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Testis |
Department of Medical Chemistry,3
Department of Pathology and Biology of Diseases,4
Department of Clinical Epidemiology,5
Department of Pharmacoepidemiology,6 Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, gametogenesis, Sertoli cells, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although stem cells are considered to have infinite self-renewal activity, there are several reasons to believe that their capacity for division is limited. First, spermatogenesis does not regenerate to the normal level from surviving stem cells if high doses of cytotoxic agents that destroy stem cells are used [4]. Second, in Steel mutant mice, germ cell number is reduced during the embryonic stage because of a defect in Steel gene expression in Sertoli cells. Although stem cells in postnatal mutant mice are functionally normal and do not express receptors for Steel factor [5, 6], their number is limited to only 5% of that in controls and they do not regenerate [6]. Third, studies of hematopoietic stem cells have shown that the self-renewal activity of stem cells declines after serial transplantation, suggesting that not all types of stem cells are capable of infinite expansion [7]. These observations strongly suggest that the self-renewal activity of spermatogonial stem cells is limited.
The development of the spermatogonial stem cell transplantation technique provides a direct method with which to assay stem cells [8]. Testis cells transferred into the seminiferous tubules of infertile recipients form colonies of spermatogenesis and produce differentiating germ cells. Although donor cells consist of various types of testis cells, including Sertoli or Leydig cells [9], only stem cells can produce this result, because they only have self-renewing ability. An important advantage of spermatogonial transplantation is that it allows identification of individual colonies arising from single stem cells [10]. The use of donor males transgenic for the E. coli lacZ gene makes it possible to detect donor-derived spermatogenesis by staining of all donor-derived cells after incubation with substrate [10], and the number of stem cells present in any cell population can be quantified. Thus the spermatogonial transplantation technique allows assessment of the self-renewal activity of the stem cells in any cell population.
Although stem cell numbers are kept constant under steady-state conditions, their numbers are considered to increase under several artificial conditions; for example, in busulfan-treated donor animals or following serial transplantation of donor-derived single colonies into host animals. In both models, spermatogenesis is regenerated from stem cells and produces spermatozoa. The first of these models depends on the use of the cytotoxic drug busulfan to deplete stem cells [4, 11, 12]. Unlike other chemicals that destroy differentiated spermatogonia, busulfan is a potent agent that preferentially kills spermatogonial stem cells of several species [11]. It is an alkylating agent and does not have any effect on DNA synthesis; however, it causes inhibition of the next mitosis when it intoxicates the cells in the G1 phase [12]. Although high-dose administration eliminates stem cells and makes animals permanently sterile, administration at a low dose reduces the number of stem cells and spermatogenesis can recover from the surviving stem cells. In addition, the duration of sterility following busulfan treatments is dependent on the extent of stem cell depletion [4]. Based on these observations, it has been suggested that surviving stem cells must undergo self-renewing division to restore their own populations and differentiating divisions to produce cells that will differentiate into spermatozoa [3, 13]. Studies have shown that for a short time (10 d or less) after cytotoxic treatment, stem cells may either not divide or produce only additional stem cells [13–15]. Although the first is almost always a self-renewing division to produce additional stem cells, differentiating cells can develop within 6 d and the probability of a self-renewing division further decreases within the first 6–10 d after irradiation [13–15]. Thus stem cells change their mode of replication at certain time points as they regenerate following cytotoxic damage. This provides a useful system for observation of self-renewing division, as the surviving stem cells are actively dividing under these conditions [3].
The second model of stem cell expansion relies on the clonal assay of individual stem cell colonies. This model is based on the assumption that if a single colony originated from a single stem cell, the self-renewal activity of a single stem cell can be evaluated by serially transplanting the colony into secondary recipients. Serial transplantation of colonies was originally performed in a hematopoietic system to evaluate the self-renewal activity of putative hematopoietic stem cells in splenic colonies [16]. Similar to the spleen colony assay, individual colonization events can be observed by spermatogonial transplantation, and the kinetics of stem cell proliferation can be determined independently for each colony [10]. Therefore, serial transplantation of a single colony provides a novel opportunity to analyze the proliferative potential of a single stem cell.
