Hormonal suppression restores fertility in irradiated mice from both endogenous and donor-derived stem spermatogonia

Abstract

Irradiation interrupts spermatogenesis and causes prolonged sterility in male mammals. Hormonal suppression treatment with gonadotropin-releasing hormone (GnRH) analogues has restored spermatogenesis in irradiated rats, but similar attempts were unsuccessful in irradiated mice, monkeys, and humans. In this study, we tested a stronger hormonal suppression regimen (the GnRH antagonist, acyline, and plus flutamide) for efficacy both in restoring endogenous spermatogenesis and in enhancing colonization of transplanted stem spermatogonia in mouse testes irradiated with a total doses between 10.5 and 13.5 Gy. A 4-week hormonal suppression treatment, given immediately after irradiation, increased endogenous spermatogenic recovery 1.5-fold, and 11-week hormonal suppression produced twofold increases compared with sham-treated irradiated controls. Furthermore, 10-week hormonal suppression restored fertility from endogenous surviving spermatogonial stem cells in 90% of 10.5-Gy irradiated mice, whereas only 10% were fertile without hormonal suppression. Four- and 11-week hormonal suppression also enhanced spermatogenic development from transplanted stem spermatogonia in irradiated recipient mice, by 3.1- and 4.8-fold, respectively, compared with those not given hormonal treatment. Moreover, the 10-week hormonal suppression regimen, but not a sham treatment, restored fertility of some 13.5-Gy irradiated recipient mice from donor-derived spermatogonial stem cells. This is the first report of hormonal suppression inducing recovery of endogenous spermatogenesis and fertility in a mouse model treated with anticancer agents. The combination of spermatogonial transplantation with hormonal suppression should be investigated as a treatment to restore fertility in young men after cytotoxic cancer therapy.

FIG. 1.

Schematics of the four experimental protocols used. Mice were irradiated at week 0 with total doses as indicated. Hormonal suppression treatment was started immediately after irradiation and continued for 4, 10, or 11 weeks. In Exps 2 and 4, transplantation was performed at 3 weeks after irradiation.

FIG. 2.

Hormonal suppression improved spermatogenic recovery from both endogenous and donor-derived stem spermatogonia in irradiated mouse testis in Exp. 1 (A) and Exp. 2 (B). The TDI is the percentage of tubule cross sections with more than three differentiated germ cells. “Endogenous” indicates the differentiated germ cells that were derived from surviving stem spermatogonia of the recipient. “Donor” indicates the differentiated germ cells that were derived from GFP-positive transplanted stem spermatogonia. The statistical analyses of 12- and 13.5-Gy donor TDI data in panel (B) were conducted by a nonparametric method because the data are not normally distributed. N = 3–4 (panel A) and N = 4–18 (panel B). “a” and “b”, p < 0.05 versus the sham- or 4-week–treated groups, respectively.

FIG. 3.

Evaluation of the recipient testes. GFP-expressing donor cell colonization was visualized at 11 weeks after transplantation by fluorescence microscopy in irradiated recipient testes that were sham treated (A) or given 4-week GnRH-ant plus flutamide (B) or 11-week GnRH-ant plus flutamide (C) treatments. Immunohistochemical staining for GFP in fixed tissue cross sections from (D) sham-treated mice, (E) 4-week GnRH-ant plus flutamide treatment, or (F) 11-week GnRH-ant plus flutamide treatment was performed to distinguish tubules with donor-derived spermatogenesis (brown) from those with endogenous spermatogenesis (blue). Note that there is some nonspecific staining of cytoplasm of the late spermatids. GFP-positive tubules were separated from recipient testis and imaged by fluorescence (G) for measurement of colony length or by bright field (H) to clearly show the tubule. Bars, 200 μm (A, B, C, G, and H) and 100 μm (D, E, and F).

FIG. 4.

Time course of accessory sexual organ or spermatogenic recovery in mice irradiated with 10.5 Gy (A–E) from Exp. 3 or 13.5 Gy (F–J) from Exp. 4, as measured by SV weight (A and F), epididymal weight (B and G), testis weight (C and H), testicular sperm head count (note log scale) (D and I), and cauda epididymal sperm count (E and J). Groups are designated as Sham, Hormone (GnRH-ant + flutamide only, no transplantation), Transplant (transplantation only), Hormone + Transplant (GnRH + flutamide and spermatogonial transplantation). N = 10 for 46-week time point data and 5 for all other time points for both irradiation doses. “a,” “b,” and “c”, p < 0.05 versus values in the sham group, hormone group, and transplant group, respectively. Ellipses in (K) identify those mice that were fertile.

FIG. 5.

TDIs in testes of mice irradiated with 10.5 Gy (A) from Exp. 3 or 13.5 Gy (B, endogenous and C, transplanted) from Exp. 4, with and without hormonal suppression. Furthermore, in the transplanted testes, the TDI of the endogenous cells and donor cells were scored separately. N = 10 for 46-week time point group and 5 for all other time point groups for both irradiation doses. The statistical analysis of 46-week TDI data in panel (B) was conducted by a nonparametric method because the data are not normally distributed. “a” and “c”, p < 0.05 versus values in the sham group and transplant-only group, respectively.

FIG. 6.

Time course of fertility recovery expressed as probability of having returned to fertility for male mice irradiated with 10.5 Gy with or without hormone suppression (A) or irradiated with 13.5 Gy with or without hormone suppression or spermatogonial transplantation (B) by Kaplan-Meier survival analysis. Relative fecundity (fraction of males that were fertile × litters per fertile male per 5-week time period × average litter size) for male mice irradiated with 10.5 Gy, with or without hormone suppression (C), or irradiated with 13.5 Gy, with or without hormone suppression or spermatogonial transplantation (D). Note, for comparison, that the calculated Relative fecundity for our unirradiated, non-hormonally treated control mice was 21.4. Symbols in panels (A and B) represent times at which individual mouse became fertile or were censored (i.e., the time of euthanasia before they became fertile). *p < 0.05 compared with values of sham-treated group. #p < 0.05 compared with values of sham, hormonal suppression, or transplant groups.