BOR Papers in Press. Published on February 5, 2014 as DOI:10.1095/biolreprod.113.115071
Functional Role of the Bovine Oocyte-Specific Protein JY-1 in Meiotic Maturation, Cumulus Expansion, and Subsequent Embryonic Development1 Running title: JY-1 REGULATION OF OOCYTE COMPETENCE Kyung-Bon Lee3,5, Gabbine Wee3,5, Kun Zhang3,5, Joseph K. Folger3,5, Jason G. Knott4,5 and George W. Smith2,3,5,6 3
Laboratory of Mammalian Reproductive Biology and Genomics, Michigan State University, East Lansing, Michigan 4 Laboratory of Developmental Epigenetics, Michigan State University, East Lansing, Michigan 5 Department of Animal Science, Michigan State University, East Lansing, Michigan 6 Department of Physiology, Michigan State University, East Lansing, Michigan 1
Supported by a National Research Initiative Competitive Grant no. 2008-35203-19094 from the USDA National Institute of Food and Agriculture.
2
Correspondence: George W. Smith, 1230D Anthony Hall, Michigan State University, East Lansing, MI 48824. Tel: (517) 432-5401; Fax: (517) 353-1699, Email: [email protected]
ABSTRACT Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine embryogenesis. Given JY-1 is present in oocytes throughout folliculogenesis and oocyte-derived JY-1 mRNA is temporally regulated post fertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 siRNA was microinjected into cumulus-enclosed germinal vesicle stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation, was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion and embryonic development after in vitro fertilization. Depletion of JY-1 protein during in vitro maturation effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8-16 cell stage and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion and early cleavage, but did not rescue development to 8-16 cell and blastocyst stages. However, effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion and subsequent embryonic development. Summary sentence: The protein JY-1, which is specifically produced by bovine oocytes, is required for proper oocyte maturation, cumulus expansion and early embryogenesis. Keywords: JY-1, Oocyte, Cattle, Embryo, In Vitro Fertilization, Cumulus
Copyright 2014 by The Society for the Study of Reproduction.
INTRODUCTION Proper oocyte maturation is a prerequisite for successful early embryonic development [1, 2]. Oocyte maturation is commonly referred to in two distinct phases: 1) cytoplasmic maturation wherein the oocyte acquires the mRNAs and proteins necessary to foster nuclear maturation and subsequent embryonic development, and 2) nuclear maturation encompassing the first meiotic division and arrest of the oocyte at metaphase of the second meiotic division [3-5]. Given the minimal transcriptional activity characteristic of mature oocytes and early embryos prior to embryonic genome activation, maternal factors play a prominent active regulatory role in facilitating meiotic maturation, cumulus expansion, fertilization and the initial cleavage divisions [6-8]. However, the specific maternal (oocyte-derived factors) that promote nuclear maturation and early embryogenesis remain poorly described especially in domestic animals, including cattle. We have previously performed analysis of expressed sequence tags (ESTs) from a bovine oocyte cDNA library to identify novel oocyte-specific genes [9]. Of interest, several ESTs were identified encoding a novel oocyte-expressed transcript: JY-1. Subsequent studies demonstrated that JY-1 mRNA and protein are ovary-specific, detected throughout follicular development, and restricted exclusively to the oocyte [10]. Studies also suggest an important paracrine and autocrine regulatory role for JY-1. Recombinant JY-1 protein (rJY-1) is biologically active and JY-1 plus FSH treatment decreases granulosa cell numbers and estradiol production but stimulates progesterone production [10]. Abundance of JY-1 mRNA is temporally regulated during embryogenesis in a similar fashion to other maternal-effect genes and JY-1 is not transcribed in early embryos [10]. Functional assays revealed that injection of JY-1 siRNA into zygotes inhibits blastocyst formation with most JY-1 depleted embryos arrested prior to 8-16 cell stage, suggesting a requirement of JY-1 in early embryogenesis [10]. Recently, analysis of linkage disequilibrium also shows that multiple SNPs within the JY-1 gene are associated with reproductive traits, such as the occurrence of early pregnancy in cattle [11, 12]. However, the functional requirement of JY-1 for critical developmental events prior to fertilization has not been determined. Given JY-1 mRNA and protein are present in oocytes from the primordial through preovulatory stages of follicular development and oocyte-derived JY-1 mRNA is temporally regulated during initial cleavage divisions post fertilization [10], we hypothesized that JY-1 also plays a functional role in meiotic maturation and/or the initial cleavage divisions following fertilization in vitro. Thus, the objective of the present studies was to determine the functional role of JY-1 in regulation of meiotic maturation, cumulus expansion and subsequent embryonic development after in vitro fertilization.
MATERIALS AND METHODS Materials All chemicals and reagents used were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. In Vitro Maturation, Fertilization, and Embryo Culture In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed as reported previously with modifications [8]. Briefly, cumulus-oocyte-complexes (COCs) that have more than three layers of cumulus cells were collected from bovine ovaries that were obtained from a local slaughterhouse. COCs were cultured in four-well dishes (NUNC) containing maturation medium (Medium-199, 10% FBS (Gibco-BRL, Grand Island, NY), 1 IU/ml FSH, 5 IU/ml LH and 1 μg/ml estradiol-17β. To arrest meiotic maturation and allow sufficient time for JY-1 knockdown to occur, COCs were initially incubated for 48 h in maturation medium supplemented with 50 μM S-roscovitine (Calbiochem, La Jolla, CA) as reported previously [13]. Then COCs were washed several times with fresh maturation medium and incubated for 24 h in maturation medium minus S-roscovitine at 38.5°C 2
under 5% CO2 in humidified air. This 72 h interval is hereafter referred to as the oocyte culture period to describe window of meiotic maturation and prior 48 h preincubation with S-roscovitine used to facilitate JY-1 knockdown. Preliminary experiments verified efficacy of 48 h treatment with 50 μM S-roscovitine in inhibiting germinal vesicle (GV) breakdown relative to control oocytes cultured in the absence of Sroscovitine (n = 10 oocytes/treatment; n = 6 replicates). Matured COCs (50 COCs/well) were fertilized with spermatozoa purified from frozen-thawed semen by Percoll gradient. After 20 h at 38.5°C under 5% CO2 in humidified air, putative zygotes were stripped of cumulus cells by vortexing for 5 min and placed in potassium simplex optimization medium (KSOM; Specialty Media, Phillipsburg, NJ) supplemented with 0.3% BSA. Presumptive zygotes were cultured at 38.5°C under 5% CO2 in humidified air. At 72 h post insemination (hpi), 8-16 cell embryos were removed, washed and incubated in fresh KSOM with 0.3% BSA and 10% fetal bovine serum until d 7. Microinjection of siRNA into Cumulus Enclosed Bovine Oocytes and Validation of Oocyte JY-1 Knockdown Previously validated [10] JY-1 siRNA cocktail (species 1 and 2, 25 μM concentration) was microinjected into cumulus-enclosed GV stage oocytes using an inverted Nikon microscope (Mager Scientific, Dexter, MI) equipped with a micromanipulator system (Narishige International, Long Island, NY). Control oocytes were uninjected, injected with similar volume of water or injected with 25 M negative control siRNA (nonspecific; Ambion universal control #1). Validation of JY-1 knockdown specificity using this negative control siRNA was reported previously [10]. After injection, all oocytes were cultured in the presence of 50 μM S-roscovitine for 48 h and then subjected to meiotic maturation as described above. Efficiency of COC microinjection was validated in preliminary studies by microinjection of Texas Red dye coupled to Dextran and examination using fluorescent microscope. Efficiency of JY-1 siRNA in ablation of JY-1 mRNA and protein was determined by real-time PCR (n = 6 pools of 10 oocytes per treatment) and Western blot analysis (n = 50 oocytes per treatment; n = 3 