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Department of Obstetrics and Gynecology, and Division of Experimental Medicine, McGill University, Montreal, Quebec, Canada H3A 1A1
ABSTRACT
Spermatogonial stem cells (SSCs) continuously support spermatogenesis after puberty. However, accumulating evidence suggests that SSCs differ functionally during postnatal development. For example, mutant mice exist in which SSCs support spermatogenesis in the first wave after birth but cease to do so thereafter, resulting in infertility in adults. Studies using a retroviral vector have shown that the vector transduces pup SSCs more efficiently than adult SSCs, which suggests that pup SSCs divide more frequently. Thus, it is hypothesized that the SSCs in pup and adult testes have different characteristics. As an approach to testing this hypothesis in the present study, we investigated the proliferation kinetics of pup SSCs (6–9 days old) and their self-renewal/differentiation patterns for the first 2 mo after transplantation, and compared them to those of adult SSCs. Using serial transplantation, we found that the number of pup SSCs declined over the first week after transplantation. Thereafter, it increased ~4-fold by 1 mo and ~9-fold by 2 mo after transplantation, which indicates that pup SSCs continuously proliferate from 1 wk to 2 mo after transplantation. Compared to the proliferation of SSCs derived from adult intact testes, that of pup SSCs was lower at 1 mo but similar at 2 mo, indicating the delayed proliferation of pup SSCs. However, the pup SSCs regenerated spermatogenic colonies at 1 mo that were similar in length to those of SSCs from adult intact testes. Therefore, these results suggest that some functional differences exist in SSCs during postnatal development, and that these differences may affect the abilities of SSCs to self-renew and differentiate.
developmental biology, spermatogenesis, testis
Spermatogonial stem cells (SSCs) are responsible for the lifelong production of male gametes. These cells can self-renew continuously, as well as differentiate to committed germ cells, which ultimately develop into spermatozoa. Thus, even if spermatogenesis undergoes damage, it can be regenerated thanks to SSC function [1, 2]. In fact, the abilities to regenerate and maintain complete spermatogenesis represent the functional definition of SSCs [3].
Although SSCs always exist in the testis after birth and continuously support spermatogenesis after puberty, accumulating evidence suggests that the biologic characteristics of SSCs do not remain constant throughout the life of an individual but change in an age-dependent manner. For example, in mice, SSCs are quiescent at birth but over the first postnatal week, initiate active proliferation and the production of daughter cells that are committed to differentiation [4, 5]. Thus, SSCs actively and rapidly undergo proliferation and differentiation during the early period of postnatal development. The first wave of spermatogenesis ensues, and the first appearance of spermatozoa is observed at ~35 days of age [4, 5]. In contrast, SSCs replicate only slowly in adult testes, so as to maintain steady-state spermatogenesis, which may be followed by a reduction in SSC numbers with age [3, 6, 7].
Both SSCs and spermatogenesis appear to be different during the first wave after birth than in later periods of life. One example of this is juvenile spermatogonial depletion (jsd) mutant mice, in which spermatogenesis is completed in the first wave but becomes arrested thereafter [8]. In these mutant testes, only primitive spermatogonia remain in the adult testes, resulting in male infertility. This cessation of spermatogenesis occurs due to a cell-autonomous defect in the spermatogonia [9–11], which suggests that these cells have different biological properties during the first wave after birth than during subsequent waves.
More specifically for SSCs, we have previously demonstrated that SSCs derived from pup (6–8 days old) testes are more efficiently transduced by a Moloney murine leukemia virus-based retroviral vector than SSCs derived from adult testes [12, 13]. Following a 7-day exposure to the viral vector in vitro, the proportion of SSCs that incorporated the viral gene into their genomes relative to the total SSCs remaining in the culture was significantly higher for pup SSCs than for adult SSCs. Since the retroviral vector used requires target cell replication for the transfer of viral genes, these results indicate that pup SSCs divide more frequently than adult SSCs.
