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BOR - Papers in Press, published online ahead of print November 7, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.063891
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BIOLOGY OF REPRODUCTION 78, 316–323 (2008)
DOI: 10.1095/biolreprod.107.063891
© 2008 by the Society for the Study of Reproduction, Inc.

Pregnancy; 

Decidual Heparanase Activity Is Increased During Pregnancy in the Baboon (Papio anubis) and in In Vitro Decidualization of Human Stromal Cells1

Sonia S D'Souza 3, Asgerally T Fazleabas 4, Prajna Banerjee 5, J. Robert A Sherwin 6, Andrew M Sharkey 7, Mary C Farach-Carson 4 8, and Daniel D Carson 2 8

Departments of Chemistry and Biochemistry3 and Material Sciences,4 University of Delaware, Newark, Delaware 19716 Department of Obstetrics and Gynecology,5 University of Illinois, Chicago, Illinois 60612 Department of Obstetrics and Gyneacology,6 The Rosie Hospital, Cambridge CB2 2SW, United Kingdom Department of Pathology,7 University of Cambridge, Cambridge CB2 1QP, United Kingdom Department of Biological Sciences,8 University of Delaware, Newark, Delaware 19716

ABSTRACT

Implantation is a complex process involving interactions between the embryo and the uterus. Adhesion, remodeling of the maternal vasculature, and decidualization are crucial events necessary for successful implantation to occur. Heparanase (HPSE), an endo-β-D-glucuronidase, cleaves heparan sulfate at specific sites, leading to release of growth factors that may be involved in decidualization and remodeling of the maternal vasculature. HPSE also can function as a cell adhesion molecule. The aim of this study was to determine the expression of HPSE in the uteri of nonpregnant and pregnant baboons as well as in human stromal fibroblasts decidualized in vitro. We examined the localization and expression of HPSE using immunohistochemistry, Western blotting, RT-PCR, and activity assays. In nonpregnant baboon uteri, HPSE expression was localized to the apical surface of the glandular epithelia and in glandular secretions. However, in pregnant baboon uteri, HPSE was localized primarily in decidua. Uteri obtained at midpregnancy had higher heparanase activity compared with the nonpregnant uteri. A slight increase in HPSE expression was observed in human stromal fibroblasts decidualized in vitro. HPSE and HPSE2 mRNA transcripts were present in both decidualized tissue and cells. Increases in heparanase activity in the decidua from pregnant baboon uteri compared with tissue from nonpregnant animals and in human stromal fibroblasts decidualized in vitro suggest that HPSE plays a role in extracellular matrix remodeling and in increasing heparin-binding growth factor release during embryo implantation.

heparanase, implantation, in vitro human decidualization, primate

INTRODUCTION

Implantation involves multiple, complex interactions between the embryo and the uterus [1, 2]. For successful implantation to occur, these interactions must be well coordinated. The first step during implantation is the apposition and adhesion of the blastocyst to the luminal epithelia. Trophoblasts must then invade the uterus and remodel the maternal vasculature. In primates, including the baboon, initial adhesion of the blastocyst occurs between Day 8 and Day 10 after ovulation [3, 4]. In nonhuman primates, chorionic gonadotrophin (CG), synthesized and secreted by trophoblasts, serves as a major embryonic signal [5]. In response to CG, early in the implantation process in the baboon uterus, luminal and superficial glandular epithelia form epithelial plaque while uterine stromal cells undergo differentiation to form decidual cells [6].

Heparanase (HPSE), an endo-β-D-glucuronidase that cleaves heparan sulfate (HS) at specific sites [7], has been identified in a wide variety of tissues and cells, including the placenta and cells of the immune system [8–13]. Initially synthesized as a catalytically inactive 65-kDa proenzyme, HPSE is processed to an active heterodimer composed of 50-kDa and 8-kDa subunits [14]. Human HPSE has been cloned by several groups [15–18]. HPSE displays optimal catalytic activity at pH 5.0 [18]; however, at pH 7.0 it can function as a cell adhesion molecule due to its relatively weak catalytic activity at this pH range [19, 20]. The catalytically inactive 65-kDa form of HPSE also supports cell adhesion [20].

