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a Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia,Pennsylvania 19104-6009
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-satellite DNA. Donor germ cells were present in the testes of all but one recipient. Donor germ cells predominantly formed chains and networks of round cells connected by intercellular bridges, but later stages of donor-derived spermatogenesis were not observed. The pattern of colonization after transplantation of cultured cells did not resemble spermatogonial proliferation. These results indicate that fresh and cryopreserved germ cells can colonize the mouse testis but do not differentiate beyond the stage of spermatogonial expansion.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Xenogeneic spermatogenesis after transplantation of rat germ cells to mouse testes was supported by mouse Sertoli cells, and only minor defects were occasionally observed in rat spermatogenesis in mouse testes [12, 13]. We recently reported successful transplantation of hamster germ cells to mouse seminiferous tubules, resulting in the production of hamster sperm by recipient mice [14]. However, morphological abnormalities in spermiogenesis were frequently observed after transplantation of hamster cells. It appears that the efficiency of xenogeneic spermatogenesis was decreased with increasing phylogenetic distance between donor and recipient species. Until now, xenogeneic spermatogonial transplantation has been studied only between rodent species. The mouse has been well established as a recipient animal, and immunodeficient mice that accept xenogeneic germ cell transplantations are readily available. Therefore, the generation of spermatogenesis from species other than rodents in mouse testes, namely from economically important animal species, would have great economical and logistic advantages over the use of larger animals as recipients. In combination with genetic modification of the donor germ cells in vitro before transplantation to recipient testes, this would allow for potential modification of the male germ line. An important step toward this goal was accomplished recently when it was shown that spermatogenesis occurred after mouse spermatogonial stem cells were maintained in culture for several months before transplantation into recipient mice [15].
A central problem in studying transplantation of testis cells from different species was the absence of a detection system for xenogeneic cells in the recipient testis. Donor animals transgenic for the lacZ marker gene are available only in mice and rats. Reliance on sperm morphology as employed in the hamster-to-mouse transplantations [14] can detect only later stages of donor-derived spermatogenesis and will therefore underestimate colonization efficiency, or will be unable to detect colonization by donor cells in cases in which spermatogenesis does not progress at least to the spermatid stage. Even unequivocal identification of donor spermatogenesis after homologous transplantation is problematic [9, 10].
The objectives of the present study were to develop a technique to detect donor cells in the recipient testis and to investigate the effect of increased phylogenetic distance between rabbits and dogs as donors and mice as recipient animals on the outcome of spermatogonial transplantation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testis cells were collected from rabbits (3–5 mo old) and dogs (1–5 yr old) and transplanted into the seminiferous tubules of recipient mice. Testis cells were either transplanted immediately after collection (6 donor rabbits for 25 recipient mice, and 3 donor dogs for 15 recipient mice) or they were cryopreserved and transplanted at a later date (2 donor rabbits for 11 recipient mice, and 2 donor dogs for 7 recipient mice). In separate experiments, testis cells from one donor rabbit and one donor dog were maintained in culture for 1 mo and subsequently transplanted to recipient mice (7 recipient mice for cultured rabbit testis cells, 11 recipient mice for cultured dog testis cells). From 1 to 6 mo after transplantation, the testes from 1 to 4 recipient animals per donor and treatment were analyzed for the presence of donor-derived spermatogenesis by whole-mount immunohistochemistry (for rabbit and dog cells) and polymerase chain reaction (PCR; for dog cells). After transplantation of freshly collected testis cells, some recipient animals were maintained for more than 1 yr before analysis.
Donor Cell Preparation
New Zealand White rabbits (Covance Research Products, Denver, PA) between 3 and 5 mo of age, and mixed breed dogs between 1 and 5 yr of age were used as the source of donor testis cells. Spermatogenesis was not yet fully developed in rabbit testes, whereas donor dogs were sexually mature. All experimental procedures were approved by the Animal Care and Use Committee at the University of Pennsylvania. Testis cells were collected by a two-step enzymatic digestion previously described [16, 17] with minor modifications. Briefly, the tunica albuginea was manually removed from the testes. The exposed seminiferous tubules were then dissociated with collagenase (2 mg/ml; type IV, Sigma, St. Louis, MO) in 10 ml of Hanks' balanced salt solution without calcium and magnesium (HBSS) at 37°C for 5–10 min. DNase I (7 mg/ml) in HBSS was added as needed (approximately 100 µl) to further dissociate tubules. After being rinsed 2–4 times in HBSS, the tissue was digested with 0.25% trypsin and 1 mM EDTA in 10 ml of HBSS at 37°C for 5–10 min. Fetal bovine serum (1–2 ml) was added to stop enzymatic digestion. The resulting cell suspension was filtered through a nylon mesh with 60-µm pore size (Tetko Inc., Kansas City, MO), and the cells were collected by centrifugation, 600 x g for 5 min at 16°C. The pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 6 mM glutamine, 6 mM lactate, 0.5 mM pyruvate, 30 mg/L penicillin, and 50 mg/L streptomycin (designated DMEM-C) to a final concentration of 98–228 x 106 cells/ml. Viability of cells was greater than 95% as determined by trypan blue exclusion.
