Shosei Yoshida

National Institute for Basic Biology | NIBB · NIBB

During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocy...

Mammalian spermatogenesis is a heat-vulnerable process that occurs at low temperatures, and elevated testicular temperatures cause male infertility. However, the current reliance on in vivo assays limits their potential to detail temperature dependence and destructive processes. Using ex vivo cultures of mouse testis explants at different controlle...

Continuity of spermatogenesis in mammals is underpinned by spermatogenic (also called spermatogonial) stem cells (SSCs) that self-renew and differentiate into sperm that pass on genetic information to the next generation. Despite the fundamental role of SSCs, the mechanisms underlying SSC homeostasis are only partly understood. During homeostasis,...

In mouse testis, a heterogeneous population of undifferentiated spermatogonia (Aundiff) harbors spermatogenic stem cell (SSC) potential. Although GFRα1+ Aundiff maintains the self-renewing pool in homeostasis, the functional basis of heterogeneity and the implications for their dynamics remain unresolved. Here, through quantitative lineage tracing...

The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte...

A remarkable feature of tissue stem cells is their ability to regenerate the structure and function of host tissue following transplantation. However, the dynamics of donor stem cells during regeneration remains largely unknown. Here we conducted quantitative clonal fate studies of transplanted mouse spermatogonial stem cells in host seminiferous t...

This article reviews the physical principles of stem cell populations as active many-particle systems that are able to self-renew, control their density, and recover from depletion. We illustrate the dynamical and statistical hallmarks of homeostatic mechanisms, from stem cell density fluctuations and transient large-scale oscillation dynamics duri...

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell...

Pancreatic islets play an essential role in regulating blood glucose level. Although the molecular pathways underlying islet cell differentiation are beginning to be resolved, the cellular basis of islet morphogenesis and fate allocation remain unclear. By combining unbiased and targeted lineage tracing, we address the events leading to islet forma...

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detail...

The existence of cytoplasmic passages between germ cells and their potential function in the control of the spermatogenic process has long been an intriguing question. Evidence of the important role of such structures, known as intercellular bridges (ICB), in spermatogenesis has been implicated by the failure of spermatogenesis in Tex14 mutant mice...

Mouse spermatogenesis is supported by spermatogenic stem cells (SSCs). SSCs maintain their pool while migrating over an open (or facultative) niche microenvironment of testicular seminiferous tubules, where ligands that support self-renewal are likely distributed widely. This contrasts with the classic picture of closed (or definitive) niches in wh...

Cell morphology changes dynamically during embryogenesis, and these changes create new interactions with surrounding cells, some of which are presumably mediated by intercellular signaling. However, the effects of morphological changes on intercellular signaling remain to be fully elucidated. In this study, we examined the effect of morphological c...

Stem cells maintain tissues by generating differentiated cell types while simultaneously self-renewing their own population. The mechanisms that allow stem cell populations to function collectively to control their density, maintain robust homeostasis and recover from injury remain elusive. Motivated by recent experimental advances, here we develop...

Table S3. Primers Used in this Study for qRT-PCR and Genomic qPCR, Related to STAR Methods

Mammalian testes produce a huge number of sperm over a long period. This process, essential for the continuity of life, depends on the delicate balance of self-renewal and differentiation of resident stem cells, termed spermatogenic (spermatogonial)stem cells or SSCs. SSCs have motivated many researchers to query their identity, behavior, and regul...

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly...

In mammalian testes, robust stem cell functions ensure the continual production of sperm. In testicular seminiferous tubules, spermatogenic stem cells (SSCs) are highly motile and are interspersed between their differentiating progeny, while undergoing self‐renewal and differentiation. In such an “open niche” microenvironment, some SSCs proliferate...

Author summary Paternal genetic information is transmitted to the offspring via sperm. The unique cell morphology of the sperm plays essential roles in sperm transport through the female reproductive tract and in fertilization with oocytes. Sertoli cells are somatic cells located in the seminiferous tubules of the testis and are known to contribute...

