterwards RNA concentration and quality were determined by measuring the absorbency at 260 nm in a Shimadzu UV-1201 spectrophotometer and by 1% agarose denaturing gels stained with ethidium bromide. Samples were labeled using the Amino Allyl Labeling protocol provided by the OCI (http://www.microarrays.ca/support/proto.html) with modifications. Briefly, each 15 Pg total RNA sample in a volume of 17.5 PL was combined with a master mix containing 8 PL 5X First Strand Buffer (Invitrogen Corp.), 4 PL 0.1 M DTT (Invitrogen Corp.), 3 PL 20 mM dNTP without dTTP (6.7 mM each of dATP, dCTP and dGTP, Invitrogen Corp.), 3 PL 2 mM dTTP, 3 PL 2 mM Amino Allyl dUTP (Sigma-Aldrich) and 1.5 PL of 100 pmol/PL AncT primer (5’ T20VN 3”) (custom primer, Invitrogen Corp.), incubated at 65qC for 5 min and quenched in ice. A total MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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84 – SHORT COMMUNICATIONS of 2 PL of Superscript II RT (Invitrogen Corp.) was added and the reverse transcription reaction took place at 42qC for 2.5 hours. The reaction was stopped by heating at 95qC for 5 min and RNA was hydrolyzed adding 8 PL of 1M NaOH and heating at 65qC for 15 min, after which 8 PL 1M HCl and 4PL 1M Tris-HCl (pH 7.6) were added to neutralize the solution. 38 PL of nuclease-free water (Ambion Inc.) were added and targets were purified using the QIAprep Spin Miniprep Kit columns (Qiagen). Targets were eluted twice by adding 50 PL of water (pH 8.5), incubating 5 min at room temperature and centrifuging 1 min at 18 000 g for a final volume of 100 PL. RNA was precipitated by adding 10 PL 3M NaOAc (pH 5.2), 1 PL glycogen (20Pg/PL) and 100 PL isopropanol after overnight incubation at -20qC. The next day, targets were collected by centrifugation at 11 200 g, 4qC for 10 min, washed with 70% cold ethanol, centrifuged again for 5 min, air dried 5 min and resuspended in 10 PL nuclease-free water (Ambion Inc.). At this point, the samples were split in two aliquots of 5 PL each for reciprocal labeling, 3 PL of 0.3M NaHCO3 (pH 9) and the respective aliquots of 2PL Cy3 or Cy5 (Amersham Bioscience) diluted in 100% DMSO (Sigma-Aldrich) were added to each sample. Labeling took place in the dark, at room temperature for 1 h. The labeling reaction was quenched by adding 4.5 PL 4M hydroxylamine and incubating 20 min at room temperature. The samples were then mixed with 60 PL nuclease-free water and 30 PL 100 mM NaOAc (pH 5.2), and purified as described before with three washes of 70% cold ethanol using the QIAprep Spin Miniprep Kit columns (Qiagen). Elution was performed three times in EB buffer for a final elution volume of 150 PL. To precipitate the targets 15 PL 3M NaOAc (pH 5.2), 1.5 glycogen (20Pg/PL) and 150 PL isopropanol were added and samples were incubated at -20qC for at least 30 min, then centrifuged and washed in 70% ethanol, pellets were allowed to air dry for 5 min and re-suspended in 3 PL of nuclease-free water and the Cy3 – Cy5 pairs were properly combined.

Hybridization procedures and parameters The hybridization mix was prepared by mixing 10 PL yeast tRNA (10 mg/mL, Invitrogen Corp.), 3 PL salmon sperm (10 mg/mL, Invitrogen Corp.) and 200 PL DIGeasy hybridization buffer (Roche), heating at 65qC for 2 min and then placed at 37qC. The hybridization chamber containing 15 mL of DIGeasy hybridization buffer (Roche) was pre-heated at 37qC. Each target Cy5-Cy3 mix was resuspended in 75 PL of the hybridization mix, heated at 65qC for 2 min and then placed at 37qC. For hybridization, each pair of microarray slides were placed with the spotted surfaces facing MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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each other and the hybridization mix was pipetted between the slides, transferred to the humid hybridization chamber and placed in the oven at 37qC for approximately 20 h. After incubation the slides were submerged once in 1X SSC at room temperature to separate the slides, then washed 3 times at 50qC in 1X SSC – 0.1% SDS for 10 min and finally rinsed in 0.1X SSC. The slides were dried by centrifugation at 900 g for 4 min, stored and scanned as soon as possible.

Measurement data and specifications The slides were scanned using a GenePix 4000 (Axon Instruments Inc.) and Tiff images were initially analyzed using GenePix Pro 4.0 (Axon Instruments Inc.). Normalization was performed using R [48] for overall slide and across slides normalization and final statistical analysis was performed using Gene Spring 4.0™ (Silicon Genetics).

Results and discussion After analysis we found consistent high hybridization and low background even using the same astringency washing parameters that are recommended for homologous hybridization of these human arrays. We were able to detect 188 upregulated ESTs in the bovine female samples, of which 36 (19.1%) were unknown, 147 (78.2%) were autosomal and only 5 (2.7%) were X-linked. Of these X-linked genes, two were found to be candidates for escaping X – inactivation in humans using an X chromosome specific microarray [45]: R63205 (IMAGE:138505, Hs.5367) and ANT3 (IMAGE:361123, Hs.164280). The differences in transcription of the candidate X-linked genes identified will be confirmed using real-time quantitative RT-PCR. Our results show that as has been previously demonstrated for other species, the use of heterologous hybridization is a useful tool for the screening of bovine genes [44]. Based on this, we are currently working to increase the starting amounts of RNA to work in the profiling of differential X-linked transcription of bovine female and male embryos.

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Avner P, Prissette M, Arnaud D, Courtier B, Cecchi C and Heard E. (1998). Molecular correlates of the murine Xce locus. Genet Res 72:217-224 Avner P and Heard E. (2001). X-chromosome inactivation: counting, choice and initiation. Nat Rev Genet 2(1):59-67. Band MR, Larson JH, Rebeiz M, Green CA, Heyen DW, Donovan J, Windish R, Steining C, Manyuddin P, Womack JE and Lewis HA (2000) And ordered comparative map of cattle and human genomes. Genome Res, 10: 1359 – 1368. Baugh LR, Hill AA, Brown EL and Hunter CP (2001) Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res, 29(5): e29. Baylis MS, Wayte DM, Owen JB. (1984) An XO/XX mosaic sheep with associated gonadal dysgenesis. Res Vet Sci 36(1):125-6. Bhatia S, Shanker V. (1990) A case report on XO/XX/XXX mixoploidy in a goat. Vet Rec 126(13):312-3. Boumil RM and Lee JT. 2001. Forty years of decoding the silence in X-chromosome inactivation. Hum Mol Genet. 10(20):2225-32. Review.Buoen LC, Zhang TQ, Ruth GR, Weber AF, Kittleson SL. (1993) Sterility associated with an XO karyotype in a miniature horse mare. Equine Vet J 25(2):164-5. Brown CJ and Robinson WP (2000) The causes and consequences of random and non-random X chromosome inactivation in humans. Clin Genet, 58: 353 – 363. Carrel L, Cottle AA, Goglin KC and Willard HF. (1999). A first-generation X-inactivation profile of the human X chromosome. Proc Natl Acad Sci USA 96(25): 14440-14444. Chureau C, Prissette M, Bourdet A, Barbe V, Cattolico L, Jones L, Eggen A, Avner P, Duret L. (2002) Comparative sequence analysis of the

