The Mechanism Of The Effect Of The Disruption Of Light Cycles On Estrogen Secretion

At molecular levels,the key component of the circadian clock is a network of transcription-translation feedback loop made up by interacting clock genes.With the regulation of circadian system,the behavior activities of mammalian and the transcription of numerous genes oscillate rhythmically with a period about 24 h.The growth,development and reproductive physiology of female mammalian are deeply integrated with circadian clock system.The synthesis of estrogen in granulosa cells a main body cell in ovarian follicles plays vital roles on estrus cycles.And it has been suggested that the biosynthesis of E2 is under control of brain and muscle ARNT-like(Bmal1),a core clock gene.Besides,by combination with Leptin receptor(Lepr),Leptin possess the ability to regulate the secretion of E2 in granulosa cells.By analyzing the promoter sequences of Lepr,we found multiple potential binding sites of Bmal1/Clock heterodimers.But,it is not clear whether the expression of Lepr is regulated by circadian clock.And whether the secretion of E2 is influenced by Lepr remains to be investigated.Therefore,it is experimental to explore whether the disruption of light/dark cycles affects E2 secretion mediated by Lepr.In this study,we first analyzed the rhythm features of E2 secretion and Lepr as well as Bmal1 transcription in mice ovary under 12 h light/12 h dark detected by ELISA and RT-qPCR.Then the variation of E2 concentration in blood and the expression of Bmal1 and Lepr in ovaries after treated with continuous light were analyzed by ELISA,RT-qPCR and Western blot.Next,we treated granulosa cells with Lepr siRNA or Bmal1 siRNA with the detection of E2 concentration in cell medium and Lepr expression.Then granulosa cells were cultured with Leptin at different doses.Finally,granulosa cells were co-treated with Leptin and Lepr siRNA or Bmal1 siRNA and with the test of E2-biosynthesis genes at transcription level.Results are shown as follows:1.The secretion of E2 and the transcription of Bmal1 and Lepr in ovaries displayed circadian rhythm with a peak appearing at ZT16.The treatment of continuous light impaired E2 secretion and the expression of Bmal1,Lepr and CYP19a1 in ovaries at ZT16(P<0.05).2.Lepr siRNA treatment significantly reduced the release of E2 and the expression of CYP19a1 which is a key enzyme of E2 synthesis.3.Bmal1 siRNA treatment suppressed the expression of Lepr and E2 synthesis-related genes accompanied with the decrease of E2 secretion in granulosa cells(P<0.05).4.Exogenous Leptin regulated the secretion of E2 in dose-dependent manner.Leptin at 0.5ng/ml promoted the transcription of E2-synthesizing genes in granulosa cells,but Leptin at high dose(10ng/ml)inhibited the expression of CYP19a1(P<0.05).5.Co-treatment with Leptin(0.5ng/ml)and Bmal1 siRNA or Lepr siRNA blocked Leptin-induced the transcription of E2-synthesizing genes.In conclusion,it was proposed that the secretion of E2 was closely associated with light/dark cycles and continuous light reduced the secretion of E2.The expression of Lepr in ovaries was regulated by circadian clock.With the stability of circadian clock was destroyed by constant light,the expression of Lepr was inhibited which blocked the regulation of Leptin on E2 secretion.