Regulation And Mechanism Of Melatonin And Inhibin On Apoptosis Of Granulosa Cells In Mice

Melatonin performs a critical role in the regulation of the animal reproductive system,particularly in follicular growth.However,the mechanism of melatonin and its receptor 1(MT1)in regulating hormones associated with reproduction remains unclear.This study was designed to examine the physiological role of constitutive MT1 silencing and follicle stimulating hormone(FSH)treatment in reproduction,making use of mouse granulosa cells(m GCs)as a model.To understand the constitutive role of MT1 in ovarian physiology,the RNAi-Ready p SIREN-RETROQ-Zs Green Vector mediated recombinant psh RNA was used to silence MT1 gene expression.The results showed that the expression of MT1 was successfully inhibited both at the protein and m RNA levels(P < 0.001).We demonstrated that RNAi-B-mediated MT1 down-regulation significantly promoted apoptosis(P < 0.001),inhibited proliferation,and regulated the cell cycle at the S-phase;Similarly,FSH treatment partially aided the apoptotic effect,but conversely improved proliferation although it just show a significant effect at the Sphase of cell cycle.Transitory knockdown of MT1 proved essential in the function of m GCs,as it significantly decreased cyclic adenosine monophospahte(c AMP)level and increased cell apoptosis.Following knockdown of MT1,the expression of Bax was significantly up-regulated(P < 0.001),but Bcl-2 was slightly down-regulated,both at the transcriptional and at translational levels.Moreover,the silencing of MT1 and its constitutive effect on FSH significantly promoted an increase in estradiol(P < 0.001)and slightly decreased the concentration of progesterone.Together,our data indicates that MT1 suppression leads to interference in the normal physiological function of the ovary by enhancing follicular apoptosis,inhibiting proliferation,and influencing hormonal signaling,whereas constitutive FSH treatment counteracted the negative down-regulatory effects of MT1 on m GCs.Inhibin,an protein complex is dimeric in structure,is produced in the gonads,pituitary gland,placenta,corpus luteum and other organs.Inhibin downregulates FSH synthesis and inhibits FSH secretion.Inhibin α knockout(Inha-/-)female mice improve sex cord-stromal ovarian cancer with whole penetrance.Present researches have validated that Inha-/-ovarian follicles grow obviously to the premature antral stage in prepubertal mice starved of any growth in serum follicle stimulating hormone(FSH),which recommended that in the absence of inhibins,granulosa cells discriminate abnormally.To check the hypothesis,we stimulated immature wild-type and Inha-/-female mice with human chorionic gonadotropin(h CG)and examined h CG induced gene expression changes in granulosa cells.Furthermore,to perform the analysis,we firstly found out differentially expressed genes(DEGs)separately in WT/ Inha-/-with and without h CG stimulation samples,took the overlapping genes as the characteristic genes in h CG.Then weighted gene coexpression network analysis(WGCNA)algorithm was used to detect related modules,genes,related mi RNAs(mmu-m IR-124 a,mmum IR-224,mmu-m IR-520 a,mmu-m IR-9,mmu-m IR-30 a,mmu-m IR-24,mmu-m IR-145,mmu-m IR-520f),Transcription factors(PAX4,MAZ,MYC,NFAT,FOXO4,SP1,LEF1),and regulatory network were utilized to get regulated DEGs.Finally,Principal component analysis(PCA)to determine significance of 14 regulated genes.The analysis result suggested that Inhibin α knockout and h CG stimulation can down-regulate the expression of Jup and up-regulate Psmc3 ip.Therefore,we determined that Jup and Psmc3 ip genes had close relationships in both expression pattern and functions in the Inha-/-h CG stimulation female mice granulosa cells.