HPSE has been implicated in a number of normal and pathological process, including angiogenesis, tumor metastasis, and cell invasion [21–23]. HPSE mRNA and catalytic activity have been detected in both human and bovine placenta [24–28]. Overexpression of human HPSE in mice suggests a role for heparanase in a number of processes, including embryonic implantation, angiogenesis, and tissue remodeling [29]. Studies in vivo and in vitro showed that HPSE increased the number of implanting embryos in mice [30]. HPSE also is present in the normal human endometrium, with highest expression during the late proliferative phase [31] and secretory phase [32]. Heparanase-2 (HPSE2), a member of the heparanase family, has been cloned, and its tissue distribution was identified at the mRNA level in several tissues, including the human uterus [33]. HPSE2 mRNA can encode up to three different protein isoforms through alternative splicing [33]. However, it is not known whether any form of HPSE2 is expressed at the protein level or has enzymatic activity.

Ethical and moral constraints limit the study of the implantation process in humans. For these reasons, we used a nonhuman primate implantation model to study the expression of heparanase during pregnancy. Recently, using a well-established simulated pregnant baboon model, it was found that heparanase mRNA was upregulated in the endometrium in response to human CG infusion [34]. Consequently, we extended these studies by examining the expression of HPSE in the baboon uterus during implantation. Our aim was to define the pattern of HPSE expression and identify changes in heparanase activity during pregnancy. Using a number of experimental approaches, we show that HPSE and HPSE2 mRNA are present in both decidual tissues from pregnant baboons and in human stromal fibroblasts decidualized in vitro. In uteri from receptive nonpregnant baboons, HPSE was present in glandular epithelia and glandular secretions. In contrast, HPSE was detected in the decidua of pregnant baboon uteri. HPSE and heparanase activity also increase in human stromal fibroblasts decidualized in vitro. Our data demonstrate that active heparanase is present in critical regions of the uterus, where it can contribute to morphogenetic events that take place during the implantation process in primates.

MATERIALS AND METHODS

Animals and Tissue Collection

Uterine tissues were obtained from adult female baboons (Papio anubis) either at hysterectomy or endometriectomy, as previously described [35, 36]. Immunocytochemical localization of heparanase was also done using archived human implantation sites obtained from elective terminations of first-trimester pregnancies (approximately 8–10 wk) under approved protocols at the Cook County Hospital and the University of Illinois at Chicago Institutional Review Boards [37]. Tissue was obtained from normally cycling females on Days 9 and 10 after ovulation as well as from pregnant baboon uteri. Uterine tissues were harvested on the indicated days for analysis. Tissue was fixed in tissue-freezing medium (Electron Microscopy Sciences, Hatfield, PA) for immunohistochemistry. Tissue was also collected for Western blotting and heparanase activity assay. All experimental procedures were approved by the Animal Care Committee of the University of Illinois, Chicago.

Materials

Acrylamide, agarose, ammonium hydroxide, bovine serum albumin (BSA), glycine, phenylmethylsulfonyl fluoride (PMSF), potassium dichromate, sodium azide, sodium dodecyl sulfate, sodium chloride, Tris base, Tween 20, Triton X-100, and urea were purchased from Fisher Scientific (Pittsburgh, PA). Phosphate-buffered saline (PBS), L-glutamine, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Dextran blue, β-mercaptoethanol, ethidium bromide, HEPES, leupeptin, magnesium chloride, pepstatin A, and sodium bicarbonate were purchased from Sigma Aldrich (St. Louis, MO). Na235SO4 (carrier-free) was obtained from MP Biochemicals (Solon, OH). Tissue culture plates were purchased from Corning (Corning, NY). All chemicals used were reagent grade or better. PI-88 was provided by Progen Pharmaceuticals Ltd.

Isolation and Culture of Endometrial Stromal Cells

Decidualized uterine endometrium maintains a proliferating population of fibroblastic cells, which closely resemble the stromal cells [38]. Human stromal fibroblasts were isolated from decidua parietalis dissected from the placental membranes after normal vaginal delivery at term [39]. These studies were approved by the Institutional Review Board of the University of Illinois. Briefly, scraped cells were digested in 0.1% (w/v) collagenase, 0.02% (w/v) deoxynuclease in calcium- and magnesium-free Hanks balanced salt solution. Cells were plated in four 100-mm culture dishes (Becton Dickinson and Co. Labware, Franklin Lakes, NJ) and placed into an incubator at 37°C, a humidified atmosphere of air/CO2 mixture (95:5, v/v). The next day, the plates were extensively washed with PBS to remove nonadherent (mainly decidual) cells. At confluence, cells were trypsinized and used for experiments in passage numbers 3–5. Cell purity was assessed by immunocytochemistry using antibodies against cytokeratin (DAKO Corp., Carpenteria, CA) and vimentin (Zymed Laboratories Inc., San Francisco, CA). The purity of the fibroblast cell preparations used in studies was more than 95%. Briefly, cells were grown in RPMI 1640 medium containing 10% (v/v) FBS. When cells reached the desired confluency (80%), cells were maintained in RPMI 1640 containing 2% (v/v) FBS. Cells were treated with 36 nM 17β-estradiol and 1 µM medroxyprogesterone acetate. A second treatment group was done in the presence of 36 nM 17β-estradiol, 1 µM medroxyprogesterone acetate, and 100 µM cAMP. Media was changed every 2 days, and treatments were maintained for either 3, 6, or 12 days, as indicated. Criteria for differentiation in these cultures were as described previously [39].

