Establishment of Etv5 gene knockout mice as a recipient model for spermatogonial stem cell transplantation

Abstract

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 (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.

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes

“…Transplantation of SSC into the sterile testes of males has been achieved in Dazl KO mice and rats ( Speed et al, 2003 ; Richardson et al, 2009 ), Etv5 KO mice ( Zhang et al, 2021 ), NANOS2 KO mice, boars, bucks, and bulls ( Ciccarelli et al, 2020 ; Latham et al, 2023 ), NANOS3 KO boars ( Wang et al, 2023 ), and Tscd22d3 KO mice ( Zhou et al, 2019 ). Germline complementation via embryo complementation has been achieved in NANOS3 KO male mice ( Miura et al, 2021 ), a NANOS3 KO heifer of fetal-age ( Ideta et al, 2016 ), Prdm14 KO rodents ( Kobayashi et al, 2021 ), and Tscd22d3 KO mice ( Koentgen et al, 2016 ).…”

E26 transformation-specific transcription variant 5 in development and cancer: modification, regulation and function

“…ETV5 expressed in Sertoli cells contributes to the integrity of the blood-testis barrier and immune response in the testes [ 78 ]. ETV5 −/− mice exhibit a Sertoli cell-only phenotype, with a lack of spermatogenic cells in the testes and no sperm in the epididymis [ 79 ]. Missense mutations of ETV5 also lead to a Sertoli cell only phenotype and increased embryonic lethality through the regulation of CXCR4 and C-C motif chemokine ligand 9 (CCL9), which are involved in the maintenance SSCs [ 80 ].…”

Application of CRISPR/Cas Technology in Spermatogenesis Research and Male Infertility Treatment

“…After being transplanted back into the testis, the repaired SSCs successfully restored the natural fertility of the mice. Xianyu Zhang et al also attempted to establish a SSC transplant recipient mouse model to achieve more effective SSC transplantation [ 128 ]. Although the Etv5 −/− mice that were successfully constructed could adopt and support foreign SSCs and produce donor-derived sperm, the efficiency of this process was very low.…”

“…In addition, the combination of gene editing and SSC transplantation offers new ideas for the treatment of male infertility, but further research is needed to establish the optimum transplantation process [ 128 ]. However, in the absence of extensive basic research and clinical trials, this therapeutic idea can be only a new direction.…”