In the experiments described here, we used the spermatogonial transplantation technique to evaluate the level of self-renewal activity of stem cells in both systems. The number of stem cells was determined, and the kinetics of stem cell expansion were examined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wild-type C57BL/6J (designated as B6) and WBB6F1-W/Wv (designated as W) mice were purchased from Japan SLC (Hamamatsu, Japan). The transgenic mouse line B6-TgR(ROSA26)26Sor (designated as ROSA26) was purchased from The Jackson Laboratory (Bar Harbor, ME). This mouse line expresses the E. coli lacZ transgene in all cells within the seminiferous tubule [10, 17]. Donor cells used for the serial transplantation experiments were isolated from the testes of transgenic mice of the line C57BL/6 Tg14(act-EGFP)OsbY01 (designated as Green) provided by Dr. M. Okabe (Osaka University, Japan) [18]. The spermatogonia and spermatocytes of these mice express the EGFP gene, the level of expression of which decreases gradually after meiosis [18]. All animal experimentation protocols were approved by the Institutional Animal Care and Use Committee of Kyoto University.
Busulfan Injection
In the first set of experiments with busulfan-induced regeneration, busulfan (Sigma, St. Louis, MO) was first dissolved in dimethyl sulfoxide (Sigma), and then an equal volume of sterile distilled water was added to provide a final concentration of 15, 30, and 45 mg/kg [19]. Adult mice (>6 wk of age) received a single intraperitoneal injection of busulfan. In each experiment, two to three animals received busulfan at each dose. In the second set of experiments with serial transplantation, busulfan (45 g/kg) was used to deplete endogenous germ cells to prepare B6 recipients. They were used as recipient mice for transplantation at least 4 wk after busulfan treatment.
Donor Cells and Transplantation Procedure
Donor cells were collected from the testes of ROSA (first experiment) or GFP transgenic mice (second experiment), and single-cell suspensions from a donor testis were prepared by two-step enzymatic digestion [19]. For the first experiment, the testes were collected at 3, 15, 42, and 70 days after treatment, and single-cell suspensions were obtained from each testis by enzymatic digestion. Cells were suspended at a concentration of 108 cells/ml in Dulbecco modified Eagle medium/10% fetal calf serum, supplemented as described previously (DMEM/FCS) [19].
For serial transplantation into secondary recipients, donor-derived colonies were identified under a fluorescence microscope (Leica, Tokyo, Japan), and segments of the seminiferous tubules containing a single GFP-positive colony were dissected out and dissociated in Eppendorf tubes. The cells were suspended in a volume of approximately 20 µl of DMEM/FCS and transplanted into two recipient testes. Viability of cells was greater than 95% as determined by trypan blue exclusion.
For testicular injections, testis cells were maintained on ice and then microinjected into the efferent ducts of recipient testes. In the first set of experiments, approximately 3 µl of the donor cell suspension was introduced into the seminiferous tubules of W testes, whereas in the second experiment 10 µl was introduced into the tubules of B6 testes through the efferent duct [19]. The injection filled 75%–85% of the tubules in each recipient testis (a visual estimate).
Analysis of Recipient Testes
In the first set of experiments, the testes of busulfan-injected animals were recovered at 35 and 70 days after injection. The testes were fixed in 10% neutral-buffered formalin (Wako Pure Chemical Industries, Osaka, Japan) and processed for paraffin sectioning. All sections were stained with hematoxylin and eosin. Two histological sections were made from the testes of each animal, and each slide was viewed at a magnification of x400 to determine the extent of spermatogenesis. The number of tubule cross-sections showing spermatogenesis (defined as the presence of multiple layers of germ cells in entire circumference of the seminiferous tubule) or not showing spermatogenesis were recorded for one section from each testis.
