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A Game of Cat and Mouse: Xenografting of Testis Tissue From Domestic Kittens Results in Complete Cat Spermatogenesis in a Mouse Host

From the Center for Animal Transgenesis and Germ Cell Research, Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania. Present address: Department of Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, Saskatchewan, S7N 5B4 Canada.

Correspondence to: Dr Ina Dobrinski, Center for Animal Transgenesis and Germ Cell Research, 147 Myrin Building, New Bolton Center, University of Pennsylvania, 382 West Street Road, Kennett Square, PA 19348 (e-mail: dobrinsk{at}vet.upenn.edu).

Received for publication April 2, 2004; accepted for publication June 27, 2004.

	Abstract

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Loss of genetic diversity because of infertility or the premature death of valuable individuals is a significant problem in the conservation of rare and endangered felid species, as well as in the maintenance of lines of cats used to study inherited feline and human disease. Attempts to overcome loss of genetic diversity have focused on freezing sperm; however, sperm cannot be collected from immature males. Previously, we reported completion of spermatogenesis in testis tissue from newborn pigs and goats grafted ectopically into host mice. The objective of this study was to extend the technique of testis tissue xenografting to the domestic cat as a model for felid species. Testes from 1- to 5-week-old domestic shorthaired kittens (n = 9) were cut into small fragments (about 0.5–1 mm³ each), and up to 8 fragments were grafted under the back skin of each castrated immunodeficient host mouse (n = 16). Histologic examination of the testis xenografts was performed between 5 and 54 weeks posttransplantation. At the time of grafting, the seminiferous cords of the donor testis tissue contained only immature Sertoli cells and gonocytes. At 14 weeks after grafting, tubular expansion was evidently caused by the proliferation of Sertoli cells and tubular lumen formation. By 18 weeks after transplantation, the seminiferous epithelium contained spermatocytes, and by 20 weeks, round spermatids were the most advanced types of germ cells. By 36 weeks after transplantation, xenografts of cat testis tissue had completed spermatogenesis. These results demonstrate the potential of xenografting to achieve full spermatogenesis in testis tissue from kittens. Therefore, sperm production in a mouse host can provide an alternative for germ line preservation from immature felids where sperm cryopreservation is not an option.

     Key words: Sperm, testis grafting, felid species, preservation

Previously, we reported completion of spermatogenesis in testis tissue from neonatal pigs and goats grafted ectopically into host mice (Honaramooz et al, 2002). The advantages of testis tissue xenografting are twofold: it serves as a powerful system for the study of spermatogenesis and testicular maturation and provides a previously unavailable system to obtain spermatozoa from immature animals. Continuous proliferation and differentiation of germ cells occur in a delicate balance with other testicular compartments, especially that of the supporting Sertoli cells (Russell et al, 1990). Testis tissue xenografting promotes natural spermatogenesis by keeping the donor testis microenvironment intact while allowing accessibility to the tissue for study and manipulation of testis function and retrieval of sperm to be used in assisted reproduction.

Live progeny were obtained from sperm extracted from ectopic allografts of immature mouse testes by intracytoplasmic sperm injection (ICSI) into mouse oocytes and subsequent embryo transfer (Schlatt et al, 2003). Sperm isolated from xenografts of immature rhesus monkey testis also supported embryo development in vitro to the blastocyst stage (Honaramooz et al, 2004). The successful generation of fertile male and female mouse pups and monkey embryos indicates that the sperm recovered from xenografts are capable of supporting normal embryonic development; therefore, progeny of other species could likely be produced using sperm originating from neonatal testis grafts.

Because of the similarities in reproductive biology between the domestic cat and nondomestic felid species, the objective of this study was to extend the technique of testis tissue xenografting to the domestic cat as a model animal for felid species to provide a novel method for male germ line preservation.

	Materials and Methods

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Testes from 9 domestic shorthaired kittens aged 1–5 weeks were obtained at euthanasia from animals at the University of Pennsylvania Medical Genetics research colony. Male immunodeficient mice (NCr nu/nu [n = 11] or severe combined immunodeficiency (SCID) [n = 5]) 7–11 weeks of age were castrated, and during the same surgery, fragments of cat testicular tissue (0.5–1 mm³) were implanted under their back skin (5–8 grafts per mouse). According to donor testis size, testis tissue was transplanted as follows: from two 1-week-old kittens and a 4-week-old kitten to 1 mouse each; from two 2.5-week-old kittens to 1 and 2 recipients, respectively; from 1 kitten each at 3, 3.5, and 5 weeks of age to 2 recipients each; and from 2 kittens 4.5 weeks old to 2 recipients each. Previous work on xenografting of pig testes was performed in NCr nu/nu mice (Honaramooz et al, 2002), whereas SCID mice were used as recipients for monkey testis xenografts (Honaramooz et al, 2004). Therefore, it could be expected that either recipient strain would be suitable. However, preliminary work in other species had shown that an interaction of donor species and recipient mouse strain is possible (Dobrinski, unpublished data). Therefore, both recipient strains were used in this study. Recipient mice were sacrificed from 5 to 54 weeks after grafting in order to assess graft survival and stage of spermatogenesis. Grafts were recovered, fixed in Bouin solution, and processed for histology. Each graft from each recipient mouse was examined histologically to determine the most advanced germ cell type present in each tubule cross section. On average, 242 tubule cross sections per recipient mouse were examined, and the germ cell types were identified on the basis of their morphology. Percentage of tubule cross sections containing a specific germ cell type was derived from the number of tubules containing this cell type compared with the total number of cross sections examined. Weight of seminal vesicles was recorded as an indicator of the presence of bioactive testosterone originating from the feline xenografts. All animal procedures were performed in accordance with and under control of the Institutional Animal Care and Use Committee at the University of Pennsylvania.