replicates) of denuded oocytes collected 72 h after siRNA microinjection. Effect of Oocyte JY-1 Ablation on Nuclear Maturation and Cumulus Expansion Microinjection of JY-1 siRNA and controls were performed as described above (n = 30 oocytes injected per treatment; n = 6 replicates). At the end of oocyte maturation, COCs were classified into one of three categories based on degree of cumulus expansion (non expanded, partially expanded and fully expanded). Then oocytes were removed of surrounding cumulus cells and treated with 4 μg/ml Hoechst 33342 dye for 20 min. Denuded oocytes were washed, placed on a microscope slide and covered lightly with a cover glass. Meiotic phase (metaphase I, anaphase I, telophase I and metaphase II) of each oocyte was determined under a fluorescent microscope (Mager Scientific, Dexter, MI). Effect of Oocyte JY-1 Ablation on Subsequent Embryonic Development JY-1 siRNA-injected and control COCs were subjected to IVM, IVF, and IVC as described above (n = 20 oocytes/treatment; n = 6 replicates). Effects of oocyte JY-1 ablation on early cleavage, total cleavage rates, development to 8-16 cell and blastocyst stages were determined at 30 h, 48 h, 72 h and 7 d post insemination, respectively. Effect of JY-1 Ablation Pre- Versus Post-Fertilization and rJY-1 Protein Replacement To determine if effects of oocyte JY-1 ablation can be reversed following replacement with rJY-1 protein, cumulus-enclosed GV stage oocytes were microinjected as described above with JY-1 siRNA, universal negative control siRNA or served as uninjected controls. JY-1 siRNA-injected oocytes were cultured for 72 h as described above (48 h culture in 50 μM S-roscovitine followed by IVM) in the presence of increasing concentrations of rJY-1 protein (0, 0.1, 0.5. 1 or 10 ng/ml). Effects of treatment on progression to metaphase II were then determined as described above (n = 10 oocytes/treatment; n = 6 replicates) and the optimal dose for rJY-1 replacement determined. Additional cumulus-enclosed JY-1 siRNA injected, universal negative control siRNA-injected and uninjected oocytes were subjected to 72 h 3
oocyte culture in the presence or absence of 1 ng/ml rJY-1 protein followed by IVF and IVC in the absence of rJY-1 and effects of treatments on early cleavage, total cleavage rates, development to 8-16 cell and blastocyst stages were determined (n = 20 oocytes/treatment; n = 5 replicates). For JY-1 ablation post fertilization, presumptive zygotes (16-18 hpi) were microinjected with JY-1 siRNA, universal negative control siRNA or were used as uninjected controls (n = 25-30 embryos per replicate; n = 4 replicates) as described previously [10], and JY-1 siRNA injected embryos cultured in the presence or absence of rJY-1 protein (1 ng/ml) for 72 h, then in the absence of rJY-1 protein until 7 d post insemination. Effects of treatments on development to 8-16 cell and blastocyst stages were determined as described above. Real-Time PCR Total RNA isolation, cDNA synthesis, and real-time PCR were performed as described previously [8]. The primer sequences used were reported before [10]. Relative amount of JY-1 mRNA was calculated using the formula 2−ΔΔCT. JY-1 mRNA abundance was normalized to endogenous control (RPS18). Statistical Analysis Data were analyzed using one-way ANOVA. All percentage data were subjected to arc-sin transformation before analysis. Differences in treatment means were compared by using Tukey’s HSD test.
RESULTS Efficient Depletion of JY-1 by Microinjection of siRNA into Immature Oocytes Microinjection of siRNA designed against specific transcripts has been widely used as a robust approach to silence genes of interest in oocytes/early embryos [7, 10, 14, 15]. To test the effect of depletion of JY-1 in cumulus-enclosed oocytes on oocyte maturation and subsequent early embryonic development following IVF, we first established procedures for siRNA-mediated ablation of gene expression in bovine oocytes using JY-1 siRNA that was previously validated for specificity and efficacy of JY-1 ablation in bovine early embryos [10]. In order to allow efficient siRNA-mediated reduction of JY-1 mRNA and protein, siRNA-injected oocytes were pharmacologically arrested at GV stage for 48 h prior to initiation of IVM. Culture of oocytes for 48 h in the presence of S-roscovitine resulted in maintenance of GV arrest in 92% of oocytes relative to control (untreated oocytes) which all underwent spontaneous GV breakdown. As depicted in Figure 1A, microinjection of JY-1 siRNA greatly reduced endogenous JY-1 mRNA abundance by > 98% in bovine oocytes collected at 72 h post injection relative to uninjected and sham-injected oocytes (P < 0.01). Furthermore, JY-1 protein was decreased by ~48 % in JY-1 siRNAinjected oocytes compared with controls (P < 0.05; Figure 1B and 1C). JY-1 Deficiency Compromised Maturation of Bovine COCs and Inhibited Subsequent Early Embryonic Development To pinpoint the functional role of maternal JY-1 in oocyte maturation, effects of oocyte JY-1 ablation on meiotic maturation and cumulus expansion were examined. Microinjection of JY-1 siRNA reduced the proportion of oocytes progressing to metaphase II stage compared to uninjected and sham-injected oocytes (P < 0.05; Figure 2). In contrast, further analysis of the nuclear stage of JY-1 siRNA-injected and control oocytes showed that increased numbers of JY-1 siRNA oocytes were arrested between metaphase I and telophase I stages (Figure 2). JY-1 siRNA injection also greatly decreased the percentage of oocytes with a fully expanded cumulus layer after 24 h IVM compared with uninjected and sham-injected oocytes (P < 0.05; Figure 3). It is well established that oocyte maturation is critical for subsequent embryonic development after fertilization. However, progression to metaphase II stage was not totally blocked for JY-1 siRNA-injected embryos. Therefore, we hypothesized that JY-1 deficiency in bovine oocytes also retards early embryonic 4
development after IVF. Thus, JY-1 siRNA-injected and control oocytes were fertilized with bovine sperm and early cleavage, total cleavage as well as blastocyst formation examined. After fertilization, a pronounced decrease in the percentage of embryos that cleaved early (within 30 hpi) was noted for JY-1 siRNA-injected groups relative to uninjected and sham control oocytes (P< 0.05; Table 1). Similarly, total cleavage rate (48 hpi) was also dramatically reduced in response to JY-1 siRNA injection compared with uninjected and sham injection (P < 0.05; Table 1). In addition, JY-1 knockdown decreased the proportion of embryos developing to 8- to 16-cell stage (72 hpi) and blastocyst stage (d 7) relative to uninjected and sham control groups (P < 0.05, Table 1). A decrease was also noted when rates of development to 8- to 16-cell stage and blastocyst stage were calculated as a proportion of cleaved embryos (P < 0.05; Table 1). Evidence Supporting the Specificity of Effects of JY-1 siRNA on Oocyte Maturation and Developmental Competence To further ensure the specificity of JY-1 siRNA in preventing oocyte maturation and early embryonic development after fertilization, JY-1 siRNA-injected oocytes were treated with increasing concentrations of exogenous rJY-1 protein during 72 h oocyte culture prior to IVF. A dose-responsive beneficial effect of rJY-1 on the proportion of oocytes progressing to metaphase II stage was noted for JY-1 siRNAinjected oocytes. The proportion of JY-1 siRNA-injected oocytes progressing to metaphase II was rescued to levels comparable with both controls when exposed to 0.5-1 ng/ml rJY-1 protein (Figure 4A). Therefore, 1 ng/ml was used in subsequent experiments to determine effects of rJY-1 replacement on development of embryos produced from JY-1-deficient oocytes. As illustrated in Figure 4B-D, exposure to rJY-1 protein during oocyte culture totally rescued rates of early cleavage and total cleavage and partially rescued development to 8- to 16-cell stage following IVF for JY-1 siRNA-injected oocytes when compared to uninjected and negative control siRNA-injected embryos. However, rJY-1 supplementation of JY-1 siRNA- injected oocytes during oocyte culture did not rescue development to blastocyst stage following IVF. In contrast, supplementation of JY-1 siRNA-injected zygotes with rJY-1 protein during first 72 h of embryo culture totally rescued negative effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages (Figure 5A and B).
