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Testis |
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous studies, we demonstrated that donor spermatogonial stem cell engraftment is enhanced when endogenous germ cells are absent because of genetic mutation or have been removed by ablative strategies. The best mouse recipient model identified to date is the dominant white spotting (W) homozygous mutant male, which is congenitally infertile and lacks endogenous germ cells because of a mutation in the c-kit receptor tyrosine kinase [18, 20, 22, 23]. Wild-type donor germ cells transplanted into adult W mouse recipients generate complete spermatogenesis and fertility [18]. Using the W mutant model, we have demonstrated that recipient age has a dramatic impact on donor cell engraftment; pup recipients support nearly 40-fold greater donor-derived spermatogenesis than do adult recipients from the same number of transplanted stem cells [20]. We recently took advantage of the favorable recipient environment in W mouse pups to introduce genetic modifications into the germline by transplanting mouse spermatogonial stem cells after transduction with a viral vector [21, 24]. Complete spermatogenesis and fertility were established from donor stem cells, and a lacZ transgene was transmitted to 4.5% of progeny, thus constituting a new transgenic technology [21]. Juvenile spermatogonial depletion (jsd) mutant mice are also infertile because of a germ cell defect, although they can support spermatogenesis from transplanted testis cells [25, 26]. Generation of fertility in these recipients has not been reported, and direct comparisons between W and jsd recipients have not been performed. Although germ cell-deficient mutant mice provide valuable recipient models, commercially available W and jsd animals (Jackson Laboratory, Bar Harbor, ME) are expensive, and complex breeding strategies are required to generate homozygous mutants. Furthermore, germ cell-deficient mutants are not available to serve as transplantation recipients in other species.
Although donor spermatogonial stem cells can colonize normal recipient testes, the efficiency of donor engraftment is increased following ablative treatments that remove endogenous stem cells and increase niche accessibility [27–30]. Local radiation treatment is one method for removing endogenous germ cells, and this approach has been used to prepare adult mouse [31, 32] and monkey [13] recipients for testis cell transplantation. Although high levels of donor colonization have been reported using this technique [32], acquisition of fertility and passage of the donor genotype to progeny have not yet been demonstrated. Furthermore, the radiation source and housing are prohibitively expensive and not universally available.
An alternative method for removing endogenous germ cells from the testes of wild-type animals and creating space for donor stem cell engraftment is treatment with a sublethal dose of the chemotherapeutic agent busulfan [1, 2]. Busulfan-treated adult mice are prepared with little difficulty and provide reliable recipients that allow quantitative assessment of stem cell activity in different donor testis cell populations [3]. However, restoration of fertility in busulfan-treated adult mice is inefficient (unpublished observations), perhaps because of damage to the testicular environment caused by the ablative therapy. We reported previously that rat-to-rat testis cell transplantations are possible using busulfan-treated recipients, and like the mouse, rat pup recipients appeared to support higher levels of donor-derived spermatogenesis than did adults [5]. However, the chemotherapeutic preparation of adult and pup rat recipients is plagued by problems of high sensitivity to the toxic effects of busulfan and incomplete removal of endogenous spermatogenesis [5]. Therefore, busulfan treatment regimens will have to be developed on a species by species basis.
