, 2.1.1., . . : 3r :4"? ‘39; l'“ \>§..,._. :- 4“. » is... ‘ 1.. x 2‘13: :4. ‘ ~1me‘ue -.1 -. Hu‘rt \ lo ....r..:.0t%1 o . luau”: at . a . x 1 1 11.1%.; - ~ - file-M Eu. . u .131: i of} . .1” .6 - Vault.» oh»... #6... An. . .rs aid} A0 .1. D\l: h I O ~11. 3.. . «1 1.1.1} :I.I,fl.h}1r.$s,nm+h?rnru«lu 5.3.... Z~ H \fludnf. (c-1111olf . t Vii. 1 15.3.5. 3. 1 1 . . 1).: I w: .. £11.”; . Jr $11.24 1 I KS! .Wun..n.n.s.nnln ”3.10. 2.1 .1.Vu1.s!| r? '1...) 111,12... W1 t {was / c9007 11] LIBRARY gan Sta University IChI This is to certify that the dissertation entitled STRUCTURE, FUNCTION AND TRANSCRIPTIONAL REGULATION OF JY-1: A NOVEL GENE SPECIFIC TO BOVINE OOCYTES/EMBRYOS presented by ANILKUMAR BETI'EGOWDA has been accepted towards fulfillment of the requirements for the PhD. degree in Animal Science [ . Wflm/A Mafor Professor’s Signature 571? 9/,/ ? Date MSU is an affinnative-action, equal-opportunity employer .. _._.-.-.—.-.-.-1-.— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/07 p:lClRC/DateDue.indd-p.1 STRUCTURE, FUNCTION AND TRANSCRIPTIONAL REGULATION OF JY -1: A NOVEL GENE SPECIFIC TO BOVINE OOCYTES/EMBRYOS By Anilkumar Bettegowda A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 2007 ABSTRACT STRUCTURE, FUNCTION AND TRANSCRIPTIONAL REGULATION OF JY-l: A NOVEL GENE SPECIFIC TO BOVINE OOCYTES/EMBRYOS By Anilkumar Bettegowda The oocyte plays key roles in regulation of female fertility. A growing body of evidence suggests that genes expressed in the oocyte are important for normal folliculogenesis, oocyte function and early embryogenesis and thus required for subsequent establishment of pregnancy. However, the identities of the oocyte expressed genes regulating above processes and required for fertility are limited, especially in monotocous mammals and farm animal species. During initial analysis of expressed sequence tags (ESTs) from a bovine oocyte cDNA library, an abundant novel oocyte expressed transcript (designated as JY-l) was identified. Additional cloning and sequence analysis indicated that the J Y-l sequence is totally novel and encodes for a secreted protein of unknown function. Expression of JY-l mRNA is ovary specific, intraovarian expression of J Y-l mRNA and protein is restricted exclusively to the oocyte and immunoreactive JY-l protein of ~ 11,000 Mr is detectable within lysates of bovine oocytes. Furthermore, JY-l mRNA is temporally regulated during the window of meiotic maturation through embryonic genome activation and embryonic JY-l mRNA is oocyte derived. Based on its oocyte specific expression and temporal and spatial expression pattern in embryos, I hypothesize that the JY-l gene is conserved in other species and may have an important ftmction during bovine folliculogenesis and early embryogenesis. To test this hypothesis, I will a) determine the gene structure of bovine JY-l and investigate presence of orthologous genes to J Y-l in other species b) determine the requirement of J Y-l for early embryonic development and 0) determine the ability of J Y-l to modulate FSH induced regulation of granulosa cell function. Genomic Southern blot analysis detected putative JY-l genes in bovine, sheep, pig and human genomic DNA. Genomic library screening and mining of sequence from available genome databases revealed that the JY-l gene is located on bovine chromosome 29, approximately 16 kb in length, and encoded by 3 exons (25, 92 and 1400 bp in length) separated by two introns (12.8 and 1.5 kb in length). JY-l like sequences were identified in DNA fragments on human chromosome 11 and syntenic regions in other species (chimpanzee chromosome 11, dog chromosome 21, mouse chromosome 7 and rat chromosome 1). Expression of a putative human mRNA ortholog of JY-l was detected in human ovary. Extensive sequence analysis of the putative human mRNA ortholog of JY-l and the conserved JY-l-like sequences in the genome of other vertebrate species did not reveal an open reading frame. Disruption of JY-l mRNA and protein in early embryos by siRNA mediated gene knockdown perturbed embryo development to the blastocyst stage. Treatment of granulosa cells with recombinant J Y-l (rJY-l) stimulated progesterone synthesis in a dose dependent manner, but decreased the FSH stimulated increase in granulosa cell number and estradiol concentrations. Based on these results, I conclude that JY-l is a novel protein encoding maternal effect gene restricted to the genome of cattle and potentially other ruminant species with important roles in regulation of folliculogenesis and early embryogenesis. ACKNOWLEDGEMENTS I am grateful for my supervisor Dr. George W. Smith for his support, guidance and for all the training I have received in his research program. I wish to thank my doctoral committee members Dr. Jose B. Cibelli, Dr. Paul M. Coussens, Dr. James J. Ireland and Dr. William S. Spielman for their valuable feedback, critical input and suggestions. I thank all co-workers, Dr. Qinglei Li, Dr. Aritro Sen, Dr. F ermin Jirnenez-Krassel, Larry Chapin, Dr. Kyung Bon Lee, Lihua Lv for the various important contributions to this study. Special thanks to Dr. J ianbo Yao for providing technical training at the beginning of this study. iv TABLE OF CONTENTS LIST OF TABLES .............................................................................. viii LIST OF FIGURES ............................................................................. 11‘ LIST OF ABBREVIATIONS ................................................................. “11 CHAPTER 1 .................................................................................... 1 Introduction ...................................................................................... 1 CHAPTER 2A .................................................................................... 5 LITERATURE REVIEW I ...................................................................... 6 Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development .......................................................................... 6 Table of contents ................................................................................ 6 Abstract .......................................................................................... 7 Introduction ...................................................................................... 8 Post-Transcriptional Control Mechanisms for Maternal mRNA Repression ........... 10 Deadenylation of maternal mRNA ...................................................... 10 Maternal mRN A masking: Nonspecific interaction of Y-box proteins ............ 11 Sequence specific interaction of maternal mRNA binding proteins ............... 13 MicroRNA and repeat-associated small interfering RNA: Regulators of maternal mRN A degradation .......................................................... 20 Mechanisms of Translational Activation of Maternal mRNA .......................... 24 Cytoplasmic polyadenylation: Role of embryo specific poly (A) - binding proteins ..................................................................................... 24 Translation regulation of maternal histone mRNA: Role of stem-loop binding proteins ................................................................................... 27 Translational Regulation in Early Embryos: The Unclear Story ...................... 30 Summary and Perspectives ................................................................. 34 Acknowledgements ......................................................................... 35 CHAPTER 2B ................................................................................. 36 LITERATURE REVIEW II ................................................................. 36 Oocyte Specific Genes Regulating Primordial Follicle Formation and Activation / Survival ..................................................................................... 36 Oocyte Secreted Factors that Regulate Progression of Follicular Development and Somatic Cell Functions ................................................................ 38 Additional Oocyte Specific Genes Described to Date and their Requirement for Fertility; .................................................................................. 47 Maternal Effect Genes and their Role in Embryonic Genome Activation and Early Embryogenesis .................................................................. 48 CHAPTER 3 .................................................................................... 56 Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro ...................... 57 Abstract ......................................................................................... 58 Introduction ................................................................................. L . . 59 Material and Methods ........................................................................ 60 Materials ..................................................................................... 60 Oocyte recovery and in vitro maturation ................................................ 61 In vitro fertilization and embryo culture ................................................. 62 In vitro transcription and RNA quantification .......................................... 63 RNA extraction and reverse transcription ............................................... 64 Quantitative real time RT-PCR ........................................................... 65 Statistical analysis .......................................................................... 67 Results .......................................................................................... 67 Validation of real time RT-PCR procedures ............................................ 67 Quantification of abundance of GAPDH, B—actin, RPL-15, CYC-A, PGK and GUSB mRNAs during oocyte maturation .............................................. 71 Temporal regulation of mRNA for GAPDH, B-actin, RPL-l 5, CYC-A, PGK and GUSB during early embryonic development .................................... 71 Dynamic regulation of Histone H2A mRNA during bovine oocyte maturation and early embryogenesis .................................................................. 78 Discussion ....................................................................................... 78 Acknowledgements ............................................................................ 88 CHAPTER 4 .................................................................................. 89 J Y-l, a novel oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle ............................................................ 89 Abstract ....................................................................................... 90 Introduction ................................................................................... 91 Materials and Methods ...................................................................... 92 Northern blot procedure .................................................................. 92 Bovine fetal ovary cDNA library construction ........................................ 93 In situ hybridization ....................................................................... 93 Immunohistochemistry ................................................................... 94 Western blotting ........................................................................... 94 Effect of recombinant JY-l on granulosa cell functlon 95 Quantification of JY—l mRNA in oocytes and early embryos ...................... 96 Synthesis of siRNA species ............................................................. 96 Partheno genetic activation ............................................................... 97 vi Validation of siRNA species by microinjection ....................................... 97 Genomic Southern blot analysis ......................................................... 99 Genomic library screening and bioinformatics analysis .............................. 99 Cloning of putative human J Y 1 cDNA ................................................ lOO Statistical analysis ......................................................................... 100 Results ......................................................................................... 101 Tissue distribution and characterization of JY -1 mRN A transcripts ................ 101 Oocyte specific localization of J Y-l mRNA and protein within ovarian follicles. 105 Characterization of JY -1 protein ......................................................... 105 Effect of recombinant JY-l protein on cell number and production of Estradiol and progesterone by cultured granulosa cells .......................................... 110 Quantification of J Y-l mRNA during oocyte-to-embryo transition and effect of JY -1 mRN A knockdown on early embryonic development .......................... 113 Identification of J Y-l like sequences in other species and cloning of putative mRNA ortholog of JY-l .................................................................. 115 Discussion ..................................................................................... 121 Acknowledgements .......................................................................... 124 CHAPTER 5 .................................................................................... 126 Summary and Future Directions ............................................................. 126 APPENDIX .................................................................................... 132 REFERENCES ................................................................................ 166 vii LIST OF TABLES CHAPTER 3 Table 1: Details of primers used in real-time PCR ............................................. 69 lviii LIST OF FIGURES Images in this dissertation are presented in color CHAPTER 2A Figure 1. Schematic model for regulation of pumilio-2 (Pum-2) bound RINGO/Spy mRNA translation during oocyte maturation ................................................. 15 Figure 2. Schematic model for regulation of cytoplasmic polyadenylation element (CPE)-containing maternal mRNA during oocyte maturation .............................. 18 Figure 3. Schematic model for regulation of histone mRNA during oocyte maturation. 29 Figure 4. Salient features involved in maternal mRN A regulation during mammalian early development ................................................................................. 33 CHAPTER 2B Figure 5. Oocyte regulates folliculogenesis and cumulus-granulosa cell function ....... 39 Figure 6. Salient features during bovine early embryonic development ................... 53 CHAPTER 3 Figure 7. Quantitative real time RT-PCR analysis of amounts of green fluorescent protein (GF P) RNA in oocyte and embryo samples spiked prior to RNA isolation. . . 70 Figure 8. Quantitative real time RT-PCR analysis of polyadenylated RPL-15, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of germinal vesicle (GV) and metaphase (MII) stage bovine oocytes .................................... 72 Figure 9. Quantitative real time RT-PCR analysis of total RPL-15, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of germinal vesicle (GV) and metaphase (MII) stage bovine oocytes ........................................... 74 Figure 10. Quantitative real time RT-PCR analysis of polyadenylated RPL-lS, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of in vitro derived bovine embryos collected at pronucleus (PN), 2-cell (2-C), 4-ce11 (4-C), 8-cell (8-C), 16-cell (16-C), morula and blastocyst stages .................................. 76 Figure 11. Quantitative real time RT-PCR analysis of H2A mRNA in oocytes and in vitro derived embryos ................................................................... 79 ix CHAPTER 4 Figure 12. Characterization of the number and size of J Y-l mRNA transcripts ...... Figure 13. Intraovarian localization of J Y-l mRNA and protein ........................ Figure 14. Western blot detection of JY-l protein in bovine oocytes using antisera generated against recombinant J Y-l protein (mature form lacking signal peptide; rJY-l) .................................................................................. Figure 15. Effect of recombinant JY-l protein (rJY-l) on granulosa cell numbers and estradiol (E) and progesterone (P) production ......................................... Figure 16. Quantification of J Y-l mRNA abundance during oocyte maturation and early embryogenesis and effect of J Y-l knockdown on blastocyst development ..................................................................................... Figure 17. Detection of J Y-l like sequences in the genome of multiple species, structure of J Y—l gene and cloning of a putative human mRNA ortholog of bovine J Y-] ........................................................................ APPENDIX Figure A. 1. Quantitative real time RT-PCR analysis of amounts of green fluorescent protein (GFP) RNA in oocyte and embryo samples spiked prior to RNA isolation ................................................................................... Figure A.2. Quantitative real time RT-PCR analysis of amounts of far-red fluorescent protein (HcRedl) RNA in oocyte and embryo RNA samples spiked prior to cDNA synthesis .............................................................. Figure A.3. RT-PCR analysis of J Y-l mRNA transcripts in multiple bovine tissues. Figure A.4. Intraovarian localization of JY-l mRNA ...................................... Figure A.5. Quantitative real-time RT-PCR analysis of total versus polyadenylated J Y-l mRN A transcripts within in vitro derived early bovine embryos .................. Figure A.6. Quantitative real-time RT-PCR analysis of JY-l mRNA within in vitro derived embryos cultured with or without the RNA polymerase II inhibitor a-amanitin ............................................................ Figure A.7. Validation of oocyte/embryo microinjection procedure ..................... Figure A.8. Validation of JY-l siRNA species for efficacy of JY-l mRNA 103 106 108 111 116 119 133 135 137 139 141 143 145 knockdown in samples of 4-cell embryos .................................................... 147 Figure A.9. Validation of JY-l siRNA species for efficacy of JY-l mRNA knockdown in samples of 2-cell embryos .................................................... 149 Figure A. 10. Validation of JY-l and universal negative control siRNA species for specificity of target mRNA recognition ...................................................... 151 Figure A.11. Effect of negative control siRNA injection on blastocyst quality ........ 154 Figure A. 12. Effect of JY-l siRNA microinjection on JY—l protein abundance in 8-16 cell embryos .............................................................................. 156 Figure A. 13. Effect of J Y-l knockdown on bovine early embryonic development ..................................................................................... 1 5 8 Figure A. 14. Genomic organization and characterization of putative cis-elements in the 5’-flanking region of the bovine J Y-l gene .......................................... 160 Figure A. 15. Characterization of JY-l like sequences in the human genome .......... 162 Figure A.16. Characterization of J Y-l-like sequences in the genome of additional species .............................................................................. 