Donor Cell Freezing
Several aliquots of rabbit and dog testis cells collected as described above were frozen and kept in liquid nitrogen for transplantation at a later date. The freezing procedure was essentially the same as for cultured somatic cells [8, 18]. First, testis cells were suspended in DMEM-C at a concentration of 6 x 107 cells/ml. Freezing medium (FBS, DMEM-C, dimethyl sulfoxide in a ratio of 1:3:1) was added slowly at a volume equal to the original cell suspension, and mixed. Cells were aliquoted 1.0 ml per freezing vial, placed in an insulated container at -70°C for at least 12 h, and stored in liquid nitrogen (-196°C). The cells were thawed by swirling in a 37°C water bath, and DMEM-C was added slowly to three times the volume in the vial. This cell suspension was centrifuged at 600 x g for 5 min at 16°C, and the pellet was resuspended in DMEM-C to a cell concentration of 121–160 x 106 cells/ml for transplantation. Viability of cells following freezing and thawing was 63–82%.
Culture of Donor Testis Cells
Testis cells were collected from one rabbit and one dog as described above and cultured on mitomycin-treated STO (mouse fibroblast cell line) feeders [18] essentially as described previously [15]. Feeder cells were seeded in 75-cm2 tissue culture flasks at 5 x 104 cells/cm2 1–3 days before testis cells were added. Cultures were maintained in DMEM-C. Testis cells were added to feeder cultures at 10 x 106 cells per 75-cm2 tissue culture flasks. Medium was changed every other day, and cultures were maintained at 32°C in 5% CO2 in air. After 1 mo, cells were harvested by trypsin digestion (0.25%), washed in DMEM-C by centrifugation, and resuspended in medium at a concentration of 83 x 106 cells/ml (rabbit) or 35 x 106 cells/ml (dog) for transplantation to recipient testes.
Recipient Mice and Donor Cell Transplantation
NCr Swiss nude (nu/nu) mice (Taconic, Germantown, NY) 10–20 wk of age were used as recipient animals to avoid immunological rejection of donor cells. The mice were treated with busulfan (44 mg/kg) at least 4 wk before donor cell transplantation to deplete endogenous germ cells in the testes [6]. Because this dose of busulfan is toxic to the hematopoietic system, some mice received bone marrow transplantation from nontreated nude mice. Donor bone marrow cells were collected from a nude mouse by flushing the marrow of femurs and tibiae with DMEM-C. A volume of 0.1 ml, containing 3–6 x 106 cells/ml, was injected into the jugular vein of recipient mice three days after busulfan treatment [11]. Within 4 wk before transplantation, recipient mice received 7.6 mg/kg of the GnRH agonist leuprolide-acetate s.c., a treatment that previously had been shown to improve colonization after spermatogonial transplantation in mice [19]. For transplantation of testis cells, the seminiferous tubules of recipient mice were filled with approximately 10 µl/testis of the donor cell suspension by injection through the efferent ducts as described previously [17].
Analysis of the Recipient Testes
Between 30 and 473 days after donor cell transplantation, the recipient mice were killed by CO2 inhalation, and both testes were recovered. The tunica albuginea was removed, and the seminiferous tubules were gently dispersed with collagenase (type IV, 1 mg/ml) to remove interstitial cells. The dispersed tubules were washed in Dulbecco's PBS (DPBS) and fixed in freshly prepared 4% paraformaldehyde for 2 h at 4°C. Samples were then stored in DPBS at 4°C until analysis.