	Results

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Grafts were recovered from 11 recipient mice, whereas no grafts could be recovered in 5 recipients. Three of these 5 mice were systemically ill because of an unrelated infectious disease. No differences in graft survival or development were observed when nude or SCID mice were used as hosts. Recovered grafts had grown to approximately 10–60 times the volume of the original grafted fragments. Graft recovery and appearance of specific germ cell stages at different time points after grafting is outlined in the Table, and the histologic appearance of donor and graft tissues is illustrated in the Figure. Testis tissue from all donor kittens contained spermatic cords consisting of only gonocytes and Sertoli cells (Figure, panel A). At 5 weeks after grafting, there was evidence of some tubular expansion, but germ cells could only be identified in less than 10% of tubule cross sections. By 14 weeks after grafting, the seminiferous tubules had formed a lumen, and spermatogonia were present at the basement membrane (Figure, panel B). Pachytene spermatocytes were first seen at 19 weeks in grafts of 3-week-old kitten testes (Figure, panel C). Round spermatids were seen at 20 weeks in grafts of 3-week-old kitten testes and after 26 weeks in grafts of 5-week-old kitten testes. Elongating spermatids or spermatozoa were first detected after 35 weeks (Figure, panel D; 2.5-week-old donors). Spermatogenesis was sustained until 54 weeks after grafting (end of study, Figure, panel E), and cat spermatozoa could be recovered from the xenografts (Figure, panel F). The weight of the seminal vesicles as an indicator for production of bioactive testosterone from xenografts was restored to precastration values (>200 mg) in recipient mice in which grafts could be recovered, whereas seminal vesicles remained small (<100 mg) in recipients without graft survival.

View larger version (156K): [in this window] [in a new window]   Histologic appearance of immature domestic cat testis tissue before and after grafting into mice (hematoxylin and eosin stain). Three-week-old donor kitten testis at the time of grafting (A) and grafted tissue at 14 weeks (B), 20 weeks (C), 35 weeks (D), and 54 weeks (E). Cat spermatozoa (F) were recovered from the graft shown in Panel E. Note the changes from seminiferous cords (A) to fully developed seminiferous tubules with complete spermatogenesis (D). (Panels A–E), bar = 30 µm; (Panel F), bar = 20 µm.

	Discussion

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Grafting of small fragments of testis tissue from immature domestic kittens into immunocompromised laboratory mice provided an adequate environment for growth and differentiation of the germ cells and supporting somatic cells, ultimately resulting in complete spermatogenesis. Mature testicular feline spermatozoa could be extracted from these xenografts. Development of functional sperm in grafts of testis tissue has been reported in a variety of species, including mice, rabbits, hamsters, pigs, goats, primates, and now cats (Honaramooz et al, 2002, 2004; Shinohara et al, 2002; Schlatt et al, 2003). The apparent applicability of the technique across broad taxa makes it likely that this approach can be applied successfully to nondomestic felids. The timing of development of cat testis xenografts was similar to the normal course of testicular development during sexual maturation in domestic cats (Concannon, 1991). This is in contrast to our previous findings in xenografts from pigs or primates transplanted into mouse hosts in which testicular maturation was slightly or significantly accelerated compared with the donor species (Honaramooz et al, 2002, 2004). Although cat testis tissue grafts grew severalfold compared with the original grafted fragments, the increase in volume was less dramatic than that observed for porcine and caprine testis xenografts (Honaramooz et al, 2002). This likely reflects species differences in testicular growth rate. It remains to be determined why testicular maturation was not accelerated in cat testis grafts. In the immature primate testis, the onset of spermatogenesis is suppressed because of a lack of adequate gonadotrophic support (Plant, 1994; Marshall and Plant, 1996). It could be speculated that the timing of testicular maturation in the cat is inherent in the testis and was therefore not modified by exposure to adult levels of mouse gonadotropins or that species-specific differences in the interactions of mouse gonadotropins with the xenografted tissue could account for differences in timing of testicular maturation. It is less likely that gonadotropic support from the mouse host was insufficient because complete spermatogenesis did ultimately occur.

