【作者】 闫真；

【导师】 旭日干；

【作者基本信息】 内蒙古大学 ， 动物学， 2009， 博士

【摘要】 大量研究证明,哺乳动物的细胞间通讯在胚胎着床前已经建立。间隙连接通道是相邻细胞之间唯一可以直接进行信息交流的细胞通讯途径。间隙连接蛋白基因(connexins)作为组成间隙连接通道的基本单位,在哺乳动物早期胚胎发育过程中广泛存在。由于connexins的多样性和时空表达的特异性,到目前为止关于相关特定基因在胚胎发育早期过程中的功能知之甚少。本研究主要采用RT-PCR、Real-time PCR、免疫组织化学和Westernblot等分子生物学技术,结合体外受精技术,检测connexins在绵羊卵母细胞和体外受精早期胚胎中表达的多样性以及connexin43和connexin45的表达规律；同时通过在胚胎单个卵裂球内注入小分子染料,观察间隙连接通讯在绵羊体外受精早期胚胎中的形成时间；并采用RNAi技术,对绵羊体外受精卵进行dsRNA注射,观察connexin43和connexin45在绵羊体外受精胚胎发育过程中的作用。一、RT-PCR方法检测多种间隙连接蛋白在绵羊体外受精胚胎中的表达从NCBI中获取六种间隙连接蛋白基因的cDNA序列并合成引物,用RT-PCR方法获得目的基因的部分cDNA序列,然后进行测序并通过序列比对以确定所获得的cDNA序列的正确性。在此基础上收集绵羊未成熟卵母细胞、成熟卵母细胞、2-细胞、8-细胞、桑椹胚以及囊胚,通过RT-PCR方法检测connexin26、connexin31、connexin32、connexin40、connexin43和connexin45等基因在绵羊未成熟卵母细胞、体外成熟卵母细胞、体外受精2-细胞、8-细胞、桑椹胚以及囊胚中的mRNA表达水平。结果发现,connexin26、connexin32、connexin43及connexin45在绵羊卵母细胞和早期胚胎发育的各个时期均有表达；但未检测到connexin31和connexin40转录子。二、Connexin 43 (Cx43)和connexin45 (Cx45)在绵羊卵母细胞和体外受精早期胚胎中转录子表达规律以及蛋白水平的检测1、Real-time PCR方法检测Cx43、Cx45在绵羊卵母细胞和体外受精胚胎中的表达对Cx43和Cx45在绵羊未成熟卵母细胞、体外成熟卵母细胞、体外受精2-细胞、8-细胞、桑椹胚和囊胚期胚胎分别进行Real-time PCR检测,结果显示：Cx43在未成熟卵母细胞、体外成熟卵母细胞、2-细胞中表达量较低,分别是8-细胞含量的0.11、0.3、0.44倍；到达桑椹胚和囊胚期时达到最高,为8-细胞含量的2.44和2.32倍；而Cx45在8-细胞时表达量最高,未成熟卵母细胞、成熟卵母细胞和2-细胞期的表达量分别为8-细胞期的0.43、0.72和0.82倍,桑椹胚和囊胚期胚胎的表达量分别为8-细胞期的0.93和0.89倍。2、免疫组化方法检测Cx43、Cx45在绵羊卵母细胞和体外受精胚胎中的表达对Cx43和Cx45在绵羊卵母细胞和体外受精胚胎中的表达进行免疫组化检测,结果发现：在绵羊未成熟卵母细胞、体外成熟的卵母细胞、体外受精的2-细胞、8-细胞、桑椹胚和囊胚等各个发育时期的胚胎中,Cx43、Cx45蛋白均有表达,并主要分布在细胞膜区域,在细胞质中分布很少,而在细胞核中未检测到。在对照组体内受精的胚胎中,Cx43和Cx45从2-细胞期到早期囊胚均有表达,两者表达部位均靠近细胞膜区域。体内受精胚胎和体外受精胚胎的表达模式基本一致,而Cx43和Cx45蛋白在体内受精胚胎中的表达量相对高一些。体内受精胚胎中转录子的含量往往高于体外受精胚胎,这是否是导致体内外受精胚胎在质量上有所不同的原因之一,尚有待进一步研究。3、蛋白印迹检测Cx43在绵羊卵母细胞和体外受精胚胎中的表达对绵羊未成熟和体外培养成熟的卵母细胞分别进行蛋白印迹检测结果发现,在未成熟和体外成熟的卵母细胞中Cx43蛋白均有表达,且蛋白电泳表现出两种磷酸化形式(P1和P2)和一种非磷酸化形式(P0)。Cx43在体外成熟卵母细胞中的蛋白表达量要高于未成熟卵母细胞。而在早期胚胎中检测结果,Cx43在2-细胞期、8-细胞期、桑椹胚期和囊胚期胚胎中均得到阳性条带,与免疫荧光染色结果完全一致。免疫印迹在膜上显示出三条带,与Cx43常规表达模式相同。胚胎在2-细胞期和8-细胞期时Cx43蛋白的磷酸化形式表达量略高于非磷酸化形式,到达桑椹胚期时非磷酸化形式含量有所增加。三、绵羊体外受精早期胚胎中间隙连接通讯的检测本实验通过显微注射技术与激光扫描共聚焦观察方法,将小分子荧光染料Lucifer yellow注射到发育到不同时期胚胎的单个卵裂球中,观察荧光染料向其它相邻卵裂球扩散情况。结果发现,注射到2-细胞、4-细胞、8-细胞、16-细胞及桑椹胚期胚胎卵裂球中的染料没有向相邻卵裂球扩散。可以推测,绵羊体外受精早期胚胎在发育早期并未建立间隙连接通讯。四、Cx43dsRNA、Cx45 dsRNA对绵羊体外受精胚胎发育的影响1、Cx43dsRNA、Cx45 dsRNA对目的基因mRNA和蛋白水平的影响将Cx43 dsRNA、Cx45 dsRNA和水分别注入绵羊体外受精卵,与未注射的空白对照组一起继续培养。通过Real-time PCR方法观察到,与对照组相比,当胚胎发育到囊胚期时Cx43mRNA和Cx45mRNA含量分别下降了74%和44%；而注水组的Cx43和Cx45 mRNA含量与未注射的对照组基本一致。同时检测到作为对照的β-actin基因、E-cadherin基因和Catenin基因的mRNA水平则在各组中基本保持一致,表明体外合成的dsRNA并未对其它对照基因mRNA的表达产生影响,能够特异而有效的降低目的基因的mRNA水平。对囊胚期胚胎进行蛋白印迹实验表明,与未注射对照组相比,向体外受精卵中注射Cx43的dsRNA能够使得Cx43dsRNA注射组中的Cx43蛋白含量下降。免疫组织化学方法结果显示,与未注射对照组相比,注射Cx43 dsRNA和Cx45 dsRNA后一定时间后,目的基因的蛋白表达均有不同程度的下降,并能够维持到囊胚期。因此,向绵羊体外受精卵中注射Cx43和Cx45 dsRNA能够使目的基因的mRNA及蛋白含量下降并持续到囊胚期。2、Cx43dsRNA、Cx45 dsRNA对绵羊体外受精胚胎卵裂率的影响。