Generation and Purification of Polyclonal Antibodies
Testis cells were isolated from a 3-mo-old rabbit and an 11-mo-old dog as described above. Testis cells were washed twice by centrifugation and resuspended in DPBS. Aliquots of 4 x 106 rabbit testis cells/0.5 ml DPBS and 8, 10, and 20 x 106 dog testis cells/0.5 ml DPBS were frozen at -70°C. In order to minimize cross-reactivity with mouse testis cells, antibodies against rabbit testis cells were generated in mouse ascites fluid essentially as described [20, 21]. Briefly, 3 female BALB/c mice were immunized with 4 x 106 rabbit testis cells in RIBI R-700 adjuvant (RIBI ImmunoChem Research Inc., Hamilton, MT) on Days 0, 14, and 28. On Days 3 and 17, mice received 0.3 ml pristane i.p. On Day 33, mice received 106 nonsecreting mouse myeloma cells (P3X63-Ag8.653; ATTC, Rockville, MD) i.p., and ascites fluid was collected beginning on Day 45, pooled, and frozen.
The polyclonal antibody against dog testis cells was generated by immunizing a female NZW rabbit by s.c. injections along the back with 20 x 106 dog testis cells in Freund's complete adjuvant on Day 0; this was followed by 3 boost injections with 10 x 106 dog testis cells and 2 additional boost injections with 8 x 106 dog testis cells in Freund's incomplete adjuvant 3 wk apart. Serum was collected 2 wk after the last immunization. Antibody production was performed by Covance Research Products.
Total IgG was extracted from mouse ascites fluid or rabbit serum using the EZ-Sep kit (Pharmacia Biotech, Piscataway, NJ) and then purified through HiTrap-Q columns (Pharmacia Biotech). Purity of the isolated IgG was confirmed by PAGE and subsequent staining with Coomassie Blue. Purified IgG was concentrated on CentriPlus spin filters (Amicon Inc., Beverly, MA), biotinylated by incubation with N-hydroxysucinimidobiotin (EZ-Link NHS-Biotin; Pierce, Rockford, IL) in 0.1 M NaCO3 for 4 h at room temperature, and dialyzed extensively against DPBS. Biotinylated IgG was incubated with 2% acetone extract of mouse testis (mouse testis powder) for > 4 h at 4°C to further reduce binding to mouse testis cells before use in immunohistochemistry.
Whole-Mount Immunohistochemistry
For detection of rabbit or dog testis cells in the seminiferous tubules of recipient mouse testes, pieces of dispersed testes representing about 1/4 to 1/3 of a recipient testis were processed for whole-mount immunohistochemistry [22]. Seminiferous tubules were incubated sequentially in 0.1 M glycine in DPBS, 0.3% H2O2 in DPBS, PBSMT (DPBS with 2% skim milk powder and 0.1% Triton X-100), and DPBS containing 5% goat serum, 1% BSA, and 0.1% Triton X-100. Endogenous biotin was blocked using a biotin-avidin blocking kit (Zymed Laboratories, Inc., San Francisco, CA), and samples were incubated with biotinylated, species-specific IgG diluted to 4 µg/ml in 5% BSA in DPBS overnight at 4°C. On the next day, samples were washed twice in PBSMT for 30 min at 4°C and once at room temperature, then washed twice in DPBS/goat serum/BSA/triton for 30 min at room temperature and incubated over night at 4°C with Z-avidin coupled to horse radish peroxidase (Zymed) diluted 1:1000 in 5% BSA in DPBS. On the following day, samples were washed through 5 changes of DPBS with 0.2% BSA and 0.1% Triton X-100 and stained with 3-amino-9-ethylcarbazole (AEC, Vector Laboratories, Burlingame, CA). Stained samples were washed with PBS and fixed in 10% neutral buffered formalin. Seminiferous tubules were spread on microscope slides, mounted under a coverslip with a small amount of DPBS and examined microscopically at x60–400 magnification. Images were captured into a PC-based image analysis system [23]. Positive control samples consisted of seminiferous tubules that had received injections of donor testis cells one day before tissue fixation, when donor testis cells are abundant and easily identified in the tubule lumen. Negative control samples were seminiferous tubules from busulfan-treated mice that had not received testis cells, and experimental and control samples incubated with biotinylated normal mouse or rabbit IgG instead of specific antibody.
