Submission Number: PAU-4-4-38

Abstract Number: 93

MOLECULAR CLONING OF BOVINE MEMBRANE-TYPE 1 MATRIX METALLOPROTEINASE (MT1-MMP) AND ITS EXPRESSION IN CORPUS LUTEUM (CL).

Bo Zhang* ¹, Li Yan ², Marsha A Moses ² and Paul CW Tsang* ¹

Department of Animal and Nutritional Sciences, University of New Hampshire, Durham, NH ¹
Department of Surgery, Children's Hospital, Harvard Medical School, Boston, MA. ²

Abstract:
Matrix metalloproteinases (MMPs) mediate the structural remodeling and angiogenesis that occur in the CL during the estrous cycle. Recent studies demonstrate that proMMP2, which we have reported to be present in bovine CL, is activated by MT1-MMP, a membrane-type MMP. Given the importance of MT1-MMP, the objectives of this study were to clone bovine MT1-MMP and to investigate its expression in CL during the reproductive cycle. A bovine UNI-ZAP II cDNA library was constructed using RNA from bovine adrenal cortical capillary endothelial cells to isolate bovine MT1-MMP cDNA. CL were collected from regularly cycling, nonlactating dairy cows on day 4, 10 and 16 of the estrous cycle (day 0=estrus; n=3 for each age) and were used to determine the temporal and spatial expression of MT1-MMP. The bovine MT1-MMP cDNA contained an open reading frame of 1749 basepairs, which encoded a predicted protein of 582 amino acids, sharing 95.7%, 95%, 92.3%, and 95.9% sequence homology to the human, mouse, rabbit, and rat enzyme, respectively. Northern blotting revealed no difference (p0.05) in MT1-MMP mRNA levels between the three ages of CL. Immunoblotting showed that two distinct bands, the 60 kD active and the 63 kD latent forms, were observed in all samples. However, the level of the latent form was higher (p0.05) in the early than in the mid- and late CL. In contrast, the active form was lower (p0.05) in the early CL than the mid- and the late stages, corresponding to the elevated levels of the active MMP-2 in the mid- and late CL, as revealed by gelatin zymography. Furthermore, immunocytochemistry revealed endothelial and luteal cell-associated MT1-MMP expression in the early CL. However, this expression was primarily detected in mid-cycle large luteal cells, and in fibroblast-like cells in the late CL. Taken together, our results indicate that MT1-MMP is differentially expressed in various cellular compartments of bovine CL to activate proMMP2. (Supported by ACS-RPG-97-013-01-CB to MAM, and Hatch343 to PCWT).

Keywords: MT1-MMP, corpus luteum, bovine

This abstract is being presented at: 8:00 AM in session:
Corpus Luteum