An intriguing variation of the busulfan treatment protocol was described nearly four decades ago [33]. Treatment of pregnant female rats with a single dose of busulfan between 13 and 18 days of gestation resulted in the live birth of male progeny that were permanently infertile [33]. The current study was designed to 1) determine whether infertile male mice can be prepared using a similar strategy, 2) evaluate the potential for mouse pups that were treated with busulfan as fetuses (fetal busulfan-treated pups) to function as transplantation recipients and to support spermatogenesis from donor germ cells, and 3) ascertain whether fetal busulfan-treated mouse pup recipients become fertile and pass the donor haplotype to progeny. Development of new recipient preparation protocols in mice is justified because the transplantation technique is well established in this species. This work will lay the foundation for developing similar strategies in other species for homologous or autologous testis cell transplantations to restore fertility and/or modify the germline.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Donor testis cells were obtained from cryptorchid adult mice expressing the LacZ transgene or the green fluorescent protein (GFP) reporter transgene (GFP) in several tissues, including cells of the male germline. For quantitative and histological studies designed to characterize donor cell colonization, cells were from ROSA26 mice (designated B6; 129S-Gtrosa26; Jackson Laboratory) that express the Escherichia coli lacZ transgene. For fertility studies, transgenic donor mice were used that express a GFP reporter gene under the control of the chicken ß-actin promoter and cytomegalovirus immediate early enhancer (CB-GFP). Two CB-GFP lines, both originally derived by Okabe et al. [34], were obtained from Eisen and Sugawa (Massachusetts Institute of Technology, Cambridge, MA) and the Jackson Laboratory (C57BL/6-TgN(ACTbEGFP)1Osb). Cryptorchid testes, which were produced as previously described [35], lack all differentiated germ cells and are enriched 25-fold for spermatogonial stem cells by the time of donor cell collection, 2–3 mo after the cryptorchid operation. Single-cell suspensions from ROSA26 and CB-GFP cryptorchid adult donor testes were produced by enzymatic digestion [1, 36, 37]. Isolated cells were suspended at concentrations of 5–60 x 106 cells/ml in Dulbecco modified Eagle medium containing 10% fetal bovine serum. Lower donor cell concentrations were used to prevent merging of donor-derived colonies and to facilitate quantitative studies. Higher donor cell concentrations were used to generate fertility in recipient animals. Transplanted donor testis cells that express the lacZ or GFP transgenes can be readily identified in recipient testes by staining with the ß-galactosidase substrate 5-bromo-4-chloro-3-indolyl ß-D galactoside (X-gal) or using an epifluorescent microscope, respectively.
Recipient Preparation and Transplantation Procedure
To generate 129 x C57 (F1) fetal busulfan-treated mouse pup recipients for spermatogonial transplantation, C57BL/6 female mice were mated to 129/SvCP males and subsequently treated with busulfan (40 mg/kg i.p.) on Days 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, or 15.5 postcoitum; male germ cells are actively proliferating throughout this time period [38]. Day of vaginal plug was considered 0.5 days postcoitum (dpc). Two control recipient preparation protocols were also evaluated for comparison: untreated 129 x C57 mouse pups that have normal endogenous spermatogenesis and W mutant [22] (Wv/W or W54/Wv; Jackson Laboratory) mouse pups that lack endogenous spermatogenesis because of a mutation in the c-kit receptor tyrosine kinase [23]. Untreated wild-type and W mutant control mice were transplanted and evaluated at the same ages as fetal busulfan-treated mice (9.5–15.5 dpc). Transplantations into untreated, fetal busulfan-treated and W mutant mouse recipients were performed at 5–12 days postpartum (dpp). These pup recipients were placed on ice to cause hypothermia-induced anesthesia [39], and approximately 2 µl of donor cell suspension were introduced per testis by efferent duct injection [37]. Animals that were clearly unhealthy on the day of the experiment were not used. The Institutional Animal Care and Use Committee of the University of Pennsylvania approved all experimental procedures in accordance with the Guide for Care and Use of Laboratory Animals from the National Academy of Sciences (assurance no. A3079-0).
Analysis of Recipients for Colonization and Fertility
For quantitative analysis of donor testis cell colonization, recipient testes were collected 2 mo after transplantation and stained with X-gal to visualize donor-derived colonies of spermatogenesis [40]. A donor spermatogonial stem cell is defined by its ability to produce and maintain blue colonies of spermatogenesis in recipient seminiferous tubules. Each colony of donor spermatogenesis is believed to arise from clonogenic proliferation and differentiation of a single stem cell [40, 41]. Other types of testis cells do not generate colonies of spermatogenesis, and endogenous stem cells do not express the lacZ transgene.
