IN VIVO CHANGES IN PROTEIN EXPRESSION OF THE GELATINASES, MATRIX METALLOPROTEINASES -2 AND -9 (MMP-2 AND MMP-9), AND TISSUE INHIBITORS OF METALLOPROTEINASES -1 (TIMP-1) IN THE OVINE CORPUS LUTEUM (CL) AFTER MULTIPLE PULSES OF PROSTAGLANDIN F2ALPHA (PGF2A)

Abstract

A model was established in sheep which mimics the frequency of prostaglandin F2alpha (PGF2a) pulses during the course of natural luteolysis. Using this model, we previously demonstrated that after one and two pulses of PGF2a, protein mediators of the extracellular matrix (ECM), tissue inhibitors of matrix metalloproteinases (TIMPs) -1 and -2, decreased dramatically in luteal tissue within one hour following each PGF2a pulse, which was accompanied several hours later by an increase in matrix metalloproteinase-2 (MMP-2) activity. The objective of the present study was to study the temporal protein expression patterns of the gelatinases, matrix metalloproteinases (MMP) -2 and MMP-9, and TIMP-1, in corpora lutea of sheep which received three systemic pulses of PGF2a (20ug/min/1hr) during the mid-luteal phase of the cycle. Intact cycling ewes were given 2 injections (7.5 mg) of Lutalyse and housed with a vasectomized marker ram to detect estrus (day 0). Sheep were then housed in metabolism cages, and infused with PGF2a via the right jugular vein to mimic the endogenous uterine pulses during luteolysis. Corpora lutea (day 10–12; n=4 sheep for each time point) were removed surgically one hr (-1hr) before the third pulse of PGF2a and at 1, 8, 16, and 24 hrs after the third infusion of PGF2a. Gelatin zymographic analysis identified the presence of three bands with relative molecular masses of approximately 85, 70, and 64 kDa. These bands co-migrated with MMP-9 (85kDa) and MMP-2 (70 and 64 kDa) gelatinase family members present in the positive control, which was conditioned medium of the fibrosarcoma cell line, HT-1080. An analysis of variance followed by Tukey's revealed no differences in the expression of the 64 kDa MMP-2 species, but the expression of the 70 kDa MMP-2 species between the 16 and 24 hr time points differed significantly (p<0.05). In our previous studies with one or two pulses of PGF2a, no differences were observed for MMP-9. In the present study, after three pulses of PGF2a, expression of the 85 kDa MMP-9 species tended to increase (p= 0.07) at 16 hrs relative to the pre- infusion (-1hr) time point. A band of approximately 30 kDa, which comigrated with a TIMP-1 recombinant protein, was identified by immunoblot analysis. Although expression of TIMP-1 fluctuated greatly, the variation among samples for each time point negated any differences (p>0.05) in the temporal expression of this protein. In summary, the increase of the 70 kDa MMP-2 species accompanied by an increasing trend of the 85 kDa MMP-9 species and varying expressions of TIMP-1 indicate changes in protein mediators of the CL in response to the third pulse of PGF2a. These results, taken together with our previous findings, indicate a continuum of changes in protein regulators of the ECM within the CL as luteolysis progresses. (Supported by USDA Grant #2004-35203- 14176) (poster)