Abstract
The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), may mediate the dramatic structural and functional changes in the corpus luteum (CL) over the course of its life span. In addition to regulating MMP activity, TIMPs are also involved in a variety of cellular processes, including cell proliferation and steroidogenesis. In a series of initial studies, we determined that matrix metalloproteinase inhibitory activity was present in protein extracts from early (4 days old, estrus = day 0), mid (10–12 days old) and late (16 days old) CL (n = 3 for each stage). Reverse zymography revealed four metalloproteinase inhibitory protein bands with relative molecular masses that are consistent with those reported for TIMP-1 to -4. In order to gain a better understanding of TIMPs and their role in luteal function, we further characterized this inhibitory activity with a particular focus on the temporal and spatial expression of TIMP-1 and TIMP-2 in the bovine CL. Northern blotting revealed that the TIMP-1 transcript (0.9 kb) was expressed at a higher (p < 0.05) level in early and mid cycle CL than in the late stage. In contrast, two TIMP-2 mRNA species, one major 1 kb species and one minor 3.5 kb species, were significantly (p < 0.05) increased in the mid and late cycle CL than in the early. Western blotting analyses demonstrated no differences in TIMP-1 (29 kDa) protein levels between early and mid stages, while its levels decreased (p < 0.05) from the mid to late stage CL. Conversely, TIMP-2 (22 kDa) protein was detected at a low level in the early CL, but significantly (p < 0.05) increased in the mid and late stages. Immunohistochemistry revealed that both TIMP-1 and -2 were localized to large luteal cells from all three ages of CL. TIMP-1 was also localized in capillary smooth muscle cells, while TIMP-2 was restricted to the endothelial cells in the capillary compartment. In conclusion, the different temporal expression patterns of TIMP-1 and TIMP-2 suggest that TIMP-1 may be important for luteal formation and development, while TIMP-2 may play significant roles during luteal development and maintenance. Furthermore, the distinct localization of these two inhibitors in the vascular compartment indicates that they may serve diverse physiological functions during different stages of luteal angiogenesis.
Highlights
The corpus luteum (CL) is a transient, dynamic endocrine gland, which develops from the postovulatory follicle [1]
Metalloproteinase Inhibitor Activities in the Bovine CL The radiometric matrix metalloproteinases (MMPs) assay for tissue inhibitors of metalloproteinases (TIMPs) activity revealed that the protein extract from all three ages of CL possessed
Four bands possessing MMP inhibitory activities were observed in luteal samples. These bands correspond to TIMP-1 (~30 kDa), TIMP-3 (~27 kDa), TIMP-4 (~24 kDa), and TIMP-2 (~22 kDa), respectively
Summary
The corpus luteum (CL) is a transient, dynamic endocrine gland, which develops from the postovulatory follicle [1]. Dramatic structural and functional changes are associated with the development, maintenance and regression of the CL [1]. These remodeling events require the participation of matrix metalloproteinases (MMPs), a growing family of zinc and calcium dependent proteolytic enzymes that collectively digest all known macromolecules constituting the extracellular matrix [2,3]. The catalytic activity of the MMPs is highly regulated at three levels, gene expression, proteolytic activation of latent proenzymes, and inhibition of activity by binding of endogenous tissue inhibitors of metalloproteinases (TIMPs) to the catalytic domain [2,4]. In CL from a variety of species, the expression of TIMP-1 and TIMP-1like proteins and messenger RNA has been determined, including cow [14,15], sheep [16], rat [17], mouse [18], monkey [19], and human [20]
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