Overexpression of CDC42 causes accumulation of DNA damage leading to failure of oogenesis in triploid Pacific oyster Crassostrea gigas

Triploid Pacific oyster Crassostrea gigas exhibits notable differences in fecundity, with the majority being sterile individuals, referred to as female β, which produce few oocytes, while a minority are fertile individuals, referred to as female α, which produce abundant oocytes. However, the molecular mechanisms underlying these differences in triploid fecundity remain poorly understood. CDC42 has been implicated in processes related to increased DNA damage and genomic instability. Here, we investigate the crucial role of CDC42 in DNA damage repair during oogenesis in triploid C. gigas. Immunofluorescence analysis of γH2AX, a marker for DNA double-stranded breaks, showed significantly higher levels of DNA damage in gonadal cells of triploids compared to diploids, particularly in female β. Histological and ultrastructural analyses revealed abnormal germ cells, termed β gonia, characterized by giant nuclei condensed into irregular chromosome-like chromatin, present in triploid gonadal follicles. RNAseq and proteomic analyses revealed significantly elevated CDC42 expression in triploid gonads compared to the diploids. Inhibition of CDC42 activity in triploids using ZCL278, a CDC42-specific inhibitor, resulted in a significant reduction in DNA damage, increased oocyte numbers, and a decrease in β gonia count. Transcriptome profiling revealed that CDC42 inhibition upregulated the PI3K-AKT signaling pathway along with DNA repair activation. Overall, our findings suggest that overexpression of CDC42 during oogenesis in triploid C. gigas impedes DNA repair, leading to the accumulation of DNA damage, and consequently, oogenesis blockade and abnormal germ cell differentiation. Conversely, inhibition of CDC42 activity activates the PI3K-AKT signaling pathway and promotes DNA repair, thereby mitigating DNA damage and facilitating oogenesis in triploids. This study provides new insights into the molecular mechanisms of sterility in female triploid C. gigas.