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X-inactivation center region in mouse, human, and bovine. Genome Res 12(6):894-908. Colantuoni C, Jeon OH, Hyder K, Chenchik A, Khimani AH, Narayanan V, Hoffman EP, Kaufmann WE, Naidu A and Pevsner J (2001) Gene expression profiling in postmortem Rett syndrome brain: differential gene expression and patient classification. Neurobiology of disease, 8: 847 – 865. Coussens PM, Nobis W. (2002) Bioinformatics and high throughput approach to create genomic resources for the study of bovine immunobiology. Vet Immunol Immunopathol 86(3-4):229-44. De La Fuente R, Hahnel A, Basrur PK and King WA. (1999). X inactive specific transcript (Xist) expression and X chromosome inactivation in the preattachment bovine embryo. Biol Reprod 60(3): 769-775. Disteche CM. (1999). Escapees on the X chromosome. Proc Natl Acad Sci 96(25):14180 – 14182. Eggan K, Akutsu H, Hochedlinger K, Rideout III W, Yanagimachi R and Jaenisch R. (2000) X-chromosome inactivation in cloned mouse embryos. Science, 290: 1578 – 1581. Farazmand A, Koykul W, King WA, Basrur PK. (2000) Expression of X inactive specific transcript (Xist) and testicular morphogenesis in bovine fetuses. Anim Biotechnol 11(1): 51-61. Farazmand A, Koykul W, Peippo J, Baguma-Nibasheka M, King WA, Basrur PK. (2001) Sex-linked genes are not silenced in fetal bovine testes expressing X-inactive specific transcript (XIST). J Exp Zool 290(4):327-40. Freeman WM, Robertson DJ and Vrana KE (2000) Fundamentals of DNA hybridization arrays for gene expression analysis. BioTechniques 29: 1042 – 1055. Geschwind DH, Gregg J, Boone K, Karrim J, Pawlikowska-Haddal A, Rao E, Ellison J, Ciccodicola A, D’Urso M, Woods R, Rappold GA, Swerloff R and Nelson SF (1998) Klinefelter’s syndrome as a model of anomalous cerebral laterality: testing gene dosage in the X chromosome pseudoautosomal region using a DNA microarray. Develop Genet 23: 215 – 229. Goto T and Monk M. (1998). Regulation of X-chromosome inactivation in development in mice and humans. Microbiol and Mol Biol Rev 62(2):362-378.

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88 – SHORT COMMUNICATIONS Graves JA. (1996) Mammals that break the rules: genetics of marsupials and monotremes. Annu Rev Genet 30:233-60. Gygi SP, Rochon Y, Franza BR and Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol, 19(3): 1720 – 1730. Heard E, Clerc P and Avner P. (1997). X-chromosome inactivation in mammals. Annu Rev Genet 31: 571-610. Hedge P, Qi R, Abernathy K, Gay C, Dharap S, Gaspard R, Hughes JE, Snesrud E, Lee N and Quackenbush J (2000) A concise guide to cDNA microarray analysis. BioTechniques 29: 548 – 562. Kelley RL and Kuroda MI. (2000). The role of chromosomal RNAs in marking the X for dosage compensation. Curr Opin Genet Develop 10(5): 555-61. Kochhar HP, Peippo J, King WA. (2001) Sex related embryo development. Theriogenology 55(1):3-14. Lee JT, Davidow LS and Warshawsky D. (1999) Tsix, a gene antisense to Xist at the inactivation center. Nature genetics 21: 400-404. Lee JT. (2000) Disruption of imprinted X inactivation by parent-of-origin effects at Tsix. Cell 103: 17-27. Lockhart DJ and Winzeler EA (2000) Genomics, gene expression and DNA arrays. Nature, 405: 827 – 835. Lyon M. (1999) X-chromosome inactivation. Curr Biol 9(7): R235-R237. Maughan NJ, Lewis FA and Smith V (2001) An introduction to arrays. J Pathol, 195: 3 – 6. Meller VH .(2000). Dosage compensation: making 1X equal 2X. Trends in cell biology 10: 54- 59. Meyers-Wallen VN. (1993) Genetics of sexual differentiation and anomalies in dogs and cats. J Reprod Fertil Suppl 47:441-52. Migeon BR, Lee CH, Chowdhury AK and Carpenter H. (2002) Species differences in TSIX/Tsix reveal the roles of these genes in X-chromosome inactivation. Am J Hum Genet 71(2): 286-93. Mlynarczyk SK and Panning B. (2000) X inactivation: Tsix and Xist as yin and yang. Curr Biol. 10: R899-R903. Natale DR, De Sousa PA, Westhusin ME, Watson AJ. (2001) Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR. Reproduction 122(5):687-93 MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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Nakao M (2001) Epigenetics: interaction of DNA methylation and chromatin. Gene 278: 25-31. Niemann H, Wrenzycki C, Lucas-Hahn A, Brambrink T, Kues WA, Carnwath JW. (2002) Gene expression patterns in bovine in vitro-produced and nuclear transfer-derived embryos and their implications for early development. Cloning Stem Cells 4(1): 29-38. Paget S, Ducos A, Mignotte F, Raymond I, Pinton A, Seguela A, Berland HM, Brun-Baronnat C, Darre A, Darre R, Tamzali Y, Bergonier D, Berthelot X. (2001) 63,XO/65,XYY mosaicism in a case of equine male pseudohermaphroditism. Vet Rec 148(1):24-5 Park Y and Kuroda MI. (2001) Epigenetic aspects of X-chromosome dosage compensation. Science 293(5532):1083 – 1085. Rajeevan MS, Ranamukhaarachchi DG, Vernon SD, Unger ER. (2001) Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods 25(4):443-51 Rizos D, Lonergan P, Boland MP, Arroyo-Garcia R, Pintado B, de la Fuente J, Gutierrez-Adan A. (2002) Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality. Biol Reprod 66(3):589-95. Schmittgen TD (2001) Real-time quantitative PCR. Methods 25:383 – 385 Storey (2002) Natural hypothermic preservation: the mammalian hibernator. Cell Preservation Technology 1(1):1 – 17. Sudbrak R, Wieczoreck G, Nuber UA, Mann W, Kirchner R, Erdogan F, Brown C, Wöhrle D, Sterk P, Kalscheuer VM, Berger W, Lehranch H and Ropers HH. (2001) X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications. Hum Mol Genet 10(1): 77-83. Tsuchiya KD and Willard HF. (2000) Chromosomal domains and escape from X inactivation: comparative X inactivation analysis in mouse and human. Mamm Genome 11(10): 849 – 854. Wrenzycki C, Lucas-Hahn A, Herrmann D, Lemme E, Korsawe K, Niemann H. (2002) In vitro production and nuclear transfer affect dosage compensation of the X-linked gene transcripts G6PD, PGK, and Xist in preimplantation bovine embryos. Biol Reprod 66(1):127-34. Yang YH, Dudoit S, Luu P, Lin D, Peng V, Ngai J and Speed TP. (2002) Normalization for cDNA microarray data: a robust composite method MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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90 – SHORT COMMUNICATIONS addressing single and multiple slide systematic variation. Nucleic Acid Res. 30(4): e15 Xue F, Tian XC, Du F, Kubota C, Taneja M, Dinnyes A, Dai Y, Levine H, Pereira LV, Yang X. (2002) Aberrant patterns of X chromosome inactivation in bovine clones. Nat Genet 31(2):216-20.

