Reader Karen L, Pratt Isabella G, Lawson Georgia L, Woolley Robert J
Department of Pathology, University of Otago, Dunedin, New Zealand.
Confocal Microscopy Unit, Research Infrastructure Centre, University of Otago, Dunedin, New Zealand.
Reprod Fertil Dev. 2024 Dec;37. doi: 10.1071/RD24131.
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 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.
背景 在大多数物种中，已通过透射电子显微镜描述了卵母细胞囊泡或液泡。在绵羊和牛的卵母细胞中，囊泡占细胞质的比例高达30%，其体积在成熟过程中减小，并且在质量较差的卵母细胞中更低，这表明它们对卵母细胞的发育能力很重要。然而，这些细胞器的组成和功能尚不清楚。目的 本研究旨在确定卵母细胞囊泡的内容物，并研究不同固定方法对这些细胞器大小和保存的影响。方法 将绵羊卵母细胞离心以分离细胞器，然后用细胞器特异性荧光染料（尼罗红、溶酶体示踪剂、Fluo-4-AM和TMRM）染色，并通过活细胞共聚焦显微镜成像。卵母细胞用戊二醛或多聚甲醛固定，并制备用于电子显微镜检查，以确认细胞器的分布，并比较超微结构和细胞器大小。主要结果 尼罗红染色表明，囊泡含有与电子显微镜观察到的锇染色脂滴不同的脂质。与活细胞成像相比，制备用于电子显微镜检查时，脂滴和囊泡明显更小。与成熟后的卵母细胞（77%）相比，成熟前的卵母细胞在离心后细胞器不太可能完全分离（20%；P结论 卵母细胞囊泡是储存脂质的细胞器，可能对卵母细胞质量很重要。意义 本研究强调了脂质对卵母细胞质量的重要性，以及需要进一步研究以确定体外成熟培养基的最佳脂肪酸含量和卵母细胞发育能力。
