Regulating Effects Of Roscovitine(ROS), Cycloheximide (CHX) And Culture Temperature On Meiotic Resumption Of Goat Oocytes

It is recognized that the developmental capacity of in vitro matured oocytes is markedly lower than that of their in vivo matured counterparts. It is known that oocytes need to undergo cytoplasmic maturation as well as nuclear maturation to become able to support successful development. In vivo, oocytes acquire cytoplasmic maturity after a long series of preparatory processes involving transcription and translation of transcripts during the meiotic prophase. In vitro, however, a premature meiotic resumption without adequate cytoplasmic maturation is induced by transfer of oocytes from follicles into culture medium. Recently, to improve the quality of IVM oocytes, attempts were made to prevent precocious meiotic resumption and allow for adequate cytoplastic maturation in vitro. However, the results of many papers did not improved the development competence of oocytes. One of important reasons is the toxiferous effects of drugs on oocytes. So this method should be improved. Metabolism slowed down when cells were cooled. We therefore hypothesize that culture of oocytes at a lower temperature would maintain meiotic arrest, or at least it would reduce the dose of drugs needed to maintain meiotic arrest. Previous study has shown that roscovitine did not inhibit germinal vesicle breakdown (GVBD) of goat oocytes until the concentration increased to a high dose. However, the development of oocytes inhibited with high dose ROS for a long time decreased. For the first time, we decrease the dose of ROS used for inhibition of germinal vesicle breakdown (GVBD) by culturing oocytes at lower temperatures to formulate an efficient and safe protocol for GVBD inhibition. Cycloheximide (CHX) was proved to be a good inhibitors to block meiotic resumption of oocytes. Studies of CHX on goat ccoytes was few and simple, further studies have not been detected. Goat oocytes were cultured at different temperatures in medium containing different concentrations of roscovitine (ROS) or cultured alone in medium containing different concentrations of CHX. At the end of inhibition, oocytes were either matured or processed for light/confocal microscopy. The matured oocytes were either activated chemically, fertilized in vitro for embryo development or processed for light/confocal microscopy. The results were summarized as follows:1 Culture at 5℃could inhibited meiotic resumption of goat oocytes, and the maturation competence of goat oocytes cultured in M199 containing 25 mM Hepes medium did not decrease, while culture in M2 and mPBS medium impaired oocytes maturation. At the same time, Culture at 5℃didn't affect the GV chromatin configuration of goat oocytes.2 Meiotic arrest was successfully maintained for 24 h with 0, 50 and 200μM ROS at 5, 20 and 38.5 oC, respectively, without impairing the competences of oocytes maturation and activation.3 Following chemical activation, morulae/blastocysts (M/B) rates similar to untreated oocytes were obtained in oocytes that had been inhibited for 24 h at 5 oC without ROS (Protocol 5C) or at 20 oC with 50μM ROS (Protocol 20C) or for 8 h at 38.5 oC with 200μM ROS (Protocol 8h), but no blastulation was observed after oocytes were inhibited at 38.5 oC with 200μM ROS for 24 h.4 Following fertilization, however, while M/B rates similar to controls were achieved in oocytes treated with protocols 5C and 20C, few oocytes inhibited with Protocol 8h developed into morulae, due to a high incidence of polyspermy.5 Changes in GV chromatin configuration were not observed after inhibition with Protocol 5C, but were apparent after inhibition with protocols 20C and 8h, leading to a precocious GVBD during subsequent maturation.6 Cortical granule (CG) migration and the formation of microtubule organizing centers occurred during inhibition and were more obvious in the absence of ROS. Significantly more oocytes inhibited by protocols 5C and 20C than by Protocol 8h completed CG migration after maturation.7 Meiotic arrest was successfully maintained for 24 h with 1μg/ml CHX, and maturation rate of CHX treatment was same to that of controls. Following chemical activation, blastocysts (M/B) rate of the treated oocytes was similar to untreated oocytes.8 Following fertilization, however, few oocytes inhibited with CHX developed into morulae/blastocysts, due to a high incidence of polyspermy.9 Cortical granule (CG) migration occurred during inhibition, but CHX inhibition impaired CG migration, significantly no oocytes inhibited by CHX completed CG migration after maturation.10 CHX inhibition had no effects onα-microtubles and microfilaments of goat oocytes.In conclusion, the amount of ROS can be reduced by culture at lower temperatures and culture at 5 oC was less harmful than culture with high dose ROS and CHX inhibition for GVBD inhibition of goat oocytes. Active cytoplasmic activity under lower temperatures might be utilized to improve oocyte cytoplasmic maturation because desired cytoplasmic maturation happen while their nuclear maturation is inhibited.