Whether or not this active cell division of pup SSCs is directly associated with self-renewal and proliferation is not known. It seems reasonable to assume that active pup SSC division would lead to robust expansion of the stem cell pool. Alternatively, pup SSCs may divide to produce more daughter cells that are committed to differentiation, rather than expanding the stem cell population.
One approach to addressing these questions involves serial transplantation, which is based on a series of consecutive transfers of donor cells into two generations of recipients (Fig. 1A). The serial transplantation technique has been used extensively to study the biology of hematopoietic stem cells (HSCs) [14–19]. The effect of HOXA9 on the expansion in vivo of the HSC pool and the importance of telomere length for the replicative capacity of HSCs were revealed using this method [14–16]. It was also used to determine so-called seeding efficiency, which is the proportion of HSCs that have colonized recipients after transplantation [17–19].
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Using serial transplantation, we have previously demonstrated the proliferation kinetics of SSCs derived from adult intact testes (hereinafter referred to as adult intact SSCs) for 2 mo after transplantation [20]. The number of adult intact SSCs increased rapidly from 1 wk to 1 mo after transplantation, and thereafter, proliferation was reduced up to 2 mo. In the current study, we conducted serial transplantation analyses and evaluated the proliferation kinetics of pup SSCs for 2 mo after transplantation, and we then compared these results to the proliferation kinetics of adult intact SSCs. Since SSCs can only be detected retrospectively based on their regeneration activities after transplantation, and as definitive SSC markers are currently not available, the serial transplantation technique provides a unique opportunity to analyze SSC proliferation potential.
Pup (6–9 days postpartum) and adult donor testes cells were obtained from B6;129S-Gtrosa26Sor (designated as ROSA26; Jackson Laboratory, Bar Harbor, ME) transgenic mice, which express β-galactosidase in all types of male germ cells [21]. In some experiments, ROSA26 x C57BL/6 (B6) F1 heterozygous pups were used as donors. Since there was no significant difference between the ROSA26 and ROSA26 x C57BL/6 F1 mice in terms of SSC colonization, these results were combined. Adult cryptorchid mice were generated by suturing the testes to the abdominal wall for 2 mo, thus exposing the tested to a high core-body temperature, which eliminates differentiating germ cells [20, 22]. Consequently, cryptorchid testes were enriched ~25-fold for SSCs compared to adult intact testes [20, 22]. As recipients, B6 x 129 F1 hybrid mice were treated with busulfan (50 mg/kg) at 4 wk of age, to destroy endogenous spermatogenesis [21]. These mice were used as recipients at least 4 wk after the busulfan treatment. All protocols for animal handling and care were approved by the Animal Care and Use Committee of McGill University.
Donor Cell Preparation and Transplantation
Donor testes were obtained from pup, adult cryptorchid, and adult intact mice and were digested with collagenase IV, which separates interstitial cells from the seminiferous tubules. Following sedimentation, the seminiferous tubules were treated with trypsin-EDTA and DNase I, to yield a single cell suspension [21]. This suspension, which contained a heterogeneous population of testis cell types, was counted, passed through a cell strainer mesh (70-µm pore size), recounted to obtain an estimate of mesh recovery, and transplanted into recipient testes. Cell viability was assessed by trypan blue exclusion.
