Introduction
BMPs and other TGFβ family members are firmly implicated as intraovarian regulators of ovarian follicle development and steroidogenesis. Whilst multiple TGFβ family ligands and receptor subtypes are known to be expressed in ovarian theca (TC) and granulosa cells, information on which ligand-receptor interactions are important for particular physiological actions is limited. Here we used a primary bovine theca cell culture model to examine whether RNAi-mediated knock down of individual BMP receptors affects androstenedione (A4) secretion and cell proliferation/survival.
Methods
TC from 4-6mm follicles were cultured for 7 days in serum-free medium with LH (150 pg/ml) present from day 3-7. On days 4 and 5 cells were exposed to RNAi duplexes (100nM) targeting bovine BMPR1A (ALK3), BMPR1B (ALK6) or BMPR2; controls included cells treated with non-silencing control RNAi, transfection reagent (TR) alone or no treatment. A4 secretion during day 6-7 was measured by ELISA and viable cell number was determined by neutral red assay at the end of culture. RNA extracts were harvested from representative wells for examination of target gene knockdown using RT-qPCR (β-actin normalisation control). Results are based on 4 independent batches of cells.
Results and Discussion
RT-qPCR indicated ~85% knockdown of BMPR1A and BMPR1B and ~75% knockdown of BMPR2 mRNA expression by the relevant RNAi; non-silencing controls and TR-only controls had similar expression levels to untreated control cells. A4 secretion was raised (P<0.01) by 3.6, 2.6 and 3.2-fold in cells treated with BMPR1A, BMPR1B and BMPR2 RNAi respectively while corresponding cell number was reduced (P<0.05) by 40, 18 and 35%. The results indicate that endogenous TC-derived TGFβ-family ligands that signal via BMPR1A, BMPR1B and/or BMPR2 exert autocrine/paracrine role to suppress thecal androgen production and enhance cell proliferation and/or survival. Further experiments will examine the extent to which knockdown of individual receptors affects responsiveness to exogenous BMP ligands.