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Stem cells support tissue maintenance by balancing self-renewal and differentiation. In mice, it is believed that a homogeneous stem cell population of single spermatogonia supports spermatogenesis, and that differentiation, which is accompanied by the formation of connected cells (cysts) of increasing length, is linear and nonreversible. We evaluated this model using lineage-analysis and live-imaging and found that this putative stem cell population is not homogeneous. Instead, the stem cell pool that supports steady-state spermatogenesis is contained within a subpopulation of single spermatogonia. Also, cysts are not committed to differentiation and appear to recover stem cell potential by fragmentation. Lastly, the fate of individual spermatogonial populations was dramatically altered during regeneration following damage. Thus, there are multiple and reversible paths from stem cell to differentiation, which may also occur in other systems.
Maintenance of adult tissues is supported by a small number of undifferentiated stem cells that self-renew to maintain their population and produce differentiating progeny for normal tissue function. It has generally been accepted that differentiating daughter cells progress uni-directionally towards terminal differentiation. This view has been recently challenged by data suggesting that under some circumstances differentiating cells can revert to the self-renewing stem cell pool (1–8). This apparent plasticity may add robustness to maintenance of the stem cell population during normal tissue maintenance and may play a crucial role in tissue regeneration following injury. However, the nature of the self-renewing stem cells and the plasticity of differentiating cells in the maintenance of tissue homeostasis and regeneration are mostly unknown, particularly in mammals.
Germ cells share a characteristic feature across all animal species. While the most primitive cells in adult gonads are singly isolated, their differentiating progeny remain connected by intercellular bridges to form syncytial cysts of 2n cells (9, 10). Thus, the length of the cysts reflects their cell division history or lineage. This unique feature has made the germline one of the most tractable systems to study adult stem cell self-renewal and differentiation (2, 3). The study of the spermatogenic stem cell compartment in mammals also relies on the heterogeneity in the cyst length (9, 11, 12). In the mouse testis, the most primitive subset of diploid germ cells (spermatogonia) includes Asingle (As, single isolated spermatogonia), Apaired (Apr, interconnected spermatogonial pairs), and Aaligned (Aal, interconnected 4, 8, or 16 spermatogonia; specifically termed Aal-4, Aal-8, and Aal-16, respectively). A vast majority of stem cell function, if not all, resides in this population. These cells transform without cell division into more differentiating A1 spermatogonia, which subsequently undergo 6 mitotic and 2 meiotic divisions to form haploid spermatids (10, 13) (Fig. S1).
The prevailing rodent stem cell model (14, 15) (Fig. 1A) assumes that the stem cell population resides in the As population and that cyst length reflects the extent of differentiation in a linear manner (9, 11). A corollary of this ‘As model’ is that As spermatogonia are functionally homogeneous, that all As cells are stem cells, and that all cells are equivalent in each morphological category (9, 10). This model, proposed in 1971, has provided the framework for years of germline stem cell research in mice and other animals. Despite its simplicity and attractiveness, the lack of appropriate molecular markers and experimental tools has hindered its critical evaluation.
In recent years, substantial progress has been made in identifying genes that are expressed in As cells and cysts of Apr and Aal (e.g. GFRα1, PLZF, E-Cadherin [E-CAD], and NGN3) (16–23). Heterogeneity in gene expression among cysts of the same length has suggested possible functional heterogeneity within cells of the same cyst length (21–23). In the present study we have used gene expression, cyst length, lineage analysis (6) and live-imaging (24) to revisit the long-held assumptions of the functionality of the spermatogonial population in mice.
Comparison of expression patterns of genes that mark the As, Apr and/or Aal population (16–23) by whole-mount double-staining of seminiferous tubules, the spermatogenic center of the testis, revealed that the two genes PLZF (17, 18) and E-CAD (21) have essentially identical expression patterns and are found in eventually all the As, Apr and Aal spermatogonia (Fig. S2 and Text S1). In contrast, two other genes, GFRα1 and NGN3, were expressed in minor and major subpopulations of the E-CAD+ total As, Apr and Aal population, respectively, with the same gene expression observed in all the cells within an individual cyst (Fig. 1B, C). Intriguingly, all the E-CAD+ cysts expressed either or both of these genes (Fig. 1E). Thus, spermatogonial cysts were heterogeneous in the expression of GFRα1 and NGN3 even in the same morphological fraction, except for Aal-16, which was essentially all NGN3+ (Fig. 1D).
