Matsubara Shin, Shiraishi Akira, Osugi Tomohiro, Kawada Tsuyoshi, Satake Honoo
Bioorganic Research Institute, Suntory Foundation for Life Sciences, Kyoto 619-0284, Japan.
Research Fellow of Japan Society for the Promotion of Science, Tokyo, Japan.
Bio Protoc. 2020 Apr 5;10(7):e3577. doi: 10.21769/BioProtoc.3577.
Ascidians are the closest living relatives of vertebrates ( Delsuc , 2006 ; Satoh , 2014 ) and are important for the evolutionary study of the ovarian follicle development including oocyte maturation and ovulation. However, neither the endogenous factors nor the molecular mechanisms underlying the oocyte maturation and ovulation had been elucidated mainly due to the lack of efficient procedure for isolating ovarian follicles. Here, we present the protocol for the effective fractionation and isolation of the ovarian follicle of type A using stainless steel sieves with various particle size-meshes, and the simple incubation method of follicles for evaluating oocyte maturation and ovulation. Combined with the RNA-seq data from each fraction, the current methods lead us to investigate ovarian follicle development including oocyte maturation and ovulation in a stage-specific manner.
海鞘是现存与脊椎动物亲缘关系最近的生物（德尔叙克，2006年；佐藤，2014年），对于包括卵母细胞成熟和排卵在内的卵巢卵泡发育的进化研究具有重要意义。然而，由于缺乏分离卵巢卵泡的有效方法，卵母细胞成熟和排卵的内源性因素及分子机制均未得到阐明。在此，我们介绍了使用具有不同粒径筛网的不锈钢筛对A型卵巢卵泡进行有效分级分离的方案，以及用于评估卵母细胞成熟和排卵的卵泡简单孵育方法。结合各分级的RNA测序数据，当前方法使我们能够以阶段特异性的方式研究包括卵母细胞成熟和排卵在内的卵巢卵泡发育。
