Testis Tissue Xenografting Research Articles

Xenografting of human fetal testis tissue: a new approach to study fetal testis development and germ cell differentiation

BACKGROUNDAbnormal fetal testis development can result in disorders of sex development (DSDs) and predispose to later testicular dysgenesis syndrome (TDS) disorders such as testicular germ cell tumours. Studies of human fetal testis development are hampered by the lack of appropriate model, and intervention systems. We hypothesized that human fetal testis xenografts can recapitulate normal development.METHODSHuman fetal testes (at 9 weeks, n = 4 and 14–18 weeks gestation, n = 6) were xenografted into male nude mice for 6 weeks, with or without hCG treatment of the host, and evaluated for normal cellular development and function using immunohistochemistry, triple immunofluorescence and testosterone assay. The differentiation and proliferation status of germ cells within xenografts was quantified and compared with age-matched controls.RESULTSXenografts showed >75% survival with normal morphology. In the first-trimester xenografts seminiferous cord formation was initiated and in first- and second-trimester grafts normal functional development of Sertoli, Leydig and peritubular myoid cells was demonstrated using cell-specific protein markers. Grafts produced testosterone when hosts were treated with hCG (P = 0.004 versus control). Proliferation of germ cells and differentiation from gonocytes (OCT4+) into pre-spermatogonia (VASA+) occurred in grafts and quantification showed this progressed comparably with age-matched ungrafted controls.CONCLUSIONSHuman fetal testis tissue xenografts demonstrate normal structure, function and development after xenografting, including normal germ cell differentiation. This provides an in vivo system to study normal human fetal testis development and its susceptibility to disruption by exogenous factors (e.g. environmental chemicals). This should provide mechanistic insight into the fetal origins of DSDs and TDS disorders.

Effects of recipient mouse strain, sex and gonadal status on the outcome of testis tissue xenografting

The aim of the present study was to examine factors that may affect the outcome of testis tissue xenografting. Recipient factors were examined by grafting small fragments of testis tissue from newborn piglets under the back skin of immunodeficient mice of different strains (severe combined immunodeficiency (SCID) v. nude), sex (male v. female) and gonadal status (intact v. gonadectomised) using a factorial design (eight groups; n = 7 mice per group). Recipient mice were killed after 8 months to compare the gross and histological attributes of the recovered grafts. Overall, approximately 94% of grafts were recovered. Gonadectomy of male or female recipients did not affect any of the measured outcomes of testis tissue xenografting, therefore data were pooled. Overall, in terms of sex, male mice and, in terms of strain, SCID mice tended to show higher gross and histological development of grafts. The group of female nude mice had the lowest graft recovery rate (75%) compared with the other groups (95-100%; P < 0.05). The grafts from male SCID mice were, on average the largest and had the highest percentage of spermatozoa-containing seminiferous tubules among all the groups (P < 0.05). These results suggest that male SCID mice provide a suitable recipient model for testis tissue xenografting and that the mice do not need to be castrated for optimal results.

