137 DYNAMIC SHIFT IN CYTOPLASMIC LIPIDS IN CAT OOCYTES DURING OVARIAN FOLLICLE DEVELOPMENT

Abstract

Cytoplasmic lipids play key roles during oocyte and embryo development. However, there is little information on the changes in lipid types during intraovarian follicular and oocyte growth. Here, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to understand lipid composition in the cat oocyte during differing stages of folliculogenesis. Follicles at different developmental stages were mechanically isolated from cat ovaries within 4 h after routine ovariohysterectomy. Oocytes with granulosa cells were recovered from secondary (preantral stage, n = 387), early antral (<0.5 mm diameter, n = 177), small antral (0.5–1 mm, n = 144), and antral (>1 mm, n = 120) follicles, then subjected to lipid extraction and MALDI-TOF analysis (n = 3 replicates). Resulting mass spectra data were evaluated using MALDIquant in R package to eliminate baseline (background) and to identify peak values. The latter maximal values for each follicle stage were selected and subjected to principal component analysis to identify similarities and differences in mass spectra profile among oocytes from varying developmental stages. Peaks were compared to those calculated molecular formulae available in the Lipid MAP database. Error estimates were calculated using Excel (Microsoft Corp., Redmond, WA), and lipid species identification performed using Lipid MS Predict. Twenty-nine lipid species from six glycerophospholipids groups (glycerophosphates, PA; glycerophosphoserines, PS; glycerophosphoinositols, PI; glycerophosphoglycerols, PG; glycerophosphoinositol monophosphates, PIP; and glycerophosphoethanolamines, PE) were identified, 15 being found in more than 1 developmental stage. Two species in particular, [PI(29:4)+Na]⁺ and [PIP(23:0)+Na]⁺, were the most abundant lipids and were identified in oocytes from all developmental stages. There were dynamic shifts in lipid species expressed at different follicle stages. Oocytes from secondary and antral follicles contained more lipid types (15 and 22, respectively) than early (10) and small antral (4) counterparts. Four (PA, PS, PI, and PIP) of the 6 glycerophospholipids were found only in oocytes from secondary and antral follicles. Oocytes from small antral follicles also lacked PA, PS, and PG, whereas PG was not found in early antral stage oocytes. In summary, we showed for the first time in the cat that, similar to goats and rats, there are temporal changes in lipid types within the oocyte during folliculogenesis. We suspect that these changing dynamics, including shifts in presence or absence of lipid species with follicle stage, may be playing key roles in oocyte growth and viability. Our findings also serve as in vivo benchmarks for parallel studies focused on enhancing an in vitro culture system for early-stage ovarian follicles to preserve fertility of genetically valuable domestic and wild felids.