Study On Expression And Function Of Bmal1 In Granulosa Cells Of Yak

Bmal1,is Aryl hydrocarbon receptor nuclear translocator-like(ARNTL),a core component of mammalian circadian clock transcription and translation feedback loop.Bmal1 gene exists in mammalian reproductive organs and participate in the regulation of animal reproductive performance,but this gene has rarely been reported in bovine reproduction.In order to explore the distribution characteristics and regularity of Bmal1 gene expression in yak ovaries,the effect on the secretion and apoptosis of yak ovarian granulocyte cells of this gene.This study took healthy yak ovaries from the Yutai slaughterhouse in Xining.Immunohistochemistry was used to explore the localization and distribution of BMAL1 protein in yak ovaries;Ovarian granulosa cells were cultured in vitro and regular expression of Bmal1 gene at different culture time points in vitro by qRT-PCR.The expression of Bmal1 gene in granular cells was knocked down by siRNA technology.The content of estradiol(E2)and progesterone(P4)in the cell culture medium was detected by ELISA;the expression of hormone receptor genes,synthetic genes were detected by qRT-PCR and Western blot.Finally,the apoptosis of granulosa cells was detected by Annexin V staining,and the expression of apoptosis factors Bax and Bcl-2 was detected by qRT-PCR and Western blot.My research results are as follows:(1)In the yak ovary,BMAL1 protein is mainly expressed in the reproductive epithelium and the granular cell layer of follicles of different sizes.(2)Bmal1 gene was rhythmically expressed in yak granulosa cells at different culture time points within 24 hours after dexamethasone simulation treated,.(3)Compared with the control group: the contents of E2 and P4 in the cell culture medium were significantly reduced;the relative expression of hormone secretion-related genes was detected by mRNA and protein levels and found that the hormone receptor genes(Fshr and Er?)in granular cells,the expression of hormone synthesis genes(Cyp19a1 and Star)were down-regulated,after inhibiting the expression of Bmal1 gene.(4)After inhibiting the expression of Bmal1 gene,the number of apoptotic granulosa cells increased(P <0.01);the expression of pro-apoptotic gene Bax was up-regulated,and the expression of apoptotic gene Bcl-2 was down-regulated.According to the above research results,it is concluded that the Bmal1 gene and the clock rhythm molecular mechanism exist in yak ovarian granulosa cells;the interference of Bmal1 gene can inhibit the hormone secretion of yak granulosa cells and promote cell apoptosis.