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A Profile of Gene Expression in Bovine Oocytes Generated by Heterospecific cDNA Array Screening R. Dalbiès-Tran1 & P. Mermillod Physiologie de la Reproduction et des Comportements, UMR 6073 INRA/CNRS/Université François Rabelais, Tours-Nouzilly (France). 1) [email protected]

Abstract. The oocyte’s ability to sustain genome reprogramming and early embryonic development is usually attributed to cytoplasmic accumulation of an adequate pool of macromolecules. Characterization of the oocyte transcriptome should therefore reveal an invaluable tool to identify genes involved in the acquisition of developmental competence. We have developed heterospecific cDNA array screening, hybridizing probes generated from bovine oocytes onto membranes spotted with human cDNA fragments. Total RNA was purified from pools of oocytes, either immediately after collection from slaughterhouse cow ovaries or following in vitro maturation (IVM). Radiolabelled PCR amplified cDNA probes were generated using the Smart system (Clontech) and hybridized onto Atlas 1.2 human cDNA arrays (Clontech). Membranes were exposed to a PhosphorScreen scanned with a Storm840 (Amersham Biosciences). ImageQuant software (Molecular Dynamics) was used for image analysis. First, we undertook a systematic transcriptome analysis of bovine oocytes before IVM. Three different membranes were screened, representing a total of 3500 genes. About 900 hybridization signals were detected over the background. The proportion (25%) was in the lower range of what is expected according to the manufacturer, consistent with hybridization being gene specific. This experiment generated the largest transcription profile available so far in this model. We then compared transcription patterns in oocytes before (duplicate sample) and after IVM. Quantitative analysis allowed for an estimation of each transcript relative representation within the entire population. Overall correlation between our duplicate experiments with non-matured oocytes was 0.993. The criterion for significant variation during IVM was a consistent twofold difference. The relative representation of most transcripts was not affected by IVM; they are believed to follow the global stream of RNA degradation that accompanies maturation. 37 and 33 transcripts respectively displayed a lower and higher relative representation after maturation. The corresponding genes are involved in various biological functions (cell cycle control, DNA processing and chromatin remodeling, transcription, signaling through growth factors, ras signaling pathway, oxidative stress, protein turnover, and apoptosis). Several of these genes play a role in oocyte maturation and/or embryonic development. This comprehensive map of oocyte gene expression will open the way for analysis of the molecular basis of oocyte late differentiation and acquisition of developmental competence.

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Introduction The oocyte represents a very unique cell in the organism. It is one of the largest, with a diameter close to 100 Pm in most mammals, typically 10 fold that of a somatic cell. This spectacular size has been related to the requirement for an abundant store of biomolecules in order to sustain oocyte maturation and early embryonic development after fertilization. Accordingly, the total RNA content of a mammalian oocyte far exceeds that of any other cell. It has been estimated to peak at about 0.5 ng in mouse and 2 ng in cow. rRNA are most abundant, representing 65% of the pool, while polyadenylated messenger RNA account for about 10% (for a review, see [1]). RNA synthesis is most active during oocyte growth, as evidenced by UTP incorporation into the nucleolus and nucleoplasm, its nucleolus ultra structure showing fibrillo-granular material, and immunodetection of RNA polymerases (see reviews [1]). Transcriptional activity decreases at the end of the growth phase to a minimal level during the differentiation step that follows. In bovine and porcine species, in vitro nuclear maturation within oocyte-cumulus complexes (OCC) requires transcription by RNA polymerase II before the condensation of chromatin that accompanies germinal vesicle breakdown [2,3]. The end of the maturation process, as well as fertilization, is believed to occur in the absence of transcription. Minor embryonic genome activation has been detected as early as the 2-cell stage in several species but appears dispensable for pre-implantation development. Major activation determines the maternal to embryonic transition (MET) and occurs at a specific stage: in the 2-, 4-, 8/16-cell embryo for mouse, human, and bovine respectively (see reviews [4,5]. As a consequence, there is a period, spanning the gonadotropin-dependent phase of follicular growth, ovulation, fertilization and early development, when translation is essentially supported by RNA synthesized earlier and stored under a stable form in the ooplasm. Therefore, the oocyte transcriptome represents the potential for gene expression in the oocyte itself, but also in the embryo before the MET. Because of its delayed MET until the 8/16-cell stage, bovine appears to be an attractive model for these studies.

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Among technologies developed to characterize global gene expression, screening of DNA arrangements spotted onto plastic or glass slides or nylon membranes appears as a time and cost effective method. Arrays of pre-identified EST provide information on the presence and relative abundance of hundreds to thousands of transcripts without the necessity for massive sequencing. However, to date no such arrangement of bovine EST is commercially available. We developed heterologous macroarray screening: probes generated from bovine oocytes were hybridized onto arrays of human EST, several hundred base pairs in length, based on our hypothesis that the 80% average sequence conservation in open reading frames between both species would result in a specific and sensitive hybridization. This methodology was applied to characterize a profile of transcripts in immature oocytes and study the evolution during in vitro maturation.

Materials and methods An overview of the experiment is schematically represented in Figure 1. Bovine ovaries were obtained from a local abattoir and OCC collected from antral follicles 3-8 mm in diameter. Oocytes were denuded by mechanical treatment and frozen, either immediately or after incubation for 24 hr in TCM199 supplemented with 10ng/ml EGF. Total RNA was purified. [32P]-labeled probes were generated using the Smart technology developed by BD biosciences, with the corresponding cDNA synthesis and probe amplification kits (Ozyme, France). For labeling, random primers were preferred over specific primers for the human EST provided by the manufacturer, considering probable mismatches with the bovine target sequences. Probes were hybridized with one of the Atlas human 1.2 arrays onto nylon membranes. Each one is spotted with about 1200 EST as well as positive and negative controls. After exposure to a sensitive screen, images were acquired with a Storm 840 and analyzed with ImageQuant software (Amersham Biosciences, France).

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94 – SHORT COMMUNICATIONS Figure 1. Experiment overview

Results and discussion In our first experiment, probes generated from immature bovine oocytes were hybridized onto all three membranes, for screening of 3500 genes. Over 800 signals were detected. Together, they provide the broadest expression profile available in this model to date. A statistical analysis of these data is presented figure 2. Genes can be classified based on the subcellular localization of the corresponding protein (fig. 2a) or on their biological function (fig. 2b). It appears that genes involved in a broad range of functions are transcribed in the oocyte, reflecting its own ability, as well as that of the embryo to come, to accomplish many cellular process. This profile is currently subjected to a systematic comparison with cDNA and serial analysis of gene expression (SAGE) libraries

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generated from mouse (UniLib#1048 and 1463) and human oocytes [6], in order to assess conservation and divergence between mammalian species. Figure 2. Statistical analysis. Gene classification based on cellular localization of the corresponding protein (a) or biological function (b). (a)

Class SB00 SC00 SE00 SF00 SG00 SH00 SK00 SL00

Localization plasma membrane proteins cytoplasmic proteins endoplasmic reticulum extracellular secreted proteins golgi complex chromosome structural proteins cytoskeletal proteins lysosomal proteins

class SM00 SN00 SP00 SR00 SS00 ST00 SU00 SX00

Localization mitochondrial proteins nuclear proteins peroxisomal proteins microsomal proteins endosomal proteins extracellular matrix nuclear membranes/ nuclear matrix/nuclear pores unclassified localization

350

300

localization

250

200

326

150

gene number

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291

217 72

50

22

31 15

4

22 13

53

7

9

4

SP00

SR00

SS00

12

6

0

SB00

(b)

Class FA FB FC FD FE FF FG FH FI FJ FK FL FM

SC00

SE00

SF00

SG00

SH00

SK00

Function cell surface antigens Transcription cell cycle cell adhesion immune system extracellular transport/carrier proteins oncogenes and tumor suppressors stress response proteins membrane channels and transporters extracellular matrix proteins trafficking/targeting proteins Metabolism post-translational modification/prot. folding

SL00 SM00

class FN FO FP FQ FR FS FT FU FV FW FX FY

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SN00

ST00

SU00

SX00

function translation apoptosis associated proteins RNA processing, turnover, and transport DNA binding and chromatin proteins cell receptors (by ligands) cell signaling, extracellular communication intracellular transducers/effectors/modulators protein turnover cell receptors (by activities) cytoskeleton/motility proteins functionally unclassified DNA synthesis, recombination, and repair

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function 150

gene num ber

202

100

159

138 53 19

29 0

108

104

50

FA

FB

FC

FD

13 FE

65

59

1

49 44 35

6

FF

FG FH FI

FJ

6 FK

FL

FM FN

33 34 16

47

FO FP

FS

FQ FR

49 FT

FU FV

30

19 37

FW FX

FY

We applied the same technology to characterize the evolution of the oocyte transcriptome during maturation. The Atlas human 1.2.I array was screened with probes generated from oocytes before IVM (two independent samples) and after IVM (one sample). In the quantitative analysis, the value for each signal was corrected for the local background; all three membranes were then normalized based on the sum of intensities. For each gene, the variation factor (VF) was defined as the ratio of relative abundance (RA) before and after IVM; a reproducible twofold difference was our criteria for considering variation. In fact, the RA of a vast majority of transcripts did not appear significantly affected (0.05
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(a) before IVM

(b) after IVM

before IVM

after IVM

Differential regulation during maturation.

