Abstract
BackgroundAdministration of a single physiological dose of 17beta-estradiol (E2:40 microg/kg) to the ovariectomized immature rat rapidly induces uterine growth and remodeling. The response is characterized by changes in endometrial stromal architecture during an inflammatory-like response that likely involves activated matrix-metalloproteinases (MMPs). While estrogen is known as an inducer of endometrial growth, its role in specific expression of MMP family members in vivo is poorly characterized. E2-induced changes in MMP-2, -3, -7, and -9 mRNA and protein expression were analyzed to survey regulation along an extended time course 0-72 hours post-treatment. Because E2 effects inflammatory-like changes that may alter MMP expression, we assessed changes in tissue levels of TNF-alpha and MCP-1, and we utilized dexamethasone (600 microg/kg) to better understand the role of inflammation on matrix remodeling.MethodsOvariectomized 21 day-old female Sprague-Dawley rats were administered E2 and uterine tissues were extracted and prepared for transmission electron microscopy (TEM), mRNA extraction and real-time RT-PCR, protein extraction and Western blot, or gelatin zymography. In inhibitor studies, pretreatment compounds were administered prior to E2 and tissues were harvested at 4 hours post-hormone challenge.ResultsUsing a novel TEM method to quantitatively assess changes in stromal collagen density, we show that E2-induced matrix remodeling is rapid in onset (< 1 hour) and leads to a 70% reduction in collagen density by 4 hours. Matrix remodeling is MMP-dependent, as pretreatment with batimastat ablates the hormone effect. MMP-3, -7, and -9 and inflammatory markers (TNF-alpha and MCP-1) are transiently upregulated with peak expression at 4 hours post-E2 treatment. MMP-2 expression is increased by E2 but highest expression and activity occur later in the response (48 hours). Dexamethasone inhibits E2-modulated changes in collagen density and expression of MMPs although these effects are variable. Dexamethasone upregulates MMP-3 mRNA but not protein levels, inhibiting E2-induced upregulation of MMP-7, and -9, and MCP-1 mRNA and protein but not inhibiting the hormone-induced increase in TNF-alpha mRNA.ConclusionThe data demonstrate that E2-regulated endometrial remodeling is rapid in onset (<1 hour) and peak expression of MMPs and inflammatory mediators correlates temporally with the period of lowest stromal collagen density during uterine tissue hypertrophy.
Highlights
Administration of a single physiological dose of 17beta-estradiol (E2:40 microg/kg) to the ovariectomized immature rat rapidly induces uterine growth and remodeling
We assessed collagen matrix density using micrographs prepared from uterine sections along the entire length of the uterine horn to best represent whole tissue architecture changes and over an extended time course after administration of a single physiological dose of E2 (40 μ/kg) to study stromal collagen architecture throughout both the early hypertrophic and latter hyperplastic phases of uterine growth
Using this model we show that expression of MMP-3, MMP-7, and MMP-9 is upregulated at both RNA and protein level early within the E2-induced growth response correlating with the time point where stromal collagen matrix density reaches its lowest point (4 h)
Summary
Administration of a single physiological dose of 17beta-estradiol (E2:40 microg/kg) to the ovariectomized immature rat rapidly induces uterine growth and remodeling. The ovariectomized (OVX) immature rat has been used extensively to study hormone-specific regulatory processes in the mammalian uterus In this model, administration of a single physiological dose of 17β-estradiol (E2; 40 μg/kg) induces a biphasic uterine growth response, characterized by rapid tissue hypertrophy followed by hyperplasia. Proteinases released by inflammatory cells, including tryptase, chymase, leukocyte elastase, and MMPs, have been shown to activate proMMPs in culture [8,9,39,40], while metabolites and oxidants generated by inflammatory cells may regulate MMP activation [4143] These studies and others suggest an important role for inflammatory mediators in the activation of proMMPs in the endometrial extracellular matrix. MMPs and inflammatory mediators are components of a complex network governing uterine growth and remodeling, as well as other normal and disease processes
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