The piRNA pathway is essential for female fertility in golden hamsters and likely humans, but not in mice. However, the role of individual PIWIs in mammalian reproduction remains poorly understood outside of mice. Here, we describe the expression profiles, subcellular localization, and knockout-associated reproductive defects for all four PIWIs in golden hamsters. In female golden hamsters, PIWIL1 and PIWIL3 are highly expressed throughout oogenesis and early embryogenesis, while knockout of PIWIL1 leads to sterility, and PIWIL3 deficiency results in subfertility with lagging zygotic development. PIWIL1 can partially compensate for TE silencing in PIWIL3 knockout females, but not vice versa. PIWIL1 and PIWIL4 are the predominant PIWIs expressed in adult and postnatal testes, respectively, while PIWIL2 is present at both stages. Loss of any PIWI expressed in testes leads to sterility and severe but distinct spermatogenesis disorders. These findings illustrate the non-redundant regulatory functions of PIWI-piRNAs in gametogenesis and early embryogenesis in golden hamsters, facilitating study of their role in human fertility.

Vasectomized mice play a key role in the production of transgenic mice. However, vasectomy can cause great physical and psychological suffering to mice. Therefore, there is an urgent need to find a suitable replacement for vasectomized mice in the production of transgenic mice. In this study, we generated C57BL/6J mice (Piwil1 D633A-INS99, Piwil1^mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1^mt/+ heterozygous mice were normally fertile and that homozygous Piwil1^mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1^mt/mt males produced no Piwil1^mt/mt genotype offspring, and the number of offspring did not differ significantly from that of pseudopregnant mice following mating and breeding with ligated males. The CRISPR‒Cas9 system is available for generating Miwi-modified mice, and provides a powerful resource to replace ligated males in assisted reproduction research.