305 OPTIMIZING DONOR AND RECIPIENT FACTORS IN XENOGRAFTING OF TESTIS TISSUE

Grafting of donor mammalian testis tissue into recipient mice allows completion of spermatogenesis in the grafted tissue and therefore can serve as a new option for preservation of male germ line. For testis tissue xenografting, castrated male nude mice typically serve as recipients, each receiving 8 testis tissue fragments; however, no study has comprehensively investigated donor and recipient factors. The objective of this study was to determine the effects of strain of immunodeficient recipient mouse (nude v. SCID), gonadal status (intact v. gonadectomized), and gender (male v. female) on the outcome of testis tissue xenografting. A secondary objective was to determine the optimal number of testis tissue fragments per mouse most suitable for xenografting. Testis parenchyma from newborn piglets were cut into small fragments (5 mg each) and grafted under the back skin of different groups of immunodeficient mice. In Experiment 1, 8 groups of mice (n = 7/group) served as recipients: castrated male nude, intact male nude, ovariectomized female nude, intact female nude, castrated male SCID, intact male SCID, ovariectomized female SCID, and intact female SCID. In Experiment 2, 4 groups of mice (n = 10/group) served as recipients of 2, 4, 8, or 16 testis tissue fragments per mouse. Recipient mice were sacrificed 8 months after grafting and the weight of the grafts and vesicular glands (male mice) were compared among groups by analysis of variance. In Experiment 1, mouse gonadal status (intact v. gonadectomized) did not affect the total graft weight (P &gt; 0.05), but both the recipient mouse strain (nude v. SCID) and gender (male v. female) affected the total graft weight (2460 ± 320.9, 1420 ± 290.0, 758 ± 156.7, and 2780 ± 297.4, mean ± SEM, P &lt; 0.0001 for SCID, nude, female, and male mice, respectively). In Experiment 2, the total graft weight was highest in the group of mice receiving 8 testis tissue fragments (192 ± 76.1, 695 ± 96.5, 2443 ± 338.8, and 1458 ± 305.4, mean ± SEM, P &lt; 0.0001 for 2, 4, 8, or 16 fragment groups, respectively). These results collectively indicate that male SCID mice receiving 8 testis tissue fragments provide optimized conditions for the recovery of largest grafts. Research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Saskatchewan Health Research Foundation (SHRF) to A. Honaramooz and scholarships from the Western College of Veterinary Medicine and the International Peace Scholarship to S. Abbasi.

The effect of donor age on progression of spermatogenesis in canine testicular tissue after xenografting into immunodeficient mice

The objective of this study was to examine the effect of donor age on progression of spermatogenesis in dog (Canis lupus familiaris) testis tissue after xenografting. In Experiment 1, canine testes were obtained by surgical castration. Based on developmental pattern of spermatogenesis at the time of grafting, donors were categorized as immature, young, and adult (<4, 4 to 6, and >6 mo old, respectively). Fragments of testis tissue were implanted subcutaneously on the back of immunodeficient mice; xenografts were retrieved and analyzed 4, 6, or 8 mo later. At 4 mo postgrafting, immature and young groups had higher graft recovery rates, graft weights, vesicular gland indices, seminiferous tubule numbers, and larger seminiferous tubular diameters compared with those of adult donor xenografts. At 8 mo postgrafting, immature donor xenografts had maintained growth and development as exhibited by greater graft weights, vesicular gland indices, seminiferous tubule numbers, and tubular diameters compared with those of adult donor xenografts. At this time point, growth and development of xenografts did not differ between immature and young donors, whereas those from young donors had greater seminiferous tubule numbers and diameters compared with those of adult donor xenografts. Elongated spermatids were the most advanced germ cell type present at 4 and 8 mo postgrafting in xenografts of immature age groups. In Experiment 2, the longer-term efficiency of spermatogenesis and the potential sperm production in xenografts from immature donor dogs were determined. Testis tissue from 2-mo-old donor dogs were grafted into recipient mice, and xenografts were retrieved after 13 mo. Complete spermatogenesis was present in 5 of 29 recovered xenografts, with isolation of fully formed sperm (up to 36.3 × 10 6 per gram tissue). In conclusion, immature and young donors (<6 mo of age) were the most promising donors for dog testis tissue xenografting. This strategy may offer an alternative for male germ-line preservation for canids that die prematurely or must be castrated before maturation.

Cryopreservation of immature porcine testis tissue to maintain its developmental potential after xenografting into recipient mice

The purpose of this study was to develop effective strategies for cooling and cryopreservation of immature porcine testis tissue that maintain its developmental potential. Testes from 1-wk-old piglets ( Sus domestica) were subjected to 1 of 12 cooling/cryopreservation protocols: as intact testes, cooling at 4 °C for 24, 48, or 72 h (Experiment 1); as fragments, programmed slow-freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol (Experiment 2); or solid-surface vitrification using DMSO, glycerol, or ethylene glycol, each using 5-, 15-, or 30-min cryoprotectant exposure times (Experiment 3). For testis tissue xenografting, four immunodeficient recipient mice were assigned to each protocol, and each mouse received eight grafts. Recipient mice were killed 16 wk after grafting to assess the status of graft development. Based on morphology and in vitro assessment of cell viability, cooling of testis tissue for up to 72 h maintained structural integrity, cell viability, in vivo growth, and developmental potential up to complete spermatogenesis comparable with that of fresh tissue (control). In frozen-thawed testis tissues, higher numbers of viable cells were present after programmed slow-freezing using glycerol compared with that after DMSO or ethylene glycol (P < 0.001). Among the vitrified groups, exposure to DMSO for 5 min yielded numerically higher viable cell numbers than that of other groups. Cryopreserved tissue fragments recovered after xenografting had normal spermatogenesis; germ cells advanced to round and elongated spermatids after programmed slow-freezing using glycerol, as well as after vitrification using glycerol with 5- or 15-min exposures, or using DMSO for a 5-min exposure.

