Characterization of ovarian and ovulation-specific proteins in the brook trout (Salvelinus fontinalis)
Coffman, M.A.; Goets, F.W. (2000). Characterization of ovarian and ovulation-specific proteins in the brook trout (Salvelinus fontinalis), in: Norberg, B. et al. (Ed.) Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish, Bergen, Norway, July 4-9, 1999. pp. 160-163
In: Norberg, B. et al. (2000). Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish, Bergen, Norway, July 4-9, 1999. International Symposium on the Reproductive Physiology of Fish, 6. Department of Fisheries and Marine Biology, University of Bergen: Bergen. ISBN 82-7461-048-2. 499 pp., more
In: International Symposium on the Reproductive Physiology of Fish. Museo Nacional de Ciencias Naturales. , more
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| Authors | | Top |
- Coffman, M.A.
- Goets, F.W.
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| Abstract |
Using stage-specific subtractive cDNA cloning, a family of cDNAs (0.8, 1.1, 1.4, 1.7 kb) that was highly upregulated at the time of ovulation was obtained and named TOPs (Trout Ovulatory Proteins). Northern blots revealed that TOP mRNA levels increase up to the time of ovulation and peak at 12 hours postovulation. Following ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Western blots of ovarian tissue and coelomic fluid confirmed that TOP proteins increase up to the time of ovulation, peak at 12-24 hours postovulation, then decrease by 4-8 days postovulation. in situ hybridization and immunocytochemistry localized TOP RNA and protein, respectively, primarily to the granulosa layer. The sequence of the 0.8 kb (TOP-1) and the 1.4 kb (TOP-2) cDNAs were homologous to mammalian antileukoproteinase, a heat- and acid-stable serine protease inhibitor. Using a chromogenic peptide substrate, the anti-protease activity of postovulatory brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activity were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 µg of total protein, respectively. Coelomic fluid could be heated to 50 degree C or treated at a pH less than 5.2 without significantly decreasing the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls, implicating TOPs in this inhibition. Using an in vitro incubation system, the regulatory effects of various agonists and antagonists on TOP RNA and protein were investigated. Of the many compounds tested, TGF alpha and the general G-protein agonist, orthovanadate, increased TOPs while corticosteroids inhibited TOPs. Finally, the coelomic fluid specifically inhibited the growth of the gram negative bacteria, Pseudomonas aeruginosa, and this inhibition was lost in fluid from which TOPs had been immunoprecipitated. Thus, one biological function of TOPs may be to protect ovulated eggs from bacterial infection. |
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