Effect Of Jasplakinolide On Bovine Oocyte Meiosis And The Subsequent Embryonic Development

Jasplakinolide (JAS) is an actin-specific reagent that promotes actin polymerization and stabilization and is usually used in the studies of the formation and morphological changes of mitotic apparatus. The knowledge about the effect of JAS on mammalian meiosis is very limited. In the present study, the bovine oocytes were incubated in JAS-containing maturation media at different concentrations, the oocytes were then treated by immunostaining and chromosomal preparation techniques to analyze the effects of JAS on meiosis spindle configuration, chromosome alignment and segregation. The subsequent development of the oocytes treated by JAS after parthenogenetic activation and somatic cell nuclear transfer were also investigated.Experiment 1: Effects of JAS on meiotic maturation of bovine oocytes(1) The results showed that the maturation of bovine oocytes affected by JAS in a dose-dependent manner. When the oocytes were cultured in JAS at 100nM, 200nM, 300nM and 400nM for 24 hr, the maturation rates were 70.3%, 53.1%, 39.7%, and 20.6%, respectively.(2) Pre-treatment of oocytes with JAS significantly affected the oocyte maturation and the formation and morphology of spindles and chromosomes. However, when the oocytes were cultured in normal medium for the first 16 hr and then the oocytes were transfer to JAS-containing media and incubated to 24 hr, there were no obvious changes were observed in the oocyte maturation rates.(3) JAS treatment affected the dynamic changes of the cytoskeleton including microtubules and microfilaments of bovine oocytes. Abnormal spindles such as two spindles, one large spindle and multi-polar spindles occurred. When the oocytes were incubated in 400 nM JAS-containing medium for 16hr, 20hr and 24hr, a higher proportion of oocytes with abnormal spindles were 71.8%, 72.8%, and 64.0%, respectively. JAS treatment of oocytes disorganized the microfilament organization and irregular microfilament distribution was observed.(4) Chromosomal analyses of oocytes showed that treatment of oocytes by JAS altered the chromosomal alignment and homogenous chromosome separation. After IVM of oocytes in JAS media for 24hr multi-ploid oocytes were observed. In the oocytes with no Pb1 extrusion, the most of them maintained at tetraploid state. In particular, 91.9% and 100% of the oocytes incubated in 200nM and 300nM JAS media were tetraploidy, respectively.Experiment 2: The effects of JAS treatment of oocytes on the parthenogenetic and NT embryo development in bovine(1) When the oocytes were matured in JAS at concentrations of 200nM and 400nM, the parthenogenetic activation of oocytes with pb1 resulted in similar cleavage and blastocyst rates, which were significantly lower than the control. The cleavage rate of the oocytes with no pb1, which cultured in 400nM JAS medium, had significantly higher cleavage rate than the oocytes treated by 200nM. The blastocyst rates, however, was similar.(2) Treatment of the oocytes with JAS decreased the development of embryos derived from somatic cell nuclear transfer (SCNT). Much lower blastocyst development were obtained when the JAS-treated oocytes were used as oocyte recipients in SCNT.