Testis

Testis Tissue Explant Culture Supports Survival and Proliferation of Bovine Spermatogonial Stem Cells

Abstract
The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2wk period. Explants of calf testicular parenchyma were placed on 0.45mm pore membranes in culture and maintained for 1-2wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P0.05) during culture when compared to t0, yet germ cell differentiation was not observed. Testosterone was present in medium throughout the culture period, indicating functional Leydig cells. Sertoli, spermatogonial, and spermatogonial stem cell viability was evaluated by RT-PCR for cell specific gene expression of stem cell factor, protein gene product 9.5, and GFR1, respectively. Results demonstrated the expression of all genes at t0, 1wk, and 2wk of culture. Single cell suspensions were prepared from the testicular tissues at t0 and during culture and transplanted into nude mouse testes to investigate spermatogonial stem cell viability. One-month after transplantation, colonies of round bovine cells were identified in all mouse testes analyzed, indicating survival of spermatogonial stem cells. The average number of resulting colonies in recipient testes was significantly (P0.05) higher following 1wk of culture compared to t0, and was numerically higher at 2wk of culture compared to t0. This increase in colony numbers over time in culture indicates spermatogonial stem cell proliferation in vitro. This explant culture system appears to provide an environment that supports survival and proliferation of bovine spermatogonial stem cells.

Key words: Testis • Gametogenesis • Spermatogenesis

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