About two thirds of all terrestrial vertebrate species reproduce by oviposition, while the rest of the vertebrates (with few exceptions) by parturition. Prostaglandins (PGs) are among the key components of the mechanism regulating both oviposition and parturition.
The specific aims of this application are a) to test the hypothesis that uterine production of PGs in the domestic hen (Gallus domesticus) is triggered by an oviposition inducing factor (OPIF) of ovarian origin which causes a rapid breakdown of phosphatidylinositol (PI) to phosphatidate (PA), followed by a Ca-dependent activation of phospholipase A2 and the liberation of arachidonic acid resulting in PG-synthesis; b) to examine the regulation of PG receptors in avian and mammalian (rat) uterus in relation to impending oviposition and parturition and c) to define the effects of PGs on the phosphorylation of myometrial cell membrane proteins as well as myosin as possible steps in the contractile action of these powerful biologic substances. Chromatographic (gel-filtration and HPLC) and radioimmunologic methods will be combined with bioassay to isolate and identify the putative OPIF. The proposed effects of OPIF and other agonists will be investigated by following the fate of phospholipids in uterine preparations (prelabeled with [32P] orthophosphate or [3H]arachidonic acid) by means of thin layer radiochromatography and high performance liquid chromatography (HPLC). PG-binding will be studied in myometrial cell membranes prepared by differential and gradient centrifugation of specimens obtained at different times prior to oviposition or parturition. In addition, the effects of steroid hormones on PG-receptors will be examined in vivo and in myometrial slices cultured in vitro. Phosphorylation of uterine proteins will be assessed by determining the incorporation of [32P] or Gamma-32ATP into proteins in the presence of PGs and other agonists. One and two dimensional SDS gel ectophoresis will be applied for the separations of proteins with appropriate molecular markers to estimate the size of the labeled moieties. Successful completion of these studies will provide a unified concept on the role and mechanism of action of PGs in a fundamental process of reproductive biology. Such knowledge will be instrumental for the better understanding of the physiology of labor and pathophysiology of premature labor.
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