Susanne E Ulbrich
email: ulbrich@wzw.tum.de

Swanhild U Meyer

Karina Zitta

Stefan Hiendleder

Fred Sinowatz

Stefan Bauersachs

Mathias Büttner

Thomas Fröhlich

Georg Arnold

Horst-Dieter Reichenbach

Eckhard Wolf

Heinrich H D Meyer

Bovine endometrial metallopeptidases MMP14 and MMP2 and the metallopeptidase inhibitor TIMP2 participate in

Keywords: matrix metallopeptidases, metallopeptidase inhibitor, preimplantation, pregnancy, (Bos taurus), (endometrium)

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A c c e p t e d M a n u s c r i p t [A]

[B]

[C]

A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t [A] TIMP2

[B] MMP2

A c c e p t e d M a n u s c r i p t [START_REF] Thatcher | Embryo health and mortality in sheep and cattle[END_REF]. 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) [START_REF] Visse | Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry[END_REF]. 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 [START_REF] Nagase | Structure and function of matrix metalloproteinases and TIMPs[END_REF]. The transcription of MMPs may be stimulated through cytokines, growth factors, hormones, and cell-to-cell or cell-matrix interactions [START_REF] Nagase | Structure and function of matrix metalloproteinases and TIMPs[END_REF][START_REF] Nagase | Matrix metalloproteinases[END_REF]. 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 preproenzymes 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 [START_REF] Nagase | Structure and function of matrix metalloproteinases and TIMPs[END_REF].

Due to the presence of specific inhibitors, proteolytic degradation, binding to a specific substrate or to a specific site of action and endocytosis [START_REF] Remacle | Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface[END_REF] [START_REF] Itoh | MT1-MMP: a potent modifier of pericellular microenvironment[END_REF]. 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 [START_REF] Curry | The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle[END_REF].

Recently we have applied a holistic transcriptomic approach to define changes in endometrial gene expression during the bovine estrous cycle [START_REF] Mitko | Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle[END_REF] and to compare pregnant and non-pregnant endometrium prior to implantation [START_REF] Bauersachs | Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity[END_REF][START_REF] Klein | Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period[END_REF]. 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.

A c c e p t e d M a n u s c r i p t 5

MATERIALS AND METHODS

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 [START_REF] Ulbrich | Quantitative characterization of prostaglandins in the uterus of early pregnant cattle[END_REF]. In brief, animals were observed for sexual behavior to determine standing heat. Blood samples were taken at day 0 of the estrous cycle and just before slaughter to determine serum progesterone (P 4 ) levels [START_REF] Prakash | Development of a sensitive enzymeimmunoassay (EIA) for progesterone determination in unextracted bovine plasma using the second antibody technique[END_REF].

Animals of the pregnant groups (n=5 per group) were inseminated with cryo-preserved sperm (ejaculate + diluter 1:10) at estrus and slaughtered either at day 15 or at day 18 after insemination. Animals only entered the study if an intact conceptus was visually detected in the uterine lumen. Control animals (n=5 per group) were inseminated with the supernatant of centrifuged sperm and slaughtered at estrus or 3.5, 12, 15, or 18 days after insemination. No concepti were present in control uteri. The obtained uteri were flushed with 100 mL PBS (~21 °C). Flushing fluid was centrifuged at 800 g for 10 min at 5 °C and stored at -20 °C until further investigation. The uterine horns were opened longitudinally as described previously [START_REF] Bauersachs | Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes[END_REF]. Samples were carefully taken from the intercaruncular endometrium of the middle part of the ipsilateral uterine horn with a scalpel and immediately transferred into liquid nitrogen (zymography) or vials containing 4 ml RNAlater (Ambion, Cambridgeshire, UK) and incubated at 4°C overnight (quantitative PCR). Samples were then stored at -20°C until further processing. For immunohistochemistry, uterine tissue samples were transferred to Bouin's fixation solution prior to embedding in paraffin. Tissue samples for qPCR, immunohistochemistry and zymography were collected from the same animals.

Study B

In a confirming survey, five monozygotic twin pairs generated by embryo splitting were sampled as described previously [START_REF] Klein | Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period[END_REF]. Briefly, seven days after standing heat, two in vitro-produced bovine blastocysts (Day 7 after in vitro fertilization) in 200 µL culture medium were transferred into the ipsilateral uterine horn of one twin of each pair. In vitro production of embryos was performed as described previously (Stojkovic et al., 1999b), with minor modifications [START_REF] Klein | Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period[END_REF]. The corresponding twin served as a control and received a sham transfer of the same amount of transfer medium without embryos. The A c c e p t e d M a n u s c r i p t 6 animals were monitored for serum progesterone as described above, and the two groups showed no differences (mean 3.7 ng/ml). At day 18 after estrus the animals were slaughtered. Samples from the ipsilateral intercaruncular endometrium were recovered and processed for mRNA analyses as described above.

RNA extraction and reverse transcription

Total RNA from endometrial tissue samples was isolated using Trizol reagent (Invitrogen, Karlsruhe, Germany) as described previously [START_REF] Bauersachs | Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity[END_REF]. Quantitative realtime PCR (qPCR) was performed to evaluate the influence of the different extraction methods on the mRNA yield according to the MIQE guidelines [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. The integrity of total RNA was determined by the Agilent 2100 Bioanalyzer (RNA 6000 Nano Assay Kit). RNA integrity numbers (RIN) ranged between 7 and 10. Quantity was spectrophotometrically determined at 260 nm by the Nanodrop 1000. A two-step qPCR was undertaken as described recently [START_REF] Bauersachs | Embryo-induced transcriptome changes in bovine endometrium reveal species-specific and common molecular markers of uterine receptivity[END_REF]. Quantitative real-time PCR reactions were performed using the LightCycler® DNA Master SYBR Green I protocol (Roche). Specific primers were used to amplify fragments referring to the genes of interest as depicted in Table 1. All amplified PCR fragments were commercially sequenced to verify the resulting PCR products (MWG-Biotech, Ebersberg, Germany). Thereafter the specific melting point of the amplified product determined within the LightCycler® standard qPCR protocol served as verification of the product identity. The cycle number required to achieve a definite SYBR Green fluorescence signal was calculated by the second derivative maximum method (LightCycler software version 3.5.28).