To examine the fertility status of the three different recipient animal models (fetal busulfan-treated pups, untreated pups, and W pups), mice transplanted with CB-GFP donor testis cells were mated with nontransgenic C57BL/6 x SJL female mice. At least 100 progeny were produced from each fertile recipient mouse and were analyzed for GFP expression (green fluorescence) using a hand-held long-wave ultraviolet light (365 nm). All remaining breeding males were killed 8 mo after transplantation, and testis weights were recorded.
Statistics
ANOVA was used to test for differences among groups in litter size, gonocytes/100 Sertoli cells, testis weight, and days to first fertility. Dunnett t-tests were calculated for all treatment versus control pairwise comparisons. When all possible pairwise comparisons were of interest, including treatment versus treatment, Tukey studentized range tests were used. Student t-tests were used to compare fertile versus infertile testis weights and mean colonies/105 cells between treated and control recipients. Short-term and posttransplantation survival was compared between busulfan-treated and control groups using logistic regression. The overall Wald chi-square test was used to examine differences among groups, and Bonferroni adjustments were made to the P values to correct for multiple comparisons. Pearson correlation coefficients were calculated to examine the linear association between busulfan treatment day and number of gonocytes, testis weight, colonies/105 cells, days from transplant to first fertility, and percentage of GFP progeny.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, pregnant female mice were treated with busulfan at different times during gestation to determine whether their male progeny would provide good spermatogonial transplantation recipients. In the initial experiments, pregnant females were treated with the same dose of busulfan (55 mg/kg i.p.) that is routinely used in our laboratory to prepare adult males of the same strain (129 x C57) for spermatogonial transplantation. However, a high rate of pregnancy failure was observed with this treatment; therefore, a lower dose of busulfan (40 mg/kg i.p.) was employed in these studies. Following busulfan treatment, pregnant females delivered litters of normal size (6.2 ± 0.2 pups/litter) compared with untreated controls (7.4 ± 0.5 pups/litter); treatment day had no effect on litter size (P = 0.17). In contrast, the short-term (1 wk) survival rates were significantly reduced for all fetal busulfan treatment groups, except for 15.5 dpc, compared with untreated controls (P < 0.001). Short-term survival rates were 58% (9.5 dpc), 47% (10.5 dpc), 30% (11.5 dpc), 38% (12.5 dpc), 44% (13.5 dpc), 60% (14.5 dpc), 81% (15.5 dpc), and 94% (untreated control). Although the later busulfan treatment groups (i.e., 14.5 and 15.5 dpc) had the highest short-term survival, long-term survival following germ cell transplantation was significantly reduced (P < 0.03) in 13.5 (37%), 14.5 (27%), and 15.5 (27%) dpc fetal busulfan-treated recipients compared with untreated (88%) or W (89%) controls. Posttransplantation survival was high in the 9.5 (70%), 10.5 (71%), 11.5 (83%), and 12.5 (82%) dpc fetal busulfan-treated recipients, similar to untreated and W controls (P > 0.05).
Morphological Analyses of Mouse Testes Following Fetal Busulfan Treatment
Histological examinations of the testes of control or fetal busulfan-treated male progeny were performed 1–2 dpp (Fig. 1 and Table 1). Busulfan treatment of pregnant females on 9.5, 10.5, 11.5, or 12.5 dpc resulted in a significant decrease (P < 0.05) in the number of gonocytes (spermatogonial stem cell precursors) observed per 100 Sertoli cells in the seminiferous tubules of male progeny compared with untreated controls (Table 1, column 2 and compare Fig. 1A with Fig. 1, B and C). In contrast, busulfan treatment on 13.5, 14.5, or 15.5 dpc did not affect the number of gonocytes in male progeny at birth (Table 1 and compare Fig. 1, A and D) (P > 0.05).
