Fully grown immature oocytes are 170-180 µm in diameter. Smaller oocytes fail to mature when exposed to the maturation hormone.
The maturation hormone 1-methyladenine was identified by Kanatani, Shirai and co-workers. It was the first maturation hormone to be identified. It is present in the follicle cells. Its release from the follicle cells is stimulated by an as yet uncharacterised factor which can be isolated from the radial nerve of starfish arms.
To work with GV intact oocytes, the oocytes must be released from the ovary. A natural release, for instance to induce spawning by injecting 1-methyladenine into the starfish body cavity, will result in the release of mature oocytes. This is not desirable, since the GV will have broken down.
One method is to remove the ovary from the animal and cut it into small pieces in cold calcium free sea water. This releases oocytes; the CFSW has the further function of causing the follicle cells to fall off. After around 5 min, CFSW is replaced with sea water. Depending on how well the procedure works, there will be some spontaneous maturation as well as some oocytes with follicle cells still attached, and some oocyte death.
Another way is to make a cut in the underside, remove a piece of ovary - (what it looks like in a dish) - and put it into a beaker with CFSW. After 15 min, replace with fresh CFSW. After another 15 min, put ovary into a 35 mm dish with 2 ml SW + 10 µM acetylcholine (10 mM stock in water stored in refrigerator). The ovary wall is permeable, so the CFSW incubation causes the ovary extracellular Ca to come out and the follicles to fall apart. The Ca that is added back causes a contraction of the ovary wall so that immature oocytes without follicle cells are extruded out of the cut end. Acetylcholine helps to ensure a strong contraction. After being extruded, the oocytes are transferred to a large volume of SW (partly to dilute the acetylcholine).
The GV is approximately 60-80 µm in diameter and is positioned very close to the animal pole. Maturation hormone causes GVBD by 20-30 min. The first polar body is at ~60 min, polar body is at ~90 min (timing is dependent on temperature and animal). The cell cycle arrests at egg pronuclear stage. Normal fertilization occurs in the time period from ~10 min after GVBD to first polar body formation. Fertilization at times other than this will result in polyspermy.