Role And Molecular Mechanisms Of PAK1 In Regulating The Self-renewal And Apoptosis Of Human Spermatogonial Stem Cells

Objective: Spermatogonial stem cells(SSCs)can differentiate to mature sperm and self-renew to retain the pool of stem cells,which is essential for maintaining male fertility.Human SSCs are the unique adult stem cells that transmit genetic information to subsequent generations,and they have significant implication in both reproductive medicine and regenerative medicine.Infertility has become a serious disease affecting human health and reproduction,due to environmental factors,infection,inappropriate living manner and genetic factors.It has been estimated that 15% of couple are infertility,and half of them are attributed to male factor.There is no effective treatment for non-obstructive azoospermia(NOA)patients,a serious issue leading to male infertility,and notably,the pathogenesis of NOA remains unclear.It has been reported that the number of SSCs in NOA patients is reduced significantly compared to men with normal spermatogenesis and the proliferation of human SSCs in vitro is relatively slow,reflecting that there might be an association between proliferation/apoptosis of human SSCs and male infertility.However,little is known about genes in mediating the fate determinations of human SSCs,due to the limited source of human primary SSCs and difficulty in obtaining human testis tissues.Here we have explored for the first time the function and molecular mechanisms of PAK1 in regulating proliferation and apoptosis of human SSCs and its abnormal expression in NOA patients.This study can provide novel insights into the genetic and epigenetic regulation of human SSCs.Methods: RT-PCR,Western blots were used to verify the identity of human SSC line using various kinds of markers for primary human SSCs,and immunocytochemistry and immunohistochemistry were utilized to check the expression of PAK1 in human SSCs and human testicular tissues.The expression levels of PAK1 were detected of human SSC line after treatment with EGF,GDNF,FGF2,or the combinations of these three growth factors.The CCK8 assay,BrdU incorporation assay,and annexin-V/PI staining and flow cytometry were performed in human SSC cell line after PAK1-siRNAs treatment.In order to identify the targets of PAK1,we conducted RNA sequencing and miRNA arrays to compare the changes of global gene expression levels and miRNAs between PAK1 siRNA and control siRNA in human SSCs.Bioinformatic analysis and real-time PCR were carried out to find out the downstream effectors.Then CCK8 and EDU assays were used to explore the role of PAK1 downstream genes in regulating human SSCs,and Annexin-V/PI staining and flow cytometry were conducted to determine their function in cell apoptosis.Finally,we compared the levels of PAK1 among subtypes of NOA patients and OA patients with normal spermatogenesis.GraphPad prism 5.0 was used for statistical analysis,and the test of normality and homogeneity of variances was conducted before being analyzed with t test and one way ANOVA.p<0.05 was considered statistically significant.Results: RT-PCR and Western blots showed that human SSC cell line expressed numerous genes and proteins for human germ cells and human SSCs,thus confirming the identity of human SSC line.Immunohistochemistry revealed that PAK1 was expressed in human spermatogonia along the basement of seminiferous tubules in human testes,whereas it was undetected in pachytene spermatocytes or round spermatids.PAK1 level was up-regulated by EGF but not GDNF or FGF2.PAK1 promoted proliferation and DNA synthesis of human SSC line,whereas it suppressed their apoptosis both in vitro and in vivo.RNA-sequencing and miRNA arrays revealed that the levels of PDK1,KDR,ZNF367 and miR-31-5p were affected by PAK1 siRNAs.Immunoprecipitation and Western blots demonstrated that PAK1 interacted with PDK1.The proliferation abilities of human SSC line were reduced by PDK1-,KDR-,ZNF367-siRNAs and miR-31-5p mimics,whereas their apoptosis was enhanced by these RNA oligos.We also found that the levels of phos-ERK1/2 and phos-AKT were decreased by PAK1-siRNAs in human SSC line.PAK1 mediated cell cycle protein cyclin A but not cyclin B1,cyclin D1,or CDK2.The mRNA and protein levels of PAK1 was lower in NOA patients compared to OA patients with normal spermatogenesis.Conclusion: We verified the identity of human SSC line as the human SSCs in phenotype.PAK1 was identified as the first molecule that controls the proliferation and apoptosis of human SSC line through PDK1/KDR/ZNF367.miR-31-5p was involved in the growth repression induced by PAK1-knockdown.PAK1 mediates the activation of ERK1/2,AKT and cyclin A in human SSC line.Furthermore,we have unveiled an association between the levels of PAK1 and NOA patients.This study provides novel gene regulation and networks underlying the fate decisions of human SSCs and offers new targets for the applications of human SSCs in translational medicine.