Germ cell transplantation & testis tissue xenografting in domestic animals

1. Germ cell transplantation & testis tissue xenograftingin domestic animals

2. Abstract • Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice results in donor-derived spermatogenesis and transmission of the donor’s genetic material to the offspring of recipient animals. • Germ cell transplantation provides a bioassay to study the biology of male germ line stem cells, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis and treat male infertility. • Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals. In domestic animals including pigs, goats and cattle, as well as in primates, germ cells can be transplanted to a recipient testis by ultrasonographic-guided cannulation of the rete testis. • Germ cell transplantation was successful between unrelated, immuno-competent pigs and goats, whereas transplantation in rodents requires syngeneicor immuno-compromised recipients. • Genetic manipulation of isolated germ line stem cells and subsequent transplantation will result in the production of transgenic sperm. • Transgenesis through the male germ line has tremendous potential in domestic animal species where embryonic stem celltechnologyis not available and current options to generate transgenic animals are inefficient. • As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including horses and primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of drugs with the potential to enhance or suppress male fertility, and to produce fertile sperm from immature donors. • Therefore, transplantation of germ cells or xenografting of testis tissue are uniquely valuable approaches for the study, preservation and manipulation of male fertility in domestic animals.

3. Intro • Mammalian spermatogenesis is a continuous, organized process of cell proliferation and differentiation resulting in the production of virtually unlimited numbers of spermatozoa throughout the life of the male (Russell et al., 1990). • The foundation of this system is the spermatogonialstem cell which has the unique potential for both self-renewal and production of differentiated daughter cells which will ultimately form spermatozoa (Huckins,1971; Clermont, 1972; Meistrich and van Beek, 1993). • Transplantation of germ cells from fertile donor mice to the testes of infertile recipient mice resulted in donor-derived sperm production by the recipient animal (Brinster and Zimmermann, 1994) and sperm arising from transplanted donor germ cells were capable of fertilization in vivo and in vitro (Brinster and Avarbock, 1994; Goossens et al., 2003; Honaramooz et al., 2003a). • For the first time this provided a functional reconstitution assay for male germ line stem cells in the mouse. • Subsequently germ cell transplantation between rats was also successful (Jiang and Short, 1995; Ogawa et al., 1999a; Zhang et al., 2003), and it was shown that spermatogonialstem cells can be cryopreserved for prolonged periods of time before transplantation and still establish spermatogenesis in the recipient testis (Avarbock et al., 1996; Kanatsu-Shinohara et al., 2003a).

4. Transplantation of germ cells in rodents, domestic animals and primates • 2.1. Cross-species transplantation of germ cells • Using immunocompromised mice as recipient animals, rat sperm developed in mouse testes following cross-species spermatogonial transplantation from rats to mice (Clouthier et al., 1996) and transplantation was subsequently also successful from mice to rats (Ogawa et al., 1999a; Zhang et al., 2003). • Cross-species transplantation of germ cells between rats and mice established that the cell cycle during spermatogenesis is controlled by the germ cell and not the Sertoli cell (Franca et al., 1998). Hamster spermatogenesis could also occur in the mouse testis (Ogawa et al., 1999b); however, with increasing phylogenetic distance between donor and recipient species, complete spermatogenesis could no longer be achieved in the mouse. • Transplantation of germ cells from non-rodent donors ranging from rabbits, dogs, pigs, cattle, horses and ultimately non-human primates and humans, resulted in colonization of the mouse testis but spermatogenesis became arrested at the stage of spermatogonial expansion (Dobrinski et al., 1999, 2000; Nagano et al., 2001a, 2002a). • Therefore, the initial steps of germ cell recognition by the Sertoli cells, localization to the basement membrane, and initiation of cell proliferation are conserved among evolutionary divergent species. • However, when donor and recipient species are phylogeneticaly more distant than rodents, the recipient testicular environment appears to become unable to support spermatogenic differentiation and meiosis. • This incompatibility of donor germ cells and recipient testicular environment could be overcome by co-transplantation of germ cells and Sertoli cells (Shinohara et al., 2003) or by testis tissue transplantation subsequently described (Honaramooz et al., 2002a). • Although cross-species spermatogonialtransplantation did not have the envisioned immediate practical application, it nonetheless provides a bioassay for stem cell potential of germ cells isolated from other species (Dobrinski et al.,1999, 2000; Izadyar et al., 2002). • Cross-species transplantation experiments reported so far are summarized in Table 1.

