Appendix 4 - Preparation of meiosis inducing factor

Anesthetize an adult female to extinction by adding 50% ethanol dropwise to a dish containing the animal in filtered seawater, over a period of ~20 to 30 min. Place animal ventral side down on a clay surfaced dissection dish and cover with filtered seawater. Make a mid-line incision; use microscissors to cut the esophagus close to the mouth and the intestine near the anal opening. Carefully remove the esophagus, the stomach and attached pulsating heart and the entire intestine and discard. See Fig. 2. In addition, remove the paired brownish nephromixia. This exposes the ventral nerve cord (Fig. 11). Trace it anteriorly where it terminates in a bi-lobed subesophageal ganglion located just medial to the gills. This ganglion is enmeshed in a more ventral red and white multi-lobed cement gland. The bifurcated subesophageal ganglion continues anteriorly and dorsally via two circumpharyngeal connectives. Dorsal to the mouth they form two pink suprastomial cerebral ganglia embedded in the base of the prostomium. The entire complex of ganglion, cement gland and primitive brain is about 3 mm long. Excise the subesophageal-cement gland complex, drain excess fluid and freeze immediately. At least six of the ganglion-cement gland complexes are dissected. They are homogenized in a teflon glass microhomogenizer by hand in ice with a minimum of 0.45 µm filtered seawater. After centrifugation (800 G) the aqueous supernate can be used directly for inoculation.

[Refinement of the extract indicates the active component is heat labile. Dialysis of the extract against 1 to 50 v/v of calcium free artificial seawater at 4°C for 18 h (Appendix 8 ) indicated the active component is larger than 12,000 daltons. For further extraction see Tweedell, 1980]

For injection, anesthetize mature adult females (16-30 mm) by immersing them in 0.04% tricaine methane sulfonate in filtered seawater - just until body movement ceases. Each anesthetized animal is injected through the cephalic plaque (see Fig. 3) with 25-50 µl (the equivalent of 2 to 3 gland complexes) of the aqueous extract from a microliter syringe fitted with a 30 ga needle. Each animal is returned to sea water immediately after injection. Oocytes will migrate into the nephromixia 6 to 20 hours after injection; at this time the gravid nephromixia appear yellow through the dorsal body wall.

In vitro studies.

If desired, the oocytes can be removed prior to shedding into seawater by the insertion into the nephridiopore of a 32 ga flexible stainless steel needle attached to a nylon hub on a microliter syringe.