Bovine endometrial metallopeptidases MMP14 and MMP2 and the metallopeptidase inhibitor TIMP2 participate in maternal preparation of pregnancy☆
Introduction
Functional changes occurring during the bovine estrous cycle are of specific importance for the preparation of the reproductive tract hosting final gamete maturation, fertilization, and embryonic development. Embryonic losses occur predominantly during the preimplantation period (Thatcher et al., 2006). A well-synchronized maternal environment must therefore be present to allow normal development.
Matrix metallopeptidases (MMPs) and the tissue metallopeptidase inhibitors (TIMPs) are most important mediators in the process of remodeling extracellular matrices (ECM) (Visse and Nagase, 2003). Built by a complex network of fibrous matrix proteins, the ECM not only drives form and polarity of cellular arrangement, but also impairs growth and development as well as blood vessel formation and stabilization. It forms a dynamic intercellular net, predominated by collagen and proteoglycans, through which ions, nutrients, metabolites and peptides like growth factors may diffuse. As MMPs degrade ECM components and cell surface molecules, they are involved in the release and activation of cytokines and growth factors which direct cell migration, differentiation, and vascularization (Nagase et al., 2006).
MMPs and TIMPs are involved in the regulation of key steps in reproduction, such as ovulation and luteolysis, endometrium function, growth and development of fetal membranes, cervix dilation during parturition and postpartum regression of the uterus. Uterine receptivity requires timely and spatially controlled changes of the ECM in which MMPs are involved. The transcription of MMPs may be stimulated through cytokines, growth factors, hormones, and cell-to-cell or cell–matrix interactions (Nagase et al., 2006, Nagase and Woessner, 1999). The activity of the MMPs is low in tissues which are in a dynamic equilibrium.
MMPs consist of highly conserved functional domains which direct substrate specificity and comprise binding sites for MMP-specific inhibition by TIMPs. MMPs are synthesized as pre-proenzymes and processed to proenzymes which are eventually secreted into the extracellular space. The activation of the proenzyme in the ECM is of specific regulatory importance, as this is a prerequisite for exhibiting proteolytic activity (Nagase et al., 2006). Due to the presence of specific inhibitors, proteolytic degradation, binding to a specific substrate or to a specific site of action and endocytosis (Remacle et al., 2003) are control mechanisms directing MMP activity. The natural inhibitors of MMPs, TIMPs 1–4, are involved in the maintenance of balanced ECM remodeling. They bind with different affinities to the catalytically active domain and thereby exert their specific proteinase inhibiting activity. TIMP2 additionally participates in activating pro-MMP2 by binding pro-MMP2 to form a trimolecular complex with membrane bound MMP14. An adjacent MMP14 molecule is then able to cleave the TIMP2 bound pro-MMP2 (Itoh and Seiki, 2006). The concentration of all three molecules is important as pro-MMP2 may only be cleaved in the presence of a neighboring non-TIMP2 bound MMP14 molecule and free available TIMP2. In case TIMP2 is present in excess or absent, pro-MMP2 is not cleaved (Curry and Osteen, 2003).
Recently we have applied a holistic transcriptomic approach to define changes in endometrial gene expression during the bovine estrous cycle (Mitko et al., 2008) and to compare pregnant and non-pregnant endometrium prior to implantation (Bauersachs et al., 2006, Klein et al., 2006). Herein, a high number of genes related to extracellular matrix remodeling were found to be differentially expressed. In the present study, we characterized the expression of a prominent subset of candidate genes thereof in detail including their cellular distribution and protein activity. Additionally, in vitro co-culture of endometrial cells with embryos was performed to address the question whether endometrial matrix metallopeptidases and the metallopeptidase inhibitor TIMP2 contribute to maternal preparation and endometrial recognition of pregnancy.
Section snippets
Pretreatment of animals and collection of endometrial tissue samples
All experiments were performed in accordance with the International Guiding Principles for Biomedical Research Involving Animals, as promulgated by the Society for the Study of Reproduction and with the European Convention on Animal Experimentation.
Study A
Cyclic heifers (Deutsches Fleckvieh, Simmental) were cycle synchronized at diestrus as described previously (Ulbrich et al., 2009). In brief, animals were observed for sexual behavior to determine standing heat. Blood samples were taken at day 0 of
Endometrial mRNA expression of MMP14, MMP2 and TIMP2 during the estrous cycle
The day of the cycle had a significant effect on endometrial transcript abundance of MMP14 (p = 0.0002), MMP2 (p = 0.007) and TIMP2 (p < 0.0001) (Fig. 1).
The mRNA expression of MMP14 was high at estrus and day 3.5 and subsequently declined. At day 18 the transcript abundance was 2.5-fold (p < 0.01) lower compared to day 12 (Fig. 1B).
A similar mRNA expression pattern as seen for MMP14 was measured for MMP2 (Fig. 1C) showing highest expression following estrus. At day 18 the level of expression was
Discussion
Matrix metallopeptidases seem likely to participate in the preparation of a receptive endometrium in cattle, as both mRNA and protein abundances varied significantly during the estrous cycle.
MMP14 and MMP2 showed similar expression profiles during the estrous cycle, with high transcript abundance around estrus followed by a gradual decline during the luteal phase. Supporting the present findings, progesterone has shown an inhibitory influence on MMP14 mRNA and protein expression in human
Acknowledgments
We thank Dr. Katrin Mitko for help with tissue sampling, Angela Sachsenhauser and Miwako Kösters for technical assistance and Nadine Waldschmitt for excellent performance of immunohistochemistry.
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Grant support: This study was supported by the German Research Foundation (UL 350/1-2, FOR 478).
- 1
Present address: Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Campus Kiel, Germany.
- 2
Present address: JS Davies Epigenetics and Genetics Group, School of Agriculture, Food & Wine and Research Centre for Reproductive Health, The University of Adelaide, Roseworthy Campus, SA 5371, Australia.