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BOR - Papers in Press, published online ahead of print May 16, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.061317
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BIOLOGY OF REPRODUCTION 77, 433–441 (2007)
DOI: 10.1095/biolreprod.107.061317
© 2007 by the Society for the Study of Reproduction, Inc.

Heparanase Expression and Function During Early Pregnancy in Mice1

Sonia S D'Souza 3, Takiko Daikoku 7, Mary C Farach-Carson 4 5 6, and Daniel D Carson 2 4

Departments of Chemistry and Biochemistry,3 Biological Sciences,4 and Materials Sciences,5 and the Center for Translational Cancer Research,6 University of Delaware, Newark, Delaware 19716 Division of Reproductive and Developmental Biology,7 Vanderbilt University Medical Center, Nashville, Tennessee 37232

ABSTRACT

Embryo implantation is a complex process that involves interactions between cell-surface and extracellular components of the blastocyst and the uterus, including blastocyst adhesion to the uterine luminal epithelium, epithelial basement membrane penetration and stromal extracellular matrix remodeling, angiogenesis, and decidualization. These processes all involve interactions with heparan sulfate (HS) proteoglycans, which harbor various growth factors and cytokines and support cell adhesion. Heparanase (HPSE) is an endo-beta-glucuronidase that cleaves HS at specific sites. HPSE also can act as an adhesion molecule independent of its catalytic activity. Thus, HPSE is a multifunctional molecule contributing to and modulating HS-dependent processes. Exogenously added HPSE improves embryo implantation in mice; however, no information is available regarding the normal pattern of HPSE expression and activity during the implantation process in any system. Using several approaches, including real-time RT-PCR, in situ hybridization, and immunohistochemistry, we determined that uterine HPSE expression increases dramatically during early pregnancy in mice. Heparanase mRNA and protein were primarily expressed in decidua and were rapidly induced at the implantation site. Uterine HPSE activity was characterized and demonstrated to increase >40-fold during early pregnancy. Finally, we demonstrate that the HPSE inhibitor PI-88 severely inhibits embryo implantation in vivo. Collectively, these results indicate that HPSE plays a role in blastocyst implantation and complements previous studies showing a role for HS-dependent interactions in this process.

decidua, embryo, endometrium, heparanase, implantation, mice, pregnancy, trophoblast

INTRODUCTION

Embryo implantation is a highly coordinated and complex process [1, 2]. To achieve successful implantation, proper embryo-maternal interactions must be maintained. In mice, the blastocyst first attaches on Day 4 postcoitum to the uterine luminal epithelia at the antimesometrial aspect during the receptive phase [3]. Attachment is followed by invasion of the trophoblast giant cells into the endometrium. At the same time, the underlying stroma undergo proliferation and decidualization to form the primary decidual zone. This process is followed by apoptosis of the uterine luminal epithelia involved in attachment. The basement membrane underlying the luminal epithelia also must be breached so that the embryo can invade the uterus [4]. By Day 8 postcoitum, cells in the primary decidual zone have undergone programmed cell death, and the primary decidual zone no longer exists [5]. The secondary decidual zone surrounds the implantation site at this stage [5]. Another important process that is crucial for implantation is angiogenesis, i.e., the formation of blood vessels [6]. During angiogenesis, the subendothelial basement membrane also must be remodeled to facilitate migration of endothelial cells.

Metalloproteases mediate much of the tissue remodeling in the mouse uterus during the peri-implantation period [7–9]. The composition of the endometrial extracellular matrix (ECM) also changes, notably including accumulation of the basal lamina components laminin, collagen type IV, and the heparan sulfate (HS) proteoglycan (PG) perlecan in the decidual interstitial matrix. HSPGs are found on cell surfaces, the basement membrane, and the and ECM [10–14] as well as sequester growth factors including FGF2, HGF, and HBEGF. Subsequently, these HS-bound growth factors can be released by metalloproteases or heparanase (HPSE) during tissue remodeling. Released growth factors can then interact with their receptors to elicit biological responses. In some cases, optimal growth factor/growth-factor receptor interaction-mediated signaling requires the presence of HS in a ternary complex, e.g., FGF signaling complex [15].

