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DIFFERENTIAL REGULATION OF TISSUE INHIBITORS OF METALLOPROTEINASES 1-4 (TIMP1-4) IN BOVINE PERIOVULATORY FOLLICLES FOLLOWING THE LH SURGE: IMPLICATIONS FOR REGULATION OF THE OVULATORY PROCESS.
Bakke, Leanne1, Dow, Mark1,2, Cassar, Carolyn2, Peters, Mike2, Charest, Marci2, Ricke, Will3, Smith, Michael3, Pursley, J. Richard2, Smith, George1,2, 1 2 3
ABSTRACT- Matrix metalloproteinases (MMP) are metal-dependent enzymes that degrade various extracellular matrix (ECM) components. Numerous studies implicated MMPs as important mediators of the follicular ECM degradation required for ovulation. MMP activity is regulated via specific MMP inhibitors, such as the tissue inhibitors of metalloproteinases (TIMP 1-4). Regulation of TIMPs may be an important factor controlling the ovulatory process. Here we test the hypothesis that localization and expression of TIMP 1-4 are differentially regulated in bovine periovulatory follicles following a GnRH-induced LH surge. Follicle growth and timing of the LH surge were synchronized in dairy cows and ovaries containing preovulatory follicles or corpora hemorrhagica (CH) collected at 0, 6, 12, 18, 24 and 48 h (CH) after GnRH injection (n = 4-6 each). Relative levels of TIMP-1 and 2 mRNA increased within 6 h following the LH surge and remained elevated through the 48 h timepoint (P < 0.05). In contrast, TIMP-3 mRNA was not upregulated following the LH surge and decreased at 18 and 48 h (P < 0.05) relative to the 0 h (pre LH surge) timepoint. TIMP-4 mRNA was expressed during the periovulatory period at all timepoints examined. The intrafollicular localization of TIMP 1-3 mRNA was also differentially regulated during the periovulatory period. TIMP-1 mRNA was localized primarily to the granulosal layer of bovine preovulatory follicles collected following the LH surge. TIMP-2 mRNA was also localized primarily to the granulosal layer of bovine preovulatory follicles. At the 0 h (pre LH surge) timepoint, localization was predominantly to the mural granulosal cells near the basement membrane. Localization to the entire granulosal layer was noted in follicles collected after the LH surge. In contrast, TIMP-3 mRNA was localized specifically to the thecal layers and adjacent ovarian stroma. Our results indicate the TIMPs are regulated in bovine preovulatory follicles following the LH surge in a temporally and spatially specific fashion. Constitutive and (or) decreased expression of TIMP-3 in the thecal layers/tunica albuginea may favor a net increase in the MMP/TIMP ratio and subsequent degradation of the follicle wall prior to ovulation. Supported by USDA 98-35203-6226 (GWS) and 98-35203-6282 (MFS).
KEY WORDS: Tissue Inhibitors of Metalloproteinases, Ovulation, Matrix Metalloproteinases, Bovine