Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? 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. 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. 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. 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. 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. N/A. 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. 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. 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.

Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

Which cryopreservation method better protects reproductive potential: the cryopreservation of a testicular cell suspension (TCS) or the cryopreservation of testicular tissue (TET)? Two cryopreservation strategies for spermatogonial stem cells (SSCs) were compared in a mouse model: cryopreservation as TET or as TCS. Evaluated outcomes were number of viable cells after thawing, number and length of donor-derived colonies after spermatogonial stem cell transplantation (SSCT), number of litters, litter size and number of donor-derived pups after mating. Compared with cryopreserving TCS, cryopreservation of TET resulted in significantly higher numbers of viable cells after thawing (TET: 13.4 × 104 ± 7.2 × 104 versus TCS: 8.2 × 104 ± 2.7 × 104; P = 0.0002), more (TET: 47.6 ± 19.2 versus TCS: 18.5 ± 13.0; P = 0.0039) and longer (TET: 5.2 ± 1.0 mm versus TCS: 2.7 ± 1.5 mm; P = 0.0016) donor-derived colonies, and more donor-derived pups per litter (TET: 2.2 ± 0.2 versus TCS: 0.5 ± 0.1; P = 0.0008). Cryopreservation of TET is the preferred method to cryopreserve SSCs prior to SSCT in a mouse model.

Immature testis tissue (ITT) cryopreservation is the only fertility preservation option currently offered to prepubertal boys at risk of germ cell loss due to gonadotoxic treatments ( Onofre et al., 2016 x Onofre et al., 2016 Onofre, J., Baert, Y., Faes, K., and Goossens, E. Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation. Hum. Reprod. Update. 2016; 22: 744–761 Google Scholar See all References , Picton et al., 2015 x Picton et al., 2015 Picton, H., Wyns, C., Anderson, R.A., Goossens, E., Jahnukainen, K., Kliesch, S., Mitchell, R.T., Pennings, G., Rives, N., Tournaye, H., Van Pelt, A.M., Eichenlaub-Ritter, U., and Schlatt, S. On behalf of the ESHRE Task Force On Fertility Preservation In Severe Diseases. A European perspective on testicular tissue cryopreservation for fertility preservation in prepubertal and adolescent boys. Hum. Reprod. 2015; 30: 2463–2475 Google Scholar See all References ). Promising technologies utilizing testis tissue biopsies, containing spermatogonial stem cells (SSCs), are being developed: in vitro spermatogenesis, spermatogonial stem cell transplantation and testis tissue transplantation (TTT) ( Clark et al., 2011 x Clark et al., 2011 Clark, A.T., Phillips, B.T., and Orwig, K.E. Fruitful progress to fertility: male fertility in the test tube. Nature medicine. 2011; 17: 1564–1575 Google Scholar See all References , de Michele et al., 2017 x de Michele et al., 2017 de Michele, F., Poels, J., Weerens, L., Petit, C., Evrard, Z., Ambroise, J., Gruson, D., and Wyns, C. Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue. Hum. Reprod. 2017; 32: 32–45 Google Scholar See all References , Ginsberg et al., 2010 x Ginsberg et al., 2010 Ginsberg, J.P., Carlson, C.A., Lin, K., Hobbie, W.L., Wigo, E., Wu, X., Brinster, R.L., and Kolon, T.F. An experimental protocol for fertility preservation in prepubertal boys recently diagnosed with cancer: a report of acceptability and safety. Hum. Reprod. 2010; 25: 37–41 Google Scholar See all References , Goossens et al., 2013 x Goossens et al., 2013 Goossens, E., Van Saen, D., and Tournaye, H. Spermatogonial stem cell preservation and transplantation: from research to clinic. Hum. Reprod. 2013; 28: 897–907 Google Scholar See all References , Keros et al., 2007 x Keros et al., 2007 Keros, V., Hultenby, K., Borgstrom, B., Fridstrom, M., Jahnukainen, K., and Hovatta, O. Methods of cryopreservation of testicular tissue with viable spermatogonia in pre-pubertal boys undergoing gonadotoxic cancer treatment. Hum. Reprod. 2007; 22: 1384–1395 Google Scholar See all References , Sadri-Ardekani et al., 2011 x Sadri-Ardekani et al., 2011 Sadri-Ardekani, H., Akhondi, M.A., van der Veen, F., Repping, S., and van Pelt, A.M. In vitro propagation of human prepubertal spermatogonial stem cells. JAMA. 2011; 305: 2416–2418 Google Scholar See all References , Sato et al., 2011 x Sato et al., 2011 Sato, T., Katagiri, K., Gohbara, A., Inoue, K., Ogonuki, N., Ogura, A., Kubota, Y., and Ogawa, T. In vitro production of functional sperm in cultured neonatal mouse testes. Nature. 2011; 471: 504–507 Google Scholar See all References , Wyns et al., 2010 x Wyns et al., 2010 Wyns, C., Curaba, M., Vanabelle, B., Van Langendonckt, A., and Donnez, J. Options for fertility preservation in prepubertal boys. Hum. Reprod. Update. 2010; 16: 312–328 Google Scholar See all References ).

Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds.