Study Of The Function Of Cdh1Gene On The Gvbd Of Bovine Oocytes

The activation of MPF (CDK1-CyclinB1) plays an important role during the progress ofGV-arrest and resumption of meiosis. It has reported that Cdh1gene played an important rolein the GV-arrested mouse oocytes. The oocytes were arrested at GV stage becauseAPCCdh1-mediated CyclinB1degradation prevented CyclinB1from accumulating andactivating CDK1. In this study, the function of Cdh1gene on the GVBD of bovine oocyteswas explorated.In this study, Cdh1gene was cloned from cDNA sample of bovine cumulus cells andpVenus-Cdh1recombinant plasmid was constructed. And then, the progress of GVBD ofbovine oocytes released from Roscovitine (ROS) was studied. Finally, Cdh1-Venus cRNAwas microinjected into bovine oocytes to observe the localization of Cdh1protein and studythe effect of Cdh1gene on the GVBD of bovine oocytes. The results were as follows:(1) Full-length sequence of Cdh1gene CDS was cloned by RT-PCR from bovinecumulus cells. The sequence of Cdh1gene was compared with that published on NCBI andthe result showed that the homology was100%. The pVenus-Cdh1recombinant plasmid wasconstructed by connecting the Cdh1gene to the pVenus eukaryotic expression vector.(2) The oocytes were cultured in maturation medium supplemented with50μM ROS for14h. Then the oocytes were fixed at0h,1h,2h,3h and4h, respectively, after the oocyteswere released from ROS. The oocytes of control groups were cultured in maturation mediumwithout ROS and were fixed at0h,2h,4h,6h and8h, respectively. The oocytes werestained for assessment of nuclear stage and the GVBD rates were obtained. The resultsshowed that the GVBD progress of bovine oocytes released from ROS were acceleratedapproximately4h compared with control groups.(3) Cdh1-Venus cRNA was microinjected into bovine oocytes to observe the localizationof Cdh1-Venus protein in oocytes of different stages. The results showed that Cdh1-Venusprotein was localized at germinal vesicle in GV-arrested bovine oocytes and at the region ofchromosome and spindle after GVBD.(4) The oocytes of control groups were microinjected with Venus cRNA and the oocytesof experimental groups were microinjected with Cdh1-Venus cRNA. The oocytes werereleased from ROS and fixed at1h,2h and3h, respectively. The oocytes were stained forassessment of nuclear stage and the percentage of GV-stage oocytes were obtained. The results showed that the percentage of GV-stage oocytes in experimental groups was higherthan control groups, respectively. The two groups showed significant differences at2h and3h, respectively, indicated that over expression of Cdh1gene delayed the GVBD of bovineoocytes.The conclusion of this study was as follows: the GVBD progress of bovine oocytesreleased from ROS were accelerated approximately4h; Cdh1protein was localized atgerminal vesicle in GV-arrested bovine oocytes and at the region of chromosome and spindleafter GVBD; over expression of Cdh1gene delayed the GVBD of bovine oocytes.