【作者】 王立民；

【导师】 刘东军；

【作者基本信息】 内蒙古大学 ， 动物学， 2008， 博士

【摘要】 胰岛素样生长因子(insulin-like growth factor,IGF)和一些与它相关的成分构成了一个复杂的生长因子家族,它们对早期胚胎发育的调节起着重要的作用。本研究主要通过RT-PCR方法和免疫荧光标记检测了IGF-Ⅰ、Ⅱ和IGF-ⅠR、ⅡR在牛卵母细胞、早期胚胎中的表达情况;在成熟培养液和发育培养液中添加不同剂量的IGF-Ⅰ、Ⅱ,观察其对卵母细胞和胚胎发育的影响;采用RNAi的方法进一步探讨了IGF-ⅠR在牛早期胚胎发育中的作用。一、IGF-Ⅰ、Ⅱ和IGF-ⅠR、ⅡR mRNA在牛卵母细胞和早期胚胎中的表达通过RT-PCR的方法检测了IGF-Ⅰ、Ⅱ和IGF-ⅠR、ⅡR mRNA在卵母细胞、早期胚胎中的表达情况。结果显示在卵母细胞和早期胚胎中均检测到IGF-Ⅱ和IGF-ⅠR、ⅡR mRNA的表达;但是始终未检测到IGF-ⅠmRNA的表达。二、IGF-Ⅱ和IGF-ⅠR、ⅡR在牛卵母细胞和早期胚胎中的表达本研究首次对IGFs蛋白在牛卵母细胞和早期胚胎中的表达进行了定位。通过免疫荧光标记和激光共聚焦观察检测IGF-Ⅱ和IGF-ⅠR、ⅡR蛋白在卵母细胞、早期胚胎中的表达情况。结果表明IGF-Ⅱ和IGF-ⅠR、ⅡR的表达情况基本一致:它们在卵母细胞和二细胞期胚胎中主要集中在细胞边缘如膜及膜下区域;并且存在于COC的颗粒细胞中,以及8细胞期胚胎和桑椹胚的整个卵裂球细胞质中;在囊胚的滋养层细胞中可以检测到这些蛋白,但是在内细胞团中没有检测到。三、IGF-Ⅱ对牛卵母细胞成熟的影响本研究首次观察了在牛卵母细胞成熟培养液中添加IGF-Ⅱ对胚胎发育的影响。将采集到的COCs随机平均分成五组,分别成熟培养于含有0(对照)、10、20、50、100 ng/ml IGF-Ⅱ的M199+0.6%BSA的成熟培养液中,经过成熟培养、受精和发育培养后,统计胚胎的发育率和囊胚凋亡细胞数。结果表明各添加组与对照组之间对卵裂率的影响没有显著差异。但是在8细胞胚胎发育率上,20 ng/ml添加组(58.2%)明显高于对照组(44.5%)(P＜0.05);而且在囊胚发育率方面,20 ng/ml添加组最高(37.0%),明显高于其他各组(25.0%,33.3%,24.8%,21.2;P＜0.05)。20 ng/ml添加组的囊胚细胞数(126)也明显高于对照组(103,P＜0.05),但是囊胚细胞中的死细胞比率在各组间没有明显差异。四、IGF-Ⅰ和IGF-Ⅱ对胚胎发育的影响1.添加IGF-Ⅰ对胚胎发育的影响在SOF+PVA培养液中添加不同剂量的IGF-Ⅰ对早期胚胎进行发育培养,培养结果显示,添加0、1、5和10 ng/ml IGF-Ⅰ的8细胞期胚胎发育率和囊胚发育率分别为55.1%、55.1%、62.9%、60.9%和18.9%、19.6%、24.2%、22.7%,结果表明8细胞期胚胎发育率与囊胚发育率在各组之间无显著差异,5 ng/ml组发育效果最好。2.IGF-Ⅱ对早期胚胎发育的影响本研究首次观察了在发育培养液中添加IGF-Ⅱ对胚胎发育的影响。在SOF+PVA培养液中添加不同剂量的IGF-Ⅱ对早期胚胎进行发育培养,发育数据显示,添加0、50、100和150 ng/ml IGF-Ⅱ的8细胞期胚胎发育率和囊胚发育率分别为63.6%、68.9%、61.3%、63.9%和19.2%、21.7%、30%、25.9%,结果表明8细胞期胚胎发育率在各组之间无显著差异;100 ng/ml组囊胚发育率明显高于对照组,有显著性差异(P＜0.05)。五、RNAi干涉IGF-ⅠR对胚胎发育的影响1.IGF-ⅠR siRNA的设计与干涉效果检测本研究首次把siRNA用于牛胚胎发育的研究。利用软件设计siRNA干涉序列,并对合成回来的序列进行干涉效果检测。对未成熟卵母细胞注射IGF-ⅠRsiRNA和对照siRNA,然后在加有成熟抑制剂的M199培养液中培养,分别于培养6小时和24小时后对IGF-ⅠR mRNA进行定量PCR检测。结果显示注射6小时和24小时后IGF-ⅠR mRNA表达量分别降为72%和54%。对受精卵注射IGF-ⅠR siRNA,于培养24小时后检测卵裂胚的mRNA和蛋白表达,发现均有所减少。结果表明,本实验所设计的IGF-ⅠsiRNA具有良好的干涉效果,可以用于IGF-Ⅰ基因敲减的研究。2.IGF-ⅠR siRNA注射对早期胚胎发育的影响注射IGF-ⅠR siRNA的受精卵经发育培养,于受精后第八天统计发育率和囊胚细胞数;未注射组、注射对照siRNA组、注射IGF-ⅠR siRNA组的8细胞期胚胎发育率、囊胚发育率分别为48.8%、46.7%、38.9%和35.4%、33.0%、23.3%;各组囊胚细胞数分别为:116、81、64。结果表明,各组在8细胞期胚胎发育率方面没有显著差异,但是在囊胚发育率方面注射IGF-ⅠR siRNA组明显低于两对照组,有显著差异(P＜0.05);在囊胚细胞数方面,注射后的囊胚细胞数明显低于未注射组(P＜0.05),注射对照siRNA组囊胚细胞数明显比注射IGF-ⅠsiRNA组囊胚细胞数多,具有显著差异(P＜0.05)。研究结果表明,在牛卵母细胞和早期胚胎中有IGF-Ⅱ和IGF-ⅠR、ⅡR mRNA和蛋白的存在,但是无法检测到IGF-ⅠmRNA。在成熟液和发育液中添加IGF-Ⅱ可以提高胚胎发育率,而且在成熟液中添加IGF-Ⅱ可以增加囊胚细胞数,但是对于凋亡细胞数没有影响。通过注射IGF-ⅠR siRNA发现,IGF-ⅠR siRNA显著降低了胚胎发育率和囊胚细胞数。更多还原

