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'Free' inhibin ? subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production.

ABSTRACT: Inhibins are ovarian dimeric glycoprotein hormones that suppress pituitary FSH production. They are synthesised by follicular granulosa cells as ? plus ?A/?B subunits (encoded by INHA, INHBA, INHBB, respectively). Inhibin concentrations are high in follicular fluid (FF) which is also abundant in 'free' ? subunit, presumed to be of granulosal origin, but its role(s) remains obscure. Here, we report the unexpected finding that bovine theca cells show abundant INHA expression and 'free' inhibin ? production. Thus, theca cells may contribute significantly to the inhibin ? content of FF and peripheral blood. In vitro, knockdown of thecal INHA inhibited INSL3 and CYP17A1 expression and androgen production while INSL3 knockdown reduced INHA and inhibin ? secretion. These findings suggest a positive role of thecal inhibin ? on androgen production. However, exogenous inhibin ? did not raise androgen production. We hypothesised that inhibin ? may modulate the opposing effects of BMP and inhibin on androgen production. However, this was not supported experimentally. Furthermore, neither circulating nor intrafollicular androgen concentrations differed between control and inhibin ?-immunized heifers, casting further doubt on thecal inhibin ? subunit having a significant role in modulating androgen production. Role(s), if any, played by thecal inhibin ? remain elusive.

SUBMITTER: Laird M

PROVIDER: S-EPMC6930203 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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'Free' inhibin α subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production.

Laird Mhairi M Glister Claire C Cheewasopit Warakorn W Satchell Leanne S LS Bicknell Andrew B AB Knight Phil G PG

Scientific reports 20191224 1

Inhibins are ovarian dimeric glycoprotein hormones that suppress pituitary FSH production. They are synthesised by follicular granulosa cells as α plus βA/βB subunits (encoded by INHA, INHBA, INHBB, respectively). Inhibin concentrations are high in follicular fluid (FF) which is also abundant in 'free' α subunit, presumed to be of granulosal origin, but its role(s) remains obscure. Here, we report the unexpected finding that bovine theca cells show abundant INHA expression and 'free' inhibin α p ...[more]

PMID: 31874971

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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).

Dataset Information

'Free' inhibin ? subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production.

Publications

'Free' inhibin α subunit is expressed by bovine ovarian theca cells and its knockdown suppresses androgen production.

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