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| Status |
Public on Mar 15, 2013 |
| Title |
Expression data from bovine ovarian theca cells in primary culture |
| Organism |
Bos taurus |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| Overall design |
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 (± 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 manufacturer’s instructions. RNA yield and quality were evaluated by spectrophotometry at 260/280 nm and agarose gel electrophoresis before submitting 5 μg 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).
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| Contributor(s) |
Knight P, Rodgers R, Glister C |
| Citation(s) |
23530236 |
| Submission date |
Feb 27, 2013 |
| Last update date |
Apr 02, 2013 |
| Contact name |
Phil G Knight |
| E-mail(s) |
p.g.knight@reading.ac.uk
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| Organization name |
University of Reading
|
| Street address |
Hopkins Building, Whiteknights
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| City |
Reading |
| ZIP/Postal code |
RG6 6UB |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL2112 |
[Bovine] Affymetrix Bovine Genome Array |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA191062 |
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