A serial transplantation strategy was used to determine the kinetics of SSC proliferation after transplantation (Fig. 1A), as described previously [20]. Briefly, donor cells were resuspended in Dulbecco modified Eagle medium plus 10% fetal bovine serum at an average concentration of 19.8 x 106 cells/ml (pup) or 27.1 x 106 cells/ml (cryptorchid). An aliquot of the cell suspension (4–7 µl) was measured using a micropipetter, carefully transferred into an injection needle, and injected into one recipient testis. The injection needle was changed for each recipient testis. Recipient testes into which the cell suspension was injected only partially were excluded from the experiments because of leakage into the interstitial tissues. The number of donor cells injected into a recipient testis was calculated from the donor cell concentration and the volume of cell suspension injected. Following transplantation, the primary recipients were separated into control and experimental groups (Fig. 1A). Recipient mice from the control group were killed 2 mo after transplantation and analyzed for colony formation (see below). Experimental group recipients were killed at different time-points (1, 3, 7, and 14 days, and 1 mo and 2 mo) after the primary transplantation (Fig. 1A). At each time-point, a pool of primary recipient testes was digested enzymatically as above, to prepare a secondary donor cell suspension (100 x 106 cells/ml) that had 90.5 ± 1.0% mesh recovery and 98.3 ± 0.2% cell viability. A known number of donor cells was injected into the secondary recipient testes, which was determined based on the concentration and injection volume of the secondary donor cell suspension, as described above. The secondary recipient testes were analyzed for colony formation 2 mo after the final transplantation (Fig. 1A).
To quantify SSCs, the numbers of donor-derived spermatogenic colonies were counted. Since one colony arises from one SSC [23, 24], the number of colonies represents the number of functional donor SSCs that colonizes the recipient testis. The colonies were visualized by staining recipient testes with 5-bromo-4-chloro-3-indolyl β-D-galactoside (X-Gal) at 2 mo after the final transplantation [21] (Fig. 1, B–E). Colony numbers were normalized per 106 original donor testis cells injected [20]. Paraffin sections were prepared for some primary colonies after counting (Fig. 1, F and G).
To evaluate the potential of SSCs to produce differentiated germ cells during spermatogenesis regeneration, the lengths of the colonies in the primary recipients were measured at 1 mo and 2 mo post-transplantation, using an eyepiece micrometer under a dissecting microscope [21]. The morphologies of the colonies that arise from pup SSCs and adult SSCs were similar at 1 mo and 2 mo post-transplantation (Fig. 1, B–E).
All values are expressed as mean ± SEM. Statistical analyses were performed using linear regression analysis or ANOVA followed by the Fisher least significant difference multiple comparisons test. Differences were considered as significant for P 
0.05.
We analyzed the proliferation kinetics of SSCs derived from the testes of 6- to 9-day-old pups (termed pup SSCs) using a serial transplantation technique that allowed us to monitor changes in SSC numbers in recipient testes after transplantation (Fig. 1A). In this procedure, pup testis cells were first injected into the testes of animals designated as the primary recipients, some of which were analyzed 2 mo after transplantation for the formation of donor-derived spermatogenic colonies in their testes. The colony number reflects the number of functional SSCs transplanted into the primary recipient testes. At different time-points over the course of the study, the testis cells of the remaining primary recipients were transplanted into secondary recipient testes (Fig. 1A). The number of colonies in these testes was determined 2 mo later. The recolonization index was derived by dividing the number of colonies in the secondary recipient testes by the number of colonies in the primary recipient testes. Thus, the recolonization index reflects the number of functional SSCs that were present in the primary recipient testis at different time-points after transplantation, relative to the number of SSCs that was originally injected into the primary recipient (Fig. 1A).
Pup SSCs Show Similar Seeding Abilities as Adult SSCs after Transplantation
Adult intact SSCs decrease in number during the first post-transplantation week, before showing a pronounced increase in number over the following three weeks [20]. Thus, adult SSCs require 7 days after transplantation to seed and to settle in the recipient seminiferous epithelium and to begin proliferation. Therefore, in order to evaluate the proliferation kinetics of pup SSCs, it was first necessary to determine the point at which the proliferation of pup SSCs begins after transplantation. Serial transplantation analysis at 1, 3, and 7 days after the primary transplantation showed that the recolonization index of pup SSCs decreased continuously up to 7 days after transplantation (Fig. 2). Regression analysis indicated that the decrease in the recolonization index was linear from Day 1 to Day 7. Therefore, the results indicate that, similar to adult SSCs, the number of pup SSCs declines during the first week after transplantation, and the lowest recolonization index is achieved at Day 7 (11.4 ± 2.3%). Importantly, the lowest recolonization index for the pup SSCs was very similar to that obtained for adult SSCs derived from intact or cryptorchid testes (12.8 ± 5.7% and 11.2 ± 1.9%, respectively, P = 0.972) [20]. The Day-7 recolonization index of SSCs of all the donor ages combined was 12.1 ± 3.1%. Taken together, these results indicate that regardless of donor age, 12.1% of SSCs seed and survive in the recipient seminiferous epithelium after transplantation and remain active for the regeneration of spermatogenesis.
