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Expression data from bovine ovarian theca cells in primary culture

ABSTRACT: Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. We carried out a microarray analysis to examine global changes in gene expression in bovine theca cells in response to treatment with BMP6 alone and in combination with LH. There was a major effect of BMP6 treatment on the gene expression profile with a much weaker effect of LH. None of these differences in response to LH treatment was found to be statistically significant after applying Benjamini-Hochberg correction. BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis.

ORGANISM(S): Bos taurus

PROVIDER: GSE44704 | GEO | 2013/03/15

SECONDARY ACCESSION(S): PRJNA191062

REPOSITORIES: GEO

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Project description:Bone morphogenetic proteins (BMP) are firmly implicated as intra-ovarian regulators of follicle development and steroidogenesis. We carried out a microarray analysis to examine global changes in gene expression in bovine theca cells in response to treatment with BMP6 alone and in combination with LH. There was a major effect of BMP6 treatment on the gene expression profile with a much weaker effect of LH. None of these differences in response to LH treatment was found to be statistically significant after applying Benjamini-Hochberg correction. BMP6 significantly (>2-fold; P<0.01) up- or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (-43-fold) with CYP17A1 and other key transcripts involved in TC steroidogenesis including LHCGR, INHA, STAR, CYP11A1 and HSD3B1 also down-regulated. BMP6 also reduced expression of NR5A1 encoding steroidogenic factor-1 known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. Theca interna cells (TC) were isolated from adult bovine ovaries obtained from the slaughterhouse. TC pooled from approximately 50 individual 4-6mm follicles were plated out in in 24-well plates (0.5 x106 viable cells/ml/well) and cultured for 6 days under defined serum-free conditions with treatments present on days 3-6 inclusive. TC were treated for 4 days with BMP6 (10 ng/ml) under both basal and LH-stimulated conditions (M-BM-1 160 pg LH/ml). These dose-levels of BMP6 and LH were chosen because they elicited optimal responses in our previous studies. The experiment was replicated four times with TC harvested from different batches of ovaries. Total RNA was isolated using the Ribopure RNA isolation kit (Ambion) according to the manufacturerM-bM-^@M-^Ys instructions. RNA yield and quality were evaluated by spectrophotometry at 260/280 nm and agarose gel electrophoresis before submitting 5 M-NM-<g of each RNA sample (n = 16 comprising 4 biological replicates x 4 treatment conditions) to an accredited Affymetrix service provider for microarray analysis using the bovine genome array GeneChip (n=16 arrays).

Project description:The purpose of this study is to assess the potential of TOTM to induce testicular maldevelopment in the rat. We studied the effects of TOTM on the expression of genes in pathways involved in steriodogenesis and testes development. Certain phthalate esters have previously been shown to be involved in the induction of rat testicular mal-development (TMD) through effects on the expression of genes in pathways involved in steroidogenesis and testes development. In order to assess the effects of a potential alternate plasticizer, tris(2-ethylhexyl)trimellitate (TOTM), rats were exposed daily, in utero, to TOTM in order to assess the potential of this compound to induce developmental effects on the fetal testes. Pregnant rats were exposed between gestational day 12 and 19 and fetal testes RNA was analysed using whole genome microarrays. The effects of TOTM on the expression of genes in pathways involved in steroidogenesis and testes development were examined. The effects of TOTM were also compared with di(2-ethylhexyl)phthalate (DEHP), mono(2-ethylhexyl)phthalate (MEHP), an active metabolite of DEHP, and 2-ethylhexanol (2-EH), which were used as positive and negative controls, respectively. MEHP & DEHP (500mg/kg) caused a repression of genes in TMD pathways involved in cholesterol synthesis and transport (HMGCS, HMGCR, StAR, SCARB1, FDFT1, FDPS), steroidogenesis (Cyp11a, HSD3B1, SC4MOL) and testes development (INSL3, INHA). 2-EH caused minor repression of some of the genes in the TMD pathway. This was rationalised on the basis that 2-EH, a DEHP metabolite, is also a weak PPARα agonist. It has been shown that in utero treatment with DBP will repress the genes from fetal testes involved in steroidogenesis and that this effect is associated with direct DBP-mediated binding of PPARα to the promoters of these genes (Plummer et. al. 2010). TOTM did not cause a significant repression of genes in the TMD pathway. Based on these data, it is highly unlikely that TOTM will cause testicular dysgenesis in rats. Rats were exposed to TOTM in utero by the administration of daily oral gavage doses to pregnant dams between gestational day 12 and 19. The foetal testes were obtained by micro-dissection and prepared to facilitate the isolation of RNA, which was subsequently analysed using whole rat genome microarrays.

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Expression data from bovine ovarian theca cells in primary culture

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