Cell Culture

WiDr cells were kindly provided by Dr. Carlton Cooper (University of Delaware, Newark, DE); B16BL6 mouse melanoma cells were a gift from Dr. Dario Marchetti (School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA). WiDr cells were cultured in Eagle minimum essential medium (ATCC, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. B16BL6 mouse melanoma cells were cultured in DMEM-F12 (Invitrogen) supplemented with 5% (v/v) heat-inactivated FBS. Cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO2 (v/v).

RT-PCR

Total RNA was extracted from endometria with TRIzol (Invitrogen) per the manufacturer's instructions and quantified by UV absorption at 260-nm wavelength. Complementary DNA was synthesized from 500 ng total RNA using Omniscript RT kit (Qiagen, Valencia, CA). The reaction was carried out at 37°C for 1 h per the manufacturer's instructions. The RT-PCR reaction was performed using HotStart Taq DNA polymerase kit (Qiagen) per the manufacturer's instructions. Samples were cycled as follows: 15 sec at 95°C and 60 sec at 60°C for 30 cycles using Gene Amp PCR System 9700 (Applied Biosystems, Foster City, CA). Products were analyzed on a 2% (w/v) agarose gel and stained with ethidium bromide. Each experiment was performed in duplicate. The primer sequences used: HPSE (AF084467) forward primer: 5'-TGTCCTGAACCTTCCATAATGTC; HPSE reverse primer: 5'-TACGTATCCACTGGTTTCCTGA; HPSE2 (AJ299719) forward primer: 5'-GCTCTGTCTACAGGCAAGGG; HPSE2 reverse primer: 5'-GGGAGTAAGTTAGGGAGACT; ACTB forward primer: 5'-AAATCGTGCGTGACATCAAAGA; and ACTB reverse primer: 5'-GCCATCTCCTGCTCGAAGTC.

Immunohistochemistry

Formalin-fixed, paraffin-embedded baboon and human uterine 8-µm sections were deparaffinized in Clearing solvent, citrus based (Cornwell Corp., Riverdale, NJ) for three 5-min rinses. Sections were rehydrated in a graded ethanol series, followed by two 5-min rinses in doubly distilled water and two 10-min rinses in PBS. Sections were blocked in 5% (w/v) BSA/PBS for 1 h, followed by incubation with mouse monoclonal anti-human HPSE at 1:40 dilution (InSight Biopharmaceuticals Ltd., Rehovot, Israel) for 1 h at room temperature in a humidified chamber. After rinsing in PBS twice for 10 min, sections were incubated with secondary antibody, Texas red-conjugated goat anti-mouse immunoglobulin G (IgG; Invitrogen), at a 1:50 dilution at 37°C for 45 min. Draq5 (Biostatus Ltd.) was used as a nuclear stain at a 1:500 dilution. Sections were rinsed in PBS twice for 10 min and were mounted using an aqueous antifading mountant (Biomeda, Burlingame, CA). Nonimmune IgG controls were treated and imaged under identical conditions.