Porcine embryos produced after intracytoplasmic sperm injection using xenogeneic pig sperm from neonatal testis tissue grafted in mice.

Embryo development after homologous intracytoplasmic sperm injection (ICSI) with sperm from testis tissue xenografts from pigs or any other farm animal species has not been evaluated critically. Here, we report development of porcine embryos in vitro following ICSI with sperm retrieved from xenografted neonatal pig testis. Small pieces of testis tissue from newborn piglets were grafted under the back skin of castrated immunodeficient mice (n = 4) and the xenografts were collected 8 months after grafting. Spermatozoa were recovered by mincing of the grafted tissue. For comparison, testicular, epididymal and ejaculated spermatozoa were also collected from mature boars. Oocytes injected with xenogeneic spermatozoa were either fixed to determine fertilisation processes (n = 89 in five replicates) or allowed to develop in vitro (n = 143 in four replicates). Xenogeneic porcine spermatozoa were fertilisation competent (24% v. 58%, 68%, 62% or 0% for xenogeneic v. control testicular, epididymal and ejaculated spermatozoa or no spermatozoa, respectively) and embryos developed to the blastocyst stage (8% v. 22%, 27%, 25% or 0%, respectively). These results demonstrate that porcine spermatozoa derived from immature testis tissue xenografted into mice are fertilisation competent, albeit at a lower rate than testicular, epididymal or ejaculated spermatozoa from control boars, and support embryo development after ICSI.

Xenografting of adult mammalian testis tissue

Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, horse and rhesus monkey) were ectopically grafted to host mice. Grafts were recovered and analyzed at three time points: less than 12 weeks, between 12 and 24 weeks and more than 24 weeks after grafting. Histological analysis of the grafts revealed effects of species and donor tissue maturity: all grafts from species with greater daily sperm production (pig and goat) were found to have degenerated tubules or grafts were completely degenerated. None of the xenografts from mature adult bull and monkeys contained differentiated spermatogenic cells when examined more than 12 weeks post-grafting but tubules with Sertoli cells only remained. In grafts from a young adult bull, Sertoli cells persisted much longer than with the mature adult grafts. In grafts from a young adult horse, spermatogenesis proceeded to meiosis. In grafts from a young adult donkey and monkey, however, complete spermatogenesis was found in the grafts. These results show that testis tissue grafts from mature adult donors did not support germ cell differentiation but seminiferous tubules with Sertoli cells only survived in some species. The timing and progression of tubular degeneration after grafting of adult testis tissue appear to be related to the intensity of spermatogenesis at the time of grafting. Testis tissue from sub-adult donors survives better as xenograft than tissue from mature adult donors, and complete spermatogenesis can occur albeit with species-specific differences.

Analysis of Gene Expression in Bovine Testis Tissue Prior to Ectopic Testis Tissue Xenografting and During the Grafting Period1

The purpose of this study was to identify factors that contribute to bovine testis development and donor age-dependent differences in the abilities of bovine ectopic testis tissue grafts to produce elongated spermatids. We used real-time RT-PCR and microarrays to evaluate and to identify the expression of genes that are involved in Sertoli and germ cell development in bovine testis tissues. Testis tissues were obtained from 2-, 4-, and 8-wk-old bull calves and were grafted immediately. Grafted bovine testis tissue was removed from mice, RNA was isolated from the grafts, and real-time RT-PCR was used to evaluate gene expression during the grafting period. In addition, the gene expression in the donor tissue was analyzed using Affymetrix Bovine GeneChips, to identify differentially expressed genes. Examination of the testis tissue grafts indicated that Sertoli cell-specific gene expression was lower in 8-wk donor tissue grafts compared to the donors of other ages. Furthermore, the expression of KIT, which is a germ cell-specific gene, was low in testis tissue grafts. Microarray analysis of the donor tissue showed that several genes that are involved in angiogenesis or tissue growth were differentially expressed in 2-, 4-, and 8-wk-old bovine testes. The levels of expression of the genes for angiogenin, transgelin, thrombomodulin, early growth response 1, insulin-like growth factor 2, and insulin-like growth factor-binding protein 3 were lower in testis tissues from older animals. Using these data, it will be possible in the future to manipulate the testis xenograft microenvironment so as to improve the efficiency of sperm production within the graft.