164 xi ACE BMP6 BMP15 BSA CPSF CPE CPEB cDNA COCs CDK CYC-A DAN DAZL DNA 6-DMAP EGA ePABs E eIF Eif4eloo eIF4E-BP eCPE ABBREVIATIONS Adenylation control elements Bone morphogenetic protein 6 Bone morphogenetic protein 15 Bovine serum albumin Cleavage and polyadenylation specificity factor Cytoplasmic polyadenylation element CPE binding protein Complementary DNA Cumulus oocyte complexes Cyclin dependent kinase Cyclophilin-A Deadenylating nuclease Deleted in Azoospermia like Deoxyn'bose nucleic acid 6-dimethylaminopurine Embryonic genome activation Embryonic poly (A) binding proteins Estradiol Eukaryotic initiation factor Eukaryotic translation initiation factor 4E like Eukaryotic initiation factor 4E-binding protein Embryonic-type CPE xii EST Fig (1 FBF F BS FSH FRGY2 GLD2 GV GC GF P GDP 9 GUSB GAPDH H100 HECM Hsfl H2A HcRed 1 IAK— 1 IgG IGF- 1 ICSI KSOM LINE Expressed sequence tag Factor in the germline alpha F em-3 mRN A binding factor Fetal bovine serum Follicle stimu1ating hormone Frog (Xenopus) germ cell specific Y-box protein 2 Germline development deficient Germinal vesicle Granulosa cells Green fluorescent protein Growth differentiation factor 9 Beta-glucuronidase Glyceraldehyde 3-phosphate dehydrogenase Oocyte specific linker histone H1 HEPES buffered hamster embryo culture medium Heat shock factor 1 Histone H2A F ar-red fluorescent protein The murine homolog of aurora-A related kinase-1 Immunoglobulin G Insulin-like growth factor-1 Intracytoplasmic sperm injection Potassium simplex optimization medium Long interspersed nuclear elements xiii LTR th8 LH Mater ' mRN A miRNA MAPK MOS MSY2 MZdicer MII MEM mHR6A NPM2 Nobox OCT4 ORF OAS 1D PARN PABP Long terminal repeat LIM homeobox protein 8 Luteinizing hormone Maternal anti-gen that embryo require Maternal ribonucleoprotein particles Maturation promoting factor Messenger ribonucleic acid MicroRN A Mitogen activated protein kinase Serine threonine kinase Mouse Y-box protein 2 Maternal zygotic dicer Metaphase II Minimal essential medium Repair of DNA damage (RAD)-6-related Relative molecular mass Nucleoplasmin 2 Newborn ovary hoineobox gene POU-domain transcription factor Open reading frame 2 ’ -5 ’ -oligoadenylate synthetase-like protein- 1 D Poly (A) specific ribonuclease Poly (A) binding protein xiv PGK PBS Pol-I Pol-II PBE Pum-2 PUF PN RINGO rasiRNA rJY-l RF PL4 RT-PCR RPL-15 RISC RNAi siRNA SINE Sohlhl SLBP Phosphoglycerokinase Progesterone Phosphate buffered saline RNA polymerase I RNA polymerase II Pumilio binding element Pumilio 2 Ptnnilio and F BF Pronucleus Radioimmunoassay Rapid inducer of G2/M progression in oocyte Repeat-associated small interfering RNA Recombinant J Y-l Ret finger protein like 4 Reverse transcription-polymerase chain reaction Ribosomal protein L-15 RNA recognition motif RNA-induced silencing complex RNA interference Small interfering RNA Short interspersed nuclear elements Spermatogenesis and oogenesis basic helix-loop-helix Stem loop binding protein SCP tPA TE TCA TKDP TL UTR ZP Synaptonemal complex protein Tissue plasminogen activator Transposable elements Tricarboxylic acid Trophoblast kunitz domain protein Tyrode’s lactate Untranslated region Zygote arrest 1 Zona pellucida xvi Chapter 1 INTRODUCTION The oocyte is a specialized cell essential for regulating ovarian function and female fertility. During oogenesis and until ovulation, the oocyte is apposed to somatic cumulus-granulosa cells for nourishment within an ovarian follicle. At the time of ovulation, dependent on the animal species, a single or multiple cumulus-oocyte- complexes (COCs) are delivered into the oviduct to undergo fertilization and embryogenesis. Apart from the female genetic contribution to the formation of an embryo, the oocyte also provides the cellular and metabolic machinery to sustain initial embryo development to the stage at which the embryonic genome becomes fully functional and takes control of the newly formed embryo. The genetic components and regulatory factors that dictate oocyte development and function are therefore cardinal to the study of reproduction. The coordinate mechanisms driving the development of an oocyte and ovarian follicle have garnered significant attention among researchers in the field of reproductive biology in recent years. A rapidly growing body of evidence points to the oocyte as a major active regulator of fertility [1-4]. Oocytes have a decisive capacity to organize and dictate the overall rate of ovarian follicular development [5]. Oocyte derived mRNAs and proteins also play important roles during meiotic maturation, fertilization and early embryogenesis [6-9] critical to fertility. Numerous studies have elucidated the regulatory role of the oocyte in control of folliculogenesis. Oocytes develop in an environment fostered by the supporting follicular somatic cells that interact with the oocyte through autocrine/paracrine mechanisms as well as direct cell-to-cell communication [2]. Such interaction between the oocyte and companion somatic cells has a pivotal effect on stage specific progression of folliculogenesis [2]. Compelling evidences indicate the oocyte has a dramatic influence on gene expression and functions of the granulosa cells [1, 2]. In return, the surrounding cumulus-granulosa cells provide nourishment to the oocyte (during folliculogenesis) required for the oocyte to attain full competency to develop into an embryo following fertilization [IO-12]. The specific oocyte derived regulatory molecules that control folliculogenesis have not been fully elucidated. However, a few oocyte-derived factors such as factor in the germline alpha (Figla/Fig a), growth differentiation factor 9 (GDF9) and bone morphogenetic protein (BMP15) have been implicated in the regulation of follicular development. [1, 3, 13-15]. The oocyte also plays a fundamental regulatory role in the earliest phase of mammalian development postfertilization. The oocyte’s role is critical during the window between fertilization and the so called “maternal embryonic transition” also termed “embryonic genome activation” (EGA), when transcriptional activity of the embryonic genome becomes fully fimctional [6, 9, 16]. The developmental program prior to embryonic genome activation is supported by the pool of maternal mRNA and proteins synthesized and stored during oogenesis. As opposed to somatic cells, the oocyte has developed a series of strategies for storing mRNA and proteins in a quiescent form and their timely mobilization during oocyte maturation and early embryonic development [16]. The mechanisms of maternal mRNA storage are generally negative, and the target mRNAs are generally repressed until specifically activated. Such repressive mechanisms are generally mediated by transcript deadenylation, association of maternal mRNA transcripts with RNA binding proteins in a non-specific or sequence specific manner and mRNA degradation. In certain instances, mRNA repression/activation is facilitated by the competitive binding of negative or positive regulators of translation within the secondary structure of maternal mRNA. The specific signals obtained during meiotic maturation and early embryogenesis displaces the repressive factors, and consequently the maternal mRNAs are either translationally activated or degraded, ensuing normal embryo development. The translational regulatory mechanisms coordinating the progression of an oocyte into an embryo is an area of active research and many questions remain unresolved. The oocyte has a fundamental role in determining the phenotype of an early embryo. Maternal effect genes encode for mRNAs and proteins that are stored in the oocyte and required for early embryo development. A few of these maternal effect genes such as zygote arrest-1 (Zarl), nucleoplasmin 2 (NPM2) and maternal antigen that embryos require (Mater) have been identified and their functions determined [17-19]. However, intricate knowledge of the battery of maternal effect genes required for early embryogenesis and their mechanism of action in promoting the initial cleavage divisions following fertilization is limited. The purpose of the following literature review is to 1) describe the mechanisms of maternal mRNA regulation during mammalian oogenesis and early embryonic development (Chapter 2A) 2) present evidence for a regulatory role of the oocyte in female germ cell development, folliculogenesis including follicle formation and establishmeni of cumulus-granulosa cell phenotype and function and the specific genes involved and 3) review the role of maternal effect genes in embryonic genome activation and early embryogenesis (Chapter 2B). Most of the information available to date is derived from studies in rodents and lower vertebrate species (Xenopus and zebrafish). However, data from studies of domestic ruminants and humans will be emphasized when available. (Zhaann'Zyi Bettegowda A and Smith GW. Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development. Frontiers in Bioscience 2007 May 1; 12: 3713-3726R 1This chapter has been published by “Frontiers in Bioscience.” Chapter 2A LITERATURE REVIEW I Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Post-Transcriptional Control Mechanisms for Maternal mRNA Repression 3.1. Deadenylation of Maternal mRNA 3.2. Maternal mRNA Masking: Nonspecific Interaction with Y-Box Proteins 3.3. Sequence-Specific Interaction of Maternal mRNA-Binding Proteins 3.4. MicroRN A and Repeat-Associated Small Interfering RNA: Regulators of Maternal mRN A Degradation 4. Mechanisms for Translational Activation of Maternal mRN A 4.1. Cytoplasmic Polyadenylation: Role of Embryo-Specific Poly (A)-Binding Proteins 4.2. Translational Regulation of Maternal Histone mRNA: Role of Stem-Loop Binding Proteins 5. Translational Regulation in Early Embryos: The Unclear Story 6. Summary and Perspective 7. Acknowledgements 8. References 1. ABS T RA C T Mammalian oocytes accumulate a large pool of mRNA molecules that orchestrate subsequent embryonic development. The transcriptional machinery is silent during oocyte meiotic maturation and early embryogenesis, and thereby the early decisive events in embryo development prior to initiation of transcription from the embryonic genome are directed by the translation of pre-existing maternal mRNAs. Oocytes display remarkable post-transcriptional regulatory mechanisms that control mRNA stability and translation. The regulatory mechanisms are generally negative, and target mRNAs are either subjected to degradation or repressed from undergoing translation until specifically activated. Such negative regulatory mechanisms generally are mediated by transcript deadenylation, interaction of transcripts with RNA-binding proteins in a nonspecific or sequence-specific fashion, and/or potentially via actions of microRNA and repeat- associated small interfering RNA, which degrade maternal RNA transcripts. In contrast, translational activation is initiated via cytoplasmic polyadenylation of maternal transcripts facilitated via the binding of embryo-specific poly (A)-binding proteins (ePABs). In certain instances, translational regulation (positive or negative) is dictated by the balance of positive and negative trans-acting factors that compete for specific sequence motifs present in maternal transcripts. Coordinate post-transcriptional regulation of the oocyte mRNA pool is critical for normal progression of early embryonic development. 2. INTRODUCTION The growing mammalian oocyte arrested at prophase I of meiosis is transcriptionally active and produces a store of mRNA and proteins required for early embryogenesis. During oocyte meiotic maturation and the initial stages of embryonic development, the transcriptional machinery is silent, and in mammals, depending on the species, embryonic genome activation (EGA) occurs coincident with the first one to four cell cycles following fertilization [9, 20, 21]. Genome activation initiates transcriptional activity within the embryonic nucleus, and subsequent development is dependent upon newly synthesized mRNA and protein. Prior to EGA, key developmental events in the oocyte, pre- and post-fertilization (e.g., meiotic maturation, fertilization, initial cleavage divisions, and programming of EGA), are totally dependent on maternally derived mRNA and proteins [16]. The maternal mRNAs are translated temporally during these critical stages into functional proteins to ensure normal embryonic development. The mechanisms whereby the maternal mRNAs are stored and translationally repressed in the prophase-l- arrested oocyte, and precisely how the ultimate fate of maternal transcripts is decided in the developing embryo, are the focuses of this review. These post-transcriptional regulatory mechanisms tightly control the translation of maternal mRNAs during early embryonic development. The maternal mRNAs are translationally repressed until specifically activated, and the repression is promoted by transcript deadenylation, association of maternal transcripts with RNA-binding proteins in a nonspecific or sequence-specific manner, and mRNA degradation. The degradation of the untranslated maternal mRNA pool is presumed critical to early embryonic development, and recent evidence suggests it may be achieved via microRNA (miRNA) or repeat-associated small interfering RNA (rasiRNA) pathways [22, 23]. During initiation of translation, the repressive effects of RNA-binding proteins are overcome via signals triggered during progression of meiosis and (or) fertilization. As a result, the maternal mRNAs are translationally activated by cytoplasmic polyadenylation. In certain scenarios, translational regulation is facilitated by the competitive binding of positively or negatively acting trans-factors at sequence-specific motifs present within the secondary structure of the maternal mRNA transcript. During early embryonic development, the inhibitory factors are displaced, and consequently the positive factors mediate translational activation of maternal transcripts. How the above events are coordinated, culminating in the progression of an oocyte into a developmentally competent embryo following fertilization is an area of active investigation, and many questions remain unresolved. Biochemical and mechanistic information on the majority of the RNA- binding factors implicated in translational regulation is derived from studies of oocytes and embryos of lower vertebrates. Accumulating data indicates that most of these factors seem to have similar yet distinct roles in oocytes of evolutionarily distant mammals, like the mouse. The following sections of this review will discuss the translational regulatory mechanisms critical to early development, with emphasis primarily on mechanistic information obtained from mammalian model systems. Gaps in knowledge and established mechanisms in other vertebrate models are also highlighted for comparison. Components of the general translational machinery pertinent to described mechanisms of translational regulation in oocytes and early embryos are outlined where essential, but the reader is referred to other reviews [24, 25] for a detailed discussion of the translational machinery and how translation is mediated. 3. POST-TRANSCRIPTIONAL CONTROL MECHANISMS MEDIA TING MA TERNAL mRNA REPRESSI ON 3.]. Deadenylation of maternal mRNA The translational potential of a maternal mRNA transcript is determined by the length of the poly (A) tail. Accordingly, an increase in translation is associated with poly (A) tail elongation, whereas translational repression correlates with shortening of the poly (A) tail [26]. Most cellular mRNAs receive a poly (A) tail in the nucleus and associate with ribosomes for translation after export to the cytoplasm [27]. However, some maternal mRNAs are translationally silenced through a cytoplasmic deadenylation mechanism involving shortening of the poly (A) tail at the 3’ end of the mRNA [28]. Regulation of tissue plasminogen activator (tPA) mRNA in growing mouse oocytes is a classical example of translational repression by cytoplasmic deadenylation. The newly synthesized tPA transcript receives a long poly (A) tail [~300 nucleotides (nt)] and subsequently undergoes poly (A) shortening (40-60 nt) in the cytoplasm, preventing translation [29]. Until resumption of meiosis, tPA mRNA with a short poly(A) tail is stored in the cytoplasm in a dormant form [29]. The mechanisms targeting specific maternal mRNAs for deadenylation in mammalian oocytes and the mediators involved are not known. In Xenopus oocytes, truncation of the poly (A) tails of cyclin B1 and Mos mRNAs is catalyzed via a cytoplasmic poly (A)-specific ribonuclease (PARN), also termed deadenylating nuclease (DAN) [30]. Although a truncated poly (A) tail generally interferes with translation initiation, it is insufficient to completely prevent translation of 10 certain maternal transcripts. For example, maternally expressed histone B4 mRNA in immature Xenopus oocytes has a short poly (A) tail, but the protein accumulates to a substantial level during oogenesis [31, 32]. The mechanisms whereby such transcripts escape translational inhibition in the presence of a short poly (A) tail are unknown. 3. 2. Maternal mRNA masking: Nonspecific interaction of Y-box proteins During oogenesis, the oocyte genome is transcriptionally active, and the newly synthesized maternal mRNAs are either translated or stored in a dormant form. For instance, the mRNAs for the zona pellucida genes (ZPl, ZP2 and ZP3) are actively transcribed and translated during mouse oogenesis and are barely detectable at ovulation, when eggs are transcriptionally inert [33]. The dormant mRNAs are recruited for translation at defined periods of early development in response to physiological cues [16]. In Xenopus, 80 percent of maternal mRNAs are dormant and are associated with maternal ribonucleoprotein (mRNP) particles, which repress translation by preventing the binding of mRNA to polyribosomes via a process referred to as “masking of maternal mRNAs” [28, 34-37]. The major constituent of mRNP particles is the Y-box proteins, a group of nonspecific RNA-binding proteins [28, 38-40]. The germ-cell-specific Y-box protein FRGY2 in Xenopus binds to nonpolysomal RNA with an average density of one protein molecule per 40 to 50 nucleotide residues [41]. FRGY2 is phosphorylated by the mRNP-associated protein kinase, and this increases the stability of the F RGY2—RNA complex [42, 43]. Microinjection of protein kinase inhibitors into growing Xenopus oocytes stimulates the rate of endogenous protein synthesis by two to threefold [44]. This may be due to destabilization of the RNA-protein interaction resulting in the release of oocyte mRNAs accessible for translation [42, 44]. Therefore, it appears that 11 phosphorylation of F RGY2 may inhibit translation and the dephosphorylation of F RGY2 may initiate translational activation of maternal mRNAs [44]. Masking of maternal mRNA may be a conserved mechanism of translational repression in mammals, because Y-box proteins (MSYl, MSY2 and MSY4) have been identified in mouse oocytes and early embryos [45-47]. MSY2, the murine homologue of Xenopus FRGY2, is specifically expressed in male and female germ cells and is maternally inherited in early embryos; both the mRNA and protein are degraded in two- cell embryos concomitant with activation of the embryonic genome [46, 48]. The loss of MSY2 at embryonic genome activation coincides with bulk degradation of maternal mRNAs, suggesting that MSY2 may play a role in the storage, stability and regulation of maternal mRNAs. Targeted disruption of the MSY2 gene results in female infertility in mutant animals, with early loss of oocytes and defects in ovulation [49]. MSY2 constitutes 2 percent of the total protein in fully grown mouse oocytes, and 75 percent of this protein is localized to the cytoplasm. Recombinant MSY2 protein inhibits translation of luciferase reporter mRNAs to a modest level in an in vitro (rabbit reticulocyte lysate) translation system [50], providing further support for a potential role in translational repression of maternal mRNAs in the mouse oocyte. Biochemical evidence also supports a potential role for MSY2 in RNA masking. The N-terminal domain of MSY2 has a cold-shock domain and a basic/aromatic amino acid island in the C-terminus. Therefore, 75 percent of the MSY2 associates with the Triton- insoluble portion of mouse oocytes, suggesting that MSY2 sequesters maternal mRNA from the translational machinery [51]. RNase-A treatment of the Triton-permeablized mouse oocytes or microinjection of RNase-A into oocytes releases MSY2 proteins bound 12 to mRNAs [51], which indirectly supports a role for MSY2 in binding and stabilization of maternal transcripts. Murine MSY2 contains several potential phosphorylation sites for protein kinases, as observed in FRGY2 [46, 48], and the MSY2 protein is phosphorylated during meiotic maturation and dephosphorylated following fertilization [48]. However, the mediators of the above post-translational modifications of MSY2 and the fimctional significance of such modifications are unclear. It is not clear whether MSY2 binds to a selective class of maternal transcripts or has a global role in binding to multiple mRNAs in the oocyte. MSY2 is implicated in storage of paternal protamine mRNAs in mouse spermatids [47, 52]. Protamines are small arginine- rich proteins involved in condensation of DNA in the nuclei of mature spermatids. MSY2 protein recognizes a consensus sequence (UCCAUCA) in the 3’-untranslated region (3’ UTR) of protamine mRNA, raising the possibility that murine MSY2 may have a previously unknown sequence-specific RNA-binding activity [52]. In contrast, another study demonstrated that mouse recombinant MSY2 protein binds at multiple sites in vitro, with limited or no sequence specificity, to full-length protamine mRNA [50]. Further, MSY2 has approximately tenfold higher affinity for binding to full—length synthetic protamine-1 mRNA compared to the short protamine mRNA sequences derived from its 3’ UTR [50], indirectly indicating that length of the mRNA maybe a prerequisite for its action. Specific transcripts bound by MSY2 in mouse oocytes have not been described. 3. 3. Sequence-specific interaction of maternal mRNA -binding proteins The translation of specific maternal mRNA is one of the hallmarks for activation of meiosis in mammalian oocytes. The maternal transcripts required for progression of meiosis are translationally repressed by sequence-specific interaction with RNA-binding 13 proteins. In response to gonadotropins, several events are initiated in the prophase-I- arrested oocyte [53]. For example, synthesis of cyclin B1 (a cofactor of maturation- promoting factor) and activation of cyclin-dependent kinase (CDK) and maturation- promoting factor (MPF) are the early events during meiotic maturation [53]. MPF activity further stimulates translation of specific maternal transcripts such as Mos (a serine threonine kinase), which in turn is required for activation of mitogen-activated protein kinase (MAPK), which enables the oocyte to progress through meiosis and finally arrest at metaphase II awaiting fertilization [53]. During meiotic maturation, the maternal mRN As are translated in a sequential, well-controlled manner with a complex network of translational regulatory mechanisms, but the chronological events are not characterized in mammals. In the Xenopus oocyte, a rapid inducer of G2/M progression in oocytes known as RINGO/Spy is implicated as one of the earliest maternal mRNAs to be translated. RINGO/Spy translation precedes Mos synthesis because overexpression of RTNGO/Spy in Xenopus oocytes induces Mos synthesis, MAPK activation and maturation [54, 55]. In contrast, these events are blocked if RINGO/Spy mRNA is knocked down in progesterone-stimulated Xenopus oocytes [54]. Further, RINGO/Spy protein can bind and activate CDK1 and CDK2 and has a role in cell cycle regulation [55, 56]. The mRNA encoding a protein related to RINGO/Spy has been detected in mouse oocytes, but the mechanism of its action during meiotic maturation is not clear [57]. Translational repression of RINGO/Spy and Mos mRNA in oocytes is conferred by sequence-specific interactions of repressive factors bound to the 3’ UTRs, with the proteins of the eukaryotic initiation factor 4E (eIF 4E) family bound to the 5’ end of the mRNA. The molecular mechanism for translational repression of RIN GO/ Spy mRNA in 14 Translational Repression Meiotic maturation -* Translational Activation Figure 1. Schematic model for regulation of pumilio-Z (Pum-2) bound RINGO/Spy mRNA translation during oocyte maturation. The RINGO/Spy mRNA is repressed by the binding of Pum-2 to the pumilio-binding element (PBE) in the 3’ untranslated region (UTR). Pum-2 resides in a complex consisting of embryonic poly (A)-binding protein (ePABP) and the RNA binding protein deleted in Azoospermia-like (DAZL) and interferes with the interaction between translation initiation factors (e.g., eIF 4G) and the 5’ end of the mRNA. During meiotic maturation, Pum-2 dissociates from the protein complex bound to mRNA, and thereby ePABP interacts with eukaryotic initiation factor-4G (eIF4G) at the 5’end, resulting in translational activation. mammalian oocytes is not known but is well described in Xenopus oocytes. Two pumilio-binding elements (PBEs), UGUAUAAA and UGUAAAUA, residing at 3’ UTR of Xenopus mRNA are necessary for the repression (Fig. 1). The PBEs are bound by pumilio-2 (Pum-2), a member of the pumilio and FBF (Fem-3 mRNA-binding factor) (PUF) family of RNA-binding proteins [58]. PUF proteins belong to a family of evolutionarily conserved translational regulators in eukaryotes [59]. Pum-2 is expressed in oocytes both before and after maturation, and injection of Pum-2 antibody relieves the repression and induces synthesis of endogenous RINGO/Spy even in the absence of progesterone stimulation [5 8]. Likewise, injection of the dominant negative form of Pum- 2 stimulates RINGO/Spy protein synthesis [58]. Pum-2 not only interacts with PBE in oocytes, but also interacts with two other proteins: DAZL (Deleted in Azoospermia-like), an RNA-binding protein, and embryonic poly (A)-binding protein (ePABP) [58]. Human Pum-2 also interacts with human DAZ and mouse DAZL (mouse homologue to DAZ) [60], but the effects of such interactions are not clear. DAZL expression is exclusive to germ cells of gonads, and gene-targeting of DAZL disrupts fertility, with loss of germ cells in both male and female mice [61, 62]. DAZ/DAZL has a highly conserved RNA recognition motif (RRM) and resides in a complex of Pum-2, DAZL and ePAB bound to RINGO/Spy mRNA in Xenopus oocytes (Fig. 1) [7, 58]. Although ePAB classically binds to the poly (A) tail and interacts with eukaryotic initiation factor 4G (eIF4G) to stimulate translation of polyadenylated transcripts, Pum-2 binding at PBE may have overriding effects to prevent translation of RINGO/Spy mRNA in prophase-I-arrested oocytes [58]. 