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Regular Article |
a 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, gamete biology, male reproductive tract, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.02% of the total testis cell population) of undifferentiated and quiescent spermatogonial stem cells that have an unlimited capacity for self-renewal [1, 2] as well as the ability to produce type A spermatogonial progenitor cells destined to differentiate and give rise to the entire spermatogenic lineage. Type A spermatogonia are contained in the transit-amplifying compartment, where approximately 9–11 divisions occur before the initiation of meiosis in spermatocytes [3]. Spermatocytes move away from the basement membrane through Sertoli cell tight junctions and enter the adluminal (differentiation) compartment, where two meiotic divisions and cell maturation result in the elaboration of spermatozoa into the lumen of the seminiferous tubules [4]. Therefore, spermatogonial stem cells are the source of all cellular elements of the spermatogenic lineage. Maintenance of the stem cell compartment involves complex interactions with other stem cells, differentiated germ cells, somatic cells, and extracellular matrix components of the testis. Together, these interactions create a microenvironment, or niche, for the spermatogonial stem cell that is located on the basement membrane of the seminiferous tubule [5]. Several other self-renewing systems in the body, including the epidermis, the intestinal epithelium, and hematopoiesis, are maintained by resident stem cells, presumably located in specific niches. The niche theory was originally proposed to describe the behavior of transplanted hematopoietic stem cells (HSCs) [6] but has now been extended to stem cells in several organs [7–11]. The basic tenets of the niche theory are that a stem cell resides in a specialized microenvironment that promotes survival and that prohibits differentiation [6]. By definition, if the number of niches is limited, then the number of stem cells will also be limited, and if all niches are occupied by resident stem cells, then efficient donor engraftment will not take place [6, 12, 13]. Moreover, the niche may play a role in the transdifferentiation of stem cells [14–16]. Studies have begun to characterize the stem cell niche in Drosophila [8, 10, 11]. However, little is currently known regarding the spermatogonial stem cell niche in mammals, because stem cells are rare and difficult to identify in vivo.
Among stem cell systems, the HSC and its niche have been the most extensively studied, in part because a stem cell transplantation technique that allows the functional identification of HSCs has been available for 40 years [17, 18]. This functional assay enabled investigators to evaluate HSC/niche interactions. A functional transplantation assay for spermatogonial stem cells was described more recently [19, 20]. Results from spermatogonial and hematopoietic transplantation studies have demonstrated that characteristics of stem cells and their niches change during development [21, 22]. Recent studies have shown that the immature mouse testis provides a more hospitable recipient environment, possibly characterized by better niche accessibility than in the adult testis [22]. In contrast, adult testes are a richer source of stem cells in comparison to immature testes [22].
Early transplantation studies in both hematopoietic [17, 18] and spermatogenic [19, 20] systems utilized ablative therapies to prepare the recipient environment, because endogenous stem cells and their niches were presumed to be tightly coupled, which would preclude donor cell engraftment. Indeed, Micklem et al. [12] and Takada et al. [13] reported that only a limited degree of donor cell engraftment was detected in normal, nonablated recipients. However, evidence in both stem cell systems suggests that ablative therapies can cause long-term, detrimental effects in the host tissue and alter the biology of the recipient microenvironment [23–25]. Now, several reports have demonstrated that large doses of donor HSCs can successfully engraft nonablated animals, and in the best case, such transplantations can lead to long-term, multilineage reconstitution [26–28]. It has been suggested that the final level of hematopoietic chimerism may be determined by the ratio of donor to host stem cells rather than by an effect of opening space for donor stem cell engraftment. Nonablative stem cell transplantations are now being clinically applied in high-risk patients for whom harmful treatment is not desired [29]. Because many similarities have been noted between hematopoiesis and spermatogenesis, including common molecular markers such as c-Kit [30, 31] and telomerase [32] and highly productive stem cell compartments [3], we hypothesized that the dynamics of stem cell/niche interactions may be similar in both systems.
