间隙连接蛋白在绵羊早期胚胎中的表达和作用研究
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。
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-PCR
PCR 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 PCR
The 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 development
4.1 Effects of dsRNA injection on gene expression of preimplantation embryos
Cx43 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.
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。
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-PCR
PCR 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 PCR
The 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 development
4.1 Effects of dsRNA injection on gene expression of preimplantation embryos
Cx43 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.
引文
[1]Bou Shorgan.Fertilization of goat and ovine Ova in vitro by ejaculated spermatozoa after treatment with ionophore A23187.Bull.Nippon vet.and Zooltech co,1984,33:219-221
[2]Iritani A, Sato E, Nishikawa Y. Secretion rates and chemical composition of oviduct and uterine fluids in sows. Journal of Animal Science,1974,39:582-588
[3]Tay JI, Rutherford AJ, Killick SR, et al. Human tubal fluid:production, nutrient composition and response to adrenergic agents. Human Reproduction,1997,12:2451-2456
[4]Figueroa XF, Duling BR. Gap junctions in the control of vascular function. Antioxid Redox Signal,2009,11(2):251-266
[5]Furuya S, Furuya K. Subepithelial fibroblasts in intestinal villi:roles in intercellular communication. Int Rev Cytol,2007,264:165-223
[6]Nakase T, Naus CC. Gap junctions and neurological disorders of the central nervous system. Biochim Biophys Acta,2004,1662(1-2):149-158
[7]Pfarrer CD, Heeb C, Leiser R. Expression of gap junctional connexins 26,32 and 43 in bovine placentomes during pregnancy. Placenta,2006,27(1):79-86
[8]Tanmahasamut P, Sidell N. Up-regulation of gap junctional intercellular communication and connexin43 expression by retinoic acid in human endometrial stromal cells. J Clin Endocrinol Metab,2005,90(7):4151-6. Epub 2005 Apr 5
[9]Disha pant., Lawrence P Reynolds., Justin S Luther., et al. Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep. Reproduction, 2005,129:191-200
[10]Kate Hardy, Anne Warner, Robert M.L.Winston, et al. Expression of intercellular junctions during preimplantation development of the human embryo. Molecular Human Reproduction,1996, 2:621-632
[11]R. Boni, E. Tosti and B. Dale, et al. Intercellular Communication in In Vivo-and In Vitro-Produced Bovine Embryos. Biology of Reproduction,1999,61:1050-1055
[12]Fire A, Xu S, Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature,1998,391(6669):806-811
[13]Chase D, Serafinas C, Ashcroft N, et al. The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans. Genesis,2000, 26(1):26-41
[14]Kuznicki KA, Smith PA, Leung-Chiu WM, et al. Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans. Development,2000,127(13):2907-2916
[15]Svoboda P, Stein P, Hayashi H, et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development,2000,127(19):4147-4156
[16]Grabarek JB, Plusa B, Glover DM, et al. Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.Genesis,2002,32(4):269-276
[17]Lazar S, Gershon E, Dekel N. Selective degradation of cyclin B1 mRNA in rat oocytes by RNA interference (RNAi). J Mol Endocrinol,2004,33:73-85
[18]Cabot RA, Prather RS. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol Reprod Dev,2003,64(3):292-301
[19]Nganvongpanit K, Muller H, Rings F, et al. Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. Reproduction,2006,131(5):861-874
[1]Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res,2004,62(2): 228-232
[2]Kumar NM, Gilula NB. The gap junction communication channel. Cell,1996,84(3):381-388
[3]Willecke K, Eiberger J, Degen J, et al. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem,2002,383(5):725-737
[4]Gabriel H D, Jung D, Butzler C, et al. Transplacental uptake of glucose in decreased in embryonic lethal connexin 26-deficient mice. J Cell Biol,1998,140:1453-1461
[5]Simon A M, Goodenough D A, Li E, et al. Female infertility in mice lacking connexin 37[J]. Nature,1998,385:525-529
[6]Granot I, Dekel N. Developmental expression and regulation of the gap junction protein and transcript in rat ovaries. Mol Reprod Dev,1997,47(3):231-239
[7]Grazul-Bilska AT, Redmer DA, Bilski JJ, et al. Gap junctional proteins, connexin 26,32, and 43 in sheep ovaries throughout the estrous cycle. Endocrine,1998,8(3):269-279
[8]Khan-Dawood FS, Yang J, Dawood MY. Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum. J Clin Endocrinol Metab,1996,81(2):835-842
[9]Granot I, Bechor E, Barash A, et al. Connexin43 in rat oocytes:developmental modulation of its phosphorylation. Biol Reprod,2002,66(3):568-573
[10]Granot I, Dekel N. Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem,1994,269(48):30502-30509
[11]Lawrence TS, Beers WH, Gilula NB. Transmission of hormonal stimulation by cell-to-cell communication. Nature,1978,272(5653):501-506
[12]Ackert CL, Gittens JE, O'Brien MJ, et al. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol,2001,233(2):258-270
[13]Franchesca D Houghton. Role of gap junctions during early embryo development. Reproduction,2005,129:129～135
[14]孙大业,郭艳林,马力耕等.细胞信号转导(第二版).北京：科学出版社,2001,11-14
Sun Daye, GuoYanlin, Ma Ligeng, et al. Signal Transduction(Ⅱ).Beijing:Science Press, 2001,11-14.(in Chinese)
[15]S.Wang, Y.liu, G.R.Holyoak. et al. Aprotocol for in vitro maturation and fertilization of sheep oocytes. Small Ruminant Research,1998,29:83-88
[16]Nganvongpanit K, Muller H, Rings F, et al. Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. Reproduction,2006,131(5):861-874
[17]De Sousa PA, Valdimarsson G, Nicholson BJ, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367
[18]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[19]Bloor DJ, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25
[20]Hardy K, Warner A, Winston RM, Becker DL. Expression of intercellular junctions during preimplantation development of the human embryo. Mol Hum Reprod,1996,2(8):621-632
[21]Lee S, Gilula N B, Warner A E.Gap junctional communication and compaction during preimplantation stages of mouse development. Cell,1987,51:851-860
[22]Bevilacqua A, R Loch-Caruso, and R P Erickson. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc Natl Acad Sci U S A,1989,86(14):5444-5448
[23]Becker, D L, Evans W H, Green C R, et al. Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions. J Cell Sci,1995,108: 1455-1467
[24]De Sousa PA, Juneja S C, Caveney S,et al. Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J Cell Sci,1997,110:1751-1758
[25]Reuss B, Hellmann P, Dahl E, et al. Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat. Dev Dyn,1996,205:172-182
[1]Javed Q, Fleming T P, Hay M, et al. Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development. Development,1993,117(3):1145-1151
[2]Ghassemifar M R, Eckert JJ, Houghton F D, et al. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol Hum Reprod, 2003,9(5):245-252
[3]Thomas F C, Sheth B, Eckert J J, et al. Contribution of JAM-1 to epithelial differentiation and tight-junction biogenesis in the mouse preimplantation embryo. J Cell Sci,2004,117(23): 5599-5608
[4]Bloor D J, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25
[5]De Sousa P A, Valdimarsson G, Nicholson B J, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367
[6]Reaume A G, De Sousa P A, Kulkarni S, et al. Cardiac malformation in neonatal mice lacking connexin43. Science,1995,267(5205):1831-1834
[7]Bruzzone R, White T W and Paul D L. Connections with connexins:the molecular basis of direct intercellular signaling. Eur J Biochem,1996,238(1):1-27
[8]Feranil J B, Isobe N and Nakao T Expression of gap junction protein connexin 43 during follicular atresia in the ovary of swamp buffaloes. J Reprod Dev,2005,51(5):675-681
[9]Houghton F D, Barr K J, Walter G, et al. Functional significance of gap junctional coupling in preimplantation development. Biol Reprod,2002,66(5):1403-1412
[10]Wrenzycki C, Herrmann D, Carnwath J W, et al. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil, 1996,108(1):17-24
[11]Alcolea S, Theveniau-Ruissy M, Jarry-Guichard T, et al. Downregulation of connexin 45 gene products during mouse heart development. Circ Res,1999,84(12):1365-1379
