Vozzi C, Formenton A, Chanson A, Senn A, Sahli R, Shaw P, Nicod P, Germond M, Haefliger J A
Reproductive Medicine Unit, Department of Obstetrics and Gynecology, CHUV-1011 Lausanne, Switzerland.
Reproduction. 2001 Oct;122(4):619-28.
In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.
在卵巢卵泡中，卵丘细胞为卵母细胞提供允许其生长和控制成熟的小分子。这些营养物质通过卵丘细胞与卵母细胞之间以及卵丘细胞之间存在的缝隙连接通道到达生殖细胞。在本研究中，研究了由缝隙连接通道介导的细胞间通讯对体外培养的牛卵丘 - 卵母细胞复合体（COCs）卵母细胞成熟的影响。通过Hoechst 33342染色确定卵母细胞成熟阶段，结果显示，置于成熟培养基中24小时的COCs中有90%发育至中期II阶段。最初使用正构烷醇破坏牛COC缝隙连接通讯，正构烷醇会抑制任何通过缝隙连接的物质传递。在1 - 庚醇（3 mmol l(-1)）或辛醇（3.0 mmol l(-1)和0.3 mmol l(-1)）存在的情况下，只有29%的COCs达到中期II。去除解偶联剂与卵母细胞成熟的恢复相关，这表明用正构烷醇处理既无细胞毒性也非不可逆。使用表达反义Cx43 cDNA的重组腺病毒特异性降低了COCs中主要缝隙连接蛋白连接蛋白43（Cx43）的浓度。在用表达绿色荧光蛋白的重组腺病毒感染后评估的卵丘细胞中，腺病毒感染效率> 95%。对从Ad - asCx43感染的COCs中分离的总RNA进行RT - PCR显示，大鼠Cx43 cDNA被转录。蛋白质印迹分析显示，在表达Cx43反义RNA的COCs中，Cx43表达下降了三倍。用荧光黄注射卵丘细胞进一步证明，感染的COCs中产生的较低量的Cx43与卵丘细胞之间偶联程度降低两倍相关。此外，在感染的COC培养物中，卵母细胞成熟降低了50%。这些结果表明，Cx43介导的卵丘细胞间通讯在牛卵母细胞成熟中起关键作用。
