Studies On Cortical Granules Behavior And Control Of Meiosis In Mouse Oocytes

Embryo technology (such as in vitro production of embryos and animal cloning) needs large quantities of high quality oocytes. However, the quality of in vitro matured oocytes is generally lower than that of the in vivo matured. The poor quality of in vitro matured oocytes was thought to be due to a premature meiotic resumption without adequate cytoplasmic maturation induced by transfer of oocytes from follicles into a suitable culture medium. The distinguishing features of IVO oocytes that confer greater developmental potential remain obscure. Changes in the distribution of cortical granules (CGs) during oocyte maturation could be used as an important criterion to evaluate cytoplasmic maturation. On the other hand, it is important to learn the mechanism of oocytes maturation for improving the condition of in vitro culture and the quality in vitro matured oocytes. In this study, chronological changes in CG distribution and nuclear progression during maturation and the competence of CG release and embryo development after activation were compared among mouse oocytes that had been matured under different conditions to search for the distinguishing features of IVO oocytes that confer greater developmental potential. And the effect of combination cycloheximide (CHX) with gonadotropin on meiotic maturation of mouse oocytes was also studied. The results are summarized as follows:1. The timing of nuclear maturation differed between IVO and IVM oocytes, and oocytes cultured in WF was closer to the IVO oocytes in meiotic progression than oocytes cultured in MB or MF.2. Six stages of CG distribution were observed during mouse oocyte maturation. Although oocytes underwent similar changes of CG distribution during IVO and IVM, the tempo of changes was much slower in the IVMoocytes than that of the IVO oocytes. The rank order of the 4 treatments in the tempo of CG redistribution was as follows: in vivo, WF, MF and MB.3. Analysis of the relationship between meiotic progression and timing of CG redistribution showed that (a) during both IVO and IVM, the GV stage lasted about 1 h longer than the stage I of CG distribution; (b) the MI stage began 1 h earlier than the initiation of the CGFD formation (stage Ⅲ) during both IVO and IVM; (c) most events lasted markedly longer during IVM than during IVO, leading to a general slowness of both nuclear progression and CG redistribution during IVM. However, the first CGFD (stage Ⅳ) persisted significantly shorter in the IVM oocytes than in the IVO ones.4. Although the cortical localization of CGs continued without GVBD, HX block of GVBD postponed CG migration and prevented the formation of CGFD. CHX facilitated both GVBD and CG migration but it inhibited the formation of CGFD.5. According to the different ability of CG exocytosis upon activation, three types of CG exocytosis were observed: no (NCE,), partial (PCE) and full CG exocytosis (FCE). (a) oocytes matured under different conditions showed different capability of CG exocytosis upon activation, with oocytes matured in vivo > in WF > in MF > in MB; (b) different methods of activation were different in the efficiency of inducing CG exocytosis, with in vivo fertilization > in vitro fertilization > pulsing > ethanol; (c) oocytes matured in the presence of serum showed a higher capability of CG release than those matured in its absence; (d) percentages of nuclear activated oocytes were directly proportional to the percentages of oocytes undergoing FCE; (e) oocytes matured in the absence of serum showed a much more severe and much earlier ZP hardening in comparison with their counterparts matured in vivo or in the presence of serum.6. The ovulated oocytes produced significantly higher rates of activation, cleavage and blastocysts than the IVM oocytes. While activation and cleavage rates were not different significantly between oocytes matured in MF and WF,oocytes matured in WF showed a significantly higher rate of blastocysts than oocytes matured in MR7. When oocytes were cultured in MFHX medium (M199+28.6mg/L HX), their timing of nuclear maturation and CG redistribution, competence of CG exocytosis and embryo development were all similar to those of oocytes cultured in the WF medium(containing 29mg/L HX).8. 10 μ g/ml CHX did not prevent meiotic resumption in mouse cumulus-oocytes complexes(COCs), but it inhibited extrusion of polar body. However, combination 10 μ g/ml CHX with lOIU/ml PMSG inhibited GVBD in mouse COCs significantly, but the inhibitory effect decreased gradually with time.9. FSH in combination with CHX also inhibited GVBD of mouse ooyctes, but LH did not so.10. The inhibitory effects of CHX-PMSG was meditated by cumulus cells and the relationship between cumulus cell and oocytes was required.11. When returned to normal culture after 3h of CHX-PMSG treatment, the meiotic progression of oocytes was accelerated, maturation of oocytes was not affected; but maturation rate decreased significantly after 12h of CHX-PMSG treatment. This result indicated that the inhibitory effect of CHX-PMSG was reversible, but long exposure to CHX-PMSG may cause damage to oocytes.12. In presence of 10IU/ml PMSG, at least 5 μg/ml CHX was needed to inhibit GVBD of mouse COCs efficiently. The addition of 0.5IU/ml PMSG to medium containing 10 μ g/ml CHX result in significant inhibition of GVBD in mouse COCs.