325 ACTIVITY OF MATURATION-PROMOTING FACTOR (MPF) AND MITOGEN-ACTIVATED PROTEIN KINASES DURING IN VITRO MATURATION OF BUFFALO OOCYTES (BUBALUS BUBALIS)

Abstract

The maturation promoting factor (MPF) and mitogen-activated protein kinases (MAPK) are the key regulators of both meiotic and mitotic cell cycles. The absence of data on the activity of the major cell cycle kinases in buffalo oocytes during meiotic progression provided the bases for this study. More specifically we assayed the MPF and MAP kinase activity of buffalo oocytes during meiosis. Abattoir-derived cumulus-oocyte complexes (COCs) with a compact, non-atretic cumulus and a homogeneous cytoplasm were utilized for the study. The COCs (n = 293, over four replicates) were matured in vitro in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 0.5 ¼g/mL FSH, 5 ¼g/mL LH, 1 ¼g/mL 17²-estradiol, 50 ¼M of cysteamine, and 50 ¼g/mL kanamycin (B199). In vitro maturation (IVM) was carried out at 38.5°C under a controlled gas atmosphere of 5% CO₂ in humidified air. At scale times during the culture (0, 3, 6, 9, 12, 15, 18, 21, 24 h) groups of oocytes were stained with Hoechst 33342 to assess chromatin configuration and stored according to the maturation stage (GV, GVDB, MI, and MII) at -80°C pending protein analysis. SDS-polyacrylamide gel electrophoresis wase performed using Laemmli discontinuous buffer system (Laemmli 1970 Nature 227, 680) with a 12% running gel. Groups of oocytes were analyzed for MPF activity (n = 65) by histone H1 kinase activity (Naito and Toyoda 1991 J. Reprod. Fertil. 93, 467-473) and for MAPK activity (n = 48) by myelin basic protein assays (Chesnel et al. 1995 Biol. Reprod. 52, 895-902). The activity of both MPF and MAP kinases was quantified by measuring the density of the bands on the autoradiographic film with a densitometer. Differences in the levels of the kinases among groups were analyzed by ANOVA. It was assumed that the value of MPF and MAPK was 100% in metaphase II (MII) stage oocytes. The lowest levels of MPF and MAPK activities were found in the oocytes at the GV (0-6 h post-IVM: 40% and 17.2%, respectively) and at the GVBD (6-9 h post-IVM: 41.2% and 18%) stages. The activities increased at metaphase I (MI) stage (9-15 h post-IVM) and at MII (21-24 post-IVM). Interestingly, although similar levels of MAP kinases were found at MI and MII stages (95.1% vs. 100%), MPF levels were significantly lower (P < 0.01) at the MI stage compared to those detected at MII (82.8% vs. 100%). The fluctuations of the MPF levels in buffalo appear different compared to those observed in other species; in particular, no differences were recorded between the GV and the GVBD stages whereas a significant increase of the MPF levels was found at MII compared to the MI stage. It seems that MPF and MAPK could differently guide meiotic resumption and progression to the MII arrest in this species. To our knowledge, this is the first report on biochemical analysis of the cell cycle regulation in buffalo oocytes.