Data Analysis of qPCR

Results are presented as means ± SEM (n=4) of relative mRNA expression levels. The crossing point (CP) determined for the target genes were normalized against the housekeeping gene Histone H3 (∆CP) [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF]. Results are presented as % expression compared to day 12 (Fig. 1) and to day 15 controls (Fig. 4). For statistical analysis the SAS program package release 9.1.3 (2002; SAS Institute, Inc., Cary, NC, USA) was used. The normal distribution was tested by the Kolmogorow-Smirnov method. The effect of the cycle day on mRNA expression was analyzed using one-way ANOVA and localized using the Least Significant Difference test (LSD). Student´s t-test was employed to compare the transcript abundance in endometrium samples of pregnant versus control group at day 15 and day 18, respectively. Differences were considered significant at P<0.05.

Graphs were plotted with SigmaPlot 8.0 (SPSS Software GmbH, Munich, Germany).

Immunohistochemistry

Serial sections (4 µm) were cut on a Leitz microtome. To expose antigenic sites dewaxed sections were heated four times to 95°C in a 600-W microwave oven in 10 mM sodium citrate buffer (pH 6.0) for 5 min. Endogenous peroxidase activity was then eliminated by incubation in 0.5 % (v/v) H 2 O 2 solution in absolute methanol for 15 min at 20°C. Non-specific protein binding was eliminated by incubation in 10 % normal goat serum in phosphate buffered saline (PBS) for 1 h at 20°C. Sections were then incubated with, a polyclonal rabbit antibody against MMP14 (RB-1544), a monoclonal mouse antibody against MMP2 (MS-806, Ab-4) (all purchased from Lab Vision, Newmarket, Suffolk, UK) [START_REF] Kliem | Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum[END_REF] as well as a monoclonal mouse antibody against TIMP2 (clone 3A4, antibodies-online GmbH, Aachen, Germany). Each antibody was used at a dilution of 4.0 µg/mL. Incubation was performed for 18 h at 4°C in a humidified chamber. This was followed by incubating the sections with HRPlabeled anti-rabbit or anti-mouse IgG at a concentration of 2.5 µg/mL (Sigma-Aldrich) for 1 hr.

The secondary antibody was made visible by reaction with 0.05 % 3,3-DAB and 0.01 % H 2 O 2 in PBS for 15 min. Sections were counterstained in Mayer's hematoxylin, dehydrated, cleared, and mounted. Controls were performed by either replacing primary antibody with buffer or non-immune serum, or incubating with DAB reagent alone to exclude the possibility of non-suppressed endogenous peroxidase activity. Lack of detectable staining in the controls demonstrated the specificity of the reactions. Images were captured on an inverse microscope (LEICA DM IRB, Wetzlar, Germany) equipped with an Olympus DP-72 camera (Hamburg, Germany) using differential interference contrasting technique.

Western blotting

Prior to Western blot analysis, uterine flushings were concentrated using Amicon Ultra-4 (Millipore, Bedford, MA) ultrafiltration devices. The devices were prepared according to the manufacturers instructions, pre-rinsed with 4000 µl water and centrifuged for 10 min at 4000 g using a Beckman GS-6KR (Beckman Coulter, Brea, CA, USA) centrifuge. 2000 µl of uterine flushing was applied to the prepared centrifugal filters and centrifuged for 15 min at 4000 g to obtain approximately 150 µl of concentrate. Protein concentration within the concentrate was determined using the Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]) assay. Aliquots of the samples were subjected to gel electrophoresis and Western blotting as described in [START_REF] Ulbrich | Quantitative characterization of prostaglandins in the uterus of early pregnant cattle[END_REF]. As a control, the transferred proteins were visualized by Ponceau S staining (0.2 % w/v Ponceau S, 3 % trichloroacetic acid, 3 % sulfosalicylic acid in water). The nitrocellulose membrane was blocked overnight in 5 % BSA PBS-T (80 mM di-sodium hydrogen phosphate dehydrate, 20 mM sodium dihydrogen phosphate monohydrate, 100 mM sodium chloride and 0.1 % Tween 20). For immunodetection, a 1:500 dilution of TIMP2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA. U.S.A.) in PBS-T was used.

Mass spectrometric identification of TIMP2

The SDS gel for TIMP2 identification was prepared using exactly the same conditions as for the Western blot detection of the protein. Instead of a semidry transfer the gel was Coomassie stained overnight (50% v/v methanol, 0.05% w/v Coomassie brilliant blue R-250, 10% v/v acetic acid). After destaining (5% (v/v) Methanol with 7% (v/v) acetic acid) the relevant gel band was excised using a scalpel. Tryptic in gel digestions and extraction of the peptides was performed as described in [START_REF] Ulbrich | Quantitative characterization of prostaglandins in the uterus of early pregnant cattle[END_REF]. Chromatographic separation of peptides was performed on a nano-HPLC system (Ettan MDLC, GE Healthcare). Peptides

Gelatine zymography

A c c e p t e d M a n u s c r i p t 9 Zymography was performed as described previously [START_REF] Kleiner | Quantitative zymography: detection of picogram quantities of gelatinases[END_REF].

