Postpubertal spermatogonial stem cell transplantation restores functional sperm production in rhesus monkeys irradiated before and after puberty

Abstract

Background: Cancer treatment of prepubertal patients impacts future fertility due to the abolition of spermatogonial stem cells (SSCs). In macaques, spermatogenesis could be regenerated by intratesticular transplantation of SSCs, but no studies have involved cytotoxic treatment before puberty and transplantation after puberty, which would be the most likely clinical scenario.

Objectives: To evaluate donor-derived functional sperm production after SSC transplantation to adult monkeys that had received testicular irradiation during the prepubertal period.

Materials and methods: We obtained prepubertal testis tissue by unilaterally castrating six prepubertal monkeys and 2 weeks later irradiated the remaining testes with 6.9 Gy. However, because spermatogenic recovery was observed, we irradiated them again 14 months later with 7 Gy. Three of the monkeys were treated with GnRH-antagonist (GnRH-ant) for 8 weeks. The cryopreserved testis cells from the castrated testes were then allogeneically transplanted into the intact testes of all monkeys. Tissues were harvested 10 months later for analyses.

Results: In three of the six monkeys, 61%, 38%, and 11% of the epididymal sperm DNA were of the donor genotype. The ability to recover donor-derived sperm production was not enhanced by the GnRH-ant pretreatment. However, the extent of filling seminiferous tubules during the transplantation procedure was correlated with the eventual production of donor spermatozoa. The donor epididymal spermatozoa from the recipient with 61% donor contribution were capable of fertilizing rhesus eggs and forming embryos. Although the transplantation was done into the rete testis, two GnRH-ant-treated monkeys, which did not produce donor-derived epididymal spermatozoa, displayed irregular tubular cords in the interstitium containing testicular spermatozoa derived from the transplanted donor cells.

Discussion and conclusion: The results further support that sperm production can be restored in non-human primates from tissues cryopreserved prior to prepubertal and post-pubertal gonadotoxic treatment by transplantation of these testicular cells after puberty into seminiferous tubules.

Keywords: GnRH-antagonist; intracytoplasmic sperm injection; radiation; spermatogenesis; transplantation.

FIGURE 1.

Study design. The monkeys were evaluated before unilateral castration and periodically after exposure to two doses of radiation, hormone suppression, and transplantation. Evaluation included sampling of serum and measurements of testis volume. In addition, periodic semen analysis was performed after the animals reached puberty. Starting immediately after second exposure to testicular irradiation, three monkeys underwent GnRH-ant-mediated hormone suppression for 8 weeks; the other 3 received only sham injections. At the end of the 8-week period, they received allogeneic transplantation of cryopreserved testis tubular cells into one testis, followed by 9 months of immune suppression.

FIGURE 2.

Changes in testis volumes (A) and serum testosterone levels (B) in monkeys during the study. The vertical red and blue dashed lines represent the times of the two doses of irradiation and of transplantation, respectively. The average values for the 3 monkeys receiving GnRH-ant treatment (filled cicle) (n=3) or sham injections (open circle) (n=3) before transplantation are plotted. The grey shaded area represents the duration of the GnRH-ant treatment. For statistical analysis the axis was divided into three time segments, after initial irradiation, after the second irradiation during GnRH-ant treatment, and after transplantation, during which there were 15, 6 and 13 comparisons, respectively. The only statistical difference between the two treatments groups (marked with asterisks) was decreased serum testosterone during the GnRH-ant treatment.

FIGURE 3.

Spermatogenic endpoints in individual monkeys. Testis weights (A), tubule differentiation indices (B) and yield of sperm from the cauda epididymis (C) are shown for the monkeys treated with GnRH-ant (hatched bars) and those receiving only sham injections. Testes with abnormal tubules (dilated or de novo) are indicated in (B). The portion of the columns filled with green in (C) shows the numbers of spermatozoa that were donor-derived.

FIGURE 4.

Testis histology at tissue harvest, 44 weeks after transplantation. (A) Most tubules only contain Sertoli cells (B) Normal Sertoli-only tubules (*) and tubules showing regeneration of spermatogenesis (†). Note that the Sertoli cells in normal tubules have columnar appearance with a small lumen often with the presence of cytoplasmic processes. (C-F) Abnormal dilated Sertoli-only tubules (‡) with low epithelial heights and large empty lumens and some adjacent normal Sertoli-only tubules (*). Monkey numbers are indicated beside panels. Scale bars: A: 200 μm; C, E: 100 μm; B,D,F: 50 μm.

FIGURE 5.

Histology of the testis of a monkey that showed donor-derived sperm in the epididymis. Representative PAS-Hematoxylin stained testis sections at the end of the study from monkey #114. Tubules showing differentiating germ cells in (A) are indicated by asterisks. Note the presence mature spermatids (arrows in C) indicating complete spermatogenesis. Scale bars: A: 200 μm; B: 50 μm; C:10 μm.

FIGURE 6.

(A) Region of irregular de novo tubular cords with interspersed endogenous Sertoli-cell only tubules (*). De novo cords with spermatogenic development to the spermatocyte (†) and spermatid (‡) stages are indicated. (B) Higher magnification of region from A showing round spermatids (arrowheads) and elongated spermatids (arrows). (C) Region of de novo cords (DN) showing that it is adjacent to the rete testis area. Interspersed normal tubules that are Sertoli-cell-only (*) and with recovery spermatogenesis (¶) are indicated. Scale bars: A &B: 50 μm; C: 200 μm.

FIGURE 7.

Embryo produced by ICSI with epididymal sperm from a monkey (#124) with high percentage of donor-derived sperm. (A) Compact morula resulting from in vitro culture of the fertilized oocyte. (B) Microsatellite DNA analysis of one donor-derived embryo and comparison with the oocyte, SSC donor and recipient male profiles. Alleles specific for the oocyte donor (represented by purple font and arrow), transplant donor (represented by black font and arrow), and transplant recipient (represented by green font and arrow) are indicated on the electropherogram panels. The presence of the alleles at 201 and 292 nucleotide pairs in the embryo demonstrates the paternal origin as being from the donor.