Long-term survival of human spermatogonial stem cells in mouse testes Makoto Nagano, D.V.M., Ph.D., Pasquale Patrizio, M.D., Ralph L Brinster, V.M.D., Ph.D.  Fertility and Sterility  Volume 78, Issue 6, Pages 1225-1233 (December 2002) DOI: 10.1016/S0015-0282(02)04345-5

FIGURE 1 Detection of human germ cells transplanted into seminiferous tubules of recipient mouse testes using a baboon testis-specific antibody. (A), Whole-mount immunohistochemistry of recipient mouse tubules 1 day after transplantation. 129Sv × C57BL/6 (129/B6) F1 mouse was pretreated with busulfan to destroy endogenous spermatogenesis. Donor human testis cells are stained red by the baboon-specific antibody detection procedure, indicating that the antibody reacts well with human testis cells. (B), The seminiferous tubules of intact 129/B6 mouse without busulfan pretreatment stained with baboon-specific antibodies. No mouse cells react with the antibody. (C), Immunohistochemistry of recipient nude mouse seminiferous tubules 1 month after transplantation of donor human testis cells. Donor cells were freshly transplanted at the high cell concentration without leuprolide treatment of recipient mouse. Human germ cells are located on the basement membrane of mouse tubules and present as single cells and paired cells, which is an identifying characteristic of undifferentiated spermatogonia. A cluster of 22 donor human spermatogonia is seen in this panel suggesting that human spermatogonia can proliferate during the first month after transplantation. (D), Donor human cells in mouse seminiferous tubule 2 months after transplantation. Donor cells were freshly transplanted at the high concentration. The recipient mouse was treated with leuprolide. Most donor cells are present as single or paired cells and do not form a cluster like shown in C. (E), Donor human spermatogonia in mouse tubule 4 months after transplantation. Donor cells were cryopreserved for 3 months and transplanted at the low concentration followed by leuprolide treatment of the recipient mouse. This panel shows that cryopreserved cells also colonize mouse testes as observed with freshly transplanted cells. (F), Paired human spermatogonia in mouse testis 5 months after transplantation. Donor cells were freshly transplanted at the low concentration without leuprolide treatment of the recipient mouse. Only single or paired cells can be found at this time point. Bar = 100 μm (A–C & E) and 40 μm (D & F). Nagano. Human spermatogonial stem cells in mice.Fertil Steril 2002. Fertility and Sterility 2002 78, 1225-1233DOI: (10.1016/S0015-0282(02)04345-5)

FIGURE 2 Combined effects of donor cell cryopreservation, donor cell concentration, and recipient treatment with leuprolide on percentage of mouse testes colonized by human spermatogonia to total mouse testes injected. Absolute number of recipient mouse testes colonized by human spermatogonia is shown as a fraction of recipient testes injected with human cells in each bar. Analysis of variance indicated no difference in colonization percentage among these combined condition groups (P = .400). Cells were either cryopreserved for 3 months before transplantation or transplanted immediately after cell preparation (Fresh). The concentration of donor cell suspension was 23.7 ± 2.0 × 106/mL (mean ± SEM; n = 3 donor cell preparations) for low and 49.7 ± 1.7 × 106/mL (mean SEM; n = 3 donor cell preparations) for high concentration. Leuprolide treatment (+ = with; − = without) of recipient mice was made every 4 weeks at 7.6 mg/kg body weight. Nagano. Human spermatogonial stem cells in mice.Fertil Steril 2002. Fertility and Sterility 2002 78, 1225-1233DOI: (10.1016/S0015-0282(02)04345-5)

FIGURE 3 Linear regression analysis showing human spermatogonial stem cell colonization of recipient mouse testes from 1 to 6 months after transplantation of donor human testis cells. Survival of human spermatogonia in recipient mouse testes is expressed as a percentage of mouse testes colonized by human stem cells in a total number of mouse testes injected with human testis cells. The survival decreased between 1 and 6 months following transplantation (P = .05). Analysis was performed using the arcsine transformation that gave the regression line equation, y = −0.159x + 1.799. The untransformed percentage at each time point was 100% at 1 to 3 months after transplantation, and 50%, 71%, and 67% at 4, 5, and 6 months, respectively. The total number of recipient mouse testes examined was 1, 2, 3, 6, 7, and 3 at each time point from 1 to 6 months, respectively, after transplantation. Nagano. Human spermatogonial stem cells in mice.Fertil Steril 2002. Fertility and Sterility 2002 78, 1225-1233DOI: (10.1016/S0015-0282(02)04345-5)