334 DNA SYNTHESIS DURING THE FIRST CELL CYCLE OF PORCINE OOCYTES FOLLOWING DIFFERENT ACTIVATION TREATMENTS

Abstract

Inhibitors of protein synthesis and phosphorylation have been widely used for oocyte activation and have been reported to induce abnormalities in nuclear ploidy due to aberrant DNA synthesis (DNAS). The present experiment was designed to compare the DNAS during the first cell cycle of porcine parthenotes following different activation treatments. Cumulus–oocyte complexes were cultured in TCM-199 supplemented with 0.5 µg mL^-1 LH and FSH, 10 ng mL^-1 EGF, and 0.1% PVA for 22 h, and additionally cultured in media without LH and FSH for 22 h. MII oocytes were then electrically pulsed twice in 0.28 M mannitol containing 0.05 mM CaCl₂ and 0.1 mM MgSO₄ at 1.8 kV cm^-1 for 30 µs (group 1), followed by 7.5 µg mL^-1 cytochalasin B (CCB, group 2), 10 µg mL^-1 cycloheximide (CHX, group 3), or 1.9 mM 6-dimethylaminopurine (6-DMAP, group 4) for 3 h. Eggs were incubated with 100 µM 5-bromo-222-deoxyuridine (BrdU) for 1 h at 0, 2, 4, 6, 8, 10, and 12 h after activation to evaluate DNAS (Adenot et al. 1997 Development 124, 4615–4625) by determining the BrdU signal under a fluorescence microscope. Experiments were replicated 4 times; results were expressed as mean ± SD and analyzed using one-way ANOVA by SPSS 10.0. The percentage of DNAS was calculated by dividing the number of BrdU-positive eggs by the total number of eggs used. DNAS in groups 1, 3, and 4 initiated at 2–3 h post-activation (hpa) but at 4–5 hpa in group 2. In group 1, DNAS was faint until 3 hpa, gradually increasing thereafter until 11 hpa (20.7 ± 19.6, 29.4 ± 17.0, 41.3 ± 16.7, and 64.4 ± 6.2, at 4–5, 6–7, 8–9, and 10–11 hpa, respectively). There was a significant (P < 0.05) increase in DNAS at 10–11 h, but a significant (P < 0.05) decrease (34.3 ± 6.4) at 12–13 hpa. In groups 2 and 3, after 4–5 hpa, DNAS gradually increased until 7 h (6.2 ± 1.4 and 29.8 ± 16.6 at 4–5 hpa, and 21.1 ± 13.7 and 40.0 ± 18.7 at 6–7 hpa, respectively), but a DNAS peak was observed at 8–9 h (44.6 ± 9.0) in group 2 and at 10–11 h (40.5 ± 22.1) in group 3. Interestingly, group 4 parthenotes showed a different DNAS pattern compared with other groups, as it started at 2–3 hpa (24.8 ± 11.7), reached a significantly (P < 0.05) high level at 4–5 hpa (56.3 ± 9.0), and gradually decreased at 6–7 hpa (42.7 ± 10.3) until 12–13 hpa (29.5 ± 14.9). In conclusion, CCB, CHX, and 6-DMAP used for oocyte activation exhibited different patterns of DNA synthesis during the first cell cycle of porcine parthenotes. Therefore, further experiments are required to evaluate the molecular signaling that regulates DNAS, embryonic developmental velocity, and ploidy of 2-cell parthenotes.