Regeneration of Spermatogenesis and Production of Functional Sperm by Grafting of Testicular Cell Aggregates in Zebrafish (Danio rerio)1

Abstract

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.

Subordinate male cichlids retain reproductive competence during social suppression

“…BrdU is commonly used across vertebrates to examine spermatogenesis because it is incorporated into DNA during the S-phase of mitosis, and thus is an indicator of cell proliferation [44][45][46].…”

Differentiation of zebrafish spermatogonial stem cells to functional sperm in culture

“…Dissociated hyperplasia cells from fish harboring a vas::egfp transgene (Krøvel and Olsen, 2002) were cultured for 4 weeks as previously described (Kawasaki et al, 2012). GFP-positive cells were then mixed with testicular aggregates derived from the inbred IM line and transplanted subcutaneously into IM recipients, as described previously (Kawasaki et al, 2010). After 3 months, we performed artificial insemination using sperm from the transplanted aggregates and obtained fertilized eggs.…”

“…As the host of transplantation we chose rag1 t26683 mutant zebrafish (hereafter rag1 mutant), which harbors a nonsense mutation in the catalytic domain of the encoded RAG1 protein resulting in loss of mature functional T and B cells (Wienholds et al, 2002;PetrieHanson et al, 2009). To examine whether the rag1 mutant endures surgery and accepts allografts, we grafted wild-type testis fragments into wild-type and rag1 mutant fish as described previously (Kawasaki et al, 2010). Grafts of testis fragments to wild-type host were immediately rejected and fragments were not found in hosts after 4 weeks; however, when testis fragments were transplanted under the abdominal skin of rag1 mutants, the graft was found in 8 out of 10 transplants (Table S1).…”

“…To transplant fragments of normal testes and testicular hyperplasias, we applied the testis transplantation method (Kawasaki et al, 2010) with slight modifications. Briefly, 1 day prior to transplantation, male recipients were maintained without feeding.…”

Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants

“…However, this in vitro differentiation assay for premeiotic germ cells cannot exclude the possibility that mitotic spermatogonia were already affected by the defective somatic cells and lost competency to progress through meiosis before culturing. Further experiments, such as germ cell transplantations (Kawasaki et al, 2010;Nóbrega et al, 2010), should be performed to elucidate in which cell types these genes are required for normal progression of spermatogenesis.…”