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:

Secretary:

HARA, Kenshiro
KITADATE, Yu
NAKAMURA, Yoshiaki
MIZUGUCHI-TAKASE,Hiroko
IKAMI, Kanako
TOKUE, Moe
ICHIKAWA, Rie
INADA, KanaMARUYAMA, Ayumi
SUGIMOTO, Ryo
KUBOKI, Yuko

Mammalian testes produce numerous sperm for a long
period in a constant manner, which is supported by the robust
behaviors of the stem cell population. Decades of research,
including 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 the mouse sperm stem cell system in vivo, i.e., inthe context of testicular tissue architectures. We haverevealed a number of characteristics of this potent stem cellsystem, including: 1) Cells believed to have irreversiblycommitted for differentiation still retain the self-renewing
potential and can contribute to stem cell pool maintenance(“potential stem cells”). 2) “Reversion” from potential stemcells occurs at a higher frequency when testicular tissue isdamaged and regeneration is induced. 3) The undifferentiatedspermatogonia (Aundiff) population that includes both “actual”
and “potential” stem cells localized to vasculature (vascularassociated niche). 4) Stem cells turn over frequently andstochastically even under steady-state situations. (Nakagawaet al., Dev. Cell 2007, Science 2010; Yoshida et al., Science
2007; Klein et al., Cell Stem Cell 2010)

Figure 1. Architecture of seminiferous tubules and seminiferousepithelium. The seminiferous epithelium exhibits a stratified organizationof the differentiating germ cells (numbers in circle), which are nourishedby Sertoli cells. Bar, 20 mm.

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In 2011, we revealed the molecular mechanisms underlying
the temporal regulation of differentiation and self-renewal ofstem cells (Aundiff) in mouse seminiferous epithelium, which
are comprised of germ cells –both stem and differentiatingcell types- and supporting somatic cells. Our study proposedthat local retinoic acid (RA) metabolism plays importantroles for the timed stem cell differentiation and coordination
with somatic cells that support the differentiation process tospermatozoa (Sugimoto et al., Mech. Dev. 2011).

I. Architecture and cyclic dynamics ofseminiferous epithelium

Spermatogenesis occurs inside the seminiferous tubule ofthe testis, where differentiating germ cells and supporting
somatic Sertoli cells comprise a composite epithelium,
termed seminiferous epithelium (Fig. 1). Sertoli cells arehuge cells that form typical epithelium. All the stages ofgerm cells (from stem cells to spermatozoa) are nourished by
Sertoli cells in a striated manner. This stratification isestablished as a result of the periodic differentiation of Aundiff
into A1 spermatogonia with an interval of 8.6 days, which isfollowed by programmed differentiating process toward
spermatozoa that takes 35 days (Fig. 2). Consequently, theresultant combination of the differentiating germ cell
observed at a particular region appears in an 8.6-day cyclicmanner, known as ‘seminiferous epithelial cycle’. In mice,
this cycle is divided into stages I to XII, while Aundiff-to-A1
differentiation occurs at stage VII to VIII.

Figure 2. Coordination of periodical events in mouse seminiferous
epithelium. (A) Germ cell differentiation, which takes 35 days from Aundiff
to spermatozoa. (B) 8.6-day cycle of gene expression in Sertoli cells. (C)Coordination of germ cell differentiation and Sertoli cell cycle.

Sertoli cells, while quiescent in their cell cycle, cyclicallychange their gene expression so that they can nourish theappropriate stages of germ cells. It is an interesting question
but remains a mystery how this periodic differentiating ofAundiff is achieved and the cycle of Sertoli cells is coordinatedwith germ cell differentiation.

II. Role of RA and regu lation o f RA
metabolism

It has been known that RA plays important roles in bothdifferentiation of A undiff and control of the seminiferous
epithelial cycle. This notion was primarily derived fromobservations made after RA signaling blockade by deficiencyof the dietary vitamin A (VA) -the only precursor of RA- andby disruption of retinoic acid receptor genes. However,results of these and other preceding studies were somewhat
puzzling and it remained a challenge to reveal how RA is
involved in the genesis of the coordinated cycling of theseminiferous epithelium.
We revisited the classical VAD model and discovered that
artificial elevation of RA signaling elicits not only
differentiation of Aundiff into A1 spermatogonia but also a resetof the cycle of Sertoli cells’ function to the appropriate stage
that supports the A1 spermatogonia in transition between
Aundiff and A1 (stage VII). Then we asked how the RA-related
metabolism is regulated in the seminiferous epithelium basedon the expression of genes encoding enzymes involved in thesynthesis of RA from VA and inactivation of RA. Based on
the data from a previous study of Manuel Mark’s group andour own analyses, it was strongly suggested that the modesof RA metabolism dynamically shift in accordance with theseminiferous epithelial cycle, with a prominent increase ofRA concentration across stage VII (Fig. 3). This is also
supported by the fact that artificially increased RA induced
Aundiff-to-A1 differentiation and reset Sertoli cells to stage VII.
Interestingly, RA metabolism involves multiple enzymes(Fig. 3A) that are expressed separately among different celltypes including meiotic and haploid germ cells and Sertolicells. Given that RA and its precursors are able to translocate
across adjacent cells rather freely, the different cell types
seem to cooperate in the regulation of RA metabolism. Then,the next question was raised: How is coordinated expressionof these genes among different cell types achieved? We
discovered that RA metabolism-related genes expressed in
Sertoli cells are regulated by RA signaling, forming positive
and negative feedback loops; while those expressed inmeiotic and haploid germ cells hardly respond to RA
signaling: Perhaps a differentiation step-related mechanism
controls their expression. Thus, control of RA metabolismrelated genes show a good contrast between Sertoli cells andgerm cells.