HPSE is an endo-ß-glucuronidase capable of cleaving HS at specific sites [16]. HPSE has been identified in a wide variety of normal and metastatic tissues and cells, including placenta, platelets, monocytes, lymphocytes, and neutrophils [17–22]. HPSE is synthesized as an inactive 65-kD form and is processed to an active heterodimer composed of 50-kD and 8-kD subunits [23]. Both human and mouse HPSE have been cloned and are 77% identical at the amino acid level [24–28]. HPSE displays optimal activity at pH 5.0 [28, 29]. At pH 7, it has been suggested that HPSE functions as a cell-adhesion molecule due to its relatively weak catalytic activity at this pH [30, 31]. In this regard, the 65-kD form of HPSE has been shown to facilitate adhesion [31]. HPSE has been implicated in a number of normal and pathological conditions, including its role as a mediator of angiogenesis in tumor metastasis [32–34]. Hpse mRNA and activity have been identified in the human and bovine placenta [35–39]. Moreover, overexpression of human HPSE in mice suggests a role for HPSE in mammary gland morphogenesis, embryonic implantation, and tissue remodeling [40]. Recently, HPSE was shown to improve embryo implantation in mice both in vitro and in vivo [41]. In addition, HPSE has been detected in the normal human endometrium, with highest expression during the late-proliferative phase [42]. Splice variants of Hpse have recently been identified [43]. Another member of the heparanase family, heparanase-2 (Hpse2), has been cloned, and its tissue distribution was identified at the mRNA level in several tissues, including the human uterus [44]. Hpse2 may encode three different proteins through alternative mRNA splicing [44]; however, it is not known if any form of HPSE is expressed at the protein level or has enzymatic activity.

The normal pattern of HPSE expression and function during implantation and early pregnancy has not been reported in any system. The aim of our study was to define the pattern of HPSE expression and changes in activity during early pregnancy in mice. Using multiple approaches, we show that Hpse mRNA and protein increase locally at the implantation sites and accumulate in decidua. Consistent with these observations, HPSE activity increases during early pregnancy. Finally, we used the HPSE inhibitor PI-88 [45] to define the requirement for HPSE activity during implantation. Collectively, our data demonstrate that uterine HPSE expression greatly increases during the implantation process, and that active HPSE is required to support key events during early pregnancy in mice.

MATERIALS AND METHODS

Materials

Heparin, sodium chloride, Tris base, glycine, BSA, PMSF, sodium azide, Triton X-100, 20 mM ammonium hydroxide, and Tween 20 were purchased from Fisher Scientific (Pittsburgh, PA). PBS, L-glutamine, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Hepes, sodium bicarbonate, and magnesium chloride 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.

Cell Culture

WiDr cells (human colon carcimona) were kindly provided by Dr. Carlton Cooper (University of Delaware, Newark, DE) and 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 (American Type Culture Collection, Manassas, VA) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. B16BL6 mouse melanoma cells were cultured in Dulbecco Modified Eagle Medium/Nutrient Mix F-12 (Invitrogen) supplemented with 5% (v/v) FBS. Cells were grown at 37°C in a humidified atmosphere of 95% air/5% CO2 (v/v).

Animals

All protocols involving animals were approved by the Institutional Animal Care and Use Committee at the University of Delaware. Mice used in the experiments were housed in the University of Delaware Animal Facility at 25°C with controlled light cycles (12L:12D). Adult ICR mice aged 6–7 wk were purchased from Taconic (Germantown, NY). Females were mated with males of the same strain to induce pregnancy. Day 1 of pregnancy was defined as the day on which the vaginal plug was observed. Mice were killed on Days 1, 3, 5, and 8 at 1030 h. Endometria were collected by scraping the inner wall of the uteri with a razor. On Day 8 of pregnancy, decidua was removed by surgical dissection. The endometria were divided into three parts. RNA was extracted from the first part. Protein was collected from the second part to test for HPSE activity. A portion of the uterine horn was frozen in Tissue Freezing Medium (TFM) (Electron Microscopy Sciences, Hatfield, PA) for immunohistochemistry.