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Fertility Assessment in Fetal Busulfan-Treated Mice
The fertility status of fetal busulfan-treated male mice was determined by mating them with wild-type females. The results shown in Table 1 (column 3) indicate that males from all busulfan treatment groups except 12.5 dpc became fertile. Acquisition of fertility in 9.5 (57 dpp), 10.5 (82 dpp), 11.5 (73 dpp) or 15.5 (100 dpp) dpc fetal busulfan-treated males occurred in a time course similar to that for untreated controls (68 dpp, P > 0.05). In contrast, fertility was significantly delayed in 13.5 (172 dpp) and 14.5 (156 dpp) dpc fetal busulfan-treated males (Table 1) (P < 0.05). Although many of the busulfan-treated males became fertile, testis weights for all fetal busulfan-treated males were significantly less than those for untreated controls (Table 1) (P < 0.01). In addition, mean testis weights in fertile fetal busulfan-treated males (43.1 mg) were significantly greater than those in infertile males (8.7 ± 0.8 mg; Table 1, compare mean testis weights for busulfan-treated animals in columns 4 and 5) (P < 0.001). Therefore, regardless of treatment, testis weight is a good predictor of fertility status. In contrast, no correlation was observed between the number of gonocytes in the testes of newborn animals and the size (r = 0.13, P > 0.75) or fertility status (r = 0.23, P > 0.58) of adult testes. Among four 12.5 dpc fetal busulfan-treated male progeny, none became fertile, and all eight testes analyzed were small (9.6 ± 1.2 mg; Table 1).
Evaluation of Recipient Mouse Testis Environments by Germ Cell Transplantation
Spermatogonial transplantation was used to evaluate and compare the recipient mouse testis environments in fetal busulfan-treated pups, untreated pups, busulfan-treated adults, and untreated adults (Fig. 2; adult data are from a previous study [30] and are included to provide comparisons). Our previous studies using W mutant mice demonstrated that the pup recipient environment supports higher levels of donor-derived spermatogenesis than does the adult recipient environment [20]. Therefore, fetal busulfan-treated male progeny received transplants as pups between 5 and 12 dpp. Although the results in Table 1 indicate that fetal busulfan treatment failed to completely remove endogenous spermatogenesis, the data in Figure 2 indicate that fetal busulfan-treated pups supported higher levels (
13.5-fold) of donor germ cell engraftment than did untreated control pups (mean, 75 colonies/105 cells vs. 5.5 colonies/105 cells; P < 0.001), demonstrating the benefit of the chemoablative therapy. In addition, there was a significant trend by busulfan treatment day (9.5–15.5 dpc) for increasing donor colonization (r = 0.36, P = 0.005). All fetal busulfan-treated recipients supported levels of donor-derived spermatogenesis that were at least as high as those in busulfan-treated adults (Fig. 2) (P > 0.3). Although donor-derived spermatogenesis was observed even in untreated recipients, busulfan treatment to remove endogenous germ cells improved donor cell engraftment in pup and adult recipients (Fig. 2) [30]. Macroscopic and histological examination of recipient testes 2 mo after transplantation revealed important differences in testis weights and donor versus endogenous spermatogenesis. Testis weights were reduced in all fetal busulfan-treated recipient testes compared with untreated control recipients (Fig. 2) (P < 0.01) at 2 mo after transplantation, and there was a significant trend by busulfan treatment day for decreasing testis weight (Fig. 2) (r = 0.39, P = 0.002). Histological evaluation suggested that the higher testis weights from controls and recipients treated earlier in gestation (e.g., 9.5 dpc) could be attributed to higher levels of endogenous (not blue) and overall (endogenous + donor derived) spermatogenesis (Fig. 3, E and F). In contrast, endogenous spermatogenesis was limited in fetal busulfan recipients treated later in gestation (e.g., 12.5 and 15.5 dpc), and these recipients had a greater proportion of donor-derived spermatogenesis (Fig. 3, G and H). Therefore, the level of donor colonization is inversely related to testis weight and the level of endogenous spermatogenesis.
