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First published online 4 April 2006
doi: 10.1242/jcs.02892

Journal of Cell Science 119, 1715-1722 (2006)
Published by The Company of Biologists 2006
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Ablation of connexin43 in uterine smooth muscle cells of the mouse causes delayed parturition

Britta Döring1, Oksana Shynlova2, Prudence Tsui2, Dominik Eckardt1, Ulrike Janssen-Bienhold3, Franz Hofmann4, Susanne Feil4, Robert Feil4,5, Stephen J. Lye2,6,7 and Klaus Willecke1,*

1 Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Römerstr. 164, 53117 Bonn, Germany
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
3 Neurobiologie, Carl von Ossietzky Universität Oldenburg, Oldenburg, Germany
4 Institut für Pharmakologie und Toxikologie, Technische Universität München, München, Germany
5 Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany
6 Institute of Medical Science, University of Toronto, Toronto, ON, Canada
7 Departments of Obstetrics and Gynecology and Physiology, University of Toronto, Toronto, ON, Canada


Figure 1
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  		Fig. 1. LacZ activation upon conditional Cx43 deletion. (A) A silent lacZ reporter gene (NLS-lacZ) was integrated into the floxed Cx43 allele. After Cre/loxP-mediated rearrangement of the Cx43-coding region, indicated by loxP sites (triangles), the lacZ gene is activated and the ß-galactosidase protein is expressed. (B) The CreERT2 fusion protein is specifically transcribed under control of the SM22{alpha} promoter in smooth muscle cells. In the cytosol, CreERT2 is associated with the heat shock protein 90 (HSP 90). After application of the ligand (tamoxifen) this complex dissociates and CreERT2 is translocated into the nucleus, where deletion of the floxed coding region occurs.

 

Figure 2
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  		Fig. 2. LacZ staining and immunofluorescence analyses of the uterus of different genotypes. (A-C) LacZ staining of untreated (A), tamoxifen-treated (B) Cx43fl/fl:SM-CreERT2 and Cx43del/+ (C) uterine tissue was performed according to standard protocols. (D,E) Cryosections of untreated (D), tamoxifen-treated (E) Cx43fl/fl: SM-CreERT2 uterus were analysed by immunofluorescence using antibodies to Cx43 (red) and smooth muscle actin (green). Inserts represent magnified images. CM, circular muscle layer; LM, longitudinal muscle layer; S, stroma. Bar, 50 µm.

 

Figure 3
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  		Fig. 3. Immunofluorescence analyses of untreated (A,C,E) and tamoxifen-treated (B,D,F) Cx43fl/fl:SM-CreERT2 uterus. (A,B) Cryosections of uterine tissue were analysed using antibodies to Cx26 (red) and smooth muscle actin (green). Most of the signals were detected in the glandular epithelium (GE). (C,D) Immunofluorescence of Cx40 (red) and smooth muscle actin (green) in uterine cryosections revealed only sparse signals in myometrial SMCs but abundant staining of blood vessels (BV). (E,F) Staining of uterine tissue using antibodies to Cx45 (red) and smooth muscle actin (green) revealed extensive expression in myometrial smooth muscle cells. The colocalization of connexin and smooth muscle actin signals is visualized in yellow. BV, Blood vessel; CM, circular muscle layer; GE, glandular epithelium; LM, longitudinal muscle layer; S, stroma. Bar, 100 µm.

 

Figure 4
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  		Fig. 4. Immunoblot analyses of uterine tissue homogenates of two untreated and tamoxifen-treated Cx43fl/fl:SM-CreERT2 mice. Membranes were probed with primary antibodies to Cx26, Cx40, Cx45 and Cx43. Normalization to ß-actin levels revealed that the amount of Cx43 protein was reduced by 69±5% in tamoxifen-treated Cx43fl/fl:SM-CreERT2 animals, whereas the levels of other connexin proteins investigated remained unchanged (n=3).

 

Figure 5
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  		Fig. 5. Representative images of microinjections in confluent cultures of myometrial cells with the gap junction permeable tracer Lucifer Yellow (A,C) and phase-contrast images of the same injection (B,D). Dye transfer shown in cultured untreated (A,B) and tamoxifen-treated (C,D) Cx43fl/fl:SM-CreERT2 SMCs 5 minutes after Lucifer Yellow microinjection into a single cell (asterisks). The restricted transfer in tamoxifen-treated cells contrasts to the basal transfer in untreated cells. (E,F) Microinjected cells (green) of tamoxifen-treated Cx43fl/fl:SM-CreERT2 mice were fixed and stained using antibodies to Cx43 (red). Dye transfer was only present in Cx43-expressing cells (white arrows in E) but absent in non-expressors (F). The overlay of Cx43 and Lucifer Yellow dye is visualized in yellow. Bar, 50 µm (A-D); 20 µm (E,F).

 

Figure 6
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  		Fig. 6. (A,B) Immunofluorescence of Cx43 (red) and smooth muscle actin (green) in cultured myometrial cells from untreated (A) and tamoxifen-treated (B) Cx43fl/fl:SM-CreERT2 mice. Decrease in immunoreactive Cx43 in tamoxifen-treated Cx43fl/fl:SM-CreERT2 cultured SMCs is shown in contrast to untreated control cells. The colocalization of Cx43 and smooth muscle actin bundles is shown in yellow. (C) Immunoblot analyses of protein extracts (100 µg) of untreated and tamoxifen-treated Cx43fl/fl:SM-CreERT2 SMC cultures using antibodies to Cx26, Cx40, Cx45 or Cx43. Normalization to ß-actin levels revealed a reduction of Cx43 protein by 65±3%, levels of other connexin proteins investigated remained unchanged. Bar, 50 µm.

 

Figure 7
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  		Fig. 7. Effect of Cx43 deletion on the length of gestation. Pregnant mice were monitored for delivery of the first pup. Delivery until 8 a.m. was considered normal, delivery after 9 a.m. was classified as a prolonged birth process. (A) Percentages of normal and prolonged parturition of Cx43del/+ and untreated (ut.) as well as tamoxifen-treated (t.) Cx43fl/fl and Cx43fl/fl:SM-CreERT2 mice. (B) Untreated and tamoxifen-treated Cx43fl/fl:SM-CreERT2 animals are shown as the moving average of animals delivering at certain time points between day 19 and day 22.

 

Figure 8
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  		Fig. 8. Relative OTR (A), FP (B) and Fos (C) mRNA levels during pregnancy and labour as well as changes in myometrial plasma progesterone (D). Samples were taken from mice at 10-10.30 a.m. on day 16 and during labour. Data are expressed as mean ± s.e.m. (n=5).

 




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