| Background: Premature ovarian failure(POF)is a female reproductive endocrine disease characterized with abnormal levels of estrogen and gonadotropin.In recent years,its incidence has been increasing year by year,and developing to the younger age.POF reduces a woman’s fertility,but also causes a lot of harm.The etiology of POF is complicated,the pathogenesis is not clear yet,and the effect of clinical intervention is limited.Therefore,it is very important to find new ideas and treatment methods for early prevention and treatment of POF from basic research.Animal models of POF can simulate the clinical features of POF patients and help to understand the pathogenesis of POF.Many animal models of POF have been successfully constructed according to different etiology.D-galactose metabolism can lead to metabolic disorder,oxidative stress damage,large amount of reactive oxygen species damage ovarian function,and induce granulosa cells to accelerate apoptosis,atresia follicles increase,and finally cause POF.Some studies have shown that m6 A modification is regulated by oxidative stress,but the specific mechanism is not clear.N-6-methyladenosine(m6A)is the most abundant epigenetics in eukaryotes and plays an important role in regulating gene expression.Methyltransferase 3(METTL3)as the core catalytic enzyme of m6A-modified methyltransferase complex,is widely involved in the processes of gamete formation,embryonic development,organ senescence and tumorigenesis.Reported that METTL3 expression was up-regulated in POF mouse model induced by alkylates.It has been shown that m6 A modification is closely related to POF.However,the effect of METTL3 on POF has not been reported.The purpose of this study is to investigate the role of METTL3 in POF.Objectives: To explore the mechanism of methyltransferase METTL3 in POF by constructing POF mouse model,and to provide theoretical basis for the study of pathological mechanism and clinical treatment of POF.Methods: The POF mouse model was established by subcutaneous injection of d-galactose into C57BL/6 female mice,and the estrous cycle was evaluated by collecting vaginal exfoliated cells The pathological changes of ovarian tissue in POF mice were observed by HE staining and the changes of sex hormones in peripheral blood and ovarian tissue were detected by ELISA,to verify the modeling success.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in peripheral blood and ovarian tissue were measured by WST-1 and TBA Superoxide dismutase respectively.The expression of METTL3 was detected by q RT-PCR and Western blots,and the apoptosis was detected by TUNEL staining.The primary granulosa cells of C57BL/6 female mice were isolated and cultured to construct lentivirus with METTL3 overexpression and knockdown and its no-load control The transfection efficiency of lentivirus in granulosa cells was detected by q RT-PCR and Western blots,and the apoptosis of granulosa cells was detected by FITC/PI double staining method,and the proliferation of granulosa cells was detected by CCK-8 method.Results: In this study,POF mouse model was established by d-galactose,and estrous cycle disorder was found in POF mice.The results of HE staining showed that in POF group,the ovarian volume decreased significantly,the total number of follicles decreased and the atresia follicles increased.Elisa showed that FSH levels in peripheral blood and ovarian tissue of POF group were higher than those of control group,and E2 levels were significantly lower than those of control group.The pathological changes of ovarian tissue and the changes of sex hormones in the above POF mice were similar to the clinical manifestations of POF patients,indicating that d-galactose can successfully construct the POF mouse model.At the tissue level,we found that the level of SOD was lower and the content of MDA was higher in the ovarian tissue of mice than that of the control group by the methods of WST-1 and TBA,q RT-PCR and Western blots were used to detect the expression level of METTL3 in ovarian tissue of POF mice,and the results showed that the expression level of METTL3 in ovarian tissue of POF mice was significantly higher than that in control mice.In cell experiments,we successfully constructed mouse granulosa cell stable transcripts with overexpression and knockdown of METTL3.q RT-PCR and Western blots showed that the transfection effect of METTL3 overexpression and knockdown lentivirus was significant,and FITC/PI double staining showed that up-regulation of METTL3 expression could promote granulosa cell apoptosis CCK-8 detection showed that METTL3 overexpression significantly inhibited cell proliferation.Conclusion(s):(1)The POF mouse model was constructed by d-galactose,and the changes of sex hormones and ovarian histopathology were found to be similar to the clinical manifestations of POF patients,it laid a foundation for the later research.The method is simple and easy to use,and has a high success rate.It is an ideal method to construct POF mouse model.(2)The ovarian tissue of POF mice showed oxidative stress injury,the expression level of METTL3 was significantly up-regulated,and the apoptosis of granulosa cells was increased.The mechanism may be that hypergalactose induces metabolic disorder of mouse ovarian tissue,oxidative stress injury,METTL3 expression,granulosa cell apoptosis,and increase of atresia follicles,leading to POF.Abnormal expression of METTL3 may be a potential cause of POF.(3)Upregulation of METTL3 expression in granulosa cells at the cellular level was found to promote granulosa cell apoptosis,confirming that METTL3 may be involved in the progression of POF by promoting granulosa cell apoptosis. |
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