Effect of sphingosine-1-phosphate on cryopreserved sheep testicular explants cultured in vitro

Abstract

Complete spermatogenesis has been achieved in vitro in mouse testicular explants with resulting sperm used to produce pups after Intra Cytoplasm Sperm Injection and Embryo Transfer. In the present study, we evaluated the influence of sphingosine-1-phosphate (S1P) on spermatogenesis of frozen-thawed lamb testis explants in vitro. Thawed testicular pieces were cultured for 12 d on agarose blocks in serum-free growth medium containing 0, 2, 5 or 10 μM S1P. At the end of D6 and D12, some pieces were fixed and processed for histology. Other pieces were processed for RNA isolation and quantitation of proliferation (PCNA, Ki67) and differentiation (PLZF) markers and genes involved in S1P signaling (S1PR1, SGPL1, SGPP1, AKT1 and NFKBIA) by qPCR. Histology revealed an increase (P < 0.05) in seminiferous cord (SC) diameter under all culture conditions, except 5 and 10 μM S1P by D6. In the presence of 5 μM S1P, percentage of gonocytes decreased (P < 0.05) by D6 (control, 24.9% vs. S1P, 10.3%) with a concomitant increase (P < 0.05) in spermatogonia formation (control, 74.4% vs. S1P, 88.1%). S1P induced PCNA or Ki67 expression by D6, whereas PLZF was up-regulated (P < 0.05) by D6 in 2 μM S1P and D12 in 5 & 10 μM S1P. Expression of SGPL1 and SGPP1 increased 4–12-fold in tissues cultured in 10 μM S1P by D12 compared to D12 control. AKT1 and NFKBIA mRNA expression was low (P < 0.05) in 5 and or 10 μM S1P treatments on D6. These results demonstrate that S1P promotes germ cell proliferation during first week of culture and may exert an anti-apoptotic influence on the seminiferous cord in sheep testicular explants in vitro.