Germ cell transplantation into mouse testes procedure.

OBJECTIVE: To illustrate the step-by-step protocol followed to assay germ cell transplantation into the seminiferous epithelium of mouse testes.

DESIGN: Video presentation of an animal model for research in reproductive and regenerative medicine.

SETTING: Research laboratory.

ANIMAL(S): Male nude mice (NU-Foxn1(nu)).

INTERVENTION(S): Mice were chemically sterilized with alkylant compounds (busulfan) followed by gonadal microsurgery to inject donor germ cells.

MAIN OUTCOME MEASURE(S): Donor cells should be labeled with reporter genes, such as green fluorescent protein (GFP), lactose operon (LacZ), or alternatively design an effective strategy with specific antibodies to track them within the recipient testes. Sperm detection in the ejaculate can also be used as a read out. However, in this case detection of the donor genotype in the sperm is mandatory to elucidate their origin.

RESULT(S): In the present study we describe the complete protocol for germ cell transplant by efferent duct injection, including the preparation of recipient mice, surgery for the germ cell transplant, and analysis of recipient testes. The main strength of this technique is that it constitutes the gold standard for a functional test of the germ cell potential as only spermatogonial stem cells are able to properly colonize the seminal lumen. Both fresh and frozen/thawed testicular cells are suitable for this technique as donor germ cells. Also, enrichment of living spermatogonial stem cells, previous to the transplant, seems to improve the efficiency of colonization. For proper colonization of germ cells, the niche should be available and thus mouse strains that lack endogenous spermatogenesis such as W/W(v) mutant mice are usually used. In the case of nonmatched donor cells, seminiferous epithelium of immune-suppressed recipient mice should be germ cell depleted before the transplant. One limitation of this technique is that the procedure can take up to 3 months. Also, in contrast to the full recovery of spermatogenesis in mouse-to-mouse transplants, xenotransplantation of germ cells from phylogenetically distant species, such as humans into mouse recipients, results in colonization of donor cells and spermatogonial expansion, but fail in their spermatogenic progression due to evolutive incompatibilities with the recipient niche. Xenografting of pieces of donor testis tissue under the skin of mouse hosts is an alternative approach that is currently being investigated to try to solve this limitation.

CONCLUSION(S): Transplantation of spermatogonial stem cells into the seminal lumen of mouse testes is a functional assay that defines this cellular subpopulation by its ability to colonize it. This technique can be used as a model to elucidate the insights of spermatogonial stem cells, to produce transgenic animals by genetically manipulating donor cells before transplantation, but also it has potential applications in fertility preservation in cattle and humans as it is feasible in large animals, as recent reports have demonstrated with rhesus monkeys, that recovered spermatogenesis after allogenic transplantation, and even from human cadaver testes. Therefore spermatogonial stem cells isolated from prepuberal boys, who are treated with alkylant chemotherapy, could be returned to their testis to regenerate spermatogenesis in the future.