Cx43dsRNA、Cx45 dsRNA注射48小时后胚胎发育情况是：在Cx43 dsRNA处理试验中dsRNA注射组、水注射组和未注射组的总卵裂率分别为83.7%、84.5%和81.5%；其中2-细胞期胚分别为5.6%、4.7%和4.6%,4-细胞期胚分别是14.6%、10.8%和10.3%,8-细胞期胚分别是64.5%、68.9和66.6%；在Cx45处理实验中dsRNA注射组、水注射组和未注射组的总卵裂率分别为81.7%、76.9%和77.5%;其中2-细胞期胚分别为7.6%、3.6%和3.8%；4-细胞期胚分别是13.9%、11.6%和7.2%；8-细胞期胚分别是60.3%、61.8%、66.5%。通过统计学方法分析以上结果可以看出,dsRNA处理组与水注射组和未注射组之间总卵裂率及8-细胞期发育率无显著差异(P＞0.05),表明对Cx43或Cx45基因进行RNA干涉并不影响绵羊体外受精卵的早期发育。3、Cx43dsRNA、Cx45 dsRNA的注射对绵羊体外受精胚胎囊胚发育率的影响。Cx43dsRNA、Cx45 dsRNA注射后体外受精卵的囊胚发育情况是：在Cx43处理实验中dsRNA注射组、水注射组和未注射组的囊胚发育率分别为20.3%、21.7%和34.5%,囊胚孵化率分别是19.2%、37.5%、41.3%,囊胚细胞数分别为76、74、83,囊胚细胞死亡数和死亡率分别为18.7(24.6%)、11.7(15.4%)、15.1(18.2%)；在Cx45处理实验组dsRNA注射组、水注射组和未注射组的囊胚发育率分别为20.8%、19.4%和32.5%,囊胚孵化率分别是18.5%、25.0%和34.9%；囊胚细胞数分别为79、71和72；囊胚细胞死亡数和死亡率分别为16.7(21.1%)、12.7(17.9%)、11.3(15.7%)。通过统计学分析可以看出Cx43 dsRNA处理组的囊胚发育率和囊胚细胞数与未处理组相比差异不显著(P＞0.05)；而囊胚孵化率显著高于未处理组(P＜0.01),且囊胚细胞死亡率也略高于未处理组。Cx45 dsRNA处理组的囊胚发育率、囊胚细胞数、囊胚孵化率与对照组相比差异不显著(P＞0.05)。表明Cx43 dsRNA处理并不能影响绵羊体外受精胚的囊胚发育率,但可能对囊胚质量有一定的影响；Cx45 dsRNA处理并不影响绵羊体外受精胚的囊胚发育率及囊胚质量。更多还原

【Abstract】 There is some evidence that gap junction intercellular communication has been built early before implantation. As the only distinct communication pathway between adjacent cells, connexins exist widely during mammalian preimplantation embryo development. However, the diversity and the temporal and spatial expression of connexins family members make the functional research for a single connexin has been complex and difficult. In this research, RT-PCR, Real-time PCR, immunochemistry and western blot, combined with in vitro fertilization technique, had been employed to detect the diversity of connexins expressed in ovine preimplantation embryos and the expression profiles of connexin43 and connexin45. Second, small molecular fluorescent dye was injected into single blastomere to observe the onset of gap junction intercellular communication. Then, connexin43 and connexin45 dsRNA were injected into zygote to explore the function of these two connexins during ovine preimplantation embryos in vitro. 1 Expression of connexin26, connexin31, connexin32, connexin40, connexin43 and connexin45 mRNA in oocytes and preimplantation embryos by RT-PCRPCR primers of all six connexins were designed according to sequences published on NCBI. Partial cDNA fragment were gained from RT-PCR and confirmed by sequence blast. Then, connexin26, connexin32, connexin43 and connexin45 had been found to express during oocyte maturation and embryos development by RT-PCR, and connexin31 and connexin40 had not been detected.2. The expression profiles of connexin 43 (Cx43) and connexin 45 (Cx45) transcripts and protein.2.1 Detection by Real-time PCRThe results indicated that Cx43 and Cx45 expressed