Effects of temperature on the development of spermatogenesis in-vitro using 3-dimension culture; possible regulation through Sertoli cell functionality

Areej Jorban ^1,2,3 Eitan Lunenfeld ⁴ Mahmoud Huleihel ^1,2,3

¹The Shraga Segal Dep. of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Israel
²The Center of Advanced Research and Education in Reproduction (CARER), Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel
³Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel
⁴Faculty of Medicine, Ariel University, Israel

Introduction: Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages. It occurs in-vivo at 34 0C-35 0C. Sertoli cells play a crucial role in the development of normal spermatogenesis.

Aim: to examine the effect of temperature on the development of spermatogenesis in-vitro, and evaluating the possible involvement of Sertoli cell functionality under these conditions.

Material and methods: Seminiferous tubules cells were enzymatically isolated from testes of 7-day-old sexually immature mice. The cells were cultured in methylcellulose [as a 3-dimension (3D) in-vitro culture system] and incubated in CO2 incubator at 35 0C or 37 0C. After 2-6 weeks the development of different stages of spermatogenesis were evaluated by immunofluorescence staining (IF) or qPCR analyses using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate functionality of Sertoli cells were examined by qPCR.

Results: Our results show that under 350C, the percentages and/or the expression levels of the developed pre-meiotic (VASA, PLZF, GFR-a), meiotic (BOULE, CREM) and post-meiotic cells (ACROSIN and PROTAMINE) were significantly higher compared to 370C. The expression levels of androgen receptor, FSH receptor, transferrin, androgen binding protein and glial-derived nerve growth factor were higher at 35 0C compared to 37 0C.

Conclusion: Our results show that optimal conditions for development of spermatogenesis in-vitro is 35 0C, similar to in-vivo. This could be related, at least partially, to Sertoli cells activity. These findings may deepen our understanding of the mechanisms behind optimal conditions of normal spermatogenesis in-vivo and in-vitro.