Western Blotting

Total protein from baboon uteri were extracted in sample extraction buffer containing 0.05 M Tris, pH 7.0, 8 M urea, 1% (v/v) SDS, 1% (v/v) β-mercaptoethanol, and 0.01% (w/v) PMSF, and protease cocktail inhibitor (Sigma-Aldrich). Protein concentrations on trichloroacetic acid precipitates were determined using a Lowry method, as described previously [40]. Ten micrograms of total protein extracts was mixed with Laemmli sample buffer (BioRad Laboratories, Hercules, CA) in a 1:1 (v/v) ratio and boiled for 5 min. Protein samples were electrophoresed through acrylamide on a 10% (w/v) Porzio and Pearson gel [41] for 2 h at 100 V. The gel was transferred to a Protan Pure Nitrocellulose and Immobilization Membrane (transfer buffer; Schleicher and Schuell Bioscience Inc., Keene, NH) for 5 h at 40 V in a cold room (4°C–6°C). After the transfer, the blot was blocked in 5% (w/v) nonfat dry milk prepared in 0.1% (v/v) Tween 20/PBS (PBS-T) at 4°C to prevent nonspecific binding. The membrane was incubated overnight at 4°C with monoclonal human anti-HPSE antibody (InSight Biopharmaceuticals). The antibody was diluted 1:5000 in 3% (w/v) BSA in PBS-T. Unbound antibody was removed by rinsing three times in PBS-T for 5 min at room temperature. The blot then was incubated for 2 h at 4°C with donkey anti-mouse IgG horseradish peroxidase conjugate (Jackson ImmunoResearch Lab Inc., West Grove, PA) at a final dilution of 1:200 000 in 3% (w/v) BSA in PBS-T. Unbound antibody was removed by rinsing thrice in PBS-T for 5 min at room temperature. The signal was developed using enhanced chemiluminesence reagent (Pierce, Rockford, IL). Recombinant human HPSE (kindly provided by Dr. Israel Vlodavsky, Technion, Haifa, Israel) was used as a positive control. All data from the Western blot were normalized to ACTB levels, detected with rabbit anti-ACTB (Abcam Inc., Cambridge, MA) diluted 1:5000 in 3% (w/v) BSA in PBS-T. Goat anti-rabbit IgG (Sigma-Aldrich) was used to detect ACTB and was incubated for 2 h at 4°C at a final dilution of 1:200 000 in 3% (w/v) BSA in PBS-T. A secondary antibody-only control was performed to verify antibody specificity. All samples and blots were obtained and treated under identical conditions. Each experiment was performed in duplicate.

Preparation of Radiolabeled Extracellular Matrix-HS Proteoglycans

HS proteoglycans (HSPGs), 35S-labeled, were prepared from human colon carcinoma cells (WiDr cells). HSPG2 is the predominant HSPG produced by WiDr cells [42]. Briefly, WiDr cells were cultured in Eagle minimum essential medium (ATCC; Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. After the second passage, the cells were plated in a 24-well plate. When the cells were subconfluent, the media were removed, and the cells were rinsed with low-sulfate media containing RPMI-1640 (Invitrogen), 3.3 mM MgCl2, 1.5 mM HEPES, 1.2 g/l sodium bicarbonate, and 100 U/ml penicillin, as well as 100 µg/ml streptomycin. The pH was adjusted to 7.3. Cells were cultured in 1 ml low-sulfate media containing 3.7 MBq/ml Na235SO4. After 48 h, cells were washed four times with Mg2+/Ca2+-free PBS to remove unincorporated Na235SO4. The wells were treated with PBS containing 0.5% (v/v) Triton X-100 and 20 mM ammonium hydroxide for 10 min to solubilize the cell layer, followed by four washes with Mg2+/Ca2+-free PBS. The extracellular matrix (ECM)-H[35S]PGs remained intact and firmly attached to the tissue culture wells. The plates were used immediately to test for heparanase activity in the indicated samples.

Heparanase Activity Assay

Uterine tissue and B16BL6 cell extracts (50 µg) were homogenized in a buffer containing 10 mM Tris-buffered saline, pH 7.2, 0.5% (v/v) Triton X-100, 0.1 µg/ml (w/v) leupeptin, 0.1 µg/ml (w/v) pepstatin, and 0.2 mM PMSF. The heparanase activity assay protocol was modified from a previous method [43], and its characterization was described in detail previously [44]. Briefly, samples were incubated on sulfate-labeled H[35S]PG-ECM-coated, 1.5-mm dishes in 0.5 ml heparanase reaction buffer (50 mM sodium acetate, pH 5.0) for 24 h at 37°C. The incubation medium containing sulfate-labeled degradation fragments released from the ECM-H[35S]PG was analyzed by molecular exclusion column chromatography on a Superose 12 PC 10/300 GL column (Amersham Biosciences, Piscataway, NJ). The fractions were eluted with PBS/0.02% (w/v) sodium azide. Heparan sulfate degradation fragments eluted near the Vt (total volume) of the column. Identity of the released 35S-labeled HS was confirmed by nitrous acid degradation and β-elimination (data not shown). Radioactivity in each fraction was determined by liquid scintillation counting. Dextran blue and potassium dichromate were used to determine Vo (void volume) and Vt, respectively. For the mixing experiment, 25 µg of extracts with low activity and 25 µg of extracts with high activity were used in the heparanase activity assay and compared to the original activity in 50 µg of extract. All experiments were done in triplicate. Statistical analyses were performed by ANOVA, followed by a Tukey-Kramer secondary multiple comparisons test.