To assess this possibility, we used the transplantation assay as a functional test to compare colonization in wild-type (i.e., not germ cell-ablated) and busulfan-treated (i.e., germ cell-ablated) adult recipients and also to examine the relative ability of donor stem cells from different developmental ages to colonize the wild-type pup recipient testis. These results demonstrate that 1) donor spermatogonial stem cells can compete with endogenous stem cells for colonization of the wild-type testis, 2) a dynamic interaction occurs between the germ line stem cell and its niche, and 3) colonization efficiency is dependent on donor/recipient stem cell number and/or developmental age. The number and pattern of developing colonies in the wild-type recipient were remarkably different compared to those seen in germ cell-depleted recipients, suggesting an interaction between donor and endogenous germ cells in the seminiferous tubule. These principles will facilitate the in vivo characterization of stem cell/niche interactions in the natural context, and they may have practical implications for the development of spermatogonial transplantation methods in other species or for clinical applications in which destructive treatment of the testis is not desired.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first experiment, cryptorchid and wild-type adult donor testis cells were obtained from the transgenic mouse line B6.129S7-Gtrosa26 (designated ROSA26; The Jackson Laboratory, Bar Harbor, ME) that express the Escherichia coli LacZ gene in many cell types, including all stages of spermatogenesis [33]. Neonate (0–2 days postpartum [PP]), pup (5–9 days PP), and cryptorchid adult (14–20 wk of age) donor testis cells used in the second experiment were collected from the transgenic mouse line B6;;thSJL-TgN(c177lacZ)227Bri (designated ZFlacZ; The Jackson laboratory), in which a zinc finger (ZF) promoter is coupled to the lacZ structural gene. The rationale for using ZFlacZ donors for the second set of experiments was that this line expresses the lacZ transgene in differentiated germ cells only; undifferentiated germ cells and somatic cells do not express the transgene [34]. Therefore, proliferation of Sertoli cells from neonate donors evaluated in this experiment cannot be mistakenly identified as spermatogenic colonies. Donor-derived colonies of spermatogenesis from both donors can be readily identified in recipient testes by staining with 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal).
Experimental cryptorchid testes were produced as previously described and are enriched for spermatogonial stem cells because they lack all differentiated germ cells by the time of donor cell collection (2–3 mo after the cryptorchid operation) [35, 36]. Cell suspensions from neonate, pup, and cryptorchid adult donor testes were prepared by enzymatic digestion [19, 37, 38]. Donor cells were suspended in Dulbecco modified Eagle medium/10% (v/v) FBS at concentrations ranging from 5 to 100 x 106 cells/ml [39]. Cell concentrations were adjusted for each experiment to produce, ideally, less than 40 colonies/testis, thus allowing individual colonies to be easily distinguished and counted.
Recipient Mice and Transplantation Procedure
In the first experiment, wild-type or cryptorchid ROSA26 donor testis cells were transplanted into immunologically compatible C57BL/6 recipient mice, which were either untreated (i.e., containing complete endogenous spermatogenesis) or treated with busulfan (44 mg/kg i.p.) at 4–6 wk of age. Busulfan-treated recipients were devoid of endogenous spermatogenesis at the time of transplantation (6 wk after busulfan treatment) [19]. For the second experiment, C57BL/6 x SJL F1 (B6/SJL) hybrid pup (5–9 days PP) recipient mice were used as immunologically compatible recipients of neonate, pup, and cryptorchid adult ZFlacZ donor testis cells. Adult recipient mice were anesthetized by Avertin (640 mg/kg i.p.). Pup recipients were placed on ice to cause hypothermia-induced anesthesia [40]. Approximately 10 µl of donor cell suspension were introduced into the seminiferous tubules of adult recipient mice, and 2 µl were injected into pup recipient testes.
