PARENT SESSION
PLATFORM SESSION 9: VASCULAR AND INTRACELLULAR SIGNALS IN CORPUS LUTEUM
Chair: Davis, John¹, ¹
Co-chair: Valdezk, Kelli¹, ¹
Grand Ballroom V
2:00 PM-4:00 PM

(214) COLLAGENASE 3 (MMP-13): EXPRESSION IN THE BOVINE CORPUS LUTEUM AND ITS REGULATION BY CYTOKINES IN LUTEAL-DERIVED ENDOTHELIAL CELLS .

Zhang, Bo¹, Ambrosi, Dominic¹, Pru, James², Davis, John³, Rueda, Bo², Tsang, Paul¹, ¹ Department of Animal & Nutritional Sciences, Durham, NH² Vincent Center for Reproductive Biology, Boston, MA³ Olson Center for Women's Health, Department of Obstetrics and Gynecology, Omaha, NE

ABSTRACT- Angiogenesis and other structural remodeling events in the corpus luteum (CL) are mediated by matrix metalloproteinases (MMPs), which are regulated, in part, by cytokines. Previously, we demonstrated that the expression patterns of membrane type 1 metalloproteinase (MT1-MMP) and gelatinase A (MMP-2) were correlated to the development of the bovine CL. Although MT1-MMP is implicated as an activator of MMP-2 and MMP-13, surprisingly little is known about MMP-13 in the ruminant ovary. Therefore, the aims of the present study were (1) to determine the temporal and spatial expression of collagenase 3 (MMP-13) in the bovine CL during the estrous cycle, and (2) to study the regulation of MMP-13 in endothelial cells isolated from the bovine CL. In the first experiment, luteal tissues were collected from dairy cows on days 4, 10, and 16 (n=3/day; day 0 = estrus) of the estrous cycle. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the MMP-13 transcript did not vary (p>0.05) in the CL on the days examined. Western blotting detected a 52 kDa protein band, corresponding to the latent form of MMP-13. The intensity of this band was similar (p>0.05) in all ages of CL. Based on immunohistochemistry, MMP-13 was restricted to endothelial cells in all CL evaluated. Therefore, in a second experiment, endothelial cells were isolated from bovine CL and treated in the absence (control) or presence of tumor necrosis factor (TNF, 100ng/ml), interferon (IFN, 200 units/ml), or a combination of these two cytokines (100ng/ml TNF and 200 units/ml IFN) for 24 hours. MMP-13 mRNA and protein expression were assessed by RT-PCR and Western blotting. While TNF increased (p<0.05) the mRNA and protein levels of MMP-13 in endothelial cells, IFN treatment had no effect. When combined, TNF and IFN increased (p<0.05) MMP-13 mRNA and protein levels above control, but IFN attenuated the effect observed with TNF alone. In summary, although MMP-13 mRNA and protein levels did not vary in d 4, d 10, and d 16 CL, evidence is provided that MMP-13 is localized to endothelial cells in vivo and TNF stimulates expression of MMP-13 in luteal-derived endothelial cells in vitro. (Supported by the USDA (98-35208-6654) and the Northeast Regional Project NE-161 to PCWT, the NIH (HD 35934) to BRR and JSD, and the VA to JSD)

KEY WORDS: matrix metalloproteinases, corpus luteum, endothelial cells, cytokines