Effect of hormone modulations on donor-derived spermatogenesis or colonization after syngeneic and xenotransplantation in mice

Abstract

Background: Cytotoxic cancer treatments, such as irradiation, can cause permanent sterility in male mammals owing to the loss of spermatogonial stem cells. In animal models, spermatogenesis could be restored from transplanted spermatogonial stem cells. Previously, we showed that transient suppression of FSH, LH, and testosterone in the recipient with a gonadotropin-releasing hormone antagonist (GnRH-ant), given immediately after irradiation, enhanced spermatogenesis from transplanted spermatogonial stem cells in mice and monkeys.

Objectives: To explore improvements in the preparation of the recipient for efficient and reliable spermatogenic recovery from spermatogonial stem cell transplantation, so that it can be used effectively in clinical practice.

Materials and methods: In mouse recipients, we evaluated the effects of hormone suppression given after germ cell depletion was complete, which is a more clinically relevant model, and also the importance of total androgen ablation and maintenance of FSH levels. Three regimens, GnRH-ant, GnRH-ant plus flutamide (androgen receptor antagonist), and GnRH-ant plus FSH, were administered prior to and around the time of transplantation of testis cells from immature mice or from prepubertal monkeys.

Results: Treatment with GnRH-ant resulted in a fourfold increase in spermatogenic recovery from GFP-marked transplanted mouse cells. Total androgen ablation with the addition of flutamide, started two weeks before transplantation, did not further enhance recovery. Surprisingly, FSH supplementation, started around the time of transplantation, actually reduced spermatogenic recovery from transplanted spermatogonial stem cells in GnRH-ant-treated mice. When prepubertal monkey testicular cells were transplanted into nude mice that were given the same hormone treatments, the numbers of donor-derived colonies were independent of hormone treatment.

Discussion and conclusion: The enhancements in spermatogenic recovery may only occur when syngeneic or closely related donor-recipient pairs are used. These results are useful in further investigations in choosing a hormone suppression regimen in combination with spermatogonial transplantation as a treatment to restore fertility in primates after cytotoxic therapy.

Keywords: GnRH-antagonist; irradiation; spermatogonia; transplantation.

FIG. 1.

Schematic of the protocol used in the syngeneic and xenotransplantation experiments. Mice were irradiated at week 0 with total doses of 13.5 Gy. Hormonal suppression treatment was started at 4 weeks after irradiation. Transplantation was performed at 8 weeks after irradiation. Mice were euthanized at 8 weeks after transplantation for analysis.

FIG. 2.

Representative histology of the testis in B6 mice at 4 weeks (A&B) and 8 weeks (C&D) after testicular irradiation with total doses of 13.5 Gy. B and D are the magnified views of areas in A and C, respectively. Asterisks in C &D represent the tubules showing differentiated germ cells derived from a few endogenous radioresistant A spermatogonia. Note that at 4 weeks, the tubules with differentiated germ cells are almost absent. The bars indicate 200 μm in A&C and 30μm in B&D.

FIG. 3.

Testicular histology (A-C) and analysis of tubules with spermatogenesis (D-E) after syngeneic (B6 GFP mouse to B6 mouse) transplantation. (A) Testicular section 8 weeks after transplantation. Donor colonies were immunolocalized with anti-GFP followed by DAB staining and then counterstained with hematoxylin. (B) Another section at higher magnification with tubules showing endogenous (†) and donor-derived (*) spermatogenesis. (C) Higher magnification of tubule with donor derived spermatogenesis showing the presence of spermatid and sperm. Percentages of tubules with (D) donor-derived and (E) endogenous spermatogenesis after transplantation to recipients with different hormone manipulations. The number of recipient testes analyzed in each group were 10–12. Significant differences in the tubule differentiation indices between different groups and irradiated-only mice by ANOVA and post-hoc Tukey’s test are indicated (* = P<0.05). In addition, the differentiation of donor cells in the group receiving GnRH-ant plus FSH was significantly lower than in the group only receiving GnRH-ant by non-parametric tests. The bars indicate 350 μm in A, 100 μm in B and 25μm in C.

FIG. 4.

Whole mounts of seminiferous tubules from xenotransplantation recipients and their analyses. Representative whole mounts of seminiferous tubules from immunostained with (A) anti-nHP and (B) anti-VASA showing the donor-derived colonies. (C) Overlay of nHP and VASA immunofluorescence. Note the variability in the intensity of VASA staining. (D) Donor-derived nHP-positive, VASA-positive colonies in xenotransplanted recipients with different hormone manipulations. The numbers of recipient testes analyzed in each group were between 5 to 10. There were no significant differences in the numbers of colonies in the different groups. (E) Example of ovoid-shaped, primate donor-derived multicellular structure, stained for nHP-antigen (green), within mouse seminiferous tubules. (F) Three donor-derived (nHP-antigen+) germ cells (VASA+) within multicellular structure. (G) Overlay of A and B. Bars represent 25μm.

FIG. 5.

Sections of normal adult rhesus monkey testis, demonstrating VASA staining of spermatogonia. (A) VASA-stained seminiferous tubule at about Stage VI. (B) DAPI fluorescence with 3 spermatogonia identified (c, d, e). (C, D, E) Enlarged images of the 3 spermatogonia identified in B. (C’, D’, E’) VASA staining of spermatogonia shown in C, D, and E. The bar in A represents 25 μm.