Rat spermatogenesis in mouse testis

Abstract

Recently, transplantation of mouse donor spermatogonial stem cells from a fertile testis to an infertile recipient mouse testis was described. The donor cells established spermatogenesis in the seminiferous tubules of the host, and normal spermatozoa were produced. In the most successful transplants, the recipient mice were fertile and sired up to 80 per cent of progeny from donor cells. Here we examine the feasibility of transplanting spermatogonial stem cells from other species to the mouse seminiferous tubule to generate spermatogenesis. Marked testis cells from transgenic rats were transplanted to the testes of immunodeficient mice, and in all of 10 recipient mice (in 19 of 20 testes), rat spermatogenesis occurred. Epididymides of eight mice were examined, and the three from mice with the longest transplants (> or = 110 days) contained rat spermatozoa with normal morphology. The generation of rat spermatogenesis in mouse testes suggests that spermatogonial stem cells of many species could be transplanted, and opens the possibility of xenogeneic spermatogenesis for other species.

FIG. 1

Generation of rat spermatogenesis in mouse testes. a, Testis from 8-week-old transgenic rat (line 14-2) incubated with 5-bromo-4-chloro-3-indole-β-D-galactosidase (X-gal). The transgene was composed of the mouse metallothionein-1 promoter and flanking regions (MT) fused to the Escherichia coli lacZ (lacZ) structural gene, which results in expression of β-galactosidase (β-gal) in the liver and testes of rats, causing blue staining of these tissues after incubation with X-gal (refs ,, and unpublished results). In rats, Sertoli cells are uniformly stained (light areas) and all germ cells will stain blue depending on substrate penetration (dark areas). b, Testis from 8-week-old non-transgenic rat which does not stain blue after incubation with X-gal. c, Testis from 16-week-old immunodeficient nude (nu/nu) mouse treated with busulphan (32 mg per kg) to destroy endogenous mouse spermatogenesis^,,. d, Testis from busulphan-treated nude mouse that was injected with transgenic rat testis cells (Left testis of 777, Table 1). Enlarged area is site of injection from which some tubules were extruded. Blue tubules represent areas of rat spermatogenesis in the mouse testis. Scale bars: a, b, 5 mm; c, d, 1 mm. METHODS. Transgenic rats were generated as previously described. Technique for isolating testis cells^,, microinjection into recipient mouse seminiferous tubules and analysis of testes have also been described^,. Roughly 0.5 ml of cell suspension was used to inject the testes of each recipient. Recipient Swiss nude mice were injected with bone marrow cells (~ 3 × 10⁶ per mouse) into the tail vein, 3 days after busulphan treatment, to reduce mortality.

FIG. 2

Microscopic appearance of rat spermatogenesis in mouse seminiferous tubules. a, Seminiferous tubule of transgenic (MT-lacZ) rat. Sertoli cells along basement membrane are uniformly stained. All germ cell stages stain depending on substrate penetration. Centre tubule shows staining of pachytene spermatocytes. Stain, X-gal and neutral fast red (NFR). b, Seminiferous tubule of control rat. No cells are stained blue by X-gal. The long, thin heads of mature rat spermatozoa are evident. Stain, X-gal and NFR. c, Seminiferous tubule of nude mouse treated with busulphan. No germ-cell stages are present; only Sertoli cells can be seen on the basement membrane of the tubule. Stain, NFR. d, Seminiferous tubule of busulphan-treated recipient nude mouse showing rat spermatogenesis from transplanted cells (left testis from mouse 777). Rat pachytene spermatocytes and round spermatids are heavily stained blue. Sertoli cells (arrowheads) do not stain blue. Stain, X-gal and NFR. e, Seminiferous tubule from left testis of mouse 777. Rat preleptotene and pachytene spermatocytes are stained blue. Stain, X-gal plus periodic acid Schiff and haematoxylin, which emphasizes acrosome and head shape of rat spermatozoa. f, Seminiferous tubule from left testis of mouse 775. Rat preleptotene and pachytene spermatocytes are stained blue. Sertoli cells (arrowheads) do not stain blue. Stain, X-gal plus haematoxylin and eosin to emphasize normal morphology of spermatogenesis arising from transplanted rat stem cells. Scale bar, 50 μm.

FIG. 3

Epididymal spermatozoa. a, c and e are phase contrast and b, d and f are fluorescence photomicrographs. a, b Normal mouse spermatozoa, with short, thick, sickle-shaped head. c, d Normal rat spermatozoa, with long, thin head, and tail that is longer and thicker than mouse. e, f Spermatozoa from epididymides of recipient mouse 767, with distinct rat and mouse morphologies. Ratio was 1 rat to 39 mouse spermatozoa in this animal. METHODS. To examine and count spermatozoa, epididymides were placed in phosphate-buffered saline and cut in several places. Mouse and rat spermatozoa were distinguished by their characteristic head morphology and tail size. For fluorescence photography, the spermatozoa solution was treated with 20 μg per ml Hoecsht 33258 dye. Scale bar, 25μm.