PCR for Canine-specific -Satellite DNA
To detect the presence of canine cells in recipient mouse testes, genomic DNA extracted from recipient testes at different time points after transplantation of dog testis cells was amplified by PCR using primers specific for canine -satellite DNA [24]. Immediately after collection, recipient testes were split; one half was fixed for immunohistochemistry and one half was used for DNA extraction using the QIAmp tissue kit (Qiagen Inc., Santa Clarita, CA). Genomic DNA from a noninjected mouse testis was prepared as a negative control sample in each extraction. Genomic canine DNA served as positive control. One microgram of DNA was amplified with 20 pmol primers for 30 cycles (94°C, 1 min; 64°C 1 min; 72°C 1 min). PCR products (324 basepairs [bp]) were visualized on ethidium bromide-stained 2% agarose gels. To demonstrate successful DNA extraction, all samples from noninjected mouse testes and a subset of positive and negative experimental samples were subjected to PCR with primers specific for the mouse Sry gene [25]. To determine the sensitivity of the assay, canine testis cells were diluted in mouse testis cells from 5 x 105 canine cells to 10 canine cells in 12.5 x 106 mouse cells, and DNA was extracted and amplified with the canine-specific primers as described above.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unequivocal detection of transplanted donor cells in recipient testes was of major importance for the development of inter-species spermatogonial transplantations in which donor animals carrying a marker gene are not available. We chose to approach this problem by generating species-specific antibodies to donor testis cells, which would enable us to detect all types of donor germ cells in the recipient mouse testes.
The antibodies generated against rabbit and dog testis cells specifically recognized transplanted cells in the seminiferous tubules of recipient mice and did not cross-react with mouse testis cells. The staining pattern of noninjected, untreated, or busulfan-treated recipient mouse seminiferous tubules (negative controls), and of rabbit cells in mouse seminiferous tubules one day after transplantation (positive control) are illustrated in Figure 1. The staining pattern of dog cells in mouse testes with the antibody against dog testis cells raised in the rabbit as host was similar (not shown); however, background staining was somewhat higher than with the antibody against rabbit testis cells raised in mouse hosts. Availability of these species-specific antibodies provides the needed tool to study colonization of recipient mouse testes by transplanted rabbit or dog testis cells.
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Transplantation of Rabbit Cells Into Mouse Testes
Once a detection system was available, testis cells from donor rabbits were transplanted into immunodeficient recipient mouse testes to investigate whether they could colonize the mouse testis, and to describe the colonization pattern over a 12-mo observation period. In addition, both freshly collected and cryopreserved rabbit testis cells were used for transplantation to study whether cryopreservation of rabbit testis cells before transplantation would affect colonization of recipient testes. Recipient mouse testes were analyzed from 1 mo to > 12 mo (473 days) after transplantation. Using the species-specific antibody against rabbit testis cells, we could demonstrate the presence of rabbit cells in the seminiferous tubules of all recipient mice (Table 1, Fig. 2).
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Rabbit germ cells proliferated to form chains of cells connected by intercellular bridges or formed more elaborate mesh structures (Fig. 2, A–E). This pattern of colonization is similar to the early stages of spermatogonial colonization described by Nagano et al. [26] when transgenic mouse donor cells were transplanted to mouse recipients (Fig. 3). However, this pattern of colonization did not change noticeably over the 12-mo observation period. Therefore, our observations indicate that rabbit spermatogenesis was arrested during horizontal expansion and did not differentiate into the adluminal compartment of the recipient mouse tubule. Similar results were obtained after transplantation of cryopreserved rabbit testis cells (Fig. 2F). To examine whether maintenance in culture affected the colonization potential of rabbit testis cells, we harvested cells from the testes of a 5-mo-old rabbit and placed them on STO feeder cells using techniques similar to those used for mouse embryonic stem cells or testis cells [15]. After 1 mo in culture, these cells were injected into recipient mouse seminiferous tubules. Beginning 1 mo after transplantation, the recipient testes were analyzed, and in every case rabbit cells could be identified (Table 1). However, the pattern of colonization was distinctly different from that found after transplantation of fresh or cryopreserved cells. No pairs, chains, or mesh structures were formed as seen after transplantation of mouse cells or fresh or cryopreserved rabbit cells (see Fig. 2, A–F; Fig. 3). The cultured rabbit cells remaining in recipient seminiferous tubules after transplantation were more elongated or of stellate appearance (Fig. 2, G and H). Thus, rabbit cells surviving after 1 mo in culture did not appear to be spermatogonial stem cells but rather somatic cell types. In contrast, transplantation of fresh or cryopreserved rabbit testis cells resulted in colonization of the recipient mouse seminiferous tubules by rabbit cells that exhibited the morphological appearance of proliferating spermatogonia.
