Functionally redundant roles of ID family proteins in spermatogonial stem cells

Spermatogonial stem cells (SSCs) are essential for sustained sperm production and male fertility, but SSC regulatory mechanisms and molecular markers remain poorly defined. Previous studies have suggested that the Id family transcriptional regulator Id4 is expressed at high levels in SSCs and involved in SSC maintenance. Here we have used knock-in reporter and inducible knockout models to define expression pattern and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and ID4 protein are found throughout the GFRa1+ fraction, comprising the self-renewing population. However, acute deletion of Id4 is tolerated by adult SSCs while revealing unexpected roles in maintenance of meiotic spermatocytes. Cultures of undifferentiated spermatogonia were also successfully established and expanded following Id4 deletion. Importantly, ID4 loss in undifferentiated spermatogonia triggers ID3 upregulation, and both ID proteins associate with transcription factor partner TCF3 in wildtype cells. Moreover, combined inhibition of ID proteins in cultured spermatogonia disrupts the stem cell state and blocks proliferation. Our data therefore demonstrate critical but functionally redundant roles of ID family proteins in the maintenance of SSC function.