| Background:With the development of science and technology and the intensification of industrial pollution,the threat of environmental pollution to reproductive health is becoming more and more serious.Endocrine disrupting chemicals(EDCs),also known as environmental endocrine disruptors,refers to the physiological effects of natural hormones or natural hormones,thereby destroying their ability to maintain the stability and regulation of exogenous compounds.Because of the widespread existence of EDCs in people’s production and life and its great harm to human reproductive health,it is now more and more attention.Triclosan(TCS)is a kind of EDCs.In the previous study,TCS exposure indicated that the expression of Nanog gene was decreased by the increase of microRNA-134 expression,which resulted in the decrease of mouse embryonic stem cells The decline of the ability.However,there is no corresponding study on the effects and mechanisms of TCS on preimplantation embryo development.Objective:1.To investigate the role of miR-134 in the development of pre-implantation embryos and its mechanism by using mice as model animals.2.Further search for other target genes and related pathways of miR-134 in early embryos.Methods:1.The preimplantation embryos were exposed to 2μM,20μM,200μM triclosan exposure solution,respectively,by embryo 1-cell site d0.5,dl.5,d2.5,d3.5,d4.5 were observed Count the number of mouse embryos 2-cell,4-cell,morula and blastocyst.The expression of Nanog protein in the morula of TCS was observed by Real-Time PCR and the expression of Nanog protein was observed by immunofluorescence assay.2.MiR-134 was overexpressed in mouse fertilized eggs by microinjection of miR-134 mimic,and the mouse embryos 2-cells were counted at d1.5,d2.5,d3.5,d4.5,Morula and blastocyst number.The embryos were collected at each stage and the expression levels of miRNA-134 at different stages of implantation were calculated by Real-Time PCR.The expression of Nanog in the morula of overexpression of miRNA-134 was calculated by Real-Time PCR,and the expression of Nanog protein was observed by immunofluorescence assay.3.MiR-134 inhibitor and Nanog mRNA fragment were injected into the mouse fertilized eggs by microinjection technique.Then,the fertilized eggs were cultured in TCS high concentration medium,and d1.5,d2.5,d3.5,d4.5 were counted to count the number of mouse embryos 2-cell,4-cell,morula and blastocyst.4.The transcriptions of miR-134 overexpression were analyzed by single cell RNA-Seq high throughput sequencing.Results:1.The blastocyst formation rate of the mouse fertilized eggs was significantly decreased in the different concentrations of TCS,and the embryonic development was mainly blocked in the morula stage(p<0.05),and the expression of miR-134 was significantly increased when exposed to 200μM TCS(p<0.05).2.MiR-134 was expressed in all stages of early embryos,and blastocyst stage was the highest.3.The expression of miR-134 overexpression in mouse fertilized eggs showed a decrease in blastocyst formation rate(p<0.05),and embryonic development was also blocked in the morula period,consistent with that after exposure to TCS.4.The expression of Nanog was significantly decreased in miR-134 overexpression of preimplantation embryos and preimplantation embryos after TCS exposure,and the protein expression was also significantly decreased(p<0.05).5.Injection of miR-134 inhibitor and Nanog mRNA fragment could restore embryonic blastocyst formation after TCS exposure(p<0.05).6.The single-cell RNA-Seq high-throughput sequencing technique was used to analyze the transcriptions of miR-134 overexpression:(1)223 differentially expressed genes were identified in miR-134 overexpression group compared with the control group,which contained 187 upgrade genes and 36 downgrade genes.(2)miR-134 may be involved in the regulation of p53 gene signaling,regulation of neuronal action potential and other biological processes,translation initiation factor activation,calcium ion binding and other molecular functions,lamellar,interleukin-12 receptor complex,Mitochondrial outer membrane and other cell components play a role in the above biological events are likely to be miR-134 in early embryonic mechanism of action.Conclusion:This study confirms the early embryonic development of TCS exposure,and the mechanism may be that the upregulation of miR-134 causes down-regulation of downstream target gene Nanog,which has an effect on embryonic development.Through the exploration of the function of miR-134 in early embryos,we can provide some theoretical basis and scientific basis for further reduction of early embryonic development and reduction of potential reproductive developmental toxicity of TCS. |
PDF Full Text Request