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Transplantation of Dog Cells Into Mouse Testes
In companion experiments, fresh, cryopreserved, or cultured testis cells from dogs were transplanted into the seminiferous tubules of recipient mice to compare colonization of mouse testes by germ cells from a different, phylogenetically distant donor species. We could show the presence of dog cells in recipient mouse testes by immunohistochemistry using a dog-specific polyclonal antibody (Table 2, Fig. 4). Dog cells were present in pairs, short chains, and small mesh structures in the recipient seminiferous tubules after transplantation of fresh and cryopreserved testis cells at all time points analyzed; however, colonization appeared less extensive than that following transplantation of rabbit cells. After transplantation of cultured cells, too few dog cells could be detected to describe a distinct colonization pattern.
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To confirm the specificity of the immunohistochemical detection method by a second technique, DNA extracted from recipient mouse testes was analyzed in parallel by PCR using species-specific primers directed against canine-specific -satellite DNA [24]. Dilution series of dog testis cells in mouse testis cells followed by DNA extraction and PCR established that one dog cell was detected in 106 mouse cells under the conditions employed in the present study (Fig. 5A). No amplification was detected in DNA from control mouse testis. However, this result was not caused by low quality of DNA preparation, because the mouse Sry gene was successfully amplified in all samples (not shown). Results for amplification of DNA extracted from recipient mouse testes at different time points after transplantation of fresh, frozen, or cultured testis cells are illustrated in Figure 5B. The results of PCR and immunohistochemistry were in agreement in all testes analyzed (Table 3). These experiments showed that transplantation of testis cells from another, phylogenetically distant donor species to mouse testes resulted in essentially the same colonization pattern as observed for rabbit germ cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the species-specific antibodies in whole-mount immunohistochemistry of recipient mouse seminiferous tubules, we could demonstrate early stages of spermatogonial expansion as identified by cell morphology and spatial arrangement. To corroborate our histochemical findings in the case of dog testis cell transplantations, we took advantage of primers specific for amplification of canine -satellite DNA [24]. These primers do not cross-hybridize with mouse DNA and could therefore be used to detect the presence of dog cells in mouse tissue with high specificity and sensitivity. Results of PCR for dog DNA were in agreement with immunohistochemistry for dog testis cells in all samples analyzed, thereby validating the immunohistochemical detection method.
Our results show that germ cells from rabbits can colonize the seminiferous tubules of immunodeficient recipient mice and will be maintained for periods exceeding 1 yr. Transplanted rabbit germ cells undergo early spermatogonial expansion as judged by their close morphological resemblance to mouse spermatogonia [29], proliferating in recipient testes during the first month after spermatogonial transplantation [26]. However, no further differentiation of donor testis cells in recipient mouse testes was observed. This colonization pattern did not change significantly over the 12-mo observation period.
Our observations after the transplantation of dog testis cells were similar to those seen using rabbits as donor animals, although the efficiency appeared lower in dog compared to rabbit germ cells. From the present experiments it is not possible to determine why transplantation of dog germ cells resulted in lower levels of colonization than transplantation of rabbit germ cells. With the exception of the experiment involving culture of testis cells, all donor dogs were sexually mature, whereas the donor rabbits did not yet have fully developed spermatogenesis. It has been estimated that there are about 2 x 104 stem cells in 108 cells of a mouse testis [5, 33]. Assuming similar relationships for testes of other species, it can be argued that testis cells recovered from mature animals will contain proportionally fewer stem cells than cells isolated from testes in which spermatogenesis is not yet complete. Therefore, using sexually immature donor animals might increase colonization efficiency.
Results were comparable after the transplantation of frozen-thawed and freshly collected testis cells. This finding confirms our previous observations that cryopreserved testis cells are capable of colonizing the recipient mouse testis [8, 14]. Confirming this phenomenon in such diverse species as rabbits and dogs provides strong evidence that spermatogonial stem cells of many mammalian species can be preserved for long periods. Cryopreservation protocols for mature spermatozoa have to be empirically determined for each species, whereas cryopreservation of spermatogonial stem cells follows a simple procedure established for cultured somatic cells, and the same protocol appears to be similarly applicable to all the species examined [8, 14]. This finding is immediately applicable in that the stem cells of valuable or unique males can now be frozen for later use after techniques of transplantation and culture are improved.
