| Background:Histone deacetylase(HDAC)is an epigenetic modified protein that binds to negatively charged DNA tightly,making chromatin compact and coiled,and inhibiting gene transcription.Studies have shown that when Luteinizing hormone(LH)binds to its receptors,LH signals rapidly inhibit transcription of C-type natriuretic peptide(CNP)and natriuretic peptide receptor 2(NPR2)genes.At the same time,the expression of Epidergrowth factor(EGF)and Epidergrowth factor receptor(EGFR)activation signal pathway was stimulated to promote the expansion of cumulus cells around oocytes and enable oocytes to mature.It was found that the epigenetic modification protein HDAC3,a member of the HDACs family,regulated oocyte maturation by influencing the secretion of EGF-like growth factor AREG in granulosic cells in response to LH signaling.Both HDAC 1/2 and HDAC3 are members of the first HDAC family,and numerous studies have shown that HDAC 1/2 plays an important role in oocyte maturation and embryonic development in mice.It is speculated that HDAC 1/2 may have a similar effect to HDAC3,but whether HDAC 1/2 can mediate ovarian granulosa cells of Tan sheep to affect oocyte maturation remains unclear.Objective:To investigate the expression of HD AC 1/2 in ovarian follicles of Tan sheep and the effect of HDAC 1/2 on the secretion of EGF-like growth factor in ovarian granulosa cells of Tan sheep and the maturation of oocytes.The purpose of this study is to provide important theoretical support for improving the in vitro maturation rate of oocytes of Tan sheep,and also to provide a certain reference for the future application of embryo engineering to achieve rapid expansion of Tan sheep population.Methods:In this study,Tan sheep was selected as the research object,and the ovaries of Tan sheep were collected.Immunohistochemistry,immunofluorescence and western blotting were used to explore the localization and expression of HDAC 1/2 in the ovaries under physiological conditions.Ovarian granulosa cells were obtained by suction method.After HDAC 1/2 inhibitor treatment,protein western blotting,enzyme-linked immunoassay and immunofluorescence were used to explore the changes of EGF-like growth factors in granulosa cells treated by HDAC 1/2 inhibitor.To explore the effect of HDAC1/2 on the expression and secretion of EGF-like growth factor.The co-culture system of granulosa cells and oocytes was used to explore whether HDAC1/2 could replace LH to promote oocyte maturation.At the same time,protein western blotting was used to detect the changes of acetylation sites in inhibitor-treated granulosa cells to explore the potential mechanism affecting oocyte maturation.Results:1.HDAC1 and HDAC2 were expressed in granulosa cells and oocyte nuclei of growing follicles at all levels,and the expression levels of HDAC1 and HDAC2 were significantly upregulated with the development of follicles to 6mm.2.The protein expression levels of HDAC1 and HDAC2 were up-regulated after the addition of follicle-stimulating hormone(FSH)in granulosa cells,and the protein expression of HDAC2 was down-regulated with the increase of LH treatment time.After inhibiting HDAC1 in granulosa cells,the expression level and secretion level of EREG protein were down-regulated,while the secretion level of betacellulin(BTC)was up-regulated.After inhibiting HDAC2 and HDAC1&2,the protein expression and secretion of epiregulin(EREG)in granulosa cells were significantly up-regulated,while the expression of BTC was down-regulated.3.Inhibition of HDAC1 can inhibit oocyte maturation and cumulus diffusion,and overexpression of HDAC1 can promote oocyte maturation.Inhibition of HDAC2 can promote oocyte maturation and cumulus diffusion instead of LH.Inhibition of HDAC1&2 also promoted oocyte maturation and cumulus diffusion,but the promotion rate of oocyte maturation was between that of HDAC1 and HDAC2 inhibitor treatment groups.4.Inhibition of HD AC1 in granulosa cells significantly increased the acetylation levels of H3K9,H4K5,H4K12 and H3K14.Inhibition of HDAC2 in granulosa cells significantly increased the acetylation level of H4K12.Inhibition of HDAC1&2 in granulosa cells significantly upregulated the acetylation levels of H3K9,H4K5,H4K12 and H4K16.Conclusions:In ovarian granulosa cells of Tan sheep,HDAC1 is not regulated by gonadotropin LH,but can promote oocyte maturation independently of LH.HDAC2 is regulated by LH,and may affect the expression of EREG and thus the maturation of oocytes by regulating the modification of the H4K12 acetylation site. |
