piRNA-19128 regulates spermatogenesis by silencing of KIT in chicken.

Spermatogenesis is a complex process. Some studies have shown that Piwi-interacting RNAs (piRNAs) play an important role in spermatogenesis. To verify the evaluate between piRNAs and PIWI proteins in chicken and its possible role in spermatogenesis and reproductive stem cell proliferation and differentiation, we performed immunoprecipitation and deep sequencing analyses and determined the expression profiles of small RNAs in primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia (Sa) cells, and spermatozoa. Length analysis showed that piRNAs bound to PIWIL1 mainly contained 23-30 nt. Base preference analysis showed "1U-10A"; moreover, base preference of piRNAs was obvious in all of germline cells. Here we reported the TE family of gallus gallus, and targeted by piRNA. Target gene of piRNA annotation enrichment analysis identified candidate genes KIT, SRC, WNT4, and HMGB2. Kyoto Encyclopedia of Genes and Genomes analysis showed that these genes were associated with steroid hormone biosynthesis, Notch signaling pathway, and melanogenesis. These results indicate that chicken piRNAs perform important regulatory roles during spermatogenesis similar to mice piRNAs. Chicken piRNAs interacted with PIWI proteins and regulated spermatogenesis and germ cell proliferation and differentiation. Further, we observed a negative correlation between piRNA-19128 and KIT expression. Results of dual-luciferase reporter assay confirmed that piRNA-19128 directly interacted with KIT, suggesting that it plays a key role in the regulation spermatogenesis by inhibiting KIT expression. Thus, the present study provides information on the length and base preference of chicken piRNAs and suggests that piRNA-19128 regulates spermatogenesis in chicken by silencing KIT.