Preparation of the Cortical Reaction: Maturation-Dependent Migration of SNARE Proteins, Clathrin, and Complexin to the Porcine Oocyte's Surface Blocks Membrane Traffic until Fertilization

Abstract

The cortical reaction is a calcium-dependent exocytotic process in which the content of secretory granules is released into the perivitellin space immediately after fertilization, which serves to prevent polyspermic fertilization. In this study, we investigated the involvement and the organization of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins in the docking and fusion of the cortical granule membrane with the oolemma in porcine oocytes. During meiotic maturation, secretory vesicles that were labeled with a granule-specific binding lectin, peanut agglutinin (PNA), migrated toward the oocyte's surface. This surface-orientated redistribution behavior was also observed for the oocyte-specific SNARE proteins SNAP23 and VAMP1 that colocalized with the PNA-labeled structures in the cortex area just under the oolemma and with the exclusive localization area of complexin (a trans-SNARE complex-stabilizing protein). The coming together of these proteins serves to prevent the spontaneous secretion of the docked cortical granules and to prepare the oocyte's surface for the cortical reaction, which should probably be immediately compensated for by a clathrin-mediated endocytosis. In vitro fertilization resulted in the secretion of the cortical granule content and the concomitant release of complexin and clathrin into the oocyte's cytosol, and this is considered to stimulate the observed endocytosis of SNARE-containing membrane vesicles.

Membrane Fusions During Mammalian Fertilization

“…After fertilization the Ca 2+ mobilization and related signaling and cytoskeletal rearrangements [18,115] cause the trans to cis ternary configuration of the trimeric SNARE complex is turned on and explains the cortical reaction. Remarkably complexin and clathrin dissociate from the cortex and relocate to intracellular structures and this is probably required to re-establish endocytosis and membrane recycling [107]. In case of polyspermic fertilization of pig oocytes, we observed a normal cortical reaction but this is not followed by a release of clathrin which may indicate that at the level of the oolemma fusion the inhibition of endocytosis may have a relationship with the fusion properties of the oolemma [107].…”

“…Nevertheless, some of the secretory granules already reside in the cortex of GV oocytes but fail to be competent to fuse with the oocyte surface at that stage which appears to depent on too low activity of calcium/calmodulin dependent kinase II activity which becomes activated in MII oocytes [113] and related to this MAPK activity seems to be inviolved in activating the cortical granule exocytosis as well [114]. In addition we have shown recently that cortical granules become docked at the oocytes plasma membrane during these two meiotic maturation stages [107]. The SNARE proteins SNAP23, VAMP-1 and syntaxin 2 are involved and probably form a similar trimeric trans complex prior to fertilization (which therefore is analogous to acrosome docking during sperm capacitation).…”

“…These factors induce intracellular Ca 2+ events and the oocyte plasma membrane depolarization and both are triggers for the cortical granule exocytosis [18]. The secretory granules that reside in the cortex (the area just under the oocyte plasma membrane) fuse with the oocyte plasma membrane and the content of the granules is released into the perivitelline space [106,107]. Although the contents of the cortical granules are very poorly characterized [106,[108][109][110][111], it is known that the release of these materials results in the cleavage of ZP2 and ZP3 into the truncated ZP2 f and ZP3 f forms [20][21][22].…”

“…The main difference between the two exocytosis events is that cortical granule exocytosis is a series of single point fusion events of many cortical granules with the oolemma, while the acrosome exocytosis is a multiple point fusion event of one acrosome with the sperm plasma membrane. The majority of secretory granules migrate towards the oocyte plasma membrane during pre-ovulatory maturation of oocytes somewhere between the germinal vesicle stage (GV, oocytes that are arrested at the prophase of meiosis I) and the arrested stage at metaphase of meiosis II (MII) [107,112]. Nevertheless, some of the secretory granules already reside in the cortex of GV oocytes but fail to be competent to fuse with the oocyte surface at that stage which appears to depent on too low activity of calcium/calmodulin dependent kinase II activity which becomes activated in MII oocytes [113] and related to this MAPK activity seems to be inviolved in activating the cortical granule exocytosis as well [114].…”

“…Remarkably complexin and clathrin dissociate from the cortex and relocate to intracellular structures and this is probably required to re-establish endocytosis and membrane recycling [107]. In case of polyspermic fertilization of pig oocytes, we observed a normal cortical reaction but this is not followed by a release of clathrin which may indicate that at the level of the oolemma fusion the inhibition of endocytosis may have a relationship with the fusion properties of the oolemma [107]. This observation confirms the finding that the polyspermy block is at least not immediately dependent on cortical exocytosis (see Section 5.6.2).…”