Mechanism Of CLOCK Regulating Proliferation And Estradiol Synthesis Of Porcine Ovarian Granulosa Cells

Reproduction is crucial link in swine production.Enhancing the reproduction of sows is vitally important to the economic performance of piggery and the development of swine industry.The development state of the follicle is the key factor that affects the reproductive ability of sows.GCs are the most considerable cell population in developing follicles,which perform critical physiological functions,including proliferation and estrogen synthesis that will affect the normal development and atresia of the follicle.Therefore,it is crucial to investigate the factors,which regulate the physiological function of granulosa cells.CLOCK is the core regulatory factor of the biological clock system,which forms a dimer complex with BMAL1 protein and combines with the E-box sequence of the target gene promoter to promote transcription.Previous studies have reported that CLOCK is concerned with the physiological function of granulosa cells,but the specific molecular mechanism remains unclear.Therefore,in this study,porcine ovarian granulosa cells were selected as the research object.To clarify the role of CLOCK in granulosa cell proliferation and estradiol synthesis and its potential molecular mechanism.Molecular biological detection methods and transcriptome sequencing were used after overexpression and interference of CLOCK treatment,including immunohistochemistry,cellular immunofluorescence,RT-q PCR,western blotting,dual luciferin reporting,Ch IP,ELISA,transmission electron microscopy detecting and so on.The results of this study are as follows.1.CLOCK is expressed in ovary and granulosa cells.CLOCK was widely expressed in preantral follicles and antral follicles.The expression of CLOCK was the highest in granulosa cells.CLOCK was expressed in cytoplasm and nucleus in granulosa cells.CLOCK was rhythmically expressed in granulosa cells.2.CLOCK inhibits granulosa cell proliferation.Overexpressed CLOCK inhibited the proliferation of granulosa cells,and significantly inhibited the transcription and translation of CCNB1 and CCNE1,and up-regulated CDKN1 A levels(P < 0.05);Interference with CLOCK promoted granulosa cell proliferation,dramatically promoted the transcription of CCNB1,CCNE1,CDK1 and CDK4,and up-regulated the protein level of CDK4(P < 0.05).3.CLOCK inhibited estradiol synthesis in granulosa cells.In granulosa cells,CLOCK overexpression markedly reduced estradiol level,inhibited the transcription and translation of CYP19A1,St AR and CYP11A1,and down-regulated the protein level of CYP17A1(P < 0.05);Interference with CLOCK significantly promoted the synthesis of estradiol,promoted the expression of CYP19A1 and St AR at the m RNA and protein levels,and up-regulated the protein levels of CYP17A1(P < 0.05).4.CLOCK inhibits the proliferation of granulosa cells by targeting ASB9.Transcriptome sequencing was performed after overexpression of CLOCK.ASB9 is a newly-identified target of CLOCK.Overexpression of ASB9 significantly inhibited granulosa cell proliferation,and interference with ASB9 significantly promoted granulosa cell proliferation(P < 0.05).The results of double luciferase reporter assay and chromatin immunoprecipitation assay showed that CLOCK could bind the E-box element on the ASB9 promoter to promote the transcription of ASB9.5.CLOCK regulates mitochondrial function and inhibits estradiol synthesis.Overexpression of CLOCK significantly decreased ATP content,mitochondrial copy number and mitochondrial membrane potential(MMP)in granulosa cells,and significantly increased reactive oxygen species(P < 0.05).Overexpression of CLOCK damages the morphology and biological function of mitochondria,resulting in mitochondrial crest fracture and stroma swelling.In summary,CLOCK inhibits granulosa cell proliferation by targeting ASB9,while CLOCK inhibits estradiol synthesis by regulating mitochondrial function.