The Effect Of Dnmt3a Conditional Knockout Of The Endometrium On The Endometrial Function Of Early Peri-implantation Mice

Objective:Embryo implantation is an important part of the mammal's reproductive process,and it is the key to pregnancy.It includes the process of embryo identification,adhesion,and invasion of the maternal endometrium.The successful implantation of embryos requires many conditions,such as adequate hormone secretion,formation of syncytiotrophoblast cells,and synchronous development and interaction of blastocysts with endometrium.However,this process is very complicated and its mechanism remains to be studied.DNA methylation can regulate the expression of eukaryotic genes.It is one of the most widely studied methods of epigenetic modification and is catalyzed by DNA methylation transferases.Dnmt3a is a type of DNA methylation transferase.Recent studies have found that DNA methylation may play an important role in the procedural changes of embryo implantation endometrium.In the previous study,we found that the Dnmt3a gene and protein in the endometrium of early pregnant mice showed regular temporal and spatial expression.Therefore,We speculate that Dnmt3a may be involved in the regulation of endometrial function during mouse embryo implantation.In this study,we used the loxP-Pgr^Cre/+system to construct a uterine Dnmt3a conditional knockout mouse model to investigate the role of Dnmt3a in the regulation of endometrial function during embryo engraftment.Methods:1.Endometrium Dnmt3a conditional knockout mouse model construction:Dnmt3aloxP/loxPPgrCre/+uterine conditional knockout mouse model?cKO?was constructed using Dnmt3aloxp/loxp and PgrCre/+mice,and Dnmt3aloxP/lox P mice were used as Control group.After mating with 6-8 week-old male C57BL/6J male mice,the expression of Dnmt3a in the endometrium of mice was detected by Western blot and immunohistochemistry to determine Dnmt3a endometrial conditional knockout mice.Whether the model was built successfully.2.To investigate the effects of conditional knockout of mouse endometrium Dnmt3a on ovarian function:Ovarian and oviductal tissues were harvested from mice at D5 days of pregnancy,HE staining was used to observe the morphology;serum of mice at D5 and D7 days of pregnancy was collected and detected by Elisa method.Serum E2,P4 levels.3.To investigate the effects of conditional knockout of mouse endometrium Dnmt3a on endometrial receptivity in early pregnant mice:Counting and visualizing the number of embryo implantation sites after tail vein injection with trypan blue on day 5 of pregnancy;Western Blot and Immunohistochemistry was used to detect the expression of ER?,MUC1,PR in the endometrial receptivity-related genes of mice on day 4 of pregnancy.4.To investigate the effects of conditional knockout of mouse endometrium Dnmt3a on decidualization of endometrial uterus in early pregnant mice:?1?Count the naked eye and observe the number of embryo implantation sites on day 7 of pregnancy;Western Blot and immunohistochemistry Expression of HOXA10,MMP2,MMP9 and BMP2genes related to endometrial decidualization in early pregnancy mice;?2?Artificially induced decidualization model in pseudopregnant mice;normal and cKO mice on the fourth day of pseudopregnancy?PD4?corneal oil injection induced decidualization on one side of uterine horn,and the other side of the uterine horn was not treated as a control,and uterine material was collected on the eighth day of pseudopregnancy?PD8?.The uterine appearance of the decidualization induced side of the pseudo-pregnant mice cKO group and the control group was visually observed,and the success rate of artificially induced decidualization was counted.5.To investigate the effect of conditional knockout of mouse endometrium Dnmt3a on reproductive performance:female mice of cKO and control groups were caged with male mice of normal C57BL/6J male mice respectively,and observed until pregnancy.After the weaning mice were weaned,the rats were divided into cages with their mother rats,and the mothers continued to co-operate with the male rats.The above steps were repeated until 6 months.The weight,number,and proportion of male and female offspring were measured in two groups.Results:1.The endometrial Dnmt3a conditional knockout mouse model was successfully constructed:Western blot and immunohistochemistry results showed that compared with the control group,Dnmt3a expression in the endometrium of cKO mice was significantly reduced on the 5th day of pregnancy,cKO mice There was no significant difference in the expression of ovarian Dnmt3a compared with the control group.2.Conditional knockout of mouse endometrium Dnmt3a had no significant effect on ovarian function:HE staining showed that compared with the control group,there was no significant difference in ovarian morphology and fallopian tube morphology in cKO mice at D5 days of pregnancy;Elisa detected pregnancy D5 Serum levels of E2 and P4 did not change significantly in the cKO group compared with the control group.3.No significant changes in endometrial receptivity of early pregnant mice after conditional knockout of mouse endometrium Dnmt3a:Visual observation revealed that compared with the control group,the number of embryo implantation sites in the cKO group of mice at day 4 of pregnancy was not increased.Obvious changes;Western Blot and immunohistochemistry results showed that compared