Effect Of CDC20on PBE Ⅰ And Cleavage In Bovine Oocytes Cultured In Vitro

CDC20(cell division cycle20), also named IPl, FZY or p55CDC, is an activatorof E3Ubiquitin ligase which can ubiquit securing and cyclinB1. CDC20is also thetarget of spindle checkpoint, which functions during the precise separation ofchromosomes, and it is tightly related to cell cycle. SAC can preventmetaphase-anaphase transition by means of transporting inhibitive signals toAPC/C^CDC20until all chromosomes precisely attach to spindle and align to equator.SAC was inactived when all chromosomes precisely attach to spindle, at the sametime securing and cyclinB are ubiquitied which was mediated by APC/C^CDC20anddegradated by26s proteasome. The degradation of securing and cyclinB is critical forcell cycle and chromosome separation. Cohesion is split opened by separase which isactived via degradation of securing. The degradation of cyclinB in anaphase leads tocell cycle dependent kinas (CDK1) inactive. During the metaphase-anaphasetransition, securing is ubiquted by APC/C and its activator CDC20. Securing is theinhibitor of separase, Scc1subunit of cohesion is split opened by separase whensecurin is degredated. CDC20is not only the target of SAC, but also the activator ofAPC/C. Consequently, the abnormal expression of CDC20inevitably will cause errorsduring meiosis division and derangement in cell cycle.This study used bovine oocyte as object, investigate the function of CDC20during meiotic division. Firstly, the expression of cdc20in different phase is detectedvia realtime PCR. Secondly, cdc20gene of bovine was cloned and eukaryoticexpression vector pcdc20-venus was constructed. cdc20-venus mRNA which usinglinearized pcdc20-venus as template was obtained through in vitro transcription.Meanwhile the CDC20morpholino antisense oligos (CDC20MO) was synthesizedfor down-regulating cdc20. cdc20was over-expressed or down regulated bymicroinjecting cdc20-venus mRNA or CDC20MO. Via the over expression and downregulation of cdc20in bovine oocytes, we conclude as follows:1. Via real time PCR detection cdc20mRNA levels, we found that cdc20mRNAexists in oocytes during the whole meiosis. The level of mRNA set up from GV stage to pre-MⅠstage and peaked in pre-MⅠstage, and is in the low point at2-cell stage.2. Total RNA was extracted from bovine fibroblast cells, and cdc20gene was clonedvia RT-PCR, then eukaryotic expression vector pcdc20-venus was constructed.Hela cells were transfected with pcdc20-venus mediated by Liposome2000, thegreen fluorescence distribute around the nuclear.3. pcdc20-venus was linearized and pcdc20-venus mRNA was obtained via in vitrotranscription. Once pcdc20-venus was injected into oocytes, the allothigenousCDC20protein accumulated in oocytes with the passage of time. The proteinrapidly synthetized during1-2h, slowly synthetized during2-4h, and reachedstationary phase at about4h.4. When the pcdc20-venus mRNA was microinjected into cytoplasm of bovineoocytes, it has no significant effect on the first polar body extrusion (PBEⅠ)(P＞0.05). Oocytes cultured8-10h was microinjected with CDC20MO. The resultsshowed that the rate of PBEⅠsignificantly reduced(P<0.01), but no significantdifference between control MO injection group and control group(P＞0.05).Immune fluorescent staining showed that severely impaired spindles appeared inoocytes injected with CDC20MO but not appeared in control MO group andcontrol group.5. In addition, pcdc20-venus mRNA was microinjected into oocytes with first polarbody, the fluorescence intensity in nuclear of each blastomere was higher than theintensity in cytoplasm. The statistics results showed that there was no significantchange in cleavage rate (P＞0.05). Then CDC20MO was injected into oocytewith first polar body, and the cleavage rate showed no significant change (P＞0.05).