in mature oocytes higher than immature ones. During embryos development, Cx43 transcript was gradually increased from 2-cell to 8-cell stage and maintaining higher mRNA level at morula and blastocyst stages; the expression level of Cx45 gene was not so obvious in each period of embryonic development.2.2 Detection of Cx43 and Cx45 in oocytes and preimplantation embryos by immunochemistry.The results showed that Cx43 and Cx45 proteins existed in immature oocytes, mature oocytes and embryos at all the developmental stages. After antibody staining, green fluorescent that denoted connexins could be observed near the cell membrane, slight fluorescent trace could be found in cell plasma and no trace in cell nucleus. The patterns of expression of Cx43 and Cx45 protein in embryos produced in vivo were similar to the embryos produced in vitro. It seemed that more abundant transcripts of Cx43 and Cx45 had been detected in embryos derived in vivo than that in embryos derived in vitro, and higher transcripts volume in the former always indicated better embryo quality.2.3 Expression of Cx43 detected by westernblot.Three confirmation of Cx43 were detectable by westernblot, containing two phosphorylation forms (PI and P2) and one disphosphorylation form (PO). Total volume of Cx43 expressed higher in mature oocytes compare to immature ones. The abundant of P1 and P2 expressed higher in 2-cell and 8-cell stages. P0 began to increase at morula, then a bit decrease in blastocyst stage. That might be attributed to gap junction gating correlated with some physiological function.3.Observation of gap junction intercellular communication in ovine preimplantation embryos derived in vitro.Lucifer yellow, a small molecular fluorescent dye, was injected into single blastomere of embryos at 4-cell,8-cell and morula stages by microinjection, respectively. The results showed that no dye diffused to adjacent cells could be observed when Lucifer yellow was injected to 4-cell or 8-cell. Then the diffusion phenomenon could be observed at morula stage. It revealed that gap junction intercellular communication began after 8-cell stage.4. Effects of Cx43 and Cx45 on preimplantation development4.1 Effects of dsRNA injection on gene expression of preimplantation embryosCx43 dsRNA, Cx45 dsRNA and water were injected into zygotes, respectively. Control group was cultured without injection. The results detected at blastocyst stage indicated the amounts of Cx43 and Cx45 transcripts had been decreased to 26% and 56% compared to control group, respectively. The abundant of housekeeping gene and relative genes showed no obviously difference among experiment groups, it is indicated that the synthetic dsRNA we