[12]Miquerol L, Dupays L, Theveniau-Ruissy M, et al. Gap junctional connexins in the developing mouse cardiac conduction system. Novartis Found Symp,2003,250:80-98; discussion 98-109, 276-9
[13]Naus C C, Zhu D, Todd S D, et al. Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol,1992,12(2):163-175
[14]Kanno Y and Loewenstein W R. Cell-to-cell passage of large molecules. Nature,1966, 212(5062):629-630
[15]Lonergan P, Rizos D, Gutierrez-Adan, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod,2003,69(4):1424-1431
[16]Crow D S, Beyer E C, Paul D L, et al. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol,1990,10(4): 1754-1763
[17]Lampe P D and Lau A F. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys,2000,384(2):205-215
[18]Lampe P D, TenBroek E M, Burt J M, et al. Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol,2000,149(7):1503-1512
[19]Li W, Hertzberg E L and Spray D C. Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia. J Biol Chem,2005,280(9):7941-7948
[20]Hendix E M, Mao S J, Everson W, et al. Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev,1992,33(1):27-38
[21]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[22]Becker D L, Leclerc-David C and Warner A. The relationship of gap junctions and compaction in the preimplantation mouse embryo. Dev Suppl,1992:113-118
[23]Hardy K and Handyside A H. Metabolism and cell allocation during parthenogenetic preimplantation mouse development. Mol Reprod Dev,1996,43(3):313-322
[24]Reima I, Lehtonen E, Virtanen I, et al. The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig. Differentiation,1993,54(1):35-45
[25]Prather R S and First N L. Cell-to-cell coupling in early-stage bovine embryos:A preliminary report. Theriogenology,1993,39(3):561-567
[26]Wrenzycki C, Herrmann D, Keskintepe L, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod,2001, 16(5):893-901
[27]Rizos D, Gutierrez-Adan A, De La Fuente J, et al. Bovine embryo culture in the presence o(?) absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68(1):236-243
[1]Adona P R, Pires P R, Quetglas M D, et al. Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine. Anim Reprod Sci,2008,104(2-4):389-397
[2]Combelles C M, Cekleniak N A, Racowsky C, et al. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod,2002,17(4):1006-1016
[3]Rizos D, Ward F, Duffy P, et al. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo:implications for blastocyst yield and blastocyst quality. Mol Reprod Dev,2002,61(2):234-248
[4]Rizos D, Lonergan P, Boland M P, et al. Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems:implications for blastocyst quality. Biol Reprod,2002,66(3):589-595
[5]Lonergan P, Rizos D, Gutierrez-Adan A, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod,2003,69(4):1424-1431
[6]Wrenzycki C, Herrmann D, Carnwath J W, et al. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol Reprod Dev,1999,53(1):8-18
[7]Betteridge K J, Smith C, Stubbings R B, et al. Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro. J Reprod Fertil Suppl,1989,38:87-98
[8]Prather R S and First N L. Cell-to-cell coupling in early-stage bovine embryos:A preliminary report. Theriogenology,1993,39(3):561-567
[9]Wrenzycki C, Herrmann D, Carnwath J W, et al. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil, 1996,108(1):17-24
[10]Wrenzycki C, Herrmann D, Keskintepe L, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod,2001, 16(5):893-901
[11]Rizos D, Gutierrez-Adan A, De La Fuente J, et al. Bovine embryo culture in the presence or absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68(1):236-243
[12]Larue L, Ohsugi M, Hirchenhain J,et al. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci U S A,1994,91(17):8263-8267
[13]Riethmacher D, Brinkmann V and Birchmeier C. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc Natl Acad Sci U S A,1995,92(3): 855-859
[14]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391(6669):806-811
[15]Svoboda P, Stein P, Hayashi H, et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development,2000,127(19):4147-4156
[16]Wianny F and Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol,2000,2(2):70-75
[17]Cabot R A, Hannink M and Prather R S. CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage. Biol Reprod,2002,67(3):814-819
[18]Cabot R A and Prather R S. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol Reprod Dev,2003,64(3): 292-301
[19]Nganvongpanit K, Muller H, Rings F, et al. Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA. Mol Reprod Dev,2006,73(2):153-163
[20]MacIntyre D M, Lim H C, Ryan K, et al. Implantation-associated changes in bovine uterine expression of integrins and extracellular matrix. Biol Reprod,2002,66(5):1430-1436
[21]Lykke-Andersen K, Gilchrist M J, Grabarek J B, et al. Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition. Mol Biol Cell,2008,19(10): 4383-4392
[22]Paradis F, Vigneault C, Robert C, et al. RNA interference as a tool to study gene function in bovine oocytes. Mol Reprod Dev,2005,70(2):111-121
[23]Hu X, Hipolito S, Lynn R, et al. Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus. Nucleic Acids Res,2004, 32(15):4609-4617
[24]Lee S, Gilula N B and Warner A E. Gap junctional communication and compaction during preimplantation stages of mouse development. Cell,1987,51(5):851-860
[25]Bevilacqua A, Loch-Caruso R and Erickson R P. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc Natl Acad Sci U S A,1989,86(14):5444-5448
[26]Ghassemifar M R, Eckert J J, Houghton F D, et al. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol Hum Reprod, 2003,9(5):245-252
[27]Billy E, Brondani V, Zhang H, et al. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci U S A,2001,98(25):14428-14433
[28]Yang S, Tutton S, Pierce E, et al. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol,2001,21(22):7807-7816
[29]Svoboda P, Stein P and Schulitz R M. RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA. Biochem Biophys Res Commun,2001,287(5):1099-1104
[30]Wei Q, Marchler G, Edington K, et al.RNA interference demonstrates a role for nautilus in the myogenic conversion of Schneider cells by daughterless. Dev Biol,2000,228(2):239-255
[31]Kobayashi S, Sakatani M, Kobayashi S, et al. Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells.J Reprod Dev,2007,53(6):1305-1311
[32]Gap junctions and follicular physiology. Res Reprod,1979.11(2):p.4
[33]De Sousa P A, Juneja S C, Caveney S, et al. Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J Cell Sci,1997,110 (Pt 15):1751-1758
[1]Naus C C, Zhu D, Todd S D, et al. Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol,1992,12:163-175.
[2]Robertson J D, T S Bodenheimer, and D E Stage. The ultrastructure of mauthner cell Synapses and nodes in goldfish brains. J Cell Biol,1963,19:159-199
[3]Benedetti E L and P Emmelot. Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J Cell Biol,1965,26(1):299-305.
[4]Revel J P and M J Karnovsky. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol,1967,33(3):C7-C12
[5]Makowski L, Caspar D L, Philips W C, et al. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol,1977,74(2):629-645
[6]Lal R, John S A, Laird D W, et al. Heart gap junction preparations reveal hemiplaques by atomic force microscopy. Am J Physiol,1995,268(4 Pt 1):C968-977
[7]Kumar N M and N B Gilula. The gap junction communication channel. Cell,1996,84(3): 381-388
[8]Phelan P and T A Starich. Innexins get into the gap. Bioessays,2001,23(5):388-396.
[9]Panchin Y, Kelmanson I, Matz M, et al. A ubiquitous family of putative gap junction molecules. Curr Biol,2000,10(13):R473-474
[10]Henderson D, H Eibl, and K Weber. Structure and biochemistry of mouse hepatic gap junctions. J Mol Biol,1979,132(2):193-218
[11]Manjunath C K, G E Goings, and E Page. Isolation and protein composition of gap junctions from rabbit hearts. Biochem J,1982,205(1):189-194
[12]Paul D L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol,1986, 103(1):123-134
[13]Beyer E C, D L Paul, and D A Goodenough. Connexin43:a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol,1987,105(6 Pt 1):2621-2629
[14]Sohl G and K Willecke. Gap junctions and the connexin protein family. Cardiovasc Res,2004, 62(2):228-232
[15]P D Lampe. Analyzing phorbol ester effects on gap junction communication:A dramatic inhibition of assembly. J Cell Biol,1994,127:1895-1905.
[16]D S Crow, E C Beyer, D L Paul, et al. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts, Mol Cell Biol,1990,10: 1754-1763.