5. 2.1. Cross-species transplantation of germ cells

6. Transplantation of germ cells in rodents, domestic animals and primates • 2.2. Germ cell transplantation in large animals • To date, germ cell transplantation has been reported in pigs, goats, cattle and monkeys (Schlatt et al., 2002; Honaramooz et al., 2002b, 2003a,b; Izadyar et al., 2003; Table 2). • Application of germ cell transplantation technology to non-rodent species required the development of a new technical approach. While direct injection of donor cells into rodent seminiferous tubules is possible via the efferent ducts, this is not feasible in larger species. • Instead, we developed a combination of ultrasonographic-guided cannulation of the centrally located rete testis with delivery of germ cells by gravity flow for germ cell transplantation in pigs and goats (Honaramooz et al., 2002b, 2003a,b), and a similar approach was described in cattle and monkeys (Schlatt et al., 2002; Izadyar et al., 2003). • Using this approach, we transplanted germ cells from transgenic donor goats into the testes of immunocompetent, prepubertal recipient animals. After these recipient goats became sexually mature, they produced sperm carrying the genetic makeup of the donor as evidenced by transmission of the transgene to the offspring of the recipient. • This provided proof-of-principle that germ cell transplantation results in donor-derived sperm production and fertility in a non-rodent species (Honaramooz et al., 2003a). Importantly, donors and recipients were unrelated and immunocompetent while in rodents, syngeneic, immunocompromised or immunosuppressed animals (Zhang et al., 2003; Kanatsu-Shinohara et al., 2003b) were required as recipient animals. • The testis is considered to be an immune privileged site, but it is unclear why transplantation between unrelated, immunocompetent animals is possible in domestic animal species but not in rodents. • In the only report of germ cell transplantation in cattle, histological evidence of spermatogenesis was only achieved after autologous transplantation but not between different bulls (Izadyar et al.,2003). • Whether this could be attributed to the use of older recipient animals than in our studies of pigs and goats or, whether this represents a species-specific difference, cannot be determined due to the few numbers of animals involved. Germ cell transplantation has not yet been reported in horses. It has been suggested that mules would provide a naturally occurring recipient animal model for germ cells from horses and donkeys (Neves et al., 2002). While the male mule generally is infertile due to the chromosomal imbalance causing failure of chromosome pairing during meiosis (Chandley et al., 1975) the mule testis nonetheless appears capable of supporting spermatogenesis (Neves et al., 2002). • The development of germ cell transplantation technology in domestic animal species is at least in part driven by the desire to develop alternate approaches for the production of transgenic animals through the manipulation of the male germ line. • While this may not be of interest to the equine industry, transplantation from valuable or immature donors to mule or horse recipients could serve as a novel means for preservation of fertility.

8. 2.3. Recipient animal preparation • The efficiency of colonization of seminiferous tubules by the transplanted germ cells can be improved if the recipient testes have little or no endogenous spermatogonia. • Busulfan, a DNA alkylating agent that destroys proliferating cells, is frequently used in rodents to deplete recipient germ cells prior to germ cell transplantation. • However, the sterilizing dose of busulfan is species- and strain-specific and treatment can be lethal due to severe bone marrow depression (Ogawa et al., 1999a; Brinster et al., 2003). • We recently explored the effectiveness of in utero treatment of pigs as previously described in the mouse (Brinster et al., 2003). • Administration of busulfan to pregnant sows at days 98 and 108 of gestation (a period of high proliferation of fetal gonocytes) resulted in depletion of gonocyteswith no observed adverse effects on the piglets or on sow health or fertility (Honaramooz et al., 2004a). • As an alternative to cytotoxic treatment irradiation of the testes will also result in depletion of endogenous germ cells (Creemers et al., 2002; Schlatt et al., 2002). • Provided a suitable radiation source is available, local testicular irradiation appears to be the method of choice in species where the anatomical position of the testes facilitates shielding of the body from irradiation, thereby minimizing systemic effects. • We recently explored this option for the preparation of recipient goats and demonstrated that fractionated testicular irradiation with 6Gy resulted in depletion of germ cells in goats (Behboodi et al., 2004) and reduction of germ cell numbers was also reported after testicular irradiation in rams (Oatley et al., 2005). • If mules are used as recipients for equine germ cell transplantation, strategies to reduce the number of endogenous germ cells would have to be applied to allow the donor germ cells widespread access to the stem cell niche because >95% of seminiferous tubules in mules contain at least spermatogonia (Hernandez-Jauregui and Monter, 1977; Neves et al., 2002).