Thus, the As, Apr and Aal population can be stratified by both morphology (cyst length) and gene expression (GFRα1 single-positive, GFRα1/NGN3 double-positive, and NGN3 single-positive). These two parameters are mutually correlated: shorter cysts have a greater probability of being GFRα1 single-positive while longer cysts tend to be NGN3 single-positive.
The observation that GFRα1+ cells are largely As or Apr, while NGN3+ cells are mainly Aal (Fig. 1D), suggests the transition of cells from GFRα1+ → NGN3+ → A1 spermatogonia. To investigate this, we used pulse-chase and live-imaging experiments.
First, we analyzed the fate of NGN3+ cells that were irreversibly labeled with GFP by a single administration of 4OH-tamoxifen (Fig. 2A) (6). Two days after the pulse, a majority of the GFP-tagged cells were E-CAD+/GFRα1−/KIT− (KIT, a marker for A1 spermatogonia through early spermatocytes (25) (Fig. 2B–D)). The labeling efficiency of the NGN3+ cells was ~30% for all morphological fractions (Fig. S3 and Text S2). Ten days after the pulse, most of the GFP-tagged cells were present in large cysts (>16 cells) that were E-CAD−/GFRα1−/KIT+ (Fig. 2E–H), indicating that most NGN3+ cells left the E-CAD+ compartment and went on to differentiate. Eventually all KIT+ spermatogonia were derived from NGN3+ cells, as they retain the GFP in the transgenic mice that express GFP under the control of the Ngn3 regulatory sequence (Ngn3/GFP) (16) (Fig. S4). Second, live-imaging of the testis of the same Ngn3/GFP mice (16, 24) was used to directly show that a gain of GFP signal, which reflects an induction of NGN3, was frequently observed in GFP− (i.e. GFRα1-single positive) As, Apr, and Aal spermatogonia (Figs. 2I and S5, Mov. S1). The minor GFRα1/NGN3-double positive cells are likely to be in transition from GFRα1 single-positive to NGN3 single-positive cells, for both signals were weaker in double-positive cells than in either single-positive population.
We then asked whether As spermatogonia are functionally heterogeneous. After a single pulse of 4OH-tamoxifen to permanently label NGN3+ cells with GFP, the contribution of the GFP-tagged cells to the entire As population rapidly decreased (Fig. 3A), indicating that the majority of the NGN3+ As cells left the As compartment. Live-imaging showed that NGN3+ As cells mostly became NGN3+ Apr, whereas a few of them divided into two As cells (Figs. 2I and S5, Mov. S1). In addition, two days after the pulse, a small number of labeled single cells were KIT+ (Fig. 3C), suggesting direct conversion of Ngn3+ As into A1 spermatogonia, although they might represent a novel and unique population. We conclude that As cells are not equivalent a vast proportion of NGN3+ As are essentially transit amplifying cells rather than self-renewing stem cells.
However, by day 20, a small number of GFP-tagged cells were still present in the As compartment (Fig. 3A). Interestingly, a majority of these persisting GFP-tagged As cells were also GFRα1+ (Fig. 3B). These data suggest that a small fraction of the NGN3+ cells can revert into GFRα1+ As. Theoretically, such “reverted” GFRα1+ As could be derived from NGN3+ As or from NGN3+ Apr and Aal, although the latter events would require cyst fragmentation.
Live-imaging of Ngn3/GFP transgenic mouse testes, in which NGN3+ cells were labeled with GFP, provided direct evidence of spermatogonial cyst fragmentation (Figs. 4 and S5), whose frequency was much lower than the normal divisions that double the cyst length (Fig. S5B–C). In one case out of the three observed, cyst #1 (Fig. 4 and Mov. S2), a GFP+ (i.e., NGN3+)Aal-8 cyst divided synchronously and then fragmented into two pairs of interconnected cells, which later divided into Aal-4, while the remainder of the 12 cell-cyst underwent synchronized death. The other two cysts showed different patterns of fragmentation, excluding the possibility of a stereotypic fragmentation pattern (Fig. S5C, Movs. S3 and S4). In these instances, the GFP signal decreased in many of the resultant shorter cysts and As, prompting us to postulate that they might have become GFRα1 single positive. Cysts with lengths other than 2n cells were also generated as a result of cyst fragmentation.