For evaluation of colony number, recipient mouse testes were recovered 2 mo after donor cell transplantation and analyzed by staining for the lacZ gene product, ß-galactosidase, with the substrate 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-gal; Wako; first experiment) [10], or by observation of fluorescence under UV light (second experiment) [18]. These methods allowed the specific identification of donor germ cells, because endogenous host testis cells did not stain with X-gal and had no endogenous fluorescence. The tunica was removed from each testis, and the seminiferous tubules were dissected apart using fine forceps [10]. The efficiency of colonization was evaluated by counting the total colony number under a stereomicroscope. Statistical analysis was performed using Student t-test or Z-test (first experiment), chi-square test or Mann-Whitney U-test (second experiment).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first set of experiments, we measured the changes in the number of stem cells during busulfan-induced regeneration. We initially determined the optimal dose to partially deplete stem cells using wild-type C57BL/6 mice. Adult B6 mice received intraperitoneal injection of busulfan at three different doses (15, 30, and 45 mg/kg), and the mice were killed after 35 or 70 days to determine the extent of ongoing spermatogenesis. As the length of time from initiation of stem cell division to formation of spermatozoa is 35 days in mice [1, 3], this period corresponded approximately to one- to two times the duration of mouse spermatogenesis and would provide sufficient time for recovery of spermatogenesis in surviving stem cells.
Experiments were performed in triplicate, and a total of at least six animals were analyzed for each dose and time point. In agreement with the results reported previously [4], injection of busulfan decreased the testis weight (Fig. 1A) and depleted spermatogenesis in a dose-dependent manner (Fig. 1B). Counts at 35 days after treatment revealed that, whereas only 0.8% of seminiferous tubules showed spermatogenesis in animals treated with busulfan at a dose of 45 mg/kg, spermatogenesis was observed in 8.8% and 63% of the tubules of animals given busulfan at doses of 30 and 15 mg/kg, respectively (Fig. 1, B and C). Spermatozoa were observed in the epididymides of all animals. These observations indicated that the effects of busulfan were dose-dependent.
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Seventy days after busulfan treatment, the increase in weight of the testis was most apparent in animals treated at a dose of 15 mg/kg (Fig. 1A; P < 0.01), suggesting extensive regeneration of spermatogenesis at this dose. Histological analysis showed that most testes contained no germ cells in animals treated with busulfan at 45 mg/kg, and that regeneration occurred in a few tubules. On the other hand, regeneration of spermatogenesis was more significant in animals given the drug at 15 and 30 mg/kg, with averages of 98.9% and 47.3% of tubules showing spermatogenesis, respectively (Fig. 1, B and D; P < 0.01). Particularly in animals treated with busulfan at 15 mg/kg, virtually all seminiferous tubules showed apparently normal spermatogenesis with mature spermatozoa, and the size of the testis was almost comparable to that in uninjected controls (data not shown). These results indicated that a significant proportion of spermatogonial stem cells were eliminated at 45 mg/kg, and that surviving stem cells can regenerate to show spermatogenesis following injection of 15 and 30 mg/kg of busulfan. We selected a dose of 15 mg/kg for further analysis, because the stem cell regeneration, as suggested by the testis weight and the number of repopulated tubules, occurred most extensively at this dose.
Determination of Stem Cell Number in the Busulfan-Treated Testis by Spermatogonial Transplantation
To determine the stem cell number during the course of regeneration after busulfan injection, we next performed spermatogonial transplantation experiments. In spermatogonial transplantation, each colony is believed to be derived from a single stem cell [10], and the use of lacZ-marked ROSA26 donor cells facilitates the quantification of stem cells after transplantation into nontransgenic recipients by allowing visualization of spermatogenic colonies [10].
Donor ROSA26 mice received busulfan injection at 15 mg/kg. As expected from the results of the histological study described in the preceding section, the number of cells that could be recovered from each testis changed markedly during regeneration (Fig. 2A). Although no significant reduction in cell number was noted at 3 or 15 days after busulfan injection, the recovery reached its minimum value, 32% of that in wild-type controls (P < 0.01), at 42 d after treatment. However, on Day 70 after treatment, cell recovery had increased to 84% of the wild-type level.
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To determine the stem cell number, the testis cell suspensions thus recovered were then microinjected into the seminiferous tubules of infertile W recipient mice. W mice are congenitally infertile [20] and lack all stages of differentiating germ cells due to mutations in the c-kit receptor tyrosine kinase [21], but they are capable of spermatogenesis and producing offspring from transplanted testis cells [5, 6]. Three to four experiments were performed for each time point. In each experiment, all recipients received the same donor cell population at the same time, and at least 13 testes were injected. Two months after transplantation, the recipient testes were analyzed by X-gal staining to determine the number of colonies of donor-derived cells.
