The Expressions Of GATA-4and GFRα-1in Mouse Seminiferous Epithelium

Spermatogonial stem cell (SSCs) is unique adult stem cell that can transfer thegenetic information to the progeny naturally. It can guarantee the continuousproceeding of spermatogenesis in male life by maintaining the stability ofself-renewal and differentiation. In the male reproductive system, GATA-4is mainlyexpressed in Sertoli cells, while the glial-derived neurotrophic factor receptor α-1(GFRα-1) is mainly expressed in As-SSCs and serves as the specific marker moleculeof SSCs. This study aims to investigate the expressions of GATA-4and GFRα-1inmouse spermatogenic epithelium and its impact on the self-renewal anddifferentiation of SSCs.The normal mice in different developmental stages and busulfan-treated mousemodel were used in this study. Morphological observation and quantitative analysis ofmouse seminiferous tubules and tissue sections were carried out using5-ethynyl-2’-deoxyuridine (EdU), floating fluorescent immunohistochemistry andhematoxylin-eosin (HE) staining, etc. in order to explore the expressions of GATA-4and GFRα-1in seminiferous epithelium and its relationship with SSCs.In this study, we established free-floating immunofluorescence staining methodof mouse seminiferous tubules and used PLZF, E-cad and GFRα-1to label thespermatogonium in seminiferous epithelium at different developmental stages. Theresults showed that the microscopic imaging at low background can be obtained byconventional incubation time and regular concentration of antibodies. As, Aprand Aalspermatogonial cells can be successfully labeled, which laid a foundation forfollow-up experiments to distinguish different SSCs lineage.In the seminiferous epithelium of normal mice at different developmental stages,the expression of GATA-4showed an increase trend firstly and then stabilized with the changes of mice age in days. It showed an upward trend from5to15days,reached the highest value on day15and then basically stabilized. Statistical analysisindicated that the expression of GATA-4on day15is significantly higher than that ofother ages in days (P<0.01). The number of GFRα-1-positive SSCs showed agradually rising trend from5to15days, reached the highest value on day15, andthen began to decline on day20, but was relatively stable from20to30days,continued to reduce from40to70days and then stabilized.Busulfan can be used to eliminate endogenous germ cells and is conducive to themigration of spermatogonial cell after transplantation. In this study, alkylatingagent-busulfan was used to inject mice intraperitoneally to make endogenousspermatogenic cells-eliminated mouse model. The results showed that the number ofSSCs was sharply reduced after Busulfan treatment. On day5, the number ofGFRα-1-positive SSCs was significantly reduced, which reached a minimum on day8and then began to recover. The statistical analysis showed that, compared with day0,the postoperative number of GFRα-1+cells within testis on days5and8wassignificantly lower than that of preoperation (P <0.01). The results of proliferatingcells EdU fluorescence intensity in busulfan model group were also consistent withabove results, which was also declined firstly, rose again and reached a minimum onday5. Statistical analysis results showed that the expression of proliferating cells EdUwithin testis on days3,5,8and10was significantly lower than that of preoperativeday0. In busulfan model, the expression of GATA4reached a minimum onpostoperative day3and the highest value on postoperative day5, showed an upwardtrend from8to18days and gradually returned to normal levels after day18.To sum up, the change trends of GFRα-1positive SSCs and GATA4expressed bysupport cells both in the normal development of testis and busulfan model testis werebasically the same, demonstrating that supporting cells played an important role in regulating the development of SSCs as the important composed cells of SSCsmicroenvironment.