321 OOCYTE DIAMETER INFLUENCES THE MEIOTIC RESUMPTION AND PROGRESSION INDUCED BY OKADAIC ACID IN DOG

Abstract

The acquisition of meiotic competence, in the bitch as in many other mammalian species, is related to the oocyte diameter. This study was designed to determine the effect of okadaic acid (OA), a potent inhibitor of seronine/threonine 1 and 2A phosphatases, on meiotic resumption and progression in canine oocytes with different diameters. In two experiments, healthy cumulus-oocytes complexes were collected from ovaries of bitches at various stages of the estrous cycle and divided, by diameters, into three treatment groups for in vitro maturation: <110 ¼m, 110-120 ¼m, and >120 ¼m. In Experiment 1, oocytes were pre-incubated for 1 h in TCM-199 + 20% estrous canine serum (SCE) + cysteamine + OA (0.5 ¼M). Then, oocytes were cultured for 48 h in the same medium without OA at 38.5°C, 5% CO₂ in air. As a control group, oocytes were matured in vitro under the same conditions but without pre-incubation with OA. In Experiment 2, to determine if the effect of OA is mediated by cumulus cells, >120 ¼m oocytes were denuded from cumulus cells, incubated with or without OA, and cultured in vitro as previously described. At 48 h, all oocytes were stained and fixed with glycerol-Hoechst 33342 to assess the stage of meiotic maturation. In Experiment 1, OA induced a significantly higher incidence of meiotic resumption in oocytes <110 ¼m (16/108, 14.8%; P < 0.05) and 110-120 ¼m (70/130, 53.8%; P < 0.01) as compared to that of oocytes in the <110 ¼m and 110-120 ¼m control groups (2/58, 3.4%; 24/82, 29.3%). The percentage of oocytes in the 110-120 ¼m OA group that underwent in vitro maturation to metaphase II (MII) was significantly higher than in the 110-120 ¼m control group (18/130, 13.8% vs. 4/82, 4.9%, respectively; P < 0.05). In contrast, smaller oocytes (<110 ¼m) did not develop to MII with or whitout OA. Meiotic resumption rate of >120 ¼m OA group (64/78, 82.0%) was similar to the >120 ¼m control group (56/72, 77.8%), but a significantly higher proportion of the oocytes pre-incubated with OA progressed to MII than did the control oocytes (40/78, 51.3% vs. 12/72, 16.7%, respectively; P < 0.01). Low rates of meiotic resumption were observed in denuded >120-¼m oocytes with (7/63, 11.1%) or without OA (7/55, 12.7%) and none of them progressed to MII. In conclusion, the results of the present study indicate that treatment of fully grown (>120 ¼m) oocytes with okadaic acid at the onset of in vitro maturation can result in a higher frequency of meiotic maturation than previously reported. Also, we determined that the beneficial effect of okadaic acid was mediated by cumulus cells.