Transplantation of bovine germinal cells into mouse testes

Abstract

To develop techniques for spermatogonial transplantation in bulls, it is essential to have an effective bioassay procedure to evaluate the transplantation efficiency of spermatogonial stem cell collection, purification, and culture techniques. The objective of the present study was to develop a mouse bioassay model to evaluate transplantation efficiency of fresh and cultured bovine germ cells. Bull calves of four ages (1, 2, 3, and 4 mo) were used as a source of donor testes cells. Two calves were used for each age point, one calf was experimentally made cryptorchidistic at 1 wk of age and the other left normal. A STO (mouse fibroblast) feeder cell line was used to culture bovine testes cells for 2 wk preceding transfer into recipient testes. Immunodeficient nude mice (nu/nu) in which endogenous spermatogenesis had been abolished by busulfan treatment served as recipient animals for transplantation. Donor bovine germ cells were microinjected into mouse seminiferous tubules. Mouse testes were analyzed 2 wk after transplant with the use of a bovine-specific antibody and whole-mount immunohistochemistry for the presence of bovine donor germ cells. Bovine testis cells were present in all recipient mouse testes analyzed. Fresh bovine testes cells were observed as colonies of round cells within mouse seminiferous tubules, indicating spermatogonial expansion and colonization; however, cultured bovine testes cells appeared as fibrous tissue and not as spermatogenic colonies. The average number of colonies resulting from donor cryptorchid testes was not different (P > 0.05) from noncryptorchid, 56+/-4 and 78+/-7, respectively. Fresh donor cells from calves older than 1 mo gave rise to a greater average number of colonies within recipient testes (P <0.05) (1 mo, 33+/-4; 2 mo, 70+/-8; 3 mo, 63+/-6; 4 mo, 87+/-9). Fresh bovine germ cells are capable of colonization in the busulfan-treated nude mouse testis, making it a suitable model for evaluation and development of spermatogonial transplant techniques in bulls.

THY1 is a conserved marker of undifferentiated spermatogonia in the pre-pubertal bull testis

“…Currently, methods for efficiently culturing and transplanting SSCs are only available for rodents (Brinster & Avarback 1994, Brinster & Zimmermann 1994 and have yet to be fully established for livestock species, though some initial progress has been made (Dobrinski et al 2000, Izadyar et al 2003, Oatley et al 2002, 2004b, Aponte et al 2008. Development of these procedures in rodents has been aided greatly by identification of surface markers expressed by undifferentiated spermatogonia, allowing for isolation of cell fractions enriched for these cells compared to the total testis cell population.…”

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

“…Bovine SSCs were successfully cultured on a monolayer of STO (SIM 6-thioguanine resistant ouabain resistant) feeder cells [24], bovine embryonic fibroblast cells (BEF) [25] and Sertoli cells [26]. Bovine SSCs were also cultured for short term on laminin coated plates in vitro [11].…”

Spermatogonial stem cells from domestic animals: progress and prospects

“…The spectacular discoveries impel scientists to investigate the potential of SSCs from large animals in this respect. As reported, SSCs from domestic species could colonize and proliferate in host mouse testes after transplantation, but could not give rise to entire spermatogenesis , Izadyar et al 2002, Oatley et al 2002, Kim et al 2006; Table 2), probably due to the phylogenetic disparity between donors and hosts. Later, allogeneic transplantation of SSCs was tested in boars, bulls, goats, sheep, and dogs (Honaramooz et al 2002a, Izadyar et al 2003, Herrid et al 2006, 2009b, Kim et al 2008, and the development of the donor germ cells was observed in those recipients of homogeneous species ( Fig.…”