The Roles And Mechanisms Of MKK7 In The Proliferation And Apoptosis Of Human Spermatogonial Stem Cells

Non-obstructive azoospermia(NOA)is one of the most severe male infertility disorders.However,effective treatments are still lacking.Spermatogonial stem cells(SSCs)are the origin of male spermatogenesis,which has the ability to restore the germlines in the testis.However,the developmental regulatory mechanism of SSCs is not clear,which limits its application in regenerative medicine and male infertility treatment.Objective: In this study,we explored the key regulatory molecules of human SSCs by analyzing publicly available single-cell sequencing data of human normal testis.We also performed cell experiments to validate their functions and molecular basis,and in this way,further explored the mechanisms of human SSCs fate determination.Methods: In this study,we screened and obtained genes highly expressed in human SSCs by analyzing data from nine human testis monocytes derived from GSE149512 and GSE112013.We verified the expression of these genes in human testes with normal spermatogenesis using Western Blot,IHC,and co-IF.Subsequently,we used small interfering RNA to knock down MKK7 expression in immortalized human spermatogonial stem cells,and examined the effect of MKK7 downregulation on cell proliferation and apoptosis using Western Blot,CCK-8 cytotoxicity assay,EDU assay,TUNEL,and flow cytometric analysis.Next,we predicted the interacting proteins of MKK7 by STRING,Gene MANIA and Hit Predict databases,and validated the predictions using immunoprecipitation and Western blot.Finally,we utilized small molecule inhibitors to validate the proliferative and apoptotic effects of MKK7 target proteins in SSCs lines.In addition,we examined the distribution of MKK7 in azoospermic patients to analyze the correlation between MKK7 and spermatogenesis dysregulation.Results: The analysis of human testis single-cell sequencing showed that MKK7 was predominantly expressed in SSCs.The results of IHC and Western blot showed that MKK7 was localized near the basement membrane of the varicose seminiferous tubules,and about 70% of GFRA1-positive spermatogonial stem cells expressed MKK7.After knockdown of MKK7 in human spermatogonial stem cell lines using small interfering RNA,cell proliferation and DNA synthesis capacity were significantly downregulated,and apoptosis was significantly increased.Protein interaction prediction and validation results indicated that MKK7 interacted directly with JNK and was involved in the regulation of JNK phosphorylation.After inhibition of JNK phosphorylation using SP600125,cell proliferation was significantly downregulated and apoptosis was significantly increased,with a phenotype similar to that of MKK7 deficiency,suggesting that JNK is a functional target of MKK7.In addition,the results of co-IF and Western blot showed that the positive rate and expression of MKK7 were significantly down-regulated in some testicular tissues with impaired spermatocyte maturation and impaired spermatogonia maturation,implying that abnormal expression of MKK7 may be correlated with impaired spermatogenesis.Conclusion: In this study,we discovered that MKK7 was highly expressed in human SSCs by single-cell RNA sequencing analysis and IHC,and revealed the mechanism by which MKK7 affected human SSCs proliferation and apoptosis through regulating JNK phosphorylation.These results enrich the understanding of SSCs fate determination and may provide a theoretical basis for the diagnosis and treatment of male infertility.