DISCUSSION In the present studies, we demonstrated for the first time that oocyte-expressed JY-1 is required for nuclear maturation and cumulus expansion with implications for subsequent embryonic development. In previous studies, we characterized the functional role of JY-1 in early embryogenesis by microinjecting JY-1 siRNA into presumptive zygotes, which can efficiently silence maternal JY-1 mRNA. Such studies clearly demonstrated a functional role for oocyte-derived JY-1 post fertilization in promoting early embryogenesis [10]. However, the specific regulatory role of maternal JY-1 in oocyte maturation and cumulus expansion was previously unclear given the timing of JY-1 depletion. A number of oocyteexpressed genes that are essential for follicular development, oocyte maturation or early embryonic development have been identified in mice, especially through gene targeting studies [16-20]. However, to our knowledge, a functional requirement role for a single oocyte-expressed gene in promoting meiotic maturation and cumulus expansion pre-fertilization, coupled with an additional requirement of the same gene post-fertilization for normal early embryogenesis, has not been demonstrated previously. Demonstration of a functional role for JY-1 pre-fertilization was dependent upon establishment of a viable model system capable of supporting JY-1 ablation prior to initiation of oocyte maturation. Previous studies demonstrated that culture of bovine oocytes in the presence of 50 M S-roscovitine for 48 h can maintain an intact GV in over 95% of oocytes [13]. Thus, to specifically knockdown maternal JY-1 mRNA and protein, we established a novel RNAi system in bovine oocytes via microinjection of JY-1 siRNA into cumulus enclosed GV stage oocytes followed by meiotic arrest for 48 h (to allow siRNAmediated reduction of mRNA and accompanying reduction/ lack of translation of JY-1 protein from 5
mRNA stores) before IVM. Maintenance of the cumulus layer after microinjection is critical to success of functional studies given cumulus cells are needed for optimal IVF [21] and embryo development [22] in cattle. Our results demonstrated that this strategy can efficiently maintain meiotic arrest and attain effective ablation of endogenous JY-1 mRNA and protein by 72 h of oocyte culture. Such approach should be of further utility to investigate the functional role of additional oocyte- expressed genes in meiotic maturation, cumulus expansion and subsequent early embryogenesis. Results of the present studies suggest oocyte-expressed JY-1 is required for at least two important aspects of oocyte maturation: nuclear maturation and cumulus expansion. It is well documented that nuclear/meiotic maturation is required for normal development, including fertilization and early embryogenesis [23-25]. Cumulus expansion is essential for normal ovulation and fertilization [3, 26]. Both are triggered by the LH surge in vivo and induced by the presence of FSH or EGF in maturation medium in vitro [27]. Oocyte-secreted factors play an active role in regulating the process of cumulus expansion [28]. It is currently unclear whether maturation- and cumulus expansion-promoting effects of JY-1 are mediated directly or indirectly via regulation of intrafollicular paracrine/autocrine signaling networks, which are potentially linked to LH-induced meiotic maturation and cumulus expansion in vivo [29, 30]. Based on results of current studies and previous results [10], we propose that JY-1 functions as an important paracrine and autocrine regulator of key aspects of follicular development and early embryogenesis in cattle. The ability of JY-1 protein to influence granulosa cell steroidogenesis when added to culture media has been described previously [10]. Furthermore, the predicted amino acid sequence of JY-1 contains a putative signal peptide [10], suggesting the JY-1 protein is secreted from the oocyte. We also showed in the current studies that the observed effects of oocyte JY-1 depletion on cumulus expansion and meiotic maturation, and of zygote JY-1 depletion on embryonic development, can be rescued by addition of rJY-1 protein during oocyte and embryo culture, respectively. Hence, results suggest that JY-1 is secreted and acts in a paracrine and autocrine fashion to regulate oocyte maturation, cumulus expansion and early embryonic development in vitro. Whether such effects of JY-1 are receptormediated and identification of potential signaling pathways involved will require further investigation. A number of studies in humans have suggested early cleavage rate is a reliable predictor of early embryonic developmental competence [31, 32]. Our previous studies also indicate early-cleaving bovine embryos have a greater capability to develop to blastocyst stage than late-cleaving counterparts [33, 34]. In the current studies, the rates of early cleavage and total cleavage were also reduced following IVF of JY-1-depleted oocytes. However, approximately 40% of JY-1-depleted oocytes were successfully fertilized and underwent the first cleavage division, but < 2% of cleaved embryos derived from JY-1depleted oocytes developed to the blastocyst stage. Progression to metaphase II, early cleavage rates and total cleavage rates following IVF for JY-1-depleted oocytes were fully rescued by addition of rJY-1 protein during oocyte culture (prior to fertilization) but development to 8- to 16-cell stage was only partially rescued and blastocyst rates were not impacted. Hence, these results support an important functional role for JY-1 protein prefertilization but indicate that rescue of JY-1-depleted oocytes with rJY-1 protein during oocyte culture alone is not sufficient to restore normal rates of blastocyst development following IVF. However, supplementation of JY-1-depleted zygotes with rJY-1 protein during initial 72 h of embryo culture totally restored development to the blastocyst stage. Collectively, such results further support a functional role for JY-1 protein both pre- and post- fertilization. In summary, the present studies strongly support a functional role of oocyte-expressed JY-1 in meiotic maturation and cumulus expansion and further support a role for JY-1 in promoting embryonic development to the blastocyst stage. Sirard et al. defined oocyte developmental competence as the capacity of the oocyte to resume meiosis, cleave after fertilization, help promote embryonic development and implantation, and bring a pregnancy to term in good health [1]. Hence, results of the current and 6
previous studies suggest a requirement of JY-1 for bovine oocyte competence and provide clear implications for understanding the genetic and physiological regulation of follicular development and early embryogenesis in cattle. Recent identification of genetic polymorphisms of the JY-1 gene associated with fertility traits in cattle [11, 12] supports potential relevance of the JY-1 gene to fertility in vivo. Further insight into regulation of JY-1 expression/activity and function has broad general implications for understanding regulation of follicular development, oocyte maturation and early embryonic development and potential improvements in assisted reproductive technologies in cattle.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo quality. Theriogenology 2006; 65:126136. Rodriguez KF, Farin CE. Gene transcription and regulation of oocyte maturation. Reproduction, Fertility, and Development 2004; 16:55-67. Edson MA, Nagaraja AK, Matzuk MM. The Mammalian Ovary from Genesis to Revelation. Endocrine Reviews 2009; 30:624-712. Eppig JJ, Schultz RM, O'Brien M, Chesnel F. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Developmental Biology 1994; 164:1-9. Watson AJ. Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo developmental competence. J Anim Sci 2007; 85:E1-3. Li L, Zheng P, Dean J. Maternal control of early mouse development. Development 2010; 137:859-870. Tripurani SK, Lee KB, Wang L, Wee G, Smith GW, Lee YS, Latham KE, Yao JB. A Novel Functional Role for the OocyteSpecific Transcription Factor Newborn Ovary Homeobox (NOBOX) during Early Embryonic Development in Cattle. Endocrinology 2011; 152:1013-1023. Lee KB, Bettegowda A, Wee G, Ireland JJ, Smith GW. Molecular Determinants of Oocyte Competence: Potential Functional Role for Maternal (Oocyte-Derived) Follistatin in Promoting Bovine Early Embryogenesis. Endocrinology 2009; 150:2463-2471. Yao JB, Ren XN, Ireland JJ, Coussens PM, Smith TPL, Smith GW. Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis. Physiological Genomics 2004; 19:84-92. Bettegowda A, Yao J, Sen A, Li Q, Lee KB, Kobayashi Y, Patel OV, Coussens PM, Ireland JJ, Smith GW. JY-1, an oocytespecific gene, regulates granulosa cell function and early embryonic development in cattle. Proceedings of the National Academy of Sciences of the United States of America 2007; 104:17602-17607. de Camargo GrMF, Costa RB, de Albuquerque LGo, Regitano LCdA, Baldi F, Tonhati H. Association between JY-1 gene polymorphisms and reproductive traits in beef cattle. Gene 2014; 533:477-480. de Camargo G, Baldi F, Regitano L, Tonhati H. Characterization of the Exonic Regions of the JY-1 Gene in Zebu Cattle and Buffaloes. Reproduction in Domestic Animals 2013; 48:918-922. Coy P, Romar R, Payton RR, McCann L, Saxton AM, Edwards JL. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro. Reproduction 2005; 129:19-26. Choi I, Carey TS, Wilson CA, Knott JG. Transcription factor AP-2gamma is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis. Development 2012. Lin CJ, Conti M, Ramalho-Santos M. Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development. Development 2013; 140:3624-3634. Wu X, Viveiros MM, Eppig JJ, Bai Y, Fitzpatrick SL, Matzuk MM. Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nature Genetics 2003; 33:187-191. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean J, Nelson LM. Mater, a maternal effect gene required for early embryonic development in mice. Nature Genetics 2000; 26:267-268. Zheng P, Dean J. Role of Filia, a maternal effect gene, in maintaining euploidy during cleavage-stage mouse embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 2009; 106:7473-7478. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 1996; 383:531-535. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Molecular Endocrinology 2001; 15:854-866. Shioya Y, Kuwayama M, Fukushima M, Iwasaki S, Hanada A. In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro. Theriogenology 1988; 30:489-496. Thomas WK, Seidel GE, Jr. Effects of cumulus cells on culture of bovine embryos derived from oocytes matured and fertilized in vitro. J Anim Sci 1993; 71:2506-2510.
