Fractionation of Ovarian Follicles and in vitro Oocyte Maturation and Ovulation Assay of Ciona intestinalis Type A.

Ascidians are the closest living relatives of vertebrates ( Delsuc et al., 2006 ; Satoh et al., 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 Ciona intestinalis type A using stainless steel sieves with various particle size-meshes, and the simple incubation method of Ciona 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.