Male infertility of mDia1/3 DKO mice. (A and B) Micrographs (A) and fertilization rate (B) of IVF of ZP-intact oocytes with sperms from WT or mDia1/3 DKO mice. Scale bar, 50 μm. Data represented mean ± SEM. Data are the average of three independent experiments. ***P < 0.001 (Student t test). DKO, double knockout; IVF, in vitro fertilization; mDia1/...

Increased apoptotic cells in mDia1/3 DKO seminiferous tubule. (A) Apoptotic cells (green) in the mDia1/3 DKO seminiferous tubules. Nuclei (magenta) were stained with Hoechst. Scale bar, 100 μm. (B) Quantification of the number of apoptotic cells per seminiferous tubule. Data represented mean ± SEM (91 seminiferous tubules from four WT mice and 99 s...

F-actin architecture of Sertoli cells in intact seminiferous tubules. (A) Transduction of LifeAct-EGFP expressing lentivirus in the testis of WT mouse. Seminiferous tubule was microinjected with LifeAct-EGFP expressing lentivirus and analyzed at 1 wk after injection under fluorescence stereomicroscope. (B) Observation strategy of LifeAct-EGFP expre...

Nectin-2 expression in the seminiferous tubules throughout the spermatogenic cycles. (A) Immunohistochemistry staining for nectin-2 (green) and Hoechst staining (magenta) of testis sections from WT mice. Strong nectin-2 signals were observed at the apical ectoplasmic specialization junction. In addition, nectin-2 signals were observed at the adhere...

Specificity of anti-mDia1 and anti-mDia3 antibodies for immunohistochemistry. (A) Immunohistochemistry staining for mDia1 (green) and vimentin (magenta) as a marker for Sertoli cells in testis sections from WT and mDia1 KO adult mice. Positive mDia1 signals at the vimentin-positive Sertoli cells observed in WT mice were abolished in mDia1 KO mice....

Reduced F-actin staining of mDia1/3 DKO primary cultured Sertoli cell. (A) Confocal images of actin filaments of WT (left) and mDia1/3 DKO (right) primary cultured Sertoli cells. The lines (magenta and green) were used to quantify the fluorescence intensity by line scan, and the fluorescence intensity profiles along these lines are shown in the rig...

Reduced cortical F-actin meshwork in mDia1/3 DKO Sertoli cells was rescued by expression of EGFP-mDia3. (A) mDia1/3 DKO primary cultured Sertoli cells transfected with pEGFP-mDia3 (green) were stained with phalloidin (magenta). The cell on the right is EGFP-mDia3 positive. The magenta and green dotted line was used to quantify the fluorescence inte...

Model of the molecular mechanism of mDia1/3 function in spermatogenesis through the regulation of F-actin and cell adhesions between Sertoli cell and germ cell. F-actin, filamentous actin; mDia1/3, mammalian diaphanous homolog1/3. (TIF)

F-actin dynamics observed with SDSRM. EGFP-LifeAct was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 2 s. EGFP, enhanced green fluorescent protein; F-actin, filamentous actin; SDSRM, spinning disk superresolution microscopy; WT, wild-type. (MOV)

Fast polymerization of cortical actin filament meshwork observed with SDSRM. EGFP-LifeAct was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 2 s. EGFP, enhanced green fluorescent protein; SDSRM, spinning disk superresolution microscopy; WT, wild-type. (MOV)

Single-molecule observation of fast movement of EGFP-mDia3 in a primary cultured Sertoli cell with TIRF microscopy. EGFP-mDia3 was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 200 ms. EGFP, enhanced green fluorescent protein; mDia3, mammalian diaphanous homolog3; TIRF, total internal reflection; WT, wild-typ...

