Abstract
An oocyte’s developmental competency and reproductive success are inextricably linked to its ability to execute cytoplasmic and nuclear maturation. While numerous investigations have been conducted on oocyte organelles, little attention has been paid to understanding oocyte vesicles. Prior studies have shown that vesicle volume decreases following oocyte maturation, suggesting the contents are being utilised for maturation. Evidence is scarce surrounding the contents of oocyte vesicles, and they have yet to be well defined within the literature; however, it has been hypothesised that they contain lipids. This study seeks to address the knowledge gap by developing methods to identify the composition of lipid droplets and vesicles in oocytes.
Sheep oocytes obtained from the abattoir were employed as the experimental model, with adult oocytes serving as good quality and lamb oocytes as poor quality. The oocytes were subjected to in vitro maturation and centrifugation for density-based organelle separation. Oocytes were fixed with paraformaldehyde or glutaraldehyde and analysed using transmission electron microscopy (TEM). With a focus on lipids, spinning disk confocal microscopy was conducted using a Nile Red stain to visualise the organelle layers through fluorescence. A lipidomic analysis was conducted using mass spectrometry, wherein the quantity of metabolites identified in lamb oocytes were compared to adult oocytes.
Lipids were confirmed to be in the vesicles based on the fluorescence of Nile Red. The vesicle’s lipid profile was identified as independent of the lipid droplet population. Comparison of lipid droplet and vesicle ultrastructure showed that the average diameter of each analysed via confocal microscopy was 4.3m and 1.85m, respectively; in comparison, TEM analysis was 3m and 0.95m, respectively. These measurements highlight the significant difference in quantitative analysis through different techniques. This study identified abundant saturated free fatty acids such as palmitic acid, stearic acid, behenic acid, and pentadecanoic acid in adult and lamb oocytes. Seventy-three lipid metabolites such as pentadecanoic acid, tetracosenoic acid, and heneicosadienoic acid were significantly decreased in lamb oocytes compared with adults. Interestingly, norethisterone acetate, a form of progesterone, was identified to be significantly lower in lamb oocytes than in adult oocytes.
These findings corroborated that oocyte vesicles contain alternative lipids to the lipid droplets, which are significant to oocyte development. Thus, these findings suggest the utilisation of these lipids during oocyte maturation and draw attention to their unique contributions to oocyte developmental competency, thus supporting our hypothesis. Decreased lipid metabolites in lamb oocytes, compared to adult, could be indicative of their importance in oocyte quality and, therefore, could be targeted for the enrichment of IVM media to support the successful maturation of immature oocytes.