Comparison of global gene expression between porcine testis tissue xenografts and porcine testis in situ

Testis tissue from immature mammalian donor animals, grafted ectopically to immunodeficient mouse hosts, can undergo complete spermatogenesis with the production of fertilization-competent spermatozoa. To further characterize testis tissue xenografts as a model for testis function in situ, the objective of this study was to compare gene expression between porcine testis tissue xenografts and testis tissue in situ. Pieces of testis tissue from 1-week-old piglets were grafted onto immunodeficient male mice and a littermate piglet was raised for comparison as control. Complete spermatogenesis was present in the testis tissue xenografts at 8 months after transplantation into mouse hosts and in the 8-month-old control porcine testis tissue. Total RNA was isolated from xenografts and control tissue, and the RNA was labeled and hybridized to the porcine genome array. By analyzing the expression of 23,256 transcripts, we found that 71 genes were differentially expressed with at least a fourfold difference between xenografts and control tissue. Interestingly, none of the 56 transcripts present on the array that were annotated in porcine testis showed differential expression between xenografts and control testis. This analysis indicates that global gene expression in porcine testis xenografts appears comparable to testis tissue in situ. These findings support the hypothesis that testis tissue xenografts can provide a representative model to study mammalian spermatogenesis.

The Length of the Spermatogenic Cycle Is Conserved in Porcine and Ovine Testis Xenografts

Xenografting of immature mammalian testis tissue into mice can accelerate sperm production. To determine whether this shortened time to sperm production is because of reduced length of the spermatogenic cycle, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Small testis fragments from newborn pigs and sheep were ectopically grafted into mice. Once complete spermatogenesis was present in grafted tissue, mice were injected with BrdU and grafts were recovered at different time points thereafter. In porcine grafts, the most advanced germ cells labeled 1 hour, 9 days, 12.3 days, and 18 days after BrdU injection were stage 1 preleptotene/leptotene primary spermatocytes, stage 1 pachytene primary spermatocytes, stage 5 newly-formed round spermatids, and late stage 2 elongating spermatids, respectively. In ovine grafts, the most advanced labeled germ cells at 1 hour, 11 days, and 22 days post-BrdU injection were stage 2 preleptotene/leptotene primary spermatocytes, late stage 1 pachytene primary spermatocytes, and stage 2 elongating spermatids, respectively. These results indicate that each spermatogenic cycle in porcine and ovine xenografts lasts approximately 9 and 11 days, respectively, which is similar to their durations in situ. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. This is consistent with earlier reports showing that the cycle length is inherent to the germ cell genotype. The shortened time to sperm production in xenografts therefore appears attributable to accelerated maturation of the testicular somatic compartments. Our results suggest that testis xenografts provide a useful model to study the timing of testicular maturation and spermatogenesis in different mammalian species.

Grafting Period and Donor Age Affect the Potential for Spermatogenesis in Bovine Ectopic Testis Xenografts

Bovine testis tissue xenografts contain elongating spermatids 6 mo after grafting. The percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal varies depending on donor age and rarely exceeds 10%. These data indicate significant changes are occurring to bovine testicular cells during the first weeks of life. The objective of this research was to xenograft testis tissue from multiple ages of bull calves for 24 or 36 wk in order to gain a better understanding of early bovine testis development. Testis tissue from 1-, 2-, 4-, and 8-wk-old calves was grafted onto the backs of castrated immunodeficient mice. Testis tissue from all donor ages grew, differentiated, and produced testosterone and elongating spermatids. Testis tissue grafts from 1- and 8-wk-old calves had elongating spermatids in greater than 5.5% of seminiferous tubule cross sections at the time of graft removal regardless of grafting period. Four-week-old donor tissue never had more than 5.2% of seminiferous tubule cross sections with elongating spermatids. Extending the grafting period from 24 to 36 wk resulted in an increase in the percentage of seminiferous tubule cross sections with elongating spermatids from 2% to 10% in 2-wk donor tissue. These data demonstrate that both donor age and grafting period may be important factors regulating the maturation of bovine testis xenografts, indicating that intrinsic differences exist within testis tissue at these donor ages. These data provide the framework for further study of bovine spermatogenesis using ectopic testis xenografting.

