Screening And Identification Of Differential Gene Expression And Histone Post-translational Modification In Ovarian Aging

ObjectiveScreening and identifying the differentially expressed genes and histone post-translational modifications related to ovarian aging,and exploring the expression changes of related regulatory enzymes,so as to provide reference for further analyzing the molecular mechanism of ovarian aging and searching for potential molecular targets.Methods1.The ovarian function flourishing stage(3M),ovarian function decline stage(12M)and ovarian function failure stage(17M)were selected as the research subjects in female mice.RNA-seq was used to detect the m RNA expression levels of different stages of the ovary and screen differentially expressed genes related to ovarian function.Furthermore,the results of transcriptome were verified by q-PCR;2.The post-translational modification levels in the ovaries of 3M,12 M and 17 M female mice were detected by Western blot using the pan-antibody of 16 proteins post-translational modification(PTMs),and the significantly different posttranslational modification levels were screened;3.The histone modified site specific antibodies were used to further identify the modification sites by Western blot,and the expression changes of related modification enzymes in the ovaries of 3M,12 M and 17 M female mice were detected by q-PCR and Western blot;4.The expression and localization of significantly different histone posttranslational modifications and related modification enzymes in ovarian tissues of female mice of different months were observed by immunohistochemistry;5.BUS(30 mg/kg)combined with CY(120 mg/kg)was intraperitoneally injected to establish a mouse model of premature ovarian failure.Western blot and q-PCR were used to detect the expressions of the above histone post-translational modifications and specific modification enzymes in the ovaries of mice with premature ovarian failure.Results1.Compared with the 3M group,a total of 5434 m RNA were differentially expressed in the 12 M group,among which 2822 m RNA were up-regulated and 2612 m RNA were down-regulated.A total of 7337 m RNA were differentially expressed in17 M group,among which 4113 m RNA were up-regulated and 3224 m RNA were down-regulated.Differential m RNAs were significantly enriched in the formation of cytoskeleton,cell signal transduction,energy metabolism,protein synthesis and its catalytic activity function(p < 0.05).2.PTMs pan-antibodies screening showed that only trimethylation of histone lysine was up-regulated progressively and significantly with increasing age(p <0.05).3.The expressions of KDM2 B,KDM5C,KDM1 A and KDM4 B genes as well as the methyltransferases Smyd3,Setd8,KMT2 C and SUV420H2 genes were further detected by q-PCR,and the results showed that only the expression of SUV420H2 specific to H4K20Me3 was significantly increased(p < 0.05).Western blot was used to identify specific histone modification sites,and the results showed that H4K20me3 expression was significantly increased in the histones of 12 M and 17 M mouse ovarian(p < 0.05).4.Immunohistochemistry showed that H4K20me3 and SUV420H2 were mainly localized in oocytes and granulocyte nuclei,and optical density analysis showed that the expressions abundance of H4K20me3 and SUV420H2 were enriched at 12 M and17M(p < 0.05).5.The expressions of H4K20me3 and SUV420H2 were also significantly increased in mice with premature ovarian failure(p < 0.05).ConclusionThere are lots of gene expression changes in ovarian aging,and histone post-translational modification SUV420H2-H4K20me3 may play an important role in regulating ovarian aging.