The pachytene-specific lncRNA 1700008K24Rik is essential for mouse spermatogenesis and functions as a conserved piRNA precursor.

Spermatogenesis is a complex differentiation process requiring precise spatiotemporal gene regulation, yet the functions of most testis-enriched long noncoding RNAs (lncRNAs) remain elusive. Here, we identify 1700008K24Rik as a pachytene-specific lncRNA that is essential for mouse spermatogenesis. Reanalysis of single-cell RNA-seq reveals that 1700008K24Rik expression is minimal in spermatogonia and preleptotene cells, sharply increases during the pachytene stage, and declines after meiosis, precisely coincides with the wave of pachytene piRNA production. In vivo knockdown of 1700008K24Rik in mouse testes via AAV9-shRNA severely disrupts spermatogenesis, causing a reduction in testis size, seminiferous epithelium thickness, and epididymal sperm count, suggesting its physiological necessity. Mechanistically, we demonstrate that 1700008K24Rik functions as a bona fide piRNA precursor: it harbors numerous piRNA sequences, and its overexpression in GC-2 spd(ts) cells specifically upregulates corresponding overlapping piRNAs. This processing relies on the core piRNA machinery, and co-overexpression of A-MYB, together with MOV10L1 or MIWI, promotes piRNA production. Notably, this piRNA precursor function is evolutionarily conserved, as the human homolog TDRG1, which is linked to asthenozoospermia, also generates piRNAs when expressed in GC-2 spd(ts) cells. Transcriptomic analysis following 1700008K24Rik overexpression in GC-2 spd(ts) cells reveals dysregulation of genes involved in metabolism and signaling, with several spermatogenesis-related genes (e.g., Msh5, Stard6) predicted as direct targets of 1700008K24Rik-derived piRNAs. Our study identifies 1700008K24Rik as a pachytene-specific lncRNA essential for male fertility and demonstrates that it functions as a conserved piRNA precursor. These findings provide mechanistic insight into how an individual lncRNA contributes to the piRNA pathway and suggest that it may operate through dual mechanisms in mammalian spermatogenesis.