In Situ Hybridization

In situ hybridization followed the protocol as previously described [46]. In brief, frozen sections (10 µm) were mounted onto poly-L-lysine-coated slides and fixed in cold 4% (w/v) paraformaldehyde in PBS. Sections were prehybridized and hybridized at 45°C for 4 h in 50% (v/v) formamide hybridization buffer containing 35S-labeled antisense or sense cRNA probes. RNase A-resistant hybrids were detected by autoradiography. Sections were post-stained with hematoxylin and eosin (H&E). Sections hybridized with sense probes did not exhibit any positive signals and served as negative controls.

Hybridization Probes

The cDNA clones for Hpse were generated by RT-PCR using primers 5'-TTTTGATCCGGACAAGGAAC and 5'-CACATAAAGCCAGCTGCAAA. For in situ hybridization, sense and antisense 35S-labeled cRNA probes were generated using Sp6 and T7 polymerases, respectively. Probes had specific activities of 2 x 109 dpm/µg.

Real Time RT-PCR

Total RNA was extracted from endometria with TRIzol (Invitrogen) per the manufacturer's instructions and quantified by UV absorption. From 500 ng total RNA, cDNA was synthesized using Omniscript RT kit purchased from Qiagen (Valencia, CA). The reaction was carried out at 37°C for 1 h per the manufacturer's instructions. The real-time RT-PCR reaction was performed using SYBR green PCR master mix (Applied Biosystems, Foster City, CA) on an ABI 7700 sequence detector. The primer sequences used were as follows: Hpse forward primer: 5'-TGTCCTGAACCTTCCATAATGTC; Hpse reverse primer: 5'-TACGTATCCACTGGTTTCCTGA; Gapdh forward primer: 5'-TTCCCTCTTCCCAGATGATG; Gapdlt reverse primer: 5'-ATGGGTTTAAGCCGAGTGTG. Samples were cycled for 15 sec at 95°C and 60 sec at 60°C for 45 cycles. The relative amounts of Hpse mRNA were identified using the comparative threshold cycle method by ABI Prism 7000 SDS software (Applied Biosystems).

Immunohistochemistry

Uterine horns were frozen in TFM, and 8-µm sections were prepared at –20°C on a Reichert Jung cryostat (Leica CM3050 S Cryostat [Leica Microsystems, Inc.]). Sections were fixed in methanol for 10 min at room temperature, rehydrated in PBS for 5 min at room temperature, and blocked in 5% (w/v) BSA/PBS for 1 h. Sections were incubated with primary antibody for 1 h at room temperature in a humid chamber. After rinsing in PBS twice for 10 min, sections were incubated with secondary antibody for 1 h at room temperature in a humid chamber. For double staining, sections were stained first with monoclonal human anti-HPSE antibody (InSight Biopharmaceuticals, Rehovot, Israel) and then with rat monoclonal antibody against perlecan domain IV (RDI, Concord, MA). Monoclonal anti-human HPSE antibody has been shown to recognize both human and mouse HPSE as provided by InSight Biopharmaceuticals Ltd., Rehevot Israel. We have also demonstrated the specificity of HPSE antibody in a number of uterine extracts (data not shown). The HPSE antibody (1 µg) was used to prepare zenon complexes (Alexa Fluor-546, Invitrogen) per the manufacturer's instructions. The anti-HPSE/zenon complex was used at a 1:40 dilution. Antiperlecan was used at a 1:50 dilution in PBS. Fluorescein isothiocyanate-conjugated donkey anti-rat IgG (Jackson Immuno Research Lab, West Grove, PA) was used as a secondary antibody to detect perlecan at a 1:40 dilution at 37°C for 45 min. Draq5 (Biostatus Ltd., Leicestershire, UK) was used as a nuclear stain at a 1:3000 dilution. An antibody to cytokeratin 19 was used at a 1:50 dilution. Alexa Fluor 568 (Invitrogen) was used at a 1:50 dilution to detect cytokeratin.