with the control group,cKO mice endometrial receptivity-related gene ER?,MUC1,PR expression did not change significantly. There was no significant change in the decidualization of endometrial uterus in mice after conditional knockout of mouse endometrium Dnmt3a:compared with the control group,there was no significant change in the number of implantation sites of uterine embryos in the cKO group of pregnant D7 days;Blot results showed that compared with the control group,the expression of MMP2 and MMP9 in the endometrium of the cKO mice was significantly reduced on the 5th day of pregnancy,and the BMP2in the uterus of the cKO group was significantly decreased on the 7th day of the pregnancy.The results of immunohistochemistry showed that they were comparable to the control group.In comparison,the expression of HOXA10 and BMP2 in the endometrium of mice at day 7 of pregnancy was not significantly different.After artificially induced decidualization,compared with the control group,the appearance of uterus and the success rate of decidualization of the decidualization side of the cKO group had no significant changes.4.The reproductive ability of mice after specific knockout of Dnmt3a in mouse endometrium was not significantly affected:6-month reproduction test results showed that compared with the control group,the offspring of the offspring of the cKO group had a slightly reduced weight,and the number of the offspring was reduced.There is no significant difference between male and female.Conclusion: After conditional denudation of uterine Dnmt3a,there was no significantchangeinendometrialreceptivityandendometrial decidualization during embryo implantation in early pregnancy mice.Objective:Embryo implantation is the process of establishing a close connection between live embryos and the recipient uterus and is a key link in pregnancy.A series of complex events occur around the endometrium of the bed,enter the state of tolerance and develop synchronously with the embryos,providing a bed microenvironment for the implanted embryos.Autophagy is another kind of programmed cell death that is different from apoptosis,and plays an important role in maintaining cell survival,renewal,substance reuse,and homeostasis.3-methyladenine?3-MA?is a common inhibitor of autophagy.The aim of this study was to investigate the effect of3-MA,an autophagy inhibitor,on engraftment of immature mouse embryos.Methods:Adult Kunming female mice were randomly divided into control group,3-MA low-dose group?15 m M?and 3-MA high-dose group?30m M?.Autophagy inhibitor 3-MA was intraperitoneally injected from mice on day D1 until sacrifice and PBS was used as a control.D1,D5,and D6 days of endometrial tissue were collected and the following experiments were performed:1.Expression of autophagy-related marker molecules in endometrium of early pregnant mice after intervention with autophagy inhibitor 3-MA:Real-time PCR detection of autophagy-related markers Cathepsin B and P62 m RNA in the endometrium of early pregnant mice Expression;Western blot was used to detect the expression of Atg5 and LC3 proteins in the endometrium of early pregnant mice.2.The effect of autophagy inhibitor 3-MA on the number of implantation sites of encircling mouse embryos: morphological observation of the number of embryo implantation sites in control group and 3-MA autophagy inhibitor group.3.Expression of PR and ER? in the mouse endometrium on the day of pregnancy D5 after intervention with autophagy inhibitor 3-MA: Real-time PCR detection of receptor-associated factors PR and ER m RNA in mouse D5 implantation days The expression;immunohistochemical detection of receptor-associated factor PR expression.Result:1.After the intervention of autophagy inhibitor 3-MA,the expression of autophagy-related marker molecules in the endometrium of early pregnant mice was significantly reduced: Real-time PCR results showed that after intervention of autophagy inhibitor 3-MA,autophagy related The expression of Cathepsin B and P62 m RNA in the endometrium of early pregnancy mice was significantly lower than that of the control group.Western blot results showed that the protein of Atg5 and LC3 was detected in the endometrium of early pregnant mice after injection of autophagy inhibitor 3-MA.Expression was significantly lower than that of the control group.2.After the intervention of autophagy inhibitor 3-MA,the number of embryo implantation sites in the 3-MA autophagy inhibitor group was significantly reduced compared with the control group.3.After the intervention of autophagy inhibitor 3-MA,the expression of PR and ER? in the mouse endometrium at D5 was significantly lower than that of the control group.Real-time PCR showed that after the intervention of 3-MA,the autophagy inhibitor.The expression of receptor-associated factors PR and ER m RNA in the implantation site of mice at day 5 of pregnancy was significantly lower than that of the control group.Immunohistochemistry results showed that compared with the control group,the expression of PR in the 3-MA inhibition group was significantly reduced.Conclusion:Under the intervention of autophagy inhibitor 3-MA,the expression of endometrial receptivity-related factors was decreased when mouse embryos were implanted,suggesting that autophagy inhibitor 3-MA may affect endometrial receptivity when mouse embryos are implanted.The impact,the mechanism for further study.