used were specific and efficient. The protein levels were estimated by westernblot and immunochemistry. Westernblot result showed that protein level of Cx43 dsRNA injected group decreased visible compared to water and control groups, and results of immunochemistry showed that the fluorescent intensity of both dsRNA injected groups displayed not as brightness as water and control groups after staining.4.2 Effects of Cx43 and Cx45 dsRNA injection on cleavage rates of preimplantation embryos.The cleavage rates of embryos were statistical at 48 hours after culture in vitro. Clevage rates of embryos were 83.7%,84.5%,81.5%; 2-cell rates were 5.6%,4.7%, 4.6%; 4-cell rates were 14.6%,10.8%,10.3% and 8-cell rates were 64.5%,68.9% and 66.6% from the Cx43 dsRNA injected group, water group and control group, respectively. The statistical datas of Cx45 were showed as Cx43 were 81.7%,76.9%, 77.5%; 7.6%,3.6%,3.8%; 13.9%,11.6%,7.2% and 60.3%,61.8%,66.5%, respectively. Except 2-cell rates of Cx45 dsRNA injected group were significantly higher than that of water and control groups (P<0.01) and 4-cell rates of Cx45 dsRNA injected group and water group were significantly higher than that of control group (P<0.01), there were no significantly differences between the rest groups (P>0.05). However, microinjection of Cx43 or Cx45 dsRNA could not affect the total cleavage rates (P>0.05).4.3 Effects of Cx43 and Cx45 dsRNA injection on development rates and embryo qualities of preimplantation embryos.The in vitro development competence of 4-and 8-cell stages embryos was assessed at day 8 after fertilization, and the hatched rate was assessed at day 8-10. The results indicated that blastocyst and hatched blastocyst rates of 4-and 8-cell stages embryos were 20.3%,21.7%and 34.5%and 19.2%,37.5%and 41.3%from the Cx43 dsRNA group, water group and control group, respectively; there was no significant difference of blastocyst rates in groups(P>0.05), but hatched blastocyst rate in control and water group were significantly higher than Cx43 deRNA group (P<0.01). Blastocyst rates and hatched blastocyst rates of Cx45 groups were 20.8%, 19.4%,32.5%and 18.5%,25.0%,34.9%, respectively; there were no significant difference between every two groups (P>0.05). Cell numbers and means of dead cell rates of blastocyst were 74,76,83 and 24.6%,15.4%,18.2%of Cx43 groups and 79, 71,72 and 21.1%,17.9%,15.7%, respectively; no significant difference existed in every two groups (P>0.05). The results showed that Cx43 could affect the quality but did not affect the developmental rate of ovine embryos derived in vitro; Cx45 did not affect both the quality and the developmental rates of ovine embryos derived in vitro.更多还原