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 unidirectionally 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 2 n 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).* To whom correspondence should be addressed. shosei@nibb.ac.jp. The study of the spermatogenic stem cell compartment in mammals also relies on the heterogeneity in the cyst length (9,11,12 (Fig. S1).The prevailing rodent stem cell model (14,15) (Fig. 1A) assumes that the stem cell population resides in the A s population and that cyst length reflects the extent of differentiation in a linear manner (9,11). A corollary of this 'A s model' is that A s spermatogonia are functionally homogeneous, that all A s 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 simplici...

To clarify the mechanisms that support the continuity of actively cycling tissues of long-lived organisms, we investigated the composition of a mouse spermatogenic stem cell system by pulse-chase of the undifferentiated spermatogonia, the population responsible for stem cell functions, in combination with transplantation and regeneration assays after pulse-labeling. We demonstrate that in addition to "actual stem cells," which are indeed self-renewing, a second population ("potential stem cells") also exists, which is capable of self-renewing but do not self-renew in the normal situation. Potential stem cells rapidly turn over in normal testes, suggesting that they belong to the transit-amplifying, rather than the dormant, population. During the long natural course, actual stem cells are occasionally lost and compensated for by progeny of their neighbors. In this process, potential stem cells are postulated to shift their modes from transit amplification to self-renewal, thus playing an essential role to ensure spermatogenesis integrity.

SummaryThe identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing “As model,” stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFRα1+ cells and find that, during steady-state spermatogenesis, the entire GFRα1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFRα1+ cells during steady state and postinsult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis.

Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3 -lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3 + lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3 + lineage) or differentiating spermatogonia (Ngn3 -lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.

In cycling tissues, adult stem cells may be lost and subsequently replaced to ensure homeostasis. To examine the frequency of stem cell replacement, we analyzed the population dynamics of labeled stem cells in steady-state mouse spermatogenesis. Our results show that spermatogenic stem cells are continuously replaced, on average within 2 weeks. The analysis exposes a simple and robust scaling behavior of clone size distributions that shows stem cell replacement to be stochastic, meaning that stem cells are equipotent and equally likely to be lost or to multiply to replace their neighbors, irrespective of their clonal history. The surprisingly fast rate of stem cell replacement is supported experimentally by 3D clone morphology and by live-imaging of spermatogonial migration. These results suggest that short-lived stem cells may be a common feature of mammalian stem cell systems and reveal a natural mechanism for matching the rates of cell proliferation and loss in tissue.

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Summary In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on “mitogen competition.” We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

This study investigated the effects of green tea polyphenol on the serum antioxidative activity and cholesterol levels of cholesterol-fed rats and compared them with those of probucol, an antioxidant hypocholesterolemic agent. To evaluate the antioxidative activity, the susceptibility to oxidative modification of low-density lipoprotein (LDL) isolated from the serum of cholesterol-fed rats was measured, as was the serum antioxidative activity using the spontaneous autoxidation system of brain homogenate. Administration of green tea polyphenol effectively inhibited LDL oxidation and elevated serum antioxidative activity to the same degree as probucol. However, higher amounts of polyphenol than probucol needed to be administered to reduce the total, free, and LDL cholesterol levels. Furthermore, green tea polyphenol increased the levels of high-density lipoprotein (HDL) cholesterol, leading to dose-dependent improvement of the atherogenic index, an effect that was not seen with probucol. Thus, green tea polyphenol may exert an antiatherosclerotic action by virtue of its antioxidant properties and by increasing HDL cholesterol levels.