National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:
Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellow:
Technical Assistants:
Visiting Scientist:
Secretary:

KITADATE, Yu
MIZUGUCHI-TAKASE,Hiroko
HARA, Kenshiro
YOSHIDA, Naoko
ICHIKAWA, Rie
INADA, Kana
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 themammalian spermatogenic stem cell system, mainly using
mice. In 2009, our second year at NIBB, we have tackled anumber of important issues in mouse spermatogenic stem
cells.

evaluations.
Similarly, corollaries of this model are that all the As cells
are functionally equivalent and uniformly act as the stem
cells, and that this defined population of ‘stem cells’ plays anactive role in every aspect of the stem cell functions, i.e.,maintenance of homeostasis, post-transplantation colony
formation, and post-injury tissue regeneration.
We have previously shown that no single stem cell
population acts in every aspect of stem cell function: Cells
that support the steady-state spermatogenesis are different
from those that support colony-formation and/or regeneration(Nakagawa et al., Dev. Cell, 2007). Based on this finding, we
propose the functional hierarchy in the spermatogenic stem
cell compartments as shown in Figure. 1. We propose that inaddition to the actually self-renewing population (actual stemcells), an extended population of cells that retain the selfrenewing potential but are destined for differentiation(potential stem cells) exist, which plays an important role forthe continuity and robustness of the stem cell system. In caseof actual stem cell loss, potential stem cells might revert tothe self-renewing mode and replenish the actual stem cellpool (Figure 1).

I. Background: The mammalian spermatogenesis

Mammalian testes continually produce numerous sperm
during the reproduction period. Lines of investigations thatemerged in the 1950s and involve detailed morphologicalanalyses established the backbone of mammalianspermatogenesis research. The morphologically mostprimitive spermatogonia in the adult mouse testis are As or
Asingle 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” (or “Aundiff” hereafter),which contribute <1% of the entire testicular cells andconsist of As, Apr (Apaired; interconnected two-cell cysts), andAal (Aaligned; cysts of 4, 8, 16 or occasionally 32 cells) containstem cells.
Then, which subfraction of Aundiff consists of the actually
self-renewing stem cell compartment in homeostasis andhow do they behave (proliferate, self-renew or die) in thetestis? The prevailing, so-called “As model”, which wasoriginally proposed in 1971, proposes that As is the only cell
type that can act as stem cells, while the interconnected
population of A undiff (A pr and A al) is devoid of stem cell
capacity.

II. To w a r d t h e n a t u re o f t h e m o u s espermatogenic stem cell system that
includes reversibility

Despite that the “A s model” is comprehensive andpersuasive, this model is not based on the direct analyses ofthe cells’ behavior. Thus, the As model warrants functional

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Figure 1. Proposed model of the functional compartments in mouse
spermatogenesis (Nakagawa et al., 2007).

In 2009, by revisiting the As model, we investigated the cell
population of Aundiff. For this attempt, we introduced a gene
expression profile for describing the heterogeneity of thecells, in addition to the number of chained cells or the lengthof the cysts, on which the “As model” entirely depended on.
Several studies, including ours, have been establishing thatthe populations of cysts that compose the same number ofspermatogonia, which are classically considered to behomogeneous, are indeed heterogeneous in terms of patternsof gene expression. Therefore, a new scope has been raisedthat the entire Aundiff population is more heterogeneous thanhas previously been considered.
We have also investigated the behaviors of these A undiff
subpopulations in homeostasis (steady-statespermatogenesis) and during regeneration after tissue insult.
We revealed that a particular subset of Aundiff changes their
behavior dynamically between homeostasis and regeneration:
In steady-state, they differentiate without self-renewal, but inregeneration, they are willing to get back into a selfrenewing stem cell state. This finding provides a novel viewfor stem cell biology and explains why the stem cells canchange the behavior between homeostasis and regeneration
so smoothly. In homeostasis, the number of stem cells should
be constant, while in regeneration, they increases to recover
the stem cell pool quickly. This feature is crucial for thecontinuity of tissue functions.

III. Investigating the temporal control ofdifferentiation and establishment of thetesticular tissue

In seminiferous tubules, spermatogenic differentiation
occurs in a periodical manner, which results in the formationof beautiful stratification of different steps of differentiating
cells within the seminiferous tubules (Figure 2A). Essentially,
this is caused by the periodical differentiation of Aundiff with aregular interval of 8.6 days (white arrows in Figure 2B).
We have investigated how this periodical event is controlled.
This question can be generalized to how the timing of Aundiff
differentiation is regulated. We paid special attention to theinvolvement of Sertoli cells and the role of retinoic acid (RA)signaling. The importance of RA has been recognized for
decades based on the finding that deficiency in vitamin A(VA), the dietary precursor of RA, causes a severe
spermatogenesis defect in which differentiation of Aundiff isaffected, and differentiating cell types are all lost.
We have revisited this VA-deficient (VAD) model andrevealed the central role of RA not only in the control of thetiming of Aundiff differentiation, but also in the regulation of thefunctional change of the supporting somatic Sertoli cells,
which are huge cells that nourish the stratified germ cells. The
local retinoid metabolism in the seminiferous tubules involves
multiple cell types of germ and somatic cells. Therefore, cooperation between different cell types that are locatedadjacently may control the differentiation of Aundiff.

