Unknown,Transcriptomics,Genomics,Proteomics

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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. 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-

ORGANISM(S): Bos taurus

SUBMITTER: Phil Knight

PROVIDER: E-GEOD-44704 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the pre-ovulatory LH-surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal and granulosa cell type-specific biological functions and signaling pathways, large dominant bovine follicles were collected before and 21 hrs after an exogenous GnRH induced LH surge. Because LH receptor density varies within the granulosa cell populations, antral granulosa (aGC; those aspirated by follicular puncture) and membrane associated granulosa (mGC; those scraped from the follicular wall) were compared to thecal cell expression profiles determined by mRNA microarrays. Thecal cell gene expression was less affected in the peri-ovulatory follicle when compared to granulosa cells, as evidenced by only 2% versus 25% of the ~11,000 genes expressed changing in response to the LH surge, respectively. The majority of the 203 LH-regulated thecal genes were also LH regulated in granulosa cells, leaving a total of 58 genes as LH-regulated theca cell specific genes. Most of the 58 genes (i.e., 74%) thecal specific genes including several known thecal markers (CYP17A1, NR5A1) were downregulated, while most genes identified are new to theca. Many of the newly identified upregulated thecal genes (e.g., PTX3, RND3, PPP4R4) were also upregulated in granulosa. Minimal expression differences were observed between aGC and mGC, however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) predominated these differences. We also identified large numbers of unknown LH-regulated granulosa cell genes and discuss their putative roles in ovarian function. The single dominant ovarian follicle was collected from each cow before the LH surge or 22 hours after GnRH (used to induce LH surge). RNA was extracted from three independent cells within each follicle and there were hybridized on Affymetrix microarrays.

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Expression data from bovine ovarian theca cells in primary culture

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