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  • Title: Pituitary adenylate cyclase-activating polypeptide is an auto/paracrine stimulator of acute progesterone accumulation and subsequent luteinization in cultured periovulatory granulosa/lutein cells.
    Author: Gräs S, Hannibal J, Fahrenkrug J.
    Journal: Endocrinology; 1999 May; 140(5):2199-205. PubMed ID: 10218972.
    Abstract:
    Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4-6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10(-7) M induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 x 10(-9) M), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 microg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P < 0.05), 41.2% (P < 0.02), and 50% (P < 0.002), respectively], compared with nonimmune IgG (100 microg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P < 0.02), 69.2% (P < 0.05), 43.8% (P < 0.02), and 52.2% (P < 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.
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