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Pup SSCs Expand More Slowly than Adult SSCs after Transplantation
Having determined the starting point of SSC proliferation, we analyzed the proliferation kinetics of pup SSCs. To this end, we retransplanted testis cells into the secondary recipients 2 wk to 2 mo after the primary transplantation, and counted the colony numbers 2 mo later (Fig. 1A). The recolonization indices of the pup SSCs were 44.6% and 108.1% at 1 mo and 2 mo, respectively (Fig. 3A). As described above, 12.1% of the transplanted SSCs seeded the recipient testes after 1 wk, irrespective of donor age. Therefore, these results indicate that pup SSCs proliferate 3.7-fold (44.6/12.1) in the period from 1 wk to 1 mo post-transplantation, and 8.9-fold in the period from 1 wk to 2 mo post-transplantation. Thus, after seeding in the primary recipient testis, pup SSCs increase continuously in number in the period from 1 wk to 2 mo post-transplantation.
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A comparison of the proliferation kinetics of pup and adult intact SSCs indicates a significant difference between the two SSC populations. In a previous study [20], we demonstrated that the recolonization index of adult intact SSCs was 90.2% at 1 mo and 95.7% at 2 mo post-transplantation, indicating that adult intact SSCs initially proliferate rapidly, but show reduced proliferation thereafter, reflecting a long population doubling time (>80 days [25]). Thus, the recolonization index for pup SSCs was 2-fold (44.6% vs. 90.2%, P < 0.007) less than that for adult intact SSCs at 1 mo, which implies that the proliferation of pup SSCs is delayed after transplantation. Despite these differences, the net expansion of SSCs from 1 wk to 2 mo post-transplantation is similar between pup and adult intact SSCs (8.9-fold vs. 7.9-fold). These results collectively indicate that although SSCs proliferate more slowly in primary recipient testes than do adult intact SSCs after transplantation, they attain a similar level of expansion by 2 mo post-transplantation.
Balance Between Self-Renewal and Differentiation May Be Skewed Towards Differentiation in Pup SSCs
Since pup SSCs expand more slowly than adult intact SSCs over the first month after transplantation, it is possible that the colonies derived from pup SSCs are smaller at 1 mo than those derived from adult intact SSCs. To address this possibility, we determined the lengths of the colonies in the primary recipient testes and compared them between pup and adult intact SSCs at 1 mo post-transplantation. The colony length represents the ability of an SSC to produce differentiating germ cells for the regeneration of spermatogenesis. As shown in Table 1 (column 3), the colonies in the primary recipients at 1 mo appeared to be longer in the pup SSCs than in the adult intact SSCs, although the difference was not significant (P < 0.066). These data indicate that despite a slower rate of proliferation, pup SSCs produce spermatogenic colonies of a similar size as those produced by adult intact SSCs.