RESULTS

Detection of HPSE in Baboon and Human Endometrium

To determine the localization of HPSE expression in baboon endometrium, we used a mouse monoclonal antibody directed against HPSE. Baboon tissue sections from the receptive stage and different stages of pregnancy were stained for HPSE. In the nonpregnant baboons, HPSE was localized primarily to the apical surface or luminal secretions of the glandular epithelia during the midsecretory phase, whereas the endometrial stroma appeared negative (Fig. 1A). During early pregnancy, HPSE expression was localized in the stroma (Fig. 1B), and at a later stage of pregnancy, more intense localization of HPSE was detected in decidual tissue (Fig. 1C). At the maternal-fetal interface obtained at Day 60 of pregnancy, both the decidua and the villi appeared to express HPSE (Fig. 1E). We also determined the expression of HPSE in human endometrium during the receptive phase and at the maternal-fetal interface during the first trimester. Similar to the baboon endometrium, HPSE was localized to the apical surface as well as in the luminal secretions of the glands (Fig. 2A) during the receptive phase. Nonetheless, we observed a variation in the intensity of HPSE between the glands. Most intense staining was observed in the glands present in the deepest region of the endometrium. A low level of HPSE was detected on the apical surface of the luminal epithelia (Fig. 2B). At the maternal-fetal interface, HPSE was detected throughout the decidual compartment and the placental region with intense staining in villi. Negative controls displayed no reactivity for either baboon or human sections (Figs. 1D and 2C). These data suggest that HPSE expression is similar in baboon and human endometrium, and its localization appears to shift from the epithelial cells to the decidual tissue during pregnancy.


Figure 1
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  		FIG. 1 HPSE localization in baboon uteri. Sections from baboon uteri were stained with anti-HPSE (red) and draq5, a nuclear stain (blue). Samples were from (A) Day 10 after ovulation, (B) Day 22 of pregnancy, (C) Day 40 of pregnancy, (E) Day 60 of pregnancy at the maternal-fetal interface, and (D) Day 40 nonimmune IgG control. Note staining at the apical aspect of the glandular and luminal epithelia. Bar in D = 50 µm (A–D), and Bar in E = 100 µm. ge, glandular epithelia; l, lumen; d, decidua; s, stroma; v, villus.


Figure 2
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  		FIG. 2 HPSE localization in human uteri. Sections from receptive phase endometrium containing (A) glandular or (B) luminal epithelium were stained with anti-HPSE (red), anti-perlecan (green), and draq5, a nuclear stain (blue). Note the anti-HPSE staining at the apical surface (arrows). D) A first-trimester implantation site also was stained with anti-HPSE (red) and draq5 (blue). C) Nonimmune IgG of a receptive phase section similar to that shown in A. Bars = 50 µm (A, C, D) and 20 µm (B). ge, glandular epithelia; le, luminal epithelia, d, decidua; s, stroma; v, villus.

HPSE Expression and Heparanase Activity in Baboon Uteri

We determined the levels of HPSE in baboon tissues by Western blotting. The active form of HPSE (i.e., the 50-kDa form) was the predominant form detected in all of the uterine tissues tested (Fig. 3A). Very little inactive HPSE (i.e., the 65-kDa form) was present. To verify that HPSE was enzymatically active, we used a well-established heparanase activity assay to monitor changes in heparanase during early pregnancy in baboons. Low levels of heparanase activity were detected in uterine extracts obtained between Day 9 and Day 10 after ovulation (Fig. 4). This activity increased approximately 2- to 3-fold in uterine extracts obtained at early pregnancy and midpregnancy (P < 0.001). Highest levels of heparanase activity were detected in tissue extracts obtained at the implantation site during midpregnancy. These data suggest that HPSE is present in the baboon endometrium primarily in the active form.


Figure 3
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  		FIG. 3 Western blotting of HPSE in baboon uterine extracts. Protein extracts from Day 10 (D 10) after ovulation, early pregnancy and midpregnancy baboon uteri were collected and analyzed by Western blotting, as described in Materials and Methods. A) Anti-HPSE monoclonal antibody was used to determine the levels of HPSE in the corresponding samples. Western blotting for ACTB was used as a loading control. Recombinant human HPSE (Recom. HPSE) was used as a positive control. B) Densitometric analysis of HPSE (both the 50-kDa and 65-kDa forms) expression normalized to that of ACTB.