16 Another well-studied example of mRNA repression in mouse oocyte cytoplasm is cytoplasmic polyadenylation element (CPE) mediated regulation of Mos and tPA mRNAs [29, 63]. CPEs (also termed adenylation control elements [ACEs]) are uridine- rich sequences (consensus sequence UUUUUAU) in the 3’-UTR of some maternal mRNAs that can either repress translation by recruiting a repressive complex or direct polyadenylation and resumption of translation [29, 63]. One other mechanism by which CPE containing transcripts achieve translational repression is through deadenylation, reducing the poly (A) tail lengths to 20 to 40 nt [29, 64]. The translational repression of CPE-containing transcripts is dependent on a CPE-binding protein (CPEB), an RNA- binding protein with high affinity to CPE elements [63, 65]. Mouse CPEB mRNA is present in the ovary, testis, brain and kidney, and it encodes for a 62-kDa protein [65]. Within the ovary, CPEB mRNA expression is exclusively restricted to oocytes [65]. The additional factors required for CPEB-mediated repression are present in mouse oocytes [66], but the regulatory mechanism of CPEB action is better understood in Xenopus. In Xenopus oocytes, CPEB anchors Maskin, a repressor protein which blocks cap- dependent translation (Fig. 2) [67]. Maskin behaves as an eukaryotic initiation factor (eIF) 4E-binding protein (eIF4E-BF) and competitively binds to eIF4E at the same region as eIF4G, thereby inhibiting interaction of eIF4E and eIF4G [63, 67]. The blockade of such interaction further prevents the eIF4F group of initiation factors from initiating translation on CPE-containing mRNAs in the prophase-I-arrested oocyte. A recently described novel gene named eukaryotic translation initiation factor 4E-like (Eif4eloo) is specifically expressed in mouse oocytes and embryos [68]. Eif4eloo encodes for seven mRN A variants that arise predominantly because of alternative splicing at the 5’ end of 17 Translational Repression RINGO/Spy. CDK ? Au fora- A ————§ Translational Activation Figure 2. Schematic model for regulation of cytoplasmic polyadenylation element (CPE)-eontaining maternal mRNA during oocyte maturation. CPE-binding protein (CPEB), an RNA binding factor binds to CPE sequence and further interacts with Maskin, a repressor protein that prevents cap-dependent translation. Maskin acts as a eukaryotic initiation factor-4E (eIF4E) binding protein and competitively binds to eIF4E at the same region as eukaryotic initiation factor-4G (eIF4G) and thereby prevents the access of translation initiation factors to the 5’ end of the mRNA. CPEB further interacts with three other proteins; (a) Cleavage and polyadenylation specificity factor (CPSF), a protein bound to poly (A) signal; (b) symplekin, a scaffold protein; and (c) germline development deficient-2 (GLD2), a cytoplasmic poly (A) polymerase. During meiotic maturation, RINGO/Spy protein acts as a cofactor for cyclin dependent kinase (CDK) activity and presumably activates Aurora-A kinase. which in turn activates CPEB by phosphorylation. CPEB phosphorylation leads to activation of CPSF/Symplekin/GLDZ protein complex, which in turn elongates the short poly (A) tail of CPE-containing transcripts. Maskin undergoes differential phosphorylation at many residues by CDK and thereby releases the repressive effects of maskin at the 5’end of the mRNA. The longer poly (A) tail binds to embryonic poly (A) binding protein (ePABP) and in turn recruits elF4G to the cap-structure of the mRNA consequently activating translation. the gene [68]. The predominant Eif4eloo mRNA encodes for a 244-amino acid protein similar to eIF4E, and the mRNA is not detected beyond the early two-cell stage, indicating its maternal origin in the embryo, with a potential role in maternal mRN A translation [68]. CPEB interacts with three other factors in Xenopus oocytes; (a) symplekin, a protein that appears to act as a scaffold for other proteins to associate with; (b) CPSF (cleavage and polyadenylation specificity factor), a group of four proteins that also bind to a poly (A) signal; and (c) GLD2 (germline development deficient), a cytoplasmic poly (A) polymerase which elongates the short poly (A) tail of CPE- containing transcripts (Fig. 2) [69]. Specific roles of symplekin, CPSF and GLD2 in mediating mRNA repression are not established, but they are necessary components of CPEB during polyadenylation-induced translational activation. CPEB has several roles in development. CPEB null mice have defects in learning and memory and carry defective female germ cells that are arrested at the pachytene stage of prophase I by embryonic day (E) 16.5 [70]. Defective germ cells are attributed to failure in polyadenylation and translation of CPE-containing synaptonemal complex protein (SCPl and SCP3) mRNAs [70]. CPEB is phosphorylated at E16.5 (pachytene) at residue T171 and is dephosphorylated by protein phosphatase 1 at E18.5 [71], implying that post- translational modification of CPEB may be necessary for translation of CPE-containing SCPI and SCP3 mRNAs. Recently, CPEB has been implicated in control of oocyte growth and follicular development in the mouse [72]. The role of CPEB in oocyte growth and follicle development was delineated by the creation of transgenic mice expressing siRNA under the control of zona pellucida protein 3 (ZP3) promoter, which induces destruction of CPEB mRNA specifically in dictyate stage oocytes at the end of prophase I 19 [72]. Transgenic oocytes display atresia, premature oocyte maturation, parthenogenetic activation, precocious follicle activation, detached cumulus-granulosa cells, and granulosa cell apoptosis with a substantial decrease in fertility [72]. Further, CPEB binds many oocyte mRNAs that encode for proteins involved in signal transduction (e.g., Smadl, Smad5), cell cycle control (e.g., spindlin, Bublb and Mos), transcription (e.g., Oboxl), maintenance of methylation patterns important for imprinting (e.g., oocyte- specific DNA methyltransferase l) and also binds mRNA for growth differentiation factor-9 (GDF 9), an oocyte-expressed growth factor critical for follicular development [72]. In transgenic oocytes, GDF9 mRNA has a shortened poly (A) tail and reduced protein expression. Therefore, evidence suggests that CPEB controls the translation of GDF9, which is necessary for oocyte-follicular growth and development [72]. 3. 4. MicroRNA and repeat-associated small interfering RNA: Regulators of maternal mRNA degradation Degradation of untranslated maternal mRNA is presumed to be a critical checkpoint during early embryo development. In mouse embryos, 90 percent of maternal mRNA is degraded by the two-cell stage, coincident with complete activation of the embryonic genome [20, 73, 74]. Specific maternal transcripts that undergo rapid degradation following fertilization have been identified in mouse embryos [75]. The majority of these mRNAs are exclusively expressed in the oocyte genome (oocyte-specific linker histone H1 [Hloo], c-mos and GDF9) and not expressed during preimplantation development [75]. Maternal mRNA degradation in mouse embryos is dependent on the 3’ UTR of the mRNA transcript. For example, chimeric mRNAs composed of the c-mos coding region fused to the Hprt 3’ UTR have reduced rates of degradation following microinjection into 20 fertilized oocytes compared to transcripts composed of the Hprt coding region and c-mos 3’ UTR [75]. There is direct evidence that maternal mRNA clearance is critical for early development in other vertebrates. In Xenopus, oocyte-specific c-mos mRNA, which is essential for regulating meiosis, is degraded soon after fertilization. Persistence of c-mos is detrimental for embryo development, because injection of e-mos protein into two-cell embryos inhibits cleavage [76]. Thus, degradation of maternal mRNAs detrimental to embryogenesis represents a conserved mechanism of vertebrate development. Recent evidence from zebrafish embryos suggests that miRNAs may be the key regulatory molecules targeting maternal mRNA for degradation. MicroRNAs are evolutionarily conserved noncoding RNAs that regulate gene expression at the post- transcriptional level [77]. In animal cells, two nucleases, Drosha in the nucleus and Dicer in the cytoplasm, are important for processing longer primary and precursor miRNAs into the ~22 nucleotide mature miRNAs [78]. The mature miRNA assembles into the effector complexes called miRNPs (miRNA containing ribonucleoprotein particles), that have many features similar to RISCs (RNA-induced silencing complexes). RISCs are large ribonucleoprotein complexes that mediate the actions of small interfering RNA (siRNA) in targeting mRNA for cleavage and degradation, commonly referred to as RNA interference (RNAi). The miRNA binds to single/multiple partial complementary sequences in the 3 ’ UTR of its target mRN A, inhibiting protein synthesis and/or targeting mRNA for destruction. A seven-nucleotide seed sequence (at positions 2 through 8 from the 5’end) in miRNAs appears to be crucial for miRNA binding and action [79]. The precise mechanisms by which miRNAs silence their target mRNAs remain unclear. However, miRNAs are known to interfere with the initiation step of translation by 21 preventing binding/blocking the action of eIF4E at the cap structure of mRNA, causing translational repression [80, 81]. Abundant evidence supports an important role for miRNAs during vertebrate embryogenesis. For example, targeted disruption of the dicer gene in mice (abolishing the generation of mature miRNAs) results in embryonic lethality [82] and dicer mutant embryonic stem cells fail to differentiate both in vivo and in vitro [83]. Furthermore, zebrafish embryos mutant for matemaI-zygotic dicer (MZdicer) activity have abnormal morphogenesis and do not process precursor miRNAs into mature miRNAs [84]. The miR-430 miRNA family (miR-430a, miR-430b and miR-430c) is highly expressed during the onset of zygotic transcription in zebrafish embryos, and injection of miR-430 mature miRNAs into dicer mutant embryos rescues brain morphogenesis, further supporting an important role for miRNAs during development [84]. It is not known whether miRNAs play a regulatory role during zebrafish oogenesis, but miR-430 targets the 3’ UTR regions of maternal mRNAs at the onset of zygotic transcription, resulting in deadenylation of the poly (A) tail and degradation of the maternal mRNA pool. Less degradation of reporter mRNAs with miR-430 target sites in the 3’ UTR occurs when injected into MZdicer mutant zebrafish embryos versus wild-type controls. Microarray analysis of RNA harvested from MZdicer mutant zebrafish embryos and wild-type embryos at the onset of zygotic transcription has revealed several hundred mRNAs that are direct targets of miR-430 alone. The target mRNAs are predominantly maternally derived, and few of them are synthesized at the onset of EGA [22]. The absence of miR- 430 and the delayed clearing of maternal mRNAs does not interfere with EGA or embryo patterning in zebrafish embryos, but the persistence of maternal transcripts delays 22 development and results in abnormal morphogenesis [22]. An important question is whether miR-430-mediated degradation of maternal transcripts is conserved during mammalian EGA. To our knowledge, expression of miR-430 has not been reported in mammalian embryos, but several other novel miRNAs have been cloned in mouse oocytes [23]. However, whether the novel miRNAs have any role in maternal mRNA degradation or inhibition of translation is not established. Although evidence from mammals is limited, it is tempting to hypothesize and likely that miRNAs are involved in degradation of maternal transcripts in mammalian embryos. There is also evidence for distinct regulatory mechanisms mediating degradation of a specific class of RNA transcripts during oogenesis in the mouse. In mice, a significant portion of the maternal mRNA pool (approximately 13 to 14 percent of the oocyte transcriptome) is composed of transposable elements (TEs) and chimeric transcripts of TEs with host genes [68, 85]. The TEs act as the first exon or first few exons of the functional chimeric mRN A transcripts and also act as stage-specific alternative promoters for a number of host genes [85]. In addition, both sense and antisense transcripts of some transposable elements are co-expressed in mouse oocytes and embryos [86]. Recently, a novel class of small RNAs referred to as repeat-associated siRNA (also termed retrotransposon-derived siRNAs [rasiRNA]) have been implicated in targeting retrotransposon-derived mRNA sequences for degradation in fully grown mouse oocytes [23]. RasiRNAs are approximately 20- to 23-nucleotide molecules and have a preference for uridine and adenine residues at the first position, similar to miRN As [23]. RasiRNAs are mapped to both sense and antisense orientations of different retrotransposons and are derived from LINE (long interspersed nuclear elements), SINE (short interspersed 23 nuclear elements) and LTR (long terminal repeat) elements [23]. Reporter transcripts composed of the EGF P coding region with either sense or antisense retrotransposon sequences with target sites for rasiRNAs at the 3’ UTR are degraded rapidly in oocytes following injection [23]. Therefore, it is thought that the mRNAs with sense and antisense orientations of transposable elements may form a double-stranded structure and trigger rasiRNA to degrade mRNAs via the RNAi pathway. However, it is not known whether the action of rasiRNAs is exclusive to oocytes or they have similar actions during early embryogenesis. Furthermore, the functional significance of rasiRNA- mediated maternal mRN A degradation in mouse oocytes is not understood. Are there are any discrete loci within the cytoplasm where the maternal mRNAs are degraded? A hint about another potential mechanism of miRNA action comes from localization of miRNA-repressed mRNA complexes within or adjacent to P-bodies [79]. P bodies (cytoplasmic processing bodies) are large cytoplasmic aggregates known to contain translationally masked mRNA that also serve as sites of mRNA degradation [87, 88]. In addition, P bodies lack ribosomes and translation initiation factors and hence may contribute to translational repression of target mRNAs [87]. It is not known whether P- bodies are sites of miRNA action or are temporary warehouses for repressed mRNAs, nor is it known how these repressed mRNAs are transported to P-bodies. Although the existence of P-bodies in vertebrate oocytes/embryos remains to be elucidated, conservation of this mechanism throughout all cell types seems likely. 4. [MECHANISMS FOR TRANSLA T I ONAL A C TI VA T I ON OF ll/[A T ERNAL mRNA 4.1. Cytoplasmic polyadenylation: Role of embryo specific poly (A)-binding proteins 24 Cytoplasmic polyadenylation is a conserved molecular regulatory step in mRNA translation both in mammals and lower vertebrates during early development. During meiotic maturation, few maternal mRNAs are relieved from the repressive effects and polyadenylated initiating translation. A classical example of cytoplasmic- polyadenylation-mediated translational activation is derived from CPE-containing maternal Mos mRNA in mouse oocytes [89]. Cytoplasmic polyadenylation of Mos mRNA requires three cis-elements in the 3’ UTR: the polyadenylation hexanucleotide (AAUAAA) and two CPE elements located upstream of the hexanucleotide [89]. Reporter mRNAs with wild-type Mos 3’ UTR are translationally recruited during meiotic maturation and polyadenylated [89]. Further, ablation of Mos mRNA with antisense oligonucleotides results in failure to progress to meiosis II [89], indicating that cytoplasmic polyadenylation-induced translation of Mos mRNA is necessary for normal development. The translational activation of CPE mRNAs during meiotic maturation occurs in a sequential order, but it is not clearly understood in mammalian oocytes. In Xenopus oocytes, CPEB-mediated cytoplasmic polyadenylation of CPE-containing maternal mRNAs requires the translation of RINGO/Spy mRNA. Progesterone-induced meiotic maturation dissociates the interaction of Pum2 with RINGO/ Spy mRNP complex (which also includes DAZL and ePAB), releasing the mRNA from repression; as a consequence, RINGO/Spy mRNA is translated (Fig. 1) [58]. Whether the activation of Xenopus RINGO/ Spy mRN A requires poly (A) tail elongation is not known. RINGO/Spy protein acts a cofactor for CDK activity, which presumably activates unknown downstream molecules which indirectly influence Aurora-A (a serine/threonine protein kinase) activity required for CPEB activation [58]. Aurora-A activates CPEB by 25 phosphorylation, and this correlates with cytoplasmic polyadenylation and translation of CPE containing Mos and other maternal mRNAs in XenOpus oocytes (Fig. 2) [90, 91]. Factors required for translation of CPE containing transcripts (CPEB, CPSF, Maskin, Ipll- and aurora-related kinase 1 [IAK1, the murine homologue of Aurora-A/Eg2]) are also found in mouse oocytes, and these mechanisms appear to be conserved among vertebrates [66]. For example, inhibiting the activity of IAKl prevents phosphorylation of CPEB and in turn blocks the progression of meiosis in mouse oocytes. Likewise, a dominant negative mutant of CPEB protein which cannot be phosphorylated by IAKI prevents cytoplasmic polyadenylation [66]. However, the intricate biochemical details of CPEB-mediated translational activation are not understood in mammalian oocytes. In Xenopus oocytes, CPEB phosphorylation leads to activation of the CPEB-associated poly (A) polymerase complex, which contains CPSF/Symplekin/GLD2, which then elongates the short poly (A) tail of CPE containing transcripts (Fig. 2) [69, 92]. The longer poly (A) tail binds to ePABP, which in turn recruits eIF4G to replace Maskin in the repressive Maskin-cap complex, resulting in translation of CPE mRNAs [7]. Maskin undergoes differential phosphorylation by CDKl at several residues, and these post-translational modifications appears to interfere with the repressive effects of Maskin at the 5’ cap structure of the mRNA [93]. The association of poly (A)-binding protein (PABP) to the poly (A) tail of the mRNA is an important step during translation. The PABP, in turn, communicates with factors at the 5’ end of the mRNA, specifically eIF4G, and regulates translational activation. Although there are structurally different groups of PABPs identified in vertebrates, this discussion will emphasize the embryonic PABPs (ePAB and ePABP2). The mouse ePAB 26 is exclusively expressed in testes, oocytes and early embryos prior to EGA [94], but its mechanism of action is not clear. In Xenopus oocytes, ePAB protects mRNA by suppressing deadenylation and can also stimulate translation of reporter mRNAs [95, 96]. The mouse ePABP2 has one RNA recognition motif, and mRNA expression is restricted to ovaries and oocytes [97, 98], but its biochemical mechanism of action is not known. Given the germ cell specific expression pattern of ePAB and ePABP2, it seems reasonable to speculate that these proteins play a functional role in the poly (A) regulation critical for expression of maternally derived transcripts during early development. 4. 