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Synaptosomal-Associated Protein 25 Gene Expression Is Hormonally Regulated during Ovulation and Is Involved in Cytokine/Chemokine Exocytosis from Granulosa Cells

Department of Applied Animal Science (M.S., Y.Yan., T.O., Y.Yam.), Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; Department of Molecular and Cellular Biology (M.S., J.S.R.), Baylor College of Medicine, Houston, Texas 77030; Department of Internal Medicine (V.S.), University of Texas Medical Branch, Galveston, Texas 77555; and Department of Neurosciences (M.C.W.), University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131

Address all correspondence and requests for reprints to: Masayuki Shimada, Ph.D., Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, 1-4-4, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan. E-mail: mashimad{at}hiroshima-u.ac.jp.

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.

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