Figure 4
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  		FIG. 4 Heparanase activity increases in pregnant baboon uteri. ECM-H[35S]PGs of WiDr cells were used as a substrate to test for heparanase activity, as described in Materials and Methods. Baboon uterine extracts were prepared as described in Materials and Methods. B16BL6 mouse melanoma extract was used as a positive control. A reaction was carried out with incubation buffer only, which served as a negative control. Error bars indicate mean ± SD of triplicate determinations in each case. ***P < 0.001. IS, implantation site; dpm, disintegrations per minute; PO, postovulation.

Expression of HPSE and Heparanase Activity During Human In Vitro Decidualization

To determine whether HPSE expression is associated with the process of decidualization, Western blotting was used to assess the presence of HPSE abundance in an in vitro human decidualization model. Low levels of HPSE were detected in Day 3 samples (Fig. 5). No changes in levels of HPSE were observed in the untreated versus treated samples throughout the 12-day time course. In contrast, heparanase activity, as measured by 35S-labeled HS release, increased in this system both as a function of time and treatment with decidualizing agents (Fig. 6). In general, samples treated with 17β-estradiol and progesterone displayed similar amounts of heparanase activity, as did untreated controls, whereas samples treated with 17β-estradiol and progesterone and cAMP displayed about twice as much activity on Days 3 and 6. This effect was blunted, but persisted through Day 12 of treatment. These data show that although HPSE levels did not change, heparanase activity increased during decidualization. We verified in other experiments that the 35S-labeled released products were HS fragments not linked to protein and, therefore, could not have been produced by protease action (data not shown). The differences in the HPSE levels and heparanase activity led us to determine whether a diffusible activator or inhibitor was present in the extracts. Therefore, we performed a mixing experiment in which we added equal amounts of uterine extracts from samples displaying high or low heparanase activity, and the mixture was assayed for activity. The resultant mixtures displayed activities representing the average expected for the two types of samples for both baboon uterine and human uterine stromal extracts (Fig. 7). These observations demonstrated that the extracts did not contain either diffusible activators or inhibitors and suggested that other factors (e.g., other isoforms of heparanase) could contribute to the increase in heparanase activity observed in these extracts.


Figure 5
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  		FIG. 5 HPSE increases in human uterine stroma decidualizing in vitro. Human uterine stromal cells were obtained, and decidualizing treatments and Western blotting were performed as described in Materials and Methods. A) Anti-HPSE monoclonal antibody was used to determine the levels of HPSE in the samples indicated. ACTB was used as a loading control. B) Densitometric analyses of HPSE expression normalized to ACTB expression. E, 17β-estradiol; P, progesterone.


Figure 6
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  		FIG. 6 Heparanase activity increases during in vitro human decidualization. Human uterine stromal cells were obtained, and decidualizing treatments and heparanase activity assay were performed as described in Materials and Methods. ECM-H[35S]PGs extracted from WiDr cells were used as a substrate to test for heparanase activity in decidualizing human stroma as described in Materials and Methods. B16BL6 mouse melanoma extract (B16BL6) and incubation buffer only (Buffer), served as positive and negative controls, respectively. Error bars indicate mean ± SD of triplicate determinations in each case. ***P < 0.001. E, 17β-estradiol; P, progesterone.


Figure 7
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  		FIG. 7 Diffusible activators or inhibitors are not detected in baboon uterine extracts and decidualizing human stroma in vitro. ECM-H[35S]PGs of WiDr cells were used as a substrate to test for heparanase activity as described in Materials and Methods. Baboon uterine extracts and human uterine decidualizing stromal were obtained and prepared, and heparanase activity assay was performed as described in Materials and Methods. B16BL6 mouse melanoma extract was used as a positive control. A reaction was carried out with incubation buffer only, which served as a negative control. Error bars indicate mean ± SD of triplicate determinations in each case. d6-C, no treatment; d6-cAMP, E + P + cAMP; Bab-low, Days 9–10 after ovulation; Bab-high, early pregnancy.

