scholarly journals OR31-04 Androgen Signaling Regulates Female Fertility Through Modulation of H3K27me3 in Granulosa Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Sambit Roy ◽  
Niharika Sinha ◽  
Binbin Huang ◽  
Holly Cline-Fedewa ◽  
Jianrong Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Target Genes ◽  
Ovarian Function ◽  
Follicular Development ◽  
Underlying Mechanism ◽  
Female Fertility ◽  
Rna Seq ◽  
Primary Mouse

Abstract Androgens play a major role in female fertility and in women’s health, in general. However, the underlying mechanism and downstream physiological targets of androgen are not fully understood. Androgen actions are mediated by “nuclear” transcriptional signals or “extra-nuclear” kinase actions. Primary androgen receptor (AR) target genes are those at which AR occupies a genomic androgen response element (ARE) and regulates gene transcription. Subsequently, androgens also regulate other genes in an AR-ARE independent fashion, involving membrane-initiated androgen signaling. In the last couple of years, we have reported that androgens may also influence gene expression through histone modifications. We have found that H3K27me3 (tri-methyl lysine 27 histone3) is a downstream target of androgen actions. H3K27me3 is a gene silencing mark, regulated by Enhancer of Zeste Homologue 2 (Ezh2), a histone methyltransferase that promotes tri-methylation of K27 and androgens inhibit the expression and activity of Ezh2. In this study, we report that androgens also regulate the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing the H3K27me3 mark. We find that in granulosa cells (GCs), androgen through the PI3K/Akt pathway, in a transcription-independent fashion, increases hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. ChIP studies reveal increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. To understand the global impact of androgens on ovarian gene expression and the contribution of androgen-induced decrease of H3K27me3, we have performed RNA-seq and ChIP-seq analysis with H3K27me3 antibody in primary mouse GCs treated with DHT or vehicle. Results show 190 significantly differentially expressed genes in DHT treated sample vs vehicle, out of which 129 and 61 genes were significantly up- and downregulated, respectively. Moreover, comparison of the RNA-seq and ChIP-seq data reveals that a number of upregulated genes have significantly lower H3K27me3 enrichment in the enhancer and/or promoter region in the DHT treated samples vs vehicle. This establishes that in the ovary, androgen-induced modulation of H3K27me3 mark through regulation Ezh2 and JMJD3 expression/activity is an important regulatory mechanism for ovarian gene expression. To delineate the physiological importance of JMJD3 in normal ovarian function and female fertility, we have also developed a GC-specific JMJD3 knockout mice. We find that GC-specific JMJD3KO mice are sub-fertile with longer estrous cycles and dysregulated follicular development. This phenotype is very similar to GC-specific ARKO mice. Thus, we propose that one of the critical actions of androgens in regulating follicular function and female fertility is through the regulation of JMJD3 expression.

scholarly journals Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle

2019 ◽  
Vol 98 (1) ◽  
Author(s):  
Breanne C Morrell ◽  
M Chiara Perego ◽  
Excel Rio S Maylem ◽  
Lingna Zhang ◽  
Luis F Schütz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Ovarian Function ◽  
Follicular Development ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Mrna Abundance ◽  
Concomitant Treatment ◽  
Ovarian Cells ◽  
Cell Proliferation And Differentiation

Abstract The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P > 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

scholarly journals β-Catenin (CTNNB1) Promotes Preovulatory Follicular Development but Represses LH-Mediated Ovulation and Luteinization

2010 ◽  
Vol 24 (8) ◽  
pp. 1529-1542 ◽  
Author(s):  
Heng-Yu Fan ◽  
Annalouise O'Connor ◽  
Manami Shitanaka ◽  
Masayuki Shimada ◽  
Zhilin Liu ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Integration Site ◽  
Gonad Development ◽  
Follicular Development ◽  
Underlying Mechanism ◽  
Female Fertility ◽  
Female Gonad ◽  
Tumor Virus

