National Institute for Basic Biology

Developmental Biology

DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei
Postdoctoral Fellow
Visiting Scientist

YOSHIDA, Naoko
SUGIMOTO, Ryo

Mammalian spermatogenesis represents a highly potent
and robust 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, as well as
their niche, remains largely unknown in the context ofhomeostatic spermatogenesis. The Division of Germ Cell
Biology, which was launched in 2008, aims to fully
understand the mammalian spermatogenic stem cell system,
mainly using the mouse system. Being a brand-new researchdivision of NIBB, our overall research view and current fociof interests will be described. These include 1) the cellular
nature of the stem cell compartment and their behaviors in
the homeostatic spermatogenesis, and 2) the anatomicalbasis and function of the mammalian spermatogenic stem
cell niche.

Ⅰ. Identifying the mouse spermatogenic stem
cell compartments
1-1. Background: The mammalian spermatogenesis
Mammalian testes continuously produce numerous sperm
during the reproduction period. Investigations that emergedin the 1950s and involve detailed morphological analyses
established the backbone of mammalian spermatogenesis
research. The morphologically most primitive
spermatogonia in the adult mouse testis are A s or A single
spermatogonia (single, isolated spermatogonia). Theirprogeny remain interconnected due to incomplete
cytokinesis, forming syncytial chains of 2n cells (2, 4, 8, 16etc.). It has been experimentally established that“undifferentiated spermatogonia” (or “A undiff” hereafter),which contribute <1% of the entire testicular cells andconsist of As, Apr (Apaired; interconnected pairs), and Aal (Aaligned;
chains of 4, 8, 16 or occasionally 32 cells) contain stem
cells.
Which fraction of A undiff consists of the actually selfrenewing stem cell compartment in homeostasis and how dothey behave (proliferate, self-renew or die) in the testis? The
so-called “As model” is currently most widely considered to
be true. This model 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. This
comprehensive model, however, is based upon “snapshots”
of fixed specimens and involves too many theoretical limits
to be entirely conclusive regarding stem cell function
because “stem cells” by definition maintain themselves
while producing differentiating progeny for a long period.
Transplantation of a single cell suspension into the26

recipient testis enables the functional and quantitative
analyses of the “stem cells” based on their repopulating
activity in the host testis. Combined with the morphologybased As model, it is often considered to be the case that all
As spermatogonia are equivalent and act as stem cells, which
support both homeostatic spermatogenesis and posttransplantation repopulation, despite the inherent limitation
due to the lack of direct functional evidence.

1-2. Heterogeneous composition of the stem cells
In order to understand the nature of the mouse
spermatogenic stem cell system in testicular tissue, we havebeen investigating the behavior and function of A undiff.
Authentic identification of Aundiff is based on morphology
under electron or high-resolution light microscopy. Using
the genetic labeling of A undiff by means of the Ngn3
regulatory sequence (Yoshida et. al., Dev. Biol. 2004), we
have established experimental systems to investigate theirlive behaviors without disturbing normal tissue architecture.
We first asked whether the “stem cells” detected bytransplantation are identical to the “stem cells” that actually
self-renew in homeostasis, or whether these two “stem cells”
represent different groups of cells. In order to address thisquestion, a tamoxifen-dependent Cre recombinase(CreERTM) was expressed in the Ngn3+ Aundiff (Yoshida et al.,Development 2006). Together with an appropriate reportertransgene, this has enabled the irreversible pulse-labeling of
A undiff spermatogonia upon transient administration oftamoxifen, and the first quantitative detection of “actual
stem cells” (i.e., the cells that persist for a long time while
producing differentiating progeny, thus supporting tissue
homeostasis). Intriguingly, the contribution of the pulselabeled subpopulation of Aundiff to “actual stem cells” and“post-transplantation colony-forming stem cells” represents
a great difference (approximately 40 times higher in thelatter than in the former). Therefore, these two “stem cells”
represent different subpopulations of Aundiff. We concluded
that, in addition to actual stem cells, an extended population
exists that does not self-renew while retaining the potentialof self-renewal, which was defined as a population of“potential stem cells” (Nakagawa et al., Dev. Cell 2007).