IV. Investigating the nature of the stem cellniche

Evidence suggests an intimate relationship between stem cells
and the niche microenvironment in seminiferous tubules. It isdifficult to identify the nature and function of the niche,
however, because seminiferous tubules do not exhibit
suspicious sub-structures. Moreover, actual stem cells can be
identified only functionally, and their histological detection
has not yet been achieved. Therefore, our current aim has been
to clarify the niche of Aundiff.

Figure 3. Localization of A undiff revealed by three-dimensional
reconstruction (Yoshida et al., Science, 2007)
Computationally reconstituted three-dimensional images of theseminiferous tubules based on 280 serial sections. Aundiff (green) show

biased localization to the blood vessel network (red) and the areaadjacent to the interstitium (yellow). (A, C) and (B, D), without orwith blood vessels, respectively. Roman numerals indicate the stageof the seminiferous epithelium.
Figure 2. Stratified tissue architecture in mouse seminiferous tubules.
(A) Appearance of a representative part of the seminiferous tubules. Notethe beautifully stratified germ cells at different steps ofdifferentiation.
(B) A scheme that explains the formation of the stratified architecture
shown in (A). The population of Aundiff (red) persists for a long period
and gives rise to A1 differentiating spermatogonia periodically with aregular interval of 8.6 days. As a result, a regular stratification of thedifferentiating germ cells is formed, as indicated by the rectangle.

The seminiferous tubules exhibit a convoluted tubularstructure with a diameter of ~200mm: Individual tubulesconnect to the common outlet of the mature sperm (rete testes)
with both ends and form loops. Spermatogenesis occurs
evenly throughout the inner surface of the tubules. Therefore,
in mouse testis, an overall ‘polarity’ that covers the entire
organ cannot be recognized, making a good contrast to theDrosophila melanogaster germline stem cell system.

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National Institute for Basic Biology

Developmental Biology

By taking advantages of live-imaging and three-dimensional
reconstruction, we previously revealed that Aundiff preferentiallylocalizes to the limited regions of seminiferous tubules thatare adjacent to blood vessels and interstitial cells that surroundthe tubules (Figure. 3). In addition, the dynamic migration of
spermatogonia from the vasculature proximity to spreadthroughout the tubules was also observed upon differentiationof Aundiff (Yoshida et al., Science, 2007). Based on thesefindings we proposed that this area may act as the niche for
Aundiff (Figure 4). We are tackling the cellular and molecular
identification of this vasculature-associated niche.

Figure 4. A proposed niche microenvironment for Aundiff.
Although the seminiferous tubule is a repeat of the same structure, theproximity to the surrounding blood vessels and interstitial cells
specializes the particular region of the basal compartment of thetubules, so that it can act as the niche for undifferentiated Aundiff
population.

Publication List

〔Original papers〕

K., Kanai-Azuma, M., Uemura, M., Shitara, H., Taya, C.,• Hara,Yonekawa, H., Kawakami, H., Tsunekawa, N., Kurohmaru, M., and•Kanai, Y. (2009). Evidence for crucial role of hindgut expansion indirecting proper migration of primordial germ cells in mouse early
embryogenesis. Dev. Biol. 330, 427-439.
Suzuki, H., Sada, A., Yoshida, S., and Saga, Y. (2009). The heterogeneityof spermatogonia is revealed by their topology and expression ofmarker proteins including the germ cell-specific proteins Nanos2 andNanos3. Dev. Biol. 336, 222-231.

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

M., Hara, K., Shitara, H., Ishii, R., Tsunekawa, N., Miura, Y.,• Uemura,Kurohmaru, M., Taya, C., Yonekawa, H., Kanai-Azuma, M., and Kanai,Y. (2009). Expression and function of mouse Sox17 gene in thespecification of gallbladder/bile-duct progenitors during early foregut
morphogenesis. Biochem. Biophys. Res. Commun. 2009 Nov. 12〔Review Articles〕

S. (2009). Spermatogenic stem cell system in the mouse testis.
• Yoshida,
Cold Spring Harb. Symp. Quant. Biol. 73, 25-32.
S. (2009). Casting back to stem cells. Nat. Cell Biol. 11, 118• Yoshida,120.

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