Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys

Abstract

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin-releasing hormone antagonist (GnRH-ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared with radiation-only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH-ant-treated monkeys and (iv) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

Keywords: gonadotropin-releasing hormone-antagonist; infertility; radiation; spermatogenesis; transplantation.

Figure 1

Overall design of the main study. Monkeys were evaluated before treatment and periodically after exposure to radiation, hormone suppression, and transplantation. Evaluation included sampling of serum and semen, measurements of testis volume and weight, and testis biopsies as indicated. All 12 monkeys were given testicular irradiation; six then underwent GnRH-ant–mediated hormone suppression for 8 weeks, while the other six did not. At the end of the 8-week period, all monkeys received autologous transplantation of GFP-lentivirus–labeled germ cells into one testis.

Figure 2

Increases in testis volume and weight suggest that combined hormone suppression and germ cell transplantation promote spermatogenic recovery. Testicular volume (expressed as a percentage of the pretreatment volume) (A) and testis weight at the end of the study, at 44 weeks after irradiation (B) are shown in radiation-only monkeys and monkeys that received GnRH-ant, for both the testes that did or did not receive transplantation at 8-weeks after irradiation. The shaded area represents the period of hormone suppression. Recovery of testis volume during the period from week 24 to week 44 and the testis weight at week 44 was compared between various groups, and significant (P<0.05) differences are shown by different letters, as follows: a: between the transplanted and sham-transplanted testes of GnRH-ant–treated monkeys (Wilcoxon test); b: between the transplanted and sham-transplanted testes of radiation-only monkeys (Wilcoxon test); c: between the transplanted testes of GnRH-ant–treated and radiation-only monkeys (Mann-Whitney test). The differences between the sham-transplanted testes of GnRH-ant–treated and radiation-only monkeys were not significant.

Figure 3

Combined hormone suppression and germ cell transplantation results in spermatogenic recovery. The three panels show histology of representative monkey testes. (A) Testis from the unirradiated untreated monkey used in the preliminary experiment. (B) Testis of a monkey that received radiation 44 weeks previously and neither GnRH-ant treatment nor transplantation. Very few of the tubules contain germ cells (marked with *) in this radiation-only monkey. (C) Testis of a monkey that received radiation 44 weeks previously and underwent both GnRH-ant treatment and germ cell transplantation. Note the large cluster of tubules containing germ cells. Bar represents 100 µm.

Figure 4

Combined hormone suppression and germ cell transplantation induced greater development of germ cells in monkey testes than either approach alone. The average (A) and individual (B) percentages of tubules showing differentiated germ cells (tubule differentiation indices) in radiation-only and irradiated GnRH-ant–treated monkeys that received autologous transplantation of testicular cells to one of the testes. Tubule differentiation indices were compared between various groups at week 24 and at week 44, and significant (P<0.05) differences are shown as follows: *: between the transplanted testes of GnRH-ant–treated and radiation-only monkeys (Mann-Whitney test); †: between the sham-transplanted testes of GnRH-ant–treated and radiation-only monkeys (Mann-Whitney test). (B) The tubule differentiation indices for individual monkeys, with the individual animal numbers indicated, were obtained at 44 weeks after irradiation. Only the transplanted testis of monkey #5 is shown, as the sham-transplanted testis became necrotic after week 24.

Figure 5

Serum hormone levels are suppressed by treatment with GnRH-ant and return to normal levels when the treatment is stopped. Serum FSH (A), LH (B), and testosterone (C) levels in radiation-only and irradiated monkeys treated with GnRH-ant for 8 weeks are shown. The shaded area represents the period of hormone suppression. Dashed lines indicate minimum levels of detection. The serum hormone levels after irradiation with or without GnRH-ant treatment were compared to the respective values before irradiation, and significant (P<0.001) differences are shown for the values lower ( *) and higher (†) than the pre-irradiation values (Dunnet test).

Figure 6

Treatment with GnRH-ant stimulates recovery of sperm counts in monkeys transplanted with germ cells. Sperm counts in radiation-only and GnRH-ant–treated irradiated monkeys that received autologous transplantation of testicular cells to one of the testes are shown. The shaded area represents the period of hormone suppression. For the purpose of averaging log-transformed value, azoospermic counts were set at 1,000/ml which is the lower limit of detection of sperm with hemocytometer counting. Recovery of sperm counts was compared between the two groups during the period from week 24 to week 44, and significant (P<0.05) differences are shown by the symbol:* (Mann-Whitney test).