Surface Ultrastructural Alterations of Bovine Oocytes after Parthenogenetic Activation

Abstract

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.

Specific activation requirements of in vitro‐matured sheep oocytes following vitrification‐warming

“…Real‐time PCR analyses also showed that MPF inactivation occurred at similar rates in the oocytes of both groups. While we found no previous study on vitrified versus activated oocytes to compare our results to, the results of this study are in agreement with other studies that have been performed separately on either parthenogenetic (Suzuki et al, 2000) or vitrified (Saunders and Parks, 1999; Diez et al, 2005) oocytes. Therefore, the question arose as to whether vitrified‐warmed oocytes need a similar or different activation protocol from unvitrified oocytes, although it remains to be understood if premature oocyte activation is a direct consequence of the vitrification‐warming process or a phenomenon that occurs following oocyte damage.…”

Zinc sparks induce physiochemical changes in the egg zona pellucida that prevent polyspermy

“…This phenomenon has been previously observed upon activation of bovine eggs. 51 Zinc has a similar effect to activation, with both 50 µM and 500 µM ZnSO 4 incubation resulting in thicker bundles within the ZP when compared to MII eggs (Figure 4a–ii and iii). The average thickness of fibril bundles within these images was quantified using an automated image analysis protocol (see supporting information for details).…”

“…These ultrastructural methods have been used previously to characterize the ZPs of mammalian eggs and zygotes and demonstrate increased protein interactions following egg activation. 49–51 ZPs from MII and Sr-activated eggs were compared with ZPs from MII eggs exposed to both 50 µM and 500 µM ZnSO 4 . In these experiments, zinc treatments were performed on intact eggs.…”

Improvement of Cloned Embryos Development by Co-Culturing with Parthenotes: A Possible Role of Exosomes/Microvesicles for Embryos Paracrine Communication

“…This may be because of the handling of oocytes and embryos during denuding and culture that affects the zona pellucida integrity and porosity. Interestingly, the zona reaction and subsequent changes in the zona surface differs between IVF and PA embryos; the zona pellucida resumed its porous characteristics after parthenogenetic activation of bovine oocytes (Suzuki et al, 2000). Molecule permeability through the zona depends on the molecule's size and physicochemical factors, such as hydrophilic-lipophilic properties; lipidcontaining molecules in particular penetrate the zona pellucida with relative ease (Turner and Horobin, 1997).…”