Analysis of Recipient Testes
Testes of recipient mice were collected either at 1, 2, and 6 mo (first experiment) or at 3 mo (second experiment) following donor cell transplantation, stained with X-gal to visualize donor-derived spermatogenesis [41], and analyzed with a computer-assisted imaging system [42]. Donor spermatogonial stem cells are defined by their ability to produce blue colonies of spermatogenesis in recipient testes, and each colony is thought to be clonally derived from a single spermatogonial stem cell [42]. Other types of testis cells do not produce colonies of spermatogenesis, and endogenous germ cells do not express the lacZ transgene. Colony number and colonized area (blue area) were determined to evaluate donor-derived spermatogenesis in recipient testes. Because the concentration of donor cells varied between experiments and between treatments, colony number was normalized to 105 cells injected per testis.
Statistics
Our analyses of experiments 1 and 2 sought to identify statistically significant differences between donor and recipient groups with regard to the number of colonies per 105 cells and the area per colony and to determine whether these relationships changed over time after transplantation (2 vs. 6 mo). Each recipient testis contributes an individual observation to the data set. To account for the repeated measures on each animal (from left and right testes), generalized estimating equations methodology was used [43]. This methodology accounts for the correlation structure between the repeats and does not delete any animal having some missing repeats (as a repeated measures ANOVA would do). The models included effects for donor, recipient, and left versus right testis. All pairwise comparisons between the donor/recipient groups were performed, and Bonferroni adjustments were made to the P values to account for multiple comparisons, thus providing conservative estimates of significant differences. The outcomes for both experiments were summarized as mean ± SEM. The distributions for the two outcomes were right-skewed, so log transformations were used to obtain approximately normal distributions. Only the results of the original data are presented here, because the conclusions did not differ when using the log-transformed data.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether competitive colonization occurs between donor spermatogonial stem cells and endogenous stem cells already occupying a niche in the adult testis, two adult donor populations (cryptorchid and wild-type) containing high and low concentrations of spermatogonial stem cells were transplanted into two adult recipients (wild-type and busulfan-treated) that contained high and low numbers of endogenous stem cells (Fig. 1). Cryptorchid testes are enriched for spermatogonial stem cells, because they lack all differentiated germ cells at the time of donor cell collection [35]. Donor germ cells express the lacZ gene, thus allowing the unequivocal identification of donor-derived spermatogenesis in recipient testes. Colonization of recipient testes was first examined 1 mo after transplantation of donor testis cells, and differences in the pattern of colonization between the two types of recipient were striking. As we previously reported for busulfan-treated recipients [41], lacZ-expressing donor cells that produce ß-galactosidase established a monolayer network on the basement membrane of the seminiferous tubule. These colonies had a uniform, symmetrical appearance and rarely exhibited the multiple layers of germ cells indicative of differentiation and migration toward the lumen of the tubule (Fig. 2A). In contrast, donor-derived colonies in wild-type recipients exhibited asymmetrical and variable patterns of expansion 1 mo following transplantation. Some monolayer colonies were observed (Fig. 2B), but many colonies were characterized by irregular margins at both ends and, occasionally, had dark-stained regions, indicating vertical expansion and differentiation toward the tubular lumen (Fig. 2C). Because differentiation of germ cells into the tubular lumen is not typically observed in busulfan-treated recipients after 1 mo, differentiation of donor-derived germ cells occurs more quickly in wild-type testes already containing complete spermatogenesis.
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Analysis of recipient testes 2 mo after transplantation demonstrated that cryptorchid donors could produce spermatogenic colonies in all wild-type recipient testes, and the colony number was 4.7% of that found in busulfan-treated recipients (1.6 ± 0.3 vs. 33.7 ± 7.9 colonies per 105 cells injected, P < 0.001) (Table 1). Wild-type donor testis cells produced 2.7 colonies per 105 cells injected into busulfan-treated recipients but failed to produce any colonies of spermatogenesis in wild-type recipients (Table 1, lines 3 and 4). Therefore, regardless of the donor testis cell population, more spermatogenic colonies were produced in the germ cell-depleted, busulfan-treated testis than in the wild-type testis. Cryptorchid adult donors produced 12.5-fold more colonies than wild-type donors in busulfan-treated testes (P < 0.001) (Table 1, lines 1 and 3). The number of spermatogenic colonies per testis did not change significantly between 2 and 6 mo after transplantation for any donor/recipient combination (P = 0.7). Collectively, these data suggest that large numbers of donor stem cells can colonize nonablated recipient testes and that endogenous germ cells modulate donor cell colonization.