During steady-state spermatogenesis, the As, Apr and Aal subpopulations are by definition constant. However, during regeneration following damage, self-renewal is favored over differentiation (11). We investigated the fate of the As, Apr and Aal subpopulations during regeneration after administration of busulfan, a drug preferentially toxic to spermatogonia including stem cells (Fig. 5A–D). Approximately 8 d after busulfan treatment, most E-CAD+ cells had died, although some had formed small regenerating clusters. By day 18, such clusters became prominent, and the local density of E-CAD+ spermatogonia had recovered to a level comparable to untreated testes, although their average density was still low (Fig. 5B). Interestingly, during the recovery period we found that both cyst length and gene expression were altered: the average cyst length became shorter and a greater percentage of cells were GFRα1+ (Fig. 5C–D).
A change in these two parameters during recovery could reflect preferential elimination of longer NGN3+ cysts, a decrease in their formation, an increase in cyst fragmentation, and/or reversion from NGN3+ to GFRα1+. Although the first possibility is not excluded, fate analysis of the pulse-labeled NGN3+ cells is compatible with the other three scenarios (Fig. 5E–I). A higher percentage of labeled GFP+ cells (which had been NGN3+ at the time of labeling) were retained in the E-CAD+ population during regeneration than during steady-state spermatogenesis (Fig. 5F). Moreover, a significantly higher percentage of the labeled cells contributed to the E-CAD+ As fraction (Fig. 5G), and were positive for GFRα1 (Fig. 5H). We also found that the percentage of labeled cells in the GFRα1+ As population increased during regeneration (Fig. 5I). These findings indicate that either the labeled NGN3+ As remained as As and “reverted” back to being GFRα1+, or alternatively, NGN3+ Apr or Aal gave rise to GFRα1+ As through cyst fragmentation.
In addition, the frequency of the GFRα1+ Aal-8 and Aal-16, which were observed in steady-state only rarely, was elevated during regeneration (Fig. S7). This indicates either a delay of the GFRα1+ → NGN3+ transition, reversion from NGN3+ → GFRα1+ in long cysts, or both. Finally, as observed previously (6), the contribution of the labeled cells to the long-term stem cell pool in regeneration was much higher than in steady-state spermatogenesis.
We conclude that the As, Apr and Aal spermatogonial subpopulations dramatically change their behavior during regeneration thus enabling a quicker recovery of the stem cell pool.
Our results demonstrate a variety of different properties of the As, Apr and Aal subpopulations that comprise the stem cell compartment (Fig. 6). Differentiation does not follow a strictly linear process where gene expression is coupled to lineage (cyst length), but includes multiple pathways along the two parameters. For example, NGN3+ Aal-4 can be generated either by division of NGN3+ Apr or by gain of NGN3 expression in GFRα1+ Aal-4.
This model proposes a number of important extensions to the As model (Fig. 1A). First, regardless of cyst length, the NGN3+ subpopulations including As are destined for differentiation. Thus, not all As cells act equivalently as stem cells. Second, Apr and Aal spermatogonia are not committed unidirectionally to differentiation but are capable of reverting to As or shorter cysts by clone fragmentation. In some cases this may also be accompanied reversion in gene expression. Clone fragmentation has been previously proposed in mice (11, 26) and primates (27, 28) and demonstrated in Drosophila (4, 5), and this study has directly observed this event by live-imaging in vivo. Third, of the two parameters, gene expression appears to be the better indicator of the fate of individual cells over the cyst length. For example, GFRα1+ cells transform directly into Kit+ spermatogonia only occasionally (Fig. S4), and eventually all the KIT+ spermatogonia are generated from the NGN3+ population, regardless of their cyst length. Based on the increasing transformation frequency into A1 spermatogonia along the cyst length, it has been proposed that the differentiation potential increases gradually (Fig. 1A) (29). Our data suggest that this is a reflection of the increasing content of NGN3+ cells.
It has been generally assumed that As cells represent the entire spermatogenic stem cell population and that they support both steady-state spermatogenesis and regeneration after tissue damage or transplantation (9, 12, 30). However, our findings challenge this assumption.
First, this study demonstrated that NGN3+ cells can revert to being GFRα1+ As cells and act even as long-lasting stem cells. The frequency of these events dramatically increases when tissue is damaged and regeneration takes place. This suggests that these “differentiating” cells can act as “potential stem cells”, defined as cells that do not normally self-renew during steady-state spermatogenesis but nonetheless retain latent self-renewing potential (6, 31). Live-imaging of clone fragmentation suggests that the potential stem cells include not only the NGN3+ As, but also Apr and Aal (either NGN3+ or GFRα1+) cells. Indeed, the NGN3+ cell population, most of which are present in connected cysts, exhibit significant colony formation and contribution to regeneration (6).