Effect of Vascular Endothelial Growth Factor and Testis Tissue Culture on Spermatogenesis in Bovine Ectopic Testis Tissue Xenografts

Bovine ectopic testis tissue grafting is a technique that can be used to study bovine spermatogenesis and for the production of germ cells for a variety of applications. Approximately 10% of seminiferous tubule cross sections in testis grafts contain spermatids, providing a unique tool to investigate what regulates germ cell differentiation. We hypothesized that manipulation of testis tissue grafts would increase the percentage of seminiferous tubule cross sections undergoing complete germ cell differentiation. To test this hypothesis, bovine testis tissue was treated with vascular endothelial growth factor (VEGF) at the time of grafting or explant cultured for 1 wk prior to grafting. For the VEGF experiment, 8-wk donor tissue and graft sites were treated with 1 microg of VEGF in order to increase angiogenesis at the graft site. For the testis tissue culture experiment, 4-wk-old donor testis was cultured for 1 wk prior to grafting to stimulate spermatogonial stem cell proliferation. Testis tissue grafts were removed from the mice 24 wk after grafting. VEGF treatment increased graft weight and the percentage of seminiferous tubule cross sections with elongating spermatids at the time of graft removal. Cultured testis tissue grafts were smaller and had fewer seminiferous tubules per graft. However, there was no difference in the percentage of seminiferous tubule cross sections that contained any germ cell type between groups. These data indicate for the first time that bovine testis tissue can be manipulated to better support germ cell differentiation in grafted tissue.

Advances and applications of germ cell transplantation

When male germ line stem cells are transplanted from the testis of a fertile donor animal to the testis of an infertile recipient they can establish donor-derived spermatogenesis in the recipient testis, and the resulting sperm can transmit the genotype of the donor to the offspring of the recipient. Germ cell transplantation provides a bioassay to study the biology of these stem cells, to develop systems for spermatogonial stem cell isolation and culture, to examine defects in spermatogenesis and to correct male infertility. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. A potential clinical application is restoration of fertility in patients that underwent cytotoxic treatments for cancer. As an alternative to transplantation of isolated germ cells to a recipient testis, ectopic grafting of testis tissue from diverse mammalian donor species, including primates, into a mouse host represents a novel possibility to study spermatogenesis, to investigate the effects of toxins or 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 mammalian species.

