THE TIMING OF CORTICAL GRANULE FUSION, CONTENT DISPERSAL, AND ENDOCYTOSIS DURING FERTILIZATION OF THE HAMSTER EGG - AN ELECTROPHYSIOLOGICALAND HISTOCHEMICAL-STUDY

To determine the temporal relationship between cortical granule exocyt osis and the repetitive calcium transients, which are characteristic o f mammalian fertilization, we monitored membrane addition from exocyto sis during fertilization of hamster eggs. Continuous measurement of me mbrane capacitance by applying a 3.1-nA alternating current at 375 Hz showed addition of cortical granule membrane. Simultaneous measurement of membrane potential revealed each calcium transient by the appearan ce of transient hyperpolarizing responses due to calcium-activated pot assium channels in the egg. The initial membrane capacitance of the eg gs averaged 736 +/- 44 pF (mean +/- SD; n = 7) and an increase in capa citance of 61 +/- 19 pF occurred within 4 sec of the start of the firs t hyperpolarizing response (HR) after fertilization. Immediately after the first increase in capacitance there was a gradual decline in memb rane capacitance in all eggs and in five/seven eggs the capacitance re turned to the unfertilized level in 7.8 +/- 4.4 min. The gradual decli ne in capacitance after the first increase indicated endocytosis, whic h was confirmed by the internalization of fluorescently labeled dextra n. Superimposed on the gradual decline in membrane capacitance were sm aller increases in capacitance that occurred with the second and later HRs. The total increase in capacitance from the first three events av eraged 72 +/- 19 pF, representing an average increase in capacitance o f about 10% of the capacitance of the unfertilized egg. By labeling eg gs before and after permeabilization with two different fluorochromes attached to Lens culinaris agglutinin, we demonstrate that the dispers al of the cortical granules contents does not occur immediately after exocytosis. Our results demonstrate that cortical granule exocytosis i n hamster eggs is closely coupled to the periodic increases in calcium , that the contents of the cortical granules are slow to disperse, and that after exocytosis, the surface area of the egg returns to the unf ertilized level because of a period of endocytosis. (C) 1994 Academic Press, Inc.