The effect of rapamycin administration on mouse spermatogenic cells

Spermatogenesis is a complex process of cellular renewal and differentiation. mTOR signaling pathway serves as a central regulator of cell metabolism, proliferation and survival. mTOR forms two protein complexes, mTORC1 and mTORC2, by interacting various proteins. Rapamycin sensitive-mTORC1 positively controls protein synthesis by phosphorylating the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase 1 (p70 S6K1). Tuberous sclerosis complex (TSC/Tuberin) is a negative regulator of mTORC1. mTOR inhibitors are immunosuppressive drugs used in organ transplantation patients. Recently, several studies have emphasized potential impact of rapamycin on male gonadal function by decreasing testosterone levels and sperm counts. Recently, we have shown immunohistochemical distributions of mTOR signalling proteins in early spermatogenic cells of adult mice. Thus; we aimed to investigate the effect of rapamycin administration to spermatogenic cells utilizing seminiferous tubule cultures from adult mice. We grouped adult mice as control, vehicle and rapamycin. We used 200 nmol rapamycin. We cultured vehicle group mice with ethanol (solvent of rapamycin) and control group seminiferous tubules with %0,1 BSA and RPMI medium for 24 hours. After culture cell viability assay were performed at 0. And 24 hours. Western blot and immünohistochemistry were applied for p-S6K, PCNA, Stra8, VASA, Sycp3 and proteins associated mTOR signaling pathway. For all groups TUNEL was performed for cell death. mRNA levels were evaluate with RT-PCR. Our results have indicated that cell viability was similar between groups. For western blot analysis p-S6K, PCNA and Stra8 expression was decreased in rapamycin administrated group significantly. No differences were observed for VASA expression between the groups. For all groups the number of apoptotic cells was similar. 4EBP1 levels decreased in rapamycin group. mRNA levels of VASA and PCNA increased significantly in rapamycin administrated group. This is the first functional study that reports mTOR signaling pathway may have an important role in spermatogonial proliferation and differentiation. Our findings would also contribute to stem cell studies as well.