DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:
NIBB Research Fellow:
Technical Staff:
Graduate Student:
Technical Assistant:
Secretary:

HARA, Kenshiro
KITADATE, Yu
NAKAMURA, Yoshiaki
MIZUGUCHI, Hiroko
IKAMI, Kanako
TOKUE, Moe
NONAMI, Yuta
INADA, KanaMARUYAMA, Ayumi
SUGIMOTO, Ryo
KUBOKI, Yuko

Mammalian testes produce numerous sperm for a long
period. This property that is fundamental for continuity oflife across generations is supported by the robust stem cellsystem, which is defined by the ability of proliferation that isassociated with self-renewal (maintenance of their ownundifferentiated population) and differentiation (productionof different cell type(s)),
The Division of Germ Cell Biology, which was launched in
2008, aims to fully understand the mouse sperm stem cellsystem in vivo under the context of tissue architecture oftestis. So far, we have revealed some characteristics of thispotent and interesting stem cell system. First, differentiating
germ cells that had been believed to be irreversiblycommitted to differentiation still retain the self-renewing
potential and can contribute to stem cell pool maintenance(“potential stem cells”). Secondly, “reversion” from potential
stem cells occurs at a higher frequency when testicular tissue
is damaged and regeneration is induced. Thirdly, theundifferentiated spermatogonia population that includes both
“actual” and “potential” stem cells localized to thevasculature (vascular- associated niche). We also discoveredthat stem cells turn over in an unexpectedly frequent andmathematically stochastic manner under steady-state
situations that continuously produce sperm. (Nakagawa etal., Dev. Cell 2007, Yoshida et al., Science 2007; Nakagawaet al., Science 2010; Klein et al., Cell Stem Cell 2010).
In 2012, we have extended these investigations for thedeeper understanding of this stem cell system. Firstly, we
have investigated the nature and the behavior of the stem
cells at a single cell resolution. Secondly, we have challenged
the cellular and molecular nature of the vascular-associatedniche. Thirdly, a mechanism underlying the coordinatedspatio-temporal regulation of stem cells has been proposed.

I. Stem cell behavior at a single cell resolutionin undisturbed testicular tissue
For the purpose of function-based investigation of stem
cells, it is essential to analyze the behavior of cells over time.
We have previously established experimental systems tostudy stem cell behavior in mouse spermatogenesis overtime, without affecting the tissue architecture, namely liveimaging and pulse-labeling experiments. For live-imaging,taking advantage of the fluorescent protein-labeling, we haveachieved continual filming of particular subsets ofspermatogonia in the testes (Figure 1: Yoshida et al., Science2007). For pulse-labeling, we have also establishedirreversible labeling of a particular spermatogonial
population using tamoxifen-dependent activation of cre
recombinase fused with a domain of the mutated humanestrogen receptor (Figure 2: Yoshdia et al., Development
2006, Nakagawa et al., Dev. Cell 2007, Nakagawa Science2010).

Figure 1. Live-imaging of fluorescence-labeled cells. An example of liveimaging observation of spermatogonia in a living testis. Spermatogonia
labeled with GFP under Ngn3 promoter (white) were continuously
filmed, shown cell division (30h and 76h) and active migration. A part of
selected frames are shown. Numerals indicate the elapsed time in hours.
(From Yoshida et al. Science 2007)

Figure 2. Pulse-labeling of subset spermatogonia with marker expression.
(A) Principle of pulse-label of a stem cell. When a self-renewing stem
cell is pulse-labeled, its descendants (shown in blue here) include bothstem and differentiating cells. Then, the clone will persist in the tissue, as
shown in the actual experiments in the following panels. (B)Spermatogonia labeled with LacZ expression (colored in blue) observed
2 days after induction of labeling with Tamoxifen administration. Asingle(1), Apair (2), Aaligned-4 (4), Aaligned-8 (8) are shown. (C) Three months after
the pulse, labeled cells that are descendant of the induced spermatogonia
form patches occupying a continual segment of the tubules includingstem and differentiating cells. This reflects the stem cell function of theoriginally labeled cell. (From Yoshida Reproduction 2012 and Nakagawa
et al., Dev. Cell 2007)29

