The Extraction of a Tissue Collagenase Associated with Ovulation in the Rat1

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 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative 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.

Association of immunolocalization of matrix metalloproteinase 1 with ovulation in hCG-treated rabbit ovary

“…Increased collagenolytic activity in the preovulatory follicles of rabbit ovaries was demonstrated (Kawamura et al, 1981). Curry et al (1985) also reported the preovulatory increase of MMP-1 activity in the rat ovary.…”

Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases

“…Of course other MMPs with overlapping substrate specificities, such as MMP-2, may account for the lack of a more dramatic ovarian phenotype in the MMP-9 null mice. Based on increased collagenase (against collagen I) activity in ovaries of ovulating rats (21,22) and bovine follicles (62), it is highly likely that MMP-13 plays a role in ovulation. However, the temporal pattern of MMP-13 expression in the rodent ovary (24) and its presence in PRKO mice (J.S.R., unpublished data) indicate that it is not a PR-regulated gene.…”

“…The matrix metalloproteinases (MMPs) have received considerable attention as candidate proteases controlling ovulation (19). Of the 23 known MMPs (20), collagenases (MMP-13 and MMP-8), which degrade collagen type I, gelatinases (MMP-2 and MMP-9), which degrade collagen types IV͞V, and stromelysin-like enzymes (MMP-19) have been shown to be present in the ovary (21)(22)(23)(24)(25). However, their relationship, if any, to each other or to ovulation has remained speculative.…”

Differential distribution of gelatinases and tissue inhibitor of metalloproteinase-1 in the rat ovary

“…Both serine proteases (Danø et al 1985) and matrix metalloproteinases (MMPs) (Birkedal-Hansen et al 1993) and their respective inhibitors have been shown to regulate homeostatically the extent and rate of extracellular matrix (ECM) reorganization associated with tissue remodeling. These enzymes or their inhibitors have been shown to be present in ovaries of the cow ( Juengel et al 1994), hamster (Ichikawa et al 1983), human (Curry et al 1990, Duncan et al 1996a, monkey (Duncan et al 1996b), mouse (Waterhouse et al 1993, Inderdeo et al 1996, pig (Smith et al 1994b), rabbit (Espey & Coons 1976), rat (Morales et al 1983, Reich et al 1985, Curry et al 1985, Roche & Ryan 1986, Hirsch et al 1993, Butler & Woessner 1994, Okada et al 1994, Nothnick et al 1995, Balbin et al 1996, and sheep (Murdoch & McCormick 1992, Smith et al 1994a, Russell et al 1995, McIntush et al 1996. The expression of the metalloproteinases and their inhibitors have been primarily studied by biochemical techniques.…”