Anilkumar Bettegowda

Identification of Novel Bovine Cumulus Cell Molecular Markers Predictive of Oocyte Competence: Functional and Diagnostic Implications

Abstract The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and to elucidate their functional significance. Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (a model of poor oocyte quality) were identified by microarray analysis. Four genes of interest encoding for the lysosomal cysteine proteinases cathepsins B, S, K, and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real-time RT-PCR. Elevated transcript abundance for cathepsins B, S, and Z also was observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus-oocyte complexes during in vitro oocyte maturation with a cell-permeable cysteine proteinase (cathepsin) inhibitor. Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro-fertilized adult oocytes to the blastocyst stage. Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells. The present results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality.

Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development.

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.

Variation in the Ovarian Reserve Is Linked to Alterations in Intrafollicular Estradiol Production and Ovarian Biomarkers of Follicular Differentiation and Oocyte Quality in Cattle

Abstract The mechanisms whereby the high variation in numbers of morphologically healthy oocytes and follicles in ovaries (ovarian reserve) may have an impact onovarian function, oocyte quality, and fertility are poorly understood. The objective was to determine whether previously validated biomarkers for follicular differentiation and function, as well as oocyte quality differed between cattle with low versus a high antral follicle count (AFC). Ovaries were removed (n = 5 per group) near the beginning of the nonovulatory follicular wave, before follicles could be identified via ultrasonography as being dominant, from heifers with high versus a low AFC. The F1, F2, and F3 follicles were dissected and diameters determined. Follicular fluid and thecal, granulosal, and cumulus cells and the oocyte were isolated and subjected to biomarker analyses. Although the size and numerous biomarkers of differentiation, such as mRNAs for the gonadotropin receptors, were similar, intrafollicular concentrations of estradiol and the abundance of mRNAs for CYP19A1 in granulosal cells and ESR1, ESR2, and CTSB in cumulus cells were greater, whereas mRNAs for AMH in granulosal cells and TBC1D1 in thecal cells were lower for animals with low versus a high AFC during follicle waves. Hence, variation in the ovarian reserve may have an impact on follicular function and oocyte quality via alterations in intrafollicular estradiol production and expression of key genes involved in follicle-stimulating hormone action (AMH) and estradiol (CYP19A1) production by granulosal cells, function and survival of thecal cells (TBC1D1), responsiveness of cumulus cells to estradiol (ESR1, ESR2), and cumulus cell determinants of oocyte quality (CTSB).

Molecular determinants of oocyte competence: potential functional role for maternal (oocyte-derived) follistatin in promoting bovine early embryogenesis.

Previous studies established a positive relationship between oocyte competence and follistatin mRNA abundance. Herein, we used the bovine model to test the hypothesis that follistatin plays a functional role in regulation of early embryogenesis. Treatment of early embryos with follistatin during in vitro culture (before embryonic genome activation) resulted in a dose-dependent decrease in time to first cleavage, increased numbers of blastocysts, and increased blastocyst total and trophectoderm cell numbers. To determine the requirement of endogenous follistatin for early embryogenesis, follistatin ablation/replacement studies were performed. Microinjection of follistatin small interfering RNA into zygotes reduced follistatin mRNA and protein and was accompanied by a reduction in number of embryos developing to eight- to 16-cell and blastocyst stages and reduced blastocyst total and trophectoderm cell numbers. Effects of follistatin ablation were rescued by culture of follistatin small interfering RNA-injected embryos in the presence of exogenous follistatin. To investigate whether follistatin regulation of early embryogenesis is potentially mediated via inhibition of endogenous activin activity, the effects of treatment of embryos with exogenous activin, SB-431542 (inhibitor of activin, TGF-beta, and nodal type I receptor signaling) and follistatin plus SB-431542 were investigated. Activin treatment mimicked positive effects of follistatin on time to first cleavage and blastocyst development, whereas negative effects of SB-431542 treatment were observed. Stimulatory effects of follistatin on embryogenesis were not blocked by SB-431542 treatment. Results support a functional role for oocyte-derived follistatin in bovine early embryogenesis and suggest that observed effects of follistatin are likely not mediated by classical inhibition of activin activity.

Cytoplasmic and nuclear determinants of the maternal-to-embryonic transition

Although improvements in culture systems have greatly enhanced in vitro embryo production, success rates under the best conditions are still far from ideal. The reasons for developmental arrest of the majority of in vitro produced embryos are unclear, but likely attributable, in part, to intrinsic and extrinsic influences on the cytoplasmic and/or nuclear environment of an oocyte and/or early embryo that impede normal progression through the maternal-to-embryonic transition. The maternal-to-embryonic transition is the time period during embryonic development spanning from fertilisation until when control of early embryogenesis changes from regulation by oocyte-derived factors to regulation by products of the embryonic genome. The products of numerous maternal effect genes transcribed and stored during oogenesis mediate this transition. Marked epigenetic changes to chromatin during this window of development significantly modulate embryonic gene expression. Depletion of maternal mRNA pools is also an obligatory event during the maternal-to-embryonic transition critical to subsequent development. An increased knowledge of the fundamental mechanisms and mediators of the maternal-to-embryonic transition is foundational to understanding the regulation of oocyte quality and future breakthroughs relevant to embryo production.