Abstract Wingless-type mouse mammary tumor virus integration site family (WNT)/β-catenin (CTNNB1) pathway components are expressed in ovarian granulosa cells, direct female gonad development, and are regulated by the pituitary gonadotropins. However, the in vivo functions of CTNNB1 during preovulatory follicular development, ovulation, and luteinization remain unclear. Using a mouse model Ctnnb1(Ex3)fl/fl;Cyp19-Cre (Ctnnb1(Ex3)gc−/−), expressing dominant stable CTNNB1 in granulosa cells of small antral and preovulatory follicles, we show that CTNNB1 facilitates FSH-induced follicular growth and decreases the follicle atresia (granulosa cell apoptosis). At the molecular level, WNT signaling and FSH synergistically promote the expression of genes required for cell proliferation and estrogen biosynthesis, but decrease FOXO1, which negatively regulates proliferation and steroidogenesis. Conversely, dominant stable CTNNB1 represses LH-induced oocyte maturation, ovulation, luteinization, and progesterone biosynthesis. Specifically, granulosa cells in the Ctnnb1(Ex3)gc−/− mice showed compromised responses to the LH surge and decreased levels of the epidermal growth factor-like factors (Areg and Ereg) that in vivo and in vitro mediate LH action. One underlying mechanism by which CTNNB1 prevents LH responses is by reducing phosphorylation of cAMP-responsive element-binding protein, which is essential for the expression of Areg and Ereg. By contrast, depletion of Ctnnb1 using the Ctnnb1fl/fl;Cyp19-Cre mice did not alter FSH regulation of preovulatory follicular development or female fertility but dramatically enhanced LH induction of genes in granulosa cells in culture. Thus, CTNNB1 can enhance FSH and LH actions in antral follicles but overactivation of CTNNB1 negatively effects LH-induced ovulation and luteinization, highlighting the cell context-dependent and developmental stage-specific interactions of WNT/CTNNB1 pathway and G protein-coupled gonadotropin receptors in female fertility.

scholarly journals Transcriptional Regulation of the Epiregulin Gene in the Rat Ovary

Endocrinology ◽  
2002 ◽  
Vol 143 (12) ◽  
pp. 4718-4729 ◽  
Author(s):  
Toshio Sekiguchi ◽  
Tetsuya Mizutani ◽  
Kazuya Yamada ◽  
Takashi Yazawa ◽  
Hiroko Kawata ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Promoter Activity ◽  
Transcriptional Start Site ◽  
Gene Promoter ◽  
Follicular Development ◽  
Upstream Region ◽  
Ovarian Granulosa Cells

Abstract Ovarian follicular development is initiated by FSH secreted from the pituitary gland. The FSH-induced follicular development involves granulosa cell proliferation and differentiation. We demonstrated that a growth factor of epidermal growth factor (EGF) family epiregulin was rapidly induced in the primary culture of rat ovarian granulosa cells by FSH within 1 h. Epiregulin gene expression was also observed in granulosa cells of antral ovarian follicles from pregnant mare’s serum gonadotropin-primed rats in vivo. To analyze the regulation of gene expression of epiregulin, we isolated and characterized the rat epiregulin gene of 22.1 kb, including 3.8 kb of 5′-upstream region as well as all five exons and four introns. We determined the transcriptional start site of rat epiregulin gene by primer extension analysis and then characterized the upstream promoter region of the gene. By using a luciferase reporter system, deletion and mutation analyses of rat epiregulin gene promoter region revealed that 125 bp upstream of transcriptional start site was essential, and that two CT boxes and one GT box within this region were important for the gene expression. We also demonstrated by EMSAs that Sp1/Sp3 proteins were involved in the epiregulin gene expression via the upstream sequence. Involvement of Sp1/Sp3 was also demonstrated that transfection of Sp1 or Sp3 expression plasmids dramatically increased the epiregulin gene promoter activities about 90- or 7.9-fold, respectively, in Drosophila SL2 cells that lack endogenous Sp family proteins. Such an increase in the promoter activity was also observed in mammalian cells when NIH-3T3 cells were used. In conclusion, we demonstrated here for the first time that EGF-type growth factor epiregulin is rapidly and strongly induced in the ovarian granulosa cells by FSH stimulation, and that two CT boxes and one GT box present in the upstream region are essential for the promoter activity of rat epiregulin. We also demonstrated that Sp family members play crucial roles in the epiregulin promoter activity through the CT boxes. The restricted and hormonally regulated expression of epiregulin in the rat ovarian granulosa cells may correspond to the physiological relevance of this peptide growth factor to the FSH-induced ovarian follicular growth and maturation.

