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First published online 11 May 2004
doi: 10.1242/jcs.01124

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Selective assembly of connexin37 into heterocellular gap junctions at the oocyte/granulosa cell interface

Gregory I. Veitch^1,2, Joanne E. I. Gittens^1,2,5, Qing Shao⁴, Dale W. Laird^1,4,* and Gerald M. Kidder^1,2,3,5,*,

¹ Departments of Physiology and Pharmacology, The University of Western Ontario, London, Ontario N6A 5C1, Canada
² Departments of Obstetrics and Gynaecology, The University of Western Ontario, London, Ontario N6A 5C1, Canada
³ Department of Paediatrics, The University of Western Ontario, London, Ontario N6A 5C1, Canada
⁴ Departments of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario N6A 5C1, Canada
⁵ Child Health Research Institute, 800 Commissioners Road East, London, Ontario N6C 2V5, Canada

View larger version (131K): [in a new window]   Fig. 1. A new anti-Cx37 antibody is specific for Cx37 gap-junction plaques. NRK cells were infected with a retroviral vector encoding Cx37 tagged with GFP (B, green) and immunolabelled with anti-Cx37 antibodies (A, red). Co-localization is indicated by yellow color and arrows (C). Bars=10 µm.

View larger version (83K): [in a new window]   Fig. 2. Connexin localization in the cumulus-oocyte complex (COC) as detected by confocal immunofluorescence microscopy. In single-labelling experiments, Cx37 (anti-Cx37, red) was localized to gap-junction plaques (arrow) on the surface of the oocyte (O), beneath the zona pellucida (ZP) in both cultured COCs (A) and follicle sections (B). In other single-labelling studies, Cx43 (anti-Cx43, green) was localized to plaques (arrow) between contacting granulosa cells (GC) in follicle sections (C). In double-labelling experiments, no Cx37 immunostaining was detected when Cx37-deficient COCs were immunolabelled (D, note lack of red signal) or when the anti-Cx37 antiserum was competed with the immunizing peptide in cultured COCs (E, note lack of red signal). Cx43 was localized to plaques between granulosa cells in D and E. Bars=10 µm.

View larger version (99K): [in a new window]   Fig. 3. Double-immunofluorescent labelling of cultured COCs revealed that Cx37 is localized to gap-junction plaques at the oocyte surface whereas Cx43 is localized to plaques between adjacent granulosa cells. Arrows indicate punctate localization of Cx37 (anti-Cx37, red) at the border between the oocyte (O) and granulosa cells (GC). Populations of intracellular Cx37 are visible within the oocyte and at candidate gap-junction plaques between adjacent GC. Cx43 (anti-Cx43, green) is localized to plaques between granulosa cells and is rarely found at the oocyte surface. Bar=10 µm.

View larger version (111K): [in a new window]   Fig. 4. Oocyte-granulosa cell coupling is maintained in the absence of Cx43. Wild type (A,B) and Cx43-deficient (C,D) follicles were isolated, cultured, and oocytes were microinjected with the gap-junction-permeable dye, Lucifer yellow. Dye transferred throughout the wild-type follicle after oocyte injection (B), whereas dye spread to only the first layer of granulosa cells directly in contact with the oocyte in Cx43-deficient follicles (D). Bar=50 µm.

View larger version (82K): [in a new window]   Fig. 5. Cx37 is essential for coupling between the oocyte and granulosa cells. Wild-type oocytes (preloaded with calcein) transfer dye extensively to wild type granulosa cells (A,D) whereas no dye transfer occurs when Cx37 is missing from either the granulosa cells (B,E) or the oocyte (C,F). Bar=25 µm.

View larger version (43K): [in a new window]   Fig. 6. Cx37 is recruited to sites where seeded Cx37-positive oocytes contact granulosa cells. Wild-type (B) or Cx37-deficient (C) denuded oocytes were seeded onto cultured granulosa cell monolayers for 4 hours, fixed, and immunofluorescently labelled for Cx37 (anti-Cx37, red) and Cx43 (anti-Cx43, green). The oocytes became detached from the granulosa cells during fixation. Wild-type oocytes recruited Cx37 (arrows) to sites of oocyte-granulosa cell contact (B), whereas granulosa cells contacting Cx37-deficient oocytes (C) or not in contact with oocytes (control, A) exhibited little or no localized Cx37. RT-PCR using Cx37 specific primers was performed with RNA isolated from wild-type mouse lung, lung from Cx37-deficient mice, and cultured mouse granulosa cells (D). Amplicons of the predicted size were found in wild-type lung and granulosa cells, but were absent from lungs obtained from Cx37-deficient mice and when RNA templates were absent (negative control). Bar=10 µm.

View larger version (122K): [in a new window]   Fig. 7. Distribution/localization of Cx37 is not affected by BFA treatment. Cultured COCs were treated with 10 µg/ml of BFA for 5 hours to block connexin transport and gap-junction plaque regeneration. Immunostaining for anti-giantin (A, red) revealed that the Golgi apparatus was effectively disrupted upon BFA treatment (C, red). However, Cx43 gap-junction plaques between adjacent granulosa cells (A,C; anti-Cx43, green) and Cx37 at oocyte-granulosa cell interfaces (B,D; anti-Cx37, red) were not markedly altered by BFA treatment. To enhance cellular architecture, COCs were double-labelled with a non-specific antibody that provided background immunofluorescence (B,D, green). Bars=10 µm (A,C); 25 µm (B,D).