Unraveling circRNAs and ceRNA regulatory networks underlying sex-specific gametogenesis impairment in triploid Pacific oysters (Crassostrea gigas).

Abstract

Background: In the Pacific oyster Crassostrea gigas, triploidization-a well-established technique for generating sterile organisms-provides a promising model to study reproductive dysfunction, as it causes severe and sex-specific impairments in gametogenesis. Specifically, triploid males produce abundant spermatocytes and spermatids but fail to generate mature spermatozoa, while oogenesis in triploid females presents a more complex and heterogeneous picture. The molecular basis for this differential gametogenesis impairment, however, is not fully understood.

Methods: To investigate the roles of circRNA and ceRNA networks in the sex-specific gametogenesis impairment of triploid oysters, gonadal circRNAs were identified and their expression was profiled from whole transcriptome data of diploid and triploid oysters. Differentially expressed circRNAs were then subjected to functional enrichment analysis. Competitive endogenous RNA (ceRNA) networks were constructed from the interactions of differentially expressed RNAs. Key candidate genes implicated in gametogenesis impairment were then identified and characterized through integrated bulk and single-nucleus RNA-seq analyses.

Results: Differential expression analysis revealed substantial alterations in circRNAs expression between diploid and triploid gonads. Functional enrichment of circRNA source genes implicated global metabolic and cell proliferation pathways associated with oogenesis impairment, while it implicated lipid/steroid metabolism, cell proliferation, and apoptosis pathways associated with spermatogenesis disruption in triploid oysters. Sex-specific ceRNA networks were constructed for oogenesis and spermatogenesis, revealing extensive regulatory rewiring in triploid gonads. Analysis of bulk and single-nucleus RNA-seq data identified key candidate genes within these ceRNA networks. Specifically, upregulation of FoxG1, Sox11, Cyp2J6, and Tra2A in somatic cells, combined with downregulation of Uhrf1, Ccna2, Sox2, and Bre1 in germ cells, was implicated in oogenesis impairment of triploids. Similarly, the ceRNA-mediated suppression of Iqch, Ccdc173, Srebp1, and Gtpbp2 was linked to disrupted spermatogenesis.

Conclusions: These findings highlight that circRNA-mediated ceRNA dysregulation is potentially associated with gametogenesis impairment in triploid oysters, and offer novel insights into post-transcriptional regulatory mechanisms underlying molluscan reproduction.