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By 2 mo after transplantation, donor-derived spermatogenic colonies for both recipients appeared as distinct, blue stretches of tubule. In busulfan-treated recipients, colonies were symmetrical, with dark-staining regions in the middle, indicating multiple layers of donor-derived germ cells that were differentiating and migrating toward the tubular lumen, and weakly stained regions at both ends, representing monolayers of germ cells on the basement membrane (Fig. 2D). These colonies rarely contained mature spermatozoa by 2 mo after transplantation (Fig. 3A). In contrast, blue colonies in the untreated recipients were asymmetrical and the intensity of blue stain atypical, with each end of a colony showing a different pattern (Fig. 2, E and F). Many of these colonies contained mature spermatozoa in the tubular lumen (Fig. 3B). Colony size was slightly reduced in wild-type compared to busulfan-treated recipients, but this difference was not significant at 2 mo after transplantation (0.39 ± 0.02 vs. 0.51 ± 0.09 mm2/colony, P = 0.43) (Table 1). At this time, cryptorchid and wild-type donor-derived colonies were of similar size (0.51 ± 0.09 and 0.59 ± 0.13 mm2/colony, P = 0.47) (Table 1). Six months after transplantation, all colonized testes contained long, dark-blue colonies (Fig. 2, G and H), and histological examination confirmed the presence of complete and normal, donor-derived spermatogenesis in both recipient types (Fig. 3, C and D). In addition, wild-type testes contained several monolayer cell clusters, resembling the network structures seen during earlier stages of colonization (Fig. 2I), that were not found in busulfan-treated recipient testes at this time. Dramatic differences in colony size were apparent by 6 mo after transplantation. For all recipients that produced colonies, a significant increase in colony size was observed between 2 and 6 mo after transplantation (P 
0.0001), but cryptorchid donor-derived colonies in busulfan-treated recipient testes grew much faster during this period (from 0.51 ± 0.09 to 3.30 ± 0.69 mm2/colony, a 6.5-fold increase) (Table 1, line 1) than cryptorchid in wild-type testes (from 0.39 ± 0.02 to 0.98 ± 0.11 mm2/colony, a 2.5-fold increase; P = 0.01) (Table 1, line 2) or wild-type in busulfan-treated testes (from 0.59 ± 0.13 to 1.56 ± 0.33 mm2/colony, a 2.6-fold increase; P < 0.04) (Table 1, line 3).
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Neonate, Pup, and Cryptorchid Adult Spermatogonial Stem Cells Compete with Endogenous Stem Cells for Available Niches in Pup Testis
In a previous study, we reported a dramatic increase in the concentration of spermatogonial stem cells during development from neonate (0–2 days PP) to pup (5–12 days PP) to cryptorchid adult (14–20 wk of age); the cryptorchid adult donors produced 13.6-fold more colonies per 105 cells injected into busulfan-treated recipients than neonate donors [22]. In that study, little competition existed with the recipient's endogenous spermatogonial stem cells, because busulfan-treated recipients are virtually stem cell-depleted. The purpose of the present study was to determine any differences in the ability of neonate, pup, and cryptorchid adult spermatogonial stem cells to form colonies in the wild-type pup testis, in which they must compete with an increasing number of endogenous stem cells for newly forming niches. Neonate donor testis cells produced colonies of spermatogenesis in 18% (4 of 22) of recipient testes, compared with 30% (6 of 20) and 100% (21 of 21) when pup and cryptorchid adult donor cells, respectively, were used. The number of colonies formed from 105 cryptorchid donor testis cells injected into pup recipient testes (27.69 ± 3.81) was 46-fold greater than that from pup donor cells (0.60 ± 0.28, P < 0.001) and 173-fold greater than that from neonate donor cells (0.16 ± 0.08, P < 0.001) (Fig. 4). Similarly, the area per colony derived from cryptorchid adult donor cells (0.4 mm2) was 1.5-fold larger than that from pup donor cells (0.27 mm2, P = 0.75) and 3.1-fold larger than that from neonate donor cells (0.13 mm2, P < 0.001) (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Removal of endogenous germ cells by busulfan treatment of recipient testes had a significant