Behaviour of the vitelline envelope inBufo arenarumoocytes maturedin vitroin blockade to polyspermy

Abstract

SummaryDuring activation of amphibian eggs, cortical granule exocytosis causes elaborate ultrastructural changes in the vitelline envelope. These changes involve modifications in the structure of the vitelline envelope and formation of a fertilization envelope (FE) that can no longer be penetrated by sperm. InBufo arenarum, as the egg traverses the oviduct, the vitelline envelope is altered by a trypsin-like protease secreted by the oviduct, which induces an increased susceptibility of the vitelline envelope to sperm lysins. Full-grown oocytes ofB. arenarum, maturedin vitroby progesterone, are polyspermic, although cortical granule exocytosis seems to occur within a normal chronological sequence. These oocytes can be fertilized with or without trypsin treatment, suggesting that the vitelline envelope is totally sperm-permeable. Vitelline envelopes without trypsin treatment cannot retain either gp90 or gp96. This suggests that these glycoproteins are involved in the block to polyspermy and that trypsin treatment of maturedin vitrooocytes before insemination is necessary to enable vitelline envelopes to block polyspermy. The loss of the binding capacity in vitelline envelopes isolated fromB. arenarumoocytes maturedin vitrowith trypsin treatment and activated by electric shock suggests that previous trypsin treatment is a necessary step for sperm block to occur. Whenin vitromatured oocytes were incubated with the product of cortical granules obtained fromin vitromatured oocytes (vCGP), vitelline envelopes with trypsin treatment were able to block sperm entry. These oocytes exhibited the characteristic signs of activation. These results support the idea thatB. arenarumoocytes can be activated by external stimuli and suggest the presence of unknown oocyte surface receptors linked to the activation machinery in response to fertilization. Electrophoretic profiles obtained by SDS-PAGE of solubilized vitelline envelopes from oocytes maturedin vitrorevealed the conversion of gp40 (in vitromatured oocytes, without trypsin treatment) to gp38 (ascribable to trypsin activity or cortical granule product activity, CGP) and the conversion of gp70 to gp68 (ascribable to trypsin activity plus CGP activity). Taking into account that only the vitelline envelopes of in vitromatured oocytes with trypsin treatment and activated can block sperm entry, we may suggest that the conversion of gp70 to gp68 is related to the changes associated with sperm binding.