DIVISION OF GERM CELL BIOLOGY

Professor

YOSHIDA, Shosei

Assistant Professor:

KITADATE, Yu
NAKAGAWA, Toshinori
Specially Appointed Assistant Professor:
SUZUKI, Shinnosuke
Technical Staff:
MIZUGUCHI, Hiroko
JSPS Postdoctoral Fellow: IKEDA, Tatsuro
Postdoctoral Fellow:
HIRANO, Kodai
SATO, Toshiyuki
Visiting Graduate Student: BABAGUCHI, Hirotaka
Technical Assistant:
FUJITA, Miyako
NISHIMURA, Chiaki
Admin Support Staff:
KUBOKI, Yuko

As cells and syncytia together comprise a single SSC pool

Our live-imaging studies combined with mathematical andstatistical analysis evidenced that SSCs reversibly change theirmorphology between singly-isolated and syncytia. This ismediated by incomplete cell division and breakdown of syncytia(Nakagawa et al., Science 2010; Hara et al., Cell Stem Cell 2014).

Cells inclined for differentiation can revert to SSCs

It is generally thought that once stem cells have been destinedfor differentiation, they never replicate themselves again. We
found that such cells maintain the potential to self-renew up
to a particular stage of differentiation and revert to SSCs withhigh frequency, particularly during regeneration after tissue
damage (Nakagawa et al., Developmental Cell 2007).

SSCs follow variable fate behaviors

Stem cells are often thought to invariably undergo asymmetric division, which produces one stem cell and one
differentiating cell to maintain homeostasis. However, wefound that individual SSCs follow highly variable fates. Still,collectively, they achieve a perfect balance between selfrenewal and differentiation at a population level (Klein et al.,Cell Stem Cell 2010; Hara et al., Cell Stem Cell 2014).

SSCs show active motion in an “open niche”

Our research

Many animals, including mammalians, produce a large
number of sperm over a long period for reliable reproduction. On the other hand, each sperm correctly replicates andtransmits the genetic information to the next generation.
The ultimate goal of the Division of Germ Cell Biology is toreveal how seemingly contradictory, albeit essentially important, productivity and fidelity are both achieved. To this end,
we pay special attention to the sperm stem cells. We are alsointerested in other aspects of spermatogenesis.

What are the sperm stem cells?

The continual sperm production is supported by the “spermstem cells,” termed formally “SSCs” after spermatogonial or
spermatogenic stem cells. SSCs maintain a delicate balance
between self-renewal and differentiation to sustain life-longspermatogenesis. It has been a central question which fractionof spermatogonia, the “early stage of cells before meiosis, areSSCs and where and how they behave in the testis.
The backbone of mammalian SSC research was establishedfrom the 1950s to the 1970s, relying on morphologicalanalyses of fixed testis specimens. This led to a proposal of
the prevailing stem cell model called the “As model.” Thismodel proposed that singly isolated spermatogonia (A-singleor As cells) act as SSCs. On the other hand, all syncytia are
all committed to differentiation.
Our lab has introduced the scale of time using updated
technologies, i.e., live-imaging and pulse-labeling studies,
which revealed many different aspects of dynamical behaviors of SSCs (Figure. 1).

In many tissues, stem cells receive self-renewing signals indefined niche regions. We found that the mouse testis has nosuch areas, with SSCs scattered among differentiated cells
and moving around actively. Further, their distribution isbiased near blood vessels. Such microenvironment is called
an “open (or facultative) niche” in contrast to the afore-mentioned “closed (or defined) niche” (Yoshida et al., Science2007; Hara et al., Cell Stem Cell 2014).

SSC heterogeneity underpins the asymmetric fates of SSCs

In an open niche, some SSCs remain undifferentiated whileothers differentiate in response to broadly distributed signaling molecules. We found that some SSC fractions resist
differentiation-promoting Wnt signal by expressing a Wntinhibitor, Shias 6. Another subset responds to retinoic acid(RA) by upregulating an RA receptor to differentiate (Ikami etal., Development 2015, Tokue et al., Stem Cell Reports 2017).