Briefly, tissue homogenates from 100 mg endometrial samples or cell epithelial cell supernatants were homogenized in PBS using a MagnaLyzer (Roche, Penzberg, Germany).

Following a 1 min centrifugation step at 1000 g, tissue supernatants were concentrated 5-fold using 10 kDa Microcon centrifugal filter devices YM-10 (Millipore, Billerica, USA). 30 µg total protein (evaluated by BioRad protein DC assay) was separated on 7 % SDS -page gels (containing 1 mg/ml gelatine) under non-reducing conditions. After electrophoresis, the gels were soaked in 2.5 % Triton X-100 for 30 min to remove SDS and incubated in Tris-HCl (50 mmol/l, pH 7.5), containing CaCl 2 (5 mmol/l), and ZnCl 2 (1 mmol/l) overnight at 37°C. Finally, gels were stained with Coomassie blue. After Coomassie blue staining white bands of lysis indicate gelatin digestion by MMPs. Densitometric analysis was performed using the ImageJ 1.41o software (ImageJ, NIH, USA).

Endometrial cell culture

Bovine uteri, approximately 8 days after estrus, were collected at a slaughterhouse and kept on ice until further processing in the laboratory (n=5). The physiological status of the uteri was estimated by examination of ovarian morphology [START_REF] Ireland | Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum[END_REF]. The uterine horn ipsilateral to the functional corpus luteum was removed from the uterus, was washed two times with PBS without Ca 2+ /Mg 2+ (PBSA), ligated at the horn tip, and filled with 0.76 % EDTA solution. It was then placed in PBSA and incubated for 60 min at 37°C. The epithelial cells were scrapped directly from the surface and placed in 5 ml Dulbecco modified Eagle medium (DMEM) / F12 with 10 % fetal bovine serum supplemented with 10,000 U/ml penicillin and 10 mg/ml streptomycin. The samples were incubated for three hours in 5 % CO 2 in air to allow the attachment of contaminating stromal cells and to improve the purity of the epithelial cell fraction. The unattached epithelial cells were collected and seeded on 24-well culture plates.

The endometrial tissue without epithelial cells was further dissected and digested with 0.05 % collagenase (digestion activity 454 U/mg) in DMEM/F12. After 3 h of incubation the suspension was filtered to separate the cells from tissue debris using re-usable syringe filter holders (Sartorius, Göttingen, Germany) equipped with 250 µm filters (Bückmann, Mönchengladbach, Germany). The filtrate was loaded on a second filter (33 µm) to separate the stromal cells from the uterine glands. Isolated stromal and epithelial cells were >95 % viable as shown by trypan blue exclusion. Immunocytochemistry with specific antibodies against cytokeratin (for epithelial cells), and vimentin (for the stromal cells), in combination with propidium iodide staining for nuclei, showed that the purity of each cell population was greater than 90 % (data not shown). Approximately 10 6 viable cells were seeded per well.

Stromal cells were placed directly on the plastic surface of a 24-well culture plate (Nunc, Roskilde, Denmark) and epithelial cells were seeded on inserts (Nunc) coated with matrigel A c c e p t e d M a n u s c r i p t 10 (BD Biosciences) according to the manufacturer's specifications. The inserts were filled with 400 µL medium (DMEM/F12, 10 % FBS) and then placed into the wells containing the seeded stromal cells in 400 µL of the same medium. To improve purity of the stromal cell preparation, the medium was changed 18 h after plating, at which time selective attachment of stromal cells had occurred. The medium was changed every other day until confluence was reached; generally after 8 days in culture at 37°C in a humidified atmosphere of 5 % CO 2 in air.

Transmission electron microscopy

Endometrial epithelial cells were cultured for transmission electron microscopy in inserts with or without stromal cells and were subsequently fixed in 2.5 % glutaraldehyde and 2 % formaldehyde in 0.1 M Na-cacodylate buffer over night at 4°C. After washing the samples four times in 0.1 M Na-cacodylate buffer the samples were contrasted with 2 % osmium tetroxide and 1.5 % potassium ferrocyanide. The samples were then washed three times in 0.1 M Na-cacodylate-buffer, and gradually infiltrated with ethanol (50 % to 100 %). Following two changes of propylene oxide the samples were embedded in Epon for 24 h at 60°C. Semithin and ultrathin sections were sliced on a Reichert Ultracut E microtome. The ultrathin sections were stained with uranyl acetate and lead-citrate [START_REF] Reynolds | The use of lead citrate at high pH as an electron-opaque stain in electron microscopy[END_REF]. All samples were analyzed with a Zeiss 902 electron microscope.

In vitro production of bovine embryos for cell co-culture

Embryos were produced based on procedures described previously (Stojkovic et al., 1999a).

Briefly, ovaries were obtained from slaughtered cows. Cumulus-oocyte complexes (COCs) were obtained by aspiration, washed in oocyte maturation medium (TCM-199) with Earle salts containing 22 mg/ml FSH and 8 mg/ml LH and supplemented with 5 % (v/v) estrous cow serum (ECS). Oocytes were matured for 20-22 h at 39°C. After maturation, COCs were washed once in IVF-TALP (modified Tyrode stock solution supplemented with 6 mg/ml essentially fatty-acid-free BSA, 0.022 mg/ml pyruvic acid, and 0.01 mg/ml heparin) and transferred to four-well plates containing 400 µL IVF-TALP per well. Spermatozoa were purified by swim-up procedure in Sperm-TALP (modified Tyrode stock solution supplemented with 6 mg/ml BSA and 0.11 mg/ml pyruvic acid) at 39 °C in 5 % (v/v) CO 2 in humidified air for 1 h. Oocytes were fertilized with 25 μl spermatozoa suspension (~1 x 10 6 spermatozoa/ml).