Table 1. Genes (HGNC symbol) whose relative transcript level varies during IVM. RA increases during IVM NME2 CCNB1 CLK3 UBE2C ID3 RALB ATF2 RASA1 YWHAB FASTK POLB

TOP1 TOP2A PCNA POLE2 LIG1 ERCC2 ERCC3 PMS1 CHRNA3 RBBP7 TCFL1

ID2 ZNF9 GABPB2 MSX1 GATA3 HSPCA GSTA2 DTR SCYB5 IDE PSMA4

RA decreases during IVM RAP1A PHB NR2F6 KRAS2 CCNH RPS3A CDC2 TFDP2 RPS19 KDR TIE GRB2

HINT CASP4 BCL2L1 DAD1 PIN RFC5 MLH1 RAD51 HTR3A PCSK1 PCP4 MJD

COPS5 RPL6 GTF2H2 ILF2 CYP1A2 GSTT1 PRDX2 PRDX1 GSTM1 TMSB10 CRHBP PSMB1 SPINK2

The extent to which the characterized profile is affected by in vitro manipulation of the oocytes remains unknown. Nevertheless, this comprehensive map of oocyte gene expression will open the way for analysis of the molecular basis of oocyte late differentiation and acquisition of developmental competence.

Acknowledgments Financial support by EU grant QLK3-CT-1999-00104 is gratefully acknowledged.

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References

1. Picton H, Briggs D, and Gosden R. (1998), The molecular basis of oocyte growth and development. Mol Cell Endocrinol. 145:27-37. 2. Kastrop PM, Hulshof SC, Bevers MM, Destree OH, and Kruip TA. (1991), The effects of alpha-amanitin and cycloheximide on nuclear progression, protein synthesis, and phosphorylation during bovine oocyte maturation in vitro. Mol Reprod Dev. 28:249-54. 3. Meinecke B, and Meinecke-Tillmann S. (1993), Effects of alpha-amanitin on nuclear maturation of porcine oocytes in vitro. J. Reprod Fertil 98:195-201. 4. De Sousa PA, Caveney A, Westhusin ME and Watson AJ. (1998), Temporal patterns of embryonic gene expression and their dependence on oogenetic factors. Theriogenology. 49(1):115-28. 5. Memili E, and First NL. (2000), Zygotic and embryonic gene expression in cow: a review of timing and mechanisms of early gene expression as compared with other species. Zygote 8:87-96. 6. Neilson L, Andalibi A, Kang D, Coutifaris C, Strauss JF 3rd, Stanton JA, and Green DP. (2000), Molecular phenotype of the human oocyte by PCR-SAGE.Genomics, 63:13-24.

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Heteroplasmy Rates Analysis in Single Blastomeres Using Fluorocromes Stained Primers C.r. Ferreira,1 F.V. Meirelles,2 W. Yamazaki,, and J.M. Garcia1 1) Departamento de Medicina Veterinâria Preventiva e Reproduçào Animal/FCAV/UNESP- Jaboticabal, Brazil 2) Departamento de Ciências Basicas/FZEA/USP-Pirassununga, Brazil Different techniques have been proposed to study mtDNA (mitochondrial DNA) inheritance in reconstructed embryos and derived animals. The proportion of different haplotypes is usually estimated applying nested-PCR and endonuclease digestion, PCR and RFLP with radioactive labelling (1), RFLP of plasmid clones each with PCR amplified control region inserts (2) and real time-PCR (3). Here we describe a screening method adapted for estimating mitochondrial contribution in cloned embryos or blastomeres. Our aim was to estimate B. indicus and B. taurus haplotypes proportions in single blastomeres using PCR amplification with primers stained with two different fluorocromes, specific either to B. taurus or B. indicus mtDNA. To produce heteroplasmic embryos, (nuclear transfer) NT was performed using 16-cell embryo blastomeres (TN-B) or fibroblasts (TN-F) B. indicus as nuclear donors and B. taurus cytoplasts as receptors. These embryos were cultivated in SOF medium after reconstruction and taken for analysis at 2; 4; 8-cell and blastocyst stages. Embryos harbouring one nuclei in all blastomeres (Hoechst 33342) were dissociated and the single blastomeres were separated for PCR. A proteinase K lyses was performed in the blastomeres before PCR. Three primers were used in the PCR: one non-specific anti-sense primer and two specific sense primers stained with fluorocromes TAMRA (for B. indicus mtDNA) and 6-FAM (for B. taurus mtDNA). All primers were used in a 10pmol concentration, which guaranteed a surplus of primers in the PCR reaction. The amplification was carried out in 20mM tris-HCI MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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100 – POSTERS (pH 8,4), 50mM KCI, 5mM MgCl2, 600PM of each dGTP, dATP, dTTP and dCTP, and 1,25U Taq DNA polymerase in 36 amplification cycles (denaturation at 94ºC for 45s, annealing at 58ºC for 55s and extension at 72ºC for 120s). Resulted fragments had 365bp. Electrophoresis of 154 amplified samples, representing 75 embryos, was performed in 1,2% agarose gel and laser scanned (FLA-3000G) at 473-520nm (6-FAM; B. taurus haplotype) and 532-580nm (TAMRA; B. indicus haplotype). Haplotypes proportions were quantified throughout densitometry (Image Gauge v.3.12; FujiFilm; Japan) and a correction factor, based on an amplification curve with known proportions of B. taurus and B. indicus mtDNA, was applied. Data was analyzed applying variance analysis through JMP (SAS Institute. Carry, USA). The mean percentage amount of B. indicus mtDNA in the TN-F group by the 2-cell-stage was 4,87% (SD = 1,25) decreased to 2,21% (SD = 1,2) in the blastocyst stage. In the TN-B group, mtDNA B. indicus amount in the 2-cell-stage was 20, 16% (SD = 25,95) and 22,05% (SD = 20,72) in the blastocyst stage. We observed that the technique allowed the detection of blastomeres and embryos heteroplasmy at 1% level. This technique sensibility may be applied to estimate the proportion of specific sequences in samples with very low DNA or cDNA content. And therefore could be used to confirm the presence of specific mRNAs of nuclear or cytoplasmic origin in hybrids reconstructed embryos.

References 1. EVANS MJ, GURER C, LOIKE JD, WILMUT I, SCHNIEKE AE, SCHON EA. 1999. Mitochondrial DNA genotypes in nuclear transfer-derived sheep. Nature Genetics 23 :90-3. 2. HIENDLEDER S, WOLF K, BRUGGERHOFF K, WENIGERKIND H, REICHENBACH H-D, PRELLE K, STOIJKOVIC M, BREM G, ZAKHARTCHENKO V, WOLF E. 2002. Mitochondrial genotype of bovine fetuses reconstructed by Bos taurus somatic cell nuclear transfer to Bos Taurus or Bos Indicus oocytes. Theriogenology 57:419. Abstract. 3. SHITARA H, HAYASHI JI, TAKAHAMA S, KANEDA H, YONEKAWA H. 1998. Maternal inheritance of mouse mtDNA in interspecific hybrids: segregation of the leaked paternal mtDNA followed by the prevention of subsequent paternal leakage. Genetics 148:851-7. Financial support: FAPESP

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Additional Step in Genomics Studies of Bovine Embryos: Proteomics L. Massicotte and M.A. Sirard Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, Canada, G1K 7P4. The oocyte has to store mRNA, proteins and organelles to supply the embryo’s development prior to the maternal-zygotic transition to continue further development. The stock of mRNA is the most studied part of the maternal storage because the precious tools of molecular biology can furnish grateful hints of what is needed to embryo development. But the presence of mRNA doesn’t give the information of when and how many proteins will be translated. Improvement of mass spectroscopy is now allowing protein identification with a small amount of proteins and offers new possibilities in the research of proteins. As our principal goal is to find proteins implicated in developmental competence, ten competent and ten incompetent two-cell embryos (36-40h post-insemination (hpi)) and ten delayed incompetent two-cell embryos (45-49hpi) were labelled with [S35]-Methionine and [S35]-Cystein during 4 hours. Using Immobiline DryStrips of pH range of 3-10 with Ettan IPGphor from Amersham Biosciences, proteins were separated and then proceeded to the second dimension, dried and visualised by autoradiography. The experience was made at least three times. The use of ImageMaster 2D software from Amersham Biosciences allowed us to make complete and fast analysis between our different treatments. The best approach to analyse a gel is by making the census of all proteins in each replicate. Next, gels within a same replicate are compared and only proteins conserved in more than half of the replicates are considered which allows the creation of a representative protein pattern of the treatment. Protein patterns from each treatment can be compared and differences found (see Table 1).

