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

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

Tracing the cellular basis of islet specification in mouse pancreas

Abstract: 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

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

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

SHISA6 Confers Resistance to Differentiation-Promoting Wnt/β-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/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

Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes

BACKGROUND AND AIMS: 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 produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules

Germline stem cells.

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 (GSCs) often reside in a defined anatomical niche. Factors within the niche regulate a balance between GSC self-renewal and differentiation. Asymmetric division of the germline stem cell to form daughter cells with alternative fates is common. The exception to both these tendencies is the mammalian testis where there does not appear to be an obvious anatomical niche and where GSC homeostasis is likely accomplished by a stochastic balance of self-renewal and differentiation and not by regulated asymmetric cell division. Despite these apparent differences, GSCs in all organisms share many common mechanisms, although not necessarily molecules, to guarantee survival of the germline