Approximately 18 h after fertilization, presumptive zygotes were denuded of cumulus cells by vortexing in 1 ml synthetic oviduct fluid (SOF) (SOF stock was modified on the day of use by adding 5 % (v/v) ECS, 4 % (v/v) essential amino acids and 1 % (v/v) nonessential amino acids) for 3 min. Groups of 30-35 presumptive zygotes were placed in 400 µL SOF overlaid Day 8 hatched blastocysts and zonae pellucidae (day 8, n=5 per well) were added to the cultures which had been in medium without serum for 24 h. Empty zonae pellucidae (day 8, n=5 per well) were added to wells that served as controls under the same conditions. After a coculture of 24 h both blastocysts and empty zonae pellucidae were taken out of the wells. In additional co-culture wells, recombinant bovine interferon- instead of embryos was applied for 4 hours (antiviral activity 4.8 x 10 3 U/mL medium, PBL Biomedical Laboratories, Piscataway, NJ, USA) with medium containing the respective diluent serving as respective control. For RNA isolation, epithelial cells were washed with PBS before 500 µL TriZol was added to the inserts or wells that contained the cells. The solution was pipetted up und down vigorously, transferred to a 1.5 ml tube and stored at -20°C until RNA isolation.

Biological assay for Interferon- bioactivity

Interferon production was quantified by means of a highly sensitive bioassay based on the inhibition of the cytopathic effect of vesicular stomatitis virus (Indiana strain) on Madin-Darby bovine kidney (MDBK) cells [START_REF] Rubinstein | Convenient assay for interferons[END_REF]. The NIH recombinant human interferon- 2 reference preparation (No. Gxa01-901-535, NIH-Research Reference Reagent Note No. 31, 1984) was included in each assay. The antiviral activity was shown to be mediated by Interferon-, as the effects of supernatant and an appropriate control Interferon- preparation were blocked by specific anti-interferon sera (kindly provided by Dr. R.M. Roberts, University of Missouri, Columbia, MO) (Stojkovic et al., 1999a). Culture medium that did not contain blastocysts showed no antiviral activity.

RESULTS

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 3.3-fold (p<0.001) lower as compared to day 3.5.

In contrast, TIMP2 mRNA expression was low at estrus and day 3.5 (Fig 1D ), increased 5fold towards day 12, and then decreased towards day 18 (Fig. 1D).

Endometrial MMP14, MMP2 and TIMP2 protein localization

The cytoplasm of luminal epithelial cells stained positive for MMP14 protein (Fig. 2A-D), and there was an observable change of intensity during the cycle with predominant expression during the luteal phase (Fig. 2B) and a rather faint epithelial staining at estrus exemplified in Fig. 2B (insert). Glandular epithelial cells were positive for MMP14 (Fig. 2C). In both, the subepithelial stroma near the endometrial lumen and the zona basalis encompassing the glands, the tunica media of blood vessels (Fig. 2D) displayed MMP14 protein, whereas there was no staining in endothelial cells. There was abundant MMP2 in the endometrium (Fig. 2E-H), especially in the sub-epithelial stroma of the zona functionalis (Fig. 2E, 2G insert). Both luminal (Fig. 2F) and glandular epithelium (Fig. 2G, 2G insert) were devoid of MMP2 protein.

Endothelial cells as well as the tunica media of vessels in the zona basalis (Fig. 2H) displayed prominent staining.

TIMP2 protein was localized to the apical part of the glandular epithelial cells, and only faintly visible on the luminal surface of the epithelial cells (Fig. 2I-L). Stromal cells and vessels were devoid of TIMP2 protein (Fig. 2K,L).

TIMP2 protein in the luminal fluid during the estrous cycle

TIMP2 protein consistently appeared distinctly at day 15 in uterine flushing fluid (Fig. 3A) while it was less prominently present at days 12 and 18. A LC-MS-identification of the corresponding protein band verified the specificity of the antibody (Fig. 3B,C).

Endometrial MMP2 enzyme activity during the estrous cycle

The enzyme activity of active MMP2 in endometrial tissue homogenates was significantly lower at days 3.5 and 12 than at days 15 and 18 (p<0.05) (Fig. 4). Day 0 displayed an intermediate enzyme activity.

Endometrial mRNA expression of MMP14, MMP2 and TIMP2 during early pregnancy

At day 15, pregnant animals in study A already showed a 1.7-fold increase of endometrial TIMP2 mRNA compared to the non-pregnant group. At day 18 the difference in endometrial TIMP2 mRNA abundance between pregnant and non-pregnant animals was 2.0-fold (p=0.03) (Fig. 5). Neither MMP14 nor MMP2 were differentially expressed in day 15 or day 18 pregnant vs. non-pregnant endometrium in study A. In study B, the mRNA expression of TIMP2 was 2.8-fold higher (p=0.01) in day 18 pregnant animals compared to their nonpregnant sisters whereas no significant differences in mRNA abundance were detected for MMP2 or MMP14.

TIMP2 protein in the luminal fluid and endometrial MMP2 enzyme activity during early pregnancy

TIMP2 protein was distinctly present in the luminal fluid of pregnant and non-pregnant day 15 and day 18 animals (Fig. 6A) with no differences between groups. Gelatinase zymography revealed active MMP2 protein in day 18 pregnant as well as control endometrial homogenates (Fig. 6B) with no differences in protein activity between the two groups (controls 169.9±2.8 units vs. pregnant 161.8±5.7 units, p=0.4).

