BIOCHEMICAL-STUDIES ON GOAT OOCYTES - TIMING OF NUCLEAR PROGRESSION, EFFECT OF PROTEIN INHIBITOR AND PATTERN OF POLYPEPTIDE-SYNTHESIS DURING IN-VITRO MATURATION

Temporal progression of nuclear events of goat oocytes matured in vitr o was studied by adding a specific inhibitor to the culture medium at different time points, to investigate protein synthesis requirements a nd its pattern during in vitro maturation. Goat cumulus-oocyte complex es (COCs) were matured in vitro in TCM 199, fixed at different time in tervals and stained with orcein to assess nuclear changes. The germina l vesicle (GV) stage was found to be present at 0 h, chromosomal conde nsation stage was observed at 8 h, metaphase I at 12 to 14 h, and meta phase II. was begun after 16 h of maturation and was nearly completed at 24 h. Protein synthesis inhibitor, cycloheximide, blocked oocyte ma turation at germinal vesicle breakdown(GVBD), if added to the maturati on medium between 0 to 4 h, suggesting that protein synthesis is requi red for GVBD. The transition from metaphase I to metaphase II was also protein synthesis-dependent, as observed when cycloheximide was used between 8 to 10 h of culture. When cycloheximide was added from 12 h o f culture onwards, nuclear progression to metaphase II was progressive ly restored, but many chromosomal abnormalities were noted. Changes in the protein synthesis pattern were studied by radiolabeling of oocyte s with [S-35]-methionine at 0,7,12and 24 h of culture, corresponding w ith GV, GVBD, metaphase I and metaphase II stages. A polypeptide of 28 .1 KDa appeared as a major band at the GV stage, and its size decrease d greatly and disappeared after the GVBD stage. Three new polypeptides (35, 36.5 and 39 KDa) appeared at GVBD and were detectable at metapha se II. In conclusion, the synthesis of proteins is required for the ma intenance and transition of goat oocytes from GV to metaphase II durin g in vitro maturation.