195 Lipid profiling of bovine oocytes matured with different stimulation of cyclic GMP synthesis by multiple reaction monitoring profiling

Abstract

Intrafollicular lipid metabolism is very important for production species such as cattle. Lipids are essential substrates to produce energy during growth, maturation, and acquisition of high competence for the development of oocytes. However, the quantity and distribution of these lipids has been identified as responsible for hindering the process of cryopreservation of oocytes and embryos produced invitro. Previous studies have indicated that the cyclic (c) GMP pathway may be involved in the lipid metabolism of bovine cumulus-oocyte complexes (COC). The synthesis of this nucleotide can be activated through guanylate cyclases (soluble, sGC; or membrane, mGC). Therefore, the objective of this study was to investigate the lipid profile of bovine oocytes matured invitro (IVM) when stimulated by specific stimulators of sGC (protoporphyrin IX) and mGC (NPPB: peptide natriuretic type B). Pools of ovum pickup (OPU) oocytes were matured invitro for 24h in TCM-199 medium, with 15% bovine serum (BS), 0.5µgmL−1 of FSH, 5µgmL−1 of LH, 0.8mM L-glutamine, and 50µgmL−1 of gentamicin at 38.5°C and 10−5 M protoporphyrin IX or 10−6 M NPPB. The control group was matured without NPs or protoporphyrin IX. After IVM, cumulus cells (CC) were removed and oocytes (OO) collected, washed in 1:3 methanol:water (v/v) and frozen at −80°C. The lipid extraction of the samples was performed based on a standard protocol (Bligh and Dyer 1959 Can. J. Biochem. Physiol. 37, 911-917) but adapted for small samples. The samples were diluted and analysed on an Agilent 6410 QQQ (Agilent Technologies) mass spectrometer and analysed according to the multiple reaction monitoring (MRM) method described (de Lima et al. 2018 J. Mass. Spectrom. 53, 1247-52). Data for 3 replicates/group were normalized and then submitted to ANOVA statistical analysis, followed by Tukey test and principal components analysis, by Metaboanalyst 4.0, with an α-level of 5%. The results, representing the analysis of 164 lipids, showed that the lipid profile was not affected when we used the cGMP synthesis stimulators protoporphyrin IX and NPPB, maintaining the same profile of lipid classes in control and treatments. In addition, the quantitative values of the major lipid classes, sphingomyelin, triglycerides, and phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol), were not altered in the treated groups. The values for each class (ng/oocyte) for control, NPPB, and protoporphyrin, respectively, were 0.89, 0.86, and 1.12 for sphingomyelin, 5.63, 5.66, and 6.90 for phosphatidylcholine, 7.34, 6.48, and 7.89 for triglycerides, 209.0, 244.0, and 207.4 for phosphatidylserine, 3.05, 3.0, and 2.35 for phosphatidylethanolamine, 3.40, 3.34, and 3.29 for phosphatidylglycerol, and 3.47, 3.52, and 3.51 for phosphatidylinositol (P<0.05). Further, the amount of these lipids per class was not affected by cGMP synthesis when stimulated by protoporphyrin IX and NPPB, showing that the relationship of this pathway with lipid metabolism needs additional study.