2. Translational regulation of maternal hist'one mRNA: Role of stem-loop binding proteins In the mouse, replication-dependent histone mRNAs and proteins are stored in oocytes and embryos, and translation of these mRNAs is not coupled to DNA replication in early embryos [99, 100]. Matemally derived histones are required to replace the protamines of the sperm DNA after fertilization and to assemble the newly synthesized embryonic DNA into chromatin until the embryonic genome is transcriptionally active [101]. Replication-dependent histone mRNAs lack a poly (A) tail but end in a conserved stem-loop structure [102]. The only known exception, histone mRNAs in the Xenopus oocyte, have a short oligo (A) tail attached to the stem-loop sequence which is thought to have a role in translational repression [103]. The stem-loop sequence also interacts with two stem-loop binding proteins (SLBP) that bind to the 3’ end of histone mRNAs. The mammalian homologue of SLBP is xSLBPl, and xSLBP2 is specific to Xenopus oocytes. The mechanism of translational activation of histone mRNAs involves exchange of 27 SLBPs associated with the 3’ end of the mRNA. xSLBP2 inhibits translation of histone mRNAs, whereas xSLBPl activates translation (Fig. 3) [104, 105]. At oocyte maturation, the oligo (A) tail is removed, the xSLBP2 is degraded, and histone mRNAs are translated [103]. But the signal or signals that mediate the detachment of the oligo (A) tail and xSLBP2 are unknown. Reporter mRNAs ending in the stem-loop sequence, with or without an oligo (A) tail, are efficiently translated in rabbit reticulocyte lysates and are active in the presence of xSLBPl [103]. However, reporter mRNAs with an oligo (A) tail are not translated in Xenopus oocytes, even in the presence of xSLBPl [103], suggesting that the oligo (A) tail might be bound by unknown additional inhibitory factors which repress translation of histone mRNAs in the oocytes. SLBP is predicted to be the functional homologue of PABP [106, 107], directing circularization of histone mRNAs through interaction with factors at the 5’ end of the mRNA. However, the precise molecular mechanism of SLBP action in translational control is not clearly understood. In contrast to Xenopus, mouse oocytes and embryos have a single SLBP. SLBP is concentrated in the nucleus of prophase-arrested oocytes (at G2/M) [108]. After entering meiosis (M-phase), the increase in CDK-1 activity mediates SLBP phosphorylation, and the protein accumulates to a high level in the cytoplasm of MII oocytes [108]. SLBP is dephosphorylated following fertilization, and protein levels remain high in both the nucleus and cytoplasm during the first cell cycle of embryo development but decline by the four-cell stage [108]. These post-translational modifications do not alter binding of SLBP to stem-loop RNA [108], but it is unclear whether translation of histone mRNA is altered. The accumulation of SLBP protein during oocyte maturation is definitely due to translation of existing maternal mRNA and is quite similar to other genes (e.g., tPA, 28 Translational Repression Melotlc maturation Degradation Translational Activation Figure 3. Schematic model for regulation of histone mRNA during oocyte maturation. In immature Xenopus oocytes, histone mRNAs have a short oligo (A) tail attached to the conserved stem-loop structure. The stem-loop sequence is bound to two stern loop binding proteins (xSLBPl and xSLBP2). The oligo (A) tail and the binding of xSLBP2 to the stem-loop sequence inhibit translation initiation. During meiotic maturation, the oligo (A) tail is removed, the xSLBP2 is degraded by unknown mechanisms and histone mRNA is translated. 29 spindlin and cyclin B), which are regulated by CPE elements in the 3’ UTR of their mRNAs. Whether SLBP mRNA regulation requires a CPE element or any other equivalent post-transcriptional regulatory mechanism has not been resolved. Coincident with the increase in SLBP during meiotic maturation, the translation of reporter mRNAs bearing the histone 3’ UTR is increased [109]. Conversely, preventing SLBP accumulation by RNAi reduces translation of the reporter mRNA, as well as synthesis of endogenous histones in matured oocytes. A significant decrease in the size of the pronucleus and in the amount of acetylated histone in the chromatin of the zygotes is also observed [109]. Although SLBP is the main molecular determinant of histone synthesis, its mechanism of action perhaps is slightly different in mice than in Xenopus oocytes. Unlike mice, Xenopus have two different forms of SLBPs, and an oligo (A) tail at the 3’ end of histone mRNAs regulates translation. Whether mouse histone mRNAs in the oocyte have a short oligo (A) tail and whether binding of xSLBP2 homologues is a key factor in translational repression is not known. 5. TRANSLATIONAL REGULATION IN EARLY EMBRYOS: THE UNCLEAR STORY Initiation of EGA in mouse embryos is dependent on maternally inherited proteins and/or recruitment of some maternal mRNAs for poly (A) tail elongation and translation (e.g., spindlin and cyclin-A2) following fertilization [110, 111]. It is evident that new proteins are synthesized following fertilization and prior to EGA, and blocking new protein synthesis reduces transcriptional activity in embryos [112-114]. Inhibiting poly (A) tail elongation by treating one-cell mouse embryos with 3’deoxyadenosine (an adenosine analog) decreases the transcriptional activity in the embryonic nucleus [112] 30 d D and further supports the premise that some maternal mRNAs are polyadenylated and translated following fertilization. Cyclin-A2, in conjunction with CDK-2 activity, appears to be one such maternal mRNA recruited following fertilization, because targeting the maternal cyclin-A2 by RNAi or blocking CDK2 with roscovitine inhibits EGA in the one-cell embryo [115]. In mice, fertilization starts in a signaling cascade (e.g., phospholipase-C zeta and/or a truncated form of c-kit tyrosine kinase), which mediates a rise in intracellular calcium oscillations [116-118]. The rise in calcium oscillations subsequently mobilizes maternal mRNAs for translation [119]. For instance, the inhibition of mouse egg calcium release blocks fertilization-associated changes in protein synthesis [117]. Further, experimental manipulation of calcium oscillations in the mouse egg is associated with changes in protein synthesis [120]. But how the rise in calcitun oscillation is directly or indirectly linked to translational activation of maternal mRNA is not well understood. However, it is proposed that the downstream events of calcium oscillation may activate certain kinases/phosphatases [120], which may in turn post- translationally modify RNA-binding proteins, releasing the maternal mRNA from repression. On the contrary, mobilization of the maternal mRNA could be transcript-specific and more specific to the cis-elements and or trans-factors at the 3’ UTR. Computational analysis of 3’ UTRs of expressed genes from mouse oocytes and an early stage embryo cDNA library has uncovered valuable information regarding features associated with the stability of maternal transcripts during early development in mammals [68]. Comparison of transcriptomes derived from full-grown mouse oocytes and two-cell stage embryos generates two sets of maternal transcripts which differ in the size of the 3’ UTR [68]. The 31 stable mRNAs have a significantly longer 3’ UTR than the transient ones [68], indicating that the 3’ UTR is a regulatory component for the stability of maternal mRN A. A higher proportion of uracil nucleotide residues is present in the 3’ UTRs of stable transcripts than in transient transcripts, which have a higher proportion of cytosines, suggesting that stability of mRNA may be due to a difference in the nucleotide content [68]. Further, CPE and PBE motifs are prominent in the stable mRNA transcripts, implying the necessity of such motifs for stability [68]. A classical example for the importance of cis- motif at the 3’ UTR for mRNA stability during oocyte to embryo transition is derived from the spin gene. The mouse 4.1-kb spindlin transcript with an ‘embryonic-type CPE (eCPE)’ (approximately 1100 nucleotides of the poly (A) signal) in the 3’ UTR loses its poly (A) tail during meiotic maturation but regains a poly (A) tail following fertilization, resulting in activation of translation in the embryos [111]. An attractive hypothesis is that eCPE alone is sufficient to withstand global cytoplasmic polyadenylation or default degradation during meiotic maturation. Otherwise, the eCPE or transcript-specific ‘translation control element’ is bound with mRNA-specific trans-factors which are post- translationally modified following fertilization, thereby releasing the mRNA from repressive effects. This theory may not be universal across mammals, because unlike in mice, EGA occurs much later — at four to eight cells in humans and at eight to sixteen cells in cattle — which requires approximately 50 to 90 hours following fertilization. The recruitment of mRNAs and requirement of proteins may extend for two to three additional embryonic cell cycles, until the genome is activated in a stepwise manner [9]. Therefore, signal(s) initiated following fertilization may not be an indicator for global recruitment and translation of maternal mRN As in embryos of higher mammals. The best 32 LH —> Selective degradation and default deadenylation of specific mRNAs tRINGO/Spy mRNA ‘ I, translation I 7 Translation of CPE-mRNAs 529. Metaphase ll Polyadenylation of Deadenylation and ‘ mRNAs (9.9. Cyclin-A2) ‘ clearance of ,_ maternal mRNAs 1 Protein synthesis 7 1 ? — . Fertilization and early embryo development Oocyte maturation Figure 4. Salient features involved in maternal mRNA regulation during mammalian early development. The onset of meiotic maturation stimulated by the surge release of gonadotropins (Luteinizing hormone, LH) initiates a complex network of translational activation or repression of maternal mRNAs in the oocyte. RINGO/Spy appears to be the first maternal mRNA to be translationally activated. RINGO/Spy protein most likely activates translation of cytoplasmic polyadenylation element (CPE)-containing mRNAs like Mos (a serine threonine kinase) and Cyclin B]. The synthesis of Mos and Cyclin B1 further activates a cascade of signaling events required for progression of meiosis and arrest at metaphase I I awaiting fertilization. One other important event during meiotic maturation is the synthesis and phosphorylation of stem-loop binding protein (SLBP), which in turn is required for synthesis of endogenous histories. Selective degradation and default deadenylation of specific maternal transcripts also accompanies meiotic maturation. Fertilization triggers a rise in intracellular calcium (Cay) oscillations which subsequently mobilizes maternal mRNA for polyadenylation and translation. The synthesis of new proteins programs embryonic genome activation (EGA) and, presumably initiates deadenylation and degradation of untranslated maternal mRNA via activation of microRNA (miRNA) and repeat-associated small interfering RNA (rasiRNA) pathways. 33 speculation is that the translation of maternal mRNA may be selective in each embryonic cycle, based on the regulatory elements present within the mRNA or the groups of RNA- binding factors with which the maternal transcripts are associated. The translational control mechanisms in higher vertebrates are not understood but may be much more complex, well controlled and vital during early embryogenesis. 6. S UMMAR Y AND PERSPECTIVES From this brief review, it is clear that significant progress has been made in solving the regulatory mechanisms of translational control in mammalian oocytes and early embryos, but much remains to be learned. Some important insight about mechanisms of mRNA masking has been obtained. An ever growing list of identified trans-factors interacting with 3’-UTR elements indicates that the translation control process is intricate and may not be fully resolved. However, the translation control mechanisms have been studied for only a few maternal mRNAs, and the mechanisms appear to be diverse. Therefore, it is premature to postulate a universal model for mRNA regulation during early development. The known sequential events of maternal mRNA activation during meiotic maturation and fertilization (Fig. 4) critical to mammalian development and the specific mediators involved in this process need to be resolved. In mammalian early development, whether miRNAs and rasiRNAs are involved in translational inhibition of maternal mRNA merits significant investigation (Fig. 4). In higher mammals including humans where multiple cell cycles occur before EGA, unlike in mice, perhaps there are multiple well-controlled events to stabilize maternal transcripts until the fate of the mRNA is decided following fertilization. It is possible that numerous convergent 34 mechanisms/forces dictate the fate of a given mRNA and each mRNA may have a different predestined sum of forces. 7. ACKNOWLEDGEMENTS The authors wish to thank the Rackham Foundation and the Michigan Agricultural Experiment Station for their generous support. 35 Chapter ZB LITERATURE REVIEW H OOC YT E SPECIFIC GENES REG ULA TING PRIMORDIAL F OLLI CLE FORll/IA TION AND ACTIVA TION/SUR VIVAL Studies over the last decade indicate that germ cell derived gene products play important roles in regulation of folliculogenesis. From the beginning of follicle formation and continuing throughout folliculogenesis, bidirectional communication between the oocyte and surrounding somatic cells is essential for coordinated development of both the germ cell and somatic cell compartments of ovarian follicles [1, 2]. Specific mechanisms that direct follicle formation are not [completely understood. Available evidence is derived primarily from gene targeting studies in mice However, F igla/F iga, a germ cell specific basic helix-loop-helix (bHLH) transcription factor has been implicated in regulation of ovarian follicle formation (Fig. 5). Studies of Figla mutant mouse lines demonstrated that this factor is essential for primordial follicle formation [15]. Embryonic gonadogenesis appears normal in Figla mutant mice, but there is rapid depletion of oocytes resulting in shrunken ovaries and female sterility [15]. Figla also regulates the coordinate expression of the zona pellucida genes (ZPl, ZP2, and ZP3) [15, 121] whose protein products form an acellular matrix separating the central germ cell from the surrounding granulosa cells which is essential for fertilization [122, 123] Molecular characterization of F igla has revealed that this transcription factor heterodimerizes with a B helix-loop-helix E12 protein and binds to an upstream conserved E-box (CANNTG) in the promoters of zona pellucida genes and coordinates 36 their oocyte specific expression [121]. The observation that Figla null females do not express ZPl, ZP2 and ZP3 indicates that F igla plays a key role in regulating expression of multiple oocyte specific genes that encode for components of the zona pellucida [15]. Figla is also expressed in prenatal human ovaries and Figla mRNA transcripts are increased in fetal ovaries by 40 fold during primordial follicle formation (at approximately 19 weeks of gestational age) [124]. Human Figla also interacts with an E- box in the human ZP2 promoter, suggestive of conserved mechanism and function of the human and mouse Figla proteins [124]. Nobox (newborn ovary homeobox gene), a homeobox containing protein, is preferentially expressed in primordial and growing mouse oocytes [125, 126]. Nobox deficiency in mice disrupts early folliculogenesis (Fig. 5). Nobox mutant mice show postnatal oocyte loss and follicular arrest at the primordial stage resulting in infertility [125]. The Nobox knockout ovaries rapidly loose oocytes and by two weeks after birth, only a few degenerating oocytes are present. However, the mechanism of oocyte loss is unclear [125]. Genes that are preferentially expressed in oocytes, such as OCT4 (POU- domain transcription factor), GDF9, BMP15, RFPL4 (ret finger protein like 4), H100 (oocyte-specific linker histone ), Zarl and Mos (a serine threonine kinase) are drastically reduced in Nobox null ovaries [125]. However, the cause for down-regulation of many oocyte specific genes in Nobox null ovaries is not understood. Whether Nobox is a master regulator of other oocyte specific genes or early germ cell loss in Nobox null ovaries leads to down-regulation/loss of transcripts for other oocyte specific genes needs to be clarified. 37 The recently identified Sohlhl (Spermatogenesis and oogenesis basic helix-loop- helix) transcription factor has also been implicated as a critical regulator of oogenesis and early stages of folliculogenesis in mice [127]. Sohlhl is preferentially expressed in female germ cells as early as embryonic day 15.5. In the newborn ovary, Sohlhl expression is confined to oocytes of primordial follicles and oocyte clusters confined to germ cell cysts. In adult ovaries, Sohlhl expression is confined to primordial oocytes but disappears as the oocytes are recruited into primary and secondary follicular stages. Mutant mice deficient in Sohlhl are infertile with atrophic ovaries characterized by early postnatal germ cell loss (Fig. 5). Oocyte specific genes such as Nobox, Figla, GDF9, Zarl, OCT4 and Mos are down-regulated in the Sohlhl null ovaries [127]. However, whether the down-regulation of oocyte specific genes is due to defects in primordial oocyte formation or failure of transcription from the oocyte nucleus is not clear. Another transcription factor th8 (LIM homeobox protein 8) is down-regulated in Sohlhl null ovaries [127]. Adult th8 null ovaries lack germ cells and appear histologically identical to Sohlhl null ovaries, indicating that part of the phenotype observed in Sohlhl null ovaries could be due to the disruption of th8 expression [127]. OOC YT E SECRE T ED FA C TORS THA T REG ULA TE PROGRESS] ON OF F OLLIC ULAR DE VELOPAEN T AND SOll/IA TIC CELL FUNCTIONS The oocyte orchestrates and coordinates ovarian follicle formation and a developmental program intrinsic to the oocyte controls the overall rate of follicular development [5]. A very good example of the oocyte’s direct role in controlling rate of progression of follicular development is derived from studies using mouse oocytes. In comparison to . oocytes from primordial follicles, mid-growth stage mouse oocytes < D Figa Nobox, Sohlhl 35¢ l l 25: —‘ 0 ———- 3 £@ Primordial germ Primordial Primary cells follicle follicle C) l— .91 \O Ovulated Preovulatory antral Secondary oocyte follicle follicle Follicle formation Cumulus cell metabolism & development Cumulus cell Steroidogenesis , apoptosrs Cumulus cell Granulosa cell . expansron proliferation and mitosis Figure 5. Oocyte regulates folliculogenesis and cumulus-granulosa cell function. (A) Oocyte specific genes are essential for the developmental progression of ovarian follicles. In mice, deficiency of oocyte specific Figa prevents primordial follicle formation. Mouse oocytes lacking Nobox and Sohlhl are defective in the transition of primordial into primary follicles. GDF-9 is required for the formation of secondary follicles in mice. Oocyte expressed BMP-15 and GDP-9 are involved in cumulus expansion and ovulation. (B) Oocyte derived factors influences steroidogenesis, metabolism, apoptosis and mitosis in the companion cumulus-granulosa cells. 39 isolated from secondary follicles accelerated the rate of follicular development when reaggregated with the somatic cell environment of primordial follicles [5]. Further, the accelerated follicular development was accompanied by a rapid differentiation of function of both the mural and cumulus granulosa cells [5]. The specific mechanisms by which the oocyte regulates rate of follicular development is not resolved. However, evidence supports a key role for oocyte derived factors in controlling granulosa cell development and function throughout folliculogenesis until ovulation [2]. The most well studied oocyte specific factor controlling progression of follicular development and phenotype and function of surrounding somatic cells is GDF9. GDF9 is an oocyte specific member of the transforming growth factor (TGF) [3 superfamily produced by mouse, human, cow, pig, sheep and rat oocytes [128-132]. GDP 9 mRNA is first detected in oocytes of primary follicles in mice [128], but in other species (cow and sheep) the mRNA is present in primordial oocytes [130]. GDF9 mRNA persists in the oocyte through all stages of folliculogenesis until ovulation [128]. Female homozygous GDF9 mutant mice are infertile. [133]. Although mutant ovaries appear normal and primordial follicles are recruited to initiate folliculogenesis, growth is disrupted after the primary follicle stage [133]. The primary follicles of GDF9 null mutant mice exhibit structural changes such as defects/absence of theca cell precursors, and the cells of the GDF9 deficient follicles histologically resemble small corpora lutea [134]. Further, kit ligand and inhibin alpha mRNAs are upregulated in follicles of GDF9 deficient mice, suggesting that GDP 9 may be directly or indirectly regulating these genes [134]. Furthermore, the GDF9 deficient oocytes show defects in meiotic competence including germinal vesicle breakdown, spontaneous parthenogenetic activation and 40 abnormal growth rates [134, 135]. Thus, GDP 9 appears to be necessary for the primary to secondary follicle transition in mice (Fig. 5). Indirect evidence also supports a role for GDF9 in regulation of ftmction of preovulatory follicles in mice (Fig. 5). Recombinant mouse GDF9 protein induces cumulus cell expansion via induction of hyaluron synthase- 2, stimulates prostaglandin and progesterone synthesis and their signaling pathways in preovulatory cumulus-granulosa cells and regulates the differential expression of cumulus-granulosa cell expressed genes [136, 137]. GDP 9 also appears to be important for follicular development in domestic farm animal species. For example, immunoneutralization studies in sheep have demonstrated a requirement of GDF 9 for normal follicle development [138]. However, apart from merely regulating folliculogenesis, GDF9 may have important roles in establishing ovulation rate and litter size in sheep [13]. Naturally occurring point mutations in the GDF 9 gene have been identified in Belclare and Cambridge ewes [139]. Ewes heterozygous for the mutation exhibit superovulation and increased litter size, whereas homozygous mutants are sterile [139]. Comparison of the sheep model with the polyovulatory mouse model reveal basic similarities as well as important differences in the phenotypes of animals with mutations in the GDF9 gene [13]. Female mice that are homozygous for the null GDF9 allele are infertile and the phenotype resembles that of the naturally occurring homozygous point mutations for GDF 9 in sheep [13]. In contrast to the increased ovulation rate and high litter size occurring in sheep heterozygous for the GDF9 mutations, mice heterozygous for the GDF9 gene disruption exhibit no obvious phenotype [13]. However, the physiological, biochemical and genetic mechanisms 41 contributing to species specific differences in the phenotype/ovulatory quota in the two species carrying GDF9 mutations are not well understood. Bone morphogenetic protein-15 (BMP15) is another oocyte specific member of the TGF-B superfarrrily [14]. The intrafollicular expression pattern of BMP15 is similar to GDF9 in mice [140]. BMP15 null mice are subfertile with defects in ovulation and fertilization [141]. BMP15 functions in a synergistic manner with GDF9 to maintain integrity of the CDC in mice and maximize female fertility [141]. Double mutant female mice homozygous for the BMP15 null allele and heterozygous for the GDF9 null allele exhibit more severe fertility defects than do single homozygous mutant BMP15 females [141]. The oocytes from double mutant mice have low fertilization rates and delayed embryo cleavage [142]. In addition, the oocytes from double mutant mice have defects in attachment of cumulus cells to the oocyte and impaired cumulus cell expansion, most likely due to aberrant cumulus cell expression of genes such as hyaluron synthase-2 [141, 142] In contrast to mice, BMP15 has a more prominent role in female fertility in sheep and humans [13, 14]. Natural point mutations in the BMP15 gene have been described in two strains of sheep (Inverdale and Hanna). Heterozygous carriers of point mutations in the BMP15 gene [143, 144] exhibit higher ovulation rates and litter size than wild type counterparts. However, homozygous carriers of such point mutations in the BMP15 gene are infertile with streak ovaries and a block in folliculogenesis at the primary follicle stage [143]. Crucial differences in phenotype have been noted for the mutant BMP15 gene between mono-ovulatory sheep versus poly-ovulatory mice. Compared to the infertile homozygous BMP15 sheep, homozygous mutant mice are subfertile with 42 decreased fertility [13, 14]. In contrast, heterozygous mutant sheep have increased ovulation rates and high litter sizes, whereas heterozygous mutant mice display no overt phenotype [13, 14]. In women, a BMP15 mutation is associated with hypergonadotrophic ovarian failure due to ovarian dysgenesis [145]. However, women who are heterozygous carriers of the BMP15 mutation have streak ovaries resembling the phenotype of homozygous mutant ewes [145]. Recombinant human BMP15 has been utilized to elucidate the effects of BMP15 on cumulus-granulosa cell fimction in rat and mouse species. BMP15 is a potent stimulator of rat granulosa cell proliferation and mitosis [146]. In addition, BMP15 stimulates rat granulosa cell mRN A for kit ligand, a factor which is necessary for early follicle growth [147]. In return, exogenous supplementation of the recombinant mouse kit ligand protein suppresses the expression of BMP15 mRNA by rat oocytes, thus forming an oocyte- somatic cell negative feedback loop [147]. BMP15 alone does not have effects on rat granulosa cell steroidogenesis. However human BMP15 is a potent suppressor of FSH induced progesterone synthesis in primary cultures of rat granulosa cells, but has no effect on F SH stimulated estradiol production [146]. Such negative effects on progesterone synthesis in rat granulosa cells are mediated through the suppression of F SH receptor mRNA, thereby preventing the FSH induced activation of mRNAs encoding for steroidogenic acute regulatory protein, P450 side chain cleavage enzyme, P450 aromatase, 3 B-hydroxysteroid dehydrogenase, luteinization hormone receptor, and inhibin/activin subunits [148]. However, the mechanisms directing the differential regulation of the two steroid hormones (estradiol and progesterone) by BMP15 and its physiological significance are not well understood. 43 The oocyte plays an active role in establishing the functions of the surrounding cumulus cells [149]. Mouse oocyte complexes cultured in the presence of FSH undergo cumulus expansion by increasing intracellular cyclic AMP which results in increased activity of the hyaluronic acid-synthesizing enzyme system [149]. In contrast, FSH does not induce hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized cumulus oocyte complexes (oocytectomy: evacuation of oocyte content without removal of zona pellucida and ablation of cumulus-cell contacts) [149]. However, when oocytectomized complexes are co-cultured with denuded germinal vesicle stage oocytes or in medium conditioned by denuded oocytes, FSH stimulates cumulus expansion, indirectly supporting a requirement for oocyte secreted factor(s) in cumulus expansion [149]. Experimental evidence suggests that oocyte synthesized BMP15 could be one such factor regulating cumulus cell fimctions (Fig. 5). In mouse, recombinant human BMP15 induces cumulus expansion in cumulus-oocyte complexes isolated from preovulatory follicles [150]. The cumulus expansion induced by exogenous BMP15 is mediated via increased mRNA expression for EGF-like growth factors (amphiregulin, betacellulin and epiregulin) as well as an increase in mRNA abundance for several genes downstream of EGF-Iike grth factor signaling such as cyclooxygenase-2, hyaluron synthase-2, tumor necrosis factor stimulated gene-6 and pentraxin-3 in the cumulus cells [150]. However, studies of BMPl 5 knockout mice alone do not support an absolute requirement of BMP15 for cumulus expansion/fertility, suggesting that additional oocyte derived factors are involved in vivo. The oocyte also directly regulates metabolic activities of the cumulus cells [11]. Subtractive hybridization procedures have identified 6 genes (aldolase-IA isoforrn, 44 enolase-lalpha, lactate dehydrogenase, phosphofructokinase, pyruvate kinase and triosephosphate isomerase) encoding for enzymes involved in glycolysis with greater expression in cumulus versus mural granulosa cells of mouse follicles [11]. In oocytectomized complexes, the cumulus cells exhibit decreased mRNA levels for above genes encoding for glycolytic enzymes as well as a decrease in metabolic activity linked to glycolysis and the tricarboxylic acid (TCA) cycle [11]. However, glycolytic enzyme mRNA levels and metabolic activity are restored when oocytectomized complexes are co-cultured with denuded fully grown mouse oocytes, suggesting an active role for unidentified paracrine factors in regulating cumulus cell metabolic functions [11]. Information on the requirement of known oocyte specific factors described earlier for regulation of folliculogenesis in my model system of interest (bovine) is lacking. However, direct and indirect in vitro evidence supports a regulatory role of the oocyte in control of follicular granulosa cell function. Co-culture of F SH-stimulated bovine mural granulosa cells with denuded bovine oocytes results in suppression of granulosa cell production of inhibin-A, activin-A and follistatin proteins as well as major steroids such as estradiol and progesterone [151]. Similarly, denuded bovine oocytes also suppress IGF-1 (insulin-like growth factor-1)-induced secretion of inhibin-A, activin-A, follistatin and estradiol but increase IGF-induced progesterone secretion, suggesting that the oocyte is capable of modulating granulosa cell responsiveness to FSH and IGF in terms of steriodogenesis and production of inhibin related peptides [151]. Bovine oocytes also have the capability to modulate granulosa cell proliferation and control differential proliferative capacity of the mural versus cmnulus bovine granulosa cells [152, 153]. Oocytectomy of bovine COCs reduces proliferative capacity but 45 increases steroidogenic potential of cumulus cells compared to intact COCs. In the absence of F SH, oocytectomy results in 5-fold reduction in [3H] thymidine incorporation (an indicator of cell proliferation) compared to COC, and an ll-fold reduction in the presence of IGF-1 [153]. Further, progesterone production is two-fold higher in oocytectomized complexes compared to intact COCs in the presence of IGF-1 + FSH. However, co-culture of oocytectomized complexes with bovine denuded oocytes restores proliferative capacity and diminishes the steroidogenic potential of the cumulus cells similar to intact COCs [153], suggestive of an active role of the bovine oocyte in regulation of cumulus cell phenotype and function. Bovine oocytes also have differential effects on phenotype and function of cumulus cells versus mural granulosa cells. In the presence of an oocyte, COCs incorporate 2-fold (IGF-1 + F SH) to 17-fold (IGF-1) higher [3H] thymidine compared to mural granulosal cells cultured in the presence of denuded oocytes but, progesterone secretion by mural granulosal cells (FSH + IGF-1) is 10-fold higher than COCs. However, co-culture of bovine denuded oocytes with mural granulosal cells significantly increases [3H] thymidine incorporation but decreases progesterone production compared to mural granulosal cells cultured without denuded oocytes [153], suggesting that oocyte derived factors can modulate proliferation and steroidogenesis of mural granulosal cells. The specific oocyte derived factors regulating the function of bovine cumulus-granulosa cells have not been identified. However, exogenous supplementation of ovine BMP15 to in vitro cultures of bovine granulosa cells stimulates [3H] thymidine uptake [154]. Further, the combination of ovine BMP15 and ovine GDF9 or murine GDF9 inhibits F SH stimulated progesterone production from bovine granulosa cells, further supporting a role of oocyte derived grth factors in 46 regulation of steriodogenesis [154]. Recombinant rat GDF9 has also been used in studies of primary cultures of bovine granulosa cells isolated from small (1-5 mm in diameter) and large (8-22 mm) antral follicles [155]. Irrespective of follicle size, recombinant rat GDF9 enhanced the IGF-1 induced increase in cell numbers, but suppressed IGF-l induced granulosa cell estradiol and progesterone production [155]. Bovine oocyte derived factors also prevent cumulus cell apoptosis [156]. Oocytectomy significantly increases the proportion of apoptotic cumulus cells, and decreases the levels of anti-apoptotic Bc12 protein, but increases amounts of the pro- apoptotic Bax protein in comparison to intact bovine COCs [156]. However, when oocytectomized complexes are co-cultured with either denuded oocytes or recombinant ovine BMP15, the apoptotic effects of oocytectomy are reversed (decreases proportion of apoptotic cumulus cells and levels of Bax protein and increases 3ch protein), suggesting an active role for BMP15 in preventing cumulus cell apoptosis [156]. In contrast, recombinant mouse GDF9 does not suppress cumulus cell apoptosis in oocytectomized complexes [156]. Recombinant human BMP6 also can block cumulus cell apoptosis in oocytectomized complexes [156]. However, the anti-apoptotic actions of denuded oocytes are only partially antagonized when follistatin (binds BMP15 and inhibits its activity) or a BMP6 neutralizing antibody are added to cultures, suggestive of a role for additional unidentified oocyte-secreted factors in suppression of bovine cumulus cell apoptosis [156]. ADDITIONAL OOC YT E SPECIFIC GENES DESCRIBED T 0 DA TE AND THEIR REQUIREMENT FOR FERTILITY 47 The c-mos gene encodes for a germ cell specific serine/threonine kinase required for regulation of meiosis in mouse and Xenopus oocytes. Disruption of c-mos in mouse oocytes causes spontaneous parthenogenetic activation of oocytes and development of ovarian cysts leading to subfertility [157, 158]. The oocyte specific OASlD (2’-5’- oligoadenylate synthetase-like protein-1D) is predominantly expressed in growing mouse oocytes and early embryos [159]. Mutant mice lacking OASID display reduced fertility due to defects in formation of ovarian follicles, reduced efficiency of ovulation and arrest of fertilized eggs at the one-cell stage [159]. RF PL4 encodes for an E3 ubiquitin ligase implicated in targeted degradation of cyclin B1, a key regulatory protein for cell cycle control in the mouse oocytes [160, 161]. The requirement of RFPL4 for female fertility is unknown. An oocyte specific family of transcription factors, called Obox is abundantly expressed in mouse oocytes [162]. Oboxl, Obox5 and Obox6 are specifically expressed in the ovary and the mRNA is abundant in unfertilized oocytes [162]. Oogenesins are leucine-rich proteins expressed in mouse oocytes [163, 164]. Oogenesin-l is expressed in oocytes at all stages of folliculogenesis, whereas oogenesins 2-4 are expressed from the primary follicle stage onward [3, 164]. The physiological roles of oogenesins and the Obox family of genes have not yet been determined. MATERNAL EFFECT GENES AND THEIR ROLE IN EAIBR Y ONIC GEN Oil/IE A C TI VA TI ON AND EARL Y Ell/[BR Y OGENESIS Fundamental studies support an active regulatory role of the oocyte in promoting the initial cleavage divisions post fertilization prior to initiation of zygotic transcription. Studies of [3 H] uridine incorporation, changes in ultrastructure of blastomere nuclei and the pattern of protein synthesis in early embryos indicate that embryonic genome 48 activation in bovine embryos occurs by the 8-16 cell stage [165-168]. However, studies involving exposure of 2- to 4-cell bovine embryos to [3H] uridine have revealed that limited transcriptional activity can be detected earlier than the 8-16 cell stage [169-171]. The maternal zygotic transition in the rabbit and mouse occurs at the 8-16 cell stage and 2-cell stage respectively [21, 172, 173]. Some amount of transcriptional activity has been detected in 2-cell rabbit embryos [21] and a minor degree of zygotic transcription in pronuclear stage mouse embryos [174-176]. Above evidence suggests that genome activation in the mouse and other mammalian species including bovine is not a single event restricted to a specific stage of embryogenesis, but occurs in a temporal stepwise manner before major activation of transcription from the embryonic genome [9, 177]. Maternal effect genes are those genes that are expressed in oocytes and are required for early embryonic development in mammals. Mater is one such oocyte-specific maternal effect gene that regulates embryonic development in the mouse. Mater was initially identified as an oocyte antigen for premature ovarian failure [178]. Mouse lines lacking Mater are sterile [19]. Although folliculogenesis, ovulation and fertilization appear normal, early embryos lacking Mater are unable to progress beyond the first cleavage, indicating that Mater is a key maternal effect gene whose expression is required for early embryonic development past the two-cell stage [19]. Mater mRN A and protein are also expressed in bovine oocytes and early embryos [179]. Mater mRNA is detectable in bovine oocytes from the primary follicle stage onwards, decreases during meiotic maturation and early embryo development and is barely detectable at morula and blastocyst stages [179]. However, Mater protein can be detected in the oocyte and early bovine embryos through the blastocyst stage, similar to the reported results in mouse 49 embryos [19, 179]. A functional role for MATER in bovine early embryogenesis has not been established. Zarl and NPM2 are two additional oocyte-specific maternal effect genes required for normal early embryonic development in the mouse. Zarl mutant female mice are infertile and mutant embryos show defects at the one-cell stage with a pronounced inhibition of syngamy [17]. Zarl mRNA expression has also been reported in pig and bovine oocytes and early embryos [180, 181]. However, its functional significance to early embryo development in such species is unknown. NPM2 in Xenopus is known to decondense sperm DNA in vitro [182]. Mammalian NPM2 has been recently implicated in chromatin decondensation during embryogenesis in mice. NPM2 null mutant mice are subfertile, with defects in preimplantation embryonic development [18]. Sperm DNA decondensation is normal in NPM2 deficient embryos, but nuclear abnormalities such as absence of coalesced nucleolar structures and loss of heterochromatin and deacetylated histone H3 that normally circumscribe nucleoli in oocytes and early embryos are present [18]. A few additional maternal effect genes have been identified in mice including Stella, Hsfl (heat shock factor 1) and mHR6A (repair of DNA damage (RAD)-6-related). Although not oocyte specific, a contribution of above genes to regulation of early embryogenesis has been established. Stella is specifically expressed in primordial germ cells, oocytes, preimplantation embryos of mouse species, and pluripotent cells in humans [183]. Females homozygous null for the Stella gene display reduced fertility with decreased rates of blastocyst development following fertilization [183] and the abnormal effects on early embryonic development are dependent on presence of the 50 mutant Stella allele in the maternal genome. Hsfl is a transactivator of stress-inducible genes in response to environmental changes, but is also involved in early embryonic development [184]. Mouse embryos deficient in Hsfl protein are unable to develop beyond the one-cell zygotic stage, even though Hsfl null oocytes ovulate and fertilize normally [184]. The mHR6A protein is the mouse homolog of yeast RAD6 protein, a key regulator of repair of DNA damage during replication [185]. The mHR6A deficient mouse oocytes are ovulated normally but arrest at the two-cell stage following fertilization [1 85] . Basonuclin is a novel maternal effect gene identified in mouse species. Basonuclin is a zinc finger protein with expression restricted to keratinocytes and the male and female germ cells [186]. In mouse oocytes, basonuclin co-localizes with RNA polymerase I (Pol-I) activity in the nucleolus [187] and may also interact with RNA polymerase II (Pol-II) transcribed promoters in the nucleoplasm [188]. Basonuclin mRNA and protein are present in oocytes and one-cell embryos but decrease drastically to undetectable levels at subsequent stages of embryonic development [186]. Targeted disruption of basonuclin specifically in mouse oocytes using a transgenic-RNAi approach leads _ to subfertility in female mice [186]. Oocytes deficient in basonuclin display a reduced rate of Pol-I transcription, perturbation of a large number of Pol-II transcribed genes and abnormal morphology [186]. Although a few basonuclin deficient oocytes ovulate and complete meiotic maturation, embryonic development is delayed with preimplantation failure resulting in female subfertility [186]. Chromatin remodeling is thought to play an important role in embryonic genome activation [189-191]. The chromatin remodeling complexes such as SWI/SNF are 51 involved in regulating gene transcription in mammals [192]. SWI/SNF is a complex of 9 subunits recruited to promoters of target genes by sequence specific transcription factors [192]. The Brgl catalytic subunit exhibits DNA dependent-ATPase activity, and the energy derived from ATP hydrolysis alters the conformation and position of nucleosomes, thereby permitting access of RNA-polymerase II enzyme to the promoter regions of genes so that transcription can be initiated [193]. Brgl has been implicated in regulation of zygotic genome activation in mouse embryos [194]. Brgl is expressed in mouse oocytes, and an oocyte-specific Brgl mutation generated by Cre-loxP gene targeting or oocyte-specific Brgl knockdown by transgenic RNAi approach, results in developmental arrest at the 2-4 cell stages of mouse embryogenesis [194]. Further, the transcriptional activity of 30% of the genes expressed at these stages in reduced in Brgl deficient embryos [194]. All the information available to date regarding the maternal effect genes and their requirement for early embryonic development is restricted to the mouse species. Due to inherent species specific differences in the duration and number of cell cycles required for embryonic genome activation in the bovine model versus mice, the regulatory mechanisms and maternal effect genes involved may not be identical in the bovine species (Fig. 6). Knowledge of key maternal effect genes required for early embryonic development in ruminants and humans is lacking. An increased understanding of composition of the oocyte transcriptome (catalog of genes expressed by female germ cells) will help facilitate identification of the essential oocyte specific regulatory factors controlling follicular development and early embryogenesis which are critical to reproductive success. Identification and characterization of such factors may help further resolve the physiological mechanisms 52 O. O 0 OS.