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In mammalian testes, premeiotic spermatogonia respond to retinoic acid by completing an essential lengthy differentiation program before initiating meiosis. The molecular and cellular changes directing these developmental processes remain largely undefined. This wide gap in knowledge is due to two unresolved technical challenges: (1) lack of robust and reliable in vitro models to study differentiation and meiotic initiation; and (2) lack of methods to isolate large and pure populations of male germ cells at each stage of differentiation and at meiotic initiation. Here, we report a facile in vitro differentiation and meiotic initiation system that can be readily manipulated, including the use of chemical agents that cannot be safely administered to live animals. In addition, we present a transgenic mouse model enabling fluorescence-activated cell sorting-based isolation of millions of spermatogonia at specific developmental stages as well as meiotic spermatocytes.

In the mammalian testis, sustained spermatogenesis relies on spermatogonial stem cells (SSCs); their progeny remain as stem cells (self-renewal) or proliferate and differentiate to enter meiosis in response to retinoic acid (RA). Here, we sought to uncover elusive mechanisms regulating a key switch fundamental to spermatogonial fate, the capacity of spermatogonia to respond to RA. Using the developing mouse testis as a model, we found that spermatogonia and precursor prospermatogonia exhibit a heterogeneous capacity to respond to RA with at least two underlying causes. First, progenitor spermatogonia are prevented from responding to RA by catabolic activity of ‘cytochrome P450 family 26’ (CYP26) enzymes. Second, a smaller subset of undifferentiated spermatogonia enriched for SSCs exhibit catabolism-independent RA-insensitivity. Moreover, for the first time, we observed that precursor prospermatogonia are heterogeneous and comprised of subpopulations which exhibit the same differential RA responsiveness found in neonatal spermatogonia. We propose a novel model by which mammalian prospermatogonial and spermatogonial fates are regulated by intrinsic capacity to respond (or not) to the differentiation signal provided by RA prior to and concurrent with the initiation of spermatogenesis.

Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25 μm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.