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Translated protein pattern differences between competent and incompetent two-cell embryos

COC source Time of first cleavage Development rate Labelling period Average translated proteins Conserved proteins pattern Differences

Follicular size COC morphology

5-10 mm Slightly atretic Fast 48.5 ± 39.5% 36-40 hpi 295 ± 56

2-ce1l Embryos 3-5 mm Healthy Fast 24.4 ± 18.5% 36-40 hpi 332 ± 45

3-5 mm Healthy Delayed 0 ± 0% 45-49 hpi 212 ± 68

289

281

167

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46

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After identification of the protein of interest, the next step is to find the translated protein in a non-radioactive gel. The protein pattern of radioactive translated protein compared with total non-radioactive protein pattern are almost completely different. With a total of 500 two-cell embryos including 130 radioactive embryos, and with combination of silver stain and autoradiography, only 166 of the 851 translated proteins detected by the autoradiography were present in the 605 proteins visualized by the silver staining presenting an homology of 27.43%. With this homology rate, only four proteins have been identified. An increasing number of embryos will enlarge the number of detected proteins which will arise the homology rate and protein retrievement. For sequencing, around 3 000 embryos have to be used for detection with the Bio-Safe Coomassie Stain from Bio-Rad to reach a similar number of protein as with 500 embryos with silver staining. The Bio-Safe Coomassie Stain is use to make sure that enough material is available for sequencing.

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Quantification of Histone Acetyltransferases Transcripts in the Early Stages of Bovine Embryo Development S. McGraw, C. Robert, L. Massicotte, and M.A. Sirard Centre de Recherche en Biologie de la Reproduction, Department of Animal Sciences, Laval University, Québec, Canada, G1K 7P4 The mammalian oocyte is a unique totipotent cell which can erase existing gene expression patterns to create new ones. The chromatin structure must first be altered before such genomic reprogramming can take place. These modifications alter the higher order chromatin organization to render the DNA accessible to the transcription machinery. One mechanism known for this action is the acetylation of the nucleosomal histones. By means of real time quantitative RT-PCR, we established mRNA expression profiles for three histone acetyltransferases (HAT1, GCN5 and MORF) and one nucleosomal histone (H2A) throughout early bovine embryo development. Oocytes were harvested from slaughterhouse ovaries and cattle embryos were produced in vitro through standard techniques. The oocytes and embryos were mechanically stripped of their cumulus cells, washed in PBS buffer and stored at -80°C. Triplicates of 20 oocytes (GV and MII) or embryos (2-cell, 8-cell and blastocyst) were used for each of the developmental stages. Total RNA was extracted and DNase treatment was carried out simultaneously. The reverse transcriptions were performed in 20 Pl total and the cDNA corresponding to 2 oocytes or embryos (1 for H2A) were used for each of the quantitative PCR assays on a Light Cycler apparatus (Roche Diagnostics, Laval, Canada). The values obtained were normalized with the values of H2A. Our results demonstrate that the expression of HAT1 is low and relatively constant during the pre-blastocyst stages, before a major level increase in blastocyst. The patterns observed for GCN5 and MORF are unlike the one for HATl. Their highest levels are express in the MII oocytes and 2-cell embryos, and their lowest levels are in the 8-cell embryo and blastocyst. MORF mRNA transcripts are extremely more abundant in every developmental stages. Throughout the pre-blastocyst period it is 500 and 3 500 times more abundant than GCN5 and HAT1. In the blastocyst it is 440 and 320 times higher than GCN5 and HATl. These results indicate that MORF acetyltransferase enzyme maybe more important in early stage of embryonic development. It may be an important player in the regulation of the transcriptional activity. This project was supported by Natural Sciences and Engineering Research Council of Canada.

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Study of Newly Synthesized Proteins During Bovine Oocyte Maturation In Vitro by Two-Dimensional Gel Electrophoresis K. Coenen and M.A. Sirard Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, Canada, G1K 7P4 It is well known that a limited correlation between mRNA levels and proteins exists. Furthermore, modifications of the proteins which strongly influence their function cannot be deduced from DNA or RNA sequences. Thus, a combination of genomics and proteomics is required for the global comprehension of cell function. In this experiment, two-dimensional (2-D) gel electrophoresis coupled with computerized image analysis was used to study the proteome during in vitro maturation of bovine oocytes, a 24 h process. Oocytes were collected from ovaries obtained from the slaughterhouse, selected in bovine follicular fluid, washed and put in maturation. Except for the interval of maturation 0 – 4 h, maturation of oocytes occurred in two steps. In the first step, ten oocytes were cultured in maturation medium consisting of modified synthetic oviductal fluid (SOF) supplemented with BSA, MEM amino acids, glutamine, pyruvate, gentamicine and hormones. Next, the oocytes were washed, some denuded and others not, and transferred for 4 h in labelling medium i.e. maturation medium containing 1 mCi/mL of Tran35S-label (ICN Biomedicals, inc.) and PVP 40 instead of BSA. After labelling, cumulus and zona pellucida were removed and oocytes were solubilised in pools of 10 in sample buffer to proceed with the 2-D gels. Denatured proteins were first separated by their isoelectric point using Immobiline DryStrip with a pH range of 3-10 with IPGphor isoelectric focusing system (Amersham Biosciences) and then separated by their molecular weight by SDS PAGE. After 30 days of autoradiographic exposition, proteins were visualized. For each maturation time interval, 3 gels were obtained and analyzed using the ImageMaster 2D Elite image analysis software (Amersham Biosciences). The gel containing the most proteins was then chosen as the reference gel and compared with the others. An average gel was created with proteins present in at least 2 other gels. Table 1 summarizes the number of proteins seen in each average gel created for each time interval of bovine oocyte maturation in vitro. The rate MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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of synthesis is higher at the beginning of maturation until the Metaphase I is reached (12-16 h) and then it is lower and relatively stable. 3 different patterns were observed during maturation: one at the beginning of maturation (0-4 h), another one in the middle (4-16h) and the last one after the completion of Metaphase I (16-28 h). By comparison of these patterns with those observed with oocytes of different competence, it will be possible to determine which proteins were essential for a good cytoplasmic maturation and a better production of good blastocysts.