The number of colonies was lowest at 3 days after busulfan injection, but gradually increased with time (P < 0.01; Fig. 2, B and C). After 42 days, there was no significant difference between the number of colonies derived from busulfan-treated donors and from untreated donors. Although the number of colonies at 70 days after treatment showed a slight decrease as compared with that at 42 days, the difference was not significant. As the same number of cells (3 x 105 cells per testis) were injected into each recipient testis, the number of colonies generated in the recipient testes reflected the concentration of stem cells in the total testis cell suspension. Assuming that 1) each colony is produced by one stem cell, 2) that 10% of transplanted stem cells can colonize [10], 3) that the stem cells recovered at different time points after treatment were equally competent to establish colonies, and 4) that there was 100% harvesting efficiency during enzymatic digestion of donor testis cells, we estimated the changes in numbers of spermatogonial stem cells per testis during regeneration (cell recovery x colony number) from the data shown in Figures 2A and 2C (Fig. 2D). Injection of busulfan at a dose of 15 mg/kg initially decreased the number of stem cells to approximately 3.8% of that in wild-type controls by Day 3 after treatment (P < 0.001). During the next 12 days, the number of stem cells increased to 14% of that in controls. The stem cells further increased in a linear manner. After 42 days, their number reached 34% of that in wild-type controls. Thus approximately one third of the stem cells were regenerated by this time point. The regeneration continued thereafter, eventually reaching 61% of the level in untreated controls 10 wk after busulfan treatment.
Serial Transplantation of GFP-Marked Spermatogenic Colonies
In the second set of experiments, we examined the self-renewal activity of spermatogonial stem cells by serial transplantation of single colonies. For these experiments, we employed Green mice to visualize colonies, because colony formation from live germ cells can be observed in real time. The spermatogenic cells of Green mice express the EGFP gene, which facilitates detection of colonies of live germ cells under UV light [18].
Donor testis cells were microinjected into the seminiferous tubules of busulfan-treated B6 primary recipients and analyzed for fluorescence under UV light illumination at 2 and 4 mo after transplantation (Fig. 3, A and B). To determine the stem cell number in single colonies, seminiferous tubules containing GFP-positive colonies were dissected out and dispersed by enzymatic digestion. The average number of cells recovered from the tubule fragments ranged from 1.6 x 104 to 4.7 x 105 cells with an average of 1.8 ± 0.2 x 105 (mean ± SEM, n = 36) cells, depending on the size of the fragment. The cell suspensions were then transplanted into both testes of a secondary recipient. Four separate experiments were performed at 2 and 4 mo, and a total of 36 seminiferous tubule fragments containing GFP-positive colonies were transplanted from nine different testes of primary recipients. The testes of the secondary recipients were examined for donor cell colonization 2 mo after transplantation, and the number of colonies was recorded for each recipient testis.
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As shown in Table 1, 19% (3/16) and 35% (7/20) of 2- and 4-mo-old colonies produced colonies in the secondary recipients, respectively. Although the frequency of successful transplants and the average number of colonies generated from 4-mo-old colonies were higher than those from 2-mo-old colonies (1.2 vs. 0.38), the differences between the two groups were not significant. However, assuming that 1) one stem cell is responsible for the generation of each colony [10] and that 2) colonization efficiency is 10% [10], the increase in the number of stem cells from the time of transplantation (time 0) was significant for 4-mo-old colonies (P < 0.05), but not for 2-mo-old colonies. In the most successful case, a total of 10 colonies was generated in the two testes of the secondary recipients by transplantation of a single 4-mo-old colony. These results indicate that stem cells can increase in number after transplantation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study provides the first functional data in support of stem cell self-renewal, and we demonstrate the increase in stem cell numbers during regeneration and after transplantation. The results of the first experiment confirmed that regeneration after cytotoxic treatment is based on stem cell expansion, as originally proposed based on morphological criteria [3]. As busulfan destroys primitive germ cells that account for less than 1% of the total number of testis cells [11, 22], most of the differentiated progenitor cells survive the treatment and continue differentiation with normal kinetics. However, the latter gradually mature and disappear by 35 days, owing to the absence of self-renewal activity [1, 3]. Therefore, as the stem cell numbers continue to recover, the number of differentiated germ cells decreases, and the ratio of stem cells to differentiated germ cells changes markedly during regeneration. Eventually, all progenitor cells must be derived from the stem cells that initially survived busulfan injection. These processes are reflected in the changes in the weight and cell recovery observed during regeneration (Figs. 1A and 2A).