Donor cells could be documented in recipient mouse testes up to 6 mo after transplantation of testis cells that had been maintained in culture for 1 mo before transplantation. However, only a few cells were observed, and their morphologic appearance was different from that of those found after the transplantation of fresh or cryopreserved donor cells. The majority of cells did not show the round shape or chain and mesh formation characteristic of spermatogenic cells [26, 29] but was more reminiscent of testicular somatic cells. Donor testis cells were cultured on mitotically arrested STO feeders secreting murine leukemia inhibiting factor (LIF), a system previously shown to support long-term culture of mouse spermatogonial stem cells before transplantation [15]. It appears, therefore, that this culture system does not provide adequate support to maintain rabbit and dog germ cells in culture in sufficient numbers to allow for subsequent colonization of recipient testes. Recently it was reported that short-term survival of isolated mouse, rat, and pig type A spermatogonia in culture was improved in the presence of stem cell factor and granulocyte-macrophage-colony stimulating factor whereas LIF was not effective [34]. Appropriate long-term culture systems for germ cells from domestic animals will have to be determined empirically. Availability of these culture systems will be of utmost importance for the genetic modification of male germ cells in vitro.
The different results of spermatogonial transplantation from rabbits and dogs to mouse recipients compared to transplantation of rodent germ cells supports the hypothesis that increased phylogenetic distance between donor and recipient leads to incompatibilities between transplanted germ cells and Sertoli cells of the recipient or the microenvironment in the recipient seminiferous tubule. Hamster spermatogenesis in mouse testes was less efficient, and more defective spermatozoa were observed compared to rat spermatogenesis in the mouse [12, 14]. Although classification is still controversial, rabbits and rodents as well as carnivora and rodents were separated at least as early as the late paleocene epoch (approximately 60 million yr ago [35]), whereas rats and mice diverged 10–11 million yr ago, and the mouse-hamster separation was approximately 16 million yr ago [36]. It appears therefore, that the extent of xenogeneic spermatogenesis decreases with increasing phylogenetic distance between donor and recipient. Compatibility of cell surface molecule configurations [37] and the presence of growth factors, cytokines, or other paracrine signals in the recipient testis are likely to be important in determining whether donor stem cells can survive and undergo differentiation.
Rat spermatogenesis in mouse testis is supported by mouse Sertoli cells [12], and in the present study there was no indication that donor Sertoli cells were present in the recipient seminiferous tubules. Transplanted donor cell populations contain Sertoli cells, but these cells have stopped dividing [38] and have little opportunity to colonize the mouse basement membrane already occupied by endogenous Sertoli cells. Furthermore, the round germ cells of rabbit and dog in recipient seminiferous tubules were found in association with nonstained (mouse) somatic cells on the basement membrane. Thus, the mouse seminiferous tubule provides a suitable environment for germ cells from distant species, such as rabbit and dog, to interact with supporting cells and associate with the basement membrane. Proliferation of these undifferentiated germ cells occurs for more than 1 yr in both rabbit and dog, with characteristic intercellular bridges. Therefore, the important first steps of xenogeneic colonization can occur across a wide phylogenetic gap. The conditions necessary to support differentiation steps of xenogeneic spermatogenesis now must be determined.
Interspecies spermatogonial transplantation provides a unique system for studying the cellular and molecular events that regulate the sequential steps of spermatogenesis. Increasing knowledge of the factors controlling spermatogonial proliferation and differentiation will aid in understanding disturbances of spermatogenesis and will enable us to manipulate the microenvironment of recipient seminiferous tubules to support spermatogenesis of phylogenetically distant donor species. In combination with cryopreservation of germ cells and improved culture systems, spermatogonial transplantation then can be used to preserve valuable genetic material and could ultimately serve as an approach to manipulate the male germ line in domestic animals.
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ACKNOWLEDGMENTS |
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-satellite DNA, and Dr. P. Dodson for advice on species divergence. In addition, we are grateful to C. Freeman and R. Naroznowski for assistance with animal maintenance and experimentation, and to J. Hayden for photography. |
FOOTNOTES |
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2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104-6009. FAX: 215 898 0667.
3 Current address: Center for Animal Transgenesis and Germ Cell Research, Dept. of Clinical Studies, New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, 382 W. Steet Rd., Kennett Square, PA 19348.
Accepted: June 22, 1999.
Received: May 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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