Surface ultrastructural alterations of bovine oocytes after parthenogenetic activation

Oocyte activation is a critical component of the current animal cloning scheme. This study was designed to examine surface characteristics of bovine oocytes by scanning electron microscopy (SEM) after activation by calcium ionophore A23187 (A23187) and electric pulse combined with cycloheximide (CHX) or 6-dimethylaminopurine (6-DMAP) treatments. In vitro matured (IVM) oocytes were activated then harvested at 0 to 19 hours after the onset of treatments for SEM processing. The zona pellucida (ZP) of untreated IVM oocytes exhibited an open mesh structure. The ZP surface showed little changes after A23187 alone, but dramatically changed to a less porous surface 3 hours after combined treatments with CHX or 6-DMAP. The vitelline membrane of IVM oocytes was covered with well-developed microvilli (MV). The MV became shorter (0.83 vs. 1.35 microm, p < 0.01) 8 hours after A23187 treatment alone. The vitelline membrane was altered in all oocytes examined 3 hours after incubation with A23187 plus CHX or 6-DMAP. A 1.5-fold increase in the diameter of MV in the CHX group and a higher incidence of large cytoplasmic protrusions (more than 1 microm width) in the 6-DMAP group were observed. After removal of inhibitors, the surface morphologies of the ZP and vitelline membrane were returned nearly to those of untreated IVM oocytes in both groups. The present study clearly showed that surface characteristics of the bovine oocyte were more profoundly changed by a combination of agents for parthenogenetic stimulation, and that the ultrastructural effects were reversible.