On the other hand, the population of GFRα1+ As, which is positioned at the top of both hierarchies, is best related to the “actual stem cells” that support normal steady-state spermatogenesis (6, 31). In the pulse-labeling experiment in undisturbed testes, however, (Figs. 2 and and3),3), the absolute number of GFP-tagged GFRα1+ As (that were originally NGN3+ at the time of labeling) decreased by day 20. This was still larger than the number of persistent patches observed three months after the pulse, which represent the long-lasting stem cells (Fig. S6). Similarly, during regeneration after damage, the number of persisting patches, which was significantly larger than that found in steady-state, was smaller than the count of GFP-tagged GFRα1+ As (Fig. S6). Thus, only a part of the GFRα1+ As persist for a long period to serve as functional stem cells and that a majority of this population do not self-renew continually and may act as “potential stem cells”, too.
The recent report of colony-forming activity in a small fraction of KIT+ spermatogonia (8) may also represent “potential stem cells”, which could reside in the minor populations of GFRα1+/KIT+ (Fig. S4) or the single and paired KIT+ spermatogonia that have been converted directly from NGN3+ As and Apr (Fig. 3C).
In summary, reference to a single homogeneous cell population serving both steady-state and regeneration is unnecessarily constraining. Rather, cells seem to possess a variable level of potential to act as stem cells. How their potential is manifested can be greatly influenced by the state of the tissue. Steady-state spermatogenesis favors the GFRα1+ As population, which may have the greatest potential, while regeneration following transplantation or damage relies on the NGN3+ and cyst (Apr and Aal) spermatogonia as well, whose potential seems to be lower.
The molecular mechanisms governing the transition between the GFRα1+ and NGN3+ populations have yet to be defined. In this regard, the finding that GDNF (glial cell line-derived neurotropic factor), the ligand for GFRα1, regulates the spermatogonial expression of GFRα1 and NGN3 in a reciprocal manner (i.e., positively and negatively, respectively) (32) suggests that GDNF may be an important determinant of stem cell behavior. Indeed, Sertoli cells, an essential player for the maintenance of the spermatogenic stem cell population, express GDNF, which is essential for the long-term maintenance of spermatogenic stem cell activity in vivo (19) and crucial for spermatogonial cultures to maintain the ability of post-transplantation colony-formation (33, 34).
In Drosophila, male and female germ cells appear to differentiate along a linear pathway in regards to lineage and gene expression. Their differentiation state is geographically recapitulated in the polarized gonad as a consequence of localized specialized supporting cells and extracellular factors that control self-renewal and differentiation (3). In contrast, in mouse testis, the stem cell compartment (Fig. 6) does not appear to be spatially constrained. Rather, while showing biased localization to the blood vessels and interstitium (24), they are intermingled among differentiating germ cells and seemingly uniform supporting Sertoli cells. Given these anatomical differences it is not surprising that distinct controlling mechanisms exist between flies and mice.
Within mammals, the primate stem cell compartment appears to differ from that in mice. In primates, primitive spermatogonia, referred to as Adark and Apale based on nuclear morphology, are generally assumed to represent the stem cell pool (11, 28). The Adark are the presumptive reserve stem cells that rarely proliferate, while the Apale cells represent the active stem cell pool and are continuously cycling. While NGN3+ spermatogonia proliferate actively, the cell cycle status of GFRα1+ population is yet to be elucidated. It is an intriguing question whether there exists a reserve population of GFRα1+ cells in mice, equivalent to the Adark in primates, or whether a reserve population of stem cells is unique to primates.
The biological significance of the syncytial nature of spermatogonial proliferation across animal species remains a mystery. Nonetheless, it provides a powerful tool to monitor gene expression in the context of cell lineage. In other stem cell systems, especially in mammals, stem and progenitor cell compartments are often classified based on gene expression and location: correlation of lineage and gene expression has generally not been feasible. Our study demonstrates that lineage is not strictly and linearly correlated with gene expression and that there may be multiple and reversible paths from stem cell to differentiation in other systems.
We are grateful to J. Miyazaki, T. Noce, and A. Imura for materials, to T. Ogawa, R. Sugimoto, Y. Kitadate, K. Hara and H. Mizuguchi-Takase for comments, to T. Fujimori for discussion and technical advice, and to M. Sukeno for technical assistance. This work was partly supported by a Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas, “Regulatory Mechanism of Gamete Stem Cells” to S. Yoshida, and a grant from the NICHD/NIH Contraceptive Development Research Centers Program (U54 HD42454) to R. E. Braun. The Uehara Memorial Foundation and the Naito Foundation also supported this work.