**2.6 Testicular tissue cryopreservation**

Tissue cryopreservation is more complex than cellular preservation because tissue is composed of more than one cell type and thus of different water and cryoprotectant permeability coefficient values and different sensitivities to chilling and osmotic challenges. Tissue is also larger in volume and thus cryoprotectant penetration is difficult and heat transfer is not uniform, putting the center of the sample at greater risk of intracellular ice formation and death. This is true for testicular tissue, ovarian tissues and many other types of tissues and whole organs. Testicular tissue preservation can be done in one of three basic forms. The tissue can be cryopreserved for future use, it can be cultured *in vitro* for short to mid-term preservation or it can be transplanted. When preserved in the cryopreserved form, one can freeze the whole organ or even the entire animal. Recently it was demonstrated that

Genome Banking for Vertebrates Wildlife Conservation 305

generation of offspring from such tissues was demonstrated (Sato et al., 2011). In that study, neonatal mouse testicular tissue cultured *in vitro* for over two months (with or without being previously cryopreserved), generated fully competent spermatids and spermatozoa, which led to embryonic development and healthy and reproductive-active offspring production. This exciting development still needs to be evaluated in terms of accuracy of the genetic profile and absence of aneuploidy in haploid cells (Cheung & Rennert, 2011) as well as its applicability to other species. However, the fact that healthy and fertile offspring were

In comparison to the male, females' gametes pose several difficulties when it comes to preservation (Table 1). Very small number of gametes is progressing to the more advanced developmental stages during each cycle, and at best only a handful mature and ovulate. When dealing with rare and endangered species in which the number of available individuals for research is extremely limited and often spatially and temporally far apart, progress is very slow and limited by the small numbers. Oocytes and embryos are orders of magnitude larger than spermatozoa, thus bringing down the ratio between surface area and volume. The outcome is slower movement of water and cryoprotectants across the cellular membrane and elevated risk for intracellular ice formation. Unlike in males, *in vivo* collection of oocytes, and to a lesser extent - embryos, is an invasive procedure requiring anesthesia or sedation. Although production of new oocytes exists even in adulthood (Niikura et al., 2009; Tilly et al., 2009), it is very minimal. So, in general terms, the female can be considered as if it is born with a limited life-long supply. Males on the other hand produce sperm continuously, throughout their adult life, sperm that can be collected relatively easy almost any time. All these differences contribute to the fact that while the number of species in which sperm was cryopreserved is in the hundreds, the number of species in which embryo cryopreservation was reported (not all successful) is currently less than 50 and the number of species in which oocyte cryopreservation was attempted is far

Male Female

Gamete size <10µm (head) 10s-100s µm (species specific)

Production Continuous Very limited new production

Collection Almost any time In estrous (need monitoring)

Female gametes can be collected at different time points in their maturation process: 1) as mature oocytes, following ovulation (natural or chemically induced), 2) as mature and immature oocytes, by ovum pick up, either transabdominally, transvaginally or

Numbers Millions to billions Few at a time

Accessibility Easy to collect Invasive procedure

Table 1. comparison between male and female gametes in relation to gamete

produced is very encouraging.

**3. The female** 

less than that.

cryopreservation.

spermatozoa or spermatids retrieved from reproductive tissues (whole testes or epididymides) frozen for up to one year at -80ºC or from whole mice frozen at -20ºC for up to 15 years, can produce normal offspring when used, through ICSI, to fertilize mature oocytes (Ogonuki et al., 2006). This success followed a previous, failed, attempt to cryopreserve the entire testis (Yin et al., 2003). The other option is to cryopreserve testicular tissue slices. This technique is widely used today in both adult and pediatric human medicine as a mean to preserve fertility of patients undergoing cancer treatments. To cryopreserve the tissue, it is cut into tiny pieces, usually in the range of 1-2 mm3 to ensure cryoprotectant penetration, efficient heat transfer and eventual successful grafting. Other alternatives that have been proposed are to mince the tissue and then suspend it in freezing extender to achieve better cryoprotection (Crabbe et al., 1999) or to cut the testicular tissue into thin stripes (e.g. ~9×5×1 mm in sheep) to increase the total number of seminiferous tubules in each graft (Rodriguez-Sosa et al., 2010). Although such tissue samples can be obtained from every individual, infant, juvenile or adult, almost all successful studies to date used immature tissue (Ehmcke & Schlatt, 2008). Like in semen cryopreservation, there are differences between species in the reaction of their testicular tissue to cryoprotectants, chilling and cryopreservation (Schlatt et al., 2002b). The preserved testicular tissue can be handled in several ways. From these tissues, spermatozoa, spermatocytes and round and elongated spermatids can all be retrieved and used to fertilize oocytes through ICSI (Hovatta et al., 1996; Gianaroli et al., 1999). Testicular tissue can also be transplanted back to the donating individual (autografting), to another individual of the same species (allografting) or to individual of a different species, usually to nude or immunodeficient mice (xenografting). After transplantation, the graft may be lost due to tissue rejection or ischemia. If it manages to survive the critical first few days, blood supply will reach the graft, it will be supported by the recipient system and, after some time, will start producing spermatozoa, which can be harvested by surgical excision of all or part of the graft (Schlatt et al., 2002b). Although dependent on the recipient system for support, the spermatogenesis cycle length is assumed to be inherent to the spermatogonial stem cells, which are expected to preserve the donating species spermatogenesis length (Zeng et al., 2006). However other studies showed that in some species, the process is accelerated when their testicular tissue was xenografted into mice (rhesus monkeys; Honaramooz et al., 2004) while in others it is not (domestic cat; Snedaker et al., 2004). Acceleration, when identified, bears special interest for species preservation as it can shorten generation time and thus speed up population growth. This acceleration, however, may also mean abnormal spermatogenesis process that produces abnormal gametes. The sperm produced this way does not go through epididymal maturation process so the only way it can be utilized is by ICSI (Shinohara et al., 2002). One should also keep in mind that it is very costly to keep immunodeficient mice and handle them under germ-free conditions and, of course, repeated transplantations from one mouse to another are required to maintain viable tissue for many years. Still, testicular tissue cryopreservation was done in several species and pregnancies were achieved in mice (Schlatt et al., 2002b; Shinohara et al., 2002), rabbit (Shinohara et al., 2002), human (Hovatta et al., 1996), Djungarian hamsters (Schlatt et al., 2002b) and marmoset monkeys (Schlatt et al., 2002b), to name a few. Testicular tissue can also be cultured *in vitro* to give rise to mature and competent cells. Culture conditions, however, are very complex and, until recently, attempts were encouraging but still unsuccessful (Gohbara et al., 2010). Earlier this year, generation of offspring from such tissues was demonstrated (Sato et al., 2011). In that study, neonatal mouse testicular tissue cultured *in vitro* for over two months (with or without being previously cryopreserved), generated fully competent spermatids and spermatozoa, which led to embryonic development and healthy and reproductive-active offspring production. This exciting development still needs to be evaluated in terms of accuracy of the genetic profile and absence of aneuploidy in haploid cells (Cheung & Rennert, 2011) as well as its applicability to other species. However, the fact that healthy and fertile offspring were produced is very encouraging.
