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

Shin Matsubara,Tomohiro Osugi,Akira Shiraishi,Tsuyoshi Kawada,Honoo Satake

doi:10.21769/bioprotoc.3577

Abstract

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.

Highlights

[Background] Ciona ovarian follicles are classified into four major developmental stages: stage I, stage II, stage III, and stage IV follicles (Figure 1; Prodon et al, 2006; Silvestre et al, 2009)
We provide an effective fractionation method that a mixture of every developmental stage of follicles can be fractionated using a series of stainless steel sieves with various particle-size meshes (20-150 μm)
Oocyte maturation and ovulation can be evaluated by germinal vesicle breakdown (i.e., Germinal vesicle breakdown (GVBD)) and rupture of follicular cell layer, respectively (Figure 1)

Summary

Pipette tips

Positive factors of oocyte maturation and ovulation (e.g., Synthetic Ciona vasopressin (CiVP). Negative factors of oocyte maturation and ovulation, e.g., U0126 (Promega, catalog number: V1121). 1. 1-L beaker (IWAKI AGC TECHNO GLASS, catalog number: 0988-01-01-66) 2. Water pump (REI-SEA IWAKI, model: RMD-151, maximum flow rate: 19 L/min) 4. Air pump (TECHNO TAKATSUKI, model: HP-30, maximum flow rate: 30 L/min) 5. Portable pipet controller (Drummond, catalog number: 4-040-101-J) 6. Forceps (Rubis, catalog numbers: 2-5149-08, 2-5149-12) 7. Scissors (AS ONE, catalog number: 2-539-11) 8. Stainless steel sieves (TOKYO SCREEN Co., LTD., catalog numbers: TS-50-20-39 (Aperture: 180 μm); TS-50-20-41 (Aperture: 150 μm); TS-50-20-45 (Aperture: 90 μm); TS-50-20-47 (Aperture: 63 μm); TS-50-20-50 (Aperture: 38 μm); TS-50-20-53 (Aperture: 20 μm), https://www.tokyo-screen.com/.en/) 9. Microscale slide (WATSON Bio Lab, catalog number: 177-401C).

Procedure

Fractionation and isolation of Ciona follicles