The self-renewal and differentiation of spermatogonial stem cells (SSCs) is essential for the continuous production of sperm throughout life in male vertebrates. The development of a functional assay to analyze these properties in isolated SSCs remains necessary. In our current study, we have developed a transplantation method for testicular cell aggregates in zebrafish (Danio rerio) in which allogeneic SSCs can undergo self-renewal and differentiation. The immature testes from juveniles are dissociated, aggregated by cultivation, and then transplanted under the abdominal skin of the recipient fish. The grafted aggregates reconstitute the appropriate testicular structures, including the lobule structure, consisting of basement membrane and interstitial steroid-producing cells on the outside, and the cysts, which comprise germ cell clusters and surrounding Sertoli cells. Bromodeoxyuridine incorporation analysis indicated that continuous spermatogenesis is maintained for at least 6 mo in the reconstituted testis. Moreover, when the sperm generated from the aggregates at 3 mo postgrafting were used for artificial insemination, fertilized eggs were obtained that developed sexually mature fish. These results suggest that self-renewal of SSCs takes place in reconstituted testes under the abdominal skin and that their differentiating progeny can develop into functional sperm. Furthermore, allogeneic spermatogonia were also found to proliferate and differentiate into sperm in these grafts. Our method of grafting testicular cell aggregates should thus prove useful not only analyzing the stem cell ability of an individual SSC but also for the production of progeny from cultured SSCs or SSCs of sterile mutants with somatic cell defects.
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