Bmal1 interference impairs hormone synthesis and promotes apoptosis in porcine granulosa cells

Abstract

In mammals, granulosa cell proliferation, differentiation, luteinization, apoptosis, and hormone synthesis are tightly related to oocyte maturation, follicular development and ovarian function. In current study, we investigated the role of the key circadian clock gene, brain and muscle arnt-like protein-1 (Bmal1), on porcine granulosa cell hormone secretion and apoptosis. The transcription levels of circadian clock genes, including Bmal1 and period circadian clock 2 (Per2), were detected by RT-qPCR. We found that the circadian clock genes exhibited rhythmic change and were further enhanced by dexamethasone synchronization in granulosa cells. Bmal1 knockdown reduced transcriptional levels of hormone receptor genes, including follicle stimulating hormone receptor (Fshr), luteinizing hormone/choriogonadotropin receptor (Lhcgr) and estrogen receptor 2 (Esr2), and decreased the mRNA and protein levels of cytochrome P450 family 11 subfamily A member 1 (Cyp11a1), cytochrome P450 family 19 subfamily A member 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star), which are the key enzymes involved in hormone synthesis. Synthesis of progesterone and estradiol were also inhibited by Bmal1 siRNA treatment in granulosa cells. Moreover, flow cytometry analysis demonstrated suppressing Bmal1 promoted granulosa cells apoptosis. Western blot analysis showed that Bmal1 interference inactivated the PI3K/Akt/mTOR signaling pathway. In conclusion, Bmal1 plays a critical role in secretion of hormone and apoptosis of porcine granulosa cells via the PI3K/Akt/mTOR signaling pathway.