[17]L S Musil, B A Cunningham, G M Edelman, et al. Differential phosphorylation of gap junction protein connexin43 in junctional communication-competent and-deficient cell lines, J Cell Biol, 1990,111:2077-2088
[18]V M Berthoud, E C Beyer, W E Kurata, et al. The gap junction protein connexin 56 is phosphorylated in the intracellular loop and the carboxy-terminal region. Eur J Biochem,1997,244: 89-97.
[19]B J Warn-Cramer, P D Lampe, W E Kurata, et al. Characterization of the MAP kinase phosphorylation sites on the connexin43 gap junction protein, J Biol Chem,1996,271:3779-3786.
[20]A D Martinez, V Hayrapetyan, A P Moreno, et al. A carboxyl terminal domain of connexin43 is critical for gap junction plaque formation but not for homo-or hetero-oligomerization. Cell Commun Adhes,2003,10:323-328.
[21]L S Musil and D A Goodenough. Biochemical analysis of connexin43 intracellular transport, phosphorylation and assembly into gap junctional plaques. J Cell Biol,1991,115:1357-1374.
[22]Albertini D F and E Anderson. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol,1974,63(1):234-250
[23]Mayerhofer A and R E Garfield. Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary. Mol Reprod Dev,1995,41(3):331-338
[24]Granot I and N Dekel. Developmental expression and regulation of the gap junction protein and transcript in rat ovaries. Mol Reprod Dev,1997,47(3):231-239.
[25]Grazul-Bilska A T, Redmer D A, Bilski J J, et al. Gap junctional proteins, connexin 26,32, and 43 in sheep ovaries throughout the estrous cycle. Endocrine,1998,8(3):269-279
[26]Khan-Dawood F S, J Yang and M Y Dawood. Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum. J Clin Endocrinol Metab,1996,81(2): 835-842
[27]Granot I, Bechor E, Barash A, et al. Connexin43 in rat oocytes:developmental modulation of its phosphorylation. Biol Reprod,2002,66(3):568-573
[28]Granot I and N Dekel. Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem,1994,269(48):30502-30509.
[29]Lawrence T S, W H Beers and N B Gilula. Transmission of hormonal stimulation by cell-to-cell communication. Nature,1978,272(5653):501-506
[30]Ackert C L, Gittens J E, O'Brien M J, et al. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol,2001,233(2):258-270
[31]Juneja S C, Barr K J, Enders G C, et al. Defects in the germ line and gonads of mice lacking connexin43. Biol Reprod,1999,60(5):1263-1270
[32]Vozzi C, Formenton A, Chanson A, et al. Involvement of connexin 43 in meiotic maturation of bovine oocytes. Reproduction,2001,122(4):619-628
[33]Anderson E and D F Albertini. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J Cell Biol,1976,71(2):680-686.
[34]Gilula N B, M L Epstein and W H Beers. Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex. J Cell Biol,1978,78(1):58-75.
[35]Brower P T and R M Schultz. Intercellular communication between granulosa cells and mouse oocytes:existence and possible nutritional role during oocyte growth. Dev Biol,1982,90(1): 144-153
[36]Vanderhyden B C, Caron P J, Buccione R, et al. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol,1990,140(2):307-317
[37]Vanderhyden B C, J N Cohen and P Morley. Mouse oocytes regulate granulosa. cell steroidogenesis. Endocrinology,1993,133(1):423-426
[38]Webb R J, Marshall F, Swann K, et al. Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes. Dev Biol,2002,246(2):441-454
[39]Webb R J, Bains H, Cruttwell C, et al. Gap-junctional communication in mouse cumulus-oocyte complexes:implications for the mechanism of meiotic maturation. Reproduction, 2002,123(1):41-52.
[40]Simon A M, Goodenough D A, Li E, et al. Female infertility in mice lacking connexin 37. Nature,1997,385(6616):525-529
[41]Cao F, Eckert R, Elfgang C, et al. A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J Cell Sci,1998, 111 (Pt1):31-43.
[42]Dekel N. Spatial relationship of follicular cells in the control of meiosis. Prog Clin Biol Res, 1988,267:87-101
[43]Valdimarsson G, P A De Sousa and G M Kidder. Coexpression of gap junction proteins in the cumulus-oocyte complex. Mol Reprod Dev,1993,36(1):7-15
[44]Grazul-Bilska A T, L P Reynolds and D A Redmer. Gap junctions in the ovaries. Biol Reprod, 1997,57(5):947-957
[45]Kalma Y, Granot I, Galiani D, et al. Luteinizing hormone-induced connexin 43 down-regulation:inhibition of translation. Endocrinology,2004,145(4):1617-1624.
[46]Cooke B A. Signal transduction involving cyclic AMP-dependent and cyclic AMP-independent mechanisms in the control of steroidogenesis. Mol Cell Endocrinol,1999,151(1-2):25-35
[47]Sela-Abramovich S, Chorev E, Galiani D, et al. Mitogen-activated protein kinase mediates luteinizing hormone-induced breakdown of communication and oocyte maturation in rat ovarian follicles. Endocrinology,2005,146(3):1236-1244
[48]Stagg R B and W H Fletcher. The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation. Endocr Rev,1990,11(2):302-325.
[49]Li W E, Ochalski P A, Hertzberg E L, et al. Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia. Eur J Neurosci,1998,10(7):2444-2463
[50]Eppig J J, A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro. J Exp Zool,1979,209(2): 345-353
[51]Larsen W J, H N Tung and C Polking. Response of granulosa cell gap junctions to human chorionic gonadotropin (hCG) at ovulation. Biol Reprod,1981,25(5):1119-1134
[52]Sela-Abramovich S, Edry I, Galiani D, et al. Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation. Endocrinology,2006,147(5):2280-2286
[53]Rizos D, A Gutierrez-Adan, S Perez-Garnelo, et al. Bovine embryo culture in the presence or absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68:236-243.
[54]De Sousa P A, Valdimarsson G, Nicholson B J, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367.
[55]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[56]Bloor DJ, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25.
[57]Hardy K, Warner A, Winston RM, Becker DL. Expression of intercellular junctions during preimplantation development of the human embryo. Mol Hum Reprod,1996,2(8):621-632.
[58]Lonergan P, Rizos D, Kanka J, et al. Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction,2003, 126(3):337-346
[59]Garfield R E, S Sims and E E Daniel. Gap junctions:their presence and necessity in myometrium during parturition. Science,1977,198(4320):958-960
[60]Beyer E C, Kistler J, Paul D L, et al. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol,1989,108(2): 595-605
[61]Albrecht J L, Atal N S, Tadros P N, et al. Rat uterine myometrium contains the gap junction protein connexin45, which has a differing temporal expression pattern from connexin43. Am J Obstet Gynecol,1996,175(4 Pt 1):853-858.
[62]Orsino A, C V Taylor and S J Lye. Connexin-26 and connexin-43 are differentially expressed and regulated in the rat myometrium throughout late pregnancy and with the onset of labor. Endocrinology,1996,137(5):1545-1553
[63]Kilarski W M, Dupont E, Coppen S, et al. Identification of two further gap-junctional proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term. Eur J Cell Biol, 1998,75(1):1-8
[64]Winterhager E, Brummer F, Dermietzel R, et al. Gap junction formation in rabbit uterine epithelium in response to embryo recognition. Dev Biol,1988,126(1):203-211
[65]Risek B and N B Gilula. Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy. Development,1991,113(1): 165-181
[66]Winterhager E, Grummer R, Jahn E, et al. Spatial and temporal expression of connexin26 and connexin43 in rat endometrium during trophoblast invasion. Dev Biol,1993,157(2):399-409.
[67]Grummer R, Chwalisz K, Mulholland J, et al. Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biol Reprod,1994,51 (6):1109-1116
[68]Hendrix E M, Myatt L, Sellers S, et al. Steroid hormone regulation of rat myometrial gap junction formation:effects on cx43 levels and trafficking. Biol Reprod,1995,52(3):547-560
[69]Risek B, Guthrie S, Kumar N, et al. Modulation of gap junction transcript and protein expression during pregnancy in the rat. J Cell Biol,1990,110(2):269-282
[70]Hermoso M, J C Saez and M Villalon. Identification of gap junctions in the oviduct and regulation of connexins during development and by sexual hormones. Eur J Cell Biol,1997,74(1): 1-9.
