Abnormal zona pellucida and follicular development in ZP1-mutant macaques

Zona pellucida glycoprotein-1 (ZP1) is essential for maintaining oocyte structural integrity and facilitating fertilization. Mutations in ZP1 are strongly associated with primary infertility disorders such as fertilization failure and empty follicle syndrome; however, the absence of accurate experimental models has hindered mechanistic understanding and obscured the etiological basis of ZP1-related infertility. In this study, CRISPR/Cas9-mediated genome editing was employed to generate two ZP1-edited cynomolgus macaques (Macaca fascicularis), designated #ZP1-1 (male) and #ZP1-2 (female). Following sexual maturation, oocytes retrieved from #ZP1-2 through superovulation exhibited a marked increase in zona pellucida-deficient oocytes and a significant reduction in maturation rates compared to controls. Integrated analyses, including immunofluorescence staining, transmission electron microscopy, transcriptomic profiling of oocytes, and histopathological examination of ovarian tissue, revealed disrupted folliculogenesis and oocyte anomalies consistent with phenotypes observed in human empty follicle syndrome. These findings establish the ZP1-knockout cynomolgus macaque as the first non-human primate model of ZP1-related infertility, providing a valuable platform for elucidating disease mechanisms and informing the development of targeted interventions for infertility arising from ZP gene mutations.