The continuous regeneration of spermatogonial stem cells (SSCs) underpins spermatogenesis and lifelong male fertility; however, little is known about the developmental origins of the SSC pool. Here, we document that heterogeneous nuclear ribonucleoprotein U (hnRNPU) is essential for establishing the SSC pool. In male mice, conditional loss of hnRNPU in prospermatogonia (ProSG) arrests spermatogenesis and results in sterility, characterized by complete loss of germ cells around postnatal day 10, which resembles the Sertoli cell-only phenotype in humans. hnRNPU-deficient ProSG fails to differentiate and migrate to the basement membrane to establish SSC pool in infancy. Moreover, we find that the deletion of hnRNPU leads to the accumulation of ProSG and the reduction of undifferentiated spermatogonia and further disrupts the process of T1-ProSG to T2-ProSG transition. hnRNPU-deficiency in ProSG deregulates the expression of spermatogenic-related genes and destroys the alternative splicing of genes related to cell cycles, and single-cell transcriptional analyses reveal germ cells are in a mitotically quiescent state and lost their unique identity upon hnRNPU deletion. We further show that hnRNPU could interact with DDX5, SRSF3, and TRIM28 proteins and bind to Vrk1, Slx4, and Dazl transcripts with identified to be suffered aberrant alternative splicing in hnRNPU-deficient testes. These observations give important insights into SSC pool establishment and may have translational implications for male fertility.