Endometrial cell culture

The morphology of epithelial cells in monolayers (Fig. 7A) and co-cultured with stromal cells (Fig. 7B) was evaluated after 8 days of culture. Transmission electron microscopy showed the presence of epithelial cell characteristics, including microvilli on the upper cell surface and interdigitations in the lateral part between cells (Fig. 7A,B). This confirmed the polarity of epithelial cells in both culture systems. However, desmosomes and tight junctions were only present in co-cultured epithelial cells and not in epithelial cell mono-cultures (Fig. 7D-F).

Moreover, the microvilli in pure epithelial cells were smaller and fewer in comparison with cocultured cells. Importantly, the morphology of co-cultured epithelial cells was more similar to epithelial cells obtained ex vivo (data not shown): mitochondria showed large numbers of cristae and other subcellular structures like the Golgi apparatus and endoplasmic reticulum were also more frequent in co-cultured epithelial cells than in epithelial cell mono-cultures.

Overall these results indicate a positive effect of co-culture with stromal cells on morphology and activity of epithelial cells.

MMP2 activity in uterine epithelial cell co-culture

Active MMP2 protein was present in epithelial cell supernatants co-cultured with stromal cells (Fig. 7C), most probably due to the contribution of stromal cells in co-culture, as stromal cells showed a distinctly higher MMP2 activity than epithelial cells if cultured alone (Fig. 7C), The intensity of enzymatic digestion was not different due to the presence of day 8 hatched blastocysts (controls 54.7±5.8 units vs. blasocyst co-culture 45.4±5.8 units, p=0.3).

TIMP2 mRNA expression in uterine epithelial cell co-culture

In endometrial epithelial cells co-cultured with stromal cells derived from uteri of five different animals the mRNA expression of TIMP2 was 2.6-fold (p<0.01) increased in the presence of day 8 hatched blastocysts. As positive control for the stimulatory effect of the embryos in our culture system, interferon-stimulated gene 15 mRNA levels were determined [START_REF] Klein | Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period[END_REF] and found 13.0-fold (p<0.0001) increased, demonstrating the ability of cultured epithelial cells to respond physiologically to the interferon- released by the hatched blastocysts. The epithelial cells did not reveal a change in TIMP2 gene expression if stimulated with recombinant interferon- (1.1-fold, p=0.9), although interferon-stimulated gene 15 mRNA was up-regulated 57.7-fold (p<0.0001).

Antiviral activity in uterine epithelial cell co-culture supernatant

The concentration of interferon- was below the detection limit of the bioassay in control cultures of endometrial epithelial cells co-cultured with stromal cells (<0.4±0.0 U/mL), whereas significantly higher amounts were assessed in the presence of day 8 blastocysts (6.4±1.8 U/mL) (p<0.0001). 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 endometrium [START_REF] Plaisier | Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle[END_REF][START_REF] Zhang | Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium[END_REF]. Both MMP14 and MMP2 are involved in endometrial shedding during human menstruation [START_REF] Goffin | Expression pattern of metalloproteinases and tissue inhibitors of matrixmetalloproteinases in cycling human endometrium[END_REF] after the luteal phase. As the bovine endometrium does not undergo endometrial shedding, the function of MMPs may be linked to obtaining a balance between proliferation and differentiation around estrus [START_REF] Nagase | Structure and function of matrix metalloproteinases and TIMPs[END_REF]. As membranetype MMP, MMP14 is specifically involved in the proteolytic activity affecting the close surrounding in a paracrine manner in processes of cell migration and invasion [START_REF] Itoh | MT1-MMP: a potent modifier of pericellular microenvironment[END_REF][START_REF] Schenk | Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution[END_REF]. The participation of MMP14 in promoting angiogenesis in the bovine endometrium is discussed controversially, as MMP14 does not appear in endothelial cells [START_REF] Plaisier | Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle[END_REF][START_REF] Zhang | Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium[END_REF]. The remainder MMP14 localization in glands and perivascular muscle cells however is in line with findings in humans [START_REF] Plaisier | Membrane-type matrix metalloproteinases and vascularization in human endometrium during the menstrual cycle[END_REF].

