Pseudopregnant mice generated from Piwil1 deficiency sterile mice.

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, Piwil1mt/mt) with a 99-base insertion in the Miwi (Piwil1) gene using CRISPR/Cas9 technology and showed that Piwil1mt/+ heterozygous mice were normally fertile and that homozygous Piwil1mt/mt males were sterile and females were fertile. Transplantation of normal fertilized eggs into wild pseudopregnant females following mating with Piwil1mt/mt males produced no Piwil1mt/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.

The non-redundant functions of PIWI family proteins in gametogenesis in golden hamsters.

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.

The piRNA pathway is essential for generating functional oocytes in golden hamsters.

Piwi-interacting RNAs (piRNAs) are predominantly expressed in germ cells and function in gametogenesis in various species. However, Piwi-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear to be widely representative of mammals. Thus, the function of piRNAs in mammalian oogenesis remains largely unclear. Here, we generated Piwil1- and Mov10l1-deficient golden hamsters and found that all female and male mutants were sterile, with severe defects in embryogenesis and spermatogenesis, respectively. In Piwil1-deficient female hamsters, the oocytes and embryos displayed aberrant transposon accumulation and extensive transcriptomic dysregulation, and the embryos were arrested at the two-cell stage with impaired zygotic genome activation. Moreover, PIWIL1-piRNAs exert a non-redundant function in silencing endogenous retroviruses in the oocytes and embryos. Together, our findings demonstrate that piRNAs are indispensable for generating functional germ cells in golden hamsters and show the value of this model species for piRNA studies in gametogenesis, especially those related to female infertility.