The main objective of this study was to determine how genes related to the extracellular matrix are expressed in the FT throughout the menstrual cycle. We used a PCR array plate that contained 84 genes selected for their role in extracellular matrix remodeling in various cell types.
An analysis of the overall behavior of this set of genes shows that 83 of the 84 genes are expressed in the FT during at least one phase of the menstrual cycle. These genes tend to be expressed at greater levels in the periovulatory phase than in the follicular phase and show reduced expression during the luteal phase. These results probably indicate that this set of genes is regulated by estradiol and progesterone.
Statistical analysis showed that 7 genes undergo a twofold increase in expression in the periovulatory phase compared to follicular phase. Of these genes, 1 encodes a structural protein (COL7A1), 4 encode genes that modify the extracellular matrix through proteolysis (ADAMTS1, ADAMTS13, MMP3, and MMP9) and two are involved in cell adhesion (PECAM1 and THBS3).
Briefly, ADAMTS1 (disintegrin and metalloproteinase with thrombospondin motif 1) has been associated with inflammatory processes, and its activity could be related to salpingitis triggered by sexually transmitted pathogens
]. In addition, this gene is necessary for the development and functionality of the genital tract in rodents
]. ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif 13) functions as a disintegrin and metalloproteinase with a thrombospondin motif. The enzyme encoded by this gene is the protease that cleaves the von Willebrand factor (vWF). Thus, this protein may participate in leukocyte migration that occurs in the fallopian tubes under both normal and pathological conditions. COL7A1 encodes the alpha chain of collagen type VII. The collagen fiber is expressed exclusively below the basal epithelial cells, and mutations in COL7A1 are associated with structural changes
]. MMP3 is involved in the degradation of the extracellular matrix in several tissues
] and is important for embryonic development, reproduction and tissue restructuring. MMP3 has also been associated with pathological processes
], such as tumor metastasis and arthritis. PECAM1 (platelet/endothelial cell adhesion molecule 1) is expressed on the surfaces of platelets, neutrophils and T cells and at intercellular junctions in various cell types. This protein is a member of immunoglobulin superfamily and is involved in leukocyte migration, angiogenesis, and integrin activation
]. THBS3 (thrombospondin 3) is an adhesive glycoprotein that is involved in cell-cell and cell-matrix interactions.
Eight genes exhibited a twofold or greater decrease in their expression in the luteal phase compared to the periovulatory phase. These genes encode proteins with structural functions, including proteolytic functions in the extracellular matrix (MMP16, MMP9, and TIMP2), three related to integrins and cell adhesion molecules (ICAM1, ITGA8, and SELE). COL7A1 is a structural protein, and CLEC3B participates in signal transduction process. Briefly, MMP16 activates MMP2 by cleavage in other cell systems. MMP9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) degrades collagen IV and V. Murine studies suggest that it has a role in tissue remodeling associated with tumor development. TIMP2 is an inhibitor of matrix metalloproteinases and the only TIMP family member capable of suppressing the proliferation of endothelial cells. This protein is critical for maintaining tissue homeostasis by limiting tissue proliferation in response to angiogenic factors and inhibiting protease activity in tissues subjected to matrix remodeling. ICAM1 (intercellular adhesion molecule 1) is a cell surface glycoprotein that is typically expressed in endothelial cells and immune cells. This protein binds to CD11a/CD18- or CD11b/CD18-type integrins. SELE (E-selectin) is expressed on cytokine-stimulated endothelial cells and is believed to be responsible for the accumulation of leukocytes at sites of inflammation. Selectins are cell adhesion molecules that could be involved in the interaction between leukocytes and endothelium, as well as in the pathogenesis of atherosclerosis. CLEC3B (C-type lectin domain family 3, member B) is also downregulated in the luteal phase
Using immunocytochemistry, we determined that MMP2 and MMP9 are expressed in the endosalpinx in a location consistent with a role in the remodeling of the fallopian tubes. Because MMPs are one of the principal regulators of the extracellular matrix, zymograms were performed at different stages of the menstrual cycle. These zymograms showed that MMP2 and MMP7 are expressed in the fallopian tube and that their enzymatic activity increases during the periovulatory phase, which is consistent with gene expression determined by PCR screen.
According to data from our bioinformatics analysis (Figure
A), MMP9 and MMP3 negatively regulate TIMP2, suggesting that increasing the relative amount of MMP9 and MMP3 should decrease the relative amount of TIMP2, but this does not occur. Therefore, we analyzed the cellular interactions involved in the regulation of TIMP2 (Figure
B) and analyzed the relative amounts of each gene. MMP14 and MMP2 showed the highest relative expression during different phases of the menstrual cycle in the initial PCR screen. In other cell systems, MMP14 increases the expression of TIMP2, which could explain the increase in TIMP2 during the periovulatory phase and its subsequent decrease in the luteal phase.
Like all reproductive tissue, the extracellular matrix of the FT should be remodeled during the luteal phase. Therefore, we analyzed the expression of genes related to matrix remodeling in other cell models. Several metalloproteinases were found to maintain their expression through the menstrual cycle, as shown in Figure
. However, there was a decrease in the expression of MMP inhibitors (TIMPs) during the luteal phase, which could be related to extracellular matrix remodeling in human fallopian tubes during the luteal phase. This piece of information will be important for the generation of new patterns of remodeling, the normal and pathological tubal morphology.