Reduced number, abnormal morphology, and impaired motility of mDia1/3 DKO sperm. (A) HE-stained epididymal cross sections from adult WT or mDia1/3 DKO mice. Scale bar, 25 μm. (B) Total number of sperm per epididymis of WT or mDia1/3 DKO mice. Data represented mean ± SEM. n = 3 and 4 for WT and mDia1/3 DKO, respectively. **P < 0.01 (P = 0.0062, Stud...

mDia3 expression in the seminiferous tubules throughout the spermatogenic cycles. (A) Immunohistochemistry staining for mDia3 (green) and phalloidin staining (magenta) of WT testis sections. Arrowheads indicate mDia3 staining at the basal ectoplasmic junction and arrows indicate mDia3 staining at the apical ectoplasmic junction. (B) Immunohistochem...

Immunohistochemistry of espin1 together with phalloidin staining in seminiferous tubule throughout spermatogenic cycles. (A) Immunohistochemistry staining for espin1 (green) and phalloidin staining (magenta) of testis sections from WT mice. Espin1 signals were observed at the apical ES junction of elongated spermatid from stages II–V seminiferous t...

Intracellular molecular movement of EGFP-mDia3 underneath the cell cortex, observed with TIRF microscopy. EGFP-mDia3 was expressed in primary cultured Sertoli cell from WT mouse; the images were taken every 200 ms. EGFP, enhanced green fluorescent protein; mDia3, mammalian diaphanous homolog3; TIRF, total internal reflection; WT, wild-type. (MOV)

Three-dimensional reconstruction of deconvolution spinning disk confocal images of Sertoli cell F-actin in intact seminiferous tubule. Z-series images of Sertoli cell transduced by LifeAct-EGFP expressing lentivirus in intact seminiferous tubule were acquired by spinning disk confocal microscopy and processed with a deconvolution algorithm. Three-d...

The germline is the only cell type that is inherited by the next generation in many multicellular animals. For the purposes of successful reproduction, animals need to produce enough gametes for a sufficient duration of time. It is also crucial to adjust the production of gametes according to the reproduction strategy that each species uniquely dev...

The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and fu...

List of the up-regulated transcripts of Mafb-cKO Sertoli cells. (XLSX)

List of the down-regulated transcripts of Mafb-cKO Sertoli cells. (XLSX)

Sertoli and germ cell separation by FACS. Immunostaining with the Sertoli cell marker vimentin (red) before and after cell sorting. Upper panels (R1): before sorting. Middle (R2) and Lower (R3) panels: after sorting. The R2 population represents germ cells as vimentin negative, while the R2 population represents Sertoli cells as vimentin positive....

RNA-Seq validation for the transcriptome profile of Mafb-cKO Sertoli cells. Sertoli cells were sorted from three-month-old Mafb-cKO or control mice and analyzed by RNA-Seq. (A) The purity of the isolated cells was confirmed by IHC staining with the Sertoli cell-specific marker anti-vimentin antibody (red) and nuclei were counterstained with DAPI (b...

In the seminiferous tubules of mouse testes, a population of glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRα1)-positive spermatogonia harbors the stem cell functionality and supports continual spermatogenesis, likely independent of asymmetric division or definitive niche control. Here, we show that activation of Wnt/β-cate...

Although vertebrates share many common traits, their germline development and function exhibit significant divergence. In particular, this article focuses on their spermatogenesis. The fundamental elements that constitute vertebrate spermatogenesis and the evolutionary changes that occurred upon transition from water to land will be discussed. The...

Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (Aundiff) are motile and intermingle with differ...

In temperate zones, animals restrict breeding to specific seasons to maximize survival of their offspring. Birds have evolved highly sophisticated mechanisms of seasonal regulation, and their testicular mass can change a hundred-fold within a few weeks. Recent studies on Japanese quail revealed that seasonal gonadal development is regulated by cent...

The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing "As model," stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial sp...

Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized; by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of mu...

Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by t...

Spermatogenesis in mice and other mammalians is supported by a robust stem cell system. Stem cells maintain themselves and continue to produce progeny that will differentiate into sperm over a long period. The pioneering studies conducted from the 1950s to the 1970s, which were based largely on extensive morphological analyses, have established the...

Homeostasis of tissues relies on the regulated differentiation of stem cells. In the epithelium of mouse seminiferous tubules, the differentiation process from undifferentiated spermatogonia (A(undiff)), which harbor the stem cell functions, to sperm occurs in a periodical manner, known as the "seminiferous epithelial cycle". To identify the mechan...