7
23. Tian XC, Lonergan P, Jeong BS, Evans AC, Yang X. Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes. Molecular Reproduction and Development 2002; 62:132-138. 24. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biology of Reproduction 2004; 70:535-547. 25. Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent. Development 1995; 121:3259-3266. 26. Chen L, Russell PT, Larsen WJ. Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass. Molecular Reproduction and Development 1993; 34:87-93. 27. Conti M, Hsieh M, Park JY, Su YQ. Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 2006; 20:715-723. 28. Su YQ, Sugiura K, Eppig JJ. Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism. Seminars in Reproductive Medicine 2009; 27:32-42. 29. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 2004; 303:682-684. 30. Hsieh M, Lee D, Panigone S, Horner K, Chen R, Theologis A, Lee DC, Threadgill DW, Conti M. Luteinizing hormonedependent activation of the epidermal growth factor network is essential for ovulation. Mol Cell Biol 2007; 27:1914-1924. 31. Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Human Reproduction 1998; 13:182-187. 32. Shoukir Y, Chardonnens D, Campana A, Bischof P, Sakkas D. The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Human Reproduction 1998; 13:676-681. 33. Patel OV, Bettegowda A, Ireland JJ, Coussens PM, Lonergan P, Smith GW. Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction 2007; 133:95-106. 34. Bettegowda A, Lee KB, Smith GW. Cytoplasmic and nuclear determinants of the maternal-to-embryonic transition. Reprod Fertil Dev 2008; 20:45-53.
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FIGURE LEGENDS Figure 1. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on JY-1 mRNA and protein levels following in vitro maturation. (A) Quantitative realtime RT-PCR analysis of JY-1 mRNA transcript abundance in uninjected, sham injected and JY-1 siRNAinjected oocytes (n = 6 pools of 10 oocytes each per treatment) collected 72 h post injection. Data were normalized relative to abundance of endogenous control (RPS18) and expressed as mean ± SEM. (a,b; P < 0.05). (B) Representative western blot images of JY-1 protein abundance in uninjected, sham injected and JY-1 siRNA-injected oocytes (n = 50 oocytes per lane; n = 3 replicates) collected 72 h post injection. (C) Densitometric analysis of relative abundance of JY-1 protein in uninjected, sham injected and JY-1 siRNA-injected oocytes. JY-1 protein abundance was normalized relative to actin and expressed as mean ± SEM. Different letters denote statistical significance (a,b; P < 0.05). Figure 2. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on extent of meiotic progression during in vitro maturation. Proportion of oocytes progressing to metaphase I (black bars), anaphase I (white bars) , telophase I (hatched bars) and metaphase II (shaded bars) following 24 h in vitro maturation for uninjected, sham injected and JY-1 siRNA-injected oocytes (n = 30 oocytes per treatment; n = 6 replicates). Oocytes were stained with Hoechst-33342 and nuclear stage determined under fluorescent microscope. Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; x,y; A,B; X,Y; P < 0.05). Figure 3. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on expansion of the cumulus cell layer during in vitro maturation. Degree of cumulus expansion [non expanded (black bars), partially expanded (white bars), fully expanded (hatched bars)] for individual JY-1 siRNA injected, sham injected and uninjected oocytes (n = 30 oocytes per treatment; n = 6 replicates) were determined at 72 h post injection. Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; x,y; A,B; P < 0.05). Figure 4. Effect of supplementation with recombinant JY-1 protein during oocyte culture on JY-1 siRNA-induced reduction in nuclear maturation and embryonic development following in vitro fertilization. Cumulus enclosed germinal vesicle stage oocytes were microinjected with JY-1 siRNA, with negative control siRNA or served as uninjected controls. JY-1 siRNA-injected oocytes were cultured in presence of increasing concentrations of recombinant JY-1 protein (0, 0.1, 0.5, 1, 10 ng/ml) for 72 h and (A) proportion of oocytes progressing to metaphase II were determined (n = 10 oocytes per treatment; n = 6 replicates). After fertilization, proportion of oocytes undergoing (B) early cleavage (30 h post insemination; hpi), (C) total cleavage rates (48 hpi), and (D) percent development to 8- to 16-cell stage (72 hpi) and (E) blastocyst stage (7 d post insemination) were determined for each treatment group (n = 20 oocytes per treatment; n = 5 replicates). Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b,c; P < 0.05). Figure 5. Effect of zygote JY-1 depletion and supplementation with recombinant JY-1 protein on embryonic development following in vitro fertilization. Presumptive zygotes were injected with JY-1 siRNA, with negative control siRNA or served as uninjected controls. JY-1 siRNA injected and uninjected zygotes were cultured for 72 h in presence or absence of 1 ng/ml recombinant JY-1 protein and effects of treatments on rates of development to (A) 8- to 16-cell stage (72 h post insemination) and (B) blastocyst stage (7 d post insemination) were determined for each treatment group (n = 20 zygotes per treatment; n = 5 replicates). Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; P < 0.05).
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TABLE 1. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on embryonic development (%) following in vitro fertilization.* Stage Uninjected Sham JY-1 siRNA Early cleavage 17.5 ± 1.1a 18.3 ± 1.1a 7.5 ± 1.1b a a Total cleavage 77.5 ± 2.1 76.7 ± 1.1 40.8 ± 3.0b a a 47.5 ± 1.1 12.5 ± 2.1b 8-16 cells 49.2 ± 1.5 a a Blastocyst 20.0 ± 1.3 18.3 ± 1.7 0.8 ± 0.8b a a 8-16 cells/cleaved embryo 63.5 ± 1.6 62.0 ± 1.7 31.3 ± 5.7b a a Blastocysts/cleaved embryo 26.1 ± 2.3 24.0 ± 2.3 1.7 ± 1.7b *Different superscript letters within a row indicate differences between treatments (P < 0.05).
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Functional Role of the Bovine Oocyte-Specific Protein JY-1 in Meiotic Maturation, Cumulus Expansion, and Subsequent Embryonic Development1 Running title: JY-1 REGULATION OF OOCYTE COMPETENCE Kyung-Bon Lee3,5, Gabbine Wee3,5, Kun Zhang3,5, Joseph K. Folger3,5, Jason G. Knott4,5 and George W. Smith2,3,5,6 3
Laboratory of Mammalian Reproductive Biology and Genomics, Michigan State University, East Lansing, Michigan 4 Laboratory of Developmental Epigenetics, Michigan State University, East Lansing, Michigan 5 Department of Animal Science, Michigan State University, East Lansing, Michigan 6 Department of Physiology, Michigan State University, East Lansing, Michigan 1
Supported by a National Research Initiative Competitive Grant no. 2008-35203-19094 from the USDA National Institute of Food and Agriculture.
