The Effect Of BMAL1 On Steroidogenesis And Apoptosis Of Pig Granulosa Cells

In mammals, ovaries development is governed by signals of hypothalamic–hypophyseal origin, involved in folliculogenesis, follicular atresia, ovulation, luteinization, and luteolysis accompanied by proliferation, differentiation, and apoptosis of granulosa and luteal cells. Follicle-stimulating hormone(FSH) and luteinizing hormone(LH) stimulate proliferation and differentiation of granulosa cells and synthesis of sex steroid hormones. The importance of circadian rhythms in female reproduction is gradually demonstrated. Especially, brain muscle arnt-like 1(Bmal1), the core circadian clock gene, has been shown to be related to disruption of endocrine and abnormalities of reproduction, as impaired expression of steroidogenic genes, reduced ovulation, altered corpora lutea formation, and failed implantation in Bmal1 null mice. But the underlying mechanisms of Bmal1 involved in proliferation, differentiation, apoptosis, and estrogen synthesis of granulosa cells need further exploration.In this study, we used siRNA to interfere the expression of circadian clock gene Bmal1 in pig granulosa cells. The mRNA expressions of hormone receptor genes including Fshr, Lhcgr and ERβ were detected by real-time qPCR. The transcription and translation levels of CYP11a1, CYP19a1 and STARwere analyzed by real-time qPCR and western blot. Moreover, we processed sequence alignment in gene promoters to search circadian clock control elements(E-box、D-box and RORE). The concentrations of estradiol(E2) and progesterone(P4) in culture medium were detected by chemiluminescence immune assay. Finally, the protein levels and their phosphorylation levels of PI3K/AKT/mTORsignaling pathway were analyzed. The main results are as follows:1.The circadian clock system existed in pig granulosa cells. The rhythmicity expressions of Bmal1, Per2, Cry and Cyp11a1 have been shown in 24 hours and were enhanced by dexamethasone treatment.2.Interference of Bmal1 impaired expression of several circadian clcok genes, hormone receptor genes(Fshr and Lhcgr) and genes related to steroidogenesis(Cyp11a1、Cyp19a1and Star), as well as their rhythmic expressionin 24 hours. Thecircadian clock control elements(E-box、D-box and RORE) have been foud in those ovarian genes, which further confirmed that circadian clock system efects steroidogenesis through transcriptional regulation.3.Suppression of Bmal1 expression promoted apoptosis of luteinizing granulosa cells in both early and late stage.4.The activity of PI3K/AKT/mTORsignaling pathway weredecreased by suppression of Bmal1 expression.All in all, our data revealed thatBmal1 and circadian clock system involved in steroidogenesis and apoptosis of pig granulosa cells. Interference of Bmal1 impaired circadian clock system, which further decreased steroidogenesis and cell survival rate by regulation of ovarian genes though circadian clock control elements in thire our promoters and/or PI3K/AKT/mTORsignaling pathway.