【Abstract】 Insulin-like growth factors and its relatives constitute a family of growth factors that might be implicated and important in growth regulation of early embryos.This study aimed to assess expression and location of insulin-like growth factorⅠ(IGF-Ⅰ), insulin-like growth factorⅡ(IGF-Ⅱ) and their receptors insulin-like growth factor receptor typeⅠ(IGF-ⅠR),insulin-like growth factor receptor typeⅡ(IGF-ⅡR) in bovine oocytes and preimplantation embryos by using RT-PCR and immunofluorescence label technique.IGF-Ⅰ/Ⅱsupplemented in maturation and development medium with different concentration were used to evaluate their effects on oocyte maturation and embryo development.The function of IGF-ⅠR on preimplantation embryo development was further discussed by RNA interference (RNAi). 1.Expression of IGF-Ⅰ/Ⅱand IGF-ⅠR/ⅡR mRNA in bovine oocyte and preimplantation embryoIGF-Ⅰ/Ⅱand IGF-ⅠR/ⅡR mRNA expression in oocytes and preimplantation embryos was detected by using RT-PCR.The results indicated that there was expression of IGF-Ⅱand IGF-ⅠR/ⅡR mRNA in immatured oocytes,matured oocytes and embryos.The IGF-ⅠmRNA was not detected in any stage of embryos and oocytes.2.Expressions of IGF-Ⅱand IGF-ⅠR/ⅡR protein in bovine oocytes and preimplantation embryosThe protein’s localization of IGF-Ⅱ,IGF-ⅠR and IGF-ⅡR in bovine preimplantation embryo were first observed in this research.The results showed that expression of IGF-Ⅱand IGF-ⅠR/ⅡR was detected in immatured and matured oocytes,cumulus cells,fertilized oocytes and different stage preimplantation embryos.The expression patterns of IGF-Ⅱand IGF-ⅠR/ⅡR in embryos were similar. They were near the membrane in oocytes and 2-cell stage embryos,and in the whole cell in COC’s cumulus,8-cell stage embryos and morula,and they can be observed in trophectoderm(TE) cells but not in inner cell mass(ICM) at the blastocyts stage.3.Effects of IGF-Ⅱon bovine oocyte maturationThe effect of IGF-Ⅱsupplemented in maturation medium on embryo development was observed first in this research.COCs were cultured in M199+ 0.6%BSA supplemented with 0,10,20,50 and 100ng/ml IGF-Ⅱrespectively for maturation.After in vitro fertilization and culture,the rates of embryo development and the dead cells in blastocysts were counted.The results indicated that there was no significant difference in cleavage rates between treated groups and control. There was a significant difference in 8-cell stage embryo development rates between 20ng/ml treatment(58.2%) and control(44.5%,P＜0.05).And the best blastocyst development rate was obtained in 20ng/ml treatment(37.0%) compared with others(25.0%,33.3%,24.8%,21.2;P＜0.05).Cell number of blastocysts in 20 ng/ml treatment(126) was significant higher than that in control(103,P＜0.05). There was no significant difference on the rates of dead cells in blastocysts in different treatments.4.Effects of IGF-Ⅰand IGF-Ⅱon bovine preimplantation development4.1 Effects of IGF-Ⅰon bovine preimplantation embryo developmentEmbryos derived from IVF were cultured in SOF+PVA added with 0,1,5,10 ng/ml IGF-Ⅰrespectively.The results showed that the rates of 8-cell stage embryos and blastocyst were 