Culture of mouse spermatogonial stem cells

Abstract

Spermatogenesis occurs within the seminiferous tubules of mammals by a complex process that is highly organized, extremely efficient and very productive. At the foundation of this process is the spermatogonial stem cell that is capable of both self-renewal and production of progeny cells, which undergo differentiation over a period of weeks to months in order to generate mature spermatozoa. It had been thought that germ cells survive only a brief period in culture, generally less than a few weeks. However, an accurate assessment of the presence of spermatogonial stem cells in any cell population has only recently become possible with development of the spermatogonial transplantation technique. Using this technique, we have demonstrated that mouse spermatogonial stem cells can be maintained in culture for approximately 4 months and will generate spermatogenesis following transplantation to the seminiferous tubules of an appropriate recipient. Extensive areas of cultured donor cell-derived spermatogenesis are generated in the host, and production of mature spermatozoa occurs. Cultivation of the testis cells on STO feeders is beneficial to stem cell survival. These results provide the first step in establishing a system that will permit spermatogonial stem cells to be cultivated and their number increased in vitro to allow for genetic modification before transplantation to a recipient testis.

Fig. 1

Appearance of ROSA26 testis cells after 2 days in culture. A and C were cultured without STO feeders; B and D were cultured with STO feeders; A and B are phase-contrast microscopy of living cells; C and D are X-gal-stained cells. Each culture well initially received 10⁶ testis cells. Note increased number of round cells present in wells with STO feeders. Many of these round cells are likely to represent various differentiated stages of germ cells. Scale bar = 60 μm.

Fig. 2

Appearance of ROSA26 testis cells after 1 month in culture. A and C were cultured without STO feeders; B and D were cultured with STO feeders; A and B are phase-contrast microscopy of living cells; C and D are X-gal stained cells. Each culture well initially received 10⁶ testis cells. Note increased number of round cells still present in wells with STO feeders after 1 month. Germ cells are generally believed to maintain a round appearance in culture. All round cells are not likely to be germ cells. Scale bar = 60 μm.

Fig. 3

Colonization of busulfan-treated recipient mouse seminiferous tubules by cultured testis cells from ZFlacZ donor. A Control C57BL/6 × SJLF₁ testis in which germ cells do not stain with X-gal for the presence of E. coli β-galactosidase; B Transgenic ZFlacZ adult donor testis showing intense blue staining from β-galactosidase activity in spermatid stages of germ cell population. C Busulfan-treated C57BL/6 × SJLF₁ recipient testis into which ZFlacZ testis cells cultured for 111 days were transplanted (Table 1, experiment 6, left testis). Cultured donor cells were originally collected from 10-day-old mice. Recipient was analyzed by X-gal staining approximately 3.5 months after cultured cell transplantation. Blue stretches of tubules indicate individual areas of colonization by cultured spermatogonial stem cells. Tubules without spermatogenesis or tubules that have reestablished recipient stem cell-derived spermatogenesis do not stain. D Tunica albuginea has been removed to reveal the extent of colonization, and to demonstrate that at the ends of the blue stretches of tubule the blue color diminishes indicating continued spread of stem cells and colonization. A – D, X-gal stain. Scale bar = 1 mm.

Fig. 4

Donor-cell-derived spermatogenesis following transplantation of cultured testis cells to recipient mice. ZFlacZ testis cells were cultured and transplanted into the seminiferous tubules of busulfan-treated recipient mice. A Testis cells were collected from 7-week-old mice and cultured for 19 days before transplantation (Table 1, experiment 1). Recipient was analyzed by X-gal staining 110 days later. Only donor stem-cell-derived germ cells will stain blue. Cross-sections in this area of the testis demonstrate spermatogenesis from cultured donor cells. B Seminiferous tubule from the same testis as (A) demonstrating the normal appearance of spermatogenesis and many mature spermatozoa. C Testis cells were collected from 10-day-old mice and cultured for 111 days before transplantation (Table 1, experiment 6). Cross-sections in this area of the testis demonstrate cultured donor stem-cell-derived spermatogenesis. D Seminiferous tubule from the same testis as (C) showing an area of donor cell-derived spermatogenesis with atypical cellular arrangement (see Russell et al., 1996 for ultrastructural details of transplanted germ cell spermatogenesis). The intensity of X-gal staining of donor-cell-derived spermatogenesis varies among tubules because of differences in X-gal substrate penetration. Morphology of spermatogenesis is not optimal because fixation for X-gal staining does not preserve testis structure well. A–D, X-gal followed by neutral fast red stain. Scale bars: A and C = 100 μm; B and D = 20 μm.