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

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.     

Collagenase activity in the normal rat myocardium. An immunohistochemical method

Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart sections were incubated under optimal conditions for collagenase activity (37 degrees C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is the result of collagen degradation by intrinsic collagenase of the myocardium.     

The role of ovarian proteases and their inhibitors in ovulation

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.     

Collagenase activity in the normal rat myocardium

Summary Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart secctions were incubated under optimal conditions for collagenase activity (37°C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is. the result of collagen degradation by intrinsic collagenase of the myocardium.     

Effects of prolactin on ovarian plasmin generation in the process of ovulation.

The present study was undertaken to assess the effects of prolactin (PRL) on gonadotropin-induced plasmin generation in the in vitro-perfused rabbit ovary. The ovarian plasmin activity was determined by measuring plasmin bound to its major inhibitor, alpha 2-plasmin inhibitor (alpha 2 PI-Plm). In the first experiment, exposure to hCG enhanced ovarian alpha 2 PI-Plm generation from 1.2 +/- 0.3 ng/min/ovary in unstimulated ovaries to 2.9 +/- 0.3 ng within 2 h. The concentration of alpha 2 PI-Plm reached a maximum at 4 h and then declined. A second peak occurred 8 h after hCG administration; however, the ovarian alpha 2 PI-Plm generation without hCG was very low throughout the entire perfusion period. In the subsequent experiment, the addition of PRL(10-10(3) ng/ml) to the perfusate inhibited hCG-induced ovulation in a dose-dependent manner. Exposure to PRL at 10(3) ng/ml significantly (p less than 0.05) inhibited hCG-induced alpha 2 PI-Plm generation in ovaries throughout the entire perfusion period. Furthermore, PRL inhibited hCG-stimulated alpha 2 PI-Plm generation at 4 h after hCG administration in the perfused rabbit ovaries in a dose-dependent manner. In conclusion, PRL directly inhibits hCG-induced ovulation in rabbit ovary, at least in part, by a mechanism depending upon inhibition of the plasmin-generating system in the preovulatory follicles.     

Types I and IV collagenolytic and plasminogen activator activities in preovulatory ovarian follicles

During ovulation, enzymatic degradation of the extracellular matrix occurs within and around the graafian follicles. In this study, the activities of several different proteolytic enzymes were measured in the culture media of follicles taken from pregnant mare serum gonadotropin (PMSG)-primed immature rats. At 52 h after PMSG, the follicles were cultured for 2 to 15 h in media with or without human chorionic gonadotropin (hCG). Type I collagenase activity in hCG-stimulated follicles gradually increased within 6 h to 3.3-fold above that of the controls. Relatively pure populations of granulosa cells produced type I collagenase to a similar extent. Likewise, type IV collagenase increased 3.8-fold by 6 h after exposure of the follicles to hCG. In contrast, plasminogen activator activity increased by 3.9-fold at 2 h after hCG, but was negligible at 4, 6, and 15 h after incubation. These results suggest that plasminogen activator may activate both type I and type IV collagenase in hCG-stimulated ovulatory follicles.     

Clofibrate-induced increase in coenzyme A concentration in rat tissues

1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.     

A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown.

Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5-fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha-macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extraction of collagenase from the involuting rat uterus.

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Collagen, collagenase and collagenolytic activity in rat Graafian follicles during follicular growth and ovulation

Follicles were dissected from the ovaries of immature rats at intervals after subcutaneous injection of 20 IU of pregnant mare's serum gonadotropin. A surge of luteinizing hormone was observed at 54 h and ovulation occurred at 64-66 h. The follicular volume between 36 and 48 h, then doubled again shortly before ovulation. The collagen content of the follicles increased 3-fold from 35 to 56 h, but decreased significantly (25%) from 61 to 66 h. Follicle homogenates, activated with trypsin or aminophenylmercuric acetate, digested Type I collagen at 28 degrees C to produce typical of a true collagenase. Collagenolytic activity assayed against endogenous collagen at 37 degrees C did not change significantly between 38 and 66 h.     

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.     

In vitro decrease of lyase activity in rat ovarian cells during incubation: effect of hCG

From PMSG-pretreated immature rats, dispersed ovarian cells were prepared with collagenase and DNase and incubated at 37 degrees C in McCoy's 5a medium under 95% air-5% CO2 atmosphere for 4 h. The activities of C17-C20 lyase measured in the 10,000 x g supernatant fluid of the cell homogenates decreased spontaneously with the lapse of time of the incubation. N,N'-Diphenyl-p-phenylenediamine (DPPD, an antioxidant) and actinomycin D inhibited the decrease most effectively. Cycloheximide was also an effective protector. Accordingly, the spontaneous decrease of the lyase activity was caused partly by an oxygen radical-mediated process and partly by a mechanism involving de novo synthesis of RNA and protein. Addition of hCG to the cells further decreased the lyase activity to about half of the control group at 4 h. DPPD itself did not affect the hCG-induced decrease of the lyase activity. However, actinomycin D and cycloheximide prevented the effect of hCG. These results indicate that de novo synthesis of RNA and protein is involved in the latter mechanism, while oxygen radical is not concerned in this process. The decrease of the enzyme activity by hCG during incubation is in agreement with the in vivo effect of hCG upon the lyase activity. On the contrary, at the end of incubation the activity of delta 5-3 beta-hydroxysteroid dehydrogenase (coupled with delta 5-delta 4 isomerase) was more than 89% of that before incubation, and the change of the enzyme activity according to the various treatments was less than 16%.     

