Expression And Function Of Aurora-B During Meiosis And First Cleavage After ICSI In PIG Oocytes

The oocyte meiotic and early embryonic development in vertebrates were regulated by a variety of protein kinases. In recent years, research on molecular mechanism of oocyte maturation, activation, and fertilization has been made great progresses. Aurora-B is widely distributed in eukaryotic organisms and there is evidence that Aurora kinases also play a key role in the process in mammalian oocyte meiotic maturation and activation. In mammals, three Aurora kinase homologous proteins have been found:Aurora-A,-B and-C respectively. They have similar structure that relatively conservative end of variable length N and C terminal, but these three homologous proteins have different protein expression, subcellular localization and enzyme activation time. Aurora-B is a member of serine/threonine protein kinase family. Due to the Aurora-B kinase excessive expression in many solid tumors, which have the effect of oncogene, thus Aurora-B in cancer is often as one of the important targets for therapy. Recent studies have shown that Aurora-B plays an important role in the related signal pathway of oocyte meiotic process and early embryonic development.Mammalian animal oocytes block and recovery mechanisms in meiosis period and the development of fertilized eggs, especially the 1-cells to 2-cell transformation mechanism in the first embryo mitosis stage is currently one of the highlights in agro-scientific research in the reproductive and developmental biology. Research on Aurora-B on the regulation of pig oocyte meiosis and embryo first mitotic division, has the vital significance for understanding the mechanism and controlling of reproduction. Although now in Aurora-B regulation oocyte meiosis process and related signal pathways have made some achievements, the study of Aurora-B location and the function in animal oocyte meiosis and early embryonic development is less.Existed research mainly concentrated in the mouse fertilized eggs and Exnopus oocyte, but Aurora-B mRNA level, protein expression and function durying the pig oocyte meiosis and first mitosis in embryo have not been elucidated. Currently, mammalian Intracytoplasmic sperm injection (ICSI) develops very fast, and exert its potential role in many fields.Use ICSI to product embryo in vitro, which can determine the time to observe sperm egg fertilization fusion time and the time of subsequent fertilization events,so ICSI can be a experiment method used in fertilization biology research, and it is the basic research of effective fertilization experiment model.This study will combine ICSI, fluorescence quantitative PCR and immunofluorescence staining techniques together to test Aurora-B mRNA level,the dynamics of protein expression and subcellular localization durying pig oocyte meiosis and the first mitosis in ICSI embryos. Then,on that basis, through Aurora-B specific inhibitors AZD-1152 specific inhibition test, analysis the influence of Aurora-B on porcine oocyte maturation and first mitosis of embryo, provide experimental basis to understand the molecular mechanisms of pig oocyte meiotic and early embryonic development. The experimental results are as follows:Experiment 1. Aurora-B mRNA level detection durying pig oocyte meiosisDurying the process of pig oocyte meiosis, Aurora-B mRNA expression quantity are the highest in 0 h (GV stage), and in 20 h are the lowest; Although, the expression was increased in 30,36 and 44 h, still significantly lower than 0 h (P<0.05). However, the Aurora-B mRNA expression in 30 and 44 h and is higher than 20 and 36 h.Experiment 2. Aurora-B and a-tubulin location durying pig oocyte meiosisIn GV stage, the tube is not assembled, Aurora-B is uniformly distributed in the cytoplasm, didn’t detected its specificity existed. After GVBD, Aurora-B protein and a-tubulin concentration near the chromosome, emerge image shows both coincide. As the meiosis progress to MI, chromosome is allign on the equatorial plate, Aurora-B location anda-tubulin completely overlapped. On late M I, Aurora-B is still located on the spindle. Near to MII stage, Aurora-B protein location was changed with the spindle position, mainly located on the oocyte nucleus and the first polar body.Experiment 3. The effect of AZD-1152 on meiotic maturation and cytoskeleton of porcine oocytesAfter use Aurora-B specific inhibitors AZD-1152, the oocytes blocked in Pro MI-AI-TI increased significantly compared with control group (P<0.05), MII oocytes were significantly lower (P<0.05). At the same time, the proportion of MII oocyte chromosome disorder, spindle shape abnormal, and microfilament fracture and dispersed are increased.Experiment 4. The development of parthenogenetic embryos after inhibit porcine oocyte expression of Aurora-BAfter inhibit Aurora-B expression of pig oocytes, the cleavage rate, blastocyst rate and total number of blastocyst cells in the AZD-1152 treatment groups were significantly lower than control group. And the development of parthenogenetic embryos affected severely with the increasing concentration of AZD-1152.Experiment 5. The observation of first mitosis process of pig ICSI embryosAfter 18h of ICSI reconstructed embryos activated, male and female pronucleus completed fusion, nucleus formation and fertilization completed. In 20 h, sister chromatids began to separate under the action of the spindle fiber. In 22 h, chromatin was locate at the poles of the cell, formed cleavage furrow, cytoplasmic enter into division state. By 24 h, cytoplasmic division finished,and the first mitosis of embryo completed.Experiment 6.Aurora-B mRNA level detection durying the first mitosis of pig ICSI embryosDurying the first mitosis of pig ICSI embryo, the Aurora-B mRNA expression in 20,22 and 24 h were significantly increased when compared with 18 h(P<0.05). And reached the highest at 24 h, significantly higher than other groups. Compared with 20h, Aurora-B mRNA expression level decreased in 22h, but no significant difference (P>0.05)Experiment 7. Aurora-B and a-tubulin location durying the first mitosis of pig ICSI embryosAt 18 h, zygote in the first embryonic mitotic division, the Aurora-B expression is higher near the polar body. In 20 h, cells in anaphase, Aurora-B protein location overlap with spindle position. In 22 h, Aurora-B protein location overlap with chromosome position. In 24 h, cells has entered into telophase, the Aurora-B location overlaped with the microtubule.Experiment 8. The effect of reduce Aurora-B expression on the the first mitosis of pig ICSI embryosUsing specific inhibitor AZD-115 inhibit Aurora-B expression,we found that with the increase of inhibitor concentration, the single nucleus of pig ICSI embryos formation rate decreases. And the effect on the separation of cytoplasmic inhibitory is more apparent, with the increase of the inhibitor concentration, treatment group’s cleavage rate decreased significantly compared with the control group.Experiment 9. Aurora-B, Mad2 mRNA level detection after inhibit Aurora-B expression durying the first mitosis of pig ICSI embryosAfter using AZD-1152 inhibits the expression of Aurora-B, Aurora-B and Mad2 mRNA expression levels of ICSI embryos were decline in varying degrees. Compared with the control group,20 and 24 h treatment groups embryonic Aurora-B mRNA expression levels decreased significantly. Mad2 mRNA expression levels of the control group in 18,22 and 24h have little difference, but the Mad2 mRNA of 20h group are the highest. The expression of Mad2 mRNA levels in treated embryos are lower than those of the control group, the 20h group was the lowest.