Table 1. Number of newly synthesized proteins during bovine oocyte maturation in vitro Time interval Number of synthesized proteins

0-4 h

4-8 h

Bovine oocyte maturation in vitro 86-12 h 12-16 h 16-20 h 20-24 h

24-28 h

601

520

500

329

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Study of Genome Activation and Developmental Block Messages Applying Fluorescent DD-RT-PCR A.R. Caetano; Y.F.Watanabe; G.K.F. Merigue, and F.V. Meirelles LMMD/ FZEA Univ. de São Paulo-Pirassununga, SP, Brazil 13630.970 Bovine embryo development in vitro bas been studied for the lasts decades and attained good development rates. Later improvement allowed commercially application as well as application in other biotechnologies fields. Yet most of the success to improve blastocyst rates is due to culture conditions empirically tested. We believe that the knowledge in the early embryos physiology could be largely improved by applying genomic methods to uncover messages related to specific moments in embryo culture. Here in we describe ours efforts to help to characterize the early developmental block and genome activation in bovine species. Bovine embryos are well known to relay until the 3rd cell cycle in oocyte cytoplamic proteins and messages (2). However development beyond this stage depends on embryo ability to activate its genome and actively transcribe necessary messages. Embryos who fail to activate its genome are selectively split out by the first developmental bottleneck that drifts them to the developmental block. The aim of this study was to characterize messages differentially expressed in embryos with high (attaining 8 cell stage before 48 hours) and low (attaining 8 cell stage between 50 and 90 hours) competence to reach blastocyst stage (1). Oocytes recovered from zebu cows ovaries collected in local abattoirs were maturated, fertilised with zebu semen and cultured in vitro. Embryos harboring exactly 8 cells were collected, at the moment 48 and 90 hours pos insemination (hpi). Total RNA extraction was obtained from 50 embryos pools in triplicates following RNeasy extraction kit protocols (Quiagen-Germany). Recovered RNA was reverse transcript following steps recommended in superscriptII protocol (Invitogen – USA) and applying a 12mers polyT anchor primer with 2 base pairs variation in the 3’end and with a 16mers T7 sequence in the 5’end (total of 10 primers). PCR was carried out in a final volume of 20 Pl of 10mM Tris-HCI (pH 8.3); 50mM KCI, 3.0 mM MgC l2; 0.01% of gelatin; 200 mM of each dNTP (pharmacia, Piscataway, NJ.); 10 pmol of each primer and 1.25 units of taq DNA polymerase (Life Technologies). Primers MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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pairs were composed by a T7 anti-sense marked with TANRA fluorochrome and one of 20 random sense primers of 10pb each. Reactions were carried out for 40 cycles of 15s at 95°C; 30s at 56°C and 90s at 72°C. PCR Amplified cDNA were denaturated and applied to a 40x20 cm 6% denaturating acrilamide 1/29 gels. Electrophoresis were carried out at 1.5kV for 3 hours at 50 to 55 °c. Acrilamide was air dried over the open glass plate, washed and dried again until no traces of urea were visible. The glass plate was then scanned in a laser scanner FUJI FLA 3000G (Fuji film – Japan) applying 520nm laser and a 580nm long pass filter. Resulted images were printed using a high resolution pictrographic printer. Printed images were used to cut differential expressed fragments. Fragments were cut from dried acrilamyde reamplified and sequenced directly from PCR. A minority of the fragments produced good quality sequences. Preliminary results show in average 3 differential expressed fragments per primer combination. The large majority of the differential expressed fragments were present in slow development embryos. This unexpected picture may be because of death genes activation. Indeed, sequences of these fragments showed mRNAs associated with apoptose (ex: BAX and BCLx associated protein) and many mitochondrial genome sequences. Cloning and sequencing the fragments will reveal the nature of others fragments differentially expressed in fast and slow developing embryos.

References 1. Watanabe YF, Schwarz KL, Merighe GKF, Caetano AR, Meirelles FV. 2002. Developmental potential and DNA damage of IVP bovine embryos with fast and slow development. Theriogenology: 57:509. 2. De Sousa PA, Caveney A, Westhusin ME, Watson AJ. 1998. Temporal patterns of embryonic gene expression and their dependence on oogenic factors. Theriogenology; 49: 115-128. Review

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Identification of First Zygotic Transcripts Using SSH and validation of RNAi Procedure in the Rabbit Embryo V. Duranthon , S. Pacheco,1 P. Chrenek,2 C. Hennequet-Antier,4 J.F. Ondin,3 AND J.P. Renard3 1) UMR Biologie du Développement et Biotechnologies, INRA, 78352 Jouy en Josas Cédex, France. 2) Laboratoire de Génétique du Paludisme., Institut Pasteur, 25 rue du Dr Roux, 75724 Paris. France. 3) Research Institute of Animal Production, 94992 Nitra, Slovaquia. 4) Laboratoire de Mathématiques Informatique et Génome, INRA, 78026 Versailles Cédex, France. It is now well documented that changes in the microenvironment of preimplantation embryos induced by in vitro culture and manipulation can alter the pattern of gene expression with consequences on early differentiation events. These changes also probably affect post-implantation development, even after birth. In order to characterize the overall changes in the pattern of gene expression at the onset of zygotic transcription -a unique period that corresponds to the transition between cell totipotency and pluripotency- we decided to create cDNA libraries starting directly from early embryonic material. We chose rabbit embryos whose zygotic gene activation spans over at least three cell cycles. This offers a wider opportunity to thoroughly analyze environmental effects during the transition between maternal and zygotic control of development (MZT). To functionally analyze gene expression perturbation, we also analyzed the potential of RNA interference in the rabbit embryo. Embryos were obtained from New Zealand White rabbits, at 19 hours post coitum. One cell stage embryos were immediately frozen, whereas morula stage embryos were cultured in 199 CO2 (Gibco BRL) supplemented with 10% fetal calf serum medium for 50 hours. Total RNA was extracted from 500 embryos at each stage. cDNA, equivalent to the cDNA of about 250 embryo, was synthesized and PCR amplified according to the Clontech SMART procedure. SSH was then performed according to the PCR Select kit (Clontech) procedure. Differential screening was performed with morula MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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and one cell stage cDNA probes. We sequenced differentially labelled clones and determined the expression pattern of a panel of these clones over the preimplantation period by hybridization with total cDNAs obtained at each preimplantation stage. RNA interference was tested using F1 transgenic rabbit embryos obtained by mating normal females with EFlD-EGFP transgenic heterozygous male rabbits. Embryos were injected at the one cell stage with in vitro synthesized dsRNA, and EGFP expression was measured by fluorescence microscopy. We created a subtracted cDNA library corresponding to the transcripts expressed at the onset of zygotic transcription in rabbit embryos. One thousand clones of this subtracted library were subjected to differential screening, resulting in the isolation of 114 clones labelled only by the morula probe. The differential expression of the corresponding genes was confirmed on a sample of 17 clones. Anova analysis of expression patterns allowed us to identify at least two groups of zygotically-expressed genes according to their early post-MZT behaviour: gradual increase from MZT onward, and abrupt increase at MZT followed by a relative decrease in expression at the morula stage. Sequence analysis showed that the newly formed genome expressed transcripts encoding for protein synthesis or degradation and for metabolic functions. It also expresses transcripts which are still unknown or whose sequence is known but not function. Several of the isolated unknown transcripts could not be found expressed in a panel of ten adult tissues. These results provide a first step towards a versatile means of checking the effects of in vitro culture conditions on zygotic gene activation. Unknown cDNAs are currently being characterized and sequences are being elongated by 5’ and 3’ race. We also validated the RNA interference technique in the rabbit whose MZT occurs after several cell cycles and showed that this approach allowed a specific inhibition of zygotic gene expression even when the dsRNA molecules were injected in one-cell stage embryos. This expressional and functional genomics strategy is now being extended to cattle.

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Poly(A) mRNA Content and Polyadenylation Status of Specific Transcripts During In Vitro Bovine Oocyte Maturation J.M. Traverso, J. Marchandise, I. Donnay, and A. Sophie Lequarré Unité Vétérinaire, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium Introduction Early embryonic development is directed by maternal mRNAs stored during oocyte growth. Most of these RNAs are included into ribonucleoparticles and will become available for translation when oocyte maturation begins. Then the maternal stock gradually disappears. In the mouse as much as 50% of the maternal poly(A) mRNA is degraded during maturation (1), while in the rabbit no major degradation occurs before the 8 to 16-cell stage (2). There is no such information for cattle. Cytoplasmic polyadenylation plays a key role determining the fate of the mentioned stored maternal mRNAs.

