Activation of Membrane associated ATPase in Hen's Egg after Ovulation

Abstract

WE have isolated the membrane complex (vitelline and plasma membrane^1,2) from oocytes as small as 4 mm in diameter and through all subsequent stages of development in the ovary and the oviduct, including the freshly laid egg. Sixty-five membranes from ovarian oocytes, forty-five from oviducal eggs and fifty-four membranes from laid eggs have been examined. Three distinct isolation procedures are required depending on the developmental stage. Ovarian oocytes and their supporting follicles with diameters < 8 mm are placed in distilled water. An incision through half the follicle allows ejection of the oocyte's contents and its surrounding membrane complex. This is freed from adhering yolk by swirling. Resulting fragments are washed in imidazole buffer, pH 7.4, before pooling for assay. Ovarian oocytes with diameters > 8 mm do not eject outer membranes on release of the yolk. These are removed by microdissection following eversion of the ovarian follicle. In both cases the oocyte outer membrane is obtained devoid of follicular cells. The outer membranes of ovulated eggs are isolated by incising the ovum in water, cleaning by swirling and washing, and storage in 4° C buffer. Surface areas of spread membrane pieces are measured with a camera lucida.