In vivo measurement of rat ovarian collagenolytic activities

Ovarian collagenases are necessary for the process of ovulation, and they are believed to be activated by the preovulatory LH surge. This information is largely based on in vitro investigations in which the balance between inhibitory and stimulatory principles involved in the activation of collagenase are largely disrupted. Therefore, we developed a simple and reliable method to measure collagenolytic activity in vivo in freely moving rats. By the use of a microdialysis system, a peptide coupled with methyl-coumarin is perfused into the bursa of the ovary. Collagenolytic enzymes cleave this peptide, and the cleaved fragments rediffuse into the microdialysis system. The effluent is collected in frections, and the peptide-methyl-coumarin complex is cleaved, which results in liberation of fluorescent methyl-coumarin. This assay is linear over a wide range of collagenolytic activity, and other proteases, such as trypsin or plasmin, do not give any fluorescent signal. In proestrous rats, collagenolytic activity increases after the onset of the preovulatory LH surge. In animals in which the LH surge was disrupted by the surgical procedure but had a normal proestrous PRL surge, neither progesterone nor collagenolytic activity increased in the perfusate fluid. This indicates that it is only LH, not PRL, that activates follicular collagenolytic enzymes. Similar results were obtained in immature PMSG/hCG-treated animals. Using a well established zymographic assay, these results were confirmed, and it was further demonstrated that type I and type IV collagenase are active in the rat ovary.