First published online 30 October 2007
doi: 10.1242/jcs.011775
Journal of Cell Science 120, 4016-4024 (2007)
Published by The Company of Biologists 2007
In vivo analysis of undocked connexin43 gap junction hemichannels in ovarian granulosa cells
Dan Tong1,2,3,4,
Tony Y. Li1,2,3,4,
Kathryn E. Naus1,2,3,4,
Donglin Bai1 and
Gerald M. Kidder1,2,3,4,*
1 Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada
2 Department of Obstetrics and Gynecology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada
3 Department of Paediatrics, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON N6A 5C1, Canada
4 Children's Health Research Institute, 800 Commissioners Road East, London, ON, Canada
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Fig. 1. The presence of undocked Cx43 hemichannels in mouse granulosa cells demonstrated by Lucifer yellow (LY) dye uptake or ATP release triggered by divalent-cation-free (DCF) solution or mechanical stimulation. (A-C) Representative experiments show LY uptake by wild-type granulosa cells in regular extracellular solution (A), DCF solution (B) or DCF solution with 200 µM carbenoxolone (CBX; C). The left column shows blue nuclear staining with Hoechst 33342, the middle column shows intracellular green fluorescence resulting from the uptake of LY and the right column shows the overlay. Scale bar, 10 µm. (D) Quantitative data, expressed as the percentage of cells loaded with LY relative to the total number of cells in a field of view, counted after Hoechst staining (mean ± s.e.m. of four to six different coverslips under each condition) of wild-type (WT) or Cx43-deficient (Cx43-KO) cells. (E) The data are plotted as the percentage of cells loaded with LY relative to the total number of cells in the field of view (mean ± s.e.m. of three to four different coverslips under each condition) after mechanical stimulation (mech). See Results for the blockers used. (D,E) ***P<0.001 versus baseline (one-way ANOVA and Tukey's post-hoc test). (F) Representative experiments show LY (middle) and rhodamine dextran (bottom) uptake by wild-type granulosa cells after mechanical stimulation. Cell numbers were counted by nuclear staining with Hoechst 33342 (top). Scale bar, 50 µm. (G) Data represent ATP release by wild-type and Cx43-deficient (Cx43-KO) granulosa cells triggered by DCF solution with or without 200 µM CBX. The data were normalized to baseline value (wild-type cells in regular ECS, broken line). ***P<0.001 versus baseline (two-way ANOVA and Bonferroni post-hoc test).
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Fig. 2. RT-PCR analysis of the mRNAs encoding pannexins and the P2X7 receptor from whole ovary and granulosa cells. (A) Representative results of the analysis for Panx1, Panx2 and Panx3 transcripts. (B) Semiquantitative measurement of Panx1 transcripts in granulosa cells from wild-type (WT) fetuses (E18.5, WT pre-GC), wild-type 3- to 4-week-old mice (WT GC) and Cx43-deficient ovaries 3-4 weeks after grafting into adult kidney capsules (KO GC). The bar graph (bottom) shows the average relative amplicon band intensity (Panx1:Actb amplicon ratio), which was determined by densitometry (n=4; P>0.05, one way ANOVA). (C) Similarly, semiquantitative RT-PCR was used to compare P2rx7 transcript levels in wild-type and Cx43-deficient granulosa cells (n=4; P>0.05, unpaired t-test).
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Fig. 3. Cys-less Cx43 structure, expression and function in Cx43-deficient granulosa cells. (A) Cx43 protein structure showing the six cysteine sites (red) on the extracellular loops that were mutated to form cys-less Cx43. (B) Schematic structure of the AP2 retrovector showing the segment encoding either cys-less Cx43 or wild-type Cx43 followed by an IRES-linked sequence encoding EGFP. The vector control contained only the IRES and the EGFP sequence. (C) Expression of wild-type (WT) Cx43 and cys-less Cx43 in Cx43-deficient granulosa cells. Infected cells were identified by EGFP expression (left column). Cx43 immunostaining showed mainly membrane expression of wild-type Cx43 with numerous gap junction-like plaques, and both membrane and cytoplasmic localization of cys-less Cx43. The overlay is shown in the right column. (D) Representative experiments showing the transfer of LY to neighboring cells after microinjection into one cell (asterisk) among the three treatment groups. Quantification of dye transfer (percentage of cells passing injected LY to more than one neighboring cell) is shown in the bar graph. The number of cells injected is shown above each bar. Scale bars, 10 µm.
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Fig. 4. Comparison of wild-type and cys-less Cx43 hemichannel function in granulosa cells. (A) Propidium iodide (PI) uptake (red, central column) of Cx43-deficient granulosa cells infected with wild-type (WT) Cx43 (top row), cys-less Cx43 (middle row) or control vector (bottom row) under DCF conditions. Scale bar, 20 µm. (B) Quantification of PI uptake (percentage of EGFP-expressing cells loaded with PI) in regular ECS (baseline), in DCF solution or in DCF solution with 200 µM CBX in the three cell types, as shown under each group of bars. Data are expressed as mean ± s.e.m. of three to four different experiments under each condition. (C) Quantification of ATP release by Cx43-deficient granulosa cells infected with wild-type Cx43, cys-less Cx43 or control vector under regular ECS, DCF solution or DCF solution with 200 µM CBX. All data were normalized to baseline value (wild-type cells under regular ECS, broken line). n=4, 4 and 3 for the three cell groups, respectively. (B,C) ***P<0.001 versus control (two-way ANOVA and Bonferroni post-hoc test).
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Fig. 5. Folliculogenesis in reaggregated ovaries. (A) In ovaries constructed with Cx43-deficient granulosa cells infected with wild-type (WT) Cx43, follicles of all developmental stages were observed (left; white arrow points to an antral follicle), whereas, in ovaries constructed with Cx43-deficient granulosa cells infected with cys-less Cx43 (middle) or control vector (right), only primary (unilaminar) follicles were observed. Scale bars, 50 µm. Notice the different magnifications in the left, middle and right micrographs. (B) Distribution of follicle stages in the three different types of reaggregated ovaries as indicated below each group of bars. Between 22 and 72 follicles from three to four grafted ovaries were categorized for each group. A significant difference (P<0.001 wild type versus cys-less; P<0.01 wild type versus vector control, chi-square) was observed between ovaries expressing wild-type Cx43 and cys-less Cx43 in granulosa cells. Only the ovaries constructed with wild-type Cx43 were seen to contain antral follicles. (C) Expression of wild-type Cx43 and cys-less Cx43 in reaggregated ovaries. Infection was identified by EGFP expression (top row). Cx43 immunostaining (red, bottom row) showed mainly membrane expression of wild-type Cx43 with numerous gap junction-like plaques (left column) and predominately cytoplasmic localization of cys-less Cx43 (middle column). No Cx43 signal was detected in the empty-vector-infected ovaries (right column). Scale bar, 20 µm.
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© The Company of Biologists Ltd 2007
