Objective  To study the effect of intratubular injection of busulfan on ablation of pig endogenous spermatogonial stem cells (SSCs) and allotransplantation of SSCs on the recovery of the reproductive ability of the recipient after ablation of endogenous SSCs.

Method  Nine 6-week-old Large-white boars were injected through seminiferous tubules with busulfan at a dosage of 3 mg/kg, and other three pigs were injected with 2 mL DMSO as controls. After three weeks, each boar in the test group was given a second injection at the same dosage. At three weeks after the second injection, testes from test and control groups were collected to evaluate the endogenous SSC ablation conditions. At one month after the second busulfan injection, testicular single-cell suspension was isolated from the testes of 5−7-day-old Large-white piglets by two-step enzyme digestion method and purified by gelatin differential adherence method. After purification, the purity of SSCs was analyzed by immunofluorescence and flow cytometry. At four months after allotransplantation of SSCs, microsatellite marker analysis was used to detect the presence of donor-derived SSCs in recipient boar semen and testicular tissue.

Result  After treating the Large-white boar twice with busulfan at a dosage of 3 mg/kg, the results of HE staining and immunohistochemistry staining of testis tissue showed that germ cells at all levels in the seminiferous tubules of the testes in test group were ablated but the structure of sertoli cell was intact, which could support the colonization and development of transplanted exogenous SSC. The results of immunofluorescence and flow cytometry showed that the rate of UCHL-1 positive cells in the isolated testicular single-cell suspension increased from 16.3% before differential adhesion to 50.8% after purification. At four months after allotransplantation of porcine SSC, HE staining and immunohistochemical staining of testicular tissues in the transplanted group showed recovery of germ cell layers compared with the group injected with busulfan but not transplanted with donor cells. UCHL-1 positive SSCs were detected on the basement membrane of seminiferous tubules in recipient testes. Microsatellite marker analysis of recipient testis tissue showed the presence of donor SSCs, suggesting that donor SSCs transplanted into recipient testes could colonize and survive in recipient testes for more than four months. However, microsatellite marker analysis of semen did not detect donor-derived sperms.

Conclusion  Intratubular injection of busulfan into boar testes at a dosage of 3 mg/kg can effectively ablate endogenous SSCs and can be used to prepare recipient pigs for SSC transplantation. Two-step enzyme digestion and gelatin differential adhesion method can be used to successfully isolate and purify porcine SSCs. Porcine SSCs can colonize in recipient testes and survive for more than four months after allotransplantation.