Our results revealed that the number of stem cells undergoes dynamic changes after busulfan treatment. Immediately after treatment, the number of stem cells decreased and reached the lowest value at 3 days. However, stem cells then started to increase; the doubling time of the stem cells was initially 6.3 days between 3 and 15 days, increasing to 21.3 days between 15 and 42 days, and finally reaching to 33.9 days between 42 and 70 days, although the standard deviation for colony numbers at the last two points was relatively large. These results indicate the following: 1) that stem cell expansion starts immediately after treatment; and 2) that doubling time for stem cells is 1–5 wk, and this period is extended as more time passed following treatment.
Our results generally agree with those of previous morphological studies. It has been shown that the regeneration of stem cells is a very slow process [1, 3]. More severe treatment is associated with a longer delay before the beginning of regeneration. There are, however, some variations between the studies, and delays of 1–2 wk have been reported [23, 24]. In one study, the number of type A spermatogonia started to decrease between Days 2 and 4 after injection of busulfan, and reached the lowest value between Days 6 and 8 [25]. Regeneration started between Days 8 and 10, and it was estimated that stem cells could divide at least three times during this period [25]. Similar observation was made with studies using irradiation, and stem cells were shown to have a high probability of undergoing a self-renewing division, as opposed to a differentiating division [14, 15]. However, in another study, no recovery of stem cell number was found for 2 wk after irradiation [24, 26]. Our results agree with the results of van Beek et al. [14, 15], which indicated that stem cells begin to increase their number immediately after treatment. Indeed, the increase in stem cell numbers was evident between Days 3 and 15 after busulfan treatment, indicating that stem cells quickly responded to the signals triggered by the decrease in stem cell number. The variation in the morphological estimates tends to be more pronounced in the later phase of regeneration; Meistrich et al. [24] calculated a stem cell doubling time of 2 wk during refilling of C3H mice seminiferous tubules after exposure to x-rays at a dose of 1200 rad, whereas others have calculated doubling times of between 3 and 6.5 wk using different methods [27]. Thus our estimates for the doubling time for the stem cells are comparable to those reported in previous studies.
These relatively wide ranges in the morphological estimates may have arisen not only from differences between studies in the type and dose of insult used, but also from the definition and description of the stem cell clones. Because more differentiating spermatogonia are killed at higher doses of busulfan, the ratio between surviving stem cells and differentiating cells could be significantly different according to the dosage, which may affect the self-renewing division of surviving stem cells. Particularly in the later phase, it becomes more difficult to evaluate stem cells in regenerating colonies in confluence, because each collision halves the growth rate of the two confluent colonies [10, 27]. In addition, the types of assays used to quantify stem cells in the later phase, such as the number of sperm heads or enzyme activity of differentiated germ cells, are not necessarily accurate, because they neglect colonies of stem cells that do not produce differentiated cells. Indeed, it has been reported that spermatogenesis following cytotoxic damage is often accompanied by abnormal features, such as missing layers of germ cells [28] or excessive production of spermatogonia [29]. In contrast, one of the drawbacks of the transplantation assay is that it is not possible to evaluate the location or the type of division of individual stem cells during regeneration. For example, we cannot exclude the possibility that stem cells in a specific phase of the cell cycle (e.g., actively proliferating stem cells) have lower seeding efficiency after transplantation. Nonetheless, the functional assay can overcome the limitations of the morphological methods described above, and may be more useful for analyzing stem cell behavior, particularly in the later phase of regeneration. More detailed analyses of stem cell numbers in the early and later stages of regeneration, and of their responses to different types of insult, will complement the findings of previous studies and will probably provide new insight into stem cell regeneration and its mechanism of regulation.