[71]Risley M S. Connexin gene expression in seminiferous tubules of the Sprague-Dawley rat. Biol Reprod,2000,62(3):748-754
[72]Pelletier R M. The distribution of connexin 43 is associated with the germ cell differentiation and with the modulation of the Sertoli cell junctional barrier in continual (guinea pig) and seasonal breeders'(mink) testes. J Androl,1995,16(5):400-409
[73]Perez-Armendariz E M, Lamoyi E, Mason J I, et al. Developmental regulation of connexin 43 expression in fetal mouse testicular cells. Anat Rec,2001,264(3):237-246
[74]Batias C, Siffroi J P, Fenichel P, et al. Batias, C., et al., Connexin43 gene expression and regulation in the rodent seminiferous epithelium. J Histochem Cytochem,2000,48(6):793-805.
[75]Bravo-Moreno J F, Diaz-Sanchez V, Montoya-Flores J G, et al. Expression of connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development. Anat Rec, 2001,264(1):13-24.
[76]Tan I P, Roy C, Saez J C, et al. Regulated assembly of connexin33 and connexin43 into rat Sertoli cell gap junctions. Biol Reprod,1996,54(6):1300-1310
[77]Risley M S, Tan I P, Roy C, et al. Cell-, age-and stage-dependent distribution of connexin43 gap junctions in testes. J Cell Sci,1992,103 (Pt 1):81-96
[78]Varanda W A and A C de Carvalho. Intercellular communication between mouse Leydig cells. Am J Physiol,1994,267(2 Pt 1):C563-569
[79]Plum A, Hallas G, Magin T, et al. Unique and shared functions of different connexins in mice. Curr Biol,2000,10(18):1083-1091.
[80]Steger K, F Tetens and M Bergmann. Expression of connexin 43 in human testis. Histochem Cell Biol,1999,112(3):215-220.
[81]You S, W Li and T Lin. Expression and regulation of connexin43 in rat Leydig cells. J Endocrinol,2000,166(2):447-453
[82]Cyr D G, L Hermo and D W Laird. Immunocytochemical localization and regulation of connexin43 in the adult rat epididymis. Endocrinology,1996,137(4):1474-1484.
[83]Piersanti M and S J Lye. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos. Endocrinology,1995,136(8):3571-3578.
[84]Goodall H and B Maro. Major loss of junctional coupling during mitosis in early mouse embryos. J Cell Biol,1986,102(2):568-575.
[85]O'Lague P, Dalen H, Rubin H, et al. Electrical coupling:low resistance junctions between mitotic and interphase fibroblasts in tissue culture. Science,1970,170(956):464-466
[86]Stein L S, J Boonstra and R C Burghardt. Reduced cell-cell communication between mitotic and nonmitotic coupled cells. Exp Cell Res,1992,198(1):1-7
[87]Ruch R J. The role of gap junctional intercellular communication in neoplasia. Ann Clin Lab Sci,1994,24(3):216-231
[88]Bittman K, Owens D F, Kriegstein A R, et al. Cell coupling and uncoupling in the ventricular zone of developing neocortex. J Neurosci,1997,17(18):7037-7044
[89]Bittman K S and J J LoTurco. Differential regulation of connexin 26 and 43 in murine neocortical precursors. Cereb Cortex,1999,9(2):188-195
[90]Lee B A and D J Donoghue. Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction. J Cell Biol,1992,118(5):1057-1070
[91]Ankoma-Sey V. Hepatic Regeneration-Revisiting the Myth of Prometheus. News Physiol Sci, 1999,14:149-155
[92]Steer C J. Liver regeneration. Faseb J,1995,9(14):1396-1400.
[93]Fukuhara Y, Hirasawa A, Li X K, et al. Gene expression profile in the regenerating rat liver after partial hepatectomy. J Hepatol,2003,38(6):784-792
[94]Dermietzel R, Yancey S B, Traub O, et al. Major loss of the 28-kD protein of gap junction in proliferating hepatocytes. J Cell Biol,1987,105(4):1925-1934
[95]Fladmark K E, Gjertsen B T, Molven A, et al. Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes. Hepatology,1997,25(4):847-855
[96]Kren B T, Kumar N M, Wang S Q, et al. Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration. J Cell Biol,1993,123(3):707-718
[97]Temme A, Ott T, Dombrowski F, et al. The extent of synchronous initiation and termination of DNA synthesis in regenerating mouse liver is dependent on connexin32 expressing gap junctions. J Hepatol,2000,32(4):627-635
[98]Kojima T, Yamamoto T, Lan M, et al. Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes. Med Electron Microsc,2004,37(2):101-113.
[99]Lampe P D and A F Lau. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol,2004,36(7):1171-1186
[100]Koo S K, Kim D Y, Park S D, et al. PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells. Mol Cell Biochem,1997,167(1-2):41-49
[101]Solan J L, Fry M D, TenBroek E M, et al. Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation. J Cell Sci,2003,116(Pt 11): 2203-2211
[102]Xie H, Laird D W, Chang T H, et al. A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells:biochemical characterization and localization. J Cell Biol,1997,137(1):203-210
[103]Kanemitsu M Y, W Jiang and W Eckhart. Cdc2-mediated phosphorylation of the gap junction protein, connexin43, during mitosis. Cell Growth Differ,1998 9(1):13-21
[104]Lampe P D, Kurata W E, Warn-Cramer B J, et al. Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase. J Cell Sci,1998,111 (Pt 6): 833-841
[105]Fukuda M, Kitaichi K, Abe F, et al. Altered brain penetration of diclofenac and mefenamic acid, but not acetaminophen, in Shiga-like toxin Ⅱ-treated mice. J Pharmacol Sci,2005,97(4):. 525-532
[106]Fujimoto E, Sato H, Nagashima Y, et al. A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells. Life Sci,2005,76(23): 2711-2720
[107]Huang R, Liu Y G, Lin Y, et al. Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43). Mol Carcinog,2001,32(3):128-138
[108]Contreras J E, Sanchez H A, Veliz L P, et al. Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res Brain Res Rev,2004, 47(1-3):290-303
[109]Kanczuga-Koda L, Sulkowski S, Koda M, et al. Connexin 26 correlates with Bcl-xL and Bax proteins expression in colorectal cancer. World J Gastroenterol,2005,11(10):1544-1548.
[110]Skrzydlewska E, Sulkowski S, Koda M, et al. Lipid peroxidation and antioxidant status in colorectal cancer. World J Gastroenterol,2005,11(3):403-406
[111]Iacobas D A, Urban-Maldonado M, Iacobas S, et al. Array analysis of gene expression in connexin-43 null astrocytes. Physiol Genomics,2003,15(3):177-190.
