MicroRNA-7a2 Affects Estrogen Synthesis By Involving In FSH And Taurine Signalings In The Ovary Of Mouse

In female animals,estrogen is mainly synthesized and secreted by ovary granulosa cells,As a main component of estrogen,17β-estradiol(17β-estradiol,E2)plays important roles in regulatory reproductive organ development and reproductive functions in female animals.It is well known that the synthesis and secretion of E2 are mainly regulated by the hypothalamuspituitary-ovarian axis,in which pituitary follicle-stimulating hormone(FSH)is included.FSH directly acts on the granulosa cells to induce the expression of the Cyp19a1 gene and promote estrogen synthesis.Estrogen synthesis is regulated by intracellular signaling pathways,transcription factors,and some exogenous substances such as taurine.MicroRNA-7(miR-7)is a class of endogenous noncoding RNAs that are evolutionarily conserved across multiple species.It has been shown that mature miR-7 molecules are transcribed from gene sequences at three different sites(miR-7a1,miR-7a2,and miR-7b),and miR-7a2 affects reproductive function by affecting the synthesis of various hormones,FSH,LH and PRL.However,the expression of miR-7a2 in the ovary and effects on ovarian estrogen synthesis remain unclear.The present study was thus proposed to study the regulating effects of miR-7a2 on the female reproductions and estrogen synthesis in ovarian granulosa cells by uing miR-7a2 knockout(miR-7a2 KO)mouse model and related in vitro,in vivo experiments.The most important results including the following points.1.miR-7a2 promotes E2 synthesis by up-regulating the expression of Cyp19a1 mRNAIn this study,in situ hybridization and immunofluorescence methods were used to demonstrate the high expression of miR-7a in mouse ovarian granulosa cells.Subsequently,estrus cycle identification,breeding experiments,histological and morphological observations proved that miR-7a2 KO female mice developed estrus cycle disorder,infertility,smaller ovaries and increased primordial follicles,decreased antral follicles and corpus luteum.The decrease was accompanied by an increase in follicular atresia.RT-qPCR and RIA were used to detect E2 synthesis key enzyme genes Cyp17a1,Cyp19a1 and Hsd17b1 in mouse ovary,key enzymes Cyp19a1 and Hsd17b1 in granulosa cells,and E2 content in serum or cell culture medium,respectively.miR-7a2 KO reduced Cyp19a1 mRNA level and E2 content,but had no significant effect on the expression of Cyp17a1 and Hsd17b1.Furthermore,we transplanted the ovaries of miR-7a2 KO mice into wild-type mice to mimic mice with ovarian-specific deletion of miR-7a2.Our results demonstrate that ovarian-specific deletion of miR-7a2 mice has a similar phenotype to miR-7a2 KO mice,and that E2 supplementation restores the mice’s reproductive capacity.These results demonstrate that miR-7a2 promotes E2 synthesis by upregulating Cyp19a1 mRNA expression in granulosa cells,thereby maintaining reproductive function in female mice.2.miR-7a2 affects E2 synthesis by involving in FSH signalings in the ovarian granulosa cellsPrimary granulosa cells were treated with different concentrations of FSH,and then RT-qPCR and RIA were used to detect the expression levels of miR-7a and Cyp19a1 mRNA in granulosa cells and the content of E2 in cell culture medium,respectively.The results showed that FSH could significantly up-regulate the expression of miR-7a and Cyp19a1 and the content of E2 in the culture medium in a dose-dependent manner,and FSH at a concentration of 200 ng/mL had the most significant effect on E2 synthesis.MiR-7a2 KO eliminated the up-regulation of E2 synthesis by FSH.In addition,our study proved that FSH regulates the expression of miR7a by activating the JNK signaling pathway;the dual-luciferase assay method proved that miR-7a can negatively regulate the expression of Glgl.The above results suggest that FSH promotes E2 synthesis by activating JNK/miR-7a/Glgl/Cyp19a1 signalings.3.miR-7a2 affects E2 synthesis by involving in taurine signalings in the ovarian granulosa cellsIn this study,the number of antral follicles was increased by taurine treatment in mouse ovaries.In addition,the treatment of taurine on primary granulosa cells in vitro significantly inhibited the expression of Caspase 3 expression and promoted the expression of Ki-67.These suggested that taurine may acted on granulosa cells and then affect follicle development.The results of in situ hybridization and immunofluorescence showed that miR-7a was highly expressed and co-localized with Taurine transport(TauT)in ovarian granulosa cells.Subsequently,taurine was shown to promote the expression of miR-7a and Cyp19a1 mRNA,as well as E2 synthesis in granulosa cells in vitro and in vivo.These results further confirmed that taurine could directly acted on granulosa cells.Finally,we used miR-7a2 KO granulosa cells,combined with the methods of Glg1 gene regulation and signaling pathway inhibition,and found that miR-7a2 promoted Cyp19a1 mRNA expression and then the synthesis of E2 by inhibiting Glgl mRNA expression,while taurine up-regulated miR-7a expression by activating the p38 signaling pathway.These results indicated that taurine affects E2 synthesis by regulating p38/miR-7a/Glg1/Cyp19a1 signalings pathway.In conclusion,this study demonstrated that miR-7a2 mediates FSH and taurine signaling pathways to affect the synthesis of E2 in ovarian granulosa cells through in vivo and in vitro experiments,but the related molecular mechanism needs further study.