9. 2.4. Applications of germ cell transplantation in domestic animals • Transplantation of germ cells could serve to restore male fertility after an insult to the testis. • Different from cryopreservation of sperm, germ cell transplantation can be applied to pre-pubertal males, thereby offering the potential to preserve genetic material from immature individuals that are lost before they reach puberty. • In most cases, germ cells can also be harvested from testes of individuals with azoospermia. • When introduced into a permissive testicular environment, these cells can initiate spermatogenesis and produce fertile sperm (Ogawa et al., 2000). • In horses and pigs where males are routinely castrated at an early age, preservation of germ cells could provide an option to obtain sperm and offspring from males that demonstrate superior traits, making their genetics highly valuable to breeders. • Another important application is transgenesis through the male germ line using transplantation of transfected germ cells. • This approach has tremendous potential in rats and domestic animals where embryonic stem cell technology is not available and current options to generate transgenic animals are inefficient. • Transgenic mice and rats have been generated by viral transduction of germ cells prior to transplantation (Nagano et al., 2000, 2001b, 2002b; Orwiget al., 2002; Hamra et al., 2002). • The use of an adeno-associated viral vector to introduce a transgene into porcine germ cells prior to transplantation has also shown promising results (Honaramooz et al., 2003c).

10. Transplantation of testis tissue in domestic animals • As previously described, cross-species germ cell transplantation did not result in complete sperm production in species other than rodents, likely due to an incompatibility between donor germ cells and recipient testicular environment. • Co-transplantation of the donor germ cells with their surrounding testicular tissue into a mouse host would preserve the testicular environment and still allow experimentation in a small rodent. • We, therefore, developed ectopic grafting of testicular tissue under the back skin of immuno-deficient mice as an alternate pproachfor the maintenance and propagation of male germ cells that can be more readily applied to different mammalian species (Honaramooz et al., 2002a). • Xenografting of testis tissue from newborn pigs and goats resulted in production of normal, functional sperm in a mouse host (Fig. 1). • This was the first report of complete, functional cross-species spermatogenesis from species other than rodents and it also represented the first time that sperm could be obtained from neonatal donors. • Similar to isolated germ cells, testicular tissue can be stored frozen prior to grafting while retaining its developmental potential (Honaramooz et al., 2002a; Schlatt et al., 2003). • Sperm recovered from allografts (mouse to mouse) and xenografts (monkey to mouse) supported embryo development when injected into oocytes (Schlatt et al., 2003; Honaramooz et al., 2004b). • When followed by embryo transfer, mouse sperm from allografts resulted in normal, fertile progeny (Schlatt et al., 2003). • The onset of spermatogenesis in xenografted pig testis tissue occurred slightly earlier than in the donor species (Honaramooz et al., 2002a). • We subsequently demonstrated that testicular maturation and sperm production in rhesus macaques can be significantly accelerated by exposure of the testis tissue to the endocrine environment of the castrated adult mouse host (Honaramooz et al., 2004b). • Recently, we also established that testis tissue recovered from sexually immature horses can survive and mature in the mouse host (Turner et al., 2005). • In addition, using the xenograftingapproach as a bioassay for the developmental potential of germ cells, we could demonstrate that testis tissue from cryptorchid adult horses had maintained its ability to undergo spermatogenic differentiation (Rathi et al., 2005).

12. 3.1. Applications of testis tissue xenografting • Testis tissue xenografting into a mouse host maintains structural integrity of the testicular tissue and provides the accessibility essential for the study and manipulation of testis function in a controlled manner that is not feasible in the donor animal. • This in turn will allow detailed analysis of the effects of compounds to enhance or suppress male fertility in an in vivo system without extensive experimentation in the target species. • Ectopic testis tissue grafting also represents a new option for male germ line preservation. • As it provides a source of male gametes even from immature gonads, grafting of fresh or preserved testis tissue offers an invaluable tool for the conservation of fertility. • The castrated mouse host initially provides for greater concentrations of FSH as stimulus for testicular development. • Additional manipulation of the endocrine milieu in the host could aid to significantly shorten the time span required for sperm production from genetically superior males. • Transfectionof germ cells prior to grafting provides a unique tool to study genetic regulation of spermatogenesis as it will allow for induction or disruption of specific genes and subsequent close observation of spermatogenesis in an easily accessible system.

13. 4. Conclusions • Germcell transplantation and testis tissue xenografting are powerful tools to explore basic biological aspects of male germ line stem cells and testis function as well as potential causes of male infertility. • Practical applications include the introduction of genetic modifications into the germ line of domestic animals, and the preservation of fertility from valuable individuals or rare and endangered animals. • Transplantation of germ cells or testis tissue will continue to significantly enhance our understanding of testis function and our ability to control and preserve male fertility.