Hormonal changes during the early development of ovarian cysts in the rat

The daily administration of human chorionic gonadotropin (hCG) to rats with thiouracil-induced hypothyroidism results in the development of cystic ovaries. This study was undertaken to delineate hormonal changes during the first 48 h of hCG treatment. Groups of euthyroid and hypothyroid rats were injected daily with hCG or saline for up to two days and killed at 0, 12, 24, or 48 h after the initial hCG injection. Sera were analyzed for progesterone (P), testosterone (T), 17 beta-estradiol (E2), and prolactin (Prl) by specific radioimmunoassay (RIA). Serum levels of these hormones were not significantly different in the euthyroid and hypothyroid rats. However, P was significantly elevated at 12, 24, and 48 h in the hypothyroid/hCG rats. T and Prl were significantly elevated at 12 and 48 h in the hypothyroid/hCG rats. T levels were also elevated at 12 and 48 h in the euthyroid rats receiving hCG. In contrast, hCG had no effect on P and Prl levels in the euthyroid rats. E2 levels were undetectable in the euthyroid and hypothyroid rats. The administration of hCG increased E2 in both the euthyroid and hypothyroid rats at 48 h with significantly more E2 detected in the hypothyroid rats. These results show that ovarian steroids and Prl levels increase during the early stages of cyst induction and suggest they may be important in triggering ovarian cyst formation.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

Some properties of latent collagenase from human synovial fluid

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.     

Expression of matrix metalloproteinases and their tissue inhibitor messenger ribonucleic acids in macaque periovulatory granulosa cells: time course and steroid regulation.

Progesterone appears essential for ovulation and luteinization of the primate follicle, but specific gene targets of progesterone action remain elusive. Limited evidence supports a role for progesterone in the induction of collagenolytic activity in the periovulatory follicle of primate and nonprimate species. This study was designed to elucidate the pattern of expression and progesterone regulation of mRNAs for the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in macaque granulosa cells during controlled ovarian stimulation cycles before (0 h) and after (up to 36 h) administration of an ovulatory hCG bolus. Levels of mRNAs for interstitial collagenase, gelatinase A, matrilysin, TIMP-1 and TIMP-2 increased (p < 0.05) within 12 h of hCG, while gelatinase B mRNA increased later, by 36 h after hCG. Administration of a 3beta-hydroxysteroid dehydrogenase inhibitor (Trilostane [TRL]) during hCG treatment decreased (p < 0.05) mRNA levels for interstitial collagenase, gelatinase B, matrilysin, TIMP-1, and TIMP-2. Progestin (R5020) replacement during hCG+TRL treatment returned interstitial collagenase and TIMP-1 mRNAs to control levels. These data suggest that one action of progesterone, and possibly other steroids, in the cascade of events leading to ovulation and luteinization of the primate follicle is to regulate the expression of specific ovarian proteases and protease inhibitors.     

Purification and characterization of a collagenase inhibitor produced by bovine vascular smooth muscle cells

A potent polypeptide inhibitor of mammalian collagenases was purified to homogeneity from medium conditioned by bovine aortic smooth muscle cells maintained in culture. This inhibitor was purified by a series of molecular sieve and heparin-Sepharose chromatographic procedures; it had an apparent Mr of 28,500 and was a major protein secreted by the smooth muscle cells. It was found to be active against several mammalian collagenases including those obtained from rabbit and human fibroblasts and a tumor-specific type IV collagenase. In contrast, it had minimal inhibitory activity for bacterial collagenase and was inactive against the serine proteases plasmin and trypsin. The inhibitor shared many characteristics with tissue inhibitor of metalloproteinases including the ability to irreversibly inhibit susceptible proteinases, heat and acid resistance, and sensitivity to trypsin degradation and reduction-alkylation. A polyclonal rabbit antiserum with blocking activity which recognized the Mr 28,500 protein was obtained. This inhibitor, which is likely produced by bovine vascular smooth muscle cells in vivo to protect the collagen matrix of blood vessels, may play an important role in pathological conditions associated with alteration of collagen metabolism in tissues.     

Identification of hepsin and protein disulfide isomerase A3 as targets of gelatinolytic action in rat ovarian granulosa cells during the periovulatory period

The matrix metalloproteinase (MMP) family is believed to play a role in the ovulatory process because MMP inhibitors block oocyte release. However, little is known about the mechanisms by which the MMPs affect ovulation. The present study investigated the degradomic actions of the gelatinases, MMP2 and MMP9, by identifying gelatinolytic targets in periovulatory granulosa cells. Granulosa cells were collected from immature rats 48 h after equine chorionic gonadotropin treatment and were cultured with human chorionic gonadotropin (hCG) in the absence or presence of a specific MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid) for an additional 24 h. The conditioned media was analyzed for gelatinolytic activity, progesterone, and peptide profiles. Gelatinolytic activity and progesterone were induced in response to hCG; however, there was no difference in progesterone between cells treated with or without the inhibitor. Peptide fragments of proteins altered in the presence of the gelatinase inhibitor were identified by two-dimensional gel electrophoresis and mass spectrometry. Protein disulfide isomerase A3 (PDIA3), which plays a role in protein folding, was identified as a peptide that decreased in the presence of inhibitor while the serine protease hepsin, was found to increase with inhibitor treatment. Subsequent experiments established that PDIA3 and hepsin were targets of MMP2/9 action by cleavage with MMP2 and Western blot analysis, respectively. Additionally, hepsin was identified as a gelatinolytic target in ovarian cancer cells. In the present study, proteomics has identified proteins that may be involved in novel ways in the complex cascades that are mediated by gelatinolytic MMPs during the periovulatory period.