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First published online November 21, 2007
doi: 10.1242/10.1242/jcs.03488

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Rescue of oogenesis in Cx37-null mutant mice by oocyte-specific replacement with Cx43

¹ Department of Physiology and Pharmacology, Obstetrics and Gynaecology, Paediatrics, The University of Western Ontario, London, Ontario N6A 5C1, Canada
² Department of Oncology and Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario N6A 5C1, Canada
³ Children's Health Research Institute, 800 Commissioners Road East, London, Ontario N6C 2V5, Canada

View larger version (58K): [in this window] [in a new window]   Fig. 1. Production of Zp3-Gja1 transgenic mice. (A) Structure of the Zp3-Gja1 transgene (not to scale). The transgene consisted of a 6 kb fragment of the Zp3 promoter, a 1.3 kb Gja1 exon 2 cDNA containing the entire rat Cx43 coding sequence (thin line), and a cassette consisting of a MYC-tag-encoding sequence and polyadenylation signal. The solid black section of the cDNA indicates the probe for Southern blotting and arrowheads represent the primers for PCR genotyping and RT-PCR. (B-D) Functional testing of Cx43-MYC. (B) Lucifer yellow dye was injected into a single cell (asterisk) of a population of gap-junctional-communication-deficient HeLa cells expressing the transgene. The dye readily passed to neighboring cells, as revealed by fluorescence microscopy (image at right). (C) The typical result of no dye transfer after injecting Lucifer yellow into un-infected HeLa cells not expressing the transgene. (D) Dye was injected into HeLa cells expressing native (not MYC-tagged) Cx43. (E) A typical Southern blot showing detection of the transgene in a sample of offspring. Digestion of tail-snip DNA with BanI generated a 1.3 kb fragment specific to the transgene in addition to the endogenous genomic fragment at 4.3 kb. Lanes 1-7 indicate results from seven of the potential founder offspring tested; lanes 5 and 7 represent transgenic lines 2b and 4, respectively, which were analyzed in the present study. Bar, 50 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Verification of transgene expression by probing for the MYC tag. (A) RT-PCR amplified a segment of the MYC-encoding sequence (upper band, 420 bp) from oocyte RNA, but not granulosa cell RNA, of both transgenic lines. A segment of mRNA encoding glyceraldehyde phosphate dehydrogenase (GAPDH; lower band, 299 bp) was amplified as a positive control. WT, wild-type ovary; blank, negative control (no template). (B) The MYC epitope (upper band) was detected by western blotting in ovaries of both transgenic lines, but not in ovaries of Cx37-knockout (Cx37 KO) or wild-type (WT) mice. The lower band is GAPDH (loading control). (C) Immunostaining for the MYC epitope in ovary sections revealed its localization at the oocyte surface in connexin-replacement mice and its absence from wild-type or Cx37-deficient mice. Oocytes are indicated by arrows. Bar, 30 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Expression of Cx43 at the oocyte surface in connexin-replacement follicles. (A) Detection of Cx43 in isolated oocytes by western blot. Oocyte lysates from transgenic lines 2b and 4 along with lysates from Cx37-deficient (Cx37 KO) and wild-type (WT) oocytes were separated electrophoretically, blotted, and the blot probed sequentially with Cx43 antibody (top) and GAPDH antibody (bottom row). (B) Localization of Cx43 in sections of follicles. Whereas no Cx43 was detected at the oocyte surface in wild-type or Cx37-deficient follicles, small foci of Cx43 immunoreactivity typical of gap junction plaques (arrowheads) were detected at the oocyte surface in Cx37-deficient follicles carrying the Cx43-expressing transgene. Bar, 20 µm.

View larger version (110K): [in this window] [in a new window]   Fig. 4. Restoration of oocyte–granulosa-cell coupling in Cx37-deficient follicles by replacement with Cx43. Injection of Lucifer yellow into oocytes (O) of follicles from Cx37-deficient mice (A,B) did not result in dye transfer to the surrounding granulosa cells. By contrast, dye injected into oocytes of follicles from Cx37-deficient mice carrying the transgene (C,D) and from wild-type mice (E,F) passed readily into the surrounding granulosa cells. Bar, 50 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Restoration of folliculogenesis in Cx37-deficient ovaries by replacement with Cx43. Sections of wild-type ovaries (A) and ovaries from Cx37-deficient mice carrying the transgene (C) revealed all stages of folliculogenesis, including large antral follicles, whereas sections from Cx37-deficient ovaries without the transgene (B) did not contain follicles beyond early antral stages. Bar, 100 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Rates of follicle growth in wild-type, connexin-replacement and Cx37-knockout ovaries. Mean follicle diameter was determined for samples of 195 follicles of four wild-type mice, 124 follicles of five connexin-replacement mice and 84 follicles of three Cx37-knockout mice. The chi-square test was used to compare the frequency distributions of follicle types between genotypes, whereas two-way ANOVA with Bonferroni test was used to test for interaction between genotype and follicle size. Although connexin-replacement follicles achieved the same mean diameter as wild-type follicles in the early antral stage, mean follicle diameter in Cx37-knockout follicles was significantly less. Bars represent means ± s.e.m.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Rates of oocyte growth in wild-type, connexin-replacement and Cx37-knockout ovaries. Mean oocyte diameter was determined for the same follicles analyzed for Fig. 5. The chi-square test was used to compare the frequency distributions of follicle types between genotypes, whereas two-way ANOVA with Bonferroni test was used to test for interaction between genotype and oocyte size. Although oocytes of connexin-replacement follicles achieved the same mean diameter as oocytes of wild-type follicles in the early antral stage, mean oocyte diameter in Cx37-knockout follicles was significantly less (P<0.001).