SSCs compete for self-renewal factors in an open niche

We also question the mechanism that maintains a constant
SSC density in the open niche of the testis. Evidence
suggests that SSC density homeostasis is achieved throughcompeting with each other for limited amounts of selfrenewal factors (e.g., FGFs). This mechanism was proposedas the “mitogen competition model” (Kitadate et al., Cell
Stem Cell 2019).

Periodic differentiation of SSCs

Strikingly, differentiation of SSCs and subsequent differentiation to mature into sperm occur synchronously every
8.6 days. This is called the seminiferous epithelial cycle.
We propose that the periodic differentiation is triggered byperiodic synthesis and degradation of RA (Sugimoto et al.,Mechanisms of Development 2012).

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National Institute for Basic Biology

Developmental Biology

Figure 1. Different looks of mouse sperm stem cells (SSCs)
A. 3D-reconstructed seminiferous tubules, showing SSCs in green, blood vessels in red.
B. An intravital live-imaging filming of GFP+ SSC syncytia undergoing differentiation.
C. Scheme of SSC dynamics in multi-compartment, interconverting singly-isolated and syncytial states.
D. Whole-mount immunofluorescence, showing SSCs (magenta) intermingled with primed (green) and committed (blue) progenies.
E. Sectioned host testis showing repopulated by transplanted donor SSCs (green), augmented by transient RA inhibition.
F. A phase portrait showing the dynamics of homeostatic SSC density regulation in the mitogen competition model.

Clonal analysis and tuning of post-transplantation
SSC fate
When SSCs are transplanted into host animals’ seminiferous
tubules whose germ cells have been depleted, SSCs engraft
and regenerate spermatogenesis (Brinster et al., 1994). What
happens during repopulation was largely elusive. We foundthat donor SSCs follow stochastic fate with significant similarity with homeostasis. We further succeeded in tuning
the donor SSC fate to increase the repopulation efficiency,
which would promise the practical use of this technology(Nakamura et al., Cell Stem Cell 2021).

Identification of a tiny, most primitive fractionof SSCs
An important advance gained in FY2021 is that we havedetailed gene expression heterogeneity of SSCs (precisely, apopulation called “undifferentiated spermatogonia”) and theirdynamical state transitioning in supporting the homeostaticand continual sperm production (Nakagawa et al., Cell Reports
2021). This is a collaborative study with Nagasawa (Osaka) andSimons (Cambridge) labs. We identified that a gene Plvap (plasmalemma vesicle associated protein) is expressed in a tiny, mostprimitive fraction of mouse SSCs (Figure 2A).

Detailing the heterogeneity of mouse SSCs
Plvap shows overlapping expression with some other genes,
including Eomes, Pdx1, Shisa6, and T (brachyury). By
dimension reduction of single-cell (sc)RNA-seq data (Laet al., 2018), Plvap+ cells cluster at the undifferentiated endof SSCs, forming a discrete subfraction of cells expressing
established undifferentiated markers, GFRα1 and Id4. In
contrast, differentiation-related genes (e.g., Sox3, Ngn3, andRARγ) is expressed in cells near the opposite end (Figure2B). Based on these, Figure 2C summarizes the heterogeFigure 2. Heterogeneity of SSCs
A. Immunostaining of a mouse testis
section for Plvap protein (magenta), withDNA counterstained in gray. Only threecells located on the periphery of seminiferous tubules express Plvap.
B. UMAP analyses of scRNA-seq data
showing the expression of indicatedgenes.
C. A summary of expression profile of aset of genes within SSCs. Genes shownin bald have been analyzed in our lab.
Adopted from Cell Reports 37 (3),109875.

38neous expression of key SSC genes.

Contribution of multiple SSC fractions to selfrenewing pool in homeostasis
By pulse-labeling using tamoxifen-dependent CreER,
Plvap+, Sox3+, and Ngn3+ cells were found to contribute to
the self-renewing pool in homeostasis. In particular, after
being sparsely labeled with GFP expression, these cells gave
rise to cell patches comprised of GFP+ germ cells in threemonths (Figure 3).
Interestingly, while the patch-forming efficiency varied
between these fractions, the resultant individual patches
showed indistinguishable size distribution irrespective of theoriginal cell state. Further, strikingly, the same heterogeneous
SSC population was established in every such patch, again,
irrespective of the original cell state.

