Cronier L, Hervé J C, Délèze J, Malassiné A
Laboratoire de Physiologie Cellulaire, CNRS URA 1869, Université de Poitiers, France.
Microsc Res Tech. 1997;38(1-2):21-8. doi: 10.1002/(SICI)1097-0029(19970701/15)38:1/2<21::AID-JEMT4>3.0.CO;2-X.
During pregnancy, the trophoblast, supporting the main functions of the placenta, develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. Gap junction channels consisting of connexins link the cytosols of cells in contact. Gap junctional communication has been involved in the control of cell and tissue differentiation. Recently, a gap junctional communication was demonstrated in trophoblast cell culture by means of the fluorescence recovery after photobleaching (gap-FRAP) technique. This gap junctional communication appeared to be stimulated by human chorionic gonadotropin (hCG). Therefore, the specificity of hCG action and the signalling mechanisms implicated in gap junctional communication were investigated by means of gap-FRAP. In culture, cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 1-2 days after plating. During this in vitro differentiation, gap junctional communication was measured, and the maximum percentage of coupling between adjacent cells occurred on the fourth day. In the presence of 500 mIU/ml hCG, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days instead of 4 days in control conditions. The hCG action was specific, since the addition of heat-inactivated hCG of oFSH or of bTSH did not affect gap junctional communication in trophoblastic cells. The addition of a polyclonal hCG antibody decreased basal gap junctional communication as well as the response to exogenous hCG. Moreover, the presence of 8Br-cAMP (0.5 or 1 mM) mimicked the stimulation by hCG. Interestingly, H89 (2 microM), a specific protein kinase-A inhibitor, dramatically decreased the responses to hCG (500 mIU/ml) and the 8Br-cAMP (0.5 mM) stimulation of trophoblastic gap junctional communication. Calphostin (1 or 2 microM), a specific protein kinase-C inhibitor, strongly stimulated gap junctional communication. In conclusion, the demonstration by means of the gap-FRAP method of a gap junctional communication preceding cellular fusion could be considered as an objective and physiological criterion to mark the beginning of trophoblast differentiation. hCG, a hormone produced by the trophoblast, and two signalling mechanisms are implicated in this phenomenon.
在孕期，支持胎盘主要功能的滋养层由细胞滋养层细胞融合形成合体滋养层而发育。由连接蛋白组成的缝隙连接通道连接着相互接触细胞的胞质溶胶。缝隙连接通讯参与了细胞和组织分化的调控。最近，通过光漂白后荧光恢复（缝隙连接-荧光恢复后漂白，gap-FRAP）技术在滋养层细胞培养中证实了缝隙连接通讯。这种缝隙连接通讯似乎受到人绒毛膜促性腺激素（hCG）的刺激。因此，利用gap-FRAP技术研究了hCG作用的特异性以及与缝隙连接通讯相关的信号传导机制。在培养中，细胞滋养层细胞在接种后1 - 2天内发育成细胞聚集体，然后形成合体。在这种体外分化过程中，对缝隙连接通讯进行了测量，相邻细胞之间耦合的最大百分比出现在第4天。在存在500 mIU/ml hCG的情况下，在培养的各个阶段耦合细胞的百分比均增加，并且在2天后观察到耦合细胞的比例最高，而在对照条件下是4天后。hCG的作用具有特异性，因为添加热灭活的hCG、促卵泡生成素（oFSH）或促甲状腺激素（bTSH）均不影响滋养层细胞中的缝隙连接通讯。添加多克隆hCG抗体降低了基础缝隙连接通讯以及对外源hCG的反应。此外，8-溴腺苷酸环化酶（8Br-cAMP，0.5或1 mM）模拟了hCG的刺激作用。有趣的是，特异性蛋白激酶A抑制剂H-89（2 microM）显著降低了对hCG（500 mIU/ml）和8Br-cAMP（0.5 mM）对滋养层缝隙连接通讯刺激的反应。特异性蛋白激酶C抑制剂钙泊三醇（1或2 microM）强烈刺激缝隙连接通讯。总之，通过gap-FRAP方法证明在细胞融合之前存在缝隙连接通讯可被视为标记滋养层分化开始的一个客观且生理的标准。hCG，一种由滋养层产生的激素，以及两种信号传导机制参与了这一现象。
