Time course of pronuclear deoxyribonucleic acid synthesis in parthenogenetically activated bovine oocytes.
The progress of pronuclear DNA synthesis was monitored by the radioactive precursor 3H-thymidine during the first cell cycle of parthenogenetically activated bovine oocytes. Bovine oocytes were exposed to Ca2+ ionophore A23187 at 24, 30, or 36 h after the onset of in vitro maturation. Young 24-h oocytes were subsequently cultured for 6 h in the protein synthesis inhibitor, cycloheximide (CHX), to ensure similar rates of activation (96-100%) and pronuclear formation (93-97%) among all groups of oocytes. Subsequent autoradiographic experiments revealed a slightly, but not significantly, accelerated start of DNA synthesis in aged (36 h) oocytes. Maximum levels of DNA labeling were reached within 4 h regardless of oocyte maturation age and persisted for 4 h in 30-h oocytes compared to 2 h in 36-h and 24-h oocytes. The period of DNA synthesis lasted for a total of 12-14 h in all groups of oocytes, and the duration of S-phase was less than 6 h. Since rates of pronuclear formation (58%) and labeling (58%) corresponded to each other, it is argued that only a fully developed pronucleus can synthesize DNA. Oocyte labeling performed in the presence of CHX revealed the capability of CHX to inhibit DNA synthesis up to 8 h postactivation. Removal of CHX by washing when the majority (94%) of oocytes had formed a fully developed pronucleus (at 8 h postactivation) led to the synchronous start of DNA synthesis within 1.5-2 h post-CHX culture. This concomitantly defined the time required for synthesis of vital proteins needed for the entry into S-phase and/or DNA replication. The prolonged exposure of activated oocytes to CHX (10-12 h) negatively affected the pattern of DNA synthesis. The start of DNA synthesis was postponed and reduced pronuclear labeling was observed. In addition, CHX-treated oocytes often exhibited a characteristic punctate pattern of pronuclear labeling in which silver grains were accumulated into clusters. In conclusion, the present results provide knowledge about timing and a possible synchronization of DNA synthesis in parthenogenetically activated bovine oocytes.
Soloy
E
E
Department of Clinical Studies, Reproduction, Royal Veterinary and Agricultural University, Frederiksberg C, Denmark.
Kanka
J
J
Viuff
D
D
Smith
S D
SD
Callesen
H
H
Greve
T
T
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010312:Parthenogenesis / Q000502:physiology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism
D011500:Protein Synthesis Inhibitors; D004247:DNA; D003513:Cycloheximide; D013936:Thymidine