National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professors:
NIBB Research Fellow:
Technical Staff:
Graduate Students:
Technical Assistants:
Visiting Scientist:
Secretary:

HARA, Kenshiro
KITADATE, Yu
HARA, Kenshiro*
MIZUGUCHI-TAKASE,Hiroko
IKAMI, Kanako
TOKUE, Moe
ICHIKAWA, Rie
INADA, Kana
SUGIMOTO, Ryo
SUGIMOTO, Ryo*
KUBOKI, Yuko

Mammalian spermatogenesis represents a highly potent androbust stem cell system. Decades of research, including
detailed morphological examinations, post-transplantation
repopulation, and in vitro culture, have made it one of themost intensively studied mammalian tissue stem cellsystems. However, the nature of the stem cells and theircontrol, as well as their niche, remains largely unknown. The
Division of Germ Cell Biology aims to fully understand themouse sperm stem cell system.
In 2010, we revealed a couple of important features ofmouse sperm stem cells. One is the discovery of a hierarchy
between subpopulations of ‘undifferentiated spermatogonia’,in which the stem cell functions reside, and reversibility
between these subpopulations. This was a proposal thatwarrants a re-evaluation of a long-held theory in this field.
Second is the finding that stem cells are not in a state where
they always experience asymmetric division and persist for
the entire life span of an organism, but that stem cells replaceeach other frequently and support spermatogenesis as apopulation.

I. H i e r a rc h y a n d re v e r s i b i l i t y w i t h i n
undifferentiated spermatogonia

Lines of morphological investigations that emerged in the1950s established the backbone of mammalianspermatogenesis research. The morphologically mostprimitive spermatogonia in the adult mouse testis are As orAsingle spermatogonia (single, isolated spermatogonia). Theirprogeny remain interconnected due to incomplete
cytokinesis, forming syncytial cysts of 2n cells (2, 4, 8, 16etc.). It has been experimentally established that“undifferentiated spermatogonia” (Aundif), which contribute<1% of the entire testicular cell population and consist of As,
Apr (Apaired; interconnected two-cell cysts), and Aal (Aaligned;
cysts of 4, 8, 16 or occasionally 32 cells) contain stem cells.
Undifferentiated spermatogonia then transform into A1 stageof ‘differentiating spermatogonia’, as they lose their vast
self-renewing potential (Figure 1).
The prevailing “A s model” (Figure 2), which wasoriginally proposed in 1971, supposes that As is the only cell
type that can act as stem cells, while the interconnected26

Figure 1. Spermatogenic cell types found in mature mouse testis.
Spermatogonia, spermatocytes, and spermatids correspond to the mitotic,
meiotic and haploid cells respectively. ‘Undifferentiated’ spermatogoniapopulation harbors the stem cell functions. Reprinted from Develop.
Growth Differ. 52, 311-317 (2010), with permission.

population of A undiff (A pr and A al) is devoid of stem cellcapacity. Similarly, corollaries of this model are that all theAs cells are functionally equivalent and uniformly act as thestem cells, and that this population plays active roles in every
aspect of the stem cell functions, i.e., maintenance of steadystate and regeneration after tissue insult and transplantation.
However comprehensive and persuasive the “As model” is,this was not based on the direct analyses of the cells’
behavior. Therefore, the A s model warrants functional
evaluations. It was previously shown by our group that nosingle stem cell population acts in every aspect of stem cellfunction: Cells supporting the steady state spermatogenesis
are different from those that support regeneration (Nakagawa
et al., Dev. Cell, 2007). However, the precise cellular identitythat supports these stem cell functions remained to beelucidated.
In 2010, we revealed that a gene expression profile
visualizes the heterogeneous nature of the undifferentiatedspermatogonia population, in addition to the number ofchained cells or the length of the syncytial cysts (Nakagawa

Figure 2. The ‘As model’. Schematically shown on the basis of theories
proposed by the groups of Huckins, Oakberg and de Rooij. Reprinted
from Science 328, 62-67 (2010), with permission

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2010. The former title isindicated by an asterisk (*).

et al., Science 2010). We established that the populations ofcysts that compose the same number of spermatogonia areheterogeneous in their expression of GFRα1 (glial cell linederived neurotropic factor receptor 1; shown in magenta inFigure 3) and Ngn3 (neurogenin3; green) genes.
We then investigated the steady-state behavior of these
subpopulations of undifferentiated spermatogonia by meansof pulse-labeling and live imaging. It was shown that thebulk of the Ngn3+ population differentiate into longer cysts(rightward black arrows in Figure 3) and Kit+ differentiating
spermatogonia (shown in blue) as represented by downward
white arrows, and that the Ngn3+ cells are supplied fromGFRα1+ spermatogonia (downward black arrows). As alogical consequence, the GFRα1+ population is postulated toNgn3+ cells return to a self-renewing stem cell state much
more frequently than in steady state. These observations
proposes modification of the As model and explain how thestem cell population change their behavior so that theyremain constant in steady state, while increasing during
regeneration to recover the stem cell pool quickly.

II. Rapid and stochastic turnover between thesperm stem cells
It has been believed that stem cells, including that of
mammalian spermatogenesis, are preserved in steady-state
cycling tissues as they repeat an asymmetric division that
produces one self-renewing and one differentiating daughter
cell (Figure 5a). Indeed, such a “stem cell-type” division has
been observed only in a limited number of instances
including fruit fly germline stem cells, but remains to beevaluated in most systems including mouse spermatogenesis.