2
Correspondence: George W. Smith, 1230D Anthony Hall, Michigan State University, East Lansing, MI 48824. Tel: (517) 432-5401; Fax: (517) 353-1699, Email: [email protected]
ABSTRACT Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine embryogenesis. Given JY-1 is present in oocytes throughout folliculogenesis and oocyte-derived JY-1 mRNA is temporally regulated post fertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 siRNA was microinjected into cumulus-enclosed germinal vesicle stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation, was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion and embryonic development after in vitro fertilization. Depletion of JY-1 protein during in vitro maturation effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8-16 cell stage and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion and early cleavage, but did not rescue development to 8-16 cell and blastocyst stages. However, effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion and subsequent embryonic development. Summary sentence: The protein JY-1, which is specifically produced by bovine oocytes, is required for proper oocyte maturation, cumulus expansion and early embryogenesis. Keywords: JY-1, Oocyte, Cattle, Embryo, In Vitro Fertilization, Cumulus
Copyright 2014 by The Society for the Study of Reproduction.
INTRODUCTION Proper oocyte maturation is a prerequisite for successful early embryonic development [1, 2]. Oocyte maturation is commonly referred to in two distinct phases: 1) cytoplasmic maturation wherein the oocyte acquires the mRNAs and proteins necessary to foster nuclear maturation and subsequent embryonic development, and 2) nuclear maturation encompassing the first meiotic division and arrest of the oocyte at metaphase of the second meiotic division [3-5]. Given the minimal transcriptional activity characteristic of mature oocytes and early embryos prior to embryonic genome activation, maternal factors play a prominent active regulatory role in facilitating meiotic maturation, cumulus expansion, fertilization and the initial cleavage divisions [6-8]. However, the specific maternal (oocyte-derived factors) that promote nuclear maturation and early embryogenesis remain poorly described especially in domestic animals, including cattle. We have previously performed analysis of expressed sequence tags (ESTs) from a bovine oocyte cDNA library to identify novel oocyte-specific genes [9]. Of interest, several ESTs were identified encoding a novel oocyte-expressed transcript: JY-1. Subsequent studies demonstrated that JY-1 mRNA and protein are ovary-specific, detected throughout follicular development, and restricted exclusively to the oocyte [10]. Studies also suggest an important paracrine and autocrine regulatory role for JY-1. Recombinant JY-1 protein (rJY-1) is biologically active and JY-1 plus FSH treatment decreases granulosa cell numbers and estradiol production but stimulates progesterone production [10]. Abundance of JY-1 mRNA is temporally regulated during embryogenesis in a similar fashion to other maternal-effect genes and JY-1 is not transcribed in early embryos [10]. Functional assays revealed that injection of JY-1 siRNA into zygotes inhibits blastocyst formation with most JY-1 depleted embryos arrested prior to 8-16 cell stage, suggesting a requirement of JY-1 in early embryogenesis [10]. Recently, analysis of linkage disequilibrium also shows that multiple SNPs within the JY-1 gene are associated with reproductive traits, such as the occurrence of early pregnancy in cattle [11, 12]. However, the functional requirement of JY-1 for critical developmental events prior to fertilization has not been determined. Given JY-1 mRNA and protein are present in oocytes from the primordial through preovulatory stages of follicular development and oocyte-derived JY-1 mRNA is temporally regulated during initial cleavage divisions post fertilization [10], we hypothesized that JY-1 also plays a functional role in meiotic maturation and/or the initial cleavage divisions following fertilization in vitro. Thus, the objective of the present studies was to determine the functional role of JY-1 in regulation of meiotic maturation, cumulus expansion and subsequent embryonic development after in vitro fertilization.
MATERIALS AND METHODS Materials All chemicals and reagents used were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. In Vitro Maturation, Fertilization, and Embryo Culture In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed as reported previously with modifications [8]. Briefly, cumulus-oocyte-complexes (COCs) that have more than three layers of cumulus cells were collected from bovine ovaries that were obtained from a local slaughterhouse. COCs were cultured in four-well dishes (NUNC) containing maturation medium (Medium-199, 10% FBS (Gibco-BRL, Grand Island, NY), 1 IU/ml FSH, 5 IU/ml LH and 1 μg/ml estradiol-17β. To arrest meiotic maturation and allow sufficient time for JY-1 knockdown to occur, COCs were initially incubated for 48 h in maturation medium supplemented with 50 μM S-roscovitine (Calbiochem, La Jolla, CA) as reported previously [13]. Then COCs were washed several times with fresh maturation medium and incubated for 24 h in maturation medium minus S-roscovitine at 38.5°C 2
under 5% CO2 in humidified air. This 72 h interval is hereafter referred to as the oocyte culture period to describe window of meiotic maturation and prior 48 h preincubation with S-roscovitine used to facilitate JY-1 knockdown. Preliminary experiments verified efficacy of 48 h treatment with 50 μM S-roscovitine in inhibiting germinal vesicle (GV) breakdown relative to control oocytes cultured in the absence of Sroscovitine (n = 10 oocytes/treatment; n = 6 replicates). Matured COCs (50 COCs/well) were fertilized with spermatozoa purified from frozen-thawed semen by Percoll gradient. After 20 h at 38.5°C under 5% CO2 in humidified air, putative zygotes were stripped of cumulus cells by vortexing for 5 min and placed in potassium simplex optimization medium (KSOM; Specialty Media, Phillipsburg, NJ) supplemented with 0.3% BSA. Presumptive zygotes were cultured at 38.5°C under 5% CO2 in humidified air. At 72 h post insemination (hpi), 8-16 cell embryos were removed, washed and incubated in fresh KSOM with 0.3% BSA and 10% fetal bovine serum until d 7. Microinjection of siRNA into Cumulus Enclosed Bovine Oocytes and Validation of Oocyte JY-1 Knockdown Previously validated [10] JY-1 siRNA cocktail (species 1 and 2, 25 μM concentration) was microinjected into cumulus-enclosed GV stage oocytes using an inverted Nikon microscope (Mager Scientific, Dexter, MI) equipped with a micromanipulator system (Narishige International, Long Island, NY). Control oocytes were uninjected, injected with similar volume of water or injected with 25 M negative control siRNA (nonspecific; Ambion universal control #1). Validation of JY-1 knockdown specificity using this negative control siRNA was reported previously [10]. After injection, all oocytes were cultured in the presence of 50 μM S-roscovitine for 48 h and then subjected to meiotic maturation as described above. Efficiency of COC microinjection was validated in preliminary studies by microinjection of Texas Red dye coupled to Dextran and examination using fluorescent microscope. Efficiency of JY-1 siRNA in ablation of JY-1 mRNA and protein was determined by real-time PCR (n = 6 pools of 10 oocytes per treatment) and Western blot analysis (n = 50 oocytes per treatment; n = 3 replicates) of denuded oocytes collected 72 h after siRNA microinjection. Effect of Oocyte JY-1 Ablation on Nuclear Maturation and Cumulus Expansion Microinjection of JY-1 siRNA and controls were performed as described above (n = 30 oocytes injected per treatment; n = 6 replicates). At the end of oocyte maturation, COCs were classified into one of three categories based on degree of cumulus expansion (non expanded, partially expanded and fully expanded). Then oocytes were