**4. Applications of testis cryopreservation for new reproductive technologies**

Given that properly cryopreserved testis biopsies can last decades in liquid nitrogen and that most prepubertal cancer patient boys donating biopsies may not need to resort to assisted reproductive technologies for a couple of decades, it is advisable that cryopreservation of testicular biopsies be offered to such patients in a hope that our ability to use such tissues will be further improved and the options expanded in the coming years.

A number of potential applications already exist for the use of cryopreserved testicular tissue in experimental and clinical settings in reproduction medicine/science. Such technologies allow retrieval of existing sperm from mature donor samples and, more importantly, offer hope for production of sperm in samples of cryopreserved testis immature testis. If the preserved testis tissue contains endogenous spermatogenesis (*e.g.,* from obstructive azoospermic adult patients), it can be used to extract sperm, elongated spermatids, or even round spermatids to be used for fertilization of oocytes through ICSI (Rosenlund et al., 1998; Schrader et al., 2002; Gianaroli et al., 1999; Tesarik et al., 2000; Schoysman et al., 1999).

If preserved testis samples are obtained from neonatal/immature donors, they can still be used to induce spermatogenesis through the following approaches.

#### **4.1 Germ cell transplantation**

The technique for germ cell transplantation has allowed (re)establishment of spermatogenesis after introduction of donor testis cell suspensions into the seminiferous tubules of infertile recipient testes. Once deposited in the tubular lumen, donor SSCs are recognized by the host Sertoli cells and allowed passage to the stem cell niche, where new colonies of spermatogenesis can begin and expand. This approach has allowed production of sufficient numbers of sperm to allow infertile recipient mice to sire donor-derived progeny (Avarbock & Brinster, 1994; Brinster & Zimmermann, 1994). Later, the capability of cryopreserved mouse testis cells after transplantation into recipient testes to start spermatogenesis was also confirmed (Avarbock et al., 1996; Brinster & Nagano, 1998; Ogawa et al., 1999). While heterologous transplantation of human germ cells into recipient mice did not lead to completion of spermatogenesis (Nagano et al., 2002), the transfer technique has been tested

whole testes with punctured tunica albuginea was deemed more suitable than using whole testes with intact tunica, whole testes without tunica, or testis halves (Gouk et al., 2011). Mouse testes have considerably less connective tissue content than most other species; therefore, tissue fragment size is especially a concern for testis tissues from species with higher interstitial tissue density. For cryopreservation of (cryptorchid) testes from prepubertal boys, fragments sizes of 2-9 mm3 were used successfully (Wyns et al., 2007). We also reported that immature porcine testis tissues undergoing the same cryopreservation treatments were not affected by the original size of the testis tissue fragment (5, 15, 20, or 30 mg) (Abrishami et al., 2010a). Although not used for cryopreservation, no effect of tissue sample size was observed for one-wk old piglet testes (as intact or fragments of 100 or 30 mg) when used for hypothermic preservation for 6 days (Yang et al., 2010). It remains to be seen if whole human testes can be cryopreserved as has been accomplished for whole

ovaries (Courbiere et al., 2006; Jadoul et al., 2007; Martinez-Madrid et al., 2007).

Schoysman et al., 1999).

**4.1 Germ cell transplantation** 

**4. Applications of testis cryopreservation for new reproductive technologies**  Given that properly cryopreserved testis biopsies can last decades in liquid nitrogen and that most prepubertal cancer patient boys donating biopsies may not need to resort to assisted reproductive technologies for a couple of decades, it is advisable that cryopreservation of testicular biopsies be offered to such patients in a hope that our ability to use such tissues will be further improved and the options expanded in the coming years. A number of potential applications already exist for the use of cryopreserved testicular tissue in experimental and clinical settings in reproduction medicine/science. Such technologies allow retrieval of existing sperm from mature donor samples and, more importantly, offer hope for production of sperm in samples of cryopreserved testis immature testis. If the preserved testis tissue contains endogenous spermatogenesis (*e.g.,* from obstructive azoospermic adult patients), it can be used to extract sperm, elongated spermatids, or even round spermatids to be used for fertilization of oocytes through ICSI (Rosenlund et al., 1998; Schrader et al., 2002; Gianaroli et al., 1999; Tesarik et al., 2000;

If preserved testis samples are obtained from neonatal/immature donors, they can still be

The technique for germ cell transplantation has allowed (re)establishment of spermatogenesis after introduction of donor testis cell suspensions into the seminiferous tubules of infertile recipient testes. Once deposited in the tubular lumen, donor SSCs are recognized by the host Sertoli cells and allowed passage to the stem cell niche, where new colonies of spermatogenesis can begin and expand. This approach has allowed production of sufficient numbers of sperm to allow infertile recipient mice to sire donor-derived progeny (Avarbock & Brinster, 1994; Brinster & Zimmermann, 1994). Later, the capability of cryopreserved mouse testis cells after transplantation into recipient testes to start spermatogenesis was also confirmed (Avarbock et al., 1996; Brinster & Nagano, 1998; Ogawa et al., 1999). While heterologous transplantation of human germ cells into recipient mice did not lead to completion of spermatogenesis (Nagano et al., 2002), the transfer technique has been tested

used to induce spermatogenesis through the following approaches.

using human testes (Schlatt et al., 1999; Brook et al., 2001). Although autologous/homologous transplantation of germ cells for humans is currently considered purely experimental, one possibility for prepubertal human testis samples taken and frozen prior to treatments is to isolate testis cells and transfer them back to the individual. As a major problem with this approach is the risk of reseeding a systemic cancer, solutions to this (e.g., soring out tumour cells) and other safety issues are under investigation.

We have expanded the technique for germ cell transplantation into farm animals (**Fig. 4**), showed the feasibility of SSC engraftment in unrelated recipient individuals (of the same species) without a need for immune-suppression, and further demonstrated the applicability of the approach through donor-derived sperm production by the recipients and birth of progeny carrying the donor characteristics (Honaramooz et al., 2002b 2003a, 2003b; Honaramooz & Yang, 2011). Therefore, although experimental at this stage, the approach may offer promise in salvaging genetic material from cryopreserved testicular tissue from immature endangered species.

Fig. 4. Schematic overview of germ cell transplantation from a donor male into the testes of a recipient. The testes are collected from a donor animal (**A**), which could theoretically include post-mortem testis recovery from a recently deceased juvenile individual of an endangered species. The testis tissue could be cryopreserved (**B**) until conditions for its use are in place. At the time of transplantation, a single-cell suspension is prepared and the cells are infused into the seminiferous tubules of a recipient animal (**C**). Mating of the recipient (**D**) produces progeny (**E**), some of which will carry the donor genome (image modified from Honaramooz et al., 2003b).