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These results suggest that the expansion of pup SSCs is reduced in comparison to adult intact SSCs, since they produce more differentiating germ cells at the expense of self-renewal by 1 mo post-transplantation. To provide a quantitative measurement to assess this notion, we determined the size of the region supported by a single SSC within a colony in a primary recipient testis (a primary colony). As described above, pup SSCs had proliferated 3.7-fold by 1 mo post-transplantation. Since one colony originates from one SSC [23], this implies that on average, 3.7 SSCs, which are descendants of an original colonizing SSC, are present in a primary colony 1 mo post-transplantation. At this time-point, the average colony length was 1.31 mm (Table 1). Thus, a single pup SSC supported a 0.35-mm segment (1.31/3.7) of a colony at 1 mo post-transplantation. In contrast, one adult intact SSC was found to support a 0.15-mm segment of a colony (Table 1). The colony length per SSC was significantly higher for pup SSCs than for adult SSCs. These results imply that one pup SSC produces a significantly higher number of committed germ cells in the primary recipient testes by 1 mo post-transplantation, as compared to one adult intact SSC. Therefore, the balance between self-renewal and differentiation is apparently shifted toward differentiation in pup SSCs by 1 mo post-transplantation. Thus, even though pup SSCs expand at a slower pace, they are capable of producing similar size colonies as adult intact SSCs.
To determine whether the difference in the abilities of pup and adult SSCs to self-renew or differentiate is maintained at a later time post-transplantation, we conducted a similar analysis on 2-mo primary colonies. Although colony length was greater for the pup SSCs than for the adult intact SSCs (Table 2, column 3), the colony length supported by a single SSC was similar for both SSC populations (column 5). These results imply that at 2 mo post-transplantation, a single SSC derived from a pup or adult intact testis supports a similar number of daughter germ cells in a colony, and further suggest that the balance between self-renewal and differentiation is similar for both types of SSCs.
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Adult SSCs Derived from Cryptorchid Testes Show Similar Proliferation Kinetics as Pup SSCs
Finally, we examined the proliferation kinetics of adult SSCs derived from experimental cryptorchid testes (hereinafter, cryptorchid SSCs) using serial transplantation. In these testes, meiotic germ cells are eliminated, and the only germ cell types that remain are primitive spermatogonia, resulting in SSC enrichment in vivo [22]. As such, experimental cryptorchidism has been widely used to study SSC biology. For example, previous studies have reported that SSCs actively divide in cryptorchid testes [26, 27].
The results of the serial transplantation experiments showed that the cryptorchid SSCs behaved in a manner similar to the pup SSCs (Fig. 3B). The recolonization index for the cryptorchid SSCs at 1 mo was significantly lower than that at 2 mo post-transplantation. Thus, as observed with the pup SSCs and in contrast to what was seen with the adult intact SSCs, cryptorchid SSCs proliferate continuously from 1 wk to 2 mo post-transplantation. Furthermore, the length of a colony supported by one cryptorchid SSC was significantly greater at 1 mo but similar at 2 mo post-transplantation, compared to the corresponding colony lengths of adult intact SSCs (Tables 1 and 2). Therefore, SSCs derived from adult cryptorchid testes appear to proliferate more slowly and produce more committed germ cells by 1 mo post-transplantation. These results suggest that while delayed expansion of SSCs after transplantation is not observed with adult intact SSCs, it can be induced in the adult SSC population by exposure to a cryptorchid testis environment.
SSCs are the only cell population in the body that can self-renew and differentiate for a lifetime and that allows transmission of genetic information from one generation to the next. Despite these biologically important roles, the patterns of SSC proliferation and differentiation and the mechanism that regulates SSC fate decisions are largely unknown. Analyses of SSC behavior have proven difficult, partly because definitive SSCs cannot be detected prospectively in situ but only retrospectively through experimental intervention, i.e., spermatogonial transplantation. In the present study, we carried out serial transplantation experiments that allowed us to observe the proliferation potential that pup SSCs can exhibit after transplantation. The results demonstrate that pup SSCs proliferate continually from 1 wk to 2 mo post-transplantation. In addition, our results indicate that pup SSCs show delayed proliferation after transplantation, compared to adult intact SSCs, and suggest that SSCs may alter their potential to self-renew or differentiate during postnatal development.
The following observations support our conclusions. First, the proliferation of pup SSCs was significantly higher at 2 mo post-transplantation than at 1 mo post-transplantation (Fig. 3A). Second, at 1 mo post-transplantation, the pup SSCs showed markedly lower proliferation than the adult intact SSCs (Table 1). Third, the lengths of the colonies that originated from the pup and adult SSCs were similar at 1 mo post-transplantation, even though the number of SSCs in a single colony, which were descendants of a single originally transplanted SSC, was lower in a pup-derived colony (Table 1). To generate a similar size of colony with fewer SSCs, a transplanted pup SSC had to produce more daughter cells that were committed to differentiation. Therefore, the data suggest that during the first month after transplantation, pup SSCs differentiate more frequently and produce more differentiated germ cells at the expense of proliferation.
We have also found that the self-renewal/differentiation balance of pup SSCs remains constant for 2 mo after transplantation, whereas that of adult intact SSCs shifts from preferred self-renewal to differentiation over the same period. As shown in Tables 1 and 2, the colony length supported by one pup SSC was 0.35 mm at 1 mo and 2 mo post-transplantation, whereas that supported by one adult intact SSC increased 2-fold (0.15 mm vs. 0.32 mm). Thus, pup SSCs apparently maintain a fate decision balance between self-renewal and differentiation over the 2-mo period after transplantation. In contrast, adult intact SSCs show a transition in fate decision kinetics during the 2-mo study period. Initially, they appear to favor self-renewal but thereafter, they frequently commit to differentiation and produce more progenitor germ cells than during the first month after transplantation.
In order for transplanted SSCs to expand the stem cell population, they must undergo symmetric divisions, whereby one SSC produces two daughter stem cells. In contrast, asymmetric SSC division produces one SSC and one committed progenitor. This allows transplanted SSCs to initiate the regeneration of spermatogenesis but prevents them from expansion, as the number of SSCs does not change after an asymmetric cell division. The results of the present study show that although pup SSCs proliferate after transplantation, they do not expand as rapidly as adult SSCs, which suggests that pup SSCs favor asymmetric cell division. Thus, their fate decision potential appears to be skewed towards differentiation, as compared to adult intact SSCs.
An interesting observation in the present study was that adult cryptorchid SSCs behaved in a manner similar to pup SSCs after transplantation. Compared to adult intact SSCs, cryptorchid SSCs expanded at a slower rate during the first month after transplantation, while producing colonies of similar length. These observations suggest that the delayed proliferation of SSCs may be associated with donor age (pup vs. adult) and/or testis environment (intact vs. cryptorchid testes). Although the mechanism underlying this stem cell behavior remains to be elucidated, it is interesting to note that cryptorchid SSCs are known to undergo cell cycling, as they do not enter G1/G0 arrest and are efficiently labeled with anti-proliferating cell nuclear antigen antibodies [26, 27]. Therefore, both pup and cryptorchid SSCs divide actively, yet they expand slowly after transplantation, compared to adult intact SSCs. These observations suggest that cell cycle activity may be a factor that affects SSC self-renewal and differentiation. Indeed, recent studies have demonstrated that the active division of HSCs leads to a reduction or a depletion of the HSC population [28]. The loss of PTEN activity drives quiescent HSCs into the cell cycle, which results in the eventual depletion of the stem cell reserve, suggesting that increased cell cycle activity may be associated with a reduction in HSC self-renewal [29, 30]. Accordingly, PTEN mutant HSCs are unable to maintain long-term reconstitution of hematopoiesis, even though they undergo differentiation into multiple lineages [29, 30]. Thus, cell cycle activity may be closely related to the regulation of self-renewal and differentiation of various stem cell types. Further studies are needed to address this possibility regarding the SSC fate decision mechanism.
