Cortical granule exocytosis and cell surface reorganization in eggs of Brachydanio

Abstract

Cortical granules in the mature, unfertilized egg of the zebra danio (Brachydanio rerio) are spherical, membrane-bound organelles measuring 3 to 22 pm in diameter and arranged in irregular rows beneath the plasma membrane. ' h o 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 Correspondence should be addressed to Nathan H. Hart. 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 p m in diameter) pinched-off from the base of the cortical crypt. ...

hnRNP I is required to generate the Ca2+ signal that causes egg activation in zebrafish

“…In zebrafish, a similar rise of cytosolic Ca 2+ is observed upon egg activation, although egg activation in zebrafish does not require fertilization, but instead depends on a hypotonic environment, which arises when eggs are laid in normal water conditions (Hart and Fluck, 1995;Hart and Yu, 1980;Iwamatsu et al, 1997;Lee et al, 1999). As in other species, CGE in zebrafish occurs as a first response to elevated cytoplasmic Ca 2+ levels during egg activation.…”

Cortical reaction and solicitation mechanism in Hemibarbus labeo ovum

“…After the inboard cortical alveoli are released, only a few cortical alveoli are left behind the ovum. According to this criterion, the climactic period lasts out about 4 min in Hemibarbus labeo, 2 min in Carassius auratus (Gao, 1996), and 1−3min in Brachydanio rerio (Hart and Yu, 1980). The type V cortical alveoli and the other remnant cortical alveoli are released unceasingly.…”

Epigenetic reprogramming of somatic cells by nuclear transplant in zebrafish

“…However, although not structurally separated by a continuous membrane, these eggs possess a functional ooplasm cortex some 15-20 µm thick where yolk globules are excluded. When mature zebrafish eggs are discharged from the ovarian stroma and come in contact with the spawning medium, they activate (Hart and Yu, 1980;Hart and Fluck, 1995;Sakai et al, 1997). …”

“…On the other hand, when the sperm nucleus is inactivated, there is not a nuclear helper role, but there are sperm derived factors which are responsible for triggering the calcium transient in the activation of a lot of species from sea urchin and ascidian oocytes (Dale et al, 1985;Dale, 1988) to human (Tesarik et al, 1994, Dale et al, 1996. However, zebrafish egg activation does not involve the need of any spermderived factors (Swann, 1993) since when eggs come in contact with water they activate (Hart and Yu, 1980;Hart and Fluck, 1995;Sakai et al, 1997), even in absence of sperm, they undergo some abortive cleavages (Lee et al, 1996). In the present work, parthenogenetic development was not observed, neither using non motile radiated sperm (with derived-factors) as activating agent (results not shown) nor applying an electroactivation protocol to non activated eggs .…”