‘ 0 J J. I I. O. O. a. n. O... ‘0. I Embryonic genome activation Blastocyst Figure 6. Salient features during bovine early embryonic development. The ovulated oocyte undergoes cumulus expansion and is arrested at the metaphase II stage with a Fertilization initiates completion of the second meiotic single polar body (PB). division with exclusion of a second PB and formation of a diploid zygote ready for the first mitosis. The initial mitotic divisions from fertilization through embryonic genome activation (8-16 cell stages) are dependent on maternal mRNA and protein supplied by the oocyte. Specific genetic factors that regulate early developmental events such as cleavage and genome activation in bovine embryos are not known. Genome activation initiates transcriptional activity within the embryonic nucleus and subsequent events of embryo development are governed by the new transcription. Repeated mitosis and migration /allocation of embryonic cells results in a structure called a blastocyst by day 7. by which the oocyte orchestrates folliculogenesis, oocyte maturation and early embryonic development. The putative factors might also serve as novel objective molecular markers/indicators of fertility in domestic ruminants as well as in humans. The oocyte derived novel factors may also facilitate development of new methods for contraception or enhancement of fertility in a clinical or agricultural setting. A thorough understanding of the oocyte derived factors may further interpretation of infertility problems associated with female germ cells. To generate fundamental knowledge on composition of the oocyte transcriptome, we recently constructed a cDNA library from bovine oocytes [195]. During initial analysis of expressed sequence tags (EST) from this library [195], we identified an abundant oocyte- expressed transcript (which we refer to as JY-l). Our preliminary studies indicated that JY-l mRNA and protein are expressed exclusively in bovine oocytes (described further in Chapter 4). Extensive sequence comparisons indicate that JY-l is novel and the predicted protein for this gene contains a signal peptide, indicating it is a putative secreted protein. Determination of temporal changes in JY-l mRNA abundance during meiotic maturation and early embryogenesis are fundamental to studies of JY-l function. Such studies are dependent upon sensitive real-time reverse transcription polymerase chain reaction (RTPCR) procedures that generally require selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variation. However, the biology of the oocyte and early embryo is unique, since maternal pools of mRNA are gradually depleted during meiotic maturation and early embryonic development. Thus, identity of an appropriate control gene for data normalization in real-time PCR assays that is constitutively expressed throughout early embryogenesis has 54 remained elusive. Therefore, to quantify JY-l mRNA during meiotic maturation and early embryonic development by real-time RTPCR, a new reliable method was developed and validated [196]. The details for this procedure are discussed in Chapter 3. Using the validated procedures, the temporal expression pattern for JY-l mRN A was determined during meiotic maturation and early embryonic development (Chapter 4). The tissue and cell specific expression of JY-l and its temporal regulation during embryogenesis is suggestive of potential biological relevance to fertility. Based on its abundant oocyte specific expression and its temporal and spatial expression pattern in embryos, I hypothesize that the JY-l gene has an important function during bovine folliculogenesis and early embryogenesis and is conserved in other species. To test this hypothesis, I will a) determine the gene structure of bovine JY-l and investigate presence of orthologous genes to J Y-l in other species, b) determine the requirement of JY-l for early embryonic development and 0) determine the ability of JY-l to modulate F SH induced regulation of granulosa cell function. Results of these studies and their significance are detailed in Chapter 4. 55 Chapter 3 Bettegowda A, Patel OV, Ireland JJ, Smith GW. Quantitative analysis of messenger RNA abundance for ribosomal protein L- 15, cyclophilin-A, phosphoglycerokinase, beta- glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro. Mol Reprod Dev 2006 Mar; 73:267-278‘. 1Reprinted with permission of Wiley-Liss, Inc. a subsidiary of John Wiley & Sons, Inc. 56 Chapter 3 Quantitative Analysis of Messenger RNA Abundance for Ribosomal Protein L-15, Cyclophilin-A, Phosphoglycerokinase, B-glucuronidase, Glyceraldehyde 3-Phosphate Dehydrogenase, fl-actin and Histone H2A during Bovine Oocyte Maturation and Early Embryogenesis In Vitro 57 ABSTRACT Real-time reverse transcription PCR has greatly improved the ease and sensitivity of quantitative gene expression studies. However, measurement of gene expression generally requires selection of a valid reference (housekeeping gene) for data normalization to compensate for inherent variations. Given the dynamic nature of early embryonic development, application of this technology to studies of oocyte and early embryonic development is further complicated due to limited amounts of starting material and a paucity of information on constitutively expressed genes for data normalization. We have validated quantitative procedures for real time RT-PCR analysis of mRNA abundance during bovine meiotic maturation and early embryogenesis and utilized this technology to determine temporal changes in mRN A abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, B-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, B-actin and histone H2A. Quantification of amounts of specific exogenous RNAs added to samples revealed acceptable rates of RNA recovery and efficiency of reverse transcription, with minimal variation. Progression of bovine oocytes to metaphase II resulted in reduced abundance of polyadenylated, but not total transcripts for majority of above genes; however phosphoglycerokinase exhibited a significant decline in both RNA populations. Abundance of mRNAs for above genes in early embryos generally remained low until the blastocyst stage, but abundance of ribosomal protein L-15 mRNA was increased at the morula stage and histone H2A mRNA showed dynamic changes prior to embryonic genome activation. Results demonstrate a valid approach for quantitative analysis of mRNA abundance in oocytes and embryos, but do not support constitutive expression of above genes during early embryonic development. 58 INTRODUCTION The translational control of oocyte mRNAs during meiotic maturation and early embryogenesis is dependent on evolutionarily conserved processes termed cytoplasmic polyadenylation and deadenylation, which involve addition or deletion of adenosine residues on the tail of mRNAs [16, 26]. The early developmental cell fate decisions of an embryo are regulated through translational activation of maternally stored mRNAs prior to embryonic genome activation, also termed the maternal zygotic transition [16]. Embryonic genome activation (at 2-cell to 8-16-cell stages, depending on the species) will further influence embryonic development via innate synthesis of new transcripts [20, 21]. Gaining a clear insight into the molecular events controlling mammalian oocyte maturation and early embryonic development is crucial to advancements in developmental biology and improvements in assisted reproductive technologies. Technical limitations and dearth of starting material have restricted accurate, widespread quantitative analysis of mRNA abundance for genes of interest in mammalian oocytes and early embryos using traditional molecular approaches. However in recent years, real- time reverse transcription polymerase chain reaction (RT-PCR) procedures have been applied for quantitative analysis of mRNA abundance in oocytes and embryos [197-206]. Although the approach is highly sensitive, inherent technical limitations associated with limited amounts of starting material (variation in RNA recovery, efficiency of reverse transcription) may influence results generated. The most common way to account for inherent variation in real time RT-PCR assays of mRNA abundance is to use amounts of mRNA detected for a constitutively expressed (housekeeping) gene for data normalization. However, the biology of oocytes and early embryos is unique, since 59 maternal pools of mRNA are gradually depleted during meiotic maturation and early development until embryonic genome activation. Therefore, despite utilization of select housekeeping genes for data normalization in previously published studies of this nature, the identity of an appropriate control gene for data normalization in real time RT-PCR assays that is constitutively expressed throughout early embryogenesis and (or) in response to various treatments has remained. elusive. An alternative approach would be to use exogenous control RNAs added during sample preparation as sensitive indicators to account for potential variation during RNA extraction, reverse transcription procedures and for normalization of results [207, 208]. The objective of this study was to develop and validate appropriate procedures for quantitative real time RT-PCR analysis of mRNA abundance in bovine oocytes and early embryos to account for potential variation in efficiency of RNA extraction and reverse transcription. In addition, temporal changes in mRNA abundance for seven known housekeeping genes [ribosomal protein L-15 (RPL-15), cyclophilin-A (CYC-A), phosphoglycerokinase (PGK), B-glucuronidase (GUSB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), B-actin and histone H2A (H2A)] during bovine oocyte maturation and early embryogenesis were determined in an attempt to identify a suitable housekeeping gene for normalization of real time RT-PCR data for oocytes and early embryos. MA T ERIALS AND METHODS Materials All materials were obtained from Sigma Aldrich (St.Louis, MO) unless stated otherwise. 60 Oocyte Recovery and In Vitro Maturation Briefly, ovaries obtained at a local abattoir were transported to the laboratory in sterile saline solution. Upon return to the laboratory, ovaries were washed in sterile saline and cumulus-oocyte complexes (COCs) were aspirated from 2-7 mm visible follicles using an l8-gauge needle and 50-60 mm Hg of negative pressure. After sedimentation, the COCs were individually selected and washed 3-4 times in Hepes- buffered hamster embryo culture medium (HECM) [114 mM NaCl, 3.2 mM KCl, 2 mM CaClz‘2HzO, 0.5 mM MgClz'6HzO, 100 pl /ml MEM non-essential (10X) amino acids, 17 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM NaHCO3, 1 mM Hepes, 0.183 mM penicillin-G, 3 mg/ml BSA; pH 7.3-7.4; 275 i 10 mOsm/kg)] under a stereomicroscope. COCs with more than 4 compact layers of cumulus cells and homogeneous cytoplasm were matured in TCM 199 [supplemented with 0.2 mM sodium pyruvate, 5 mg/ml gentamicin sulfate, 6.5 mM L-glutamine, 156 nM bovine LH (Sioux Biochemical, Sioux Center, Iowa), 15.6 nM bovine FSH (Sioux Biochemical), 3.67 nM 17B-estradiol and 10% v/v defined FBS (Hyclone, Logan, UT)] for 24 h in groups of 50 in four-well dishes containing 400ul of maturation medium at 385°C, 5% C02 in air with maximum humidity. For germinal vesicle (GV) stage oocyte RNA samples, cumulus cells were completely removed by hyaluronidase (0.1%) digestion and repeated pipetting and denuded oocytes in groups of 10 were snap frozen in 100 pl lysis solution (RNAqueous Micro Kit, Ambion Inc, Austin, TX) and stored at -800 C until RNA isolation. For MII oocyte RNA samples, cumulus cells were removed after maturation (as described above) and mature oocytes in groups of 10 were snap frozen in 100 pl lysis solution and stored at 61 -80° C until RNA isolation. Metaphase II oocytes were selected based on the presence of a single polar body. In Vitro Fertilization and Embryo Culture For in vitro fertilization, spermatozoa from a frozen-thawed semen straw were selected through a Percoll density gradient. The semen was layered on a Percoll gradient consisting of 0.5 ml each of 30%, 60% and 90% Percoll in Hepes buffered Tyrode’s lactate (TL) sperm medium (100 mM NaCl, 25 mM NaHCO;;, 10 mM Hepes, 0.29 mM NaH2P04, 0.035 mM sodium lactate, 1.5 mM MgCl2'6H20, 2.61 mM CaCl2'2H20 supplemented with 0.2 mM sodium pyruvate, 6 mg/ml BSA and 50 pg/ml gentamicin) and centrifuged at 2000 rpm for 10 min. Then, the pellet was resuspended in 4 m1 of TL sperm medium and centrifuged at 800 rpm for 10 min. Finally, the pellet was resuspended in 100 pl of TL sperm medium and concentration determined in a hemocytometer. Matured oocytes and sperm (10°spern1/ml) were co-incubated for 20 h in groups of 50 in four-well dishes containing 400 pl of fertilization medium (114 mM NaCl, 25 mM NaHC03, 3.2 mM KCl, 0.34 mM NaH2P04, 0.183 mM penicillin-G, 16.6 mM sodium lactate, 0.5 mM MgCl2'6H20, 2.7 mM CaCl2'2H20, 0.2 mM sodium pyruvate, 6 mg/ml BSA and 1.5 U of heparin) at 385°C, 5% C02 in air with maximum humidity. Semen from the same bull was used for all experiments. To separate cumulus cells, the presumptive zygotes were vortexed for 2 min and washed three times in Hepes buffered-HECM. Embryo culture was then performed in groups of 50 presumptive zygotes in four—well dishes containing 400 pl of KSOM medium (Specialty Media, Phillipsburg, NJ) supplemented with 3mg/ml BSA under mineral oil. Culture was carried out at 385°C, 5% C02 in air with high humidity. Embryos at the 8-16 cell stage were 62 separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 3 mg/ml BSA and 10% FBS until day 7. The 2-cell embryos were collected 33 h following fertilization, 4-cell embryos 44 h later, 8-cell embryos 52 h later, l6-cell embryos 72 h later, morula 5 d later and blastocyst-stage embryos 7 (I later. All the embryos were snap frozen in 100 pl lysis solution and stored at -80° C until RNA isolation. As a control for IVF procedure, a pool of embryos in each IVF run was cultured until the blastocyst stage to assess the developmental competence of the fertilized eggs. Only embryos collected from controlled experiments with rates of development to blastocyst stage of 2 25% (d 7) were used in the analysis. For each of the oocyte and embryo stages, five pools of samples were collected from a total of twelve different IVF runs. In Vitro Transcription and RNA Quantification For synthesis of green fluorescent protein (GFP) RNA, linear DNA templates having a SP6 promoter sequence at the 5’ end and poly (T13) tail on the 3’ end were generated by polymerase chain reaction (PCR) from plasmid vector pCX-EGF P (generously provided by Jeffrey B. Kopp, NIH). The following primers 5’- TCATTTAGGTGACACTATAGAATTCGCCACCATGGTGAGCAAGG-3’ (forward) and 5’-TTTTTTT'ITI‘T1’TTI’TTTACTCCAGCAGGACCATGTGATCGCGC-3’ (reverse) were used to PCR amplify a 719 bp segment from above GFP plasmid. For PCR, 500 ng of pCX-EGFP plasmid DNA was added to 100 pl PCR mixture [10pl of 10X PCR buffer, 3 pl of 50 mM MgCl2, 5 pl of 100% DMSO, 5 pl of 10 mM dNTP mix, 5 pl each of 10 pM forward and reverse primers and 5 units of T aq DNA polymerase (Invitrogen Life Technologies, Carlsbad, CA)]. PCR conditions used were as follows: 63 initial denaturation, 15 min at 94°C; amplification 40 cycles of 30 s at 94°C, 30 s at 55°C and 45 s at 72°C. PCR amplified product was purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA). GFP RNA was synthesized by in vitro transcription fi'om PCR amplified GFP [DNA template with SP6 RNA polymerase using the RiboProbe—SP6 system (Promega, Madison, WI). After in vitro transcription, template DNA was removed with RNAse-free DNAse and reactions subjected to phenol- chloroforrn-isoarnyl alcohol extraction and ethanol precipitation. Precipitated cRNA was resuspended in RNA storage solution (Ambion) and stored at -80° C until use. Concentration and integrity of cRNA was quantified on the Agilent Bioanalyzer 2100 using RNA 6000 Nano chip (Agilent Technologies, Palo Alto, CA). Generation of far-red fluorescent protein (HcRedl) cRNA was conducted basically as described above for GFP RNA. but with different primers. The 738 bp linear DNA templates for in vitro transcription were generated by PCR from plasmid vector pHc- Redl-Nuc (BD Biosciences, San Jose, CA) using the following primers [5’- TCATTTAGGTGACACTATAGCCATGGTGAGCGGCCTGCTGAA-3’ (forward) and 5’- TTTTTT'ITI’TTTTTT’I‘TTGATCTGAGTCCGGAGTTGGCCTT-3’ (reverse) with a poly (T13) tail fused to HcRedl sequence. RNA Extraction and Reverse Transcription Total RNA was extracted from each pool of oocytes/embryos (n = 5 pools of 10 oocytes/embryos per time point), and residual genomic DNA removed by DNAse I digestion, using the RNAqueous micro kit (Ambion) according to manufacturer’s instructions, but with slight modifications. Before RNA extraction, each sample was spiked with 250 femtograms of GFP synthetic RNA as an exogenous control and 50 pg 64 of tRNA as a carrier. RNA was eluted twice from the silica-based microfilter cartridge using 10 pl volume of pre-warrned (75°C) elution solution according to manufacturer’s instructions. The RNA from each pool of oocytes and embryos was divided into two samples so that the RNA equivalent of 5 oocytes (10 p1) was primed with 1 pl (450 nanograms) of oligo dT(15) and the other half of the RNA was primed with 1 pl of 20 pM random hexamers. Before reverse transcription (RT), 250 femtograms (1 pl) of HcRedl synthetic RNA was added to each reaction. RNA/primer mixture was incubated at 70°C for 10 min and rapidly cooled to 4°C prior to RT. RT was performed at 42°C for 1 h in a final volume of 20 pl containing 8 pl RT mix [4 pl of 5X RT buffer, 2 pl of 0.1 M dithiothreitol, 1 pl of 10 mM dNTP mix and 1 pl (200 units) of Superscript II reverse transcriptase (Invitrogen, Life Technologies)] followed by incubation at 70°C for 10 min to terminate the reaction. Each RT reaction was then diluted with nuclease free water (Ambion) to a final volume of 100 pl (one oocyte or embryo equivalent = 20 pl of cDNA). Quantitative Real Time RT-PCR The quantification of all gene transcripts was done by real-time quantitative RT-PCR using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Absolute quantification using this method is described elsewhere [209, 210]. Primers were designed using Primer Express program (Applied Biosystems) and derived from bovine sequences found in GenBank (see Table 1) and the amplicon size for each of the genes studied ranged from 80-150 bp. A primer matrix was performed for each gene tested to determine optimal primer concentrations. Each reaction mixture consisted of 2 pl of cDNA (corresponding to 1/10 of an oocyte/embryo), 1.5 pl each of forward (5 pM) and 65 reverse primers (5 pM), 7.5 pl of nuclease free water and 12.5 pl of SYBR Green PCR Master Mix in a total reaction volume of 25 pl (96-well plates). Reactions were performed in duplicate for each sample in an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The thermal cycler program consisted of 40 cycles of 95°C for 15 sec and 60°C for l min. Standard curves for each gene and controls were constructed using lO-fold serial dilutions of corresponding plasmids and run on same plate as samples. Copies of GFP and HcRedl RNA in each pool were determined using standard curves constructed from the plasmid pCX-EGFP and pHc-Redl-Nuc. respectively. Real time primer sequences for GF P and HcRedl were as follows: GFP forward 5’-CAACAGCCACAACGTCTATATCATG-3’; GFP reverse 5’- ATGTTGTGGCGGATCTTGAAG-3’; HcRedl forward 5’-GCCCGGCTTCCACTTCA- 3’; and HcRedl reverse 5’-GGCCTCGTACAGCTCGAAGTA-3’. Partial cDNAs for RPL-15 and H2A were amplified from bovine GV oocytes, cloned into pCR2.1 Topo vector (Invitrogen Life Technologies) and subjected to fluorescent dye primer sequencing to confirm identity. Resulting plasmids, along with plasmids containing cDNA inserts for CYC-A, PGK, GUSB, GAPDH and B-actin [vector pCMV Sport6 (Invitrogen Life Technologies)] obtained from the MSU Center for Animal Functional Genomics NBFGC (National Bovine Functional Genomics Consortium) library were used to construct standard curves. Representative R2 for GFP, HcRedl, GAPDH, B-actin, RPL-15, CYC-A, PGK, GUSB and H2A standard curves were all > 0.98. For each measurement, threshold lines were adjusted to intersect amplification lines in exponential portion of amplification curve and cycles to threshold (Ct) recorded. For each sample, amounts of mRNA 66 (copies) for GF P, HcRedl and housekeeping genes analyzed were determined from their respective standard curves. Statistical Analysis The copy numbers of GF P and HcRedl RNA added to samples were calculated according to molecular weight of the RNA (base pair size of the RNA product x 330 Daltons) and then converted into copy numbers based upon Avogadro’s number (1 mol = 6.022 x 1023 molecules). Percent RNA recovery was determined by calculation of copies of GFP RNA detected versus number of copies added prior to RNA extraction and efficiency of reverse transcription by comparison of copies of HcRedl measured versus number of copies added to RNA samples immediately prior to reverse transcription. Copies of RNA for each individual gene of interest within each sample were normalized relative to copies of GP P RNA measured in each sample and differences in normalized RNA data across developmental stages were determined by one-way analysis of variance using the GLM procedure of SAS. Individual mean comparisons were performed using Tukey’s test. Differences of P < 0.05 were considered significant. RES UL TS Validation of Real Time RT -PCR Procedures To assess potential variation in RNA recovery and efficiency of reverse transcription, samples were spiked with 250 fg of GFP and HcRedl cRNAs prior to RNA isolation or cDNA synthesis respectively. Representative electropherogram for in vitro synthesized GFP RNA is shown in Fig. 7A. Results of real time RT-PCR assay of MII and GV oocyte cDNA generated with oligo dT primers (used for quantification of amounts of polyadenylated transcripts) indicate an acceptable rate of GFP RNA recovery (~ 50%) in 67 both sample types that was not significantly different (P > 0.05), and with minimal variation within samples (Fig. 7B). Furthermore, percent recovery of GFP RNA (~95%) was greater in GV and M11 cDNA samples primed with random hexamers (used for quantification of total transcripts), but [did not differ between samples (Fig. 7C). The copies of GFP RNA detected in oligo dT primed cDNA generated from early embryos at the pronuclear, 2-cell, 4-cell, 8-cell, 16-cell, morula and blastocyst stages also did not differ (Fig. 7D). In addition, copies of GP P RNA recovered from early embryos primed with random hexamers were greater than oligo dT but did not differ significantly (Appendix Fig. A.1). Similar results as reported in Fig. 7 were obtained when copies of HcRedl RNA in above described samples were assayed to specifically assess variation in efficiency of reverse transcription reactions (> 85%; Appendix Fig. A2). 68 Table 1: Details of Primers Used in Real Time-PCR GenBank Gene accession Species Sequence number 1. 1.... 1.