removed of surrounding cumulus cells and treated with 4 μg/ml Hoechst 33342 dye for 20 min. Denuded oocytes were washed, placed on a microscope slide and covered lightly with a cover glass. Meiotic phase (metaphase I, anaphase I, telophase I and metaphase II) of each oocyte was determined under a fluorescent microscope (Mager Scientific, Dexter, MI). Effect of Oocyte JY-1 Ablation on Subsequent Embryonic Development JY-1 siRNA-injected and control COCs were subjected to IVM, IVF, and IVC as described above (n = 20 oocytes/treatment; n = 6 replicates). Effects of oocyte JY-1 ablation on early cleavage, total cleavage rates, development to 8-16 cell and blastocyst stages were determined at 30 h, 48 h, 72 h and 7 d post insemination, respectively. Effect of JY-1 Ablation Pre- Versus Post-Fertilization and rJY-1 Protein Replacement To determine if effects of oocyte JY-1 ablation can be reversed following replacement with rJY-1 protein, cumulus-enclosed GV stage oocytes were microinjected as described above with JY-1 siRNA, universal negative control siRNA or served as uninjected controls. JY-1 siRNA-injected oocytes were cultured for 72 h as described above (48 h culture in 50 μM S-roscovitine followed by IVM) in the presence of increasing concentrations of rJY-1 protein (0, 0.1, 0.5. 1 or 10 ng/ml). Effects of treatment on progression to metaphase II were then determined as described above (n = 10 oocytes/treatment; n = 6 replicates) and the optimal dose for rJY-1 replacement determined. Additional cumulus-enclosed JY-1 siRNA injected, universal negative control siRNA-injected and uninjected oocytes were subjected to 72 h 3
oocyte culture in the presence or absence of 1 ng/ml rJY-1 protein followed by IVF and IVC in the absence of rJY-1 and effects of treatments on early cleavage, total cleavage rates, development to 8-16 cell and blastocyst stages were determined (n = 20 oocytes/treatment; n = 5 replicates). For JY-1 ablation post fertilization, presumptive zygotes (16-18 hpi) were microinjected with JY-1 siRNA, universal negative control siRNA or were used as uninjected controls (n = 25-30 embryos per replicate; n = 4 replicates) as described previously [10], and JY-1 siRNA injected embryos cultured in the presence or absence of rJY-1 protein (1 ng/ml) for 72 h, then in the absence of rJY-1 protein until 7 d post insemination. Effects of treatments on development to 8-16 cell and blastocyst stages were determined as described above. Real-Time PCR Total RNA isolation, cDNA synthesis, and real-time PCR were performed as described previously [8]. The primer sequences used were reported before [10]. Relative amount of JY-1 mRNA was calculated using the formula 2−ΔΔCT. JY-1 mRNA abundance was normalized to endogenous control (RPS18). Statistical Analysis Data were analyzed using one-way ANOVA. All percentage data were subjected to arc-sin transformation before analysis. Differences in treatment means were compared by using Tukey’s HSD test.
RESULTS Efficient Depletion of JY-1 by Microinjection of siRNA into Immature Oocytes Microinjection of siRNA designed against specific transcripts has been widely used as a robust approach to silence genes of interest in oocytes/early embryos [7, 10, 14, 15]. To test the effect of depletion of JY-1 in cumulus-enclosed oocytes on oocyte maturation and subsequent early embryonic development following IVF, we first established procedures for siRNA-mediated ablation of gene expression in bovine oocytes using JY-1 siRNA that was previously validated for specificity and efficacy of JY-1 ablation in bovine early embryos [10]. In order to allow efficient siRNA-mediated reduction of JY-1 mRNA and protein, siRNA-injected oocytes were pharmacologically arrested at GV stage for 48 h prior to initiation of IVM. Culture of oocytes for 48 h in the presence of S-roscovitine resulted in maintenance of GV arrest in 92% of oocytes relative to control (untreated oocytes) which all underwent spontaneous GV breakdown. As depicted in Figure 1A, microinjection of JY-1 siRNA greatly reduced endogenous JY-1 mRNA abundance by > 98% in bovine oocytes collected at 72 h post injection relative to uninjected and sham-injected oocytes (P < 0.01). Furthermore, JY-1 protein was decreased by ~48 % in JY-1 siRNAinjected oocytes compared with controls (P < 0.05; Figure 1B and 1C). JY-1 Deficiency Compromised Maturation of Bovine COCs and Inhibited Subsequent Early Embryonic Development To pinpoint the functional role of maternal JY-1 in oocyte maturation, effects of oocyte JY-1 ablation on meiotic maturation and cumulus expansion were examined. Microinjection of JY-1 siRNA reduced the proportion of oocytes progressing to metaphase II stage compared to uninjected and sham-injected oocytes (P < 0.05; Figure 2). In contrast, further analysis of the nuclear stage of JY-1 siRNA-injected and control oocytes showed that increased numbers of JY-1 siRNA oocytes were arrested between metaphase I and telophase I stages (Figure 2). JY-1 siRNA injection also greatly decreased the percentage of oocytes with a fully expanded cumulus layer after 24 h IVM compared with uninjected and sham-injected oocytes (P < 0.05; Figure 3). It is well established that oocyte maturation is critical for subsequent embryonic development after fertilization. However, progression to metaphase II stage was not totally blocked for JY-1 siRNA-injected embryos. Therefore, we hypothesized that JY-1 deficiency in bovine oocytes also retards early embryonic 4
development after IVF. Thus, JY-1 siRNA-injected and control oocytes were fertilized with bovine sperm and early cleavage, total cleavage as well as blastocyst formation examined. After fertilization, a pronounced decrease in the percentage of embryos that cleaved early (within 30 hpi) was noted for JY-1 siRNA-injected groups relative to uninjected and sham control oocytes (P< 0.05; Table 1). Similarly, total cleavage rate (48 hpi) was also dramatically reduced in response to JY-1 siRNA injection compared with uninjected and sham injection (P < 0.05; Table 1). In addition, JY-1 knockdown decreased the proportion of embryos developing to 8- to 16-cell stage (72 hpi) and blastocyst stage (d 7) relative to uninjected and sham control groups (P < 0.05, Table 1). A decrease was also noted when rates of development to 8- to 16-cell stage and blastocyst stage were calculated as a proportion of cleaved embryos (P < 0.05; Table 1). Evidence Supporting the Specificity of Effects of JY-1 siRNA on Oocyte Maturation and Developmental Competence To further ensure the specificity of JY-1 siRNA in preventing oocyte maturation and early embryonic development after fertilization, JY-1 siRNA-injected oocytes were treated with increasing concentrations of exogenous rJY-1 protein during 72 h oocyte culture prior to IVF. A dose-responsive beneficial effect of rJY-1 on the proportion of oocytes progressing to metaphase II stage was noted for JY-1 siRNAinjected oocytes. The proportion of JY-1 siRNA-injected oocytes progressing to metaphase II was rescued to levels comparable with both controls when exposed to 0.5-1 ng/ml rJY-1 protein (Figure 4A). Therefore, 1 ng/ml was used in subsequent experiments to determine effects of rJY-1 replacement on development of embryos produced from JY-1-deficient oocytes. As illustrated in Figure 4B-D, exposure to rJY-1 protein during oocyte culture totally rescued rates of early cleavage and total cleavage and partially rescued development to 8- to 16-cell stage following IVF for JY-1 siRNA-injected oocytes when compared to uninjected and negative control siRNA-injected embryos. However, rJY-1 supplementation of JY-1 siRNA- injected oocytes during oocyte culture did not rescue development to blastocyst stage following IVF. In contrast, supplementation of JY-1 siRNA-injected zygotes with rJY-1 protein during first 72 h of embryo culture totally rescued negative effects of JY-1 depletion post fertilization on development to 8- to 16-cell and blastocyst stages (Figure 5A and B).