1 XENOGRAFTING OF ADULT MAMMALIAN TESTIS TISSUE

Testis tissue grafting presents an option for preservation of genetic material when sperm recovery is not possible. Grafting of testis tissue from sexually immature males to immunodeficient mice results in germ cell differentiation and production of fertilization-competent sperm from different mammalian species (Honaramooz et al. 2002 Nature 418, 778–781). However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Spermatogenesis was arrested at meiosis in grafts from mature horses (Rathi et al. 2006 Reproduction 131, 1091–1098) and hamsters (Schlatt et al. 2002 Reproduction 124, 339–346), and no germ cell differentiation occurred in xenografts of adult human testis tissue (Schlatt et al. 2006 Hum. Reprod. 21, 384–389). The objective of this study was to investigate survival and germ cell differentiation of testis xenografts from sexually mature donors of different species. Small fragments of testis tissue from 10 donor animals of 5 species were grafted under the back skin of immunodeficient, castrated male mice (n = 37, 2–6/donor). Donors were pig (8 months old), goat (18 months old and 4 years old) (n = 2), bull (3 years old), donkey (13 months old), and rhesus monkey (3, 6, 11, and 12 years old). At the time of grafting, donor tissue contained elongated spermatids, albeit to different degrees (&amp;gt;75% of seminiferous tubules in testis tissue from pig, goat, bull, and 6–12-year-old monkeys, and 33 or 66% of tubules in tissue from donkey or 3-year-old monkey, respectively). Grafts were recovered &amp;lt;12 weeks (n = 14 mice), 12–24 weeks (n = 16 mice), and &amp;gt;24 weeks (n = 7 mice) after grafting and classified histologically as completely degenerated (no tubules found), degenerated tubules (only hyalinized seminiferous tubules observed), or according to the most advanced type of germ cell present. Grafts from pig, goat, bull, and 6–12-year-old monkeys contained &amp;gt;60% degenerated tubules or were completely degenerated at all time points analyzed. In contrast, in grafts from the 3-year-old monkey, only 18% of tubules were degenerated, 14% contained Sertoli cells only, 64% contained meiotic, and 4% haploid germ cells at 24 weeks after grafting. Similarly, donkey testis grafts recovered 12–24 weeks after grafting contained &amp;lt;2% degenerated tubules, 46% of tubules had Sertoli cells only, 45% contained meiotic, and 7% haploid germ cells. These results show that survival and differentiation of germ cells in testis grafts from sexually mature mammalian donors is poor. However, better graft survival and maintenance of spermatogenesis occurred in donor tissue from donkey and 3-year-old monkey that were less mature at the time of grafting. Therefore, species and age-related differences appear to exist with regard to germ cell survival and differentiation in xenografts from adult donors. This work was supported by USDA/CSREES 03-35203-13486, NIH/NCRR 5-R01-RR17359-05, the Spanish Ministry of Education, and Science (BES-2004-4112).

Germ cell transplantation and testis tissue xenografting in domestic animals

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 syngeneic or 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 cell technology is 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.

Germ Cell Transplantation

Transplantation of male germ line stem cells from a fertile donor to the testis of an infertile recipient restores donor-derived spermatogenesis in the recipient testis. The resulting sperm pass the donor genotype to the offspring of the recipient. Germ cell transplantation helped to elucidate the biology of male germ line stem cells and their niche in the testis, develop systems to isolate and culture spermatogonial stem cells, examine defects in spermatogenesis, correct male infertility, and introduce genetic changes into the male germ line. Although most widely studied in rodents, germ cell transplantation has been applied to larger mammals, including primates. Potential clinical applications include restoration of fertility in patients who underwent sterilizing treatments for cancer or targeted correction of genetic defects in testicular somatic cells. Recently, ectopic grafting of testis tissue from diverse donor species, including primates, into a mouse host has opened an additional possibility to study spermatogenesis and to produce fertile sperm from immature donors. Testis xenografts are ideally suitable to study toxicants or drugs with the potential to enhance or suppress male fertility without the necessity of performing experiments in the target species. Therefore, transplantation of germ cells and xenografting of testis tissue represent powerful approaches for the study, preservation, and manipulation of male fertility.

267 TESTIS TISSUE XENOGRAFTING AS A BIOASSAY FOR GERM CELL DEVELOPMENTAL POTENTIAL IN EQUINE CRYPTORCHID TESTES

In domestic animals, spermatogenic differentiation is blocked in abdominally retained testes exposed to core body temperature. It is not known if undifferentiated germ cells are retained in long-term cryptorchid equine testes, nor is it known whether any surviving germ cells retain their ability to progress through spermatogenesis. If functional germ cells do persist in equine abdominal testes, then the possibility exists that offspring could be derived even from bilaterally cryptorchid individuals. Previously, we reported an in vivo model where completion of spermatogenesis with production of spermatozoa capable of fertilization occurred in fragments of testicular tissue from immature mice, domestic animals, and monkeys grafted under the skin of immunodeficient mice. Therefore, spermatogenic development in testis tissue xenografts can serve as an in vivo assay system for the developmental potential of germ cells. The objective of this study was to investigate if cryptorchid horse testes that had been exposed to core body temperature for 1–3 years had retained developmentally competent germ cells. Small fragments of abdominally cryptorchid testis tissue (about 1 mm3) from three donor horses (1-, 2-, and 3-year-old Quarterhorse) were grafted under the back skin of castrated male immunodeficient mice (n = 8, 6, and 3 recipient mice, respectively). At the time of grafting, donor tissue did not contain differentiated germ cells. Histological examination of the testis xenografts was performed between 5 and 45 weeks post-transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. By 28 weeks after grafting, pachytene spermatocytes were observed in xenografts from all cryptorchid donor testes. While haploid gametes would be expected to be present in xenografted testis tissue from descended equine testes by 35 weeks after grafting, spermatogenesis did not progress through meiosis in the cryptorchid grafts. In all recipient animals where spermatogenic differentiation occurred, the weight of the seminal vesicles in the castrated host mice was restored to pre-castration values, indicating that xenografts were capable of releasing biologically active testosterone. These results indicate that even after 3 years of exposure to core body temperature, equine cryptorchid testes contain germ cells capable of differentiation. It remains to be investigated if supplementation of exogenous gonadotropins might support post-meiotic differentiation of germ cells in cryptorchid equine testes xenografts. This work was supported by USDA 03-35203-13486.

