Regulation of bovine tumor necrosis factor-alpha-induced protein 6 in ovarian follicles during the ovulatory process and promoter activation in granulosa cells.
ABSTRACT To study the regulation of bovine TNFalpha-induced protein 6 (TNFAIP6) prior to ovulation, preovulatory follicles obtained after the treatment with human chorionic gonadotropin (hCG) were used. RT-PCR analyses showed that levels of TNFAIP6 mRNA were low before hCG but significantly increased after hCG treatment in follicles. Further analyses and immunohistochemistry indicated that this increase in transcript and protein levels occurred in theca and granulosa cells. To investigate molecular mechanisms involved in TNFAIP6 transactivation, the activity of bovine TNFAIP6 promoter was studied in granulosa cell cultures. Mutant studies identified the minimal region conferring full-length promoter activity, in which activator protein-1 (AP1) and cAMP response element (CRE) elements were required for promoter activity. Overexpression of dominant-negative AP1 and activating transcription factor/cAMP response element-binding protein (CREB) inhibited forskolin-inducible promoter activity. DNA binding assays demonstrated the importance of AP1 and CRE for activity and identified JunD, FosB, Fra2, CREB1, and CREB2 as being part of the AP1 complex, and FosB, Fra2, and CREB1 for the CRE complex. Chromatin immunoprecipitation assays confirmed binding of these proteins with endogenous TNFAIP6 promoter. Treatment with forskolin, prostaglandin E2, and catalytic subunit protein kinase (cPKA) stimulated, but H89, PKA inhibitor peptide, and indomethacin inhibited, TNFAIP6 promoter activity and gene expression in granulosa cells. Collectively, this study is the first to describe that the ovulatory process in cows is associated with a gonadotropin-dependent induction of TNFAIP6 in ovarian follicles and provide the molecular basis through which AP1 and CRE sites and PKA activation played important roles in the regulation of TNFAIP6 in granulosa cells.
- [show abstract] [hide abstract]
ABSTRACT: The objective of this study was to explore whether hyperandrogenism induces epigenetic alterations of peroxisome proliferator-activated receptor gamma 1 (PPARG1), nuclear corepressor 1 (NCOR1), and histone deacetylase 3 (HDAC3) genes in granulosa cells (GCs) of polycystic ovary syndrome (PCOS) women and whether these alterations are involved in the ovarian dysfunction induced by hyperandrogenism. Thirty-two infertile PCOS women and 147 infertile women with tubal blockage were recruited. PCOS women were divided into the hyperandrogenism (HA) PCOS group (n = 13) and nonhyperandrogenism (N-HA) PCOS group (n = 19). Sixty female Sprague-Dawley rats were used for PCOS model establishment. In GCs of HA PCOS women, PPARG1 mRNA expression was lower, whereas NCOR1 and HDAC3 mRNA expression were higher than N-HA PCOS women and controls (P < 0.05). When all women were divided into successful and failed pregnancy subgroups according to the following clinical pregnancy outcome, we found lower PPARG1 mRNA levels and higher NCOR1 and HDAC3 mRNA levels in the failed subgroup of HA PCOS (P < 0.05). Two hypermethylated CpG sites in the PPARG1 promoter and five hypomethylated CpG sites in the NCOR1 promoter were observed only in HA PCOS women (P < 0.01 to P < 0.0005). The acetylation levels of histone H3 at lysine 9 and p21 mRNA expression were decreased in human GCs treated with dihydrotestosterone in vitro (P < 0.05). PCOS rat models also showed alterations of PPARG1, NCOR1, and HDAC3 mRNA expression and methylation changes of PPARG1 and NCOR1, consistent with the results from humans. Hyperandrogenism induces the epigenetic alterations of PPARG1, NCOR1, and HDAC3 in GCs, which are involved in the ovarian dysfunction of HA PCOS.Journal of Molecular Medicine 02/2012; 90(8):911-23. · 4.77 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: To identify reliable genomic biomarkers expressed in cumulus cells that accurately and non-invasively predict the oocyte developmental competence and reinforce the already used morphological criteria. Eight consenting patients were selected for ovarian stimulation and ICSI procedures. Cumulus-oocyte complexes were transvaginally punctured and individually selected based on both good morphological criteria and high zona pellucida birefringence. Following ICSI, two 3-day embryos per patient were transferred. Pregnancy outcome was recorded and proven implantation was thereafter confirmed. Differential gene expression was assessed using two microarray platforms. Further real-time PCR validation, Ingenuity pathways analysis and intra-patient analysis were performed on 17 selected candidates. Seven genes were differentially (p ≤ 0.05) associated to successful pregnancy and implantation. These biomarkers could be used to predict the oocyte developmental competence. These genomic markers are a powerful reinforcement of morphological approaches of oocyte selection. Their large-scale validation could increase pregnancy outcome and single embryo transfer efficiency.Journal of Assisted Reproduction and Genetics 10/2010; 28(2):173-88. · 1.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Follicular development and ovulation process are complex events in which a sequential process is started by two pituitary hormones, FSH and LH. Recent studies using microarray analysis have shown that numerous endocrine factors are expressed and specifically activate the signal transduction cascades to upregulate or downregulate the expression of target genes in granulosa cells and cumulus cells. Especially, the PI 3-kinase-AKT pathway during the follicular development stage and the ERK1/2 pathway after LH surge are essential for initiating the dramatic changes in follicular function. The proliferation of granulosa cells and cell survival are dependent on the FSH-induced PI 3-kinase-AKT pathway in a c-Src-EGFR dependent manner. On the other hand, the transient activation of ERK1/2 by LH surge is dependent on de-novo transcription and translation. EGF-like factors, especially amphiregulin, that are expressed in a cAMP-PKA-CREB dependent manner, act on EGFR expressed on granulosa cells and cumulus cells. The phosphorylated EGFR induces the RAS-cRAF-MEK-ERK1/2 pathway in both cell types. One of the ERK1/2 target molecules is C/ EBP that is a member of transcription factors Increasing the expression of genes involved in granulosa cell luteinization, cumulus expansion and oocyte maturation. These signaling cascades upregultaed by FSH or LH in granulosa cells play important roles in follicular development and ovulation process.Journal of Mammalian Ova Research 06/2011;