BMP15 regulates FSHR through TGF-β receptor II and SMAD4 signaling in prepubertal ovary of Rongchang pigs

Abstract

Bone morphogenetic protein 15 (BMP15) and follicle-stimulating hormone (FSH) both play important roles in mammalian ovary and follicular development. The aim of the present study is to investigate the effects of BMP15 and FSH in the prepubertal ovary of Rongchang pigs considering a possible signaling mechanism involving TβRII/ SMAD4 and FSHR in granulosa cells. For this purpose, we quantified expression levels of BMP15, SMAD2, SMAD3, SMAD4, SMAD7, TGF-β1, TGF-β2, TGF-β3, TGFβRI, TGFβRII, and FSHR via qRT-PCR at different ages in prepubertal ovaries and cultured biopsy of 90-day-old ovary in Rongchang pig. Additionally, the protein levels of BMP15, FSHR, SMAD2, SMAD4, TGFβRI, TGFβRII, TGF-β1, TGF-β2 were quantified via Western blot and the localizations of BMP15, FSHR and TGFβRII were observed via immunofluorescence confocal microscope. The results showed that expression levels of BMP15, TGF-β1, TGFβRII and FSHR increased significantly at day 60 as compared to day 30 and reached peak value at day 90 in prepubertal ovary of Rongchang pigs. We observed that BMP15, TGFβRII and FSHR was highly presented, which TGFβRII and FSHR displayed co-localization in the follicles of the prepubertal ovaries of 90-day-old Rongchang gilts. Treatment with TGFβRI/II inhibitor LY2109761 significantly decreased the expression of TGFβRI, TGFβRII and SMAD4 and TGFβRI inhibitor LY2157299 decreased TGFβRI, but increased the TGFβRII, SMAD4 and FSHR expression levels. Furthermore, the addition of rBMP15 and rFSH group significantly increased the expression of TGFβRII and FSHR proteins (P < 0.01), but no significant change in the expression of TGFβRI (P > 0.05) was observed by Western blot. In conclusion, BMP15, TGFβRII and FSHR were increased significantly in the prepubertal ovarian follicles of Rongchang pigs and FSHR expression in GCs was regulated by BMP15 and FSH through the TGFβRII.