To determine the specificity of heparanase enzymatic activity in baboon and human uterine extracts, we used a heparanase inhibitor, PI-88 [45]. We observed approximately a 65%–72% reduction in heparanase activity in the presence of 500 µg/ml PI-88, a concentration known to inhibit heparanase activity [44] (data not shown). Heparanase displays much higher activity at acidic versus neutral pH [18]. To further characterize heparanase activity in baboon and human uterine extracts, we compared the activity at pH 7.0 versus pH 5.0. As expected, heparanase activity was greatly reduced (75%) at pH 7.0 relative to pH 5.0 in both human and baboon extracts (data not shown). Since HPSE also can be secreted in vitro [46, 47], we measured heparanase activity in conditioned media from human decidualizing stroma. We observed that 17% of total heparanase activity in these cultures was secreted (data not shown). Thus, the cell-associated fraction was the major repository of active heparanase.

Expression of HPSE and HPSE2 mRNA in Baboon Uteri and During Human In Vitro Decidualization

Given the discrepancies between the levels of HPSE and heparanase activity, we performed RT-PCR to determine whether HPSE2 mRNA in addition to HPSE mRNA was detectable in baboon uteri. As expected, HPSE mRNA was readily detected in all samples derived from baboon uteri (Fig. 8A, top panel). In addition, HPSE2 mRNA also was detected in all the samples (Fig. 8A, bottom panel). Different-sized HPSE2 transcripts were observed in many samples, suggesting that splice variants may exist in baboons, as is the case in humans [29]. RT-PCR also was performed on human stromal cells decidualized in vitro. HPSE as well as HPSE2 transcripts were detected in all the samples tested (Fig. 8B). Again, different HPSE2 mRNA transcript sizes were observed in most of the human stromal cell samples tested. Consequently, discrepancies observed between levels of HPSE and heparanase activity may be due to differences in expression of HPSE2 mRNA and its variants.


Figure 8
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  		FIG. 8 HPSE and HPSE2 mRNA is present in baboon uterus and in vitro human decidualizing stroma. RNA extraction, cDNA preparation, and RT-PCR were performed as described in Materials and Methods. Samples were electrophoresed on a 2% (v/v) agarose gel and stained with ethidium bromide. A) RT-PCR results with baboon uterine samples (D, Day). B) RT-PCR results with in vitro human decidualizing stroma. Lane 1: Day 3, no treatment; Lane 2: Day 3, E + P; Lane 3: Day 3, E + P + cAMP; Lane 4: Day 6, no treatment; Lane 5: Day 6, E + P; Lane 6: Day 6, E + P + cAMP.

DISCUSSION

Heparanase action results in ECM remodeling, release of HS-bound growth factors, and promotion of angiogenesis [21–23]. These processes are essential to support many events that occur during implantation, including remodeling of the uterine ECM, decidualization, and remodeling of the maternal vasculature [3–6]. Proper coordination of these events is crucial to support the developing embryo. HPSE expression and activity have been demonstrated in human and bovine placenta [24–26], and exogenous heparanase has been shown to improve embryo implantation in mice [30]. Nonetheless, HPSE expression in primate or human uteri has not been reported. Using the baboon as a nonhuman primate model, we were able to study the expression of HPSE in the uterus during pregnancy.

We determined the expression of HPSE at three different stages: the receptive phase, early pregnancy, and midpregnancy. In baboons, blastocyst attachment occurs between Days 8 and 10 (receptive phase) and is characterized by the formation of epithelial plaque in response to CG [6]; however, a similar plaque reaction does not occur in humans. During this stage, HPSE expression is confined to the glandular epithelia and glandular secretions in the baboon and the human uterus. Previous studies indicate that in addition to its enzymatic function, HPSE may function as a cell adhesion molecule [19, 20]. Our studies show the presence of HPSE on the apical surface of the luminal epithelia in the human uterus. Early stages of mouse blastocyst attachment appear to be HS dependent [48]. Thus, it is possible that HPSE plays a role in early stages of primate blastocyst adhesion. Osteopontin is an example of an adhesion-promoting protein that is secreted by uterine epithelia [49]. In this case, osteopontin is proposed to bind to integrin receptors on the apical surface of luminal epithelia and blastocysts bridging these two cell surfaces [49]. It is not clear whether a similar situation occurs for HPSE, since no true receptors for HPSE have been identified. HPSE has a potential trans-membrane domain and could be retained at cell surfaces as an integral membrane protein. Alternatively, binding to HSPGs could retain shed HPSE ectodomains. HPSE has much reduced activity at neutral pH compared with acidic conditions [18]. The pH of human uterine fluid is around 6.6 to 7.6, depending on the stage of the menstrual cycle; however, at the time of ovulation the pH is slightly acidic [50, 51]. Therefore, it seems likely that lumenally disposed HPSE has little catalytic activity and is more likely to facilitate HSPG binding. During early pregnancy and midpregnancy in baboons, localization of HPSE changes, and it accumulates in the decidual compartments of the uterus. This epithelial-to-decidual switch in protein synthesis during pregnancy in the baboon has been reported previously for insulinlike growth factor [52]. We also noted modest HPSE staining in the stroma of receptive human uteri that was not apparent in the baboon. Since humans but not baboons undergo predecidual differentiation in the stroma, it is possible that this accounts for the differences in expression between these species.