193 TESTIS TISSUE XENOGRAFTING TO PRESERVE GERM CELLS FROM A CLONED BANTENG CALF

In April 2003, two banteng (Bos javonicus) calves were born after heterologous nuclear transfer of donor cells from a genetically valuable individual frozen in 1978. One of the cloned banteng calves died at one week of age. The calf was found to have one scrotal and one abdominally cryptorchid testis. In an attempt to preserve male germ cells from this valuable animal, parts of each testis were shipped on ice to the University of Pennsylvania for xenografting. Grafting of testis tissue from immature domestic animals and monkeys under the back skin of immunodeficient mice can result in complete spermatogenesis, albeit with different levels of efficiency in different species. The objective of this experiment was to investigate if grafting of immature banteng testis tissue would result in spermatogenesis in a mouse host. Small fragments of tissue (about 1 mm, 3 each) from both testes were grafted under the back skin (4 pieces of scrotal testis on the right side and 4 pieces of retained testis on the left side) of 6 castrated male immunodeficient mice. Histological examination of the testis xenografts was performed 3, 6, 9, 12, and 15 months after transplantation. Weight of the seminal vesicles in the host mouse was recorded as an indicator of bioactive testosterone produced by the xenografts. At the time of grafting, both testes contained seminiferous cords with immature Sertoli cells and gonocytes. At 3, 6, and 9 months after grafting, pachytene spermatocytes were present in the xenografts of the scrotal testis whereas no germ cell differentiation was observed in grafts from the retained testis. However, spermatogenesis in grafts of the scrotal testis did not proceed further through meiosis in grafts analyzed at 12 and 15 months after grafting, with pachytene spermatocytes still the most advanced germ cell type present in grafts recovered 15 months after grafting. The weight of the seminal vesicles in the castrated host mice was restored to pre-castration values showing that xenografts were releasing bioactive testosterone. These results indicate that banteng spermatogenesis was initiated in the mouse host but became arrested at meiosis as observed previously in xenografts of immature bovine or equine testis. Therefore, haploid germ cells could not be recovered. This represents the first example of trying to preserve fertility from a rare, valuable newborn animal by testis tissue xenografting. While xenografting presents a previously unavailable option for preservation of male germ cells from immature individuals, the efficiency of sperm production in testis xenografts appears to be variable and has to be determined empirically for different donor species. This work was supported by USDA 03-35203-13486.

A game of cat and mouse: xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host.

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.

Accelerated maturation of primate testis by xenografting into mice.

Testicular maturation and sperm production throughout the life of the male form the basis of male fertility. It is difficult to elucidate the intricate processes controlling testicular maturation and spermatogenesis in primates in vivo due to the long time span required for sexual maturation and also to the lack of accessible in vitro or in vivo models of primate spermatogenesis. Ectopic xenografting of neonatal testis tissue into mice provides an accessible model to study and manipulate the propagation and differentiation of male germ cells from immature donor animals. However, it was not clear whether this approach would be applicable to slowly maturing primates. Here we report that grafting of testis tissue from immature rhesus monkeys (Macaca mulatta) into host mice resulted in the acceleration of testicular maturation and production of fertilization-competent sperm in testis xenografts. The system reported here provides a powerful, practical approach to study timing and control of testicular maturation and regulation of primate spermatogenesis without the necessity for experimentation in primates. This approach could potentially be applied to produce fertile sperm from sexually immature individuals of rare or valuable primate species or from prepubertal boys undergoing sterilizing therapy for cancer.