Regulations And Mechanisms Of MiR-7 On The Synthesis And Secretion Of Gonadotropins In Pituitary Gland

Gonadotropins,including luteinizing hormone(LH)and follicle-stimulating hormone(FSH),play important roles in animal development and reproduction.Mass research evidence indicated that gonadotropins are important in hypothalamus-pituitary-gonad(HPG)axis.Recently,it is found that among all the miRNAs,miRNA-7(miR-7)is highly expressed in pituitaries of pigs.However,little is known about the function and mechanism of miR-7 in pig pituitaries.Therefore,the purpose of this research focus on miR-7 function in HPG axis of pigs,using mice as an experimental tool.Problems need to be solved include miR-7 expression in pituitaries,roles of miR-7 in gonadotropins,roles of miR-7 in the regulation of gonadotropins by gonadotropin-releasing hormone(GnRH),estrogen and zearalenone(ZEA)and a-zearalenol(a-ZOL),and related mechanisms.The results of this essay include five parts.1.Real-time PCR,in-situ hybridization and immunohistochemistry were used to detect miR-7 expression in pituitaries in pigs and mice.It was shown that miR-7 is highly expressed in pituitaries compared with other tissues and miR-7 is located in gonadotropes.2.MiR-7-mimics and miR-7-inhibitors were transfected into pituitary primary cells of pigs and mice.It was found that miR-7 could inhibit the expression of gonadotropins in pig pituitaries.And miR-7 could inhibit the expression,synthesis and secretion of gonadotropins in mice.3.GnRH was added to pig pituitary primary cells with time gradient.Real-time PCR was used for gene detection and RIA was used for hormone detection.Results showed that GnRH inhibits miR-7 while increases expression,synthesis and secretion of gonadotropins.Further investigation proved that miR-7 mediates the regulation of GnRH on gonadotropin expression.4.Technologies such as bioinformatics,dual-luciferase reporter system and Western Blot were used.Mice and L?T2 cell line model were used to investigate the mechanism of miR-7 in the regulation of GnRH on gonadotropins.Results showed that in mouse pituitaries,GnRH inhibits miR-7 expression through PKC signal pathway.MiR-7 and its target RAF1 can inhibit the downstream ERK1/2 signal pathway and further inhibit the expression,synthesis and secretion of gonadotropins.5.Pig ovariectomy model and pig primary pituitary cell culturing model were used.Results showed that miR-7 mediates the inhibitory regulation of ?-estradiol,ZEA and a-ZOL on FSH instead of LH.Further investigation showed that in pig pituitaries,ZEA promotes miR-7 expression through non-classical estrogen membrane receptor GPR30 and PKC,p38MAPK signal pathway.MiR-7 and its target c-FOS can increase the expression,synthesis and secretion of FSH.In all,miR-7 mediates the regulation of GnRH,P-estradiol,ZEA and a-ZOL on gonadotropins.This assay provides new evidence that miR-7 may function as a molecular switch in HPG axis in pigs.This research provides new evidence of the function and mechanism of pig miRNA regulating gonadotropins.Since ZEA and its metabolites damage pig production industry and human reproduction system severely,miR-7 may become the drug target treating reproduction diseases caused by estrogen and ZEA from the level of pituitaries.