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Fertility Analysis and Generation of Transgenic Progeny Exhibiting Donor Haplotype
Donor testis cells from CB-GFP transgenic mice were transplanted into mouse pup recipient testes to evaluate reproductive performance. All mice that received donor testis cells as pups, including untreated and W mutant controls, became fertile between 2 and 3 mo after transplantation. W mutant recipient mice have no endogenous spermatogenesis; therefore, these mice passed the donor haplotype to all progeny. Because the donor animals were hemizygous for the GFP transgene, 50% of progeny from W recipients were expected to carry and express GFP, and our results were consistent with this expectation (52%, Table 2). In contrast, none of the untreated control mouse pup recipients produced green progeny. Although all fetal busulfan-treated recipients became fertile, only 10.5, 12.5, 14.5, and 15.5 dpc recipients produced GFP progeny. No correlation was observed between timing of busulfan treatment and days from transplant to first progeny (r = -0.25, P = 0.18) or percentage of green progeny (r = 0.23, P = 0.22). Nonetheless, the 12.5 dpc fetal busulfan-treated mouse pups were the best recipients; seven of seven became fertile, six passed the donor haplotype to progeny, and a total of 93 of 674 pups (13.8%) expressed the GFP transgene (Table 2). Based on these results, approximately 25% of progeny from 12.5 dpc fetal busulfan-treated recipients were derived from donor germ cells (13.8%/50%). Transgenic and nontransgenic littermates are shown in Figure 4A. Compared with 12.5 dpc fetal busulfan recipients, fewer 10.5 dpc (two of six), 14.5 dpc (one of four), and 15.5 dpc (one of five) recipients produced GFP progeny; the GFP transgene was expressed in 0.4%, 0.7%, and 8% of progeny, respectively (Table 2). Green fluorescent progeny from fetal busulfan-treated recipients appeared to develop normally and passed the GFP transgene to 47.5% (19/40) of F2 progeny (an F1 GFP adult male and its F2 pups are shown in Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The highly organized spermatogenic process is well conserved in animals [44]. Despite 10–15 million years of evolutionary separation, rat [45] and hamster [7] testis cells can generate complete spermatogenesis when transplanted into mouse seminiferous tubules. Furthermore, testis cells from baboons [14] and humans [15], which diverged from mice approximately 100 million years ago, can migrate to the basement membrane of mouse seminiferous tubules to generate and maintain (for at least several months) colonies of spermatogonia (reviewed in [3]). These observations demonstrate an incredible conservation of factors needed for early spermatogenic stages and suggest that methods for spermatogonial transplantation developed in mice will be applicable in other species.
Our previous results demonstrated that the efficiency of donor-derived spermatogenesis following testis cell transplantation is critically dependent on recipient preparation. Two conditions that have proven favorable for engraftment of donor germ cells and generation of spermatogenesis in recipients are 1) removal of endogenous germ cells by cytoablative methods (e.g., busulfan or radiation treatment [30]) or genetic methods [18, 20, 21] and 2) use of preadolescent instead of adult recipients [20]. The congenitally infertile W mutant mouse provides an excellent transplantation recipient, and when these animals receive transplants as pups (8–12 dpp), fertility from donor germ cells is established in a time course (3 mo) similar to that observed in wild-type males [20]. Because infertile genetic models are not available for most other species, it is necessary to establish alternative strategies. In contrast to the W mouse pup recipient, fertility is rarely restored following transplantation into busulfan-treated adult mice [1] (unpublished observations) even though this treatment effectively removes competing endogenous germ cells [1]. In other species, chemotherapeutic treatment of prepuberal [5] (C.L. Hausler, personal communication) or adult [5] animals at levels sufficient to completely remove endogenous spermatogenesis is toxic. Treatment of pregnant females (i.e., fetal busulfan treatment) constitutes an alternative strategy, which in rats results in the live birth of male progeny that are permanently infertile [33] and could presumably receive transplants as pups. The current results demonstrate that the situation is somewhat more complicated in mice, but fetal busulfan-treated mouse recipients were generated, received transplants as pups, supported high levels of donor spermatogenesis, became fertile, and transmitted the donor haplotype.