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

The “potential stem cells” were shown to rapidly turn overin homeostasis, suggesting that they consist of a transitamplifying compartment.
Figure 1 shows our simplest interpretation of thehierarchical composition of the mouse spermatogenic stem
cell system (Nakagawa et al., Dev. Cell 2007). In case of
actual stem cell loss, potential stem cells might revert to theself-renewing mode and replenish actual stem cells. We are
currently investigating the cell-biological characteristics ofactual and potential stem cells.

Ⅱ. Testicular niche for Aundiff
2-1 Anatomy of the mouse testis and the researchbackgroundSpermatogenesis proceeds inside the seminiferous tubules,
a convoluted tubular structure with a diameter of ~200μm:
Individual tubules connect to the common outlet of themature sperm (rete testes) with both ends and form loops.
Spermatogenesis occurs evenly throughout the seminiferous
epithelium (the inner surface of the tubules). Therefore, inthe mouse testis, an overall ‘polarity’ that covers the entire
organ cannot be recognized, making a good contrast to the
Drosophila melanogaster germline stem cell system.
Evidence suggests an intimate relationship between stem
cells and the niche microenvironment in seminiferous
tubules. It is difficult to identify the nature and function of
the niche, however, because seminiferous tubules do notexhibit 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 is to clarify the niche of Aundiff.

2-2. Live imaging and the vasculature-associatedniche for Aundiff
We have developed a live imaging system in which GFPlabeled Ngn3+ Aundiff and their progeny can be continuously
filmed in undisturbed testes. It was revealed that Aundiff
preferentially localize to the area adjacent to blood vessels
and interstitial cells that surround the seminiferous tubules.
In addition, the dynamic migration of spermatogonia fromthe vasculature proximity to spread throughout the tubules
was also observed upon differentiation of Aundiff (Figure 2).
The same relocation was also supported by threedimensional reconstruction based on authentic
morphological identification of Aundiff on serial sections(Figure 3). These extend the preceding observations frommouse and rat testis sections that Aundiff shows a significant
biased localization to the interstitium, and we proposed sucharea as the niche for Aundiff (Yoshida et al., Science 2007).
We are currently investigating the cellular and molecular
identification of this vasculature-associated niche.

Figure 3. Localization of A undiff revealed by three-dimensional
reconstruction (Yoshida et al., Science 2007)
Figure 2. Localization of GFP-labeled Ngn3+ Aundiff and their relocation
upon transition into differentiating spermatogonia (Yoshida et al.,Science 2007).
(A) Live imaging of spermatogonia upon differentiation of Aundiff. Before
differentiation (0 hour; the elapsed time indicated in each panel inhours), labeled Aundiff preferentially localized to the area adjacent to theblood vessels (seen as a black line) and surrounding interstitium. Upon
differentiation, two chains of 8-cell cysts (Aal-8; indexed in yellow andorange) migrated from this position to spread all over the tubule (3660h). Subsequently, the two cysts underwent synchronous mitotic
division with 2-3 hours’ interval, resulting in the formation of two 16cell cysts of differentiating spermatogonia (73-74h).
(B-E) Examples of the vasculature-proximal localization of Aundiff. Aundiff(arrowhead) preferentially localized to areas adjacent to blood vessels,
more characteristically to their branch points. In (B-C), A undiff inneighboring seminiferous tubules show back-to-back localization overbranching vessels.

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 or withblood vessels, respectively. Roman numerals indicate the stage of theseminiferous epithelium.

Publication List
〔Original paper〕● Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S.,

Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y. (2008). Notch
mediates the segmental specification of angioblasts in somites andtheir directed migration toward the dorsal aorta in avian embryos.
Dev. Cell. 14, 890-901.

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