DISCUSSION In the present studies, we demonstrated for the first time that oocyte-expressed JY-1 is required for nuclear maturation and cumulus expansion with implications for subsequent embryonic development. In previous studies, we characterized the functional role of JY-1 in early embryogenesis by microinjecting JY-1 siRNA into presumptive zygotes, which can efficiently silence maternal JY-1 mRNA. Such studies clearly demonstrated a functional role for oocyte-derived JY-1 post fertilization in promoting early embryogenesis [10]. However, the specific regulatory role of maternal JY-1 in oocyte maturation and cumulus expansion was previously unclear given the timing of JY-1 depletion. A number of oocyteexpressed genes that are essential for follicular development, oocyte maturation or early embryonic development have been identified in mice, especially through gene targeting studies [16-20]. However, to our knowledge, a functional requirement role for a single oocyte-expressed gene in promoting meiotic maturation and cumulus expansion pre-fertilization, coupled with an additional requirement of the same gene post-fertilization for normal early embryogenesis, has not been demonstrated previously. Demonstration of a functional role for JY-1 pre-fertilization was dependent upon establishment of a viable model system capable of supporting JY-1 ablation prior to initiation of oocyte maturation. Previous studies demonstrated that culture of bovine oocytes in the presence of 50 M S-roscovitine for 48 h can maintain an intact GV in over 95% of oocytes [13]. Thus, to specifically knockdown maternal JY-1 mRNA and protein, we established a novel RNAi system in bovine oocytes via microinjection of JY-1 siRNA into cumulus enclosed GV stage oocytes followed by meiotic arrest for 48 h (to allow siRNAmediated reduction of mRNA and accompanying reduction/ lack of translation of JY-1 protein from 5
mRNA stores) before IVM. Maintenance of the cumulus layer after microinjection is critical to success of functional studies given cumulus cells are needed for optimal IVF [21] and embryo development [22] in cattle. Our results demonstrated that this strategy can efficiently maintain meiotic arrest and attain effective ablation of endogenous JY-1 mRNA and protein by 72 h of oocyte culture. Such approach should be of further utility to investigate the functional role of additional oocyte- expressed genes in meiotic maturation, cumulus expansion and subsequent early embryogenesis. Results of the present studies suggest oocyte-expressed JY-1 is required for at least two important aspects of oocyte maturation: nuclear maturation and cumulus expansion. It is well documented that nuclear/meiotic maturation is required for normal development, including fertilization and early embryogenesis [23-25]. Cumulus expansion is essential for normal ovulation and fertilization [3, 26]. Both are triggered by the LH surge in vivo and induced by the presence of FSH or EGF in maturation medium in vitro [27]. Oocyte-secreted factors play an active role in regulating the process of cumulus expansion [28]. It is currently unclear whether maturation- and cumulus expansion-promoting effects of JY-1 are mediated directly or indirectly via regulation of intrafollicular paracrine/autocrine signaling networks, which are potentially linked to LH-induced meiotic maturation and cumulus expansion in vivo [29, 30]. Based on results of current studies and previous results [10], we propose that JY-1 functions as an important paracrine and autocrine regulator of key aspects of follicular development and early embryogenesis in cattle. The ability of JY-1 protein to influence granulosa cell steroidogenesis when added to culture media has been described previously [10]. Furthermore, the predicted amino acid sequence of JY-1 contains a putative signal peptide [10], suggesting the JY-1 protein is secreted from the oocyte. We also showed in the current studies that the observed effects of oocyte JY-1 depletion on cumulus expansion and meiotic maturation, and of zygote JY-1 depletion on embryonic development, can be rescued by addition of rJY-1 protein during oocyte and embryo culture, respectively. Hence, results suggest that JY-1 is secreted and acts in a paracrine and autocrine fashion to regulate oocyte maturation, cumulus expansion and early embryonic development in vitro. Whether such effects of JY-1 are receptormediated and identification of potential signaling pathways involved will require further investigation. A number of studies in humans have suggested early cleavage rate is a reliable predictor of early embryonic developmental competence [31, 32]. Our previous studies also indicate early-cleaving bovine embryos have a greater capability to develop to blastocyst stage than late-cleaving counterparts [33, 34]. In the current studies, the rates of early cleavage and total cleavage were also reduced following IVF of JY-1-depleted oocytes. However, approximately 40% of JY-1-depleted oocytes were successfully fertilized and underwent the first cleavage division, but < 2% of cleaved embryos derived from JY-1depleted oocytes developed to the blastocyst stage. Progression to metaphase II, early cleavage rates and total cleavage rates following IVF for JY-1-depleted oocytes were fully rescued by addition of rJY-1 protein during oocyte culture (prior to fertilization) but development to 8- to 16-cell stage was only partially rescued and blastocyst rates were not impacted. Hence, these results support an important functional role for JY-1 protein prefertilization but indicate that rescue of JY-1-depleted oocytes with rJY-1 protein during oocyte culture alone is not sufficient to restore normal rates of blastocyst development following IVF. However, supplementation of JY-1-depleted zygotes with rJY-1 protein during initial 72 h of embryo culture totally restored development to the blastocyst stage. Collectively, such results further support a functional role for JY-1 protein both pre- and post- fertilization. In summary, the present studies strongly support a functional role of oocyte-expressed JY-1 in meiotic maturation and cumulus expansion and further support a role for JY-1 in promoting embryonic development to the blastocyst stage. Sirard et al. defined oocyte developmental competence as the capacity of the oocyte to resume meiosis, cleave after fertilization, help promote embryonic development and implantation, and bring a pregnancy to term in good health [1]. Hence, results of the current and 6
previous studies suggest a requirement of JY-1 for bovine oocyte competence and provide clear implications for understanding the genetic and physiological regulation of follicular development and early embryogenesis in cattle. Recent identification of genetic polymorphisms of the JY-1 gene associated with fertility traits in cattle [11, 12] supports potential relevance of the JY-1 gene to fertility in vivo. Further insight into regulation of JY-1 expression/activity and function has broad general implications for understanding regulation of follicular development, oocyte maturation and early embryonic development and potential improvements in assisted reproductive technologies in cattle.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo quality. Theriogenology 2006; 65:126136. Rodriguez KF, Farin CE. Gene transcription and regulation of oocyte maturation. Reproduction, Fertility, and Development 2004; 16:55-67. Edson MA, Nagaraja AK, Matzuk MM. The Mammalian Ovary from Genesis to Revelation. Endocrine Reviews 2009; 30:624-712. Eppig JJ, Schultz RM, O'Brien M, Chesnel F. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Developmental Biology 1994; 164:1-9. Watson AJ. Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo developmental competence. J Anim Sci 2007; 85:E1-3. Li L, Zheng P, Dean J. Maternal control of early mouse development. Development 2010; 137:859-870. Tripurani SK, Lee KB, Wang L, Wee G, Smith GW, Lee YS, Latham KE, Yao JB. A Novel Functional Role for the OocyteSpecific Transcription Factor Newborn Ovary Homeobox (NOBOX) during Early Embryonic Development in Cattle. Endocrinology 2011; 152:1013-1023. Lee KB, Bettegowda A, Wee G, Ireland JJ, Smith GW. Molecular Determinants of Oocyte Competence: Potential Functional Role for Maternal (Oocyte-Derived) Follistatin in Promoting Bovine Early Embryogenesis. Endocrinology 2009; 150:2463-2471. Yao JB, Ren XN, Ireland JJ, Coussens PM, Smith TPL, Smith GW. Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis. Physiological Genomics 2004; 19:84-92. Bettegowda A, Yao J, Sen A, Li Q, Lee KB, Kobayashi Y, Patel OV, Coussens PM, Ireland JJ, Smith GW. JY-1, an oocytespecific gene, regulates granulosa cell function and early embryonic development in cattle. Proceedings of the National Academy of Sciences of the United States of America 2007; 104:17602-17607. de Camargo GrMF, Costa RB, de Albuquerque LGo, Regitano LCdA, Baldi F, Tonhati H. Association between JY-1 gene polymorphisms and reproductive traits in beef cattle. Gene 2014; 533:477-480. de Camargo G, Baldi F, Regitano L, Tonhati H. Characterization of the Exonic Regions of the JY-1 Gene in Zebu Cattle and Buffaloes. Reproduction in Domestic Animals 2013; 48:918-922. Coy P, Romar R, Payton RR, McCann L, Saxton AM, Edwards JL. Maintenance of meiotic arrest in bovine oocytes using the S-enantiomer of roscovitine: effects on maturation, fertilization and subsequent embryo development in vitro. Reproduction 2005; 129:19-26. Choi I, Carey TS, Wilson CA, Knott JG. Transcription factor AP-2gamma is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis. Development 2012. Lin CJ, Conti M, Ramalho-Santos M. Histone variant H3.3 maintains a decondensed chromatin state essential for mouse preimplantation development. Development 2013; 140:3624-3634. Wu X, Viveiros MM, Eppig JJ, Bai Y, Fitzpatrick SL, Matzuk MM. Zygote arrest 1 (Zar1) is a novel maternal-effect gene critical for the oocyte-to-embryo transition. Nature Genetics 2003; 33:187-191. Tong ZB, Gold L, Pfeifer KE, Dorward H, Lee E, Bondy CA, Dean J, Nelson LM. Mater, a maternal effect gene required for early embryonic development in mice. Nature Genetics 2000; 26:267-268. Zheng P, Dean J. Role of Filia, a maternal effect gene, in maintaining euploidy during cleavage-stage mouse embryogenesis. Proceedings of the National Academy of Sciences of the United States of America 2009; 106:7473-7478. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 1996; 383:531-535. Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS, Dube JL, Celeste AJ, Matzuk MM. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Molecular Endocrinology 2001; 15:854-866. Shioya Y, Kuwayama M, Fukushima M, Iwasaki S, Hanada A. In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro. Theriogenology 1988; 30:489-496. Thomas WK, Seidel GE, Jr. Effects of cumulus cells on culture of bovine embryos derived from oocytes matured and fertilized in vitro. J Anim Sci 1993; 71:2506-2510.
