In vivo analysis of undocked connexin43 gap junction hemichannels in ovarian granulosa cells.
Connexin43 (Cx43, encoded by Gja1) is required for ovarian follicle development in the mouse. It is strongly expressed in granulosa cells, in which it forms intercellular gap junction channels that couple the cells metabolically. However, recent evidence indicates that undocked gap junction hemichannels can also have physiological roles such as mediating the release of small messenger molecules, including ATP. In this study, the presence of undocked Cx43 hemichannels in granulosa cells was revealed by dye uptake induced either by mechanical stimulation or by the reduction of extracellular divalent cations, both of which are known triggers for hemichannel opening. ATP release was also detected, and could be abolished by connexin-channel blockers. None of these putative hemichannel-mediated activities were detected in Cx43-deficient granulosa cells. Therefore, we hypothesized that hemichannels account for the essential role of Cx43 in folliculogenesis. To test this, a Cx43 mutant lacking the conserved cysteines on the extracellular loops (cys-less Cx43), reported to form hemichannels but not intercellular channels, was retrovirally expressed in Cx43-deficient granulosa cells. The infected cells were then combined with wild-type oocytes to make reaggregated ovaries, which were grafted into host kidneys. Although re-introduction of wild-type Cx43 rescued folliculogenesis, introduction of cys-less Cx43 did not. Therefore, although Cx43 gap junction hemichannels might play a role in ovarian folliculogenesis, their contribution does not supplant the need for intercellular gap junction channels.
Tong
Dan
D
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada.
Li
Tony Y
TY
Naus
Kathryn E
KE
Bai
Donglin
D
Kidder
Gerald M
GM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004396:Coloring Agents / Q000378:metabolism; D018031:Connexin 43 / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine; D004285:Dogs; D005260:Female; D017629:Gap Junctions / Q000378:metabolism*; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D058486:Receptors, Purinergic P2X7
D004396:Coloring Agents; D018031:Connexin 43; D009419:Nerve Tissue Proteins; C546469:P2rx7 protein, mouse; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D003545:Cysteine