Adult Human, but Not Rodent, Spermatogonial Stem Cells Retain States with a Foetal-like Signature.

Spermatogenesis involves a complex process of cellular differentiation maintained by spermatogonial stem cells (SSCs). Being critical to male reproduction, it is generally assumed that spermatogenesis starts and ends in equivalent transcriptional states in related species. Based on single-cell gene expression profiling, it has been proposed that undifferentiated human spermatogonia can be subclassified into four heterogenous subtypes, termed states 0, 0A, 0B, and 1. To increase the resolution of the undifferentiated compartment and trace the origin of the spermatogenic trajectory, we re-analysed the single-cell (sc) RNA-sequencing libraries of 34 post-pubescent human testes to generate an integrated atlas of germ cell differentiation. We then used this atlas to perform comparative analyses of the putative SSC transcriptome both across human development (using 28 foetal and pre-pubertal scRNA-seq libraries) and across species (including data from sheep, pig, buffalo, rhesus and cynomolgus macaque, rat, and mouse). Alongside its detailed characterisation, we show that the transcriptional heterogeneity of the undifferentiated spermatogonial cell compartment varies not only between species but across development. Our findings associate 'state 0B' with a suppressive transcriptomic programme that, in adult humans, acts to functionally oppose proliferation and maintain cells in a ready-to-react state. Consistent with this conclusion, we show that human foetal germ cells-which are mitotically arrested-can be characterised solely as state 0B. While germ cells with a state 0B signature are also present in foetal mice (and are likely conserved at this stage throughout mammals), they are not maintained into adulthood. We conjecture that in rodents, the foetal-like state 0B differentiates at birth into the renewing SSC population, whereas in humans it is maintained as a reserve population, supporting testicular homeostasis over a longer reproductive lifespan while reducing mutagenic load. Together, these results suggest that SSCs adopt differing evolutionary strategies across species to ensure fertility and genome integrity over vastly differing life histories and reproductive timeframes.

PRC1 directs PRC2-H3K27me3 deposition to shield adult spermatogonial stem cells from differentiation.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

Branched germline cysts and female-specific cyst fragmentation facilitate oocyte determination in mice.

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 oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation and accumulate cytoplasm and organelles from sister germ cells to become primary oocytes. Changes in cyst structure and differential cell volumes among cyst germ cells suggest that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During fetal cyst formation, E-cadherin (E-cad) junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cad-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cad knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on how oocyte fate is determined within mouse germline cysts.

Temperature sensitivity of DNA double-strand break repair underpins heat-induced meiotic failure in mouse spermatogenesis.

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 controlled temperatures, we found that spermatogenesis failed at multiple steps, showing sharp temperature dependencies. At 38 °C (body core temperature), meiotic prophase I is damaged, showing increased DNA double-strand breaks (DSBs) and compromised DSB repair. Such damaged spermatocytes cause asynapsis between homologous chromosomes and are eliminated by apoptosis at the meiotic checkpoint. At 37 °C, some spermatocytes survive to the late pachytene stage, retaining high levels of unrepaired DSBs but do not complete meiosis with compromised crossover formation. These findings provide insight into the mechanisms and significance of heat vulnerability in mammalian spermatogenesis.

Regulation of spermatogenic stem cell homeostasis by mitogen competition in an open niche microenvironment.

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, the stem cell pool remains constant while differentiating cells are continually produced to replenish the lost differentiated cells. One of the outstanding questions here is how self-renewal and differentiation of SSCs are balanced to achieve a constant self-renewing pool. In this review, we shed light on the regulatory mechanism of SSC homeostasis, with focus on the recently proposed mitogen competition model in a facultative (or open) niche microenvironment.

A multistate stem cell dynamics maintains homeostasis in mouse spermatogenesis.

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 of SSC subpopulations, we show that an ensemble of heterogeneous states of SSCs supports homeostatic, persistent spermatogenesis. Such heterogeneity is maintained robustly through stochastic interconversion of SSCs between a renewal-biased Plvap+/GFRα1+ state and a differentiation-primed Sox3+/GFRα1+ state. In this framework, stem cell commitment occurs not directly but gradually through entry into licensed but uncommitted states. Further, Plvap+/GFRα1+ cells divide slowly, in synchrony with the seminiferous epithelial cycle, while Sox3+/GFRα1+ cells divide much faster. Such differential cell-cycle dynamics reduces mitotic load, and thereby the potential to acquire harmful de novo mutations of the self-renewing pool, while keeping the SSC density high over the testicular open niche.