55.1%,55.1%,62.9%,60.9%and 18.9%,19.6%,24.2%,22.7% respectively.There was no siginificant difference between different treatments.The best development rate was observed in 5ng/ml treatment.4.2 Effects of IGF-Ⅱon bovine preimplantation embryo developmentThe effect of IGF-Ⅱsupplemented in developmental culture medium on embryo development was observed first in this research.Embryos derived from IVF were cultured in SOF+PVA added with 0,50,100,150ng/ml IGF-Ⅱrespectively.The rates of 8-cell stage embryo and blastocyst were 63.6%,68.9%,61.3%,63.9%and 19.2%, 21.7%,30%,25.9%respectively.There was no significant difference on rates of 8-cell stage embryo between different treatments.The rate of blastocysts in 100 ng/ml treatment was higher than that in control(P＜0.05).5.Effects of IGF-ⅠR siRNA injection on bovine preimplantation embryo development5.1 IGF-ⅠR siRNA design and effect detectionAs the first time,siRNA was used on bovine embryo development in this research.IGF-ⅠR siRNA designed by software was used to detect the interference effect.Immatured oocytes were injected with IGF-ⅠR siRNA or control siRNA,and then cultured in M199 added with maturation inhibitor.After 6 and 24hours,the IGF-ⅠR mRNA was detected by real-time PCR.The result showed that the IGF-ⅠR mRNA reduced to 72%and 54%.The mRNA and protein reduced both in cleavage embryos injected with IGF-ⅠR siRNA 24hours later.4.2Effects of IGF-ⅠR siRNA injection on preimplantation embryo developmentThe fertilized oocytes with no injection(control) or injected with control siRNA and IGF-ⅠR siRNA were cultured in culture medium respectively.The rates of 8-cell stage embryo and blastocyst in the all treatments were 48.8%,46.7%, 38.9%and 35.4%,33.0%,23.3%respectively;and the cell number of blastocysts were 116,81,64 respectively.The results showed that there was no significant difference on 8-cell satge embryo rates among them.Blastocyst rates of IGF-ⅠR siRNA injected treatment was siginificant lower than that of other two treatments (P＜0.05).Cell number of blastocysts in no injected treatment was significant higher than that of injected treatment(P＜0.05),and it was significant higher in injected IGF-ⅠsiRNA treatment than that of injected control siRNA treatment.The results in this research suggested that the expression of IGF-Ⅱand IGFIR/ⅡR mRNA and protein were detected in bovine oocytes and preimplantation embryos.But IGF-ⅠmRNA could not be detected.Added IGF-Ⅱin maturation and culture medium could improve the rates of embryo development.The cell number of blastocyst was higher when IGF-Ⅱwas added in maturation medium,but there was no difference on the rate of dead cell in blastocyst.Embryo development rates and cell number in blastocysts were decreased by IGF-ⅠR siRNA injection.更多还原