Experiments were designed to systematically identify an optimum fetal busulfan treatment protocol for generating infertile mouse pup recipients. These studies identified several factors that impacted recipient effectiveness, including short- and long-term recipient survival, levels of endogenous versus donor spermatogenesis, and local environmental conditions; these factors are likely to be relevant in other species. For example, our results demonstrated that 1-wk survival rates were significantly reduced for all fetal busulfan-treated pups (except 15.5 dpc) compared with untreated controls. In contrast, treatment day had a dramatic impact on posttransplantation survival, which was high (mean, 76%) in the 9.5- to 12.5-dpc fetal busulfan treatment groups (similar to untreated wild-type and W mutant controls, 88%) but was significantly decreased for the 13.5- to 15.5-dpc fetal busulfan treatment groups. Perhaps these recipients (13.5- to 15.5-dpc fetal busulfan) failed to recover from the systemic toxic effects of busulfan before the transplantation procedure, which was performed between 5 and 12 dpp.
Busulfan treatment of adult mice results in nearly complete removal of endogenous spermatogenesis, a condition known to facilitate colonization by donor germ cells [30]. Although fetal busulfan treatment caused a significant decrease in the number of gonocytes present in the testes of newborn males in the 9.5- to 12.5-dpc treatment groups, gonocyte numbers at the time of birth did not presage the level of endogenous spermatogenesis or acquisition of fertility. Males from the 9.5- to 11.5-dpc fetal busulfan treatment groups became fertile in a time course similar to that for untreated controls and significantly sooner than did males from the 13.5- and 14.5-dpc fetal busulfan treatment groups. Although the 13.5- to 15.5-dpc fetal busulfan treatment groups had normal numbers of gonocytes at the time of birth, many of these gonocytes probably suffered from occult damage, because acquisition of fertility in these animals was significantly delayed. Only the 12.5-dpc fetal busulfan treatment group failed to become fertile from endogenous germ cells, although histological examination of these animals following transplantation showed the presence of some endogenous spermatogenesis. These results reveal an important difference between the mouse and rat models; rats treated with busulfan any time between 13 and 18 dpc are nearly devoid of endogenous spermatogenesis and fail to become fertile [33]. However, fetal busulfan-treated mouse pups were significantly better spermatogonial transplantation recipients than were untreated control pups and were at least as effective as busulfan-treated adult mice. Most importantly, 10.5-, 12.5-, 14.5-, and 15.5-dpc fetal busulfan-treated mice became fertile and passed the donor genotype/phenotype to progeny, which rarely occurs using busulfan-treated adult mice as recipients (unpublished observations). Therefore, despite the fact that fetal busulfan treatment failed to remove endogenous spermatogenesis, this treatment may have delayed the initiation of the endogenous processes sufficiently to allow donor germ cells to compete for occupation of the rapidly expanding number of niches in the developing mouse testis.
