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Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.13/927259
- Matrix metalloproteinase-2 in early embryo implantation in the mouse
Roy, Tammie K.
- University of Newcastle. Faculty of Health, School of Biomedical Sciences and Pharmacy
- Research Doctorate - Doctor of Philosophy (PhD)
- In mammals the implantation of trophoblast cells into the uterus is highly invasive and requires the degradation of the extracellular matrix (ECM) of the uterine wall. It was first hypothesised more than 70 years ago that the invasiveness of the trophoblast cells was due to proteolytic activity. Matrix metalloproteinases (MMPs) are a family of zinc and calcium dependent endopeptidases that in combination are capable of degrading virtually all of the components of the ECM, both interstitial matrix and basement membrane [1, 2]. Research into implantation in rodent and ovine models has implicated numerous members of the MMP family (MMP-2, -3, -7, -9, -11, -13 and MT1-MMP) in the control of early events of embryo implantation [3-9]. The majority of these studies agree that the most important of the MMPs in implantation are MMP-2 and MMP-9. The process of embryo attachment and invasion is one of the most biologically complex processes studied in reproductive biology. While evidence exists for the presence of MMPs during embryo implantation, knowledge is limited on the exact expression patterns and regulatory pathways that control these enzymes and allow for the invasion of trophoblast cells in the maternal endometrium. Previous research, undertaken by this research group, has looked at the expression of MMP-2 and MMP-9 during embryo implantation in the mouse using a new model of mating that allows for a more accurate timing of implantation events. The results from these experiments showed that there is a significant increase in the presence of MMP-2 in the luminal flushes around 97 – 98 hours post-insemination (day 4 of pregnancy). The aim of this project was to test the broad hypothesis that MMP-2 proteins are expressed and activated at the early stages of blastocyst implantation by cells of both endometrial and embryonic origin. Additionally, studies also tested the hypothesis that the expression of MMP-2 is specific to the events of embryo implantation and requires an interaction between the embryo and endometrial cells. An optimised mating protocol was used to facilitate more accurate identification of the early stages of implantation in order to be able to further define the expression patterns of MMP-2. Immunohistochemical labelling of MMP-2 proteins in formalin fixed sections of embryo implantation sites at specific times post-insemination localised MMP-2 expression to cells of both the blastocyst and endometrium. Specifically, antibody staining was identified in trophectoderm cells and inner cell mass cells of the embryo and in endometrial epithelial cells of the lumen and in the cells of the endometrium. While these experiments demonstrated MMP-2 protein localisation, they did not allow the assessment proteinase activity. The presence of gelatinase activity was identified through the application of in situ zymography to fresh frozen sections of tissue from implantation sites collected at specific times post-insemination. Results from these experiments confirmed the presence of gelatinolytic activity at implantation sites collected between 96-102 hours post-insemination. Gelatinolytic activity was localised to the uterine lumen, luminal epithelial cells, trophoblast cells and in endometrial cells surrounding the implanting blastocyst. These results suggest that the MMP-2 protein identified by the immunohistochemistry results may be active during the early stages of embryo attachment and adhesion. Two murine pseuodopregnancy models were employed to test the hypothesis that expression of MMP-2 is specific to the events of embryo implantation and requires an interaction between the embryo and endometrial cells. Gelatin zymography of luminal flushes taken from both pseudopregnant and pregnant females showed that secretion of MMP-2 into the luminal cavity was increased in normal pregnancy. To further test this hypothesis, embryos were cultured in the presence of fibronectin on a substratum of endometrial cells extracted from pregnant female mice. Embryos cultured in the presence of fibronectin secreted both inactive MMP-9 and MMP-2. Cultured endometrial cells secreted MMP-2 in all experiments assessed and this was independent of the presence or absence of an embryo. In summary the data presented within this thesis support the hypothesis that MMP-2 protein is expressed and activated at the early stages of blastocyst implantation by cells of both endometrial and embryonic origin. Furthermore, the use of the optimised mating protocol allowed for more precise timing of MMP-2 expression throughout the early stages of embryo attachment and adhesion. The expression of MMP-2 is also specific to the events of embryo implantation and requires an interaction between the embryo and endometrial cells.
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- Copyright 2011 Tammie K. Roy