In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produc...

Mammalian spermatogenesis endures on the persistent activity of stem cells, i.e., their self-renewal and production of differentiating progeny. The normal functioning of stem cells explicitly requires a particular microenvironment within the tissue – the stem cell niche – as an indispensable element. While the mammalian spermatogenic stem cell nich...

Vitamin A is instrumental to mammalian reproduction. Its metabolite, retinoic acid (RA), acts in a hormone-like manner through binding to and activating three nuclear receptor isotypes, RA receptor (RAR)α (RARA), RARβ, and RARγ (RARG). Here, we show that 1) RARG is expressed by A aligned (A(al)) spermatogonia, as well as during the transition from...

Most vertebrates living outside the tropical zone show robust physiological responses in response to seasonal changes in photoperiod, such as seasonal reproduction, molt, and migration. The highly sophisticated photoperiodic mechanism in Japanese quail has been used to uncover the mechanism of seasonal reproduction. Molecular analysis of quail medi...

Sperm and egg production requires a robust stem cell system that balances self-renewal with differentiation. Self-renewal at the expense of differentiation can cause tumorigenesis, whereas differentiation at the expense of self-renewal can cause germ cell depletion and infertility. In most organisms, and sometimes in both sexes, germline stem cells...

In mammalian testis, numerous sperms are produced persistently for a prolonged period, which plays an essential role for the continuity of life. It is generally considered that ‘stem cells’ that are both capable of self-renewal and differentiation supports the continuity of spermatogenesis. ‘Stem cells’ are also considered to be crucial not only fo...

Mammalian testes produce a huge number of sperms for a long reproduction period. This is supported by the constant differentiation of a small subset of undifferentiated spermatogonia that makes up the stem cell compartment. In the testis, differentiation and self-renew of undifferentiated spermatogonia are beautifully coordinated both temporally an...

The switch from mitosis to meiosis is a unique feature of germ cell development. In mammals, meiotic initiation requires retinoic acid (RA), which activates meiotic inducers, including Stra8, but how the switch to meiosis is controlled in male germ cells (spermatogonia) remains poorly understood. Here we examine the role of the Doublesex-related tr...

It has been known for many decades that nonmammalian vertebrates detect light by deep brain photoreceptors that lie outside the retina and pineal organ to regulate seasonal cycle of reproduction. However, the identity of these photoreceptors has so far remained unclear. Here we report that Opsin 5 is a deep brain photoreceptive molecule in the quai...

In cycling tissues, adult stem cells may be lost and subsequently replaced to ensure homeostasis. To examine the frequency of stem cell replacement, we analyzed the population dynamics of labeled stem cells in steady-state mouse spermatogenesis. Our results show that spermatogenic stem cells are continuously replaced, on average within 2 weeks. The...

Mammalian testes continually produce a huge number of sperm over a long reproductive period. This constant spermatogenesis is supported by a highly robust stem cell system. Morphological analyses in the 1960s and 70s established the basis of mammalian spermatogenesis and the associated stem cell research. Subsequently, from the 1990s on, functional...

Stem cells support tissue maintenance by balancing self-renewal and differentiation. In mice, it is believed that a homogeneous stem cell population of single spermatogonia supports spermatogenesis, and that differentiation, which is accompanied by the formation of connected cells (cysts) of increasing length, is linear and nonreversible. We evalua...

Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein repo...

In the classical view, adult stem cells and their differentiating progeny are considered to be distinct entities in an irreversible sequence of development. However, emerging evidence suggests that this distinction may not be so definitive. Recent work in the mouse shows that a population of differentiating spermatogonia, when transplanted, can 'de...

Mouse spermatogenesis represents a highly potent and robust stem cell system. Decades of research have made it one of the most intensively studied mammalian tissue stem cell systems. These studies include detailed morphological examinations, posttransplantation colony formation, and in vitro culture of the stem cells; however, the nature of the ste...