Studies On The Function And Related Regulatory Mechanisms Of IFITM1 Gene In Murine Follicular Development And Ovulation

Follicular development and ovulation,a complex physiological process,requires multiple factors or signaling pathways,including endocrine hormones,immune regulation and metabolic signals.Nevertheless,the molecular mechanism of follicular development to ovulation is still relatively limited.In previous studies,2675 genes differentially expressed in the pre-ovulatory follicles of Large White and Chinese Meishan sows by transcriptome sequencing.Interferon-induced transmembrane protein 1(IFITM1)was significantly differently expressed,which showed 3.174 fold changes in pre-ovulatory ovarian follicles between Large White and Chinese Meishan sows.In this study,primary mouse ovarian granulosa cells(mGCs)was used as model and we applied transfection,qRT-PCR,Western blot,ELISA,EDU staining,MTT,CCK-8 and FCM(Flow CytoMetry)to uncover the function and related molecular regulation mechanism of IFITM1 gene in ovulation,which also provides new insights into whether IFITM1 gene can be used as a candidate gene for litter size.The main research results are as follows: 1.Function of IFITM1 gene in ovulation and related molecular regulation mechanism(1)IFITM1 gene affects ovulation marker gene expression and molecular mechanism.Inhibition of IFITM1 gene expression in mGCs by qRT-PCR and Western blot showed that the expression of ovulation marker genes LHR,AREG,EREG and Cyp19a1 were significantly enhanced.The rescue experiments showed that overexpression of IFITM1 gene can significantly inhibit the expression of ovulation marker gene LHR and Cyp19a1.Inhibitors of three key cell signaling pathways(ERK1/2,TGF-?,and PI3K/AKT)which regulate follicular development or ovulation were added to mGCs to inhibit the cellular signaling pathways.The results showed that inhibiting the expression of IFITM1 gene through the inhibition of ERK1/2 or TGF-? signaling pathway,significantly increased the mRNA levels of ovulation marker genes EREG,LHR by qRT-PCR;However,inhibiting the PI3 K /AKT signaling pathway did not inhibit the mRNA levels expression of ovulation markers LHR,Cyp19a1 and EREG.It showed that the protein level of p-AKT(Ser473)decreased significantly when inhibiting the expression of the IFITM1 by Western blot.These results demonstrate that IFITM1 does not affect ovulation marker gene expression through ERK1/2 or TGF-? signaling pathways,but affects the expression of ovulation marker genes through the PI3K/AKT cell signaling pathway.(2)IFITM1 gene affects the concentration of progesterone of mGCs.In mGCs,IFITM1 was overexpressed or inhibited,and then the mRNA levels of progesterone receptor was measured by qRT-PCR.The mRNA expression of PGR was significantly increased by over-expressing IFITM1;Inhibiting the expression of IFITM1 gene,the mRNA levels of PGR significantly declined,however,the mRNA levels of 3?-HSD,StAR significantly increased.pCMV-HA-IFITM1 or siRNA-IFITM1 was transfected into mGCs to measure the level of progesterone by ELISA.The results showed that there was no significant change in the production of progesterone by overexpressing IFITM1,however,the production of progesterone was significantly reduced by inhibitting the expression of IFITM1.In summary,IFITM1 gene affects ovulation process and may affect the level of progesterone in mouse granulosa cells.2.IFITM1 gene affects cell cycle and cell proliferation in mouse granulosa cells.The effect of IFITM1 gene on granulosa cells cycle was measured by Flow cytometry.The results showed that the overexpression of IFITM1 gene significantly decreased the percentage of granulosa cells in the S phase.In addition,the percentage of granulosa cells in G0/G1 phase significantly decreased by transfecting siRNA-IFITM1 into mGCs.The results of qRT-PCR showed that the overexpression of IFITM1 significantly increased the mRNA level of CCNA2 and CCND1 and the mRNA level of CCND1 decreased when IFITM1 was inhibited.IFITM1 gene can regulate follicular ovulation by influencing the cycle of granulosa cells.EDU experiment,CCK-8 assays and MTT assays were used to measure the mouse granulosa cells proliferation.The experimental results showed: The level of cell proliferation was significantly reduced by inhibiting IFITM1 gene compared with the control group.In conclusion:(1)IFITM1 gene affects the secretion of progesterone in ovarian granulosa cells,and affects follicular ovulation by influencing the activity of PI3K/AKT signaling pathway.(2)The expression of IFITM1 gene regulates the cell cycle of the mGCs by regulating the expression of CCND1 in the cell cycle.IFITM1 gene regulates the proliferation of ovarian granulosa cells.