During pregnancy, decidualization of uterine stromal cells and trophoblast invasion results in uterine remodeling of the ECM as well as the maternal vasculature. At the maternal-fetal interface, angiogenic processes establish a vasculature between the mother and the fetus to provide nourishment for the developing embryo. HPSE has been shown to promote angiogenesis by causing the release of HS-bound growth factors and by degrading the subendothelial basement membrane [21, 22]. HPSE can degrade HS of different HSPGs, such as SDC-1, a cell surface HSPG, as well as HSPG2, an HSPG present in the basement membrane and the ECM [53]. Depending on the type of proteoglycan, HPSE can release HS products that have different biological activities. HPSE can release HS-bound growth factors from both HSPG2 and SDC-1, thus facilitating a number of processes, such as angiogenesis, migration, and growth [53]. SDC-1 HS fragments generated by HPSE were able to inhibit melanoma cell invasion; however, HSPG2 HS fragments generated by HPSE do not inhibit invasion in melanoma cells [53].

Even though levels of HPSE do not change significantly in the uterus of nonpregnant and pregnant baboons, heparanase activity increases during early pregnancy and midpregnancy compared with secretory phase (P < 0.001). Similarly, while the levels of HPSE only increase slightly during in vitro decidualization of human uterine stroma, heparanase activity increases significantly in this model as well. Given the disparity between HPSE levels and heparanase activity, we concluded that factors other than changes in HPSE levels must account for these differences. Since the predominant form of HPSE detected in all samples was the 50-kDa active form, differential activation of latent HPSE cannot explain these results. Two natural inhibitors of HPSE have been identified, HIP/RPL29 and eosinophil major basic protein [54, 55]; no naturally occurring heparanase activators have been described. Mixing experiments did not detect the presence of a diffusible activator or inhibitor in extracts from either baboon uterine tissue or decidualized human stromal cells. Thus, the presence of diffusible factors that changed the inherent activity of HPSE is unlikely. We speculated that another gene product might contribute to changes in heparanase activity. Previous reports have shown that HPSE2, encoded by a distinct gene, is present at the mRNA level in many tissues, including the human uterus [33]. We detected HPSE2 mRNA in all baboon samples as well as in in vitro-decidualized human stromal cells. Unfortunately, there is no information available on whether HPSE2 is catalytically active. Nonetheless, differential expression or activation of HPSE2 or changes in expression of a novel heparanase may explain the discrepancies between the HPSE-specific Western blotting data and the activity assays. Recently, splice variants of human HPSE have been identified [56]. Human HPSE, lacking exon 5, has no enzymatic activity [56]. The biological significance and function of the other truncated forms remains to be elucidated. In summary, these observations suggest that heparanases in addition to HPSE are likely to contribute to changes in uterine heparanase activity during early pregnancy in primates and humans.

In conclusion, we show that HPSE and HPSE2 mRNA are present in both baboon uteri and in vitro-decidualized human stromal cells. HPSE is expressed in the decidua during pregnancy, and heparanase activity increases during pregnancy with the onset of decidualization. Further studies should examine the expression of HPSE during the later stages of pregnancy and placentation. In addition, we currently are examining the interplay between HPSE and its natural inhibitor, HIP/RPL29, in the release of growth factors.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Catherine Kirn-Safran, JoAnne Julian, Anissa J. Brown, Benjamin Rohe, Daniel Oristian, Rob Long, and all members of Carson and Dr. Farach-Carson laboratories for their discussions and insightful suggestions. We greatly appreciate the excellent secretarial assistance of Ms. Sharron Kingston. The authors wish to thank Progen Pharmaceuticals Ltd. (Australia) for providing PI-88.

FOOTNOTES

1Supported by National Institutes of Health grants HD25235 (D.D.C. and M.C.F.-C.) and HD42280 (A.T.F). Back

Correspondence: 2FAX: 302 831 2281; e-mail: dcarson{at}udel.edu

Received: 3 July 2007.

First decision: 31 July 2007.

Accepted: 28 October 2007.

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