RELATIONSHIP BETWEEN THE DURATION OF EQUILIBRATION STEP OF THE VITRIFICATION AND CHANGES ON ABUNDANCE AND COMPOSITION OF OOCYTE MEMBRANE LIPIDS

Abstract

Volumetric damages and cytotoxicity due to cryoprotectants exposure are time-sensitive in the vitrification process and can trigger the lipid remodeling of the plasma membrane. Here, we investigate the effects of exposure time to equilibration solution (ES) on membrane lipid profile of C57BL/6J mice oocytes using MRM-profiling, a sensitive exploratory method for lipidomics of oocytes and embryos1. We also assessed the effects of supplementing ES with antioxidants and unsaturated fatty acids. Experimental groups included oocytes equilibrated with Irvine Scientific (IRV), a commercial standard vitrification medium; Tvitri-4 (T4) produced in small scale for research by INVITRA®, and Tvitri-4 supplemented with L-carnitine (LC) and oleic and linoleic fatty acids (FA) (T4-LC/FA; Patent n. BR102019013697-9). Experimental study. Oocytes were randomly divided in 7 groups: non-exposed (NE) and exposed to either 10-minute equilibration step (ES10) according to the manufacturer’s procedure or 7-minute equilibration step (ES7), using IRV, T4, or T4-LC/FA media. After equilibration, oocytes were washed in methanol: H2O and lipids extracted with methanol. Diluted lipid extracts were flow injected into the triple quadrupole spectrometer with electrospray ionization (ESI). Relative ion intensities were used for univariate (ANOVA, fold-change, t-test, volcano plot) and multivariate analysis (PCA, PLS-DA, cluster analysis). Most informative lipids were sorted out using statistical significance of p<0.05 or partial least square discriminant analysis (PLS-DA) variables of importance (VIP) scores>1. One-way ANOVA showed PC (38:8) differently represented among NE, ES7 and ES10 groups, upregulated in T4-LC/FA oocytes. In ES7 groups, two by two comparisons between IRV, T4 and T4-LC/FA using volcano plot (p-value≤0.05; fold-change≥2.0) detected 4, 5, and 4 significant lipids between IRV vs. T4, IRV vs. T4-LC/FA, and T4 vs. T4-LC/FA, respectively, with the PEo(36:1) overrepresented in IRV (fold-change=12.5). In ES10 groups, were detected 11, 19 and 4 lipids differentially represented among IRV vs. T4, IRV vs. T4-LC/FA, and T4 vs. T4-LC/FA, respectively. PLS-DAVIP scores identified in IRV oocytes 8 free- fatty acids, phosphatidyletanolamine PEo(36:1), phosphatidylcholines PCo(32:0) and PC(36:3), phosphatidylserines PS(14:1), PS(16:0), PS(30:2), PSo(40:5), and phosphatidylinositols PI(22:4) and PI(40:8) among the top features, whereas unsaturated PC and SM predominated in T4 and T4-LC/FA oocytes. Significant lipids observed between T4 and T4-LC/FA pointed oleic (18:1) and linoleic (18:2) acids upregulated in T4-LC/FA oocytes exposed for 10 min to ES. The duration of the equilibration phase changed the abundance and composition of membrane lipids. Phospholipids were upregulated at 10-minute exposure to ES, except in IRV oocytes. The oleic and linoleic acids used as supplements in T4-LC/FA medium were promptly detected in the oocytes and seem contribute to better preserving membrane phospholipids during cell equilibration.