No abstract

Pachytene piRNA biogenesis is a hallmark of the germline, distinct from another wave of pre-pachytene piRNA biogenesis with regard to the lack of a secondary amplification process known as the Ping-pong cycle. However, the underlying molecular mechanism and the venue for the suppression of the Ping-pong cycle remain elusive. Here, we showed that a testis-specific protein, ADAD2, interacts with a TDRD family member protein RNF17 and is associated with P-bodies. Importantly, ADAD2 directs RNF17 to repress Ping-pong activity in pachytene piRNA biogenesis. The P-body localization of RNF17 requires the intrinsically disordered domain of ADAD2. Deletion of Adad2 or Rnf17 causes the mislocalization of each other and subsequent Ping-pong activity derepression, secondary piRNAs overproduced, and disruption of P-body integrity at the meiotic stage, thereby leading to spermatogenesis arrested at the round spermatid stage. Collectively, by identifying the ADAD2-dependent mechanism, our study reveals a novel function of P-bodies in suppressing Ping-pong activity in pachytene piRNA biogenesis.

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.