National Institute for Basic Biology

Developmental Biology

In addition, our results strongly suggest that within theundifferentiated group of spermatogonia consisting of Asingle,
Apaired and Aaligned cells, a small subset expressing GFRα1(glial cell line-derived neurotropic factor receptor alpha 1) is
responsible for the stem cell functions, while thoseexpressing Ngn3 (neurogenin 3, a bHLH transcription factor)
are destined for differentiation while maintaining theirpotential to get back into being GFRα1-positive and selfrenew (Nakagawa et al., 2010).
In addition, in collaboration with Professor Benjamin
Simons, a theoretical physicist of Cambridge University, we
have shown that the spermatogenic stem cells show astochastic and continuous turnover between neighbors(Nakagawa et al., Dev. Cell 2007, Klein et al., Cell Stem Cell2010): Instead of the ‘text book’ type of asymmetric
divisions that always give rise to one self-renewing and one
differentiating daughter, fate selection for stem cells either toremain undifferentiated or to differentiate appears to bestochastic. As a result, fates of stem cell-derived cohorts arehighly variable and show neutral competition between stem
cell clones.
Based on these preceding findings, we began investigating
the behavior of GFRα1+ spermatogonia in undisturbed testis
using live-imaging and pulse-labeling at a single cellresolution. These have allowed us to directly demonstrate forthe first time that this tiny subset of spermatogonia indeed
acts as the stem cell pool supporting the steady-state turnoverof spermatogenic cells. We are now intensively challenging
the problem of how to link the actual single-cell-level
behavior (such as cell division, fragmentation of
spermatogonial syncytia, fate selection, whether todifferentiate or remain undifferentiated, and death) of theGFRα1+ spermatogonia and the overall robustness of thestem cell system at a level of population, with the aid ofmathematical modeling.

II. N a t u r e o f t h e s t e m c e l l n i c h e
microenvironment in the mouse testis
It is generally believed that some specialized
microenvironment within tissue is involved in the stem cellsystem as an indispensable element that regulates the stem
cell behavior, designated as the “stem cell niche”. In contrast
to well-defined examples of the stem cell systems
accompanying anatomically defined niches where stem cells
always settle and differentiation accompanies their exit fromthe niche region, such as Drosophila gonads, mammalian
testis do not exhibit such a defined substructure. Therefore it
has long been a mystery how stem cell behavior is regulated
in the context of this tissue, namely the seminiferous tubules(Figure 3). Regarding this issue, it has been shown that,within the ‘basal compartment’ of seminiferous tubules, theundifferentiated spermatogonia show a preferential
localization to the area adjacent to blood vessels that make anetwork running through the interstitial space between theseminiferous tubules (Chiarini-Garcia, Biology ofReproduction 2001, Yoshida et al., Science 2007). However,again, lacking a discrete niche structure, this example of astem cell niche has been considered to stand on a facultativenature.

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Figure 3. Architecture of seminiferous tubules, the spermatogenic centerin the testis. Schema of seminiferous tubules. In basal compartment
between the basement membrane and tight junction between Sertoli cells,
undifferentiated spermatogonia (the population responsible for the stem
cell pool) show a preferential localization to blood vessels running
between the tubules. The differentiating progeny of the stem cells stratify
into four layers, each of which is comprised of mitotic spermatogonia,
meiotic spermatocytes, and haploid round and elongating spermatids.
(Modified from Spradling et al, 2011)

Based on these preceding observations of our own andothers, we hypothesize that the blood vessel-adjacent area isspecialized and optimal for harboring and regulating the stem
spermatogonia, and that this feature is reflected by thespecialized gene expression in this area. We therefore havebeen challenging this issue by searching for genes showing
localized expression to such a presumptive niche area. We
first collected the area of interest using microdissection
techniques on sectioned testis specimens, followed by cDNA
microarray and in situ hybridization analyses to search forgenes showing preferential expression. We have been
studying these genes with regard to the spatial relationshipbetween the sites of expression of these genes withundifferentiated (and stem) spermatogonia, as well as thepossible function of these genes in stem cell regulation.
These findings will provide basic and fundamental
information on stem cell regulation in tissues harboring
facultative niche.

