AID-mediated protein knockdown reveals the requirement of NANOS2 in prenatal gonocytes for establishing functional spermatogonial stem cells

Abstract

The RNA-binding protein NANOS2 plays a crucial role in male gonocyte development and the maintenance of spermatogonial stem cells. In the absence of the Nanos2 gene (Nanos2-KO), germ cells fail to enter G0 arrest and initiate the male differentiation program (including DNA methylation and the piRNA pathway), ultimately undergoing apoptosis before birth. Nanos2 transcription begins at embryonic day 12.5 (E12.5) and terminates at E15.5. However, as the NANOS2 protein continues to be stably expressed beyond E15.5, it is important to elucidate the function of NANOS2 during this post-E15.5 period in germ cell fate determination. To address the functional significance of sustained NANOS2 protein expression, we employed an auxin-inducible degron (AID2) system to achieve rapid degradation of NANOS2 after E15.5. Within 24 hours of 5-Ph-IAA administration, NANOS2 protein was efficiently depleted. As a result, germ cells resumed the cell cycle, exhibited aberrant gene expression patterns similar to Nanos2- KO gonocytes, and underwent apoptosis if NANOS2 depletion occurred at E15.5 or E16.5. Although some surviving cells initiated spermatogenesis and expressed PLZF and GFRA1 after birth, further spermatogenesis was not observed. These findings reveal that sustained NANOS2 protein expression during the embryonic stage is essential for establishing functional spermatogonial stem cells, highlighting a previously unrecognized regulatory mechanism in male germ cell development.