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First published online 7 December 2004
doi: 10.1242/jcs.01587

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Interplay between paracrine signaling and gap junctional communication in ovarian follicles

¹ Departments of Physiology and Pharmacology, Obstetrics and Gynaecology, and Paediatrics, The University of Western Ontario, London, Ontario N6A 5C1, Canada
² Child Health Research Institute, 800 Commissioners Road East, London, Ontario N6C 2V5, Canada
³ Center for Cancer Therapeutics, Ottawa Regional Cancer Centre, 503 Smyth Road, Ottawa, Ontario K1H 1C4, Canada

View larger version (72K): [in a new window]   Fig. 1. GDF9 expression is maintained in oocytes of Cx43 null mutant follicles. Wild-type (A), Gja1–/Gja1– (B) and Gdf9–/Gdf9– (C) ovary sections were immunostained for GDF9. Bar, 50 µm.

View larger version (163K): [in a new window]   Fig. 2. Cx43 expression is maintained in GDF9 null mutant follicles. Gdf9+/Gdf9– (A,B) and Gdf9–/Gdf9– (C,D) ovary sections were immunostained for Cx43. Punctate immunoreactivity characteristic of gap junctions is evident between the granulosa cells of both control (B) and null mutant (D) follicles. Bar, 50 µm.

View larger version (109K): [in a new window]   Fig. 3. Intercellular coupling is maintained in GDF9 null mutant follicles. Dichlorofluorescein was injected into a single granulosa cell (asterisk) of a Gdf9+/Gdf9– (A) and a Gdf9–/Gdf9– follicle (B). The extent of intercellular dye spread did not differ significantly between the two genotypes (C). These images are representative of the ten control and 13 mutant follicles that were tested. Bar, 50 µm.

View larger version (67K): [in a new window]   Fig. 4. Granulosa cell proliferation is reduced in Cx43 null mutant follicles as revealed by PCNA staining. (A,B) In wild-type ovaries of the CD1 strain, virtually every granulosa cell is positive for PCNA. (C) In Gja1–/Gja1– ovaries of the C57BL/6 strain, many granulosa cells of the arrested primary follicles lacked PCNA immunoreactivity. (D) Gdf9–/Gdf9– follicles had very little PCNA immunoreactivity. (E) In Gja1–/Gja1– ovaries of the CD1 strain, some primary follicles (single arrow) stained intensely for PCNA whereas many granulosa cells of secondary follicles (double arrows) stained weakly or were negative for PCNA. (F) Quantitative differences in the frequency of PCNA staining between mutant lines are evident. Bars represent the % of PCNA-positive cells in all follicles and error bars indicate the s.e. Different letters above the bars indicate significant differences between mutant lines within the same genetic background (P<0.01 in all cases). The number of follicles is indicated in parentheses above each bar. Bar, 50 µm.

View larger version (74K): [in a new window]   Fig. 5. Granulosa cell apoptosis is not increased in Cx43 null mutant follicles as indicated by the TUNEL assay. (A) In wild-type ovaries, apoptosis can be readily detected in some follicles, indicative of atresia. (B) Very few apoptotic cells were observed within Gja1–/Gja1– follicles, although apoptotic cells were observed in the stroma. (C) GDF9 null mutant ovaries did not display evidence of any apoptotic granulosa or stromal cells. (D) A positive control section, treated with DNase before TUNEL staining. (E) A quantitative comparison of TUNEL staining in wild-type and Cx43 null mutant follicles failed to reveal a significant difference (P>0.3). The bars indicate the total pixel intensity normalized to follicle area, the error bars indicate s.e. and the number of follicles surveyed is indicated in parentheses above each bar. Bar, 100 µm.

View larger version (120K): [in a new window]   Fig. 6. Carbenoxolone completely blocks dye coupling in cultured granulosa cells whereas its inactive analog does not. Follicles were cultured for 24 hours without drug (A,B), with 200 µM glycyrrhizic acid (C,D) or with 200 µM carbenoxolone (E,F), then tested for dye coupling using Lucifer Yellow. The asterisk in A,C and E indicates the injected cell. O, oocyte. Bar, 50 µm.

View larger version (36K): [in a new window]   Fig. 7. Exogenous GDF9 can override the impairment of proliferation in Cx43 null mutant granulosa cells (CD1 strain) that lack gap junctional coupling. The frequency of PCNA staining for each treatment group is normalized to the respective control. The error bars indicate s.e.; different letters above the bars indicate significant differences (P<0.01). The number of follicles examined is indicated in parentheses above each bar.

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