Identifying the composition of large vesicles in the cytoplasm of oocytes

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Context Oocyte vesicles, or vacuoles, have been described using transmission electron microscopy in most species. In sheep and cow oocytes, vesicles constitute up to 30% of the cytoplasm, their volume decreases during maturation and is lower in poorer quality oocytes, suggesting they are important for oocyte competence. However, the composition and function of these organelles is unknown. Aim This study aimed to ascertain the content of oocyte vesicles and examine the effect of different fixation methods on the size and preservation of these organelles. Methods Sheep oocytes were centrifuged to segregate organelles then stained with organelle-specific fluorescent dyes (Nile Red, LysoTracker, Fluo-4-AM and TMRM) and imaged by live cell confocal microscopy. The oocytes were fixed with either glutaraldehyde or paraformaldehyde and prepared for electron microscopy to confirm the distribution of organelles and compare ultrastructure and organelle size. Key results Nile Red staining has identified that vesicles contain lipid that is different to that in the osmium-stained lipid droplets observed by electron microscopy. Lipid droplets and vesicles were significantly smaller when prepared for electron microscopy compared to live cell imaging. Organelles were less likely to be fully segregated following centrifugation in oocytes prior to maturation (20%) compared to oocytes after maturation (77%; P < 0.0001). Conclusions Oocyte vesicles are lipid storing organelles that may be important for oocyte quality. Implications This study highlights the importance of lipid for oocyte quality and the need for further research to identify the optimal fatty acid content for in vitro maturation media and oocyte competence.