III. Spatio-temporal stem cell regulation byretinoic acid metabolism
In seminiferous tubules, differentiation of Ngn3-positive
undifferentiated spermatogonia into Kit-positive stage occurs
periodically with an interval of 8.6 days, which is followedby programmed differentiation process toward spermatozoa
that takes 35 days. This temporal regulation is essential forthe integrity of the tissue and continuity of spermatogenesis
in that this process supports the regular stratification of germ
cells in the wall of seminiferous tubules from stem cells tomature testis (Figure 3).
Based on the classic finding that retinoids (whoseprominent active form is retinoic acid) are essential for the
timed differentiation of undifferentiated spermatogonia, weinvestigated how the differentiation of spermatogonia isregulated by retinoic acid signaling, taking advantage ofexperimental modulation of retinoid supply and examinationof retinoid metabolism-related enzyme expression patterns(Sugimoto et al., Mech. Dev. 2012). It has been demonstrated
that a series of enzymes involved in retinoic acid metabolism
at different steps are expressed separately but in a beautifully

orchestrated manner among multiple differentiating germ celltypes (i.e., meiotic and haploid cells) and somatic Sertoli
cells (which nourish all the steps of germ cells in
seminiferous tubules), suggesting strongly that RA
concentration changes in a 8.6 day cyclic manner, and that it
increases at the same timing with that of differentiation ofspermatogonia into Kit-positive. In addition, expression ofRA metabolism-related genes in Sertoli cells are underregulation of RA signaling, forming positive and negative
regulatory loops, leading us to propose that it is RA itself thatcoordinates the orchestration of metabolism between
neighboring cell types that constitute the seminiferous
tubules (Figure 4).

identified. As well, mathematical analyses and modeling will
be quite powerful to link the observations obtained indifferent scales.

Publication List
〔Original papers〕
S., Hamasaki, H., Sekiguchi, S., Hara, K., Ishii, Y., Kyuwa,• Koyanagi,S., and Yoshikawa, Y. (2012). Effects of ubiquitin C-terminal hydrolase
L1 deficiency on mouse ova. Reproduction 143, 271-279.

Y., Usui, F., Miyahara, D., Mori, T., Ono, T., Kagami, H.,• Nakamura,Takeda, K., Nirasawa, K., and Tagami, T. (2012). X-irradiation removes••

endogenous primordial germ cells (PGCs) and increases germline
transmission of donor PGCs in chimeric chickens. J. Reprod. Dev. 58,
432-437.
Sato, T., Yokonishi, T., Komeya, M., Katagiri, K., Kubota, Y., Matoba,S., Ogonuki, N., Ogura, A., Yoshida, S., and Ogawa, T. (2012). Testis
tissue explantation cures spermatogenic failure in c-Kit ligand mutant
mice. Proc. Natl. Acad. Sci. USA 109, 16934-16938.
Sugimoto, R., Nabeshima, Y., and Yoshida, S. (2012). Retinoic acidmetabolism links the periodical differentiation of germ cells with thecycle of Sertoli cells in mouse seminiferous epithelium. Mech. Dev.
128, 610-624.

〔Review article〕
S. (2012). Elucidating the identity and behavior of• Yoshida,spermatogenic stem cells in the mouse testis. Reproduction 144, 293302.

Figure 4. Proposed model for coordination of periodic differentiation ofgerm cells linked by retinoid metabolism. (A) Particular stages of meioticcells (LP) and haploid cells (RT) send the ‘go’ and ‘wait’ signals to Aundff
and Sertoli cells, by regulating the local RA metabolism to increase anddecrease the RA concentration, respectively. (B) These signals will occur
reciprocally and periodically to maintain the seminiferous epithelial
cycle. (From Sugimoto et al., Mech. Dev. 2012)

IV. Perspectives
As described above, we have been tackling the robust stem
cell system that supports mouse spermatogenesis fromdifferent aspects and scales. Once this research is achieved itwill be quite intriguing to ask how these events are linked
with each other and integrated into the robustness of thewhole stem cell system. For that purpose, it is clear that westill need to know about molecules (and genes that encode
these factors) that play essential roles but have not been

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