Effect of Leydig and Sertoli Cells Respective Cul-ture on Expressions of Self-Renewal Biomarkers in Sper-matogonial Stem Cells of Mice

Background: The Sertoli cells-produced Glial cell-derived neurotrophic factor (GDNF) has been known to directly induce the spermatogonial stem cells (SSCs) self-renewal by interact-ing with its special receptors Gfrα1 and C-Ret. Thus, the current study was designed to investigate whether testosterone, as known co-factor in maintaining and progressing spermatogenesis, plays a role in stimulating the Sertoli cells to produce GDNF or not Materials and Methods: Therefore, the TM4 cells (line Ser-toli cells) were co-cultured with different concentrations (0.1 ng/ml, 0.2 ng/ml, and 0.4 ng/ml) of exogenous (commercial pure testosterone) and endogenous TM-3 (line Leydig cells)-produced testosterone, and consequently the TM4-produced GDNF concentration was evaluated by ELISA, immunocyto-chemistry (ICC) and qRT-PCR analyses. In the second step, the activity of produced GDNF was estimated by co-culturing the SSCs with the TM-4 derived media (containing endogenous GDNF). For this purpose, the SSCs were dissected (from 5-6 days old male neonates), purified, approved (using qRT-PCR of SOX-2, THY1, and NANOG), and finally cultured with TM-4-derived media (containing 0.1 ng/ml, 0.2 ng/ml, and 0.4 ng/ ml of GDNF). Next, the expressions of Gfrα1 and C-Ret were evaluated by ICC and qRT-PCR. The cell viability ratios were evaluated in each step of study using the MTT test.Results: Observations showed that endogenous testosterone, albeit in 0.1 ng/ml and 0.2 ng/ml, as well as exogenous testos-terone (at 3 dose levels) could fairly up-regulate the GDNF ex-pression versus non-treated Sertoli cells. The 0.4 ng/ml endog-enous testosterone-treated Sertoli cells represented diminishedexpression of GDNF versus other treated cells in the same cat-egory. In continue, Observations revealed that TM-4-produced GDNF, albeit at 3 dose levels, could up-regulate the Gfrα1 and C-Ret expression. Meanwhile, this situation was more notable in 0.1 and 0.2 ng/ml GDNF-treated SSCs.Conclusion: In conclusion, our findings showed that testoster-one plays a role in stimulating the Sertoli cells to synthetizing of GDNF. Moreover, the testosterone-induced GDNF is able to amplify its receptor’s expression both at mRNA and protein levels. Moreover, we found that GDNF solely is able to amplify its receptor expression may as a stimulator factor.

Keywords:

Testosterone , GDNF , Gfrα1 , C-Ret , SSCs