In Vivo Temporal Expression of Protein Levels of Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) During Prostaglandin F2alpha-Induced Luteolysis in Sheep.

Abstract

Multiple pulses of uterine prostaglandin F2alpha (PGF2α) are required to cause the complete functional decline and structural regression of the sheep corpus luteum (CL). We have used a physiological model in sheep that mimics the effects of PGF2α pulses to study the family of proteins known to play key roles in remodeling the extracellular matrix (ECM), the matrix metalloproteinases (MMPs) and TIMPs. Following the first, second and third pulse of PGF2α our findings to date indicate that the activities of the gelatinases, MMP-2 and MMP-9, exhibit minor fluctuations. However, TIMP-1 and TIMP-2 protein expression decreases following the first and second PGF2α pulses, while they rebound to control levels following the third pulse. Because other members of this family may also have a role in luteolysis, the objective of the present study was to determine the temporal protein expression of TIMP-3 in the sheep CL following one, two and three infusions of PGF2α. Among the four known TIMPs, TIMP-3 is the only member that is bound to the ECM and it is associated with apoptosis. During the mid-luteal phase (day 10/11), sheep were given three 1 hour-long systemic infusions of PGF2α (~0.22μg/kg/min/hr) at intervals matching endogenous pulses of PGF2α during luteolysis. Luteectomies (n=4 per time point) were performed at 0 hour (pretreatment control) and at -1, 1, 8, 16, or 24 hours after each PGF2α infusion. Immunoblotting was used to determine TIMP-3 protein expression. Densitometry of immunoblots was performed before the Mixed Procedure of SAS was used to determine differences in protein expression between time points. Two forms of TIMP-3 migrating at relative molecular masses of 48 and 24 kDa were present in sheep luteal extracts. The expression of the 48 kDa form of TIMP-3, compared to the 0 hour pretreatment control, was unchanged (p>0.05) after the first infusion of PGF2α until the 16 hour time point, when expression increased (p<0.05) by 3-fold. Thereafter, following the second and third infusions of PGF2α, TIMP-3 expression overall was increased (p<0.05) by 1.5-fold above the 0 hour pretreatment control and throughout the remaining sampling period. In contrast, the expression of the 24 kDa form of TIMP-3, compared to the 0 hour pretreatment control, decreased (p<0.05) at the 1 hour time point and remained depressed (p<0.05) throughout the remaining period and following both the two and three infusions of PGF2α. In summary, the concomitant increase and decrease in the expression of the 48 and 24 kDa forms, respectively, of TIMP-3 in the sheep CL following multiple infusions of PGF2α may be related to their roles in remodeling of the ECM and in causing apoptosis during luteolysis. Future studies are warranted to determine the functional role(s) of TIMP-3 on luteal function in the sheep. (Supported by National Research Initiative Competitive Grant # 2004-35203-14176 from the USDA Cooperative State Research Education and Extension Service). (poster)