scholarly journals Differentially expressed IGF2BP2 in ovarian disorders: strongly associates with alternative splicing regulation in human granulosa cells

2021 ◽  
Author(s):  
Feiyan Zhao ◽  
Qin Wang ◽  
Tong Chen ◽  
Xuehan Zhao ◽  
Zhimin Xin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Granulosa Cells ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulation Of Gene Expression ◽  
Follicular Development ◽  
Endocrine Disorders ◽  
Human Granulosa Cells

Abstract Genome-wide interactions between RNA-binding proteins (RBPs) and RNA targets account for an important portion of post-transcriptional regulation. IGF2BP2 is associated with type 2 diabetes (T2D) and obesity and reportedly functions as an RBP that regulates a series of target genes by binding RNA transcripts. In this study, we detected the differential expression of IGF2BP2 in granulosa cells from women with ovarian disorders and performed RNA-seq and RIP-seq experiments in immortalized human granulosa cells (KGN cells) to evaluate global transcript levels and alternative splicing on KGN cells overexpressing IGF2BP2 versus controls. Our results show that IGF2BP2 preferentially binds to the 3’and 5’UTRs of mRNAs and enriches target gene expression in KGN cells. Notably, besides the conventional GGAC motif, we found a significant enrichment of a new GAAG motif within IGF2BP2-binding regions. We demonstrate that IGF2BP2 is involved in transcription regulation and alternative splicing in genes associated with follicular development. Furthermore, IGF2BP2 partly influences the expression levels of some of these alternatively spliced genes, including MBD3 and FN1, which may lead to ovarian endocrine disorders. In conclusion, we provide a transcriptome-wide analysis that demonstrates the role played by IGF2BP2 in the regulation of gene expression, transcription and alternative splicing of a number of genes involved in the pathogenesis of ovarian endocrine diseases.

Identification of Gαs messenger ribonucleic acid splice variants in human granulosa cells

1997 ◽  
Vol 18 (1) ◽  
pp. 27-35 ◽  
Author(s):  
G N Europe-Finner ◽  
E Cartwright ◽  
J Bellinger ◽  
H J Mardon ◽  
D H Barlow ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Ovarian Function ◽  
Splice Variants ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Follicular Development ◽  
Protein Isoforms ◽  
Mrna Isoforms ◽  
Messenger Ribonucleic Acid

ABSTRACT Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by Gαs-linked receptors. In this paper we have investigated the expression of Gαs mRNA splice variants in relation to expression of Gαs protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all Gαs mRNA isoforms as well as quantifying total amounts of Gαs mRNA. Granulosa cells express the message for Gαs-Large and Gαs-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of Gαs-Large and Gαs-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in Gαs variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between Gαs variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

scholarly journals Elucidation of Regulatory Modes for Five Two-Component Systems in Escherichia coli Reveals Novel Relationships

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Kumari Sonal Choudhary ◽  
Julia A. Kleinmanns ◽  
Katherine Decker ◽  
Anand V. Sastry ◽  
Ye Gao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Target Gene ◽  
Target Genes ◽  
Rna Seq ◽  
Content Type ◽  
E Coli ◽  
Component Systems ◽  
Two Component ◽  
Two Component Systems

ABSTRACT Escherichia coli uses two-component systems (TCSs) to respond to environmental signals. TCSs affect gene expression and are parts of E. coli’s global transcriptional regulatory network (TRN). Here, we identified the regulons of five TCSs in E. coli MG1655: BaeSR and CpxAR, which were stimulated by ethanol stress; KdpDE and PhoRB, induced by limiting potassium and phosphate, respectively; and ZraSR, stimulated by zinc. We analyzed RNA-seq data using independent component analysis (ICA). ChIP-exo data were used to validate condition-specific target gene binding sites. Based on these data, we do the following: (i) identify the target genes for each TCS; (ii) show how the target genes are transcribed in response to stimulus; and (iii) reveal novel relationships between TCSs, which indicate noncognate inducers for various response regulators, such as BaeR to iron starvation, CpxR to phosphate limitation, and PhoB and ZraR to cell envelope stress. Our understanding of the TRN in E. coli is thus notably expanded. IMPORTANCE E. coli is a common commensal microbe found in the human gut microenvironment; however, some strains cause diseases like diarrhea, urinary tract infections, and meningitis. E. coli’s two-component systems (TCSs) modulate target gene expression, especially related to virulence, pathogenesis, and antimicrobial peptides, in response to environmental stimuli. Thus, it is of utmost importance to understand the transcriptional regulation of TCSs to infer bacterial environmental adaptation and disease pathogenicity. Utilizing a combinatorial approach integrating RNA sequencing (RNA-seq), independent component analysis, chromatin immunoprecipitation coupled with exonuclease treatment (ChIP-exo), and data mining, we suggest five different modes of TCS transcriptional regulation. Our data further highlight noncognate inducers of TCSs, which emphasizes the cross-regulatory nature of TCSs in E. coli and suggests that TCSs may have a role beyond their cognate functionalities. In summary, these results can lead to an understanding of the metabolic capabilities of bacteria and correctly predict complex phenotype under diverse conditions, especially when further incorporated with genome-scale metabolic models.