Figure 4. Multistate dynamics of mouse SSCs
A. A general perspective of the multistate dynamics, in which SSCs
transit between multiple states reversibly to form a single SSC pool.
B. A numerically fit dynamics of mouse SSCs in homeostasis. The flowsof cells between cell states and cell division at respective states areshown in values relative to the total GFRα1+ cells per day. Note that theGFRα1– (Ngn3+) to GFRα1+/Sox3+ state is not defined due to its low frequency in this model. Adopted from Cell Reports 37 (3), 109875.

Interestingly, such a multistate dynamics is found capableof saving the cell divisions in the self-renewing pool in
producing a certain number of differentiating cells. This isdue to the differential cell division frequencies between thestates; the more primitive the state is, the less frequently
the SSC divide. See Nakagawa et al., Cell Reports 2021 fordetail.
Furthermore, this quantitative model can explain the seemingly contradictory models proposed for the SSC dynamics,
and may provide inclusive insights into this long-heldproblem in the field of SSC research.
Figure 3. Pulse-labeling of multiple SSC fractions
A. Experimental design addressing the contribution of spermatogonia
expressing particular genes to homeostasis, following a pulse-labeling bytamoxifen injection.
B. Results of the pulse-labeling of the Plvap+, Sox3+, and Ngn3+ fractions. Note the differential patch forming efficiencies in contrast to thesimilarly-looking individual patches.

We reasoned that SSCs transit continually and reversibly
between multiple cell states, from the self-renewal-biased
Plvap+ state to the differentiation-primed Ngn3+ state (Figure3B). As a result, a heterogeneous SSC pool is always established, which both maintains itself and gives rise to irreversibly committed progenitor cells of Kit+ spermatogonia as aresult of cell division and state transition.

A multistate dynamics: Proposal of an
inclusive model of mouse SSCs
Finally, we synthesized a mathematical model that capturesthe multistate SSC dynamics in homeostasis. This model
assumes that the cells undergo cell division and state transition (either differentiation and reversion) in a stochastic (i.e.,probabilistic) manner, following particular rates specific toeach state (Figure 4).

Publication List:〔Original Papers〕

T., Jörg, D.J., Watanabe, H., Mizuno, S., Han, S., Ikeda,• Nakagawa,T., Omatsu, Y., Nishimura, K., Fujita, M., Takahashi, S., Kondoh, G.,••

Simons, B.D., Yoshida, S., and Nagasawa, T. (2021). A multistate stem
cell dynamics maintains homeostasis in mouse spermatogenesis. CellRep. 37, 109875. DOI: 10.1016/j.celrep.2021.109875
Nakamura, Y., Jörg, D.J., Kon, Y., Simons, B.D., and Yoshida, S.
(2021). Transient suppression of transplanted spermatogonial stem celldifferentiation restores fertility in mice. Cell Stem Cell 28, 1443-1456.
e7. DOI: 10.1016/j.stem.2021.03.016
Osawa, Y., Murata, K., Usui, M., Kuba, Y., Le, H.T., Mikami, N.,
Nakagawa, T., Daitoku, Y., Kato, K., Shawki, H.H., Ikeda, Y., Kuno, A.,Morimoto, K., Tanimoto, Y., Dinh, T.T.H., Yagami, K., Ema, M., Yoshida,S., Takahashi, S., Mizuno, S., and Sugiyama, F. (2021). EXOC1 plays an
integral role in spermatogonia pseudopod elongation and spermatocyte
stable syncytium formation in mice. eLife 10, e59759. DOI: 10.7554/
eLife.59759〔Review article〕

Y., and Yoshida, S. (2022). Regulation of spermatogenic• Kitadate,stem cell homeostasis by mitogen competition in an open nichemicroenvironment. Genes Genet. Syst. 97, 15–25. DOI: 10.1266/
ggs.21-00062

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