The Regulation And Mechanism Of Eif2s3y In The Proliferation Of Dairy Goat Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs)are located on the basement membrane of the seminiferous tubules of the testis and can maintain the stability of their population by selfrenewal.It is the only male mammal adult stem cells that can transmit genetic information to the next through natural reproductive processes.Capra hircus spermatogonial stem cells(GSSCs)are adult stem cells isolated from the testis of puberty or sexually mature male goats.As an important livestock in Guanzhong area,dairy goats have far-reaching significance for the study of high-quality goat breeding and research on transgenic sheep.It also provides a theoretical basis for cherishing animal protection and treatment of human infertility.Eif2s3y is a gene encoding the translation initiation factor family e IF2γ,and the formed e IF-2 polymer can function as a translation initiation.In recent years,some studies have shown that Eif2s3y plays an indispensable role in mouse sperm formation,and overexpression of this gene in the process of inducing spermatogenic cells in vitro can improve the induction efficiency.However,its intrinsic regulatory mechanisms are not clear,and it is unclear what effect Eif2s3y can have on the proliferation and differentiation of spermatogonial stem cells.What role does the gene play in the reproductive development of livestock? There are no related reports.The purpose of this study is to explore the regulation mechanism of Eif2s3y in the spermatogonial stem cells of dairy goats and find its main signaling pathway.In this study,we obtained the Eif2s3y gene CDS sequence(KP326346)from the testis of dairy goats.We found that this gene is widely expressed in various tissues and organs of goats and is highly conserved among different species.We constructed a recombinant plasmid that overexpresses and interferes with the expression of Eif2s3y in GSSCs.On the one hand,it is found that Eif2s3y can promote the proliferation and reduce the pluripotency of GSSCs.On the other hand,it is found that Eif2s3y can promote the meiosis of m GSCs and reduce the GSSCs.Apoptosis.In the mouse model of busulfan,Eif2s3y can promote the repair of seminiferous tubules and the regeneration of spermatogenic cells at all levels.At the same time,when we knocked down Eif2s3y in mouse testes,the number of spermatogenic cells in the mice decreased sharply.Further experiments showed that Eif2s3y may regulate the proliferation of spermatogenic stem cells in dairy goats through the ERK1/2 pathway.Our study reveals that Eif2s3y plays an important role in the self-renewal and sperm production of spermatogonial stem cells in dairy goats.1.Eif2s3y promotes the proliferation of GSSCsFirst,we designed a cloning primer for the Eif2s3y gene in dairy goats,and cloned the CDS sequence of Eif2s3y in the m RNA of the testis of dairy goats.We then used this sequence to design a highly specific q RT-PCR primer,which was used for different ages.The expression level of Eif2s3y in the testis m RNA of dairy goats was detected.It was found that Eif2s3y increased with the increase of goat age and reached its peak after sexual maturity.Next,we detected the high expression of Eif2s3y in the testis by detecting the m RNA of different tissues and organs of goats.Then,we isolated the spermatogonial stem cells of the dairy goats and overexpressed Eif2s3y in GSSCs to construct an overexpressing cell line,by q RT-PCR detection of overexpressing cell lines,plotting growth curve,Edu staining and flow cytometry.By other techniques,we found that overexpression of Eif2s3y promoted the proliferation rate of cells,shortened the population doubling time,and the percentage of Edu positive was significantly higher than that of the control group,and the ratio of S phase cells was also higher than that of the control group.These results confirmed that Eif2s3y can promote the expression of proliferation-related genes CCND1,PCNA,Cyclin A in GSSCs,activate the Erk1/2 pathway,and promote the proliferation of GSSCs.2.Eif2s3y activates the MEK-ERK1/2 pathwayPrevious studies have found that the Erk1/2 pathway is activated after overexpression of Eif2s3y,so we further investigated the regulation of this pathway by reconstituting inhibitors and activators that interfere with cell line binding to the MAPK-Erk1/2 pathway.Finally,it was found that the proliferation rate of cell lines that interfered with Eif2s3y decreased significantly,and the phosphorylated Erk1/2 protein decreased.After the addition of the MAPK-Erk1/2 pathway activator,the proliferation rate was restored.When an inhibitor is added to cells overexpressing Eif2s3y,the rate of proliferation is slowed and the phosphorylated Erk1/2 protein is reduced.Through the above experiments,it was confirmed that Eif2s3y did affect the proliferation of GSSCs through the Erk1/2 pathway.3.Eif2s3y can improve the anti-apoptotic ability of GSSCsFirst,we examined the m RNA levels of Eif2s3y-expressing cell lines and found that Caspase3 was significantly decreased.Next,we performed an Annexin V-FITC/PI staining and observed the apoptosis rate under the fluorescence microscope.It was found that the Eif2s3y overexpression group had a lower apoptosis rate than the control group,and the same results were obtained by flow cytometry.The results of Western blotting showed that both Caspase-3,Caspase-12 and CHOP were significantly down-regulated,and we know that the upstream of Caspase-12 is endoplasmic reticulum stress(ERS),so we speculate that Eif2s3y may reduce the apoptosis rate of cells by inhibiting endoplasmic reticulum stress.