The overall goal of the proposed studies is to improve our understanding of the mechanisms by which ovarian function is controlled by metalloproteinases and their inhibitors during the reproductive cycle of the rat. The proposal is based on the hypothesis that matrix metalloproteinases, such as collagenase and gelatinase, as well as their associated inhibitors (i.e. tissue inhibitors of metalloproteinases, TIMPs) play a fundamental role in ovarian physiology by controlling extracellular matrix remodeling during follicular development, ovulation, and formation and regression of the corpus luteum (CL). The hypothesis to be tested is that there are dynamic, coordinated changes in the expression, activity, cellular localization, and function of metalloproteinases and their inhibitors which are regulated (both positively and negatively) by hormonal signals associated with these periods of extensive ovarian connective tissue remodeling. To test this hypothesis, metalloproteinase and inhibitor mRNA, activity, regulation, and cellular localization will be determined during follicular development, ovulation and CL function, using molecular, biochemical, morphologic, and physiologic techniques. The first specific aim of this proposal is to determine the changes and regulation of collagenase, gelatinase, and TIMP mRNA and activity during follicular and luteal development. The regulation of enzyme and inhibitor expression and activity by LH-induced messenger systems, such as prostaglandins, interleukins, and prolactin, will be studied in vivo and in vitro with a granulosa and luteal cell culture model. The second specific aim will elucidate the cellular origin and determine the morphometric changes in ovarian metalloproteinases and TIMP mRNA during follicular development and rupture as well as during the life span of the CL by in situ hybridization. The third specific aim will determine the functions role of metalloproteinases and TIMPs by blocking enzyme activity, inhibiting TIMP action, stimulating with exogenous TIMP, or using TIMP-l knockout mice to examine ovarian morphology and steroidogenesis throughout follicular and luteal development. The fourth specific aim will investigate the transcriptional regulation of collagenase and TIMP expression by LH- induced messenger systems utilizing nuclear transcription run-on assays and the respective promoter fused to the bacterial reporter gene chloramphenicol acetyl transferase (CAT). Only by understanding such fundamental information will we be able to promote or inhibit ovarian events controlled by metalloproteinase action (such as ovulation or CL formation) thereby leading to future applications for the improvement of fertility or contraception. These studies will impact on other ovarian endocrine abnormalities, such apolycystic ovarian disease or luteinized unruptured follicle syndrome, where ovulation is compromised as well as other areas where metalloproteinases have been implicated such as embryonic development, implantation, placentation, cervical dilation, parturition, and cancer.