Materials and methods Cumulus-oocyte complexes (COCs) were aspirated from slaughterhouse-collected ovaries, taking follicles ranging from 3 to 10mm in diameter. COCs were carefully denuded, and pools of oocytes and cumulus granulosa cells were made before and after maturation, and frozen at –80°C until use. Maturation took place during 24h under 20% O2 in TCM-199 supplemented with 10 ng/ml EGF and 0.4 mM pyruvate, without serum. After maturation pools were made at 6, 12, 18, and 24h from the beginning of maturation. Total RNA was extracted with acid-phenol. A) Poly(A) mRNA Quantification: Pools of 50 oocytes were used in this experiment. Measures were taken using a new method based on the pyrophosphorylation property of the DNA polymerase Klenow that produces ATP in proportion to the number of poly(A) tails in the sample. This ATP production is quantified by a classic luminometric assay (Poly(A) mRNA Detection System. Promega, USA). B) Polyadenylation status of specific mRNAs: Changes in the poly(A) tail length were studied using RACE-PAT (3), a PCR-based technique, MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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where the length of the smear obtained after PCR gives an estimate of the length of the poly(A) tail. Transcripts analyzed were GDF-9, an oocyte-exclusively expressed gene that plays an important role during follicle development; and Cyclin A, involved in control of the tell cycle. Two different primers were used for each of the analyzed transcripts. Pools of 20 oocytes were used in this experiment.

Results and discussion A) According to our results an immature bovine oocyte would contain around l0pg of poly(A) mRNA. As in the mouse oocyte, there is an important decrease of this mRNA during maturation leaving only 1/3 of what is detected in the immature oocyte, and the degradation follows a linear kinetics (analyzed at 6, 12, 18, and 24h from the beginning of maturation). Consequently, the poly(A) mRNA content of an immature oocyte seems much lower of what has been expected from the literature. B) In oocytes and cumulus granulosa cells, a longer poly(A) tail was found after maturation for the Cyclin A transcript (suggesting a probable transcriptional activation), while an unexpected band appeared for the GDF-9 transcript. Kinetics of the changes at the poly(A) tail level showed that important changes occur between 12 and 18 h, corresponding to the lengthening of the poly(A) tail for the Cyclin A transcript, and the appearance of a new band for GDF-9. More detailed studies should be done to shed light on the changes observed for the GDF-9 transcript.

References 1. Paynton BV, Rempel R, Bachvarova R. 1988. Changes in state of adenylation and time course of degradation of maternal mRNAs during oocyte maturation and early embryonic development in the mouse. Dev Biol. Oct;129(2):304-14. 2. Henrion G, Brunet A, Renard JP, Duranthon V. 1997. Identification of maternal transcripts that progressively disappear during the cleavage period of rabbit embryos. Mol Reprod Dev. Aug;47(4):353-62. 3. Sallés FJ, Richards WG, Strickland S. 1999. Assaying the polyadenylation state of mRNAs. Methods. Jan;17(1):38-45.

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Temporal Expression Patterns of Transcription Factors During Cattle Early Embryogenesis C. Vigneault and M.A. Sirard Centre de Recherche en Biologie de la Reproduction, Université Laval, Québec, Canada, GIK 7P4 When mammalian embryos are cultured in vitro, a developmental block can be observed at a specific developmental stage depending on the species. This developmental block occurs at the 8-cell stage in cattle, which coincides with embryonic genome activation. Therefore, we believe that a problem in the activation of transcription at the 8-cell stage in some embryos may be implicated in that developmental block. Our hypothesis is that good oocytes or good early embryos contain or produce the transcription factors needed for the major activation of transcription that occurs at the 8-cell stage. We looked at the presence of mRNA of some transcription factors thought to be important in embryogenesis such as CREB, P300, POU5Fl, TEAD-2 and NFAR during bovine embryo development from the GV-stage oocyte to the blastocyst. Oocytes and embryos were produced using selected cumulus-oocyte complexes (COCs) aspirated from 3-5 mm follicles from ovaries obtained at the slaughterhouse. The oocytes and embryos were matured and developed in SOF medium. We used 120 oocytes or embryos of each stage (GV, MII, 2-cells, 4-cells, 8-cells and blastocyst) and pooled in groups of 40 before freezing. RNA extraction was performed using the Strataprep total RNA microprep kit (Stratagene), and reverse transcription was done using the Omniscript reverse transcriptase (QIAGEN). The quantitative amplification was performed in triplicate using the equivalent of 1 oocyte or embryo per reaction in a Lightcycler, a real-time PCR apparatus (ROCHE). For standardization of our samples, 1 pg of exogenous RNA corresponding to a fragment of the Green Fluorescent Protein (GFP) produced by in vitro transcription was added to the samples before RNA extraction. The values of each transcription factors were corrected with the values obtained from the amplification of the GFP RNA. Our results show that CREB and NFAR have a similar pattern of expression through the developmental stages examined. Their mRNA levels are relatively constant from the oocyte to the 4-cell stage. Afterwards, we MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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see a slight decrease in mRNA levels at the 8-cell stage and finally a slight increase at the blastocyst stage. In the case of P300, the mRNA levels remain relatively constant from the oocyte to the 8-cell embryo and slight increase at the blastocyst stage. Finally, for TEAD-2 and POU5Fl, the mRNA levels decrease very slowly from the oocyte to the 8-cell stage and increase dramatically at the blastocyst stage.

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Quantitative Real-time PCR Analysis of RNA and DNA in Individual Mouse Embryos and their Blastomeres C. Hartshorn, J.E. Rice, and L.J. Wangh Department of Biology, Brandeis University, Waltham MA 02454-9110, USA Introduction Fluorescent in situ hybridization (FISH) demonstrates that Xist gene expression begins early in mouse embryogenesis and that by the 8-cell stage Xist RNA is bound to one X chromosome of female embryonic cells (1). We used real-time RT-PCR to quantify Xist RNA levels in individual embryos and their blastomeres and to determine their sex. Because Sry is a single copy gene present on the Y chromosome, parallel analysis of Xist and Sry DNA provided an independent verification of embryo gender.

Materials and methods At each developmental stage (l-cell to expanded blastocyst) RNA and DNA were extracted from 15-21 individual embryos using a Stratagene Micro RNA Isolation Kit. In most cases, each sample was divided into three aliquots and analyzed for Xist genomic DNA, Sry genomic DNA, and, following reverse transcription with ThermoScriptTM (Invitrogen), Xist cDNA. Quantitative real-time PCR of both Xist and Sry was carried out in an ABI Prism 7700 Sequence Detector using SYBR£Green as the detector. The “threshold cycle” (Ct) at which each fluorescent signal was first detected above background was used as a measure of the number of templates present at the start of the reaction by comparison to a standard scale of mouse genomic DNA. In some experiments the zona pellucida of 8-cell embryos was drilled using a Zilos Laser and almost every blastomere was recovered and individually analyzed for Sry DNA and Xist DNA + RNA, using multiplex real time RT-PCR with two differently colored molecular beacons.

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Results and discussion Xist RNA was first detected in some late 2-cell stage embryos and, by the 4-cell stage, all embryos could be placed in two distinct groups on the basis of their levels of Xist RNA. Male embryos had very low levels of Xist RNA, but were positive for both Sry and Xist genomic DNAs. In contrast, female embryos had no Sry DNA, but contained Xist DNA at twice the level round in male embryos of the same stage. Xist RNA levels increased dramatically in early female embryos and reached a peak of 5800 copies per embryo (280 copies per cell) in compacted morula-stage. The average level of Xist RNA per cell decreased to 137 copies in early blastocysts and to 45 copies in hatching blastocysts. This decline can only be partly accounted for by the accumulation of undifferentiated cells of the inner cell mass, which comprises roughly one third of the 32-cell embryo (2), and may play a role in embryonic development. Xist RNA was consistently present in single female blastomeres isolated from 8-cell stage embryos via laser zona drilling, a very rapid and reliable technique. The total amount of Xist RNA recovered from all single blastomeres of one female embryo was comparable to that obtained from an intact embryo. Surprisingly, levels of Xist RNA differed considerably among the blastomeres of a single embryo and two cells consistently showed particularly high amounts of Xist RNA. In conclusion, our real-time PCR methodology proved quantitative and sensitive down to the level of one DNA molecule. The results of this study have implications for cell lineage determination in early mouse embryos. Supported by a grant from Hamilton Thorne Biosciences.