impact on donor stem cell colonization efficiency. Busulfan is one example of an ablative therapy that kills differentiating germ cells and stem cells, thus opening niches in host seminiferous tubules, a function that has been considered to be essential for spermatogonial transplantation [19, 20]. Because cryptorchid adult donor testis cell populations should contain approximately 500 stem cells per 105 cells transplanted [36], the colonization efficiency of cryptorchid testis cells in busulfan-treated recipients was 6.7% (33.7/500) (Table 1, line 1) in this experiment, which is consistent with the results of previous studies that showed an efficiency of 5–10% [22, 41, 42, 44] relative to morphometric estimates [1, 2]. By similar calculation, colonization efficiency of cryptorchid adult donor cells in wild-type (nonablated) adult recipients was only 0.32% (1.6/500) (Table 1, line 2), which is 21-fold less than that in busulfan-treated testes (Table 1, lines 1 and 2). Interestingly, donor-derived colonization of wild-type testes appears to be facilitated compared to hematopoiesis, because a single injection of cryptorchid adult donor cells was sufficient to produce colonies in all wild-type testes injected. In contrast, the hematopoietic system typically requires multiple injections of large doses of donor cells for successful colonization [12, 13, 26]. Taken together, these results demonstrate that ablative therapy is not a prerequisite for spermatogonial cell engraftment but, rather, that colonization efficiency appears to be a function of the relative number of donor and recipient stem cells.
An important characteristic of the spermatogonial transplantation assay is that donor-derived spermatogenic colonies are easily visualized in seminiferous tubules and are believed to remain in close association with the transplanted stem cell that produced them, thus allowing quantitative and qualitative analysis of individual clones [41]. Whereas colony number reflects the number of stem cells in the donor population, colony appearance, size, and growth rate reflect the impact of the recipient environment and donor cell origin. A significant finding from the first experiment was the difference in the pattern or appearance of colonies in busulfan-treated versus untreated recipient testes. Long, uniformly blue, donor-derived colonies in busulfan-treated testes contrasted with the short and asymmetrical appearance of colonies in untreated testes (Fig. 2). Thus, the pattern of donor spermatogenic colonies is influenced by endogenous germ cells, and the removal of these cells changed the mode of colony differentiation from transplanted stem cells. The interaction between different classes of germ cells was previously suggested in reports detailing testicular extracts that inhibited proliferation of undifferentiated spermatogonia [45, 46], an activity that was absent when type A spermatogonia were removed before preparation of the extracts [46]. That study [45] suggests that spermatogonia produce a negative feedback, possibly mediated by a "chalone," that regulates the proliferation and number of undifferentiated spermatogonia [45, 47]. Our results from clonal analysis support the notion of an interaction between different germ cell populations in the testis.
Effects of donor cell origin and recipient environment on colony size and growth rate are evident in Table 1. Donor-derived colonies grew faster and were significantly larger in busulfan-treated than in wild-type (nonablated) adult recipient testes by 6 mo after transplantation (P < 0.02) (Table 1, lines 1 and 2). These differences in growth rate likely reflect a competitive effect of the endogenous germ cells on the basement membrane of seminiferous tubules in nonablated recipients. Donor cell population also affected the size of the colonized area after transplantation. Cryptorchid adult donor-derived colonies grew faster and were significantly larger than wild-type adult donor-derived colonies by 6 mo after transplantation into busulfan-treated testes (P < 0.05) (Table 1, lines 1 and 3). These results suggest that cryptorchid adult and wild-type adult donor spermatogonial stem cell-derived colonies have unequal growth potentials, and these differences may reflect effects of the donor testis environment.
