Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate

Abstract

Study question: Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously?

Summary answer: Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months.

What is known already: Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful.

Study design, size, duration: In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation.

Participants/materials, setting, methods: Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids.

Main results and the role of chance: Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation.

Large scale data: N/A.

Limitations, reasons for caution: Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants.

Wider implications of the findings: In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood.

Study funding/competing interest(s): This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Keywords: fertility preservation; immature testis tissue; intratesticular transplantation; primates; spermatogenesis.

Figure 1

Marmoset testis tissue donor information. (A) Basic parameters of the testis tissue donors. (B) Representative sections of immature marmoset testis tissue prior to xenotransplantation. Histological identification of spermatogonia as the most advanced germ cell type by H/PAS and immunohistochemical confirmation by MAGE-A4.

Figure 2

Recovery of marmoset testis tissue xenotransplants and spermatogonial quantity. (A) Recovery of ectopic marmoset testis xenotransplants in immunodeficient mouse. Transplant contains seminiferous tubules with germ cells as histologically observed by H/PAS. (B) Recovery of intratesticular xenotransplants. Localisation of the transplant within the mouse testicular parenchyma by immunostaining for VIMENTIN (brown) and the histology of marmoset testis tissue on the subsequent H/PAS stained section. (C) Increased survival rates for the intratesticular marmoset testis tissue xenotransplants compared to the ectopic xenotransplants at 4 (P < 0.05) and 9 months (P < 0.0001) post-transplantation. (D) Mean number of spermatogonia per tubule in the xenotransplants. *P < 0.05; **P < 0.01.

Figure 3

Germ cell differentiation in ectopic marmoset testis tissue xenotransplants at 4 months post-transplantation. Morphological identification of spermatogonia and spermatocytes by H/PAS and VIMENTIN, and confirmation by immunostaining for MAGE-A4 and BOLL expression. Transplants were negative for the post-meiotic marker CREM-1. Arrows indicate spermatocytes in the lumen of marmoset seminiferous tubules; arrowheads indicate spermatogonia at the basal membrane.

Figure 4

Germ cell differentiation in intratesticular marmoset testis tissue xenotransplants. Morphological identification of post-meiotic germ cells by H/PAS, and on VIMENTIN and MAGE-A4 stained sections and immunohistochemical confirmation by CREM-1 expression and acrosome visualisation by immunostaining for ACROSIN. Arrowheads indicate round spermatids; arrows indicate elongating/elongated spermatids.

Figure 5

Spermatogenic progress in intratesticular marmoset testis tissue xenotransplants. Percentages of seminiferous tubules containing no germ cells, pre-meiotic (spermatogonia), meiotic (spermatocytes) and post-meiotic (spermatids and spermatozoa) germ cells in the recovered marmoset transplants at 4 and 9 months post-transplantation. *P < 0.05; **P < 0.01; ***P < 0.001.