Figure 3. RA metabolism-related genes and their cyclic expression. (A)Multiple genes act together in the RA metabolic pathways, starting fromretinol (VA). (B) These genes are expressed in different cell types and indifferent stages of the seminiferous epithelial cycle.

III. Model for the seminiferous epithelial cycle

Based on these observations, we have proposed a modelthat can explain the coordinated expression of RA
metabolism-related genes that appears to cause the cyclicchange of the RA concentration (Fig. 4, 5). Importantly, thismodel suggests that meiotic and haploid germ cells regulate
the local RA metabolism to increase and decrease the RA

Figure 4. Regulation of RA metabolism-related gene expression.
Expression patterns of these genes (Fig.3) in early (A) and late (B) stages
are explained as a result of feedback regulation in Sertoli cells but not ingerm cells.

29National Institute for Basic Biology

Developmental Biology

concentration, respectively (Fig. 4). Given that RA signaling
causes the induction of Aundiff-to-A1 differentiation and resetof Sertoli cell’s cycle, it can be said that these differentiating
germ cells play a central role in the coordination of theseminiferous epithelial cycle by sending ‘go’ and ‘wait’signals to the Aundiff and Sertoli cells (Fig. 5). Therefore, thedifferentiation program of germ cells appears to determine
the timing of stem cell differentiation and makes the local
environment appropriate for germ cell differentiation.
This model nicely explains the beautiful orchestration
between germ and Sertoli cells in the seminiferous
epithelium. Generally speaking, regulation of stem celldifferentiation by their differentiating progeny that modulate
the local environment may be a common strategy of stem
cell control in other systems.

IV. Perspectives and ongoing researchmodel and future investigations will clarify the details of thetesticular environment and the stem cell response.
Besides the seminiferous epithelial cycle, we are also
investigating a number of yet-to-be elucidated problems inspermatogenesis, especially with regard to its stem cells. In
addition to temporal regulation described here, the spatialregulation of stem cells is also a very important issue. We
previously observed that Aundiff preferentially localize to thevasculature-proximal region, and dispatch this region when
they differentiate into A1 at stage VII (Yoshida et al., Science2007), which is likely to occur in response to RA signaling.
We are also investigating the cellular and molecular nature ofthis ‘vasculature-associated niche’. We are also investigating
the behavior of Aundiff taking advantages of live imaging that
we have developed (Yoshida et al., Science 2007, Nakagawa
et al., Science 2010). We hope that these studies will throw
light to a better understanding the mouse sperm stem cellsystem.

Publication List

〔Original paper〕

T., Aiyama, Y., Ishii-Inagaki, M., Hara, K., Tsunekawa, N.,• Sato,Harikae, K., Uemura-Kamata, M., Shinomura, M., Zhu, X. B., Maeda,S., Kuwahara-Otani, S., Kudo, A., Kawakami, H., Kanai-Azuma, M.,Fujiwara, M., Miyamae, Y., Yoshida, S., Seki, M., Kurohmaru, M., andKanai, Y. (2011). Cyclical and patch-like GDNF distribution along thebasal surface of sertoli cells in mouse and hamster testes. PLoS ONE 6,e28367.

〔Original papers (E-publication ahead of print)〕

A., Raverdeau, M., Célébi, C., Dennefeld, C., Feret, B.,• Gely-Pernot,
Klopfenstein, M., Yoshida, S., Ghyselinck, N.B., and Mark, M.

•Spermatogonia differentiation requires retinoic acid receptor γ.
Reproduction 2011 Nov. 1.
Sugimoto, R., Nabeshima, Y., and Yoshida, S. Retinoic acid metabolism
links the periodical differentiation of germ cells with the cycle of Sertoli
cells in mouse seminiferous epithelium. Mechanisms of Development
2011 Dec. 19.

〔Review articles〕

A., Fuller, M.T., Braun, R.E., and Yoshida, S. (2011).
• Spradling,
Germline stem cells. In Cold Spring Harbor Perspectives in Biology
Collection, “Germ Cells”, Cold Spring Harbor Laboratory Press, 1-20.

Figure 5. Coordination of germ cell differentiation and Sertoli cells’
cycle. Particular stages of meiotic cells (LP) and haploid cells (RT) sendthe ‘go’ and ‘wait’ signals to Aundff and Sertoli cells, by regulating thelocal RA metabolism to increase and decrease the RA concentration,respectively (A). These signals will occur reciprocally and periodically tomaintain the seminiferous epithelial cycle. All the figures are reproduced
from Sugimoto et al., Mech. Dev. (2011) with permission.

The suggested model for the role of RA in the regulated
stem cell differentiation warrants farther evaluation: Whilethe central role of RA is in no doubt, it is also clear that RA
solely cannot regulate the very complex and highly
orchestrated events that occur in seminiferous epithelial
cycle. In addition, stem cells should behave in response to
the cyclically changing microenvironment. We hope that our30S. (2011). Stem cell niche system in mouse spermatogenesis.
• Yoshida,
In Stem Cell Biology and Regenerative Medicine Series, “MaleGermline Stem Cells: Developmental and Regenerative Potential”,
Humana Press, 159-175.