isolation-murine-spermatogenic-cells-using-violet-excited-cell

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

National Institutes of Natural Sciences

Graduate University for Advanced Studies (SOKENDAI)

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 analyses, which have included post-transplantation colony formation, in vitro spermatogonial culture with persisting stem cell activity, in vivo lineage tracing, and live imaging, and also lines of molecular-genetic analyses, have contributed greatly to our understanding of mammalian spermatogenic stem cells. This review will provide a brief overview of the history of this field and then go on to describe in detail the progress made in recent years.

Stem cells in mammalian spermatogenesis.

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 fundamentals of mammalian spermatogenesis and its stem cells. The prevailing so-called A(single) (A(s)) model, which was originally established in 1971, proposes that singly isolated A(s) spermatogonia are in fact the stem cells. In 1994, the first functional stem cell assay was established based on the formation of repopulating colonies after transplantation in germ cell-depleted host testes, which substantially accelerated the understanding of spermatogenic stem cells. However, because testicular tissues are dissociated into single-cell suspension before transplantation, it was impossible to evaluate the A(s) and other classical models solely by this technique. From 2007 onwards, functional assessment of stem cells without destroying the tissue architecture has become feasible by means of pulse-labeling and live-imaging strategies. Results obtained from these experiments have been challenging the classical thought of stem cells, in which stem cells are a limited number of specialized cells undergoing asymmetric division to produce one self-renewing and one differentiating daughter cells. In contrast, the emerging data suggest that an extended and heterogeneous population of cells exhibiting different degrees of self-renewing and differentiating probabilities forms a reversible, flexible, and stochastic stem cell system as a population. These features may lead to establishment of a more universal principle on stem cells that is shared by other systems.

Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis.

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 the culture method without any genetic manipulations. The Sl/Sl(d) mouse is used as a model of such male infertility. The testis of the Sl/Sl(d) mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl(d) mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.

Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice.

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 spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFRα1+ cells and find that, during steady-state spermatogenesis, the entire GFRα1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFRα1+ cells during steady state and postinsult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis. 

Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states.

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 differentiating cells in an 'open' niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)(+) and neurogenin 3 (NGN3)(+) subpopulations, both of which retain the ability to self-renew. However, whereas GFRα1(+) cells comprise the homeostatic stem cell pool, NGN3(+) cells show a higher probability to differentiate into KIT(+) spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3(+) cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3(+) cells, but not in GFRα1(+) cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1(+) cells to directly differentiate to KIT(+) cells without transiting the NGN3(+) state. Therefore, RARγ plays key roles in the differentiation competence of NGN3(+) cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals. 

Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

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 life-long continuity of spermatogenesis is supported by the function of stem cells. Series of mitotic and meiotic germ cell divisions are 'incomplete' due to incomplete cytokinesis, forming syncytia interconnected via intercellular bridges (ICBs). Throughout this process, germ cells are supported by appropriate microenvironments established primarily by somatic Sertoli cells. In anamniotes (fish and amphibians) spermatogenesis progresses in cysts, in which developing germ cell syncytia are individually encapsulated by Sertoli cells. Accordingly, Sertoli cells undergo turnover with germ cells that they nourish. This mode of cystic spermatogenesis is also observed in nonvertebrates as insects. In amniotes (reptiles, birds, and mammals), however, Sertoli cells do not turn over but comprise a persistent structure of seminiferous tubules. Sertoli cells nourish different stages of germ cells simultaneously in distinct regions of their surface. This function of Sertoli cells is spatiotemporally orchestrated, and the seminiferous epithelial cycle and spermatogenic wave make the seminiferous tubules a high-throughput factory for sperm production. Furthermore, contrary to the organized differentiating cells, undifferentiated spermatogonia that comprise the stem cell compartment exhibit active motion over the basal layer of seminiferous tubules and the frequent breakdown of ICBs. Thus, amniote seminiferous tubules represent a typical facultative (or open) niche environment without a stem cell tethering anatomically defined niche. WIREs Dev Biol 2016, 5:119-131. doi: 10.1002/wdev.204 For further resources related to this article, please visit the WIREs website. 

From cyst to tubule: innovations in vertebrate spermatogenesis.

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-catenin signaling promotes spermatogonial differentiation and reduces the GFRα1 cell pool. We further discovered that SHISA6 is a cell-autonomous Wnt inhibitor that is expressed in a restricted subset of GFRα1 cells and confers resistance to the Wnt/β-catenin signaling. Shisa6 cells appear to show stem cell-related characteristics, conjectured from the morphology and long-term fates of T (Brachyury) cells that are found largely overlapped with Shisa6 cells. This study proposes a generic mechanism of stem cell regulation in a facultative (or open) niche environment, with which different levels of a cell-autonomous inhibitor (SHISA6, in this case) generates heterogeneous resistance to widely distributed differentiation-promoting extracellular signaling, such as WNTs.

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/β-Catenin Signaling in Mouse Spermatogenic Stem Cells.

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, while others exit the stem cell compartment through differentiation; therefore, self-renewal and differentiation are perfectly balanced at the population (or tissue) level, a dynamics termed "population asymmetry." This is in stark contrast to the classical perception of tissue stem cells being cells that are clustered in a specialized "closed niche" region and that invariantly undergo asymmetric divisions. However, despite its importance, how the self-renewal and differentiation of SSCs are balanced in an open niche environment is poorly understood. Recent studies have thrown light on the key mechanism that enables SSCs to follow heterogeneous fates, although they are equally exposed to signaling molecules controlling self-renewal and differentiation. In particular, SSCs show heterogeneous susceptibilities to differentiation-promoting signals such as Wnt and retinoic acid. Heterogeneous susceptibility to the ubiquitously distributed fate-controlling extracellular signal might be a key generic mechanism for the heterogeneous fate of tissue stem cells in open niche microenvironments.

