Regulation of sperm motility by exosomes of ovarian fluid during female sperm storage in black rockfish ( Sebastes schlegelii)

Sperm storage within the female reproductive tract is a widespread phenomenon among vertebrate species, including various viviparous teleosts. The black rockfish (Sebastes schlegelii) serves as a typical example, wherein sperm remains stored inside the ovary for extended periods, spanning over four months from entry until fertilization. While previous investigations have explored the occurrence of female sperm storage and its adaptive advantages, the precise mechanisms governing sperm storage in the ovary and the maintenance of sperm activity remain incompletely understood. Recently, extracellular vesicles or exosomes have been identified as important components of the oviductal or uterine fluid, but their interactions with sperm are not fully understood. Our study represented the isolation and identification of ovarian fluid exosomes (Ov-exos) from the black rockfish, while also affirming their role in sustaining sperm activity. Based on the transcriptome and proteome of the ovaries, it was found that the levels of gene transcription and protein translation, which related to exosomes, were upregulated compared to pre-mating levels, such as CD81, CD63, CD9, ANXA1, VPS25, and RAB8A. Furthermore, our study corroborated these findings through qRT-PCR analysis. Immunohistochemical localization of CD81 suggested that exosomes were secreted at the sperm storage site within the ovary. The ultrastructure of ovaries, particularly with exosomes, was examined by transmission electron microscopy (TEM). It was further found that epithelial cells and follicular layer cells secreted exosomes, which bound to the midpiece of sperm stored in the ovaries. To validate our findings, experiments involving the co-incubation of sperm with Ov-exos were conducted. This analysis demonstrated that exosomes could bind with sperm and transfer their cargo, significantly enhancing sperm viability and motility. Notably, this study helps to reveal the complexity of sperm storage mechanisms and their adaptive implications in teleost reproduction.