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.

SummarySustained 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.

Mammalian spermatogenesis is sustained by mitotic germ cells with self-renewal potential known as undifferentiated spermatogonia. Maintenance of undifferentiated spermatogonia and spermatogenesis is dependent on tightly co-ordinated transcriptional and post-transcriptional mechanisms. The RNA helicase DDX5 is expressed by spermatogonia but roles in spermatogenesis are unexplored. Using an inducible knockout mouse model, we characterise an essential role for DDX5 in spermatogonial maintenance and show that Ddx5 is indispensable for male fertility. We demonstrate that DDX5 regulates appropriate splicing of key genes necessary for spermatogenesis. Moreover, DDX5 regulates expression of cell cycle genes in undifferentiated spermatogonia post-transcriptionally and is required for cell proliferation and survival. DDX5 can also act as a transcriptional co-activator and we demonstrate that DDX5 interacts with PLZF, a transcription factor required for germline maintenance, to co-regulate select target genes. Combined, our data reveal a critical multifunctional role for DDX5 in regulating gene expression programmes and activity of undifferentiated spermatogonia.