Analysis of the expression of PLZF and VASA genes and their protein relationships in mouse spermatogenic stem cells during the process of differentiation into sperm

Abstract

Germ cells Spermatogonial stem cells PLZF VASAAim: Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemical analysis showed PLZF-positive cells in the basement membrane of the seminiferous tubule. It seems that the PLZF germ cell marker is specifically expressed in the spermatogonial cells of the testis. In mice with genetic deletion of the VASA gene, males show reproductive deficiency with reduced sperm production. In this study, we looked at the expression of PLZF and VASA in spermatogenic tubules and germ cells in vivo and in vitro. Material and method: Isolation of spermatogonial stem cells, Cultivation of testis on STO feeder layer, Immunohistochemistry (IHC), Immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR), Network analysis of protein-protein interactions (PPI), Pathway enrichment analysis and gene analysis (GO), were used to analyze the expression of PLZF and VASA in mice testis tissue. Results: In this experimental study, whereas undifferentiated spermatogonial cells sharply express PLZF, other types of germ cells located in the seminefrous tubul were negative for this marker. In other hand, the germ cells near the basal membrane of seminefrous tubul showed expression of VASA wheras the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferetiated cells in compare to the differentiated germ cells. Fluidigm real-time RT-PCR result demonstrated a significant expression (P<0.05) of VASA in the spermatogonial stem cells compared to differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. In the present experiment, after the production of SSCs under the stimulation of growth factors FGF, EGF, and GDNF, immunocytochemical staining showed a clear expression of PLZF and VASA in SSCs compared to in-vivo conditions, and VASA is less expressed in these cells. The data obtained from IHC analysis showed that VASA is expressed in the center of testicular cords. The data set related to protein-protein interaction members was used in this research due to the lack of information about PLZF expression in different stages of spermatogenesis. The study of gene expression showed that several biological and functional pathways are involved in the expression of PLZF in different stages of spermatogenesis. Conclusion: These results clearly proved the role of PLZF as a specific marker for spermatogonial stem cells, and can be beneficial for advance reserach about in-vitro differentiation of SSCs to functional sperms. ، ‫شماره‬ 2 ، ‫تابستان‬ 1402 ‫صفحات‬ ، 153 ‫تا‬ 165