scholarly journals Female Fertility Is Reduced in Mice Lacking Ca2+/Calmodulin-Dependent Protein Kinase IV**This work was supported by an NIH Medical Scientist Training Program award (to J.Y.W.) and NIH Grants HD-07503 (to A.R.M.) and HD-1272 (to J.S.R.)

Endocrinology ◽  
2000 ◽  
Vol 141 (12) ◽  
pp. 4777-4783 ◽  
Author(s):  
Joy Y. Wu ◽  
Ignacio J. Gonzalez-Robayna ◽  
JoAnne S. Richards ◽  
Anthony R. Means
Keyword(s):  
Protein Kinase ◽  
Granulosa Cells ◽  
Critical Role ◽  
Follicular Development ◽  
Female Fertility ◽  
Female Reproduction ◽  
Dependent Protein Kinase ◽  
Medical Scientist ◽  
Dependent Protein ◽  
Medical Scientist Training Program

Abstract Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) is a serine/threonine protein kinase with limited tissue distribution. CaMKIV is highly expressed in the testis, where it is found in transcriptionally inactive elongating spermatids. We have recently generated mice deficient in CaMKIV. In the absence of CaMKIV, the exchange of sperm nuclear basic proteins in male spermatids is impaired, resulting in male infertility secondary to defective spermiogenesis. The involvement of CaMKIV in female fertility has not been addressed. Here we report that female fertility is markedly reduced in CaMKIV-deficient mice due to impaired follicular development and ovulation. CaMKIV is expressed in the ovary, where it is localized in granulosa cells. We further find that in cultured granulosa cells, CaMKIV expression and subcellular localization are hormonally regulated. As granulosa cells differentiate, CaMKIV levels decrease and the kinase translocates from the nucleus into the cytoplasm. Our results demonstrate a critical role for CaMKIV in female reproduction and point to a potential function in granulosa cell differentiation.

scholarly journals Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

2007 ◽  
Vol 10 (8) ◽  
Author(s):  
D. S. Salomon
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Target Genes ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Specific Effects ◽  
Primary Mouse ◽  
Ductal Elongation

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha. Journal of Cell Science 2007; 120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3−/− MEFs being more sensitive and Timp3−/− MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3−/− MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3−/− mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

scholarly journals TNF-α Suppressed FSH-Induced LH Receptor Expression Through Transcriptional Regulation in Rat Granulosa Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (9) ◽  
pp. 3192-3202 ◽  
Author(s):  
Kohshiro Nakao ◽  
Hiroshi Kishi ◽  
Fumiharu Imai ◽  
Hiroto Suwa ◽  
Takashi Hirakawa ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Ovarian Function ◽  
Receptor Expression ◽  
Luciferase Assay ◽  
Pelvic Cavity ◽  
Transcriptional Level ◽  
Lh Receptor ◽  
Tnf Α

