Abstract

Female infertility often arises from oogenic defects, yet the underlying molecular mechanisms remain elusive. The Piwi-piRNA pathway is crucial for gametogenesis, but its role in mammalian female fertility remains unclear, partly due to reliance on mouse models lacking PIWIL3. PIWIL3 exits in most other placental mammals and is highly expressed in human oocytes, but its function remains largely unexplored. Here, we show that rabbit PIWIL3 closely resembles its human counterpart and is the predominant PIWI protein in oocytes. Using CRISPR-Cas9 knockout, we demonstrate that PIWIL3 is essential for female fertility in rabbits, its loss leads to severe defects in oogenesis. Embryos lacking maternal PIWIL3 arrest by the 8-cell stage. Mechanistically, PIWIL3 binds ~18-nucleotide piRNAs, supports piRNA biogenesis, and regulates transcriptomic, proteomic, and transposable element dynamics during oocyte maturation and early embryogenesis. These findings establish PIWIL3 as an essential regulator of female fertility in non-rodent mammals, potentially including humans.