Reconstitution of spermatogenesis from frozen spermatogonial stem cells

Abstract

Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at -196 degrees C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal.

Fig. 1

Morphology of testes with transplanted stem cells previously stored at −196 °C. a, Testis of donor mouse (designated ZFlacZ), which carries an E. coli β-galactosidase transgene (lacZ) that allows round spermatids and later stages of spermatogenesis to be stained blue following incubation with 5-bromo-4-chloro-3-in-dole-β-D-galactosidase (X-gal)^,,. b, Testis of recipient male (C57BL/6 × SJL) F₁ treated with busulfan (36 mg/kg) to destroy endogenous spermatogenesis^,,. c, Left testis of recipient male 891 (Table 1) that received donor testis cells isolated from adult mice (8–16 weeks of age) and frozen 7 days. The testis has been bisected to allow penetration of fixative and stain, d, Left testis of recipient male 771 (Table 1) that received donor testis cells isolated from prepubertal mice (6–14 days of age) and frozen 111 days. Following incubation with X-gal, blue tubules in c and d identify areas of spermatogenesis from frozen donor cells. Scale bar, 1 mm.

Fig. 2

Microscopic appearance of spermatogenesis in recipient seminiferous tubules following microinjection of donor testis cells preserved at −196 °C. a, Seminiferous tubule of donor testis from transgenic ZFlacZ mouse. Round spermatids and more mature stages stain blue following incubation with X-gal (Fig. 1a)^,,. When staining is intense, immature stages also appear blue. b, Seminiferous tubule from recipient mouse treated with busulfan. No germ cell stages are present. Only Sertoli cells remain. c and d, Seminiferous tubules from busul-fan-treated recipient mice 891 and 771, respectively (Table 1). Blue staining of germ cells indicates their origin from transplanted donor cells that had been frozen 7 and 111 days, respectively. Background stain in all sections is neutral fast red. Scale bar, 50 μm.