Fig. 7. Immunostaining and intercellular coupling via gap junctions in primary granulosa cells. (A) Immunostaining for Cx43 (green) in granulosa cells in vivo and (B) in vitro showed only a few scattered gap junction-like plaques in Gja1^Jrt/+ x FVB granulosa cells. O, oocyte. Scale bars: 20 µm. (C,D) Lucifer dye injection (asterisks mark injected cells) revealed strong dye coupling among wild-type granulosa cells (+/+), whereas dye coupling among granulosa cells from cultured Gja1^Jrt/+ x FVB mutant follicles was severely restricted. O, oocyte. Scale bar: 50 µm. (E) Graphical representation of the mean number of neighboring cells receiving dye after injection where the number of cells tested is shown in parentheses above each bar. (F) The mean conductance of cells that were electrically coupled, as indicated by capacitative current transients, showed that coupling was severely reduced in Gja1^Jrt/+ x FVB granulosa cells. The number of cells tested is shown in parentheses above each bar. (G) Representative current transients from wild type (+/+), Gja1-null (Gja1^-/Gja1^-) and Gja1^Jrt/+ x FVB granulosa cells show that Gja1^Jrt/+ x FVB granulosa cells exhibited either very weak coupling or a complete lack of coupling (12/17 weakly coupled; 5/17 not coupled). In vivo and in vitro experiments were performed on primary granulosa cells isolated from ovaries on both genetic backgrounds with similar results. (H) Western blots reveal that the level of total Cx43 and especially the slower migrating phosphorylated species, was greatly reduced in heart and ovary from Gja1^Jrt/+ versus wild-type (+/+) mice (11 weeks). GAPDH was used as a gel loading control.