The extraction of a tissue collagenase associated with ovulation in the rat

T. E. Curry, D. D. Dean, J. F. Woessner, W. J. LeMaire

Research output: Contribution to journalArticle

53 Scopus citations

Abstract

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60°C in 50mM Tris buffer containing 100mM CaCl2. This extract contains collagenase activity and purative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9±1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7±1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7±1.9% and 16.1±1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.

Original languageEnglish (US)
Pages (from-to)981-991
Number of pages11
JournalBiology of reproduction
Volume33
Issue number4
DOIs
StatePublished - 1985

ASJC Scopus subject areas

  • Reproductive Medicine
  • Cell Biology

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