[2]Iritani A, Sato E, Nishikawa Y. Secretion rates and chemical composition of oviduct and uterine fluids in sows. Journal of Animal Science,1974,39:582-588
[3]Tay JI, Rutherford AJ, Killick SR, et al. Human tubal fluid:production, nutrient composition and response to adrenergic agents. Human Reproduction,1997,12:2451-2456
[4]Figueroa XF, Duling BR. Gap junctions in the control of vascular function. Antioxid Redox Signal,2009,11(2):251-266
[5]Furuya S, Furuya K. Subepithelial fibroblasts in intestinal villi:roles in intercellular communication. Int Rev Cytol,2007,264:165-223
[6]Nakase T, Naus CC. Gap junctions and neurological disorders of the central nervous system. Biochim Biophys Acta,2004,1662(1-2):149-158
[7]Pfarrer CD, Heeb C, Leiser R. Expression of gap junctional connexins 26,32 and 43 in bovine placentomes during pregnancy. Placenta,2006,27(1):79-86
[8]Tanmahasamut P, Sidell N. Up-regulation of gap junctional intercellular communication and connexin43 expression by retinoic acid in human endometrial stromal cells. J Clin Endocrinol Metab,2005,90(7):4151-6. Epub 2005 Apr 5
[9]Disha pant., Lawrence P Reynolds., Justin S Luther., et al. Expression of connexin 43 and gap junctional intercellular communication in the cumulus-oocyte complex in sheep. Reproduction, 2005,129:191-200
[10]Kate Hardy, Anne Warner, Robert M.L.Winston, et al. Expression of intercellular junctions during preimplantation development of the human embryo. Molecular Human Reproduction,1996, 2:621-632
[11]R. Boni, E. Tosti and B. Dale, et al. Intercellular Communication in In Vivo-and In Vitro-Produced Bovine Embryos. Biology of Reproduction,1999,61:1050-1055
[12]Fire A, Xu S, Montgomery MK,et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, Nature,1998,391(6669):806-811
[13]Chase D, Serafinas C, Ashcroft N, et al. The polo-like kinase PLK-1 is required for nuclear envelope breakdown and the completion of meiosis in Caenorhabditis elegans. Genesis,2000, 26(1):26-41
[14]Kuznicki KA, Smith PA, Leung-Chiu WM, et al. Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans. Development,2000,127(13):2907-2916
[15]Svoboda P, Stein P, Hayashi H, et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development,2000,127(19):4147-4156
[16]Grabarek JB, Plusa B, Glover DM, et al. Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation.Genesis,2002,32(4):269-276
[17]Lazar S, Gershon E, Dekel N. Selective degradation of cyclin B1 mRNA in rat oocytes by RNA interference (RNAi). J Mol Endocrinol,2004,33:73-85
[18]Cabot RA, Prather RS. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol Reprod Dev,2003,64(3):292-301
[19]Nganvongpanit K, Muller H, Rings F, et al. Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. Reproduction,2006,131(5):861-874
[1]Sohl G, Willecke K. Gap junctions and the connexin protein family. Cardiovasc Res,2004,62(2): 228-232
[2]Kumar NM, Gilula NB. The gap junction communication channel. Cell,1996,84(3):381-388
[3]Willecke K, Eiberger J, Degen J, et al. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem,2002,383(5):725-737
[4]Gabriel H D, Jung D, Butzler C, et al. Transplacental uptake of glucose in decreased in embryonic lethal connexin 26-deficient mice. J Cell Biol,1998,140:1453-1461
[5]Simon A M, Goodenough D A, Li E, et al. Female infertility in mice lacking connexin 37[J]. Nature,1998,385:525-529
[6]Granot I, Dekel N. Developmental expression and regulation of the gap junction protein and transcript in rat ovaries. Mol Reprod Dev,1997,47(3):231-239
[7]Grazul-Bilska AT, Redmer DA, Bilski JJ, et al. Gap junctional proteins, connexin 26,32, and 43 in sheep ovaries throughout the estrous cycle. Endocrine,1998,8(3):269-279
[8]Khan-Dawood FS, Yang J, Dawood MY. Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum. J Clin Endocrinol Metab,1996,81(2):835-842
[9]Granot I, Bechor E, Barash A, et al. Connexin43 in rat oocytes:developmental modulation of its phosphorylation. Biol Reprod,2002,66(3):568-573
[10]Granot I, Dekel N. Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem,1994,269(48):30502-30509
[11]Lawrence TS, Beers WH, Gilula NB. Transmission of hormonal stimulation by cell-to-cell communication. Nature,1978,272(5653):501-506
[12]Ackert CL, Gittens JE, O'Brien MJ, et al. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol,2001,233(2):258-270
[13]Franchesca D Houghton. Role of gap junctions during early embryo development. Reproduction,2005,129:129～135
[14]孙大业,郭艳林,马力耕等.细胞信号转导(第二版).北京：科学出版社,2001,11-14
Sun Daye, GuoYanlin, Ma Ligeng, et al. Signal Transduction(Ⅱ).Beijing:Science Press, 2001,11-14.(in Chinese)
[15]S.Wang, Y.liu, G.R.Holyoak. et al. Aprotocol for in vitro maturation and fertilization of sheep oocytes. Small Ruminant Research,1998,29:83-88
[16]Nganvongpanit K, Muller H, Rings F, et al. Selective degradation of maternal and embryonic transcripts in in vitro produced bovine oocytes and embryos using sequence specific double-stranded RNA. Reproduction,2006,131(5):861-874
[17]De Sousa PA, Valdimarsson G, Nicholson BJ, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367
[18]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[19]Bloor DJ, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25
[20]Hardy K, Warner A, Winston RM, Becker DL. Expression of intercellular junctions during preimplantation development of the human embryo. Mol Hum Reprod,1996,2(8):621-632
[21]Lee S, Gilula N B, Warner A E.Gap junctional communication and compaction during preimplantation stages of mouse development. Cell,1987,51:851-860
[22]Bevilacqua A, R Loch-Caruso, and R P Erickson. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc Natl Acad Sci U S A,1989,86(14):5444-5448
[23]Becker, D L, Evans W H, Green C R, et al. Functional analysis of amino acid sequences in connexin43 involved in intercellular communication through gap junctions. J Cell Sci,1995,108: 1455-1467
[24]De Sousa PA, Juneja S C, Caveney S,et al. Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J Cell Sci,1997,110:1751-1758
[25]Reuss B, Hellmann P, Dahl E, et al. Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat. Dev Dyn,1996,205:172-182
[1]Javed Q, Fleming T P, Hay M, et al. Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development. Development,1993,117(3):1145-1151
[2]Ghassemifar M R, Eckert JJ, Houghton F D, et al. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol Hum Reprod, 2003,9(5):245-252
[3]Thomas F C, Sheth B, Eckert J J, et al. Contribution of JAM-1 to epithelial differentiation and tight-junction biogenesis in the mouse preimplantation embryo. J Cell Sci,2004,117(23): 5599-5608
[4]Bloor D J, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25
[5]De Sousa P A, Valdimarsson G, Nicholson B J, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367
[6]Reaume A G, De Sousa P A, Kulkarni S, et al. Cardiac malformation in neonatal mice lacking connexin43. Science,1995,267(5205):1831-1834
[7]Bruzzone R, White T W and Paul D L. Connections with connexins:the molecular basis of direct intercellular signaling. Eur J Biochem,1996,238(1):1-27
[8]Feranil J B, Isobe N and Nakao T Expression of gap junction protein connexin 43 during follicular atresia in the ovary of swamp buffaloes. J Reprod Dev,2005,51(5):675-681
[9]Houghton F D, Barr K J, Walter G, et al. Functional significance of gap junctional coupling in preimplantation development. Biol Reprod,2002,66(5):1403-1412
[10]Wrenzycki C, Herrmann D, Carnwath J W, et al. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil, 1996,108(1):17-24
[11]Alcolea S, Theveniau-Ruissy M, Jarry-Guichard T, et al. Downregulation of connexin 45 gene products during mouse heart development. Circ Res,1999,84(12):1365-1379
[12]Miquerol L, Dupays L, Theveniau-Ruissy M, et al. Gap junctional connexins in the developing mouse cardiac conduction system. Novartis Found Symp,2003,250:80-98; discussion 98-109, 276-9
[13]Naus C C, Zhu D, Todd S D, et al. Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol,1992,12(2):163-175
[14]Kanno Y and Loewenstein W R. Cell-to-cell passage of large molecules. Nature,1966, 212(5062):629-630
[15]Lonergan P, Rizos D, Gutierrez-Adan, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod,2003,69(4):1424-1431
[16]Crow D S, Beyer E C, Paul D L, et al. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol,1990,10(4): 1754-1763
[17]Lampe P D and Lau A F. Regulation of gap junctions by phosphorylation of connexins. Arch Biochem Biophys,2000,384(2):205-215
[18]Lampe P D, TenBroek E M, Burt J M, et al. Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication. J Cell Biol,2000,149(7):1503-1512
[19]Li W, Hertzberg E L and Spray D C. Regulation of connexin43-protein binding in astrocytes in response to chemical ischemia/hypoxia. J Biol Chem,2005,280(9):7941-7948