Transplantation studies in both the hematopoietic and spermatogenic systems have demonstrated that donor stem cell engraftment is improved following ablative therapies to remove endogenous cells [27, 29, 30]. However, accumulating evidence in both systems suggests that ablative therapies can cause long-term detrimental effects and alter the biology of the recipient environment [46–48]. The homeostatic maintenance of fertile testes involves a well-orchestrated balance between the spermatogenic and endocrine compartments. Treatments, such as busulfan, that remove germ cells disrupt this balance and create an environment that is detrimental to germ cells [48] and somatic cells [49] and could hinder reinitiation of spermatogenesis in recipient testes. Two lines of evidence support this premise. First, busulfan treatment (40–50 mg/kg) of adult mice results in nearly complete removal of endogenous germ cells, creating space that allows high levels of donor cell engraftment [1, 40]. However, these animals rarely become fertile [1] (unpublished observations). In contrast, fertility was established and the donor genotype passed to progeny when recipients were treated with a lower dose of busulfan (30 mg/kg), which did not completely remove endogenous germ cells [1]. Second, busulfan treatment fails to completely remove endogenous germ cells from rat pup testes, but high levels of donor spermatogenesis can be generated in this environment [5]. Therefore, recipient preparation methods that remove most but not all endogenous spermatogenesis may be desirable. It is unclear why the W mouse pup, which is completely devoid of endogenous spermatogenesis, is the best recipient model. A likely explanation is that donor cells were introduced before deleterious effects were manifested in the recipient environment, and donor cell-derived spermatogenesis developed as the testes matured.
All fetal busulfan-treated pups were at least as effective as busulfan-treated adults at supporting donor spermatogenesis, but 12.5-dpc fetal busulfan-treated pups clearly were the best transplantation recipients, passing the donor phenotype to >25% of progeny (13.8% x 2, donor cells are hemizygous for the GFP transgene). The success of this recipient compared with that of other fetal or adult busulfan-treated recipients can be attributed to several factors. First, based on testis weight and fertility status (Table 1), 12.5-dpc fetal busulfan recipients appeared to have the lowest level of endogenous spermatogenesis to compete with donor stem cells for available niches. Second, residual endogenous spermatogenesis and other local environmental factors may contribute to a healthier testicular environment than is found following busulfan or radiation treatment of adult mice. Third, 12.5-dpc recipients had high posttransplantation survival (82%); survival dropped off rapidly for the later treatment groups (13.5–15.5 dpc). Fourth, the fetal busulfan treatment regimen enabled us to transplant donor cells into mouse pups, which have already been established as better recipients than their adult counterparts [20]. The superiority of the pup recipient environment probably results in part from the absence of Sertoli cell tight junctions before 10–12 dpp [50], which allows donor stem cells easier migratory access to the seminiferous tubule basement membrane and a hospitable recipient environment [20].
In mouse development, 12.5 dpc corresponds to the time when primordial germ cells associate with Sertoli cells of the presumptive gonad and become gonocytes. This differentiation event marks the transition from a multipotent primordial germ cell (PGC) to a gonocyte that has the restricted potential to develop the male germline [51–54]. This period may be the time when the male germline is most susceptible to disruption and a time that should be targeted when extending the transplantation technology to other species. The transition from PGC to gonocyte is defined by the appearance of seminiferous cords [54]. Therefore, this developmental juncture can be determined by morphological analysis of the developing gonad and has already been characterized in a number of species, thus facilitating selection of an optimum treatment interval in pregnant females of any species.
Preliminary evidence suggests that the fetal busulfan recipient preparation protocol described here for mice will be effective in other species. Fetal busulfan treatment of pregnant rats results in the live birth of male progeny that are completely devoid of endogenous spermatogenesis [33]. The capacity of these fetal busulfan-treated rats to serve as spermatogonial transplantation recipients is currently being evaluated. Similarly, busulfan treatment of pregnant sows on Day 98 and again on Day 108 of gestation (the period when gonocytes of the fetal pig are rapidly dividing) causes a significant decrease in endogenous spermatogenesis with no increase in mortality (C.L. Hausler, personal communication). Evaluation of these animals as transplantation recipients has not yet been reported. Other ablation strategies, including alternative chemotherapies (e.g., adriamycin, procarbazine) or radiation, may also prove effective for recipient preparation in other species.
