Molecular Mechanism Of NR1D1 Regulating Estrogen Synthesis In Porcine Ovarian Granulosa Cells

The reproduction performance of sows will directly affect the economic benefits of pig farms,and it is an important index to evaluate the production capacity of pig farms.For long term consideration,it is particularly important to improve the reproduction performance of sows.The fertility of sows is regulated by various factors.And the synthesis and secretion of steroid hormones affect the reproductive physiological process of sows,especially the key stages of follicular development and ovulation.Follicles are important sites for the synthesis and secretion of gonadal steroid hormones in the ovary.Granulosa cells,as the main follicle body cells,play an important role in the synthesis of gonadal steroid hormones.Follicle stimulating hormone(FSH)and luteinizing hormone(LH)affect the mature and mature granulosa cells respectively,which can promote the proliferation and maturation of follicular cells and the synthesis of gonadal steroids.The synthesis and secretion of FSH and LH have obvious biological rhythm.Biorhythm is a life phenomenon widely existing in nature,and has the function of adapting the body to the periodic changes of environment and behavioral activities.A large number of studies have shown that there is the expression of biological clock gene in ovary,and the biological clock function in ovary is related to steroid hormone synthesis,follicular development,follicular somatic cell differentiation and ovulation.Nuclear receptor NR1D1 combines circadian rhythm with many physiological processes.It has been reported that NR1D1 can regulate ovarian circadian rhythm and metabolism,but its specific effect on gonadal steroid hormone synthesis is not clear.Therefore,the purpose of this study is to explore the effect of NR1D1 on the synthesis and secretion of steroids and its molecular mechanism.Porcine ovarian granulosa cells were isolated and cultured,and were treated with NR1D1 activators,inhibitors and Nr1d1 siRNA.A series of cellular,molecular and biochemical methods such as Immunohistochemistry,cellular Immunofluorescence staining,Real-time quantitative PCR,Western blotting and Enzyme-linked immunosorbent assay were used to clarify the effect of NR1D1 on steroid synthesis and secretion,and explore its molecular mechanism.The main results are as follows:1: NR1D1 was widely expressed in porcine ovarian granulosa cells,thecal cells,oocytes and stromal cells.The expression of NR1D1 in granulosa cells is more than that of other cell types;cell immunofluorescence staining shows that NR1D1 is mainly in the nucleus of granulosa cells;real-time q PCR detection found that the circadian clock gene NR1D1 and steroid hormone synthesis key genes CYP19A1,CYP11A1,StAR expression in ovarian granulosa cells showed rhythm.2: The addition of NR1D1 activator GSK4112 significantly inhibited the expression of circadian clock gene Bmal1 and steroid synthesis key genes CYP19A1,CYP11A1,StAR at the m RNA level and protein level;the number of cells in S phase decreased after GSK4112 treatment,indicating that the proliferation of granulosa cells was inhibited by activating NR1D1.ELISA results prove that CYP19A1,CYP11A1,StAR,3β-HSD and hormone receptor genes FSHR and LHCGR,which are key enzymes in steroid synthesis of biological clock genes NR1D1,BMAL1 and CRY1,showed obvious rhythmic expression in the control group,but the addition of GSK4112 could significantly inhibit the synthesis of estradiol and progesterone in porcine ovarian granulosa cells.GSK4112 treatment weakened the m RNA expression rhythm of BMAL1,CRY1,CYP19A1,StAR,3β-HSD,FSHR and LHCGR,and decreased the amplitude of gene expression.3: The effect of NR1D1 siRNA and inhibitor SR8278 on porcine ovarian granulosa cells was opposite to that of GSK4112 treatment.The biological clock gene Bmal1,steroid synthesis key genes CYP19A1,CYP11A1,StAR,3β-HSD and hormone receptor genes FSHR,LHCGR and ERβ m RNA level were significantly increased.The expression level and the protein level of CYP19A1 were significantly increased;the addition of SR8278 promoted the proliferation of granulosa cells and significantly inhibited the synthesis of estradiol in porcine ovarian granulosa cells;At the same time,the circadian clock genes BMAL1,CRY1,PER1,PER2,steroid biosynthesis key genes Star and hormone receptor genes FSHR,LHCGR,ERβ increased significantly under the action of SR8278,while the rhythmic expression of CYP19A1 disappeared after SR8278 treatment,and increased significantly after 12 hours.4: In order to investigate whether CYP19A1 plays a role in the regulation of estrogen synthesis by NR1D1,CYP19A1 was over-expressed and interfered under the condition of adding NR1D1 activator GSK4112 or inhibitor SR8278 respectively.The recovery experiments showed that overexpression of CYP19A1 could eliminate the inhibitory effect of NR1D1 on estrogen synthesis.While interfering with CYP19A1 could promote the inhibitory effect of NR1D1 on estrogen synthesis.Furthermore,the double luciferase reporter activity test showed that NR1D1 regulates the transcription of CYP19A1 by binding to the RORE elements of the CYP19A1 promoter region-1228 ～-1223 and-1116 ～-1111 dotted RORE element thus affecting the synthesis and secretion of estradiol in porcine ovarian granulosa cells.In summary,in porcine ovarian granulosa cells,activation of the circadian clock regulator NR1D1 enhances the inhibitory effect of NR1D1 on transcriptional activity,recognizes and binds to the RORE element on the CYP19A1 promoter,inhibits the transcription of CYP19A1,and leads to a decrease in estradiol content;It inhibits the activity of NR1D1 and its ligand binding,thereby eliminating the transcriptional inhibition of CYP19A1,and increasing the synthesis and secretion of estradiol in porcine ovarian granulosa cells.