Comprehensive proteomic, structural and functional analysis of cryoprotectant effects on marine mussel oocytes.

Cryopreservation is a valuable tool for preserving marine genetic diversity and supporting selective breeding in aquaculture. The blue mussel (Mytilus galloprovincialis) is an important species for the aquaculture industry and a useful model for studying cryopreservation in marine invertebrates. Declining mussel seed availability over the past decade, combined with the growing demand for year-round hatchery production, has increased interest in developing effective cryopreservation methods for gametes and embryos. However, cryopreservation remains challenging, particularly for marine invertebrate oocytes, as it requires balancing cryoprotectant cytotoxicity with sufficient cell protection during freezing and thawing. Moreover, the molecular, structural, and functional changes associated with oocyte cryopreservation in invertebrates are still poorly understood. This study examines the effects of two commonly used cryoprotectants, dimethyl sulfoxide (DMSO) and ethylene glycol (EG), on M. galloprovincialis oocytes under a monitored slow freezing (MSF) protocol. Using an integrative analytical approach combining proteomics, functional assessments, and structural analyses, we provide a comprehensive understanding of cryoprotectant-induced changes. Our findings reveal that cryoprotectants cause significant proteomic alterations, with DMSO having more pronounced effects. These alterations are associated with increased oxidative stress, an insufficient antioxidant response, and disruptions in meiosis restart mechanisms, ultimately delaying larval development. Cryoprotectant-induced oxidative stress may also weaken the oocyte membrane, leading to rupture during the MSF protocol and subsequent fertilisation failure post-thawing. These results provide evidence that EG is less cytotoxic than DMSO and suggest that supplementing cryoprotectants with antioxidants and membrane stabilizers could enhance success rates in the cryopreservation of oocytes from mussels and other marine invertebrate species.