Most prominently present in stromal cells, blood vessels and the basal lamina, MMP2 is likely to be involved in vascular remodeling processes as shown in human tissue [START_REF] Zhang | Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium[END_REF]. Knockout mice have demonstrated that MMP2 is essential for angiogenesis in tumors [START_REF] Mccawley | Matrix metalloproteinases: multifunctional contributors to tumor progression[END_REF], and angiogenic stimuli like vascular endothelial growth factor VEGF and FGF increased MMP2 production from endothelial cells [START_REF] Lamoreaux | Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro[END_REF][START_REF] Zucker | Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: conversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation[END_REF]. The angiogenic action of FGF in turn can be affected by MMP2 since MMP2 is able to release the ectodomain of FGF receptor which is a prerequisite for activation [START_REF] Mccawley | Matrix metalloproteinases: multifunctional contributors to tumor progression[END_REF][START_REF] Moses | The regulation of neovascularization of matrix metalloproteinases and their inhibitors[END_REF]). In accordance to our findings in bovine, pro-MMP2 has been detected in stromal cells of the human endometrium and MMP2 was not present in luminal and glandular epithelium [START_REF] Goffin | Human endometrial epithelial cells modulate the activation of gelatinase a by stromal cells[END_REF]. Although in human endometrial stromal MMP2 transcripts appear to be constantly expressed during all phases of the menstrual cycle, a specific function has been ascribed facilitating menstrual proteolyis [START_REF] Goffin | Expression pattern of metalloproteinases and tissue inhibitors of matrixmetalloproteinases in cycling human endometrium[END_REF][START_REF] Zhang | Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium[END_REF]. The highest MMP2 enzyme activity coincides with the time of conceptus elongation. Thus, MMP2 in bovine endometrium might facilitate and regulate the release of growth factors likely of importance during the window of implantation. The temporal expression pattern of the natural MMP inhibitor TIMP2 appears inversely compared to the expression of MMP2 and MMP14. The highest mRNA levels were detected at day 12, probably due to a stimulatory effect of progesterone. The results are in line with previous findings in cattle [START_REF] Bauersachs | Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes[END_REF][START_REF] Ledgard | Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus[END_REF][START_REF] Mitko | Dynamic changes in messenger RNA profiles of bovine endometrium during the oestrous cycle[END_REF], sheep [START_REF] Hampton | Tissue inhibitors of metalloproteinases in endometrium of ovariectomized steroid-treated ewes and during the estrous cycle and early pregnancy[END_REF] and human [START_REF] Goffin | Expression pattern of metalloproteinases and tissue inhibitors of matrixmetalloproteinases in cycling human endometrium[END_REF] suggesting that ovarian hormones may control TIMP2 expression. Nuclear receptors and their ligands including retinoids and glucocorticoids may participate in the regulation of TIMP2 as well [START_REF] Schroen | Nuclear hormone receptors inhibit matrix metalloproteinase (MMP) gene expression through diverse mechanisms[END_REF]. The demonstrated protein abundance is in line with in vivo studies that found co-expression of mRNA and protein [START_REF] Freitas | Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium[END_REF]. Interestingly, TIMP2 was only marginally detectable in endometrial homogenates (data not shown), but clearly in the uterine fluid. Confirmatively, TIMP2 protein has been found abundantly in the non-pregnant and pregnant uterine luminal fluid [START_REF] Ledgard | Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus[END_REF], suggesting that TIMP2 localized on the apical edge of the glandular epithelium is directly secreted. Others [START_REF] Ledgard | Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus[END_REF] have also detected luminal epithelial and sub-epithelial stromal as well as blood vessel staining in bovine as shown for human endometrium displaying TIMP2 mostly in arterioles and fibroblasts [START_REF] Freitas | Expression of metalloproteinases and their inhibitors in blood vessels in human endometrium[END_REF][START_REF] Stetler-Stevenson | TIMP-2: an endogenous inhibitor of angiogenesis[END_REF]. This disparity may be due to the antibody used.

TIMP2 binds pro-MMP2 with high affinity, so in case of low TIMP2 concentrations at the beginning of the luteal phase only limited amounts of unbound TIMP2 might be available. This could stabilize the endometrium during the secretory phase in a definite homeostasis possibly of relevance for the early formation of the uterine fluid. Around the mid-luteal phase TIMP2 increases extensively. If TIMP2 was present at the interface of epithelial and stromal cell, this increase could allow activation of MMP2 by the TIMP2/MMP14/pro-MMP2 complex at the cell surface into the extracellular space [START_REF] Curry | The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle[END_REF]. Thus TIMP2 could stimulate the MMP2-mediated release of growth factors furthering maternal receptivity when embryo elongation commences. Towards estrus, this induction may cease as all three molecules decrease after prolonged P4 exposure at the end of the luteal phase [START_REF] Curry | The matrix metalloproteinase system: changes, regulation, and impact throughout the ovarian and uterine reproductive cycle[END_REF].

Interferon-, produced in large amounts by the elongating embryo, has been shown to inhibit the expression of pro-MMP1 and pro-MMP2 [START_REF] Salamonsen | Matrix metalloproteinases and their tissue inhibitors at the ovine trophoblast-uterine interface[END_REF] as well as the activity of MMP2 [START_REF] Hashizume | Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture[END_REF], but in our study the expression of neither MMP14 nor MMP2 mRNA was reduced due to pregnancy. In sheep, interferon- has been shown to trigger the increase of TIMP2 [START_REF] Chen | A microarray analysis for genes regulated by interferon-tau in ovine luminal epithelial cells[END_REF][START_REF] Gray | Identification of endometrial genes regulated by early pregnancy, progesterone, and interferon tau in the ovine uterus[END_REF], and TIMP2 mRNA expression has been shown to remain elevated in ovine and bovine early pregnancy (during A c c e p t e d M a n u s c r i p t 17 implantation until day 31) [START_REF] Ledgard | Bovine endometrial legumain and TIMP-2 regulation in response to presence of a conceptus[END_REF]. Because the increase in TIMP2 mRNA at day 18 was moderate in our study A, we confirmed the data using a second experimental set-up with identical twins from which one had received an embryo and the other a seminal plasma control insemination [START_REF] Klein | Monozygotic twin model reveals novel embryo-induced transcriptome changes of bovine endometrium in the preattachment period[END_REF]. Both studies thus clearly confirm an embryo induced up-regulation of TIMP2. Direct physiological consequences of elevated endometrial TIMP2 could include an effective block of proliferation, migration and invasion of endothelial cells and inhibition of endothelial growth factor stimuli [START_REF] Lambert | TIMPs as multifacial proteins[END_REF][START_REF] Murphy | Tissue inhibitor of metalloproteinases-2 inhibits bFGF-induced human microvascular endothelial cell proliferation[END_REF] thus limiting epithelio-chorial placentation. However, the endometrial TIMP2 mRNA increase was not accompanied by a distinct increase of TIMP2 protein in the luminal fluid.

