Abstract: 【Objective】 This study is designed to ascertain the expression and localization of Cdc42 protein during bovine oocyte maturation, and to clarify the effect of Cdc42 inhibition on maturation of bovine oocytes. 【Method】 Bovine oocytes in vitro matured for different times (0-24 h) were collected. Western blotting and immunocytochemical technique were adopted to detect the expression and distribution of Cdc42 protein from oocytes collected at different stages of in vitro maturation. A pair of primers with restriction enzyme sites of KpnⅠand EcoRⅠat the ends of forward primer and reverse primer respectively, was designed based on the mRNA sequence of bovine Cdc42 which was obtained from Genbank (accession No.: NM_001046332.1). The CDS of bovine Cdc42 gene was amplified by PCR, and then ligated with pMD19T vector to construct pMD19T-Cdc42WT, the cloning vector wild type of Cdc42. A pair of site-directed mutagenic primers was used in a PCR to obtain pMD19T-Cdc42T17N, the cloning vector of the dominant negative mutant of Cdc42. Both pMD19T-Cdc42WT and pMD19T-Cdc42T17N were digested by restriction endonuclease to obtain the fragment Cdc42WT (775 bp) or Cdc42T17N (775 bp). The obtained fragments were ligated to the multiple cloning site of pcDNA3.1(+) to construct the prokaryotic expression vectors. The cRNA of Cdc42WT and Cdc42T17Nwas synthesized by in vitro transcription, and then micro injected into the cytoplasm of bovine GV stage oocytes. The maturation rate of injected oocytes was detected. 【Result】 The expression level of Cdc42 protein remained constant over the whole bovine oocytes maturation process, but the distribution pattern varied as the meiosis proceeded. The distribution pattern that Cdc42 protein gathering at the cortex region was hardly found in oocytes of germinal vesicle (GV) stage, but occurred frequently and remarkably in metaphase Ⅰ (MⅠ) oocytes. The distribution pattern that Cdc42 protein gathering at the cortex region nearing meiosis apparatus rather than the other region of cortex was found in the oocytes at the stage of pro-metaphase Ⅰ (pMⅠ), and growingly frequent till to the stage of metaphase Ⅱ(MⅡ). The percentage of the pattern that Cdc42 overlaps to spindle was higher in the oocytes at the stage of anaphase Ⅰ (AⅠ) to telophaseⅠ (TⅠ), but went down when it comes to the MⅡ stage. Fragments of 785 bp were obtained via amplifying Cdc42 gene from the total cDNA of bovine oocytes. The cloning vector of wild type Cdc42 and the dominant negative mutant pMD19T-Cdc42WT and pMD19T-Cdc42T17N accorded with anticipation through the identification of restriction endonuclease digestion and sequence alignment. So bovine wild type Cdc42 gene and mutant Cdc42T17N were cloned successfully. The constructed prokaryotic expression vectors pcDNA3.1 (+) -Cdc42WT and pcDNA3.1 (+) -Cdc42T17N accorded with anticipation through the identification of restriction endonuclease digestion and sequence alignment, RNA fragments of 3079 bases and 987 bases were obtained by in vitro transcription in which the vector pcDNA3.1 (+) -Cdc42WT and pcDNA3.1 (+) -Cdc42T17N were used as the templates. The maturation rate of Cdc42T17N injected group was lower compared with Cdc42WT injected group and non-injected group (P < 0.05). 【Conclusion】 It was concluded that Cdc42 protein might be accumulated in the cytoplasm of oocytes during the follicular development, and was functional at the region of cortex and spindle during miosis of bovine oocyte. The normal activity of Cdc42 was necessary to bovine oocyte maturation and extrusion of first polar body.

Key words: Cdc42, immunocytochamical, dominant negative mutant, in vitro transcription, micro injection