RESUMEN
The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (Aundiff); however, only a fraction of this population act as stem cells in undisturbed testis and Aundiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult Aundiff fractions and dissect Aundiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing Aundiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- Aundiff states upon transplant and culture. We conclude that Aundiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Espermatogonias/metabolismo , Células Madre/metabolismo , Testículo/metabolismo , Transactivadores/genética , Animales , Diferenciación Celular , Linaje de la Célula/genética , Efecto Fundador , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Integrasas/genética , Integrasas/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteína de la Leucemia Promielocítica con Dedos de Zinc/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Análisis de la Célula Individual , Espermatogonias/citología , Células Madre/citología , Testículo/citología , Testículo/crecimiento & desarrollo , Transactivadores/metabolismo , Proteína Fluorescente RojaRESUMEN
Sustained spermatogenesis in adult males and fertility recovery following germ cell depletion are dependent on undifferentiated spermatogonia. We previously demonstrated a key role for the transcription factor SALL4 in spermatogonial differentiation. However, whether SALL4 has broader roles within spermatogonia remains unclear despite its ability to co-regulate genes with PLZF, a transcription factor required for undifferentiated cell maintenance. Through development of inducible knockout models, we show that short-term integrity of differentiating but not undifferentiated populations requires SALL4. However, SALL4 loss was associated with long-term functional decline of undifferentiated spermatogonia and disrupted stem cell-driven regeneration. Mechanistically, SALL4 associated with the NuRD co-repressor and repressed expression of the tumor suppressor genes Foxl1 and Dusp4. Aberrant Foxl1 activation inhibited undifferentiated cell growth and survival, while DUSP4 suppressed self-renewal pathways. We therefore uncover an essential role for SALL4 in maintenance of undifferentiated spermatogonial activity and identify regulatory pathways critical for germline stem cell function.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/genética , Silenciador del Gen , Genes Supresores de Tumor , Células Germinativas/citología , Proteínas Tirosina Fosfatasas/genética , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Fenotipo , Regiones Promotoras Genéticas/genética , Proteína de la Leucemia Promielocítica con Dedos de Zinc/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Regeneración , Espermatogonias/citología , Espermatogonias/metabolismo , Testículo/citologíaRESUMEN
Spermatogonia stem cell (SSC) self-renewal and differentiation are tightly regulated processes that ensure a continued production of mature sperm throughout male adulthood. In the present study, we investigated the role of glucocorticoid-induced leucine zipper (GILZ) in maintenance of the male germline and spermatogenesis. GILZ was detectable in germ cells of wild type mice on the day of birth, suggesting a role for GILZ in prospermatogonia and SSC pool formation. Gilz KO mice were generated and adult males were azoospermic and sterile. During the first wave of spermatogenesis in Gilz KO mice, spermatogenesis arrested part way through pachytene of meiosis I. Subsequent waves resulted in a progressive depletion of germ cells through apoptosis to ultimately produce a Sertoli cell-only phenotype. Further, in contrast to wild type littermates, PLZF(+) cells were detected in the peri-luminal region of Gilz KO mice at day 6 post-natal, suggesting a defect in prospermatogonia migration in the absence of GILZ. At age 30 days, transient accumulation of PLZF(+) cells in a subset of tubules and severely compromised spermatogenesis were observed in Gilz KO mice, consistent with defective SSC differentiation. GILZ deficiency was associated with an increase in FOXO1 transcriptional activity, which leads to activation of a selective set of FOXO1 target genes, including a pro-apoptotic protein, BIM. On the other hand, no evidence of a heightened immune response was observed. Together, these results suggest that GILZ suppresses FOXO1 nuclear translocation, promotes SSC differentiation over self-renewal, and favours germ cell survival through inhibition of BIM-dependent pro-apoptotic signals. These findings provide a mechanism for the effects of GILZ on spermatogenesis and strengthen the case for GILZ being a critical molecule in the regulation of male fertility.
