New Area for Testosterone and GDNF-Induced Roles in Varicocele-Related Infertility; Correlation with Spermat-ogonial Stem Cell Self-Renewal

Based on previous clinical and experimental trials, the testos-terone withdrawal in association with severe oxidative stress remarkably suppresses spermatogenesis as well as sperm count and quality in both animal models and infertile individuals. Indeed, testosterone through the classical (androgen recep-tor) and the non-classical (testosterone-mediated [Ca2+] in-flux) pathways boosts/maintains the spermatogenesis process. On the other hand, Sertoli cells are involved in maintaining/ progressing spermatogenesis and spermiogenesis processes through several pathways. Among these pathways, the Glial cell-derived neurotrophic factor (GDNF) in association with its special receptors Gfrα1 and C-RET are actively participated in spermatogonial stem cells (SSCs) self-renewal process. How-ever, this question remains that, does testosterone affect GDNF expression/synthesis in Sertoli cells or not To respond this question, a two-step study (in vitro and in vivo) was designed. In the in-vitro part; the mouse TM3 (Leydig) line cells were cultured, and the TM3-produced testosterone was included in TM4 (Sertoli) cells culture media, in a concentration (0.1 ng/ ml, 0.2ng/ml and 0.4 ng/ml)-dependent manner. As a positive control, the same concentrations (0.1 ng/ml, 0.2 ng/ml and 0.4 ng/ml) of exogenous (commercial) testosterone was included in additional four Sertoli cell groups. Following 12 hours, the GDNF expression was evaluated by ELISA, qRT-PCR and ICC techniques. Observations showed that the TM3-produced testosterone (at 0.1 ng/ml and 0.2 ng/ml concentrations) and exogenous testosterone (at three concentrations) could signifi-cantly (P<0.05) up-regulate the GDNF expression compared to those testosterone non-treated Sertoli cells. This finding clearly illustrates that the testosterone is able to directly boost GDNF expression in Sertoli cells. This pathway was investigated par-allelly in in-vivo condition by inducing experimental varicocele in rats. Observations revealed that simultaneous with Leydig cell elimination and testosterone suppression in varicocelized rats, the GDNF expression (assessed by western blot, IHC and qRT-PCR) was diminished. Therefore, we can suggest that the varicocele-suppressed testosterone production is able to nega-tively affect the GDNF in Sertoli cells (in in-vivo) similar to those in in vitro condition. Next, minding that GDNF interacts with its receptors, this hypothesis came out, does GDNF act as transcriptional/boosting factor for its receptors or not To respond this Delima different concentrations (0.1 ng/ml, 0.2 ng/ml and 0.4 ng/ml) of TM4-produced and exogenous GDNF were included in SSC culture medium. Observations showed that TM4-produced GDNF (0.1 ng/ml and 0.4 ng/ml) and the exogenous GDNF (at all concentration levels) could amplify Gfrα1 and C-RET expression in SSCs. Thus, we came close to this fact that GDNF not only interacts with its receptors to initi-ate SSC self-renewal but also boosts Gfrα1 and C-RET expres-sion, at the same time. As an in vivo part; we demonstrated that when testosterone is administrated to varicocele-induced ani-mals, the GDNF and at the same time Gfrα1 and C-RET expres-sions were increased. Thus, we can conclude that varicocele by suppressing testosterone production is able to negatively affect the SSCs self-renewal process leading to severe germ cell loss through spermatogenesis.

Keywords:

Varicocele , GDNF , Testosterone , Spermatogonial Stem Cells , Self-Renewal