Study Of The Function Of Slc9a1 Gene In The Embryonic Development Process Before Implantation In Mouse

In the process of human embryo in vitro culture,about 50%of embryos have developmental block,how to reduce in vitro embryonic development block,improve blastocyst formation rate and clinical pregnancy rate has become a hot spot in the field of reproductive medicine.Most of the processes and important biological events of preimplantation embryonic development in mice are similar to human embryonic development.Preimplantation embryonic development undergoes processes such as cell division and differentiation.Intracellular p H is maintained within a certain physiological range,which is crucial for physiological processes such as cell growth and differentiation,intracellular enzyme activity,and assembly and disaggregation of cytoskeleton.By reading the literature,we learned that NHE1(Slc9a1 encoding)is one of the main regulators of intracellular p H,so we studied the biological function of Slc9a1 during early embryonic development in mouse.As a transmembrane protein,Na~+/H~+exchangers(NHEs)belong to the solute carrier-coupled ion transporter family 9A(Slc9a).NHE1(Slc9a1 code)is the most abundant subtype in the NHE family and is found in many mammalian cell types.Involved in a variety of cellular processes and maintaining a variety of functions,including involvement in regulating intracellular p H and osmotic homeostasis,influencing cell volume,and apoptosis.Previous studies of early embryos in mice have found that NHE1 is a familial protein that plays a major role in the NHE family during the recovery of induced acidosis.However,there have been no reports of Slc9a1 knockout mice and knockdown embryo models,nor have there been any reports of more relevant biological functions in early embryos before implantation.Therefore,this experiment uses RNA interference(RNAi))technology to construct a mouse embryonic Slc9a1 gene knockdown model to explore the effect of Slc9a1 knockdown on embryos,and further supplement its function in early embryos.In this experiment,through RNAi and the use of embryonic in vitro culture,it was found that Slc9a1 knockdown would lead to a decrease in the blastocyst development rate of the embryo at E3.5 days,that is,in the early stage of blastocyst,and the development rate of E4.0 and E4.5 blastocyst did not increase significantly and accompanied by an increase in embryonic death,while there was no significant difference in embryonic morphology and development period between the 2C,4C,8C and Mo stage control groups and the Slc9a1gene knockdown experimental group.The experimental results of the incubation experiment showed that most of the experimental group embryos failed to complete the incubation.Immunofluorescence detection of CDX2 and OCT4,marker transcription factors for embryonic lineage differentiation,and Cdx2 and Oct4 m RNA test results showed that embryonic lineage differentiation in the Slc9a1 gene knockdown experimental group failed.At the same time,through statistical analysis,it was found that the total number of embryos in the Slc9a1 gene knockdown experimental group was reduced.Immunofluorescence pictures also show a decrease in embryo volume in the gene knockdown experimental group.In addition,based on the increase in blastocyst death,we performed q PCR detection of p21and p53 m RNA expression levels,and the results showed that both p21 and p53 were significantly elevated in the experimental group,indicating increased DNA apoptosis and abnormal cell cycle.Reactive oxygen species(ROS)testing showed an increase in embryo ROS in the experimental group,suggesting that Slc9a1 knockdown caused oxidative damage to embryos.Detection of pro-oxidase gene expression levels that may cause ros levels to increase also suggests and further validates increased cellular oxidation.For embryonic intracellular p H test results,the intracellular p H(p Hi)of embryos in the experimental group was higher than that of the control group embryos,indicating that Slc9a1 knockdown caused abnormal intracellular p Hi abnormalities in embryos.Based on the results of the experiment,the deletion of Slc9a1 gene caused embryonic lineage differentiation failure,abnormal p H,increased cell damage,increased embryonic death,abnormal cell cycle,increased intracellular ROS,and accompanied by a decrease in embryo volume.It is shown that Slc9a1 is essential for maintaining early embryonic development in mouse.