Establishment of gene knockout mice as a recipient model for spermatogonial stem cell transplantation.

Spermatogonial stem cell (SSC) transplantation is an alternative reproductive method to achieve conservation and production of elite animals in livestock production. Creating a recipient animal without endogenous germ cells is important for effective SSC transplantation. However, natural mutants with depletion of SSCs are difficult to obtain, and drug ablation of endogenous germ cells is arduous to perform for practical use. In this study, we used mouse models to study the preparation of recipients with congenital germ cell ablation. We knocked out (KO) Ets-variant gene 5 () in mice using the CRISPR/Cas9 system. The testicular weight of mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in mice. At 12 weeks of age, the tubules of mice lacked almost all spermatogenic cells with a Sertoli cell-only phenotype, and sperm were completely absent in the epididymis. We subsequently transplanted allogeneic SSCs with enhanced green fluorescent protein (EGFP) into 3- (immature) or 7-week-old (mature) mice. Partial restoration of germ cell layers in the seminiferous tubules and spermatogenesis was observed in all immature testes but not in mature adult testes at 2 months post-transplantation. The presence of heterologous genes and in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although mice could accommodate and support foreign germ cell transplantation, this process occurs in a quite low efficiency to support a full spermatogenesis of transplanted SSCs. However, using mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.