[20]Hendix E M, Mao S J, Everson W, et al. Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor. Mol Reprod Dev,1992,33(1):27-38
[21]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[22]Becker D L, Leclerc-David C and Warner A. The relationship of gap junctions and compaction in the preimplantation mouse embryo. Dev Suppl,1992:113-118
[23]Hardy K and Handyside A H. Metabolism and cell allocation during parthenogenetic preimplantation mouse development. Mol Reprod Dev,1996,43(3):313-322
[24]Reima I, Lehtonen E, Virtanen I, et al. The cytoskeleton and associated proteins during cleavage, compaction and blastocyst differentiation in the pig. Differentiation,1993,54(1):35-45
[25]Prather R S and First N L. Cell-to-cell coupling in early-stage bovine embryos:A preliminary report. Theriogenology,1993,39(3):561-567
[26]Wrenzycki C, Herrmann D, Keskintepe L, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod,2001, 16(5):893-901
[27]Rizos D, Gutierrez-Adan A, De La Fuente J, et al. Bovine embryo culture in the presence o(?) absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68(1):236-243
[1]Adona P R, Pires P R, Quetglas M D, et al. Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine. Anim Reprod Sci,2008,104(2-4):389-397
[2]Combelles C M, Cekleniak N A, Racowsky C, et al. Assessment of nuclear and cytoplasmic maturation in in-vitro matured human oocytes. Hum Reprod,2002,17(4):1006-1016
[3]Rizos D, Ward F, Duffy P, et al. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo:implications for blastocyst yield and blastocyst quality. Mol Reprod Dev,2002,61(2):234-248
[4]Rizos D, Lonergan P, Boland M P, et al. Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems:implications for blastocyst quality. Biol Reprod,2002,66(3):589-595
[5]Lonergan P, Rizos D, Gutierrez-Adan A, et al. Temporal divergence in the pattern of messenger RNA expression in bovine embryos cultured from the zygote to blastocyst stage in vitro or in vivo. Biol Reprod,2003,69(4):1424-1431
[6]Wrenzycki C, Herrmann D, Carnwath J W, et al. Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol Reprod Dev,1999,53(1):8-18
[7]Betteridge K J, Smith C, Stubbings R B, et al. Potential genetic improvement of cattle by fertilization of fetal oocytes in vitro. J Reprod Fertil Suppl,1989,38:87-98
[8]Prather R S and First N L. Cell-to-cell coupling in early-stage bovine embryos:A preliminary report. Theriogenology,1993,39(3):561-567
[9]Wrenzycki C, Herrmann D, Carnwath J W, et al. Expression of the gap junction gene connexin43 (Cx43) in preimplantation bovine embryos derived in vitro or in vivo. J Reprod Fertil, 1996,108(1):17-24
[10]Wrenzycki C, Herrmann D, Keskintepe L, et al. Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod,2001, 16(5):893-901
[11]Rizos D, Gutierrez-Adan A, De La Fuente J, et al. Bovine embryo culture in the presence or absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68(1):236-243
[12]Larue L, Ohsugi M, Hirchenhain J,et al. E-cadherin null mutant embryos fail to form a trophectoderm epithelium. Proc Natl Acad Sci U S A,1994,91(17):8263-8267
[13]Riethmacher D, Brinkmann V and Birchmeier C. A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc Natl Acad Sci U S A,1995,92(3): 855-859
[14]Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature,1998,391(6669):806-811
[15]Svoboda P, Stein P, Hayashi H, et al. Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference. Development,2000,127(19):4147-4156
[16]Wianny F and Zernicka-Goetz M. Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol,2000,2(2):70-75
[17]Cabot R A, Hannink M and Prather R S. CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage. Biol Reprod,2002,67(3):814-819
[18]Cabot R A and Prather R S. Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3. Mol Reprod Dev,2003,64(3): 292-301
[19]Nganvongpanit K, Muller H, Rings F, et al. Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA. Mol Reprod Dev,2006,73(2):153-163
[20]MacIntyre D M, Lim H C, Ryan K, et al. Implantation-associated changes in bovine uterine expression of integrins and extracellular matrix. Biol Reprod,2002,66(5):1430-1436
[21]Lykke-Andersen K, Gilchrist M J, Grabarek J B, et al. Maternal Argonaute 2 is essential for early mouse development at the maternal-zygotic transition. Mol Biol Cell,2008,19(10): 4383-4392
[22]Paradis F, Vigneault C, Robert C, et al. RNA interference as a tool to study gene function in bovine oocytes. Mol Reprod Dev,2005,70(2):111-121
[23]Hu X, Hipolito S, Lynn R, et al. Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus. Nucleic Acids Res,2004, 32(15):4609-4617
[24]Lee S, Gilula N B and Warner A E. Gap junctional communication and compaction during preimplantation stages of mouse development. Cell,1987,51(5):851-860
[25]Bevilacqua A, Loch-Caruso R and Erickson R P. Abnormal development and dye coupling produced by antisense RNA to gap junction protein in mouse preimplantation embryos. Proc Natl Acad Sci U S A,1989,86(14):5444-5448
[26]Ghassemifar M R, Eckert J J, Houghton F D, et al. Gene expression regulating epithelial intercellular junction biogenesis during human blastocyst development in vitro. Mol Hum Reprod, 2003,9(5):245-252
[27]Billy E, Brondani V, Zhang H, et al. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci U S A,2001,98(25):14428-14433
[28]Yang S, Tutton S, Pierce E, et al. Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells. Mol Cell Biol,2001,21(22):7807-7816
[29]Svoboda P, Stein P and Schulitz R M. RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA. Biochem Biophys Res Commun,2001,287(5):1099-1104
[30]Wei Q, Marchler G, Edington K, et al.RNA interference demonstrates a role for nautilus in the myogenic conversion of Schneider cells by daughterless. Dev Biol,2000,228(2):239-255
[31]Kobayashi S, Sakatani M, Kobayashi S, et al. Gene silencing of cyclooxygenase-2 mRNA by RNA interference in bovine cumulus-granulosa cells.J Reprod Dev,2007,53(6):1305-1311
[32]Gap junctions and follicular physiology. Res Reprod,1979.11(2):p.4
[33]De Sousa P A, Juneja S C, Caveney S, et al. Normal development of preimplantation mouse embryos deficient in gap junctional coupling. J Cell Sci,1997,110 (Pt 15):1751-1758
[1]Naus C C, Zhu D, Todd S D, et al. Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol,1992,12:163-175.
[2]Robertson J D, T S Bodenheimer, and D E Stage. The ultrastructure of mauthner cell Synapses and nodes in goldfish brains. J Cell Biol,1963,19:159-199
[3]Benedetti E L and P Emmelot. Electron microscopic observations on negatively stained plasma membranes isolated from rat liver. J Cell Biol,1965,26(1):299-305.
[4]Revel J P and M J Karnovsky. Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol,1967,33(3):C7-C12
[5]Makowski L, Caspar D L, Philips W C, et al. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol,1977,74(2):629-645
[6]Lal R, John S A, Laird D W, et al. Heart gap junction preparations reveal hemiplaques by atomic force microscopy. Am J Physiol,1995,268(4 Pt 1):C968-977
[7]Kumar N M and N B Gilula. The gap junction communication channel. Cell,1996,84(3): 381-388
[8]Phelan P and T A Starich. Innexins get into the gap. Bioessays,2001,23(5):388-396.
[9]Panchin Y, Kelmanson I, Matz M, et al. A ubiquitous family of putative gap junction molecules. Curr Biol,2000,10(13):R473-474
[10]Henderson D, H Eibl, and K Weber. Structure and biochemistry of mouse hepatic gap junctions. J Mol Biol,1979,132(2):193-218
[11]Manjunath C K, G E Goings, and E Page. Isolation and protein composition of gap junctions from rabbit hearts. Biochem J,1982,205(1):189-194
[12]Paul D L. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol,1986, 103(1):123-134
[13]Beyer E C, D L Paul, and D A Goodenough. Connexin43:a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol,1987,105(6 Pt 1):2621-2629
[14]Sohl G and K Willecke. Gap junctions and the connexin protein family. Cardiovasc Res,2004, 62(2):228-232
[15]P D Lampe. Analyzing phorbol ester effects on gap junction communication:A dramatic inhibition of assembly. J Cell Biol,1994,127:1895-1905.
[16]D S Crow, E C Beyer, D L Paul, et al. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts, Mol Cell Biol,1990,10: 1754-1763.
[17]L S Musil, B A Cunningham, G M Edelman, et al. Differential phosphorylation of gap junction protein connexin43 in junctional communication-competent and-deficient cell lines, J Cell Biol, 1990,111:2077-2088
[18]V M Berthoud, E C Beyer, W E Kurata, et al. The gap junction protein connexin 56 is phosphorylated in the intracellular loop and the carboxy-terminal region. Eur J Biochem,1997,244: 89-97.