The results from the serial transplantation study also confirmed the self-renewing activity of stem cells at the clonal level. In the present study, single colonies that were dissected from primary recipients at 2 and 4 mo after transplantation generated 0.38 and 1.2 colonies per testis, respectively. As approximately 10% of transplanted stem cells can colonize the recipient testes [10], these results indicate that 3.8 and 12 colonies were generated from one colony after 2 and 4 mo, respectively (0.38 x 10; 1.2 x 10). Thus the number of stem cells increased to approximately three-fold at 4 mo after transplantation compared with 2 mo, and the doubling time of the stem cells was 31.2 days between Time 0 and 2 mo, and 33.5 days between Time 0 and 4 mo, both of which are close to the values estimated for the later phase of busulfan-induced regeneration. Although the increase in the stem cell number was modest in the serial transplantation compared with the busulfan-induced regeneration, we speculate that this difference may reflect the fact that repopulation from the transplanted stem cells requires extra steps, such as migration through tight junctions between Sertoli cells or settlement in the "niche" [14], before initiating regeneration. Indeed, a previous study showed that the speed of colony growth from the transplanted stem cells was initially slower when compared with the later phase [10]. This may explain the lower success rate and colony number after retransplantation of 2-mo-old colonies. Nevertheless, the increase in the stem cell number from the time of transplant was significant for 4-mo-old colonies, demonstrating that stem cells underwent self-renewing division in the colony after transplantation.
Interestingly, the serial transplantation study suggested that the self-renewing activity of stem cells was remarkably heterogeneous. Although most of the primary colonies did not produce secondary colonies, a few were able to generate many colonies. Although we cannot completely exclude the possibility that it was an artifact of the experimental procedure resulting from handling small samples or from the merging of adjacent colonies [10], previous studies using spermatogonial transplantation also suggested the heterogeneity of stem cells. It was shown that the length of colonies is significantly variable after spermatogonial transplantation, and that patchy structures, which are indicative of the early colonization pattern of stem cells, can still be observed even at 4 mo after transplantation [10]. Similar variations in colony length or differentiation patterns were observed after irradiation [27]. Given that only 10% of transplanted stem cells can colonize the recipient testes [10], it is possible that stem cells in a specific phase of the cell cycle or residing in a specific part of the seminiferous tubules are more likely to have higher seeding efficiency than others. Although the precise relationship between the length of colonies and stem cell number needs to be carefully evaluated, these possibilities should be tested in future experiments.
Although our study clearly demonstrated the self-renewing activity of stem cells, previous morphological studies suggest that stem cell proliferation may be subject to complex regulation. It has been reported that non-repopulating seminiferous tubule cross-sections persisted at 1 yr after gamma irradiation at a dose of 1200 rad, and that, even after irradiation at a dose of 600 rad, the number of stem cells did not recover to the control level [24]. This indicates that, although they survive irradiation, stem cells do not complete regeneration in some cases. As another example, it is known that the regeneration of spermatogenesis does not occur after prenatal injection of busulfan into pregnant females, and empty tubules persist even in postnatal stages [30]. It remains to be determined why the remaining stem cells in tubules with a normal appearance do not regenerate to fill the empty tubules in these cases. These discrepancies from our results may have been due to damage to the Sertoli cells. However, similar mosaic patterns of spermatogenesis is often observed in other genetic models, including those with germ-cell defects [31–33], suggesting that variations in the spermatogenesis in different tubules may be caused by the cell-autonomous activity of germ cells. Analyses of stem cells in such models will likely reveal further information about regulation of the process of stem cell self-renewal.
Functional evaluation of spermatogonial stem cells during regeneration or serial transplantation has provided direct evidence for their proliferative activity. Thus unlike other self-renewing systems in the body, spermatogenesis is unique in that both functional and morphological assessments of stem cells are possible, which makes it an ideal model for studying stem cell self-renewal [34]. The identification of factors involved in the self-renewal process will contribute to our understanding of spermatogenesis and will also provide important information for the establishment of systems with which to expand these cells in vitro, which will be useful for correcting male infertility or for genetic modification of the male germline.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Takashi Shinohara, Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. FAX: 81 75 753 4388; takashi{at}mfour.med.kyoto-u.ac.jp
Received: 22 October 2002.
First decision: 8 November 2002.
Accepted: 6 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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