Open niche regulation of mouse spermatogenic stem cells.

Dynamic homeostasis: From development to aging.

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 regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.

Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche.

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 changes in Wnt-producing cells on intercellular signaling in the spinal cord. After mid-gestation, roof-plate cells stretched along the dorsoventral axis in the mouse spinal cord, resulting in new contact at their tips with the ependymal cells that surround the central canal. Wnt1 and Wnt3a were produced by the stretched roof-plate cells and delivered to the cell process tip. Whereas Wnt signaling was activated in developing ependymal cells, Wnt activation in dorsal ependymal cells, which were close to the stretched roof plate, was significantly suppressed in embryos with roof plate-specific conditional knockout of , which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in  conditional knockout mice during development and after induced spinal cord injury in adults. Therefore, morphological changes in Wnt-producing cells appear to generate new Wnt signal targets.

Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord.

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 regulation in the tissue microenvironment. The study of SSCs has a long and prominent history: Taking advantage of the unique organization of the seminiferous tubules, and the accompanying seminiferous epithelial cycle and spermatogenic wave, intricate concepts of SSC dynamics were established from the early days. This is represented by the "A model" first published in 1971. Then, numerous technical breakthroughs including transplantation, stem cell culture, organ culture, intravital live-imaging, and lineage tracing have made SSCs an invaluable model for tissue stem cell research. Further progress is likely to come from emerging technologies, such as in vitro gametogenesis and single cell omics. An ensemble of these experimental systems has lead to the identification of the dynamic and heterogeneous nature of SSCs, underpinning their context-dependent and flexible behavior. In addition, active migration of SSCs over the open (or facultative) niche microenvironment of the seminiferous tubules is in stark contrast to stem cell regulations within anatomically defined niches such as in the Drosophila ovary and testis (see chapter "Germline stem cell homeostasis" by Nelson et al.). By revealing novel mechanisms of stem cell regulation, research on mouse spermatogenesis will continually make significant contributions to the understanding of general concepts in the tissue stem-cell field.

Heterogeneous, dynamic, and stochastic nature of mammalian spermatogenic stem cells.

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 which stem cells are gathered and the ligands are highly localized. Some of the key properties observed in the dynamics of SSCs in the testicular niche in vivo, which show the flexible and stochastic (probabilistic) fate behaviors, are found to be generic for a wide range of, if not all, tissue stem cells. SSCs also show properties characteristic of an open niche-supported system, such as high motility. Motivated by the properties of SSCs, in this review, I will reconsider the potential unity and diversity of tissue stem cell systems, with an emphasis on the varying degrees of ligand distribution and stem cell motility.

Mouse Spermatogenesis Reflects the Unity and Diversity of Tissue Stem Cell Niche Systems.

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 formation in the mouse. From the statistical analysis of clones induced at multiple embryonic timepoints, here we show that, during the secondary transition, islet formation involves the aggregation of multiple equipotent endocrine progenitors that transition from a phase of stochastic amplification by cell division into a phase of sublineage restriction and limited islet fission. Together, these results explain quantitatively the heterogeneous size distribution and degree of polyclonality of maturing islets, as well as dispersion of progenitors within and between islets. Further, our results show that, during the secondary transition, α- and β-cells are generated in a contemporary manner. Together, these findings provide insight into the cellular basis of islet development.

Article	Year
Stem cells in mammalian spermatogenesis. Development, growth & differentiation\| PubMed ID: 20388168	2010
Elucidating the identity and behavior of spermatogenic stem cells in the mouse testis. Reproduction (Cambridge, England)\| PubMed ID: 22733803	2012
Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22984182	2012
Mouse spermatogenic stem cells continually interconvert between equipotent singly isolated and syncytial states. Cell stem cell\| PubMed ID: 24792118	2014
Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis. Development (Cambridge, England)\| PubMed ID: 25858458	2015
From cyst to tubule: innovations in vertebrate spermatogenesis. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 26305780	2016
SHISA6 Confers Resistance to Differentiation-Promoting Wnt/β-Catenin Signaling in Mouse Spermatogenic Stem Cells. Stem cell reports\| PubMed ID: 28196692	2017
Open niche regulation of mouse spermatogenic stem cells. Development, growth & differentiation\| PubMed ID: 30443901	2018
Dynamic homeostasis: From development to aging. Development, growth & differentiation\| PubMed ID: 30556144	2018
Competition for Mitogens Regulates Spermatogenic Stem Cell Homeostasis in an Open Niche. Cell stem cell\| PubMed ID: 30581080	2019
Wnt produced by stretched roof-plate cells is required for the promotion of cell proliferation around the central canal of the spinal cord. Development (Cambridge, England)\| PubMed ID: 30651295	2019
Heterogeneous, dynamic, and stochastic nature of mammalian spermatogenic stem cells. Current topics in developmental biology\| PubMed ID: 31155360	2019
Mouse Spermatogenesis Reflects the Unity and Diversity of Tissue Stem Cell Niche Systems. Cold Spring Harbor perspectives in biology\| PubMed ID: 32152184	2020
Tracing the cellular basis of islet specification in mouse pancreas. Nature communications\| PubMed ID: 33028844	2020

Shosei Yoshida

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