JY-1, an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle

Oocyte-specific gene products play a key role in regulation of fertility in mammals. Here, we describe the discovery, molecular characterization, and function of JY-1, a bovine oocyte-expressed gene shown to regulate both function of ovarian granulosa cells and early embryogenesis in cattle and characteristics of JY-1 loci in other species. The JY-1 gene encodes for a secreted protein with multiple mRNA transcripts containing an identical ORF but differing lengths of 3′ UTR. JY-1 mRNA and protein are oocyte-specific and detectable throughout folliculogenesis. Recombinant JY-1 protein regulates function of follicle-stimulating hormone-treated ovarian granulosa cells, resulting in enhanced progesterone synthesis accompanied by reduced cell numbers and estradiol production. JY-1 mRNA of maternal origin is also present in early bovine embryos, temporally regulated during the window from meiotic maturation through embryonic genome activation, and is required for blastocyst development. The JY-1 gene has three exons and is located on bovine chromosome 29. JY-1-like sequences are present on syntenic chromosomes of other vertebrate species, but lack exons 1 and 2, including the protein-coding region, suggestive of species specificity in evolution and function of this oocyte-specific gene.

Evidence that the preovulatory rise in intrafollicular progesterone may not be required for ovulation in cattle

Despite ample evidence pointing to an obligatory involvement of progesterone in ovulation, the mechanisms responsible for the ovulation promoting effects of intrafollicular progesterone are unclear. The objectives of this study were to determine if ovulation, luteinization and the gonadotropin surge-induced regulation of select extracellular matrix-degrading enzymes and their inhibitors, and mRNAs for prostaglandin (PG) biosynthesis and metabolizing enzymes are blocked following suppression of the intrafollicular increase in progesterone. Bovine preovulatory follicles were injected with the 3 beta-hydroxysteroid dehydrogenase inhibitor trilostane or diluent and collected at 0, 12, and 24 h after GnRH induction of the preovulatory LH surge. Intrafollicular trilostane administration blocked the preovulatory increase in follicular fluid progesterone resulting in concentrations similar to those observed at time 0 post-GnRH injection. The preovulatory increase in follicular fluid PGE(2) and PGF(2alpha) was reduced in trilostane-treated follicles and accompanied by upregulation of prostaglandin dehydrogenase mRNA in the granulosal and thecal cells. However, follicle rupture was not blocked by inhibition of the preovulatory rise in intrafollicular progesterone, and normal serum progesterone concentrations were observed during subsequent luteal development. Effects of trilostane administration on preovulatory changes in mRNA abundance and protein/activity in preovulatory follicles for most regulators of extracellular matrix remodeling examined were distinct from changes previously observed following the inhibition of intrafollicular prostaglandin synthesis. Results suggest that the preovulatory increase in intrafollicular progesterone may not be obligatory for bovine follicle rupture, luteinization, or regulation of prominent matrix-degrading proteinases and their inhibitors associated with ovulation.

Evidence Supporting a Role for Cocaine- and Amphetamine-Regulated Transcript (CARTPT) in Control of Granulosa Cell Estradiol Production Associated with Dominant Follicle Selection in Cattle

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.

148 EFFECT OF FOLLISTATIN TREATMENT POST-FERTILIZATION ON TIME TO FIRST CLEAVAGE, DEVELOPMENT TO THE BLASTOCYST STAGE, AND CELL ALLOCATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

