Oocyte cryopreservation is crucial for fertility preservation and germplasm conservation. However, low quality of vitrified oocytes due to membrane damage hampers its efficacy. Membrane damage is linked to mitochondrial dysfunction, but the mechanism is unclear. Mitochondria operate at higher temperatures than the surrounding cell, suggesting that suppressing mitochondrial activity might protect oocytes during vitrification's extreme temperature changes. This study hypothesized that reducing mitochondrial activity before vitrification, using metformin, would protect oocytes from cryoinjury. Metformin is a known mitochondrial complex I inhibitor and has shown benefits in improving oocyte quality in previous studies. This research aimed to investigate metformin's effect on porcine oocytes during vitrification, focusing on the relationship between mitochondrial activity, membrane fluidity, and cryoinjury.

Extensive research demonstrates the detrimental effects of vitrification on oocyte quality, with membrane damage being a primary cause of reduced developmental potential. Osmotic stress and cryoprotectant toxicity contribute to this damage. Membrane fluidity, a key membrane parameter, is influenced by lipids like cholesterol and long-chain fatty acids. Reduced fluidity can increase membrane stiffness, potentially protecting against damage. Mitochondria are central to lipid synthesis and metabolism, and mitochondrial dysfunction is linked to poor oocyte outcomes after cryopreservation. Studies have shown mitochondrial injuries, including decreased membrane potential and reduced ATP levels, in vitrified oocytes. Mitochondria operate at high temperatures (up to 50°C), and their sensitivity to temperature fluctuations suggests that reducing their activity might protect them during vitrification. While metformin's beneficial effects on oocyte quality have been reported, its role in protecting against cryoinjury remains largely unexplored.

Porcine oocytes were collected and matured in vitro. Mitochondrial inhibitors (metformin, rotenone, oligomycin, UK5099) were tested at various concentrations to determine their effect on mitochondrial temperature, assessed using Mito-Thermo-Yellow (MTY) dye. The most effective concentration of each inhibitor was then used to treat oocytes before parthenogenetic activation and assessment of cleavage and blastocyst rates. The optimal inhibitor (metformin) was selected based on its effect on oocyte development and was subsequently used in vitrification experiments. Oocytes were vitrified using the cryotop method and thawed using a gradient thawing method. Oocyte survival rates were evaluated using fluorescein diacetate (FDA) staining. Mitochondrial ultrastructure was analyzed using transmission electron microscopy (TEM). Mitochondrial activity was assessed by measuring mitochondrial temperature (MTY), ATP levels, and mitochondrial respiration using a Seahorse metabolic flux analyzer. Membrane fluidity was determined using fluorescence recovery after photobleaching (FRAP) with Dil dye. Transcriptome sequencing was performed to identify differentially expressed genes, followed by gene set enrichment analysis (GSEA) and gene ontology (GO) analysis to explore pathways affected by metformin treatment. Finally, lipidomic analysis using UPLC-MS/MS was conducted to identify lipid content changes in oocytes.

Metformin (400 µM) significantly reduced mitochondrial temperature compared to controls (P=0.0181). Metformin pretreatment significantly improved oocyte survival rates after vitrification (81.36% vs 71.85% in the vitrified group, P=0.0165). TEM analysis showed that metformin pretreatment reduced mitochondrial abnormalities in vitrified oocytes, including ruptured membranes, increased mitochondrial area, and decreased electron density (P<0.0001). Metformin significantly decreased mitochondrial motion, ATP levels, ATP production, basal respiration, and maximum respiration rate before vitrification (P<0.05). Metformin pretreatment significantly decreased cell membrane fluidity after exposure to cryoprotectants and vitrification, recovering to levels similar to fresh oocytes (P<0.05). Transcriptome analysis revealed that metformin pretreatment upregulated genes involved in fatty acid elongation (e.g., HACD1/3/4, ECHS1, ELOVL6/3, HADHA, HADH, HSD17B12, THEM5, ACOT4). Lipidomic analysis confirmed increased long-chain saturated fatty acid (LCSFA) content (myricinic acid (C31:0)) in metformin-pretreated vitrified oocytes compared to the vitrified group.

This study demonstrates that metformin pretreatment effectively mitigates cryoinjury in porcine oocytes. The observed improvement in oocyte survival is linked to metformin's ability to reduce mitochondrial activity, leading to decreased membrane fluidity. The reduction in membrane fluidity appears to be mediated by increased fatty acid elongation, resulting in higher levels of LCSFAs. This aligns with the established role of fatty acids in regulating membrane fluidity. The findings contrast with some previous studies on mouse oocytes, potentially due to differences in lipid content between porcine and mouse oocytes, affecting the extent of lipid phase transition during cryopreservation. Metformin's effect on mitigating cryoinjury via mitochondrial quiescence aligns with the growing body of evidence suggesting that mitochondrial quiescence is a protective mechanism against various stresses.

Metformin pretreatment before oocyte vitrification improves survival rates after thawing by decreasing mitochondrial activity and reducing membrane fluidity through modulation of fatty acid elongation. This study highlights the importance of mitochondrial activity regulation in maintaining oocyte quality during cryopreservation. Future research could focus on optimizing metformin concentration and exploring other potential mitochondrial inhibitors for enhancing oocyte cryopreservation outcomes in various species.

This study used a porcine model, and the findings might not be directly generalizable to all species. The study focused on the effects of metformin on oocyte survival; further research is needed to fully evaluate its impact on oocyte developmental competence and subsequent embryo development. The mechanisms underlying metformin's effects on fatty acid elongation warrant further investigation.