Metformin alleviates cryoinjuries in porcine oocytes by reducing membrane fluidity through the suppression of mitochondrial activity

The study addresses why vitrified oocytes exhibit plasma membrane damage coupled with mitochondrial dysfunction and tests whether pre-vitrification suppression of mitochondrial activity can protect oocytes from cryoinjury. Prior work shows mitochondria operate at elevated temperatures (~50 °C) and that dysfunction occurs after cryopreservation, but how mitochondrial activity influences membrane stability is unclear. Given extreme thermal and osmotic stresses during vitrification and the sensitivity of mitochondria to temperature, the authors hypothesized that transiently lowering mitochondrial activity (and mitochondrial temperature) before vitrification would mitigate membrane damage. Metformin, a known inhibitor of mitochondrial complex I with reported benefits on oocyte quality, was evaluated (along with other mitochondrial inhibitors) in porcine oocytes, a lipid-rich model highly susceptible to cryodamage.

Background literature indicates that oocyte vitrification reduces developmental potential, with membrane damage and mitochondrial dysfunction as major contributors. Vitrified oocytes show decreased mitochondrial membrane potential, reduced ATP, oxidative stress, and ultrastructural abnormalities by TEM. Mitochondrial thermogenesis is elevated during normal activity and previously reported to increase in some mouse oocytes post-vitrification; mitochondria can reach ~50 °C. Modulating mitochondrial activity (thermogenesis) may enable adaptive stress responses. Metformin inhibits complex I and has been reported to improve oocyte quality under various insults (arecoline exposure, aging, PCOS). Other inhibitors (rotenone, oligomycin, UK5099) also suppress mitochondrial activity. Effects of such inhibition on cryoinjury had not been established, motivating the present study.

- Model and ethics: Porcine oocytes (Landrace × Large White, ~6 months). Animal procedures approved by China Agricultural University IACUC. - Oocyte collection and IVM: Cumulus–oocyte complexes (COCs) aspirated from 3–8 mm follicles, matured 42–44 h at 38 °C/5% CO₂ in TCM-199 with glucose, pyruvate, cysteine, antibiotics, FSH (0.01 U/mL), LH (0.01 U/mL), EGF (10 ng/mL), and 10% porcine follicular fluid. - Parthenogenetic activation: Denuding with 0.1% hyaluronidase; oocytes with first polar body were electrically activated (65 V/mm, 80 µs) then cultured in PZM-3 with cytochalasin B (5 µg/mL) and cycloheximide (10 µg/mL) for 4 h; cleavage assessed day 2, blastocyst day 7. - Mitochondrial inhibitor treatments: Metformin (0, 100, 200, 400 µM in TL-HEPES), rotenone (0–1 µM), oligomycin (0–2.5 µM), UK5099 (0–2 µM). Denuded MII oocytes treated for 1 h. - Vitrification and thawing (Cryotop): Wash in DPBS + 20% FBS (BM); pre-equilibrate in E20 (20% ethylene glycol) for 3 min; equilibrate in EFS40 (12% FBS, 0.3 M sucrose, 18% Ficoll, 40% EG) for 20–30 s; load ≤1 µL droplet onto Cryotop and plunge into liquid nitrogen within 1 min from EFS40 exposure. Thaw in 1 M sucrose (37 °C, 1 min), then BM with 0.5, 0.25, and 0 M sucrose for 3, 3, and 5 min; recover 2 h in IVM medium. - Viability assay: FDA staining (2.5 µg/mL, 1 min); green fluorescence indicates live cells; survival rate calculated. - Mitochondrial temperature: Mito-Thermo-Yellow (MTY, 0.5 µM, 15 min); fluorescence imaged by confocal; intensity inversely correlates with temperature. - ATP quantification: Luminescence assay with standard curve (0–32 pmol), normalized per oocyte. - TEM: Fixation (4% paraformaldehyde 1 h, 2.5% glutaraldehyde 14 h), osmium fixation, dehydration, embedding; 70 nm sections imaged at 7,000× and 20,000×; quantify mitochondrial number per area, electron density, and area. - FRAP assays: • Mitochondrial movement: TMRM (100 nM) labeling; photobleaching (7.93 s, 100% laser); recovery imaged every 5 s for 3 min; recovery rate R=(F1−F0)/(Fi−F0). • Membrane fluidity: DiI (10 µM, 10 min); photobleaching 15.82 s; recovery measured similarly. - Mitochondrial respiration: Seahorse XFe96 Mito Stress Test on 15–50 oocytes/well in XF Base medium (1 mM pyruvate, 2 mM glutamine, 5 mM glucose). Sequential injections: oligomycin 1 µM, FCCP 2.5 µM, rotenone/antimycin A 1 µM. Basal, maximal OCR and ATP-linked respiration quantified and normalized per oocyte. - Transcriptomics: RNA-seq (Illumina NovaSeq 6000, PE150) of fresh (F), vitrified (V), and metformin-pretreated vitrified (M) oocytes; n=3 biological replicates/group. Alignment to Sus scrofa reference; DEGs by DESeq with criteria log2FC>1 and q<0.05. GSEA and GO analyses; heatmaps and clustering. - Lipidomics: UPLC–ESI–MS/MS (Thermo Accucore C30 column) quantified lipid species from F, V, M groups (n=3 replicates). Differential lipids by VIP>1 and P<0.05; K-means clustering and heatmaps; focus on free fatty acids (FFA), long-chain unsaturated (LCUFA) and long-chain saturated (LCSFA) species. - Statistics: Chi-square for survival; Student’s t-test and one-way ANOVA for other comparisons; ≥3 biological replicates; data as mean ± SEM; significance P<0.05.