Preparation of Radiolabeled Extracellular Matrix-Heparan Sulfate Proteoglycans

35S-Labeled HSPGs were prepared from WiDr cells. Perlecan is the predominant HSPG produced by WiDr cells [47]. Briefly, WiDr cells were cultured in Eagle Minimum Essential Medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. After the second passage, the cells were plated in a 24-well plate. When the cells were 80% confluent, the media was removed and the cells were rinsed with low-sulfate media containing RPMI-1640 (Invitrogen), 3.3 mM magnesium chloride, 1.5 mM Hepes, 1.2 g/L sodium bicarbonate, and 0.05% (v/v) penicillin/streptomycin solution. The pH was adjusted to 7.3. Cells were cultured in 1 ml of low-sulfate medium containing 100 µCi of Na235SO4. After 48 h, cells were washed four times with magnesium/calcium-free PBS to remove unincorporated Na235SO4. The wells were treated with 0.5% (v/v) Triton X-100 and 20 mM ammonium hydroxide in PBS for 10 min to solubilize the cell layer followed by four washes with magnesium/calcium-free PBS. The ECM-H[35S]PGs remain intact and firmly attached to the tissue culture wells. The plates were used immediately to test for HPSE activity.

HPSE Activity Assay

Endometria obtained from different days of pregnancy (Days 1, 5, and 8) and B16BL6 cell extracts were homogenized in a buffer containing 10 mM TBS (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 assay protocol was modified from that of Marchetti and Nicolson [48]. Briefly, samples (50-µg protein extracts) were incubated on sulfate-labeled ECM-H[35S]PG-coated, 1.5-mm dishes in 0.5 ml of HPSE 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 containing 0.02% (w/v) sodium azide. HS degradation fragments eluted near the Vt (total volume) of the column. HS chemical analysis was validated by nitrous acid degradation and ß-elimination. Briefly, ß-elimination was performed by incubation with 0.05 M NaOH and 1 M sodium borohydride at 45°C for 48 h followed by neutralization with acetic acid. The reaction mixture was lyophilized, dissolved in PBS, and analyzed by filtration on a Sephadex G-75 column (Amersham). Radioactivity in the eluted fractions was counted using a scintillation counter. Nitrous acid degradation was performed by incubation with a 0.5:0.5:1.0 (v/v/v) ratio of 20% (v/v) N-butyl-nitrite in 100% ethanol, 1 N HCl, and double distilled H20, respectively, at 25°C for 4 h. The reaction mixture was then lyophilized, dissolved in PBS, and analyzed by using a Sephadex G-75 column. Radioactivity in the fractions was counted using a scintillation counter. Dextran blue and potassium dichromate were used to determine Vo (void volume) and Vt, respectively.

Effect of PI-88 on Embryo Implantation

PI-88 was provided by Progen Industries, Ltd. (Brisbane, Australia). PI-88 is an oligosaccharide preparation with a molecular mass range of 1.4–3.1 kD [49–51]. PI-88 was solubilized in PBS and was injected (i.p.) into pregnant mice (n = 12) at 1000 h on consecutive Days 5, 6, and 7 using a dose of 30 mg/kg per day (assuming an average weight per mouse of 25 g), a dose shown to be effective in previous in vivo studies [45]. A second group of pregnant mice (n = 4) were injected with 10 mg/kg per day of PI-88 on consecutive Days 5, 6, and 7. Control animals (n = 16) were injected with 100 µl PBS at the same time points. The mice were sacrificed on Day 8, the number of implantation sites was counted, and then uteri were frozen in TFM for histological examination. Sections (8 µm) were stained with H&E to compare the histology of implantation sites in control versus PI-88 injected animals. A third group of pregnant mice (n = 4) were injected with 30 mg/kg per day of PI-88 on Day 4 and killed on the evening of Day 5, after injection with 1% (v/v) of Pontamine Sky Blue 6BX (Alfa Aesar, Ward Hill, MA). The number of implantation sites was counted in PBS-injected mice versus PI-88 injected mice. The statistical analysis used to analyze the implantation inhibition data was Student impaired t-test.