[19]B J Warn-Cramer, P D Lampe, W E Kurata, et al. Characterization of the MAP kinase phosphorylation sites on the connexin43 gap junction protein, J Biol Chem,1996,271:3779-3786.
[20]A D Martinez, V Hayrapetyan, A P Moreno, et al. A carboxyl terminal domain of connexin43 is critical for gap junction plaque formation but not for homo-or hetero-oligomerization. Cell Commun Adhes,2003,10:323-328.
[21]L S Musil and D A Goodenough. Biochemical analysis of connexin43 intracellular transport, phosphorylation and assembly into gap junctional plaques. J Cell Biol,1991,115:1357-1374.
[22]Albertini D F and E Anderson. The appearance and structure of intercellular connections during the ontogeny of the rabbit ovarian follicle with particular reference to gap junctions. J Cell Biol,1974,63(1):234-250
[23]Mayerhofer A and R E Garfield. Immunocytochemical analysis of the expression of gap junction protein connexin 43 in the rat ovary. Mol Reprod Dev,1995,41(3):331-338
[24]Granot I and N Dekel. Developmental expression and regulation of the gap junction protein and transcript in rat ovaries. Mol Reprod Dev,1997,47(3):231-239.
[25]Grazul-Bilska A T, Redmer D A, Bilski J J, et al. Gap junctional proteins, connexin 26,32, and 43 in sheep ovaries throughout the estrous cycle. Endocrine,1998,8(3):269-279
[26]Khan-Dawood F S, J Yang and M Y Dawood. Expression of gap junction protein connexin-43 in the human and baboon (Papio anubis) corpus luteum. J Clin Endocrinol Metab,1996,81(2): 835-842
[27]Granot I, Bechor E, Barash A, et al. Connexin43 in rat oocytes:developmental modulation of its phosphorylation. Biol Reprod,2002,66(3):568-573
[28]Granot I and N Dekel. Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J Biol Chem,1994,269(48):30502-30509.
[29]Lawrence T S, W H Beers and N B Gilula. Transmission of hormonal stimulation by cell-to-cell communication. Nature,1978,272(5653):501-506
[30]Ackert C L, Gittens J E, O'Brien M J, et al. Intercellular communication via connexin43 gap junctions is required for ovarian folliculogenesis in the mouse. Dev Biol,2001,233(2):258-270
[31]Juneja S C, Barr K J, Enders G C, et al. Defects in the germ line and gonads of mice lacking connexin43. Biol Reprod,1999,60(5):1263-1270
[32]Vozzi C, Formenton A, Chanson A, et al. Involvement of connexin 43 in meiotic maturation of bovine oocytes. Reproduction,2001,122(4):619-628
[33]Anderson E and D F Albertini. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J Cell Biol,1976,71(2):680-686.
[34]Gilula N B, M L Epstein and W H Beers. Cell-to-cell communication and ovulation. A study of the cumulus-oocyte complex. J Cell Biol,1978,78(1):58-75.
[35]Brower P T and R M Schultz. Intercellular communication between granulosa cells and mouse oocytes:existence and possible nutritional role during oocyte growth. Dev Biol,1982,90(1): 144-153
[36]Vanderhyden B C, Caron P J, Buccione R, et al. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol,1990,140(2):307-317
[37]Vanderhyden B C, J N Cohen and P Morley. Mouse oocytes regulate granulosa. cell steroidogenesis. Endocrinology,1993,133(1):423-426
[38]Webb R J, Marshall F, Swann K, et al. Follicle-stimulating hormone induces a gap junction-dependent dynamic change in [cAMP] and protein kinase a in mammalian oocytes. Dev Biol,2002,246(2):441-454
[39]Webb R J, Bains H, Cruttwell C, et al. Gap-junctional communication in mouse cumulus-oocyte complexes:implications for the mechanism of meiotic maturation. Reproduction, 2002,123(1):41-52.
[40]Simon A M, Goodenough D A, Li E, et al. Female infertility in mice lacking connexin 37. Nature,1997,385(6616):525-529
[41]Cao F, Eckert R, Elfgang C, et al. A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes. J Cell Sci,1998, 111 (Pt1):31-43.
[42]Dekel N. Spatial relationship of follicular cells in the control of meiosis. Prog Clin Biol Res, 1988,267:87-101
[43]Valdimarsson G, P A De Sousa and G M Kidder. Coexpression of gap junction proteins in the cumulus-oocyte complex. Mol Reprod Dev,1993,36(1):7-15
[44]Grazul-Bilska A T, L P Reynolds and D A Redmer. Gap junctions in the ovaries. Biol Reprod, 1997,57(5):947-957
[45]Kalma Y, Granot I, Galiani D, et al. Luteinizing hormone-induced connexin 43 down-regulation:inhibition of translation. Endocrinology,2004,145(4):1617-1624.
[46]Cooke B A. Signal transduction involving cyclic AMP-dependent and cyclic AMP-independent mechanisms in the control of steroidogenesis. Mol Cell Endocrinol,1999,151(1-2):25-35
[47]Sela-Abramovich S, Chorev E, Galiani D, et al. Mitogen-activated protein kinase mediates luteinizing hormone-induced breakdown of communication and oocyte maturation in rat ovarian follicles. Endocrinology,2005,146(3):1236-1244
[48]Stagg R B and W H Fletcher. The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation. Endocr Rev,1990,11(2):302-325.
[49]Li W E, Ochalski P A, Hertzberg E L, et al. Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia. Eur J Neurosci,1998,10(7):2444-2463
[50]Eppig J J, A comparison between oocyte growth in coculture with granulosa cells and oocytes with granulosa cell-oocyte junctional contact maintained in vitro. J Exp Zool,1979,209(2): 345-353
[51]Larsen W J, H N Tung and C Polking. Response of granulosa cell gap junctions to human chorionic gonadotropin (hCG) at ovulation. Biol Reprod,1981,25(5):1119-1134
[52]Sela-Abramovich S, Edry I, Galiani D, et al. Disruption of gap junctional communication within the ovarian follicle induces oocyte maturation. Endocrinology,2006,147(5):2280-2286
[53]Rizos D, A Gutierrez-Adan, S Perez-Garnelo, et al. Bovine embryo culture in the presence or absence of serum:implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod,2003,68:236-243.
[54]De Sousa P A, Valdimarsson G, Nicholson B J, et al. Connexin trafficking and the control of gap junction assembly in mouse preimplantation embryos. Development,1993,117(4):1355-1367.
[55]Houghton F D. Role of gap junctions during early embryo development. Reproduction,2005, 129(2):129-135
[56]Bloor DJ, Wilson Y, Kibschull M, et al. Expression of connexins in human preimplantation embryos in vitro. Reprod Biol Endocrinol,2004,2:25.
[57]Hardy K, Warner A, Winston RM, Becker DL. Expression of intercellular junctions during preimplantation development of the human embryo. Mol Hum Reprod,1996,2(8):621-632.
[58]Lonergan P, Rizos D, Kanka J, et al. Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality. Reproduction,2003, 126(3):337-346
[59]Garfield R E, S Sims and E E Daniel. Gap junctions:their presence and necessity in myometrium during parturition. Science,1977,198(4320):958-960
[60]Beyer E C, Kistler J, Paul D L, et al. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol,1989,108(2): 595-605
[61]Albrecht J L, Atal N S, Tadros P N, et al. Rat uterine myometrium contains the gap junction protein connexin45, which has a differing temporal expression pattern from connexin43. Am J Obstet Gynecol,1996,175(4 Pt 1):853-858.
[62]Orsino A, C V Taylor and S J Lye. Connexin-26 and connexin-43 are differentially expressed and regulated in the rat myometrium throughout late pregnancy and with the onset of labor. Endocrinology,1996,137(5):1545-1553
[63]Kilarski W M, Dupont E, Coppen S, et al. Identification of two further gap-junctional proteins, connexin40 and connexin45, in human myometrial smooth muscle cells at term. Eur J Cell Biol, 1998,75(1):1-8
[64]Winterhager E, Brummer F, Dermietzel R, et al. Gap junction formation in rabbit uterine epithelium in response to embryo recognition. Dev Biol,1988,126(1):203-211
[65]Risek B and N B Gilula. Spatiotemporal expression of three gap junction gene products involved in fetomaternal communication during rat pregnancy. Development,1991,113(1): 165-181
[66]Winterhager E, Grummer R, Jahn E, et al. Spatial and temporal expression of connexin26 and connexin43 in rat endometrium during trophoblast invasion. Dev Biol,1993,157(2):399-409.
