Timing of Nuclear Progression and Protein Synthesis Necessary for Meiotic Maturation of Bovne Oocytes1

Abstract

In cows, protein synthesis is required for germinal vesicles breakdown (GVBD). This study examines more closely the need for protein synthesis and the nuclear changes in the bovine oocyte during 24 h of culture. Bovine oocytes with compact and complete cumulus, were washed and incubated in groups of 10 for up to 24 h in 50- .mu.l drops of TCM-199 supplemented with follcle-stimulating hormone (NIAMADD, 0.5 .mu.g/ml), luteinizing hormone (LH) NIAMADD, 5 .mu.g/ml), estradiol-17 .beta. (1 .mu.g.ml), pyruvate (20 .mu.M), and 10% heat-treated fetal calf serum. Medium was overlaid with paraffin oil. Oocytes (n = 891) were fixed at the end of each 3-h interval from 0 to 24 h of culture, or at 24 h after addition of cycloheximide (10 .mu.g/ml at 10 different times during maturation (0, 1, 2, 3, 6, 9, 12, 15, 18, 21 h; n = 175). At each time point, the chromosomal status of oocytes was evaluated, frequencies were computed, and the time spend on each step was determined. The germinal vesicle (GV) was present from 0 to 6.6 h, GVBD at 6.6 to 8.0 h, chromatin condensation at 8.0 to 10.3 h, metaphase I at 10.3 to 15.4 h, anaphase I at 15.4 to 16.6, telophase I at 16.6 to 18.0 h, and metaphase II at 18.0 to 24 h. Cycloheximide blocked oocyte maturation at GVBD, if added from 0 to 3 h; at chromatin condensation, if present from 6 to 24 h; and at metaphase I, when present from 9 to 12 h. When cycloheximide was present from 12 to 24 h an increasing number of oocytes reached metaphase II (19% for 12 h, 36% for 15 h, 51% for 18 h, 90% for 21 h, 94% for 24 h), but many abnormalities were noted: formation of one or two "pronucleus-like" structures with or without polar bodies, multiple metaphases, and fragmentation of chromatin. Therefore, a certain amount of protein synthesis is still needed after 12 h for the majority of ooctyes to complete meiosis I. In conclusion, protein synthesis is needed at four different steps in bovine oocyte maturation.