RESULTS

Expression of Hpse mRNA in the Mouse Uterus During the Peri-Implantation Period

In situ hybridization was used to localize Hpse mRNA in the mouse uterus during early pregnancy. Low levels of Hpse mRNA were detected on Days 1 and 4 (Fig. 1, A and B). On Day 5, Hpse mRNA accumulated locally at the site of embryo implantation, i.e., the primary decidual zone (Fig. 1C). By Day 8, Hpse expression was highest in the decidualizing stroma at both the antimesometrial and mesometrial aspects of the uterus (Fig. 1D). Low levels of Hpse mRNA also were detected in the myometrium. No hybridization was observed with the corresponding sense probe (data not shown). Real-time RT-PCR was used to determine the relative levels of Hpse mRNA. Consistent with the in situ hybridization results, Hpse mRNA expression was very low on Day 1 in endometrial extracts. Endometrial Hpse mRNA increased approximately twofold by Day 5 and fourfold by Day 8 (Fig. 2). Collectively, these data demonstrate increased Hpse mRNA expression in decidual tissue during early pregnancy.


Figure 01
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  		FIG. 1 Hpse mRNA accumulates in the decidua during early pregnancy. In situ hybridization was performed as described in Materials and Methods. Hybridization signals in representative dark-field photomicrographs of longitudinal uterine sections of Day 1 (A) and Day 4 (B) and cross sections of Day 5 (C) and Day 8 (D) are shown. le, Lumina epithelium; s, stroma; myo, myometrium; m, mesometrial pole; am, anti-mesometrial pole; Dc, diciduas. Bar = 400 µm. Arrowheads indicate the location of embryos.


Figure 02
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  		FIG. 2 Endometrial Hpse mRNA levels during early pregnancy. Real-time RT-PCR assays were performed as described in Materials and Methods. The levels of Hpse in Day 1, 5, and 8 endometria as well as a B16BL6 positive control (data not shown) were determined relative to that of Gapdh. The bars indicate the mean ± SD values from triplicate determinations of duplicate samples in each case and are expressed relative to Day 1.

HPSE Protein Expression During Early Pregnancy

To determine if the HPSE protein was expressed in the mouse uterus, we used a commercial antibody directed against HPSE. Cross sections of Day 6 and Day 8 implantation site were stained for HPSE and perlecan. Relatively little HPSE immunoreactivity was observed at Day 5 implantation sites (data not shown); however, on Day 6, intense staining was observed in the primary decidual zone (Fig. 3). The stromal compartment also appeared to stain strongly for HPSE. Furthermore, we noted HPSE staining at the apical aspect of the luminal epithelia at this stage (Fig. 3B). By Day 8, HPSE protein was detected throughout the endometrium, with intense staining in the decidua (Fig. 4). In contrast, the myometrium appeared to have very little HPSE (data not shown). In addition, the embryo as well as the trophoblast giant cells displayed relatively low levels of HPSE compared to the decidua. Perlecan, an HSPG and potential target of HPSE action, was localized to the basal lamina of vascular elements and interstitial matrix surrounding decidual cells. Perlecan expression had a complementary pattern of expression relative to HPSE in many regions of the decidua. Colocalization of perlecan and HPSE was detected in the basal lamina of Reichert's membrane (arrows in Fig. 4, A and B). Interimplantation sections taken from Day 6 pregnant mouse uterus were stained with the HPSE antibody. Our results demonstrate that HPSE expression was increased locally at the implantation site (data not shown). Collectively, these observations demonstrated robust HPSE protein expression in decidua surrounding the embryo with lesser expression in other regions.


Figure 03
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  		FIG. 3 HPSE localization in Day 6 implantation sites. Frozen sections of Day 6 pregnant mouse uteri were stained by indirect immunohistochemistry as described in Materials and Methods. A) Sections stained with anti-HPSE (red), antiperlecan (green), and Draq5 (blue), a nuclear stain. Arrows indicate the perimeter of the primary decidual zone. B) Higher magnification of the luminal epithelia. Note the presence of positive anti-HPSE staining in the luminal epithelia (arrows). Bar = 100 µm. C) Nonimmune IgG control imaged under similar conditions