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. 2011 Oct 18;286(50):43193–43203. doi: 10.1074/jbc.M111.297358

FIGURE 3.

FIGURE 3.

IMD/ADM2 signaling is important for cumulus cell survival and normal cell-cell contacts in COCs. A, treatments of FSH or IMD reduced the detachment of cumulus cells from COCs cultured in vitro. The images were taken at 40 h after culture (top). The percentage of intact COCs was significantly increased by IMD or FSH at 40–50 h after culture (bottom, n = 4). *, significantly different from controls (p < 0.05). Similar results were obtained in five separate experiments. B, detection of cumulus-cell apoptosis by staining with propidium iodide (red fluorescence, 562–588 nm) and Hoechst 33342 (blue fluorescence, 461 nm). The staining of dead cells by propidium iodide was extensive in control COCs at 20 h after culture (top left). The staining in IMD-treated (top middle) or FSH-treated (top right) COCs was sparse at this time. At 40 h after culture, most oocytes in the control group were stripped of cumulus cells (bottom left panels; DF, dark field; BF, bright field). C, ablation of IMD signaling in COCs by an anti-IMD antibody led to accelerated detachment of cumulus cells from oocytes. The percentage of intact COCs was significantly reduced by the anti-IMD antibody treatment at 40 h after culture (bottom, n = 4). By contrast, the percentage of intact COCs in cultures was not affected by a control antibody. Staining with a mitochondria membrane potential sensor, JC-9, verified that COCs treated with the anti-IMD antibody have a perturbed membrane potential (orange-red fluorescence, top) at 2 h after culture, whereas COCs in the control group or cultures with 100 nm IMD exhibited normal mitochondrial membrane potentials (green fluorescence, top). D, treatment with a high dose of CGRPβ (300 nm) prevented the anti-IMD antibody-induced detachment of cumulus cells from COCs. E, IMD and FSH stimulated connexin 43 transcript expression in cumulus cells at 24 h after culture (p < 0.01). F, cotreatment with an anti-IMD antibody blocked the protective effect of FSH on the loss of intact COCs in cultures. G, cotreatment with an IMD antagonist (Ac-IMD(17–47)), but not with a CGRP antagonist (CGRP(8–37)), significantly reduced the protective effect of FSH on the loss of intact COCs in cultures. *, significantly different from controls (p < 0.05). Similar results were obtained in three separate experiments. Error bars, S.E.