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First published online September 5, 2008
doi: 10.1242/10.1242/dev.025494

Development 135, 3229-3238 (2008)
Published by The Company of Biologists 2008
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Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption

Rachael P. Norris1,*, Marina Freudzon1,*, Lisa M. Mehlmann1, Ann E. Cowan2, Alexander M. Simon3, David L. Paul4, Paul D. Lampe5,{dagger} and Laurinda A. Jaffe1,{dagger}

1 Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06032, USA.
2 Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06032, USA.
3 Department of Physiology, University of Arizona School of Medicine, Tucson, AZ 85724, USA.
4 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
5 Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.


Figure 1
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  		Fig. 1. LH reduces gap junction permeability between cumulus and mural granulosa cells. Determined by injecting Alexa Fluor 350 into the oocyte and imaging its diffusion within the follicle at 20 minutes after injection. (A,B) No LH. (C,D) LH applied 70 minutes before injecting Alexa Fluor 350. Upper panels show scanning transmission images; lower panels show Alexa Fluor 350 distribution. In A and B, Alexa Fluor 350 diffused throughout the mural granulosa cells, whereas in C and D, the Alexa Fluor 350 spread into the cumulus cells, but little or none was seen in the mural granulosa cells. (E) Regions of the follicle used for the measurements shown in F. The dashed lines indicate the approximate border between the cumulus mass and the antral space. (F) Ratio of fluorescence intensity in the mural granulosa cells to that in the inner cumulus cells, as a function of LH treatment time. Measurements were made at 20 minutes after injecting Alexa Fluor 350 into the oocyte. Bars show mean±s.e.m., and the numbers in parentheses indicate the number of follicles tested at each time point (0.5 hour=33-40 minutes, 1 hour=64-75 minutes, 2 hours=124-140 minutes, 5 hours=4.5-5.7 hours).

 

Figure 2
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  		Fig. 2. LH reduces gap junction permeability between mural granulosa cells. Determined by fluorescence redistribution after photobleaching of Alexa Fluor 488. (A) No LH. (B) LH applied 58 minutes before photobleaching. For A and B, a rectangular region was photobleached for 4.5 seconds, between the images indicated by the arrow. Each column shows images before and at various times (in seconds) after the end of the bleach. (C,D) Fluorescence intensity in the bleached region as a function of time, for the images shown in A and B. (E) Percent recovery of fluorescence intensity in the bleached region during the first minute after the bleach, as a function of LH treatment time. Bars show mean±s.e.m., and the numbers in parentheses indicate the number of follicles tested at each time point (0.5 hour=27-40 minutes, 1-2 hours=58-120 minutes).

 

Figure 3
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  		Fig. 3. LH causes MAP kinase-dependent phosphorylation of multiple serines of Cx43. (A-D) Immunoblots of follicles, ±LH for various times, probed for total Cx43, and for phosphorylation on particular serines as indicated. The fastest migrating species, marked P0, contains the unphosphorylated form, and P1, P2 and P3 are three different commonly observed phosphorylated forms. (E) Densitometric analysis of the relative amount of phosphorylation on S262 and S279/S282 of Cx43, as a function of time after LH addition. The results are expressed as the ratio of the density of the phosphorylated bands divided by the density of the bands representing total Cx43 from the same blot, and are normalized to the maximum value obtained (at 0.5 or 1 hour) for the time series. The results of four independent experiments for each antibody were combined (mean±s.e.m.). A similar time course was seen in three independent experiments with the pS255 antibody, but the signal was too low to allow meaningful quantitation. (F) Immunofluorescence images of pS279/S282 Cx43 in antral follicles with or without a 1 hour exposure to LH. The images are representative of six follicles without LH, and 11 follicles with LH. (G) Immunoblots of follicles with or without a 1 hour exposure to LH, in the presence of 10 µm of the MEK inhibitor U0126 or its inactive analog U0124. The blots were probed for phosphorylation of MAP kinase and particular serines of Cx43, and also for vinculin (lower row), to confirm that protein amounts in each lane were equivalent. Similar results were obtained in three independent experiments.

 

Figure 4
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  		Fig. 4. Immunofluorescence of Cx43 in antral follicles with or without a 1 hour exposure to LH. (A) Cumulus-oocyte region. Upper panels show scanning transmission differential interference contrast (DIC) images, middle panels show total Cx43 fluorescence, and lower panels show overlayed images. (B) Mural granulosa region, showing overlayed images as in A. The images are representative of eight follicles without LH, and seven with LH; both the cumulus and the mural granulosa regions were examined in each follicle.

 

Figure 5
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  		Fig. 5. Carbenoxolone decreases gap junction permeability and causes meiotic resumption. (A) CBX at a concentration of 100 µm reduces gap junction permeability to an undetectable level. (B) CBX at a concentration of 10 µm reduces gap junction permeability only partially. For A and B, Alexa Fluor 350 was injected into follicle-enclosed oocytes at 58-70 minutes after applying CBX, and follicles were imaged 20 minutes later. A and B are representative of the results of injections into six and 11 follicle-enclosed oocytes, respectively. See Fig. 1A,B for comparison without CBX. (C) A concentration of 100 µm CBX causes NEBD, but 10 µm CBX does not.

 

Figure 6
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  		Fig. 6. Injection of follicle-enclosed oocytes with an antibody against Cx37 decreases gap junction communication and causes meiotic resumption. (A) Immunoblot of mouse oocytes (1 µg of protein), showing the specificity of the Cx37 antibody. (B) An antral follicle in which the oocyte was injected with 2 µm of the Cx37 antibody, and then, after 5 hours, injected with Alexa Fluor 350; the follicle was imaged 20 minutes later. B is representative of the results obtained with three antral follicle-enclosed oocytes. (C) Diffusion of Alexa Fluor 350 from the oocyte to the somatic cells is inhibited only weakly when tested at 20 minutes after the Cx37 antibody injection, but is inhibited strongly at 5-6 hours. The experiments shown in C were carried out using 140-180 µm diameter preantral follicles, because these provided a technically easier system for investigating multiple conditions. Somatic cell/oocyte intensity ratios were measured at 30 minutes after injection of the Alexa Fluor 350; bars show mean±s.e.m.; n, number of follicles. (D) Injection of the Cx37 antibody causes NEBD, whereas a control IgG has no effect.

 

Figure 7
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  		Fig. 7. Inhibition of MAP kinase activation prevents gap junction closure, but does not inhibit nuclear envelope breakdown in response to LH. (A) U0126 (10 µm) inhibits LH-stimulated gap junction closure. Alexa Fluor 350 was injected at 72 minutes after applying LH, and the follicle was imaged 20 minutes later. (B) Ratio of fluorescence intensity in the mural granulosa cells to that in the inner cumulus cells, comparing follicles that had been treated with LH for 1 hour (66-72 minutes) in the presence of 10 µm U0126, and follicles without LH treatment (data from Fig. 1F). Measurements were made at 20 minutes after injecting Alexa Fluor 350 into the oocyte. Bars show mean±s.e.m. (C) A concentration of 100 µm U0126 inhibits LH-stimulated NEBD, but 10 µm U0126 does not. For B and C, n is the number of follicles tested for each condition.

 

Figure 8
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  		Fig. 8. Diagram illustrating LH-induced gap junction closure. (Left) Before LH treatment, or at 5 hours after LH application. (Right) At 0.5-2 hours after LH application.

 

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