RNA Interference As A Tool To Study The Function Of MAD2 In Mouse Oocyte Meiotic Maturation

Spindle checkpoint proteins control entry into anaphase and chromosome correct segregation. Spindle checkpoint proteins including main components(MAD1, MAD2, MAD3/BUBR1, BUB1, BUB3 and MPS1), kinetochore proteins(NDC80, ROD/ZW10, AME1 and OKP1),microtubule motors(CENP-E, DYNEIN and MCAK/XKCM1),effectors(CDC20, CDH1 and APC/C)and other players(Ipl1/Aurora kinase, Survivin, APC, MAPK, CMT2, Polo-like kinase, histone and P53). Loss of these proteins may induce genomic instability and cancer, so they have received the most attention. Previous research proved that MAD2 is the most direct inhibitor of APC or CDC20. RNA interference (RNAi) has become a well-established technique to study gene functions in several species. Our objectives were to develop an RNAi approach to study MAD2 gene functions in mouse oocytes meiosis and maturation.Objectives: Our objectives are to construct real-time PCR method for the quantitative analysis of MAD2 mRNA level in mouse oocyte using SYBR GreenⅠas reporter dye and use it to study MAD2 mRNA level in mouse meiosis maturation in vitro. Here we used MAD2 siRNA transfection approach to study MAD2 functions during mouse oocyte meiotic maturation in vitro.Methods: We have constructed real-time PCR method for quantitative analysis MAD2 mRNA level in mouse oocyte using SYBR GreenⅠas reporter dye,β-ACTIN as inner reference gene and recombined plasmid as standard curve template. We have analysed MAD2 mRNA level in mouse oocyte meiosis and maturation. MAD2 siRNA was transfected to mouse oocyte. Quantitative real-time PCR and confocal were used to analyse the changes of mRNA and protein level, and finally combined with morphological and cell apoptosis method to study MAD2 functions during mouse oocyte meiotic maturation in vitro.Results: We constructed a specific, sensitive quantitative real-time PCR method to analyse MAD2 mRNA level in mouse oocyte, and the amplification efficiencies for both MAD2 andβ-ACTIN were near 100%. We studied MAD2 mRNA level in mouse oocyte meiosis and maturation. Real-time PCR and laser scanning confocal microscopy showed that we have successfully down-regulated MAD2 transcript and protein expression. We further demonstrated that MAD2 down-regulation resulted in meiotic spindle abnormality and a shortened duration of meiosisⅠ, suggesting the function of MAD2 in mouse oocyte meiotic maturation. We also showed that MAD2 interference to some extent decreased the rate of GVBD, but increased apoptosis in mouse oocytes.Conclusions and Significance: In conclusion, our data suggest that quantitative real-time PCR using SYBR GreenⅠas reporter dye would be an ideal method for rapid analysing mRNA level. RNAi studies suggest that siRNA transfection is an effect tool to study MAD2 functions, and our results provide further evidence for the role of MAD2 as a spindle checkpoint protein in mouse oocytes.