References 1. Sheardown SA, Duthie SM, Johnston CM, Newall AB, Formstone EJ, Arkell RM, Nesterova, TB, Alghisi, GC, Rastan S, Brockdorff N. (1997) Stabilization of Xist RNA mediates initiation of X chromosome inactivation. Cell 91, (99-107). 2. Kiessling AA, Davis HW, Williams CS, Sauter RW, Harrison LW. (1991). Development and DNA polymerase activities in cultured preimplantation mouse embryos: comparison with embryos developed in vivo. J Exp Zool 258, (34-47).

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Targeting Gene Expression In the Preimplantation Mouse Embryo Using Morpholino Antisense Oligos A.J. Watson and L.S. Siddall Departments of Obstetrics and Gynecology and Physiology, The University of Western Ontario, London, ON, Canada, N6A 5C1

Introduction A greater understanding of the genetic components regulating early embryo development is essential to account for substantial embryo loss during preimplantation development. Morpholino antisense oligonucleotides, synthetic DNA analogues, that act by blocking translation of their target gene products, have been shown to be effective tools for gene targeting in several species and cell systems. The purpose of the current study was first, to elucidate the optimal treatment conditions for the use of Morpholino oligos (MOs) in mammalian preimplantation embryos, and second to use MOs to target genes and study gene function in the early embryo.

Materials and methods Different treatment methods were evaluated for efficient delivery of MOs into murine embryos by assessing embryo development, MO uptake and down-regulation of targeted gene expression by immunofluorescence analysis. Ethoxylated polyethylenimine (EPEI), Lipofectin or Lysolecithin delivery agents were employed with a nonsense, fluorescent MO, or either D-catenin or Na/K-ATPase E1 subunit specific MOs, to target genes involved in preimplantation development. MO concentrations of 1, 2, 5, 10 and 20 uM and treatment times of 3, 6, 24, 48 and 72 h were used for treatment of 2 or 8 cell embryos.

Results and discussion Treatment of 2 cell embryos with Lipofectin and 20 uM MO, with the embryos transferred to fresh MO after 24 h and maintained in MO, resulted MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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in a reduction in targeted gene expression and suppression of blastocyst development (control 42.6 +/- 1.9%; fluorescent 61.2 +/- 0.9%; D-catenin 16.7 +/- 2.3%; Na/K-ATPase E1 29.7 +/- 3.5%). These results indicate that Morpholino antisense oligonucleotides are effective tools for down-regulating gene expression during mammalian preimplantation development.

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Identification of Novel Products in Maturing Bovine Cumulus-oocyte Complexes M.D. Calder, A.J.M. Paliga, and A.J. Watson Departments of Physiology and OB/GYN, University of Western Ontario, London, ON, Canada N5W 2K4

Introduction Only 30-40% of bovine COCs recovered from antral follicles develop in vitro to the blastocyst stage. Many oocytes undergo spontaneous nuclear maturation in vitro (1, 2). But, oocytes obtained from smaller antral follicles may have less ability to develop (3) as the source of oocyte has the largest effect on the number that develop to blastocyst stage in vitro (4). Likely some oocytes have defective cytoplasmic maturation. Bovine IVF labs routinely add gonadotropins, estradiol, growth factors and/or serum components to maturation medium try to improve oocyte cytoplasmic maturation. However, few gene transcripts have been identified which may improve oocyte developmental competence. Our objective was to identify differentially expressed transcripts in COCs at varying times of maturation under different conditions.

Materials and methods: Four groups of (50) COCs were collected: a) immediately after collection, time 0; b) after 6h maturation in absence of additives, no H6; c) after 6h maturation in 10% serum, SS6; d) after 6h maturation in media with gonadotropins, estradiol and serum, FLES6 Differential display RT-PCR was performed according to established protocols (5) with one 3’ primer and one of six 5’ primers. Bands that appeared to be differentially expressed were cut from the gel, amplified and sequenced. MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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Results and discussion About 20 bands were selected and 10 have currently been sequenced. Six matched bovine, human or rodent sequences. One was repetitive bovine sequence. Three did not match any sequence in Genbank. We have identified several sequences that appear to be bovine homologues of RNAs that were not known to have roles in bovine oocyte maturation. RT-PCR will be used to confirm interesting sequences and to determine whether they are associated with oocyte quality and improved oocyte competence. ddRT-PCR is a useful tool in identifying novel transcripts or gene products that have novel roles during bovine oocyte maturation.

References 1. Pincus and Enzmann, 1935. J. Exp. Med. 62:665. 2. Harper and Brackett, 1993. Biol. Reprod. 48:409. 3. Lonergan et al., 1994. Mol. Reprod. Dev. 37:48. 4. Rizos et al., 2002. Mol. Reprod. Dev. 61 :234. 5) Natale et al., 2000. Mol. Reprod. Dev. 55: 152.

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Gene Expression and DNA Methylation of an Imprinted Gene Cluster on Ovine Chromosome 18 During Early Embryonic Development R.E. Durkin, C.A. Bidwell, A.C. Clifford, and R.L. Krisher Department of Animal Sciences, Purdue University, West Lafayette, IN 47907 USA Introduction Epigenetic modification of DNA by cytosine methylation has an important role in gene regulation through genomic imprinting, X chromosome inactivation, nuclear reprogramming and gene silencing An imprinted gene cluster on ovine chromosome 18 has been characterized in sheep due to its association with a mutation, callipyge, that results in polar overdominant inheritance of skeletal muscle hypertrophy (1,2). The objective of this experiment was to characterize gene expression in early embryos and the methylation status of CpG islands within the imprinted gene cluster in sheep gametes, preimplantation and post-implantation embryos.

Materials and metrods Methylation of CpG dinucleotides was analyzed by HpIIl digestion of DNA from abattoir-derived oocytes, sperm, in vitro derived blastocysts, 28-day conceptus samples, lymphocytes and granulosa cells. Undigested DNA, Hpall or MspI digested DNA was amplified by PCR with primers that flank CpG islands in various regions of DLKI and GTL2 genes. A CpG island from the estrogen receptor D gene was used as a control. Total RNA was isolated from 28-day conceptus tissues and expression of 5 genes from the chromosome 18 imprinted gene cluster was analyzed by northern blot analysis.

Results and discussion Six CpG islands were analyzed from the DLKI and GTL2 genes. All six were methylated in lymphocytes, sperm and blastocysts but not in oocytes. A CpG island that spans DLKI intron 5 was not methylated in any of the MAMMALIAN EMBRYO GENOMICS – 512003111P/ISBN 92-64-10426-7 © OECD 2003

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28-day conceptus tissues, however, a second CpG island in DLKI exon 6 was methylated. Two CpG islands from the 5’ flanking region of GTL2 were methylated in the 28-day conceptus tissues but a third CpG island located between them was not methylated. The fourth CpG island in GTL2 exon 1 was also methylated in all 28-day conceptus tissues. A CpG island from the promoter region of estrogen receptor D gene was unmethylated in all tissues with the exception of sperm and 28-day conceptus head DNA. Analysis of five transcripts from the imprinted gene cluster showed that DLKI was prominently expressed in the head, body and extra-embryonic membranes of the 28-day conceptus. Three other transcripts, GTL2, antiPEGII and MEG8 were expressed, but the PEGII transcript was not detected. These results show different methylation patterns for two CpG islands in postimplantation embryos relative to preimplantation embryos or adult tissues. Expression analysis indicates that this chromosomal region is transcriptionally active during early development and therefore this locus will serve as a model for epigenetic regulation of early development in sheep.

References 1. Charlier C, Segers K, Wagenaar D, Karim L, Berghmans S, Jaillon O, Shay T, Weissenbach J, Cockett N, Gyapay G, Georges M. 2001. Human-ovine comparative sequencing of a 250-kb imprinted domain encompassing the callipyge (clpg) locus and identification of six imprinted transcripts: DLK1, DAT, GTL2, PEGII, antiPEGII and MEG8. Genome Research II:850-62. 2. Cockett NE, Jackson SP, Shay TL, Farnir F, Berghmans S, Snowder GD, Nielsen DM, Georges, M. 1996. Polar overdominance at the ovine callipyge locus. Science 273 :236-238.

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