| Bovine and ovine both are very important livestock in the field of mammal reproduction study including embryo engineering,animal cloning and transgenic animal production, but the mechanism of bovine and ovine oocyte meiotic maturation is not completely clear. In order to understand the mechanisms of oocyte meiotic maturation better, to establish an in vitro serum-free physiological culture model for bovine and ovine COCs which can mimic the inhibitory follicular environment in vivo, to optimized the culture medium ,to improve the oocyte quality, the possible mechanisms that phosphodiesterase involved in bovine and ovine COCs meiotic maturation were experimented.Experiment 1.Two methods, surface cutting and aspiration, were used for recovering of the usable cumulus-oocyte complexes (COCs) from surface-visible follicles of bovine and ovine ovaries. Bovine and ovine ovaries were collected at the abattoir. The average number of COCs recovered per bovine ovary was 12 (1944 oocytes/162 ovaries) by surface cutting and 4.70 (6312 oocytes /1343 ovaries) by aspiration. The average number of COCs recovered per ovine ovary was 2.56 (10104 oocytes /3952 ovaries) by surface cutting and 1.83 (241 oocytes /132 ovaries) by aspiration. The results indicated that surface cutting is more efficient than aspiration in recovering COCs from bovine and ovine ovary.Experiment 2. The inhibitory action of hypoxanthine (HX), which has been proved to inhibit mouse and pig oocyte spontaneous maturation in vitro, was studied on bovine and ovine COCs in order to establish an in vitro serum-free culture model for bovine and ovine oocyte. Bovine and ovine COCs were cultured in HX medium (serum-free M-199 supplemented with 4mM HX), the control was serum-free M-199 medium. At the end of the incubation, COCs were denuded and oocytes were then fixed and classified according to the status of nuclear maturation. Bovine COCs, when cultured in vitro for 8 and 12 h, the GV% of experiment group was significantly different from the untreated group (P<0.01 and P<0.01 respectively). When ovine COCs were cultured in HX medium (0,2,4,6mM ) for 8 and 12 h in vitro, the GV% of experiments group were not different from the untreated group (P>0.05).The results indicated that HX only inhibit bovine and ovine COCs spontaneous maturation in vitro in the given time, which suggesting that HX might not be a physiological inhibitor of bovine and ovine COCs maturation.Experiment 3. Ovine COCs were cultured in vitro serum-free medium containing different specific PDE inhibitors, i.e. milrinone (PDE3 inhibitor), rolipram (PDE4 inhibitor) and zaprinast (PDE5 inhibitor). At the end of the incubation, COCs were denuded and oocytes were then fixed and classified according to the status of nuclear maturation. The PDE3 inhibitor milrinone but not PDE4 inhibitor and PDE5 inhibitor, prevented spontaneous meiotic maturation in vitro. The results indicated that specific PDE subtype-the type 3 PDE might serve as the physiological inhibitor involved in the maintenance of ovine oocyte meiotic arrest.Experiment 4. The effects of inhibitor of cdk and specific inhibitors of phosphodiesterase on ovine oocyte maturation were studied. Milrinone and Rolipram are specific PDE inhibitors and Roscovitine (ROSC) is a selective inhibitor of the cyclin-dependent kinase. These chemicals were selected to evaluate the effect on meiotic resumption of ovine oocytes for 24h in vitro. Oocytes treated with ROSC for 24h and subsequently allowed to undergo maturation for another 24h in the absence of the inhibitor media but contain FSH (200AU.L-1). Immature ovine cumulus oocyte complexes were recovered from ovaries collected at the abattoir. The dose of inhibitors was50μmol.L-1 in medium. At the end of the incubation, COCs were denuded and oocytes were then fixed and classified according to the status of nuclear maturation. The results revealed that both PDE3 inhibitor Milrinone and cdk inhibitor ROSC prevented oocyte spontaneous meiotic maturation. In contrast, the PDE4 inhibitor Rolipram had no effect. And R...
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