The differences in the proliferation profiles of pup, adult cryptorchid, and adult intact SSCs may arise from differences in the environments of the donor and recipient testes. Compared to adult intact testes, SSCs in pup and adult cryptorchid testes are exposed to an environment that contains few differentiating germ cells. In addition, a difference in the endocrine environment of the donor testis may affect SSC proliferation. For example, it has been reported that in mice, the intratesticular testosterone (ITT) concentration in pup testes is initially high at birth but rapidly decreases to low levels during the first postnatal week [31]. Thereafter, the ITT concentration fluctuates, with peaks at 40 and 90 days of age [31]. In contrast, the intratesticular concentrations of inhibin and activin in rats have been found to be high during the first postnatal week and to decrease subsequently to low levels in the adult testis [32]. Thus, the activities of SSCs, and perhaps, their cell cycle progression, may be affected by the cellular and/or endocrine/growth factor environment of the donor testes before transplantation. Similarly, the recipient testis environment may affect SSC activities after transplantation. Further studies are necessary to examine the effects of testis environments on the regulation of SSC activity.
We focused our discussion on the self-renewal and differentiation of SSCs, even though cell death and quiescence are also elements of SSC fate choice. The following two observations provide the rationale for our focus. First, the results of the present study suggest that SSC death is not likely to be a major factor in the slow expansion of pup SSCs. Although SSC death does occur upon transplantation, the survival rates of the adult and pup SSCs were similar on Day 7 post-transplantation (~12%), at which time-point the lowest number of colonizing SSCs was observed. Therefore, the mortality rate for pup SSCs after transplantation does not appear to exceed that for adult SSCs under the experimental conditions used in the present study. Second, previous studies have indicated that SSCs do not remain dormant upon transplantation, but continuously regenerate spermatogenesis after transplantation [21, 25]. Thus, it is unlikely that the commitment of SSCs to a quiescent state contributes significantly to the change in their expansion after transplantation. Even if some SSCs become quiescent, the data obtained in the present study imply that pup SSCs that remain active differentiate more frequently than adult intact SSCs. In addition, it is currently difficult, if not impossible, to analyze directly SSC death and quiescence, since the detection of these two states requires the prospective identification of target cells, which is not yet feasible for SSCs. Ultimately, the development of an SSC identification technique will allow us to evaluate thoroughly the SSC fate decision patterns, including SSC death and quiescence.
Finally, we have shown that pup SSCs seed the seminiferous epithelium as efficiently as adult SSCs after transplantation. A similar observation has been made for HSCs; adult HSCs and fetal liver HSCs show a similar seeding efficiency for adult bone marrow [19]. Therefore, the seeding abilities of SSCs and HSCs, as well as the machinery required for this process, may be established during the early periods of their development.
Currently, information regarding SSC fate decision patterns and the underlying mechanisms is limited. Therefore, the results obtained in the present study provide an important foundation for more intensive investigations that will lead to a better understanding of SSC biology. Further studies to compare the biological properties of pup and adult SSCs, as well as the molecules that are expressed in these two SSC populations may contribute to the elucidation of the SSC fate decision mechanism.
ACKNOWLEDGMENTS
We thank Drs. H. Clarke, R. Farookhi, and F. Clerk for suggestions regarding this manuscript.
FOOTNOTES
1Supported by the Canadian Institutes of Health Research (MOP-49444) and the Canadian Foundation for Innovation (4177). K.T.E. is supported in part by the Stem Cell Network. M.C.N. is a Fondation pour la Recherche en Sante du Quebec scholar. 
Correspondence: 2Makoto Nagano, Royal Victoria Hospital, F3.07, 687 Pine Avenue West, Montreal, Quebec, Canada H3A 1A1. FAX: 514 843 1662; e-mail: makoto.nagano{at}muhc.mcgill.ca
Received: 25 October 2006.
First decision: 29 November 2006.
Accepted: 15 January 2007.
REFERENCES
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  J. R Yeh, X. Zhang, and M. C Nagano Establishment of a Short-Term In Vitro Assay for Mouse Spermatogonial Stem Cells Biol Reprod, November 1, 2007; 77(5): 897 - 904. [Abstract] [Full Text] [PDF]
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