Journal: The Journal of Biological Chemistry
Article Title: Regulation of Oocyte and Cumulus Cell Interactions by Intermedin/Adrenomedullin 2 *
doi: 10.1074/jbc.M111.297358
Figure Lengend Snippet: Blockage of IMD/ADM2 signaling in vivo led to aberrant oocyte development and reduced cyclin D2 expression in growing follicles. A, intrabursal injection of an IMD antagonist led to increased incidences of fragmented or shrunk oocytes in secondary and tertiary follicles (right panels). By contrast, follicles of contralateral ovaries injected with PBS exhibited normal morphology (left panels). Fragmented oocytes in large antral follicles of an IMD antagonist-treated ovary are indicated by arrows (bottom right). Similar results were obtained in three separate experiments. B, ovaries that had received the intrabursal injection of PBS exhibited robust cyclin D2 expression in granulosa and cumulus cells of most growing follicles (brown stain, left). The intensity of immunoreactive cyclin D2 staining in contralateral ovaries that had received the IMD antagonist was restricted to a fraction of follicles (right). Images of representative ovaries include original photographs and the digitized versions for HistoQuest analysis (with a light blue background). C, scattergrams of staining intensity of whole ovaries that had received PBS (top) or the IMD antagonist injection (bottom). Results are displayed in dot plots, with the intensity of dots on the x axis and the DAB staining area on the y axis. Analyses of time-matched sections based on the HistoQuest software showed that the IMD antagonist treatment reduced the overall staining by more than 32%. The cut-off values for background staining were chosen manually using the forward/backward gating tool of the HistoQuest software. D, tissue cytometric analysis of staining in individual follicles of time-matched sections. Intrabursal injections of an IMD antagonist down-regulated cyclin D2 expression in secondary, small antral, and large antral follicles (p < 10−11). Eleven representative sections from 3–4 independent ovaries were analyzed in each treatment group. A total of 170 secondary, 151 early antral, and 236 large antral follicles were scanned individually and analyzed. Data are represented as the mean ± S.E. (error bars). The number of follicles analyzed in each data point is shown within or above the bar graph.
Article Snippet: Five-micron-thick paraffin sections of fixed ovaries were immunostained with mouse monoclonal antibodies against cyclin D2 (Abcam Plc., AB3087).
Techniques: In Vivo, Expressing, Injection, Staining, Software