EXOC1 plays an integral role in spermatogonia pseudopod elongation and spermatocyte stable syncytium formation in mice.

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 stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

Transient suppression of transplanted spermatogonial stem cell differentiation restores fertility in mice.

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 tubules. We found that, after a large population of donor spermatogonia settle in host testes, through stochastic fate choice, only a small fraction persist and regenerate over the long term, and the rest are lost through differentiation and cell death. Further, based on these insights, we showed how repopulation efficiency can be increased to a level where the fertility of infertile hosts is restored by transiently suppressing differentiation using a chemical inhibitor of retinoic acid synthesis. These findings unlock a range of potential applications of spermatogonial transplantation, from fertility restoration in individuals with cancer to conservation of biological diversity.

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 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 isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Tracing the cellular basis of islet specification in mouse pancreas.

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 ß-cells are generated in a contemporary manner. Together, these findings provide insight into the cellular basis of islet development.

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

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.

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

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 "As 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.

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

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 Wls, which encodes a factor that is essential for Wnt secretion. Furthermore, proliferation of these cells was impaired in Wls 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.

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

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.

Open niche regulation of 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.

mDia1/3 generate cortical F-actin meshwork in Sertoli cells that is continuous with contractile F-actin bundles and indispensable for spermatogenesis and male fertility.

Formin is one of the two major classes of actin binding proteins (ABPs) with nucleation and polymerization activity. However, despite advances in our understanding of its biochemical activity, whether and how formins generate specific architecture of the actin cytoskeleton and function in a physiological context in vivo remain largely obscure. It is also unknown how actin filaments generated by formins interact with other ABPs in the cell. Here, we combine genetic manipulation of formins mammalian diaphanous homolog1 (mDia1) and 3 (mDia3) with superresolution microscopy and single-molecule imaging, and show that the formins mDia1 and mDia3 are dominantly expressed in Sertoli cells of mouse seminiferous tubule and together generate a highly dynamic cortical filamentous actin (F-actin) meshwork that is continuous with the contractile actomyosin bundles. Loss of mDia1/3 impaired these F-actin architectures, induced ectopic noncontractile espin1-containing F-actin bundles, and disrupted Sertoli cell-germ cell interaction, resulting in impaired spermatogenesis. These results together demonstrate the previously unsuspected mDia-dependent regulatory mechanism of cortical F-actin that is indispensable for mammalian sperm development and male fertility.

MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice.

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 function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not. MAFB was localized in Leydig and Sertoli cells at embryonic day (E) 18.5 but in Leydig cells, Sertoli cells, and pachytene spermatocytes in adults. Mafb-deficient testes at E18.5 showed fully formed seminiferous tubules with no abnormal structure or differences in testicular somatic cell numbers compared with those of control wild-type mice. Additionally, the expression levels of genes related to development and function of testicular cells were unchanged between genotypes. In adults, the expression of MAFB in Sertoli cells was shown to be stage specific and induced by RA. By generating Mafbfl/fl CAG-CreER™ (Mafb-cKO) mice, in which Cre recombinase was activated upon tamoxifen treatment, we found that the neonatal cKO mice died shortly upon Mafb deletion, but adult cKO mice were alive upon deletion. Adult cKO mice were fertile, and spermatogenesis maintenance was normal, as indicated by histological analysis, hormone levels, and germ cell stage-specific markers. Moreover, there were no differences in the proportion of seminiferous stages between cKO mice and controls. However, RNA-Seq analysis of cKO Sertoli cells revealed that the down-regulated genes were related to immune function and phagocytosis activity but not spermatogenesis. In conclusion, we found that MAFB is dispensable for fetal testis morphogenesis and spermatogenesis maintenance in adult mice, despite the significant gene expression in different cell types, but MAFB might be critical for phagocytosis activity of Sertoli cells.

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

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/ß-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/ß-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.

From cyst to tubule: innovations in vertebrate 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.