“ RPL- 1 5 MM 141 Cow 1‘22 ’1ffgfiéfilflfffé’fgflfécs1 1. Phosphoglycerokinase BG688981 Cow 11:: giggigqigfigf SGZEATTCEAATGGS B‘G'Wumnidase 315751040 COW ii: 55i:Eiiziiffrccldcilhcghficciihgirt-3’ Histone H2 A BF076713 Cow F: 5’-GAGGAGCTGAACAAGCTGTTG-3’ R: 5’-TTGTGGTGGCTCTCAGTCTTC-3’ 69 Figure 7. Quantitative real time RT-PCR analysis of amounts of green fluorescent protein (GFP) RNA in oocyte and embryo samples spiked prior to RNA isolation. (A) A representative electropherogram generated by Agilent Bioanalyzer nanochip for in vitro synthesized GFP RNA. Quantification of amounts of exogenous GFP RNA in samples of germinal vesicle (GV) and in vitro matured metaphase (MII) stage oocytes (B) reverse transcribed with oligo dT(I5) or (C random hexamers (n = 5 each). (D) Quantification of amounts of exogenous GFP RNA in samples of in vitro derived embryos collected at pronucleus (PN), 2-cell (2-C), 4-cell (4-C), 8-cell (8-C), 16-cell (16-C), morula and blastocyst stages (n : 5 each) reverse transcribed with oligo dT(15). Data are shown as mean 3: SEM. Means with common superscripts are not different. A B 125 I t S a to I .0 a E 100 I E 5000 “9’ 5 I 3 95’ 0 ’ §: 25001 2 25 I U u. r . m 0 é-n—lu—y-uphnct ...- 4.-.._ -.. -,. (L5 0 ti 19 29 39 49 59 9" M11 Time (3) Stage ofmeiotic maturation C. D t— a a 522100001 a a 5.? 5000 a a a a a g E >1 5000 2 2500 l : Q1 >s 8 8 a 0 Ir 7a g 0 J T -1. -- 4 \1 ‘3 GV MI] I; of 1,91 ,0 1961‘s of 0 9‘ 96‘» Stage of meiotic maturation Stage of embryogenesis 70 Quantification of Abundance of GAPDH, ,B-actin, RPL-15, C YC—A, PGK and GUSB mRNAs During Oocyte Maturation Amounts of polyadenylated GAPDH, B-actin, RPL-15, CYC-A, PGK and GUSB mRNA (normalized relative to amounts of GFP RNA in each sample) decreased (P < 0.05) 1.5 to 5 fold during meiotic maturation (Fig. 8). However, amounts of total transcripts (polyadenylated + non-adenylated, determined by real time RT-PCR using RT reaction primed with random hexamers) for GAPDH, B-actin, RPL-15, CYC-A and GUSB genes were not decreased in M11 versus GV oocytes (Fig. 9). In contrast, total transcripts for the PGK gene were significantly decreased in MI] oocytes (P < 0.05; Fig. 9). Temporal Regulation of mRNA for GAPDH, ,B-actin, RPL-15, CYC—A, PGK and GUSB during Early Embryonic Development Quantification of polyadenylated transcripts for CYC-A, PGK, GUSB, GAPDH and B-actin genes revealed a similar temporal expression pattern, where mRNA abundance for each gene remained low through embryonic genome activation until the blastocyst stage when a significant increase in mRNA abundance was detected (Fig. 10). A similar temporal expression pattern for RPL-15 mRNA was observed, except that a significant increase in transcript abundance was observed at the morula stage and further increased at the blastocyst stage (Fig. 10). 71 Figure 8. Quantitative real time RT-PCR analysis of polyadenylated RPL-15, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of germinal vesicle (GV) and metaphase (MII) stage bovine oocytes (n = 5 each). Data are normalized relative to abundance of exogenous control (GFP) RNA and shown as mean 3: SEM. Time points without a common superscript are significantly different, P < 0.005. 72 Relative mRNA Relative mRN A Relative mRNA 0.5 . 0.4 4 0.3 : 0.2 < abundance abundance 9s>9 OOH #OON abundance O Stage of meiotic maturation RPL-15 GV MII PGK GV MII GAPDH a GV Mll OPPrrN JBOONG 0.08 0.06 * 0.04 ‘ 0.02 ‘ #l CYC-A MII GUSB MII B-ACTIN 73 GV MII Stage of meiotic maturation Figure 9. Quantitative real time RT-PCR analysis of total RPL-lS, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of germinal vesicle (GV) and metaphase (MII) stage bovine oocytes (n = 5 each). Data are normalized relative to abundance of exogenous control (GFP) RNA and shown as mean :t SEM. Time points without a common superscript are significantly different, P < 0.0001. 74 Relative mRNA Relative mRNA Relative mRN A abundance abundance abundance RPL-15 0.032 1 PGK .O Ur .9 N O P o S O\ llvj- {It GV MII Stage of meiotic maturation 75 CYC-A 0.3 ~ 0.6 * 0.4 : 0.2 t 0 03 I GUSB 0.02 0.01 B-ACTIN GV MII Stage of meiotic maturation Figure 10. Quantitative real time RT-PCR analysis of polyadenylated RPL-15, CYC-A, PGK, GUSB, GAPDH and B-actin transcripts in samples of in vitro derived bovine embryos collected at pronucleus (PN), 2-cell (2-C), 4-cell (4-C), 8-cell (8-C), 16-cell (16- C), morula and blastocyst stages (n = 5 each). Data are normalized relative to abundance of exogenous control (GFP) RNA and shown as mean :1: SEM. Time points without a common superscript are significantly different, P < 0.05. 76 CYC-A RPL-15 e... 0 b\ 0% b - é. . o .963 6: 6%- a a 09 a a I so 900765432110 in 421006.420 11 0000 C :5 x. \ 6% $0 a} éOQ} 0,0 .00 $0,, 62.85% 90% efficiency in targeting J Y-l mRN A were combined at a final concentration of 25 pM and are denoted as JY-l cocktail siRNA. As a control for siRNA injection, negative control siRNA (nonspecific; Ambion universal control #1) was also tested as described above except that blastocyst development on day 7 post injection was used as the endpoint. Quality of blastocysts derived from uninjected controls versus negative control siRNA injection was also confirmed by counting total cell numbers (by Hoechst nuclear staining) [243, 244]. Reduction of J Y-l protein in JY-l siRNA injected parthenogenetic embryos collected at the 8-16 cell stages (72 h after parthenogenetic activation) was determined by Western blot analysis. Uninjected 8-16 cells embryos generated at the same time as microinjection experiments were used as the control. Protein lysates from 185 embryos per treatment (n = 3 samples of 50 embryos each, and n = 1 sample of 35 embryos each) were loaded per lane. To determine the effects of J Y-l knockdown on blastocyst development, microinjection experiments were performed in two different in vitro models of embryo development: parthenogenesis and IV F. In each experiment (n=4-5 replicates), groups of approximately 25-30 denuded MII oocytes or denuded presumptive one cell embryos derived from IVF were randomly assigned to one of the following treatments: 1) JY-l cocktail siRNA (25 pM), 2) negative control siRNA-1 (25 pM), 3) sham water injection or 4) uninjected controls. After microinjection, groups of activated 98 oocytes or IVF embryos were cultured in 75 pl drops of KSOM supplemented with BSA (3 mg/ml). Cleaved embryos were separated and cultured in fresh drops of KSOM supplemented with 10% FBS and BSA (3 mg/ml). Cleavage rate was determined on day 3, and blastocyst rate (total no. of blastocysts / total cleaved embryos) assessed on day 7. Genomic Southern Blot Analysis Genomic DNA was extracted from cow, sheep, pig, mouse and chicken (liver tissue) and whole rainbow trout and zebrafish, followed by residual RNA digestion and DNA precipitation. RNAse treated human genomic DNA (source: liver) was purchased from a commercial vendor (Biochain Institute Inc., Hayward CA). Southern blot was prepared by digesting approximately 15 pg of genomic DNA per sample with EcoRI for 30 hr, electrophoretically separated and transferred to a Zeta-probe membrane (Bio-Rad, Hercules CA). The membrane was hybridized at 62 C in PerfectHyb solution (Sigma- Aldrich, St. Louis MO) with 32P-labeled JY-1 cDNA (450 bp, region corresponding to 5’UTR, ORF and a portion of 3’UTR). The membrane was washed twice at 60 C in 0.5X SSC/O.1% SDS for 15 minutes and subjected to autoradiography. Genomic Library Screening and Bioinformatics analysis A partial J Y—l gene structure was determined by screening a bovine genomic library (EMBL3 SP6/T7 vector; Clontech Laboratories, Inc., Palo Alto CA) using 32P-labeled JY-l cDNA (455 bp) following manufacturer’s instructions. Positive plaques were isolated and further screened by hybridization and PCR. The lambda DNA was purified using Wizard Lambda Preps DNA Purification System (Promega), subjected to restriction digestion and DNA fragments subcloned into pBluescript SK (i) vector (Stratagene) and subjected to fluorescent dye terminator sequencing. Using the partial 99 J Y-l gene sequence, the complete gene structure and chromosomal location for the J Y-l gene was determined by searching the bovine genome database at NCBI. Genomic DNA databases at NCBI for chimpanzee, dog, mouse, rat, chicken, zebrafish and drosophila were then searched with the predicted amino acid and nucleotide sequence of the 1.5 kb bovine JY-l cDNA. Likewise, the human EST database was searched with the nucleotide sequence of the 1.5 kb bovine J Y-l cDNA. Cloning of Putative Human JY -1 cDNA A single human EST sequence derived from a Hembase library (erythroid precursor cells, GenBank accession: BU656412) showing identity to a small portion of the ORF present in the bovine JY-l cDNA sequence was identified following a search of the human EST database at NCBI GenBank. Two pairs of PCR primers were designed based on the human EST sequence and a two step nested PCR was performed. cDNA was synthesized from total RNA from adult human ovary (Stratagene) and H9 human embryonic stem cells (generously provided by Dr. Jose Cibelli) as described previously [240]. Synthesized cDNA was used as template for first round PCR amplification of cDNA encoding for putative human JY-l using specific primers (F: 5’- AAATCTGTGTGGATAGCCTTATCAG-3’ and R: 5’- CCTGGTGACAAAGAGAACATACG-3’) and standard PCR procedures [240] with 3 mM magnesium chloride concentration. Negative controls reactions for cDNA synthesis incubated in the absence of reverse transcriptase enzyme were included to confirm absence of residual genomic DNA contamination. Nested PCR was performed with a second set of PCR primers (F: 5’-CCAGGCATGTTACTTATGAATAACTT-3’ and R: 5’-AGGGAGCTGAAGCTTGGAA-3’) using 1 pl of first round PCR amplification 100 product from respective RT plus and RT minus reactions. Amplification of the housekeeping gene beta-actin (IS-actin) with PCR primers (F: 5’- TCCTCCCTGGAGAAGAGCTA-3’ and R: 5’-AGTAC'ITGCGCTCAGGAGGA-3’) spanning two introns was used as a positive control to verify cDNA synthesis and to confirm absence of genomic DNA contamination. Amplified cDNA were ligated into pCR 2.1 TOPO vector (Invitrogen) and plasmids containing cDNA of interest were subjected to fluorescent dye terminator sequencing. Statistical Analysis For real-time PCR experiments, differences in mRNA abundance were determined by one-way analysis of variance using the GLM procedure of SAS. For microinjection experiments, rates of development to blastocyst stage were analyzed by one-way analysis of variance following arcsin transformation using the Mixed Linear Models procedure of SAS. Similarly, differences in P, E and cell numbers were determined by the Mixed Linear Models procedure of SAS. Individual mean comparisons were performed using Tukey’s test. The dose response relationship between rJY-l and P was determined by regression analysis. Differences of P < 0.05 were considered significant. RESULTS Tissue Distribution and Characterization of JY -1 mRNA Transcripts Screening of RNA from various tissues by RT-PCR detected JY-l mRNA only in fetal ovaries collected at 180 and 210 days of gestation but not in any other tissues examined (Appendix 1; Fig. A.3) supporting tissue specific expression of JY-l RNA. Northern analysis revealed three predominant J Y-l transcripts in RNA isolated from fetal ovaries (Fig. 12A). Further analysis of adult GV oocytes by Northern blotting confirmed 101 the presence of three major J Y-l transcripts of different length (approximately 1.8 kb, 1.2 kb, and 700 bp) (Fig. 12B). Since all 14 JY-l inserts sequenced from the oocyte library were small (the longest is ~ 455 bp in length) and could be partial cDNAs or represent the smaller predominant transcript detected by Northern analysis, a fetal ovary cDNA library was screened and two additional clones containing larger inserts were obtained. One of the clones contained an insert of approximately 1.5 kb and the other clone had an insert of approximately 1.0 kb in length. Sequence analysis of these 2 larger JY-l cDNAs as well as the 2 original smaller cDNAs (455 bp and 355) from the oocyte library revealed that the 4 cDNA clones represent 4 different transcripts of the JY-l gene (Fig. 12C). The minor differences in the length of JY-l transcripts observed in fetal ovary versus adult GV oocytes is most likely attributed to polyadenylation status of the transcripts and or resolution of the gels utilized. An identical open reading frame of 255 bp encoding for a predicted protein of 84 amino acids was identified in all 4 transcripts derived from the oocyte and fetal ovary libraries (Fig. 12C). All four transcripts differ in length of the 3’ untranslated region (UTR) but have an identical 5’UTR. The AU rich putative cytoplasmic polyadenylation elements (AUUUUAAAA and UAUUUUAAUA) were also noted in the 3’ UTR of the two longest transcripts (Fig. 12C). Publicly available Signal 1P3 program [245] predicted a signal peptide of 21 amino acids with a probability of >50%, indicating that JY-l protein could be secreted. The predicted molecular weight of JY-l is approximately 9,000 Mr, but the publicly available NetOGlyc-3.1 program [246] predicted two O-linked glycosylation sites in the deduced JY-l amino acid sequence, suggesting potential glycosylation of J Y-l protein. 102 Figure 12. Characterization of the number and size of JY-l mRNA transcripts. (A) Northern analysis of JY-l mRNA in multiple bovine tissues and (B) adult germinal vesicle stage oocytes (GVO). Three predominant J Y-l transcripts were detected in RNA from fetal ovaries and GVO. (C) Characterization of JY-l cDNA clones derived from oocyte and fetal ovary cDNA libraries. Note two shorter clones of 450 bp and 350 bp derived from the oocyte library and two larger clones of 1.5 and 1 kb from fetal ovary library representing four distinct transcripts with identical open reading frame (ORF) but differing lengths of the 3’-untranslated region (3’UTR). UA rich cytoplasmic polyadenylation elements (CPE) are present in the 3’UTR of the two largest transcripts. 103 A g S B “$3 >. > (”5 >28£3$ o .— RPL 19... -0 W1 ”-1 —A C CPE CPE 15kbAT—Im-I——J——l 3’ UTR Fetal ovary [ 5 UTR CPE l'b , ‘ my lkb 000318 [450.— bp —=—— library 350 bp—-_ Oocyte Specific Localization of JY -1 mRNA and Protein within Ovarian F ollicles Intraovarian expression of JY-l mRNA and protein was restricted exclusively to oocytes. In situ hybridization localized JY-l mRNA specifically to oocytes of preantral and antral follicles. Note prominent localization of JY-l mRNA to oocyte present in a preantral (Fig 13A and 13B) and an antral follicle (Appendix-1; Fig. A.4). No significant hybridization to additional ovarian cell types (granulosa, theca and stroma) was noted. J Y-l protein was localized to oocytes of growing follicles at the primordial (single layer, with less than 10 flattened granulosa cells), primary (single layer with cuboidal granulosa cells) through antral follicle stages (Fig. 13C-13F) in fetal ovaries collected at day 230 of gestation. Irnmunoreactivity was not detected when tissue sections were incubated with pre-irnmune rabbit IgG (Fig. 13G) or when the JY-l antibody was preabsorbed with immunogen peptide (Fig. 13H). Characterization of JY-I Protein Polyclonal antiserum raised against rJY-l protein (mature form without signal peptide) was used in Western blot analysis to detect JY-l protein. As presented in Fig. 14A, immunoreactive J Y-l protein of approximately 11,000 Mr and additional higher Mr bands were detectable in extracts of adult GV oocytes. The polyclonal antiserum utilized also readily detected the rJY-l protein (6,700 Mr, mature form lacking the signal peptide) that was used to generate the antiserum (Fig. 14A). Preincubation of J Y-l antiserum with excess antigen (rJY-l) blocked binding of the antibody specifically to the 11,000 Mr protein and to rJY-l protein, but not to the higher Mr bands (Fig. 14B) which thus represent non-specific cross reactivity. Immunoreactive J Y-l protein was only detected in adult GV oocytes but not in any other samples examined (Fig. 14C). The 11,000 Mr 105 Figure 13. Intraovarian localization of J Y-l mRNA and protein. (A) Representative bright field micrograph of a preantral follicle stained with hematoxylin and eosin. (B) The corresponding dark field micrograph of (A) demonstrating oocyte-specific localization of JY-l mRNA. (C-E) Irnmunohistochemical localization of JY-l protein to the oocytes of (C) growing follicles, (D) primordial follicles and (E) primary follicles. Arrows (D and E) indicate a primordial and a primary follicle. (F) Localization of JY-l protein to the oocyte of an antral follicle. (G-H) Adjacent section to that depicted in (F) incubated with (G) pre-immune IgG or with (H) J Y-l antibody pre-absorbed with excess antigen. A and B, Magnification, x400. C, Magnification, x200. D and E, Magnification, x1000. F-H, Magnification, x100. 106 n.5,, ,‘.-.,... I‘1" ' l . ..-.- 107 Figure 14. Western blot detection of JY-l protein in bovine oocytes using antisera generated against recombinant JY-l protein (mature form lacking signal peptide; rJY-l). (A) Representative Western blots demonstrating detection of immunoreactive J Y-l protein of approximately 11,000 Mr in lysates of ISO germinal vesicle oocytes (GVO) and immunoreactivity of rJY-l (6,700 Mr). (B) Duplicate blot to (A) incubated with JY- 1 anti-serum pre-absorbed with excess rJY-l protein. Pre-absorption of antiserum specifically blocked binding of antibody to JY-l protein in GVO and to rJY-l. (C) Representative Western blots demonstrating tissue specificity of JY-l immunoreactivity. Note absence of immunoreactive J Y-l in samples of bull serum, granulosa cells, liver and adrenal tissue. (D) Duplicate blot to (C) demonstrating absence of JY-l immunoreactivity when blot was incubated with preimmune serrun. 108 7%? use? ”a 3.5 _ . £8 803580 Edam ==m . _ 0:35 W... o>o : 0 3:83.. _ . e25 . . mace «mo—=55 Essa _am MSW—m O>O .x I new! 9!“ B-actin — "" v : JY-l -—- . 109 JY-l protein also was not detected in GV oocytes when blots were incubated with pre- immune rabbit serum (Fig. 14D). Publicly available databases were searched with the predicted amino acid sequence of J Y-l to identify functional domains and predict the structure of the J Y-l protein. No significant orthologs of the JY-l protein were found in available protein databases. A putative secondary structure for J Y-l protein was predicted using the PSIPRED program [247], but we have not identified any motifs that are indicative of functional domains using CDD [248, 249]. Additional searches of numerous databases such as pFam A and B [250] and a PSI_BLAST search of the PDB database at NCBI [251] designed to designate sequences to protein families based on homology and identify known 3D structures for homology modeling were unsuccessful. Thus, we conclude J Y-l is presumably a member of a novel protein family. Efict of Recombinant JY-I Protein on Cell Number and Production of E and P by Cultured Granulosa Cells The rJY-l (mature form lacking signal peptide) was utilized to test the ability of J Y-l to regulate bovine granulosa cell proliferation and steroidogenesis. Addition of rJY-l to cultured granulosa cells inhibited the FSH stimulated increase in granulosa cell numbers at the 0.5 ng/ml dose (P<0.05) and the response was maximal at 1 and 10 ng/ml doses (P<0.05, Fig. 15A). Addition of rJY-l at 0.1 ng/ml had no effect on granulosa cell numbers (Fig. 15A). However, in vitro production of E was inhibited by 2 fold in FSH supplemented granulosa cells (P<0.005) treated with the 0.1 ng/ml rJY-l dose where significant effects on granulosa cell numbers were not observed (Fig. 15B). Further, the inhibitory effect on E production was maximal at 0.5 ng/ml rJY-l and supplementation with 1 and 10 ng/ml rJY-l did not inhibit E production. In contrast, addition of rJY—l 110 Figure 15. Effect of recombinant JY-l protein (rJY-l) on granulosa cell numbers and estradiol (E) and progesterone (P) production. A long-term granulosa cell culture system was utilized to determine the effects of rJY-l (0, 0.1, 0.5, 1 and 10 ng/ml) on granulosa cell numbers and in vitro production of estradiol (E) and progesterone (P) for cells cultured in the presence of 25 ng/ml of FSH (n=3-7 replicates per treatment). At the end of culture (day 7), cell numbers were counted and concentrations of E and P in culture medium determined. (A) Effect of rJY-l on total granulosa cell numbers for FSH treated cells. Note decrease in cell numbers with increasing concentration of rJY-l. The maximal response was noted at 1 and 10 ng/ml rJY-l (P < 0.05). (B) Effect of rJY-l on FSH stimulated E production by bovine granulosa cells. Concentrations of E were decreased in response to 0.1 ng/ml rJY-l and the response was maximal at 0.5 ng/ml rJY- 1 (P < 0.005). (C) Effect of rJY-l on P production by FSH treated bovine granulosa cells. Note dose dependent increase in P in response to increasing concentrations of rJY-l (P < 0.01). Concentrations of E and P were normalized to 30,000 cells. Data are depicted as mean 1: SEM. 111 A Ce“ # / Well 100000 - 75000 _ Cell number B Estradiol 2500 — é: 2000I O 8 q 1500 - \ 1000 - ‘35? 