Cortical granule exocytosis and cell surface reorganization in eggs of Brachydanio

Cortical granules in the mature, unfertilized egg of the zebra danio (Brachydanio rerio) are spherical, membrane-bound organelles measuring 3 to 22 μm in diameter and arranged in irregular rows beneath the plasma membrane. Two types of cortical granules are identified in thin sections viewed under the transmission electron microscope: “dark” type (electron-dense core) and “light” type (electron-lucent core). Cytochemically, glycoproteins and acid mucopolysaccharides are constituents of the cortical granules. Differences in staining reactions with Alcian blue and toluidine blue solutions also reveal the presence of two types of cortical granules in the danio egg. The “dark” type of cortical granule stains strongly with both dyes and shows a single, central core surrounded by a zone of lighter, particulate material. The “light” type of cortical granule stains faintly with both dyes. Differences in staining responses are not related to the size of the cortical granules. Analysis of the cortical granule reaction in inseminated eggs by light microscopy reveals that breakdown and discharge of cortical granules begin after 30 seconds postactivation. Since the chorion is lifted away from the egg surface by this time, the initial elevation of this membrane occurs independently of the onset of cortical granule exocytosis. The breakdown and discharge of cortical granules is a prominent feature of egg surface activity between 1 and 3 minutes postactivation. The cortical granule reaction is completed by 5 to 6 minutes postactivation. Scanning electron microscopy has provided an unusual opportunity to observe in detail cortical granule exocytosis and the reorganization of the egg surface during the cortical reaction. Cortical granule exocytosis consists of the following sequence of morphological stages: elevation of the cortical granule above the egg surface, rupture of the membrane covering the cortical granule, and discharge of central and peripheral constituents of the cortical granule into the perivitelline space. Rupture of the cortical granule follows contact and fusion between perigranular and egg plasma membranes and is effected through the coalescence of fissures that appear in the egg surface between the equator and the top of the elevated granule. Our SEM and TEM findings suggest strongly that the mosaic surface formed upon cortical granule exocytosis is temporary and that perigranular membranes have limited residence in the egg surface. Morphological evidence presented here is consistent with the view that the zebra danio egg accommodates the excessive membrane introduced into its surface during the cortical reaction by endocytosis. It is proposed that perigranular membrane is retrieved continuously after exocytosis but is visible morphologically as a two-step process. First, perigranular membrane is removed from the egg surface into the cortical cytoplasm in the form of vesicles (0.5–1.5 μm in diameter) pinched-off from the base of the cortical crypt. Second, localized folds of the egg plasmalemma overgrow and replace, perhaps through contractile activity, remaining perigranular membrane at the egg surface.