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23. Tian XC, Lonergan P, Jeong BS, Evans AC, Yang X. Association of MPF, MAPK, and nuclear progression dynamics during activation of young and aged bovine oocytes. Molecular Reproduction and Development 2002; 62:132-138. 24. Fan HY, Sun QY. Involvement of mitogen-activated protein kinase cascade during oocyte maturation and fertilization in mammals. Biology of Reproduction 2004; 70:535-547. 25. Jones KT, Carroll J, Merriman JA, Whittingham DG, Kono T. Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent. Development 1995; 121:3259-3266. 26. Chen L, Russell PT, Larsen WJ. Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass. Molecular Reproduction and Development 1993; 34:87-93. 27. Conti M, Hsieh M, Park JY, Su YQ. Role of the epidermal growth factor network in ovarian follicles. Mol Endocrinol 2006; 20:715-723. 28. Su YQ, Sugiura K, Eppig JJ. Mouse oocyte control of granulosa cell development and function: paracrine regulation of cumulus cell metabolism. Seminars in Reproductive Medicine 2009; 27:32-42. 29. Park JY, Su YQ, Ariga M, Law E, Jin SL, Conti M. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Science 2004; 303:682-684. 30. Hsieh M, Lee D, Panigone S, Horner K, Chen R, Theologis A, Lee DC, Threadgill DW, Conti M. Luteinizing hormonedependent activation of the epidermal growth factor network is essential for ovulation. Mol Cell Biol 2007; 27:1914-1924. 31. Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Human Reproduction 1998; 13:182-187. 32. Shoukir Y, Chardonnens D, Campana A, Bischof P, Sakkas D. The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Human Reproduction 1998; 13:676-681. 33. Patel OV, Bettegowda A, Ireland JJ, Coussens PM, Lonergan P, Smith GW. Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction 2007; 133:95-106. 34. Bettegowda A, Lee KB, Smith GW. Cytoplasmic and nuclear determinants of the maternal-to-embryonic transition. Reprod Fertil Dev 2008; 20:45-53.
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FIGURE LEGENDS Figure 1. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on JY-1 mRNA and protein levels following in vitro maturation. (A) Quantitative realtime RT-PCR analysis of JY-1 mRNA transcript abundance in uninjected, sham injected and JY-1 siRNAinjected oocytes (n = 6 pools of 10 oocytes each per treatment) collected 72 h post injection. Data were normalized relative to abundance of endogenous control (RPS18) and expressed as mean ± SEM. (a,b; P < 0.05). (B) Representative western blot images of JY-1 protein abundance in uninjected, sham injected and JY-1 siRNA-injected oocytes (n = 50 oocytes per lane; n = 3 replicates) collected 72 h post injection. (C) Densitometric analysis of relative abundance of JY-1 protein in uninjected, sham injected and JY-1 siRNA-injected oocytes. JY-1 protein abundance was normalized relative to actin and expressed as mean ± SEM. Different letters denote statistical significance (a,b; P < 0.05). Figure 2. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on extent of meiotic progression during in vitro maturation. Proportion of oocytes progressing to metaphase I (black bars), anaphase I (white bars) , telophase I (hatched bars) and metaphase II (shaded bars) following 24 h in vitro maturation for uninjected, sham injected and JY-1 siRNA-injected oocytes (n = 30 oocytes per treatment; n = 6 replicates). Oocytes were stained with Hoechst-33342 and nuclear stage determined under fluorescent microscope. Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; x,y; A,B; X,Y; P < 0.05). Figure 3. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on expansion of the cumulus cell layer during in vitro maturation. Degree of cumulus expansion [non expanded (black bars), partially expanded (white bars), fully expanded (hatched bars)] for individual JY-1 siRNA injected, sham injected and uninjected oocytes (n = 30 oocytes per treatment; n = 6 replicates) were determined at 72 h post injection. Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; x,y; A,B; P < 0.05). Figure 4. Effect of supplementation with recombinant JY-1 protein during oocyte culture on JY-1 siRNA-induced reduction in nuclear maturation and embryonic development following in vitro fertilization. Cumulus enclosed germinal vesicle stage oocytes were microinjected with JY-1 siRNA, with negative control siRNA or served as uninjected controls. JY-1 siRNA-injected oocytes were cultured in presence of increasing concentrations of recombinant JY-1 protein (0, 0.1, 0.5, 1, 10 ng/ml) for 72 h and (A) proportion of oocytes progressing to metaphase II were determined (n = 10 oocytes per treatment; n = 6 replicates). After fertilization, proportion of oocytes undergoing (B) early cleavage (30 h post insemination; hpi), (C) total cleavage rates (48 hpi), and (D) percent development to 8- to 16-cell stage (72 hpi) and (E) blastocyst stage (7 d post insemination) were determined for each treatment group (n = 20 oocytes per treatment; n = 5 replicates). Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b,c; P < 0.05). Figure 5. Effect of zygote JY-1 depletion and supplementation with recombinant JY-1 protein on embryonic development following in vitro fertilization. Presumptive zygotes were injected with JY-1 siRNA, with negative control siRNA or served as uninjected controls. JY-1 siRNA injected and uninjected zygotes were cultured for 72 h in presence or absence of 1 ng/ml recombinant JY-1 protein and effects of treatments on rates of development to (A) 8- to 16-cell stage (72 h post insemination) and (B) blastocyst stage (7 d post insemination) were determined for each treatment group (n = 20 zygotes per treatment; n = 5 replicates). Data are expressed as mean ± SEM. Different letters denote statistical significance (a,b; P < 0.05).
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TABLE 1. Effect of microinjection of JY-1 siRNA into cumulus-enclosed bovine germinal vesicle stage oocytes on embryonic development (%) following in vitro fertilization.* Stage Uninjected Sham JY-1 siRNA Early cleavage 17.5 ± 1.1a 18.3 ± 1.1a 7.5 ± 1.1b a a Total cleavage 77.5 ± 2.1 76.7 ± 1.1 40.8 ± 3.0b a a 47.5 ± 1.1 12.5 ± 2.1b 8-16 cells 49.2 ± 1.5 a a Blastocyst 20.0 ± 1.3 18.3 ± 1.7 0.8 ± 0.8b a a 8-16 cells/cleaved embryo 63.5 ± 1.6 62.0 ± 1.7 31.3 ± 5.7b a a Blastocysts/cleaved embryo 26.1 ± 2.3 24.0 ± 2.3 1.7 ± 1.7b *Different superscript letters within a row indicate differences between treatments (P < 0.05).
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Figure 1.
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Figure 5.
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