The present study was conducted to evaluate the development of spermatogenesis and utility of using electroporation to stably transfect germ cells with the beta-galactosidase gene in neonatal bovine testicular tissue ectopically xenografted onto the backs of recipient nude mice. Bull testicular tissue from 4-wk donor calves, which contains a germ cell population consisting solely of gonocytes or undifferentiated spermatogonia, was grafted onto the backs of castrated adult recipient nude mice. Testicular grafts significantly increased in weight throughout the grafting period and the timing of germ cell differentiation in grafted tissue was consistent with postnatal testis development in vivo relative to the bull. Seminiferous tubule diameter also significantly increased with advancing time after grafting. At 1 wk after grafting, gonocytes in the seminiferous cords completed migration to the basement membrane and differentiated germ cell types could be observed 24 wk after grafting. The presence of elongating spermatids at 24 wk confirmed that germ cell differentiation occurred in the bovine tissue. Leydig cells in the grafted bovine tissue were also capable of producing testosterone in the castrated recipient mice from 4 wk to 24 wk after grafting at concentrations that were similar to levels in intact, nongrafted control mice. The testicular tissue that had been electroporated with a beta-galactosidase expression vector showed tubule-specific transgene expression 24 wk after grafting. Histological analysis showed that transgene expression was present in both Sertoli and differentiated germ cells but not in interstitial cells. The system reported here has the potential to be used for generation of transgenic bovine spermatozoa.

The present study was designed to evaluate the survival and proliferation of bovine spermatogonial stem cells in an explant culture system over a 2-wk period. Explants of calf testicular parenchyma were placed on 0.45-microm pore membranes in culture and maintained for 1-2 wk. Histological examinations of fresh (t0) and cultured tissues revealed morphologically normal seminiferous tubules. Germ cell numbers/tubule increased (P < or = 0.05) during culture when compared with 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 reverse transcription-polymerase chain reaction for cell-specific gene expression of stem cell factor, protein gene product 9.5, and glial cell line-derived neurotrophic factor family receptor-alpha1, respectively. Results demonstrated the expression of all genes at t0, 1 wk, and 2 wk 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 (P < or = 0.05) higher following 1 wk of culture compared with t0 and was numerically higher at 2 wk of culture compared with 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.

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.

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P< 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.