Even if a minor quantitative increase of endometrial TIMP2 protein did occur, TIMP2 did not further the cleavage of pro-MMP2 as gelatinase zymography revealed that active endometrial MMP2 remained unchanged at day 18 of pregnancy. Thus a physiological conclusion of the up-regulated endometrial TIMP2 mRNA during early pregnancy remains to be shown. However, the presence of TIMP2 in the pregnant luminal fluid was clearly demonstrated using a specific antibody as well as a mass spectrometry based approach. As mass spectrometry signals for TIMP2 peptides were intense, significant amounts of TIMP2 were present in the uterine lumen during early pregnancy, most probably in close contact with the developing embryo. As part of the uterine milk, TIMP2 may contribute to providing a shielded environment from ECM degrading enzymes allowing the rapid elongating of the trophoblast.

We furthermore performed in vitro experiments to examine whether the presence of early embryos at the blastocyst stage would locally induce TIMP2 as well and effect MMP2 enzyme activity, thus evidencing their participation in very early embryo-maternal mechanisms of communication. The rationale of developing an in vitro model was because due to its small size prior to elongation, in vivo studies are difficult to perform. Especially finding the site where the embryo possibly exerts important local paracrine interactions with the maternal epithelium is impossible. A co-culture system of primary epithelial and stromal endometrial cells was used clearly mimicking the in vivo situation as cells maintained ultrastructural characteristics and morphological hallmarks such as numerous microvilli, tight junctions and desmosomes among the cells, a monolayer with clear polarity, and well shaped organelles such as Golgi apparatus, endoplasmic reticulum and mitochondria.

Moreover, the co-culture showed higher basal active MMP2 in supernatants than epithelial cells cultured alone, thus demonstrating that the stromal cells in culture resembled those in vivo by producing the majority of MMP2. Only in such a co-culture setup the effects of an early embryo may be reasonably analyzed [START_REF] Ulbrich | In vitro systems for intercepting early embryo-maternal cross-talk in the bovine oviduct[END_REF]. After co-culture of hatched day 8 blastocysts for 24 h, cell culture supernatants contained significant amounts of A c c e p t e d M a n u s c r i p t 18 interferon- (Stojkovic et al., 1999a) inducing ISG15 ubiquitin-like modifier as internal control gene. The epithelial cells also responded to the stimulus of the co-cultured blastocysts by significantly increasing TIMP2. Because the transcriptional response did not lead to enhanced pro-MMP2 activation in the culture supernatants, TIMP2 in this context might resemble an interferon-dependent gene which does not induce further downstream effects.

But interestingly and contrary to findings in sheep endometrium [START_REF] Chen | A microarray analysis for genes regulated by interferon-tau in ovine luminal epithelial cells[END_REF], a direct stimulation of recombinant interferon- did not affect TIMP2 gene expression in our bovine cell culture, indicating species specific peculiarities [START_REF] Spencer | Genes involved in conceptus-endometrial interactions in ruminants: insights from reductionism and thoughts on holistic approaches[END_REF]. Thus, both the causing variable, most probably a factor secreted by the early embryo, and the purpose of the enhancement remain to be shown. By cautiously extrapolating in vitro findings, these results point towards an earlier maternal recognition of pregnancy appearing locally than previously described [START_REF] Evans | Use of microarray technology to profile gene expression patterns important for reproduction in cattle[END_REF]. Further studies are indicated.

The regulation of MMPs and TIMPs is a complex interplay of many participating factors. Although the impact of pregnancy is much less pronounced in ruminants than in primates due to the noninvasive placentation, the principal importance of matrix metallopeptidases during the preimplantation period is established in cattle as well [START_REF] Salamonsen | Role of proteases in implantation[END_REF]. In this study, we have characterized a subset

Figure 1 :

 1 Figure 1: Transcript abundance of matrix metallopeptidases and their inhibitor in bovine endometrium.

  Figure 2: Immunohistochemical localization of MMP14, MMP2 and TIMP2 in bovine endometrium.

Figure 3 :

 3 Figure 3: Protein abundanceof TIMP2 in selected uterine fluid samples during the estrous cycle and LC-MS identification of TIMP2 protein.

  Figure 4: Enzyme activity of MMP2 in bovine endometrium.

Figure 5 :

 5 Figure 5: Messenger RNA expression of MMP14 [A], MMP2 [B] and TIMP2 [C] in endometrial tissue during the estrous cycle (black bars) and early pregnancy (grey bars).

Figure 6 :

 6 Figure 6: TIMP2 western blot of selected uterine fluid samples [B] and gelatine zymography showing active MMP2 at 62 kDa [B] of selected endometrium samples during early pregnancy compared to nonpregnant control animals. No differences appeared.

Figure 7 :

 7 Figure 7: Representative transmission electron microscopy of endometrial epithelial cells in culture [A-B, D-F]. Gelatine zymography of MMP2 in cell culture supernatants [C].

  Figure 7

  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.

