Abstract
ABSTRACTSpermatogonial 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 (Etv5) in mice using the CRISPR/Cas9 system. The testicular weight of Etv5−/− mice was significantly lower than that of wild-type (WT) mice. The germ cell layer of the seminiferous tubules gradually receded with age in Etv5−/− mice. At 12 weeks of age, the tubules of Etv5−/− 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) Etv5−/− mice. Partially 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 Etv5 and EGFP in recipient testicular tissue and epididymal sperm by PCR indicated that sperm originated from the transplanted donor cells. Our study demonstrates that, although Etv5−/− 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 Etv5−/− mice as a recipient model for SSC transplantation is feasible, and still needs further investigation to establish an optimized transplantation process.
Highlights
Our study explored the possibility of using Ets-variant gene 5 (Etv5)−/− mice as the recipient model with congenital germline ablation to facilitate Spermatogonial stem cell (SSC) transplantation study
DNA gel electrophoresis results demonstrated that homozygous knockout (KO) mice generated a 710 bp band, heterozygotes had two bands (710 and 6326 bp), and wild-type (WT) mice had a single band of 6326 bp (Fig. 1B)
Etv5−/− male mice at 3 weeks of age were obviously smaller than WT male mice, and this difference was more pronounced at 12 weeks of age (Fig. S1A)
Summary
Spermatogonial stem cells (SSCs) are male germline stem cells that reside in the basement membrane of the seminiferous tubule in. SSC transplantation has been demonstrated in many species, including rat, goat, sheep, pig, and non-human primate (Ogawa et al, 1999; Honaramooz et al, 2003; Rodriguez-Sosa et al, 2009; Mikkola et al, 2006; Hermann et al, 2012) This technique opens new avenues for the treatment of male infertility, development of alternative livestock reproduction technology, and generation of transgenic animals for biomedical and agricultural purposes. On the basis of the above considerations, specific gene mutations that are associated with the developmental deficit of SSCs without affecting testicular somatic support cells can be used to create such a recipient model In this regard, the currently developed genome editing technology can engineer any genomic regions to generate desired genotypes and phenotypes in many species, offering a universal platform for genome engineering in various animals. If Etv5−/− mice are applicable for SSC transplantation, this strategy can be extended to large farm animals by specific gene targeting to create transplant recipients serving SSC-based reproduction and transgenesis
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