3 4 m 500 - 0 — 0 — 0 0.1 0.5 1 10 0 0.1 0.5 1 10 JY -1 (ng/ml) JY -1 (ng/ml) C Progesterone P (ng) / 30,000 cells 0 0.1 0.5 1 10 JY"Una/ml) 112 increased production of P in a dose dependent manner (P< 0.01) and the response was maximal at 1 and 10 ng/ml rJY-l (Fig. 15C). Even though the total cell numbers were decreased by approximately 50% in response to treatment with 1 and 10 ng/ml rJY-l, P production was doubled compared to cells cultured without rJY-l (Fig. 15A and 15C). No effects of rJY-l on granulosa cell numbers or E and P production were observed for granulosa cells cultured in the absence of F SH. Quantification of JY-I mRNA during Oocyte-to-Embryo Transition and Effect of JY-I Knockdown on Early Embryonic Development Given the observed oocyte specific localization of JY-l mRNA and protein, we hypothesized that JY-l mRNA is dynamically regulated during meiotic maturation and early embryonic development. Temporal changes in abundance of polyadenylated versus total J Y-l transcripts during early development were characterized by quantitative real- time PCR. Amounts of polyadenylated JY-l transcripts (cDNAs synthesized from oligo dT primers) decreased during meiotic maturation (P < 0.0001), were increased (P < 0.05) at the pronuclear and 4-cell stages relative to the M11 stage, and then decreased to nearly undetectable levels after the 16-cell stage of embryo development (Fig. 16A, Appendix 1; Fig. A.5). In contrast, amount of total J Y-l transcripts (cDNAs synthesized from random hexamers) gradually decreased from GV through l6-cell stages to nearly undetectable levels thereafter (Fig. 168, Appendix 1; Fig. A5). The difference in abundance of polyadenylated versus total transcripts is likely due to potential changes in polyadenylation status of J Y-1 mRNA (deadenylation and readenylation), a mechanism characteristic of CPE containing transcripts. Further, results of embryo culture experiments in the presence of the transcription inhibitor a-arnanitin suggest that the J Y-l 113 gene is not actively transcribed in early embryos (Appendix-1; Fig. A6) and thus the J Y- 1 mRN A detected in early bovine embryos is presumably maternal/oocyte derived. To test the requirement of J Y-1 during early embryonic development, we validated procedures for siRNA mediated gene silencing in bovine embryos. Procedures for microinjection of bovine oocytes were validated via injection of dextran-Texas red conjugate and > 90% efficiency was observed (Appendix 1; Fig. A.7). Multiple siRNA species were designed and tested for efficacy and specificity of J Y-l mRN A knockdown via microinjection into MII oocytes followed by parthenogenetic activation. Parthenogenesis was utilized as a model to test the efficacy of JY-l knockdown in embryos because it is easier to manipulate and allows for removal of cumulus cells and injection of siRNA earlier in development. The two most effective siRNA species (species 1 and 2, at 25 pM concentration) reduced J Y-l mRNA abundance by ~90% in 4- cell stage embryos (Appendix 1; Fig. A8). The cocktail of both JY-l siRNA species reduced JY-l mRNA abundance by ~ 90% in 2-cell embryos (Appendix 1; Fig. A.9). Specificity of the J Y-1 siRNA species and utility of our negative control (non specific) siRNA were confirmed via microinjection of a cocktail of both JY-l siRNAs, along with sham water and the negative control siRNA and measurement of abundance of mRNA for JY-l and several control genes in the 4-cell embryos. JY-l siRNA specifically reduced JY-l mRNA but had no effect on abundance of 188 rRNA and mRNA for 5 other control genes examined (Appendix 1; Fig. A.10). Negative control siRNA also did not reduce abundance any of the mRNA transcripts measured (Appendix 1; Fig. A.10). Further, quality of blastocysts (determined by cell number count) derived from negative control siRNA injection was not different from uninjected controls (Appendix 1; Fig. 114 A.11). Specificity of the JY-l siRNA species were further confirmed via microinjection of JY—l siRNA and measurement of JY-l protein abundance in 8-16 cell embryos. JY-l siRNA specifically reduced JY-l protein to undetectable levels compared to uninjected control embryos, but had no effect on protein abundance for actin (Appendix 1; Fig. A.12). J Y-l siRNA injection strikingly decreased the proportion of parthenogenetic embryos developing to the blastocyst stage (7.4%) relative to uninjected (31.7%), sham injected (31.5%) and negative control siRNA injected (33.7%) embryos (P<0.005, Fig. 16C). Cleavage rates of embryos were not different between the groups and were in the range of 75-79%. Similarly, JY-l siRNA injection did not affect the cleavage rates but dramatically reduced the proportion of IVF embryos developing to the blastocyst stage (4.2%) relative to uninjected (23.5%), sham injected (24.1%) and negative control siRNA injected (23.6%) embryos (P<0.01, Fig 16D, Appendix 1; Fig. A.13). Identification of JY-I like Sequences in Other Species and Cloning of Putative Human mRNA Ortholog of JY-I Southern blot analysis was utilized to investigate the presence of the lY-l gene in the genome of cattle and other species. The 450 bp JY-l cDNA strongly hybridized to an EcoRI genomic fragment in bovine genomic DNA. Relatively weaker hybridization to sheep, pig and human genomic DNA was noted (Fig. 17A). No significant hybridization to mouse, chicken, rainbow trout and zebrafish genomic DNA was detected (Fig. 17A). The structure and chromosomal localization of the bovine J Y-l gene was determined by genomic library screening and data mining approaches. Sequencing of exon- and exon- intronic junctions of two genomic clones revealed partial gene structure. Using this 115 Figure 16. Quantification of J Y-l mRN A abundance during oocyte maturation and early embryogenesis and effect of JY-l knockdown on blastocyst development. (A) Dynamic changes in relative abundance of polyadenylated JY-l mRNA transcripts during meiotic maturation and prior to embryonic genome activation [germinal vesicle (GV) and metaphase (MII) stage (oocytes), pronucleus (PN), 2-cell (2C), 4-cell (4C), 8-cell (8C), and 16-cell (16C) stage (embryos)]. (B) Gradual decline in abundance of total JY-l transcripts in same samples (n = 5 each) as depicted in (A). Oligo dT(lg) primers were used to reverse transcribe polyadenylated transcripts, whereas random hexamers were used to reverse transcribe total transcripts. Data are normalized relative to abundance of exogenous control (GF P) RNA and shown as mean :1: SEM. (C) Effect of JY-l knockdown on parthenogenetic blastocyst development. Denuded MII oocytes were subjected to one of the following microinjection treatments: (1) uninjected control, (2) sham water injection (3) J Y-l siRNA cocktail and (4) negative (-) control siRNA (n = 25- 30 embryos per treatment). Microinjected oocytes were parthenogenetically activated and rates of blastocyst development were recorded on day 7 and the experiment replicated 4X. (D) Effect of JY-l knockdown on development of IVF embryos to the blastocyst stage. Presumptive one cell IVF embryos were subjected to microinjection treatments as in (C). Microinjected embryos were cultured until day 7 and rates of blastocyst development recorded. The experiment was replicated 5X. JY-l siRNA injection dramatically decreased the proportion of parthenogenetic and IVF embryos developing to the blastocyst stage. Average rates of blastocyst development were calculated and data are shown as mean 1 SEM. Time points without a common superscript are significantly different, P<0.05. 116 Relative mRNA abundance > :~:;: be cc be b L. , GV M11 PN 2C 4C 8C 16C Stage of oocyte / embryo development 0 Day 7 blastocysts (%) w 0.1 0 Relative mRNA abundance GV MII PN 2C 4C 8C 16C Stage of oocyte / embryo development U Day 7 Blastocysts (%) 117 sequence information, remaining exonic and intronic regions of the entire J Y-l gene were identified by searching the bovine genome database. Collectively, the JY-l gene has 3 exons (25, 92 and 1400 bp in length) separated by two introns (12.8 and 1.5 kb in length) (Fig. 17B, Appendix 1; Fig. AM). The gene is 16 kb in length and located on chromosome 29 in the bovine genome. To identify putative cis-elements that may confer tissue/cell specific expression of JY-l, the 5’-flanking sequence of the JY-l gene was visually inspected. Five putative E-boxes (canonical sequence CANNTG; known to mediate oocyte specific expression [121, 252] in other species) were identified within 500 bp of the 5’flanking sequence of the bovine J Y-l gene (Appendix 1; Fig. AM). J Y-l like sequences were also found in the human genomic and EST databases. J Y- 1-like sequence was identified on human chromosome-11 (syntenic with bovine chromosome 29) with region of similarity corresponding to 187 bp of the protein coding region and 850 bp in the 3’UTR of the 1.5 kb JY-1 cDNA (Appendix 1; Fig. A.15). In the human EST database, a single EST sequence derived from a Hembase [human erythroid precursor cell (adult stem cell)] cDNA library was identified. The region of sequence similarity in the Hembase EST is 187 bp and maps to human chromosome 11 (l 1q14) (http://hembase.nidd1k.nih.gov) (Appendix 1; Fig. A.15) with 100% identity at the locus where the sequence similar to bovine JY-l is present (Appendix 1; Fig. A.15). RT-PCR analysis of adult human ovary and H9 human embryonic stem (ES) cell RNA using primers generated against the Hembase EST detected a putative human mRNA ortholog of bovine JY-l (Fig. 17C). Nucleotide sequence analysis of the amplified cDNA confirmed 100 % identity with the human Hembase EST. JY-l-like sequences were also identified in the genome of additional 118 Figure 17. Detection of J Y-l like sequences in the genome of multiple species, structure of JY-l gene and cloning of a putative human mRNA ortholog of bovine JY-l. A) Genomic Southern blot hybridized with 32P labeled 450 bp JY-l cDNA (probe) spanning the open reading frame (ORF), 5’ UTR and a portion of the 3’UTR. Southern blot was prepared with genomic DNA from cow (lane 1), sheep (lane 2), pig (lane 3), human (lane 4), mouse (lane 5), chicken (lane 6), rainbow trout (lane 7) and zebrafish (lane 8). Note strong hybridization to a bovine genomic DNA fragment. Weaker hybridization signals were also detected in sheep, pig and human genomic DNA. (B) Gene structure of bovine J Y-l. The JY-l gene has 3 exons (25, 92 and 1400 bp in length) separated by two introns (12.8 and 1.5 kb in length). All three exons in the gene are marked as E1, E2 and E3 respectively. (C) Detection of putative human mRNA ortholog of bovine JY-l in adult human ovary and H9 human embryonic stem (ES) cell RNA using RT-PCR. Amplification of the housekeeping gene beta-actin (B-actin) with PCR primers spanning two introns was used as a positive control to verify cDNA synthesis and to confirm absence of genomic DNA contamination [1, 3 = cDNA synthesis in the presence of reverse transcriptase; 2, 4 = negative control; cDNA synthesis in absence of reverse transcriptase]. 119 m=oo mm 5285 Ego BEBE _ w. 8203 Fluorescence U'J HcRedl copy number —t-— N U! N omomg ; . I . 1 > 150001 10000‘ 5000. l 0 1 I 19 29 39 49 59 Time (s) a a Stage of meiotic maturation (3 -§ 15000 a a E >. 10000 O. 8 ._ 5000 'U Q) ‘5 z: 0 ’ GV MII Stage of meiotic maturation D a E 20000 a :3 S: EIOOOOI I Ill'] 0 U 5% 14i‘ 49‘ v R9° VP @ci’ \¢O<:§} Stage of embryogenesis 136 Figure A.3. RT-PCR analysis of JY-l mRNA transcripts in multiple bovine tissues. Samples of fetal ovary tissue (an enriched source of oocytes), fetal testis, spleen, heart, muscle, lung, adult testes, uterus, thymus, kidney, liver, adrenal gland, hypothalamus, brain cortex, gut, pituitary, bone marrow (sternum and leg) and leukocytes were subjected to RNA isolation, DNAse digestion and cDNA synthesis as described previously [240]. Amplification of JY-l cDNA was performed using specific primers (F: 5’-TTGGAAC'ITCCATGGACGACC-3’ and R: 5’- TCATTTTGTGGCT'I‘CCATTCTG- 3’) and standard PCR procedures. Amplification of a 360 bp cDNA encoding for bovine RPL-19 was used as a positive control to confirm that cDNA synthesis was successful. 137 899 :96 65¢ 88: :96 am”. 68: :96 Emu mzwfl _Smu 25933 8% 5m AEzEmymv Em 29:55 SO xmtoo wsEmEEOQf 6:92 .m>_._ >952 magi”; Eoc 2:93 258 0:34 9822 tam: comaw $223 8 mm? m J RPL19 138 Figure A.4. Intraovarian localization of JY-l mRNA. (A) Representative bright field micrograph of an antral follicle hybridized with 35S-labeled antisense JY-l cRNA and stained with hematoxylin and eosin. (B) Representative dark-field micrograph of the same section depicted in (A). Note oocyte specific localization of JY-l mRNA. A and B, Magnification, x400. GC, granulosa cell layer; 00, oocyte 139 140 Figure A.5. Quantitative real-time RT-PCR analysis of total versus polyadenylated J Y-l mRNA transcripts within in vitro derived early bovine embryos. (A) Relative abundance of polyadenylated JY-l transcripts in samples collected from l6-cell (16C) through blastocyst stages (n = 5 samples of 10 embryos per sample at 16C, morula and blastocyst stage). (B) Relative abundance of total J Y-l transcripts in same samples (n = 5 each) as depicted in (A). 250 femtograms of polyadenylated GFP mRNA were added to each sample prior to RNA extraction. Oligo dT(18) primers were used to reverse transcribe polyadenylated transcripts, whereas random hexamers were used to reverse transcribe total transcripts. cDNA corresponding to 1/ 10th of an oocyte or embryo was used for real time analysis. Data were normalized relative to abundance of exogenous control (GFP) RNA and are shown as mean 1 SEM. Time points with a common superscript are not significantly different. 141 > Relative mRNA abundance 0.2 0.16 0.12 0.08 0.04 w .o u 4 Relative mRN A abundance O a a a a a a J I I I 0 ‘J l I I 16C Morula Blastocyst 16C Morula Blastocyst Stage 0f embryo development Stage of embryo development 142 Figure A.6. Quantitative real-time RT-PCR analysis of JY-l mRNA within in vitro derived embryos cultured with or without the RNA polymerase II inhibitor a-amanitin. (A) Experimental model utilized for a-amanitin treatment. Bovine embryos were cultured with a-amanitin (25 ug/ml) between 24 to 33 h or between 33 to 44 h post fertilization (during first and second bovine embryonic cell cycles). The 2-cell and 4-cell embryos from untreated control and a-amanitin treatment groups were collected at 33 and 44 h post fertilization respectively. (B) Effects of a-amanitin on blastocyst development. From each in vitro fertilization (IVF) run, a proportion of a—amanitin treated andfcontrol embryos were cultured until day 7 and percentage of embryos reaching the blastocyst stage recorded. None of the embryos treated with (IL-amanitin developed to the blastocyst stage. (C) Relative abundance of polyadenylated J Y-l mRNA transcripts in control and a-amanitin treated 2-cell and 4-cell embryos [n = 4 samples of 10 embryos each for 2-cell (2C) and 4-cell (4C) stage embryos; untreated control embryos [oi-amanitin (-)]; a- amanitin treated embryos [a amanitin (+)]. Samples were spiked with 250 femtograms of GFP RNA prior to RNA extraction. Oligo dT(18) primers were used in reverse transcription. cDNA corresponding to 1/10th of an embryo was used for red time analysis. Data are normalized relative to abundance of exogenous control (GFP) RNA and shown as mean i SEM. Means with common superscripts are not different. Results demonstrate abundance of J Y-l mRNA transcripts detected in 2-cell and 4-cell embryos is not affected by a-amanitin treatment suggesting that such transcripts are not synthesized in the embryos but rather are of oocyte origin. 143 A Experimental model a-amanitin treatment a-amanitin treatment -. 9 d -' 24 h 33 h I l l i Fertilization First Second embryonic embryonic cell cell cycle cycle B C [I a-amanitin (-) El u-amanitin (4% ° 0.3 - Blastocyst (%) g IVF Batches Control a—Amanitin E 1 26 0 3 0.2 ‘ a a a a 2 30 0 E 3 30 0 g 0.1 . 4 40 0 g 82 0 l 2C 4C Stage of embryogenesis 144 Figure A.7. Validation of oocyte/embryo microinjection procedure. Metaphase 11 stage bovine oocytes were denuded of cumulus cells by vortexing in 0.1% hyaluronidase enzyme in Hamster embryo culture medium. Each denuded oocyte was secured to the holding pipet with brief negative suction. The IC SI microinjection pipet was loaded with Texas-red Dextran dye by aspiration. During the microinjection procedure, the oocyte cytoplasm was briefly aspirated into the microinjection pipet with negative suction pressure to ensure the breakage of cytoplasmic membrane before dye injection. Microinjected oocytes were parthenogenetically activated by 4 min incubation in 5 uM ionomycin followed by 4 h incubation in 2 mM 6-dimethylaminopurine (6-DMAP) and then cultured for 7 days. Representative bright field micrographs taken at (A) 48 h and (C) day 7 of embryonic development. The corresponding dark field micrographs are presented in Panel B and D. The success rate of microinjection procedure was calculated based on total number of embryos retaining fluorescent dye at 48 hr after activation over total number of oocytes microinjected. Results demonstrate that the success rate of microinjection was greater than 90 %. 145 48h Day 7 Bright Field Dark Field 146 Figure A.8. Validation of JY-l siRNA species for efficacy of JY-l mRNA knockdown in samples of 4-cell embryos. Denuded metaphase II bovine oocytes were either microinjected with water or individual JY-l siRNA species at two different doses (25 uM and 50 pM). Microinjected oocytes were parthenogenetically activated by 4 min incubation in 5 uM ionomycin followed by 4 h incubation in 2 mM 6- dimethylaminopurine (6-DMAP) and 4-cell embryo samples were collected at 41-43 h after activation (11 = 4 samples of 5 embryos each) for RNA isolation and quantification of J Y-l mRNA abundance by real-time PCR. (A) Relative abundance of J Y-l transcripts in samples subjected to sham (water) or JY-l siRNA species-l injection. (B) Relative abundance of JY-l transcripts in samples subjected to sham (water) or JY-l siRNA species-2 injection. Oligo dT(18) primers were used to synthesize cDNA. Data were normalized relative to abundance of endogenous control 18S rRNA by the following formula: Z'MCT[269]. Results demonstrate that both siRNA species 1 and 2 (at 25 pM concentration) reduce J Y-l mRNA abundance by approximately 90 % in 4-cell embryos within 41-43 h after injection and parthenogenetic activation. 147 .2: 0m :m_om A 148 Figure A.9. Validation of JY-l siRNA species for efficacy of JY-l mRNA knockdown in samples of 2-cell embryos. Denuded metaphase II bovine oocytes were either microinjected with water or JY-l siRNA cocktail (25 pM; species 1 + 2). Microinjected oocytes were parthenogenetically activated by 4 min incubation in 5 uM ionomycin followed by 4 h incubation in 2 mM 6-dimethylaminopurine (6-DMAP) and 2-cell embryo samples were collected at 33 h after injection and parthenogenetic activation (n = 4 samples of 5 embryos each) for RNA isolation and cDNA synthesis. Relative abundance of J Y-l transcripts was quantified by real-time PCR analysis. Oligo dT(18) primers were used to synthesize cDNA. Data were normalized relative to abundance of endogenous control 18S rRNA by the following formula: Z'AACT [269]. Means without a common superscript are significantly different, P < 0.05. Results demonstrate that microinjection of the JY-l siRNA cocktail reduces JY-l mRNA abundance by approximately 90 % in 2-cell embryos within 33 h after activation. 149 QIY- 1 b “t r . Aobaoo . Basia: . A? JY-1—> 157 Figure A.13. Effect of J Y-l knockdown on bovine early embryonic development. Presumptive one cell bovine embryos derived from in vitro fertilization were subjected to one of the following treatments: 1) JY-l cocktail siRNA (25 pM), 2) negative control siRNA-l (25 pM), 3) sham water injection or 4) uninjected controls. Injected embryos were cultured for seven days and rates of blastocyst development recorded. Representative micrographs of day 7 embryos derived from (A) uninjected controls, (B) Sham water injection (C) negative control siRNA injection and (D) J Y-l siRNA cocktail injection are shown. The day 7 blastocysts are indicated by pointed arrows. Results demonstrate JY-l siRNA injection reduces the development of IVF embryos to the blastocyst stage. 158 159 Figure A.14. Genomic organization and characterization of putative cis-elements in the 5’-flanking region of the bovine J Y-l gene. (A) The JY-l gene has three exons separated by two introns. Exonic and intronic sequences at the exon-intron junctions are shown in upper-case and lower-case letters, respectively. The donor (gt) and acceptor (ag) splice sites corresponding to the first and last two bases of the intron, and are underlined. The donor (gt) and acceptor (ag) splice sites are in agreement with consensus sequences. (B) Putative cis-elements in the 5’ flanking region of the bovine J Y-l gene. A putative TATA box was detected in the 5’ flanking region and position of the cis-elements are depicted relative to the TATA box. Note five E-box motifs (canonical sequence: CANNTG) within 500 bp of the 5’-flanking region. E-box motifs have been identified in several genes with tissue-specific expression [270, 271] and they are key motifs necessary for oocyte-specific gene expression [121, 252]. 160 Exon Sizc lntron Size No. (bp) Sequence No. (kb) Sequence l 25 GCTT'I'ACAgIgagtactg I 12.8 kb gtactctcagAI I l IGGA 2 92 C CTTGAAGgIa gggataa 2 1.5 kb gtttttcca gTI‘CTTC AC 3 1400 B E-box-S E-box-4 E-box-3 E-box-Z E-box-l [CAGCTGl»—{CAGCTG?1{CAGCTG'l—lCAGCTGjl—{CACCTG'T I I fl I I - 388 —220 -212 -75 -12 -1 m l6l Figure A.15. Characterization of JY-l like sequences in the human genome. (A) Alignment of bovine JY-l cDNA with DNA fragments on the long arm of human chromosome 11 (11q14). The human genomic DNA database at NCBI was searched with the nucleotide sequence encoding for the longest bovine JY-l cDNA (1.5 kb). JY-l-like sequence was identified on human chromosome 11 with region of similarity corresponding to 187 bp of the protein coding region and 850 bp in the 3’UTR. (B) Alignment of bovine JY-l cDNA with a single human EST sequence derived from a Hembase [human erythroid precursor cell (adult stem cell)] cDNA library. The region of sequence similarity in the Hembase EST is 187 bp and corresponds to human chromosome 11 (11q14) (http://hembase.niddl_<.nih.gov). (C) Alignment of Hembase human EST with DNA fragment present on human chromosome 11. The human Hembase EST aligned to human chromosome 11 (100 % identity) identical to the locus where the sequence similar to bovine J Y-l is present. 162 A 1.5 kb - Bovine JY -1 cDNA —— Sequence identity (76%) -i.‘ :‘f Locus 1 Human genomic sequence (chromosome 11q14) B - 1.5 kb Bovine JY-l cDNA ; Sequence identity (75%) Hembase Human EST (chromosome 11q14.1) C — Hembase Human EST Sequence identity (100%) -h‘ a Human genomic sequence (chromosome 11q14) 163 Figure A.16. Characterization of J Y-l-like sequences in the genome of additional species. Genomic DNA databases at NCBI for chimpanzee, dog, mouse, rat, chicken, zebrafish and drosophila were searched with the nucleotide sequence of the 1.5 kb bovine JY-l cDNA. JY-l-like sequences were identified on chimpanzee chromosome 11, dog chromosome 21, mouse chromosome 7 and rat chromosome 1 (syntenic chromosomes to human chromosome 11). 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