The value of the spermatogonial transplantation technique for studying stem cell biology and germline development, restoring fertility, and modifying the germline has become increasingly evident during the past decade. The therapeutic potential in human cancer patients is already being examined in a clinical trial [17], even though the ability to restore fertility using this technique has not been demonstrated for any species except mice. The agricultural applications of stem cell transplantation are many, including 1) improving the health and production characteristics of food animals by modifying gene expression, 2) preserving the germline of valuable males by cryopreservation and reimplantation of testis cells, 3) developing transgenic animal bioreactors that produce therapeutic proteins in their milk, and 4) generating transgenic animals for xenogeneic organ transplantation to humans. Development of the spermatogonial transplantation technique in mice creates the foundation for extension into other species, where this technique will have a dramatic impact on practical applications and the understanding of physiological and disease processes. The recipient model identified here will greatly facilitate the extension of this technology to other species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: K.E. Orwig, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Ave., Philadelphia, PA 19104. FAX: 215 898 0667; korwig{at}vet.upenn.edu
Received: 18 February 2003.
First decision: 9 March 2003.
Accepted: 17 March 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for development or function. Endocrinology 2000 141:1273This article has been cited by other articles:
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  B. P. Hermann, M. Sukhwani, D. R. Simorangkir, T. Chu, T. M. Plant, and K. E. Orwig Molecular dissection of the male germ cell lineage identifies putative spermatogonial stem cells in rhesus macaques Hum. Reprod., July 1, 2009; 24(7): 1704 - 1716. [Abstract] [Full Text] [PDF]
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Y. M. Lee, J. G. Jung, J. N. Kim, T. S. Park, T. M. Kim, S. S. Shin, D. K. Kang, J. M. Lim, and J. Y. Han A Testis-Mediated Germline Chimera Production Based on Transfer of Chicken Testicular Cells Directly into Heterologous Testes Biol Reprod, September 1, 2006; 75(3): 380 - 386. [Abstract] [Full Text] [PDF]
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Y. Kim, V. Selvaraj, I. Dobrinski, H. Lee, M. C. Mcentee, and A. J. Travis Recipient Preparation and Mixed Germ Cell Isolation for Spermatogonial Stem Cell Transplantation in Domestic Cats J Androl, March 1, 2006; 27(2): 248 - 256. [Abstract] [Full Text] [PDF]
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A. Honaramooz, E. Behboodi, C. L. Hausler, S. Blash, S. Ayres, C. Azuma, Y. Echelard, and I. Dobrinski Depletion of Endogenous Germ Cells in Male Pigs and Goats in Preparation for Germ Cell Transplantation J Androl, November 1, 2005; 26(6): 698 - 705. [Abstract] [Full Text] [PDF]
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 K. E. Orwig and S. Schlatt Cryopreservation and Transplantation of Spermatogonia and Testicular Tissue for Preservation of Male Fertility J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 51 - 56. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, S. Toyokuni, and T. Shinohara Transgenic Mice Produced by Retroviral Transduction of Male Germ Line Stem Cells In Vivo Biol Reprod, October 1, 2004; 71(4): 1202 - 1207. [Abstract] [Full Text] [PDF]
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M. Kanatsu-Shinohara, N. Ogonuki, K. Inoue, A. Ogura, S. Toyokuni, and T. Shinohara Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells Hum. Reprod., December 1, 2003; 18(12): 2660 - 2667. [Abstract] [Full Text] [PDF]
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6) for control and 9.5-, 10.5-, 11.5-, 12.5-, 13.5-, 14.5-, and 15.5-dpc fetal busulfan-treated pup recipients were 121.6 ± 2.2, 47.9 ± 2.9, 61.6 ± 4.5, 42.8 ± 13.3, 26.2 ± 8.6, 14.0 ± 0.4, 33.7 ± 11.0, and 25.3 ± 2.0 mg, respectively. Transplantation results for busulfan-treated and untreated adult mouse recipients were reported previously [