The purpose of the second experiment was to assess competition between spermatogonial stem cells from three different donors (neonate, pup, and cryptorchid adult) and endogenous stem cells in the developing environment of the pup testis. In contrast to the adult testis, spermatogonial stem cells are actively proliferating in the pup testis [48], thereby providing a different competitive environment. In this environment, testis cells from the cryptorchid adult produced significantly more colonies per 105 cells injected than pup (46-fold more colonies) or neonate (173-fold more colonies) testis cells. Based on previous data from transplantation into busulfan-treated adults [22], the concentration of spermatogonial stem cells in testis cell populations from the cryptorchid adult is only approximately 3.3-fold higher than in pup and 13.6-fold higher than in neonate. Thus, the large differences between these donor populations observed in this study must reflect competition from endogenous stem cells and suggests that donor spermatogonial stem cells from cryptorchid adult testes are more competitive than those from neonate or pup testes. Because all donor testis cell populations were injected at a concentration of 100 x 106 cells/ml and cryptorchid adult testis cell populations contain a significantly higher concentration of stem cells than in pup or neonate [22], more stem cells were introduced when cryptorchid adult cells were injected. Therefore, the apparent competitive advantage of cryptorchid adult spermatogonial stem cells observed in this study might be due to increased number, inherent characteristics, or both. However, the possibility that cryptorchid adult donor testis cells are inherently more competitive is supported by the observation that, once seeded on the basement membrane of the seminiferous tubule, they produce larger spermatogenic colonies than pup or neonate donors. This difference between donors was only revealed in the competitive environment of pup recipient testes, because cryptorchid adult, pup, and neonate donor populations produce colonies of similar size in busulfan-treated testes (0.5 mm2/colony; [22]). Therefore, immature donor spermatogonial stem cells did not compete well for stem cell niches in the wild-type pup testis due to inherent characteristics or low numbers, and they appeared to be at a disadvantage for colony growth as well.
Comparison of results from cryptorchid adult donors in experiments 1 and 2 revealed that wild-type pup testes are more efficient recipients than wild-type adult testes (compare Fig. 4A and Table 1, line 2). Although the particular mechanisms are unknown, several important morphological and functional differences between pup and adult testes may contribute to their effectiveness as recipients. First, pup testes do not have Sertoli cell tight junctions or multiple germ cell layers to hinder the migration of donor stem cells to the basement membrane of the seminiferous tubule. Second, environmental differences, such as growth factor and hormonal stimuli, may affect colony development [49]. Third, proliferation of Sertoli cells in the immature testis, as it increases in size, likely is accompanied by a corresponding increase in the number of stem cell niches available for colonization by donor or endogenous stem cells. Collectively, results from the second experiment indicate that, as in the adult, a dynamic relationship exists between stem cell and niche in the pup recipient testis and that donor cells can compete for stem cell niches starting from the early stages of postnatal testis development.
These data have practical implications for the development of spermatogonial transplantation in other species. Techniques for testis cell transplantation have now been reported in several species, including the rat, bovine, monkey, and human [49–51], but ablation of endogenous spermatogenesis in the recipient testis has been problematic [49]. Furthermore, it has been postulated that removal of endogenous germ cells causes an imbalance between the spermatogenic and endocrine compartments and that the abnormal intratesticular hormonal milieu could inhibit normal spermatogenesis from stem cells [25]. The level of colonization achieved in the current study highlights the potential for using untreated, immature recipients for stem cell transplantation, a possibility that might be extended to other animal systems for which stem cell-depleted models are not available.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 T.S. was supported by the Japan Society for Promotion of Science. Microscopic sections were produced in the Institute for Human Gene Therapy, Cellular Morphology Core (5-P30-DK-47747-07). Financial support for the research was from the NIH (NICHD 36504), Commonwealth and General Assembly of Pennsylvania, and the Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation. 
2 Correspondence: Ralph L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104. FAX: 215 898 0667
3 Current address: Department of Medical Chemistry, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-0667, Japan
Accepted: December 10, 2001.
Received: November 1, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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