Functional Investigation of a Novel PIWIL4 Mutation in Nonobstructive Azoospermia During the First Wave of Spermatogenesis

PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that are almost exclusively expressed in germ cells where they work to silence harmful transposons to maintain genomic stability. It is proposed that the associated piRNAs guide the nuclear PIWIL4 to transposable elements where it recruits the DNA methylation apparatus and instructs de novo DNA methylation. Herein we identify a missense variant of PIWIL4 (c.805 C>T p.R269W) in two infertile males. Though male homozygous knock-in mice harboring the orthologous variant exhibit normal sperm counts and morphology, they display increased expression of transposable elements and abnormal gene expression during the first wave of spermatogenesis. Mechanistically, the mutated site impairs the piRNA-binding ability of PIWIL4 and leads to the derepression of some LINE-1 elements. In summary, we have identified a piRNA binding mutation in PIWIL4 that may be involved in human non-obstructive azoospermia. RNA-sequencing on 3-week-old wild-type, piwil4WT/R264W and piwil4R264W/R264W testes.