Transplantation of male germ line stem cells restores fertility in infertile mice

Abstract

Azoospermia or oligozoospermia due to disruption of spermatogenesis are common causes of human male infertility. We used the technique of spermatogonial transplantation in two infertile mouse strains, Steel (Sl) and dominant white spotting (W), to determine if stem cells from an infertile male were capable of generating spermatogenesis. Transplantation of germ cells from infertile Sl/Sld mutant male mice to infertile W/Wv or Wv/W54 mutant male mice restored fertility to the recipient mice. Thus, transplantation of spermatogonial stem cells from an infertile donor to a permissive testicular environment can restore fertility and result in progeny with the genetic makeup of the infertile donor male.

Fig. 1

Steel factor (Kit ligand)–Kit receptor interaction in the mouse testis, with phenotypes and testis sizes of mice. a, Black wild-type C57BL/6 male; testis weight, 107 mg. In wild-type mice, Sertoli cells (S) express both the soluble and the membrane-bound form of Steel factor. Functional Kit receptor dimers on the germ cells (G) interact with the soluble and membrane-bound form of Steel factor. Signal transduction then occurs by autophosphorylation-induced interaction with target proteins. b, White Sl mutant (Sl/Sl^d) male; testis weight, 14 mg. In Sl/Sl^d mice, Sertoli cells do not express the membrane-bound form of Steel factor because the Sl mutation deletes the entire gene, and the Sl^d mutation deletes the transmembrane and intracellular domains. Therefore, only the soluble form of Steel factor is produced to interact with Kit receptors on germ cells. Binding of the soluble form is not sufficient for normal germ cell differentiation. c, White Kit mutant (W/W^v) male; testis weight, 15 mg. In W/W^v mice, Sertoli cells express both the soluble and the membrane bound form of Steel factor. However, the W mutation is a 78-amino-acid deletion that includes the transmembrane domain, and the W^v mutation results in a defective receptor on the germ cell because of a point mutation in the tyrosine kinase domain. Therefore, neither the bound nor the soluble form of Steel factor can effectively influence germ cells. Scale bars (insets) represent 1 mm.

Fig. 2

Microscopic appearance of seminiferous tubules of W/W^v recipient mouse testes after transplantation of Sl/Sl^d germ cells. a, Wild-type C57BL/6 male with normal spermatogenesis. b, W/W^v male (recipient strain). Spermatogenesis is absent; seminiferous tubules contain almost exclusively Sertoli cells. c and d, Sl/Sl^d male (donor strain). Spermatogenesis is absent, but a few germ cells are present on the basement membrane (arrows, d). e and f, W/W^v recipient male (number 1807) 107 d after transplantation of germ cells from Sl/Sl^d donor. Spermatogenesis occurs in most seminiferous tubules. g and h, W/W^v recipient male (number 1805) 365 days after transplantation of germ cells from Sl/Sl^d donor. Spermatogenesis occurs in most seminiferous tubules. Hematoxylin and eosin stain. Scale bars represent 50 μm (a–c and g), 20 μm (d and h), 200 μm (e) and 100 μm (f).

Fig. 3

Pedigree and Southern blot analysis of seven weanling mice from the mating of a white (open symbol) W/W^v recipient male mouse (number 1803) to a black (filled symbols) wild-type (+/+, C57BL/6) female mouse. Both the Sl and Sl^d haplotype of the donor germ cells were transmitted to male and female progeny of the recipient, indicating that both alleles present in donor cells were segregated during meiosis to individual spermatozoa. Left and right margins, molecular size markers: The wild-type locus produces bands at 4.3 and 13 kb, the Sl locus produces no bands, and the Sl^d locus produces bands at 4.3 and 7 kb (ref. 10).