Previous studies from our laboratory have demonstrated a positive association of follistatin mRNA abundance with oocyte competence. Follistatin mRNA is greater in germinal vesicle stage oocytes collected from prepubertal (model of poor oocyte competence) vs. adult animals. Furthermore, follistatin mRNA abundance is also greater in early-cleaving 2-cell bovine embryos (collected prior to the maternal zygotic transition and initiation of significant transcription from the embryonic genome) than their late-cleaving counterparts. Given these results and the fact that early-cleaving embryos develop to the blastocyst stage at a greater rate, we hypothesized that follistatin has a stimulatory role in early embryonic development. To begin to test this hypothesis, we determined the effects of follistatin treatment of in vitro-produced bovine embryos (during the initial 72 h post-fertilization) on time to first cleavage, development to the blastocyst stage (Day 7), and blastocyst cell allocation (quality). Cumulus–oocyte complexes (COCs) were harvested from ovaries obtained from a local abattoir, matured, and fertilized in vitro. After 20 h of co-incubation with spermatozoa, presumptive zygotes were stripped of cumulus cells and cultured in KSOM medium supplemented with 0.3% BSA containing 0, 1, 10, or 100 ng mL-1 follistatin (n = 25 presumptive zygotes per treatment; n = 6 replicates). Proportions of embryos reaching the 2-cell stage within 30 h (early-cleaving), 30–36 h (late-cleaving), and within 48 h post-fertilization (total cleavage rate) were recorded. Embryos at the 8–16-cell stage were separated 72 h after fertilization and cultured in fresh KSOM medium supplemented with 0.3% BSA and 10% FBS until Day 7. The proportion of embryos reaching the blastocyst stage at Day 7 post-fertilization was recorded and the numbers of inner cell mass (ICM) and trophectoderm (TE) cells determined by differential staining. Follistatin treatment did not increase the rate of total cleavage and the proportion of late-cleaving embryos when compared to control. However, supplementation with 1 and 10, but not 100, ng mL-1 follistatin increased the proportion of early-cleaving embryos (26.3 and 35.3% vs. 9.5%) and development to the blastocyst stage (28.6 and 31.7% vs. 18.4%) relative to controls (P < 0.05). Treatment with 10 ng mL-1 follistatin increased total cell numbers (130.1 vs. 110.9) and proportion of trophectoderm cells (61.6% vs. 48.4%) and decreased the ICM/total cell ratio (38.4% vs. 51.5%) in Day 7 blastocysts relative to controls (P < 0.05). The results indicate that exogenous follistatin treatment during the early stages of in vitro bovine embryo development can enhance time to first cleavage, development to the blastocyst stage, and cell allocation in favor of increased trophectoderm cells, and can support a potential functional role for follistatin in early embryogenesis.

261 IDENTIFICATION OF OOCYTE- AND CUMULUS-DERIVED CO-REGULATED AND DIFFERENTIALLY REGULATED TRANSCRIPTS ASSOCIATED WITH BOVINE MEIOTIC MATURATION

Understanding the process of oocyte maturation is critical for efficient application of biotechnologies such as in vitro embryo production and nuclear transfer/cloning. Intercellular communication between the oocyte and the encompassing somatic (cumulus) cells is pivotal for successful growth of ovarian follicles and oocyte maturation. Therefore, we utilized global gene expression profiling to determine changes in the transcriptome of oocytes and their adjacent cumulus cells during meiotic maturation in vitro to identify both co-regulated and differentially regulated transcripts within the two cell compartments of the cumulus oocyte complex (COC). Germinal vesicle (GV) and in vitro matured metaphase II (MII) COC (n = 5 pools of 5 COC per group) were denuded and separated into oocytes and cumulus cells. RNA was extracted from the oocytes and cumulus cells and subjected separately to microarray analysis using a bovine cDNA array containing expressed sequence tags (ESTs) representing 15 500 unique genes. A combined total of 1045 genes displaying greater mRNA abundance in GV oocytes and associated cumulus cells compared to MII samples were detected (P < 0.05; false discovery rate (FDR) = 5%). A combined total of 711 genes displaying greater mRNA abundance in MII oocytes and enclosing cumulus cells compared to GV samples were detected (P < 0.05; FDR = 5%). Fourteen transcripts were identified that were co-regulated and of greater abundance in GV or MII oocytes and in their matching cumulus cells (P < 0.05; FDR = 5%). The co-regulated transcripts identified are implicated in metabolism (e.g. heme oxygenase-2, leukotriene B4 12-hydroxydehydrogenase), signal transduction (e.g. caveolin 1, ring finger protein 31), and cell growth (e.g. BTG family member 2, myosin regulatory light chain 2). In contrast, thirteen transcripts differentially regulated in the GV oocyte versus MII cumulus cells were identified (P < 0.05; FDR = 5%). Similarly, nine transcripts differentially regulated in the MII oocyte versus GV cumulus cells were identified (P < 0.05; FDR = 5%). Some of the identified differentially regulated transcripts encode for genes associated with the cytoskeleton (e.g. tropomyosin 1), apoptotic activity (e.g. death effector domain containing protein 2) and DNA replication (e.g. epsilon polymerase). The results provide novel insights into the identity of transcripts whose abundance is co-regulated or differentially regulated between the oocyte and cumulus cells during the transition of a COC from the GV to the MII stage. Characterization of the signaling pathways driving changes in transcript abundance for co-regulated and differentially regulated genes in oocytes versus associated cumulus cells may lead to a better understanding of regulation of meiotic maturation and potential cross-talk between germ cells and somatic cells during the oocyte maturation cascade. This work was supported by the Rackham Foundation and the MI Agriculture Experiment Station.