BmOrb2, a CPEB-family RNA-binding protein, specifically regulates late-stage eupyrene spermiogenesis and male fertility in Bombyx mori.

CPEB (Cytoplasmic Polyadenylation Element Binding protein) comprises a highly conserved family of RNA-binding proteins that serve as key post-transcriptional regulators in diverse biological processes. In Drosophila melanogaster, the CPEB family member Orb2 plays vital roles in spermatogenesis, particularly during the flagellar axoneme elongation. Bombyx mori, like other lepidopterans, produces dimorphic sperm: eupyrene sperm and apyrene sperm. However, whether CPEB functions in this spermatogenesis process remains unknown. In this study, we demonstrate that B. mori orb2 (Bmorb2) is critical for the proper progression of eupyrene spermatogenesis, particularly at the elongation stage. We found that Bmorb2 is predominantly expressed in testis, specifically in spermatocytes and early elongating eupyrene sperm, but absent from late eupyrene sperm and apyrene sperm. Functional analysis reveals that Bmorb2 depletion severely disrupts eupyrene sperm development, causing aberrant nuclear positioning and shortened flagella. Consequently, these defective eupyrene sperms fail to migrate to the spermatheca after mating, leading to complete male sterility. Partial rescue of male sterility was achieved through sequential mating of females with Bmorb2 and Sex-lethal mutant males, demonstrating that apyrene sperm from Bmorb2 mutants retain normal function. In addition, our data suggest that the spermatogenesis defects in mutants are potentially associated with disrupted energy metabolism, impaired chromatin remodeling, and dysregulated assembly of motor protein and IFT complexes. Our findings demonstrate that Bmorb2 functions as a crucial stage-specific regulator of late spermiogenesis in B. mori.