Stage-Dependent Effects of Inhibiting Ribonucleic Acids and Protein Synthesis on Meiotic Maturation of Bovine Oocytes In Vitro

Abstract

Ribonucleic acid synthesis continues at a low rate within 1h of germinal vesicle breakdown. To determine if this newly synthesized mRNA is required for the resumption of meiosis in cattle oocytes, cumulus-enclosed oocytes were removed from 2 to 4-mm antral follicles directly into Dulbecco's medium with or without the RNA inhibitor, α-amanitin, or the protein synthesis inhibitor, cycloheximide (10μg/ml). They were washed twice and matured for 28 or 48h in medium 199, with Earle's salts, 2.2g/L NaHCO3 and L-glutamine supplemented with 20% heated fetal calf serum to which were added gonadotropins (10μg/ml NIH-LH-S18; 10μg/ml NIH-FSH-S8; 20ng/ml NIH-P-S9), estradiol-17β (1.5μg/ml), solcoseryl (30 μl/ml), and Dibekacin sulfate (100μg/ml) with or without the inhibitors. After 28h of maturation, 95.8% of control oocytes had undergone germinal vesicle breakdown and formation of a metaphase plate compared with only 1.3% of those continuously exposed to cycloheximide and 38% of those continuously exposed to amanitin. Exposure to cycloheximide or amanitin for only the first 16h of culture followed by 12h of inhibitor free culture resulted in 96.6% germinal vesicle breakdown with cycloheximide but only 56.5% germinal vesicle breakdown with amanatin. We conclude newly synthesized mRNA and protein synthesis is required for both full cumulus cell expansion and germinal vesicle breakdown.