Presence of a functional endocrine compartment in the feline testis xenografts with production of physiological levels of biologically active testosterone was evidenced by restoration of the seminal vesicle weights to precastration values, whereas in castrated mice with no grafts, the seminal vesicles remained small. These findings were similar to those previously reported for mouse testis allografts in which a feedback between the endocrine cells of the allograft and the recipient mouse pituitary was established by 2 weeks after grafting (Schlatt et al, 2003).

Almost all 36 species of wild felids are facing population pressures in at least part of their range (Nowell and Jackson, 1996). Survival of these species depends on our ability to preserve their existing genetic diversity despite the loss of habitat, a lack of movement of genetic material between isolated groups of animals, the poor reproductive performance of many felids in captivity, and a high rate of prepubertal mortality in several species in captivity. To aid the conservation effort, "genome banks" preserve somatic tissue, mature sperm, embryos, and ovarian tissue. However, this approach has limitations with regard to male genetic material. First, the number of future breedings with sperm stored from an individual will be limited because sperm cannot replicate. Second, in species such as the Pallas cat (Otocolobus manul), black-footed cat (Felis nigripes), and clouded leopard (Neofelis nebulosa), young animals often die before becoming reproductively mature. The genetics of these individuals is lost forever.

Xenografting a portion of a testis from one species into an immunodeficient mouse recipient provides a way to overcome this loss of genetic potential and therefore offers a strong complement to the preservation of mature sperm. Xenografts contain a self-renewing population of male germ line stem cells, unlike spermatozoa, which cannot replicate themselves. In addition, the cells they produce can undergo genetic recombination, thereby preserving the entire potential genetic diversity of that male. Finally, they can be harvested from immature males as well as adults, offering a way to preserve genes from cats that die prior to maturity. On the female side, follicular development in xenografts of cat ovarian tissue has been reported (Gosden et al, 1994) and represents a complementary technology to the production of sperm in testis tissue xenografts demonstrated in the present study. Sperm harvested from xenografts have not undergone epididymal maturation, and the number of sperm harvested at any given point in time is limited. Therefore, xenogeneic sperm will have to be used in assisted reproductive technology, namely ICSI followed by embryo transfer. Although these technologies are established in the domestic cat (Gomez et al, 2000), their application to other felid species has been much slower, and technical problems will have to be overcome before ICSI with sperm recovered from grafts could routinely produce live offspring. Therefore, the novel approach to male germ line preservation in cats described here presently provides an additional tool for genetic management of felids, especially for the recovery of genetic material from immature animals.

In this study, grafts were not recovered from all recipient animals. Some of the recipient animals were systemically ill at the time of analysis from a viral infection that could have interfered with support of the tissue grafts. In addition to health problems in the immunodeficient recipients, unrelated to the grafting procedure, other factors like tissue handling or time from tissue collection to grafting could also affect graft survival. To assure preservation and development of germ cells from a specific donor, it would therefore seem prudent to prepare more than 1 recipient animal per donor.

This study focused on the potential of testis tissue xenografting from immature animals. Although donors ranged in age from 1 to 5 weeks, the histologic appearance of the testis in all the donor animals was similar, with the seminiferous tubules containing only Sertoli cells and immature germ cells. No attempt was made to distinguish potential differences in developmental potential of testis tissue from kittens of different ages. It remains to be explored whether xenografting of feline testis tissue from adult donors could serve to extend sperm production or to recover spermatogenesis from individuals that have undergone testicular degeneration as a result of age or health-related causes. If possible, this would provide an alternative to cloning for the genetic preservation of select valuable males from which banked sperm are not available.

The domestic cat can serve as a model to examine the physiology and pathology of reproduction in other felid species (Franca and Godinho, 2003) in which, for example, teratospermia is prevalent (Pukazhenthi et al, 2001). This study was not designed to critically evaluate efficiency of sperm production or sperm morphology. However, future experiments comparing sperm morphology between sperm recovered from donor animals and those from xenografts are likely to provide valuable insights into the pathogenesis of teratospermia in cats. In addition to their potential use in germ line conservation, feline testis tissue xenografts offer an approach to study spermatogenesis in a cat model without the necessity of experimentation in the target species.

This study demonstrated that cat spermatogenesis can occur in a mouse host and therefore provides a novel approach for the study and manipulation of feline spermatogenesis as well as for the preservation of genetic material from immature males of rare or endangered feline species.

	Acknowledgments

 
We thank Janet Turpin for animal care; Dr Mark Haskins (grant DK25759 from National Institute of Diabetes & Digestive & Kidney Disease) for providing cat testis tissue; Susan Megee for technical assistance; James Hayden for image preparation; and Drs Buddhan Pukazhenthi, Pierre Commizoli, and Alex Travis for their valuable input.

	Footnotes

 
^? Supported by grant 2003-02714 from the US Department of Agriculture, grant 1 R01 RR17359-01 from the National Center for Research Resources, and the Commonwealth and General Assembly of Pennsylvania.

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