Effect of TNFα on the development of different stages of spermatogenesis in-vitro, using spermatogonial cells isolated from normal and busulfan-treated immature mice

Nagham Ali ^1,2,3 Eitan Lunenfeld ⁴ Mahmoud Huleihel ^1,2,3

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

Introduction: TNFα is an autocrine/paracrine factor, secreted by germ and Sertoli cells. In the testis, TNFα is known to affect Leydig and Sertoli cells functions. Busulfan (BU) is a cytotoxic anti-cancer drug. It damages proliferating cells. Therefore, it may lead to sub-fertility or even to infertility.

Aims: To evaluate the effect of TNFα on the capacity of spermatogonial cells, isolated from normal and busulfan-treated immature mice, to develop different stages of spermatogenesis in-vitro.

Methodology: Seven-days-old immature mice were used as normal or were intraperitoneally-injected with busulfan (45 mg/kg), and sacrificed 10 days post-injection. Cells from seminiferous tubules (STs) were enzymatically isolated, and cultured in methylcellulose (MCS) without (CT) or with TNFα (1,10,100 pg/ml). Fresh media without (CT) or with TNFα were added from the beginning, and after two weeks of culture. Developed cells were collected after 5 weeks, and examined for different stages of spermatogenesis by specific immunofluorescence staining (IF) or qPCR analyses.

Results: Addition of TNFα to MCS that contained isolated cells from STs of normal immature mice significantly increased the development of cell of the premeiotic (VASA), meiotic (BOULE) and postmeiotic (ACROSIN) stages. TNFα also differently affected the expression levels of Sertoli cell functionality markers. The effect of TNFα on the development of spermatogenesis from STs of busulfan-treated immature mice in MCS was lower compared to normal mice.

Conclusion: Our findings indicate the possible involvement of TNFα in the development of spermatogenesis in-vitro, and thus to be used in developing future therapeutic strategies for prepubertal male fertility preservation.