  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 A c c e p t e d M a n u s c r i p t 4 trimolecular complex with membrane bound MMP14. An adjacent MMP14 molecule is then able to cleave the TIMP2 bound pro-MMP2

  A c c e p t e d M a n u s c r i p t 8 Membranes were incubated for 1 hour at room temperature, washed once for 10 min and twice for 15 min in PBS-T, and incubated for 1 hour in a 1:2000 dilution of horse raddish peroxidase conjugated anti-mouse secondary antibody (polyclonal goat anti-mouse immunoglobulins, Dako, Hamburg, Germany). The substrate (SuperSignal West Pico Chemiluminescent, Thermo Fisher Scientific , Rockford, USA) was used as recommended by the manufacturer. The membrane was exposed to X-ray film (BioMax XAR, Kodak, Rochester, USA).

  were loaded on a reversed phase trap column with a flow-rate of 10µl per min (Loading buffer: 0.1 % formic acid; Trap column: C18 PepMap 100, 5µm bead size, 300 µm i.d., 5mm length, LC Packings) and subsequently separated with an analytical column (Dr. Maisch, Reprosil-Pur C18 AQ, 3 µm; 150 mm x 75 µm, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) with a 120-min gradient (A: 0.1% formic acid, B: 84% acetonitrile and 0.1% formic acid) at a flow rate of 280 nl/min. The gradient was 0-30% B in 80 minutes, 30-60% B in 30 minutes, 100% B for 10 minutes. Mass spectrometry was performed on a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) online coupled to the nano-HPLC system. For electrospray ionization a distal coated SilicaTip (FS-360-50-15-D-20) and a needle voltage of 1.4 kV was used. The MS method consisted of a cycle combining one full MS Orbitrap scan (Resolution: 60,000; Mass range: 300-2000 m/z) with five data dependant LTQ MS/MS scans (acquisition parallel to Orbitrap scans, 35% collision energy). The dynamic exclusion was set to 30 s. Finally the MS/MS data were searched with Mascot Version: 2.1.03 (Matrix Science, Boston, USA) using IPI-bovine database (Release 3.52) and the following parameters: i) Enzyme: Trypsin, ii) Fixed Modification: Carbamidomethyl (C), iii) Variable modifications: Oxidation (M), Methyl (K), N-acetyl (protein); iv) Peptide tol.: 10 ppm, v) MS/MS tol.: 0.8 Da, vi) Peptide charge 1+, 2+ and 3+, vii) Instrument: ESI-TRAP and viii) Allow up to 1 missed cleavages.

  and cultured at 39°C in a humidified atmosphere of 5 % (v/v) CO 2 , 5 % (v/v) O 2 , and 90 % (v/v) N 2 for seven days.

  likely to participate in the preparation of a receptive endometrium in cattle, as both mRNA and protein abundances varied significantly during the estrous cycle.

  of matrix metallopeptidases involved in cleavage of different substrates and participating in various regulatory mechanisms. Taken together we find pronounced variations in their abundance during the cycle. Interestingly, a high activity of endometrial MMP2 during the elongation phase of the trophoblast may determine the allocation of growth factors supporting conceptus development.Moreover, abundant TIMP2 in the uterine fluid during trophoblast elongation may participate in regulating a well-balanced ECM integrity, thus indicating that MMPs may actively take part in maternal preparation of a receptive endometrium.

  pairs used, corresponding references as well as annealing temperatures (AT), fluorescence acquisition (FA), melting points (MP) and the resulting fragment length.

Figure 1 :

 1 Figure 1: Transcript abundance of matrix metallopeptidases and their inhibitor. Messenger RNA expression of MMP14 (A), MMP2 (B) and TIMP2 (C) in endometrial tissue (n=5-7 animals per group) during the estrous cycle is shown as means % ± SEM relative to day 12 (100 %). Different superscript letters indicate significant differences (p<0.05) among day 0, 3.5, 12, 15 and 18 following estrus, thus same superscript letters indicate that there is no significant difference.

Figure 2 :

 2 Figure 2: Immunohistochemical localization of MMP14 (A-D), MMP2 (E-H) and TIMP2 (I-L) in bovine endometrium. The luminal epithelium (B,F,J), glands (C,G,K) and vessels (D,H,L) are specifically depicted. Panel G depicts MMP2 localization in a section of the zona basalis of the endometrium, whereby the insert shows the clear absence of MMP2 in glandular epithelial cells of the sub-epithelial stroma, which are distinctly MMP2-positive. Panel K exemplifies TIMP2 localized to the apical edge of glandular epithelial cells in both zona basalis (K) and zona functionalis of the sub-epithelial stroma (K insert). The inserts in (A, F, J) represent negative controls. Black arrowheads point at immunopositive cells, white arrowheads indicates lack of staining. Tissue sections were counterstained with Mayer's haemalaun. Bar in (A), (E) and (I) = 300 µm, and 50 µm in all other figures.

Figure 3 :

 3 Figure 3: Representative Western blot of TIMP2 protein (two samples per cycle stage) of uterine fluid during the estrous cycle [A] with short (left) and longer (right) exposure time. A corresponding protein gel stained with Ponceau S is shown in [B] indicating a band segment which was cut out for the LC-MS-identification of TIMP2. Peptides highlighted in red were identified. A representative MS/MS spectrum of peptide EVDSGNDIYGNPIK (corresponding to aa 54 to 67 of bovine TIMP2 NCBI accession AAI02711) is shown in [C].

Figure 4 :

 4 Figure 4: Gelatine zymography of MMP2 enzyme activity in bovine endometrium during the estrous cycle. Band intensity units of active MMP2 are shown as means ± SEM with different superscript letters indicating significant differences (p<0.05) among day 0, 3.5, 12, 15 and 18 following estrus. Same superscript letters indicate that there is no significant difference.

Figure 5 :

 5 Figure 5: Messenger RNA expression of MMP14 (A), MMP2 (B) and TIMP2 (C) in endometrial tissue (n=5-7 per group) during the estrous cycle (black bars) and early pregnancy (grey bars). Data are shown as means % ± SEM relative to day 15 controls (100 %). * indicates a significant difference (p<0.05).

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.

Grant support: This study was supported by the German Research Foundation (UL 350/1-2, FOR 478).