148 Characterising cytoplasmic lipid profiles during in vitro maturation of porcine oocytes

Oocyte maturation is a complex process leading to acquisition of developmental competence before fertilisation. One component of maturation is cytoplasmic maturation, which is the accumulation or depletion of certain molecules and organelles in the oocyte. Lipid metabolism is key to organelle function, signalling, and cell division, but the lipid profile of oocytes is difficult to characterise beyond physical appearance. The objective of this study was to use matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry to investigate the lipid profiles of individual oocytes during IVM. In this experiment, porcine oocytes were obtained from a commercial supplier and matured in vitro for 44 h. Oocytes were collected at 0 (n = 16), 24 (n = 25), and 44 (n = 22) h, denuded in 1% hyaluronidase, and fixed in 4% paraformaldehyde prepared in Dulbecco’s PBS (DPBS). Fixed oocytes were transferred to an indium-tin-oxide (ITO) slide with as little medium as possible and sprayed with 50 mg mL⁻¹ Super-DHB suspended in a 1:1 ratio of methanol to water. Oocytes were then individually analysed using a UltrafleXreme MALDI-TOF/TOF mass spectrometer (Bruker) in positive ion mode to identify lipid species present at each time point. Mass spectra were processed using mMass software, and MetaboAnalyst 5.0 was used to perform a one-way ANOVA followed by Tukey’s honestly significant difference test, with significance detected at P < 0.05. The results identified 36 lipids significantly more abundant in immature oocytes and 18 lipids significantly more abundant in oocytes matured for 44 h. The lipid species present primarily in immature oocytes collected at 0 h of IVM were phosphatidylserines (PS), phosphatidylcholines (PC), phosphatidylethanolamines (PE), ceramides (Cer), diacylglycerols (DG), triglycerides (TG), and fatty acids (FA). These lipids function as components of cell membranes, in regulation of mitochondrial function, protein modification, cellular signalling, and regulation of cell division. Lipid species present primarily in mature oocytes collected at 44 h of IVM were TG, FA, PC, phosphatidic acids (PA), and PE. The lipids that accumulated during IVM function as long-term energy storage molecules, cell membrane components, and cell signalling molecules. Oocytes from the 24-h group had a similar profile to oocytes from both the 0-h and 44-h groups, in that they contained Cer, DG, PC, PE, and TG. These results indicate that porcine oocytes have different metabolic lipid profiles at the start of IVM and after 44 h of IVM and that the lipid species present become less diverse towards the end of IVM. Oocytes that have undergone 24 h of IVM have an intermediate profile, displaying components found at both 0 and 44 h. This work demonstrated clear changes in lipid metabolism during IVM, which could be further investigated to understand the mechanisms behind cytoplasmic maturation in oocytes.

Support for this research was provided by USDA-NIFA 2020-67016-31134.