Figure 3. A modified version of ‘As model’. Based on the proposal byNakagawa et al. (2010). See text for the detail. Arrows with asterisks are
not confirmed but sill hypothetical. Reprinted from Science 328, 62-67(2010), with permission.

function as the main body of the self-renewing compartment.
Interestingly, a very small portion of Ngn3+ cells did‘revert’ back into being GFRα1+ and shorter chains (dottedarrows in Figure 3) in steady state. This accompanies
fragmentation of syncytial cysts as shown in Figure 4: Thisphenomenon was not assumed in the mouse ‘As model’,while observed in fruit fly germlines. During regeneration,Figure 4. An example of fragmentation of A al-16 cyst spermatogonia
observed by live imaging. Two pairs of spermatogonia (magenta andorange, morphologically defined as Apr ’s) were pinched off an Aal-16
spermatogonia. Elapsed times are shown.

Figure 5. A classical view of stem cell behavior based on the idea ofasymmetric division. (a) A single stem cell undergoes a sequence ofasymmetric division that produces one self-renewing and one
differentiating daughter cell. (b) Given that all the stem cells act as in (a),the stem cell repertoire and each stem cell’s differentiating progeny(shown in different colors) will be stable over time in the tissue.

We analyzed the long-term behavior of steady-state mouse
sperm stem cells that were pulse-labeled using an inducible
cre-loxP system for over a year after pulse (Klein et al., Cell
Stem Cell 2010; Nakagawa et al., Dev. Cell 2007), incollaboration with Ben Simons (Cambridge University).
If we suppose that the individual stem cells are preserved
as a result of repeated asymmetric divisions, the stem cell
repertoire should be constant and the number and the size ofthe stem cell cohorts will also be constant (Figure 5b).
However, the number of the observed stem cell-derived
clones decreased while the surviving clones expanded in size
(Figure 6a). Mathematical analyses indicate that stem cells
disappear frequently and stochastically with a surprisingly
short average longevity of less than two weeks, and that thelost stem cells are replenished by the progeny of theneighboring stem cells (Figure 6b).
These findings represent a new idea for the functionality ofa stem cell compartment, where cells replace each other andmaintain themselves and supply differentiating progeny as apopulation.

27

National Institute for Basic Biology

Developmental Biology
meiosis decision in male germ cells. Dev. Cell 19, 612-624.

T., Sharma, M., Nabeshima, Y., Braun, R.E., and Yoshida, S.
• Nakagawa,(2010). Functional hierarchy and reversibility within the murine••

Figure 6. Actual behaviors of pulse-labeled sperm stem cells inseminiferous tubules. (a) Upper panel, a LacZ-labeled single sperm stem
cell-derived clonal patch in seminiferous tubules three months afterpulse. Visualized by X-gal reaction. Lower panels: Number (left, pertestis) and size (right, length in mm) of the surviving pulse-labeled
patches indicated periods after pulse (shown in months). (b) A schematicof stem cell behavior in steady state spermatogenesis. Stem cells show
frequent replacement by their neighbors, making the stem cell repertoire
unstable.

III. Perspectives

We feel that the above findings elucidate novel andfundamental features of mouse sperm stem cells.
Accordingly one of the next steps is to provide a molecularbasis of the reversibility underlying stem cell differentiation
and reversion, as well as the mutual replacement happening
between the stem cells.
We will be also investigating aspects of the environmental
control of the stem cell population. One is the spatialr e g u l a t i o n : We h a v e p r e v i o u s l y o b s e r v e d t h a t
undifferentiated spermatogonia preferentially localize to thevasculature-proximal region (Yoshida et al., Science 2007),
while the details of the nature of these ‘niche’ regions is still
unknown. Second is temporal regulation: The differentiationof undifferentiated spermatogonia does not occur randomly
but shows a beautiful periodicity once every 8.6 days,
representing the seminiferous epithelial cycle.
We hope these studies will shed light and lead to the better
understanding the mouse sperm stem cell system.

Publication List

〔Original papers〕

Y., and Kobayashi, S. (2010). Notch and Egfr signaling act• Kitadate,antagonistically to regulate germ-line stem cell niche formation in••

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Drosophila male embryonic gonads. Proc. Natl. Acad. Sci. USA 107,14241-14246.
Klein, A.M., Nakagawa, T., Ichikawa, R., Yoshida, S., and Simons,B.D. (2010). Mouse germ line stem cells undergo rapid and stochasticturnover. Cell Stem Cell 7, 214-224.
Matson, C.K., Murphy, M.W., Griswold, M.D., Yoshida, S., Bardwell,V.J., and Zarkower, D. (2010). The mammalian doublesex homolog
DMRT1 is a transcriptional gatekeeper that controls the mitosis versus

spermatogenic stem cell compartment. Science 328, 62-67.
Nakane, Y., Ikegami, K., Ono, H., Yamamoto, N., Yoshida, S., Hirunagi,K., Ebihara, S., Kubo, Y., and Yoshimura, T. (2010). A mammalianneural tissue opsin (Opsin 5) is a deep brain photoreceptor in birds.
Proc. Natl. Acad. Sci. USA 107, 15264-15268.
Uemura, M., Hara, K., Shitara, H., Ishii, R., Tsunekawa, N., Miura, Y.,
Kurohmaru, M., Taya, C., Yonekawa, H., Kanai-Azuma, M., and Kanai,
Y. (2010). Expression and function of mouse Sox17 gene in thespecification of gallbladder/bile-duct progenitors during early foregut
morphogenesis. Biochem. Biophys. Res. Commun. 391, 357-363.

〔Review Article〕

S. (2010). Stem cells in mammalian spermatogenesis.
• Yoshida,Develop. Growth Differ. 52, 311-317.