- Screening mitochondrial inhibitors for rapid mitochondrial temperature reduction identified effective doses: metformin 400 µM (P=0.0181), rotenone 1 µM (P=0.0093), oligomycin 0.5–2.5 µM (P<0.0001), UK5099 1–2 µM (P≤0.0092). - Developmental competence after inhibitor exposure: Oligomycin (0.5 µM) and rotenone (1 µM) reduced blastocyst rate (P=0.0184 and P<0.0001). Metformin (0.4 mM) and UK5099 (1 µM) did not significantly affect cleavage or blastocyst rates. - Vitrification outcomes with pretreatment: Metformin pretreatment significantly improved post-thaw survival versus vitrified control (81.36% [179/220] vs 71.85% [171/238], P=0.0165). UK5099 pretreatment showed no improvement (67.50% [135/200]). - Ultrastructure and mitochondrial function: Vitrification increased mitochondrial area and reduced electron density (both P<0.0001) and decreased mitochondrial number; metformin pretreatment increased mitochondrial number (P=0.0079) and rescued area and electron density toward fresh levels (P<0.001). Post-warming MTY signal indicated decreased mitochondrial temperature vs fresh (P<0.0001); metformin pretreatment elevated mitochondrial temperature compared to V (P=0.0001). Metformin did not restore reduced ATP levels after vitrification. - Suppression of mitochondrial activity pre-vitrification: Metformin reduced mitochondrial movement (FRAP, P<0.05), lowered ATP content (P=0.0246), and decreased Seahorse OCR metrics: ATP production (P=0.0058), basal respiration (P=0.0418), and maximal respiration (P=0.0358). - Membrane fluidity: Cryoprotectant exposure increased membrane fluidity; metformin pretreatment decreased membrane FRAP recovery with cryoprotectants (P=0.0061). After warming, metformin-pretreated oocytes exhibited lower membrane fluidity than vitrified controls (P=0.0126), approximating fresh levels. - Transcriptomics: Pairwise DEGs counts: V vs F (324 up, 605 down), M vs F (480 up, 927 down), M vs V (108 up, 88 down). GSEA showed an upregulation tendency in fatty acid elongation pathway in M vs V; core genes increased in M included HACD1/3/4, ECHS1, ELOVL6/3, HADH, HADHA, HSD17B12, THEM5, ACOT4. GO CC enrichment placed many in mitochondrial matrix and ER. - Lipidomics: Differential lipids: V vs F (75 up, 14 down), M vs F (59 up, 17 down), M vs V (14 up). LCUFAs generally increased with vitrification. Metformin pretreatment increased the LCSFA myricinic acid (C31:0) vs V and showed broader upward trends for LCSFAs (K-means clustering), consistent with reduced membrane fluidity.

The findings support the hypothesis that transient suppression of mitochondrial activity prior to vitrification protects oocytes. Metformin-induced mitochondrial quiescence (reduced ATP production, OCR, movement, and mitochondrial temperature) appears to enhance resilience to extreme cooling and cryoprotectant stresses. TEM indicates that metformin preserves mitochondrial membrane integrity and ultrastructure, which likely contributes to better survival. Functionally, metformin lowers plasma membrane fluidity in cryoprotectant-exposed and vitrified oocytes, mitigating membrane damage. Mechanistically, transcriptomic upregulation of fatty acid elongation genes and lipidomic increases in long-chain saturated fatty acids suggest a shift toward longer, more saturated membrane lipids, reducing fluidity and stabilizing membranes during freeze–thaw. Species-specific differences in mitochondrial temperature responses (porcine vs mouse) may relate to higher lipid droplet content and lipid phase transition severity in pig oocytes affecting mitochondria–lipid interactions. Overall, regulating mitochondrial thermogenesis/activity can modulate membrane biophysics and reduce cryoinjury, improving post-thaw oocyte viability.

Pretreating porcine oocytes with metformin (400 µM, 1 h) before vitrification suppresses mitochondrial activity and thermogenesis, preserves mitochondrial ultrastructure, reduces plasma membrane fluidity via upregulation of fatty acid elongation pathways and increased long-chain saturated fatty acids, and improves post-thaw survival. The study highlights mitochondrial temperature/activity as a lever to enhance cryotolerance and links mitochondrial regulation to membrane stability. Future work should explore translational relevance to human oocytes and embryos, optimize dosing/timing, assess long-term developmental competence and offspring outcomes, and directly manipulate fatty acid elongation components to validate causality.

- Species model limitation: results in porcine oocytes may not fully translate to human oocytes due to species-specific lipid content and cryotolerance. - Developmental assessment was limited to parthenogenetic activation; fertilization competence and in vivo developmental outcomes were not evaluated. - Metformin was primarily tested at a single effective concentration (400 µM) and short exposure (1 h); dose–response, timing windows, and washout kinetics warrant further study. - Although transcriptomics and lipidomics implicate fatty acid elongation, direct functional validation (e.g., knockdown/overexpression or enzymatic assays of ELOVL/HACD/HADH pathway) was not performed. - Mitochondrial temperature was inferred by MTY fluorescence; complementary thermometry approaches could strengthen conclusions. - Some measurements have modest sample sizes at the oocyte level; broader replication across donors could improve generalizability.