Several inflammatory cytokines regulate ovarian function. TNF-α is produced in granulosa cells under physiological conditions and has a reciprocal action on follicle development. In contrast, in pelvic inflammatory diseases, TNF-α is excessively produced in the pelvic cavity and has an adverse effect on reproductive functions. The objective of this study was to elucidate the mechanism of action of TNF-α on the expression of LH receptor (LHR) in immature rat granulosa cells. TNF-α suppressed FSH-induced LHR mRNA and protein expression and was not associated with cAMP accumulation. By using a luciferase assay, the construct containing base pairs −1389 to −1 of the rat Lhcgr promoter revealed that TNF-α decreased FSH-induced promoter activity. In response to TNF-α, nuclear factor (NF)-κB p65 was translocated to the nucleus, and the suppressive effect of TNF-α on LHR mRNA expression was abrogated by an NF-κB inhibitor. In a chromatin immunoprecipitation assay, TNF-α induced the association of NF-κB p65 with the rat Lhcgr transcriptional promoter region. NF-κB p65 and histone deacetylase (HDAC) interact to mediate expression of several genes at a transcriptional level. HDAC activity is thought to induce tight connections within local chromatin structures and repress gene transcription. Furthermore, the TNF-α–induced suppression of LHR mRNA expression was blocked by an HDAC inhibitor. Taken together, these results suggest that the interaction of NF-κB p65 with HDAC in the promoter region of rat Lhcgr might be responsible for TNF-α action on the regulation of LHR.

285 RELATIONSHIP BETWEEN FOLLICLE FLUID STEROID CONCENTRATIONS, GRANULOSA CELL GENE EXPRESSION, AND BOVINE OOCYTE DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 299
Author(s):  
S. Matoba ◽  
S. Mamo ◽  
E. Gallagher ◽  
A. G. Fahey ◽  
T. Fair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Target Genes ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Oocyte Competence ◽  
Cell Gene Expression ◽  
The Relationship

The ability to culture oocytes and embryos in an individually identifiable manner facilitates the study of the relationship between follicle param- eters and oocyte development, in order to identify markers of competent oocytes. The aim of this study was to examine the predictive value of intrafollicular steroid concentrations and granulosa cell transcript abundance on the ability of immature bovine oocytes to develop to the blastocyst stage in vitro. Individual follicles (n = 214, 11 replicates, 49 animals) were dissected from the ovaries of slaughtered animals. Following measure- ment of diameter, follicles were carefully ruptured under a stereomicroscope and the oocyte was recovered and individually processed through maturation, fertilization, and culture on the cell adhesive Cell-Tak (20 oocytes/100 μL; Matoba and Lonergan 2009 Reprod. Fertil. Dev. 21, 160). Cleavage and blastocyst rates were assessed on Days 2 and 9, respectively. Follicular fluid was recovered and stored at -80°C until analysis for concentrations of the steroids estradiol, progesterone, and testosterone by RIA. Granulosa cells were collected from each follicle for analysis of gene expression by quantitative RT-PCR. Primers were designed for 7 target genes (AMH, CYP19A, ESR1, ESR2, FSHR, HSD3B1 and LHCGR) and 2 reference genes (PPIA and H2AZ). Transcript abundance of target genes in granulosa cells associated with embryos that cleaved and developed to the blastocyst stage (competent) and those that cleaved but failed to develop (incompetent) was examined. Mean steroid concentrations were compared by ANOVA and Spearman correlations, and logistical regression were used to test the relationship between follicle size and steroid con- centration and the ability of steroid concentration to predict developmental competence. Gene expression data were analyzed using the delta-delta CT (cycle threshold) method. Values were normalized to the average values of the reference genes and means were compared by the Student’s t-test In total, 79.1% of oocytes cleaved after IVF and 28.3% developed to the blastocyst stage. The mean (±SEM) follicular concentrations of testosterone (62.8 ± 4.8 ng mL-1), progesterone (616.8 ± 31.9 ng mL-1), or estradiol (14.4 ± 2.4 ng mL-1 were not different (P ≥ 0.05) between competent and incompetent oocytes. Follicular diameter was negatively correlated with testosterone, progesterone, testosterone:estradiol, and pro- gesterone:estradiol (P ≤ 0.01) and positively correlated with estradiol (P ≤ 0.01). Logistical regression analysis showed that steroid concentrations or the ratio of steroids were not satisfactory predictors of oocyte competence. Transcript abundance of AMH, ESR1, ESR2, FSHR, and HSD3B1 was significantly higher (P ≤ 0.05) in granulosa cells associated with competent compared with incompetent oocytes. In conclusion, follicular steroid concentrations were not associated with oocyte development. In contrast, granulosa cell gene expression may be a useful predictor of oocyte competence. Supported by Science Foundation Ireland (07/SRC/B1156).

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