[67]Grummer R, Chwalisz K, Mulholland J, et al. Regulation of connexin26 and connexin43 expression in rat endometrium by ovarian steroid hormones. Biol Reprod,1994,51 (6):1109-1116
[68]Hendrix E M, Myatt L, Sellers S, et al. Steroid hormone regulation of rat myometrial gap junction formation:effects on cx43 levels and trafficking. Biol Reprod,1995,52(3):547-560
[69]Risek B, Guthrie S, Kumar N, et al. Modulation of gap junction transcript and protein expression during pregnancy in the rat. J Cell Biol,1990,110(2):269-282
[70]Hermoso M, J C Saez and M Villalon. Identification of gap junctions in the oviduct and regulation of connexins during development and by sexual hormones. Eur J Cell Biol,1997,74(1): 1-9.
[71]Risley M S. Connexin gene expression in seminiferous tubules of the Sprague-Dawley rat. Biol Reprod,2000,62(3):748-754
[72]Pelletier R M. The distribution of connexin 43 is associated with the germ cell differentiation and with the modulation of the Sertoli cell junctional barrier in continual (guinea pig) and seasonal breeders'(mink) testes. J Androl,1995,16(5):400-409
[73]Perez-Armendariz E M, Lamoyi E, Mason J I, et al. Developmental regulation of connexin 43 expression in fetal mouse testicular cells. Anat Rec,2001,264(3):237-246
[74]Batias C, Siffroi J P, Fenichel P, et al. Batias, C., et al., Connexin43 gene expression and regulation in the rodent seminiferous epithelium. J Histochem Cytochem,2000,48(6):793-805.
[75]Bravo-Moreno J F, Diaz-Sanchez V, Montoya-Flores J G, et al. Expression of connexin43 in mouse Leydig, Sertoli, and germinal cells at different stages of postnatal development. Anat Rec, 2001,264(1):13-24.
[76]Tan I P, Roy C, Saez J C, et al. Regulated assembly of connexin33 and connexin43 into rat Sertoli cell gap junctions. Biol Reprod,1996,54(6):1300-1310
[77]Risley M S, Tan I P, Roy C, et al. Cell-, age-and stage-dependent distribution of connexin43 gap junctions in testes. J Cell Sci,1992,103 (Pt 1):81-96
[78]Varanda W A and A C de Carvalho. Intercellular communication between mouse Leydig cells. Am J Physiol,1994,267(2 Pt 1):C563-569
[79]Plum A, Hallas G, Magin T, et al. Unique and shared functions of different connexins in mice. Curr Biol,2000,10(18):1083-1091.
[80]Steger K, F Tetens and M Bergmann. Expression of connexin 43 in human testis. Histochem Cell Biol,1999,112(3):215-220.
[81]You S, W Li and T Lin. Expression and regulation of connexin43 in rat Leydig cells. J Endocrinol,2000,166(2):447-453
[82]Cyr D G, L Hermo and D W Laird. Immunocytochemical localization and regulation of connexin43 in the adult rat epididymis. Endocrinology,1996,137(4):1474-1484.
[83]Piersanti M and S J Lye. Increase in messenger ribonucleic acid encoding the myometrial gap junction protein, connexin-43, requires protein synthesis and is associated with increased expression of the activator protein-1, c-fos. Endocrinology,1995,136(8):3571-3578.
[84]Goodall H and B Maro. Major loss of junctional coupling during mitosis in early mouse embryos. J Cell Biol,1986,102(2):568-575.
[85]O'Lague P, Dalen H, Rubin H, et al. Electrical coupling:low resistance junctions between mitotic and interphase fibroblasts in tissue culture. Science,1970,170(956):464-466
[86]Stein L S, J Boonstra and R C Burghardt. Reduced cell-cell communication between mitotic and nonmitotic coupled cells. Exp Cell Res,1992,198(1):1-7
[87]Ruch R J. The role of gap junctional intercellular communication in neoplasia. Ann Clin Lab Sci,1994,24(3):216-231
[88]Bittman K, Owens D F, Kriegstein A R, et al. Cell coupling and uncoupling in the ventricular zone of developing neocortex. J Neurosci,1997,17(18):7037-7044
[89]Bittman K S and J J LoTurco. Differential regulation of connexin 26 and 43 in murine neocortical precursors. Cereb Cortex,1999,9(2):188-195
[90]Lee B A and D J Donoghue. Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction. J Cell Biol,1992,118(5):1057-1070
[91]Ankoma-Sey V. Hepatic Regeneration-Revisiting the Myth of Prometheus. News Physiol Sci, 1999,14:149-155
[92]Steer C J. Liver regeneration. Faseb J,1995,9(14):1396-1400.
[93]Fukuhara Y, Hirasawa A, Li X K, et al. Gene expression profile in the regenerating rat liver after partial hepatectomy. J Hepatol,2003,38(6):784-792
[94]Dermietzel R, Yancey S B, Traub O, et al. Major loss of the 28-kD protein of gap junction in proliferating hepatocytes. J Cell Biol,1987,105(4):1925-1934
[95]Fladmark K E, Gjertsen B T, Molven A, et al. Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes. Hepatology,1997,25(4):847-855
[96]Kren B T, Kumar N M, Wang S Q, et al. Differential regulation of multiple gap junction transcripts and proteins during rat liver regeneration. J Cell Biol,1993,123(3):707-718
[97]Temme A, Ott T, Dombrowski F, et al. The extent of synchronous initiation and termination of DNA synthesis in regenerating mouse liver is dependent on connexin32 expressing gap junctions. J Hepatol,2000,32(4):627-635
[98]Kojima T, Yamamoto T, Lan M, et al. Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes. Med Electron Microsc,2004,37(2):101-113.
[99]Lampe P D and A F Lau. The effects of connexin phosphorylation on gap junctional communication. Int J Biochem Cell Biol,2004,36(7):1171-1186
[100]Koo S K, Kim D Y, Park S D, et al. PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells. Mol Cell Biochem,1997,167(1-2):41-49
[101]Solan J L, Fry M D, TenBroek E M, et al. Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation. J Cell Sci,2003,116(Pt 11): 2203-2211
[102]Xie H, Laird D W, Chang T H, et al. A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells:biochemical characterization and localization. J Cell Biol,1997,137(1):203-210
[103]Kanemitsu M Y, W Jiang and W Eckhart. Cdc2-mediated phosphorylation of the gap junction protein, connexin43, during mitosis. Cell Growth Differ,1998 9(1):13-21
[104]Lampe P D, Kurata W E, Warn-Cramer B J, et al. Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase. J Cell Sci,1998,111 (Pt 6): 833-841
[105]Fukuda M, Kitaichi K, Abe F, et al. Altered brain penetration of diclofenac and mefenamic acid, but not acetaminophen, in Shiga-like toxin Ⅱ-treated mice. J Pharmacol Sci,2005,97(4):. 525-532
[106]Fujimoto E, Sato H, Nagashima Y, et al. A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells. Life Sci,2005,76(23): 2711-2720
[107]Huang R, Liu Y G, Lin Y, et al. Enhanced apoptosis under low serum conditions in human glioblastoma cells by connexin 43 (Cx43). Mol Carcinog,2001,32(3):128-138
[108]Contreras J E, Sanchez H A, Veliz L P, et al. Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res Brain Res Rev,2004, 47(1-3):290-303
[109]Kanczuga-Koda L, Sulkowski S, Koda M, et al. Connexin 26 correlates with Bcl-xL and Bax proteins expression in colorectal cancer. World J Gastroenterol,2005,11(10):1544-1548.
[110]Skrzydlewska E, Sulkowski S, Koda M, et al. Lipid peroxidation and antioxidant status in colorectal cancer. World J Gastroenterol,2005,11(3):403-406
[111]Iacobas D A, Urban-Maldonado M, Iacobas S, et al. Array analysis of gene expression in connexin-43 null astrocytes. Physiol Genomics,2003,15(3):177-190.