Tehran University Medical Journal

Background: Spermatogenesis is a complex and highly organized process of proliferation and differentiation of spermatogonial stem cells. Spermatogonial stem cells (SSCs) as a unique stem cell have the potential to self-renewal, differentiation and transmit genetic information to the next generation and play a vital role in maintaining fertility. Sertoli cells as the only somatic cells within the seminiferous epithelium play central roles in the formation of niche and balance between self-renewal and differentiation by secrete many growth factors. Given the importance and widespread use of SSCs, particularly in the treatment of infertility, the aim of this study was to create an optimal environment for the proliferation of SSCs. So we decided to study of undifferentiated (ID4) and differentiated (c-Kit) gene expression in SSCs followed by co-culture with Sertoli cells for a one-month.

Methods: This experimental study was conducted from November 2013 to December 2014 in Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, on immature NMRI mouse (6-3 days old). Initially, Sertoli cells and SSCs were isolated from neonates mouse testes during the two-step enzymatic digestion characteristics Sertoli cells with vimentin marker and SSCs with promyelocytic leukemia zinc-finger (PLZF) marker were confirmed. Then SSCs were cultured in two groups: co-culture with Sertoli and without co-culture (control). Undifferentiated (ID4) and differentiation (c-Kit) gene expression were evaluated by Real-time PCR technique.

Results: Spermatogonial stem cells purity was obtained 66.91% by flow cytometry. The relative expression levels of gene ID4 in co-culture group at the end of each week, compared to the control group showed a significant increase (P<0.05). While the expression of this gene significantly decreased in each group over time (P<0.05). The results of the comparison of the relative expression of c-Kit gene in co-culture group are indicated significant decrease than the control group at the end of each week (P<0.05). In addition, this gene expression was showed significant increase in each group individually over time (P<0.05) ID4 gene expression showed a significant (P<0.05) increase toward the control group, while in the expression of c-Kit was observed a significant (P<0.05) decrease compared with the control group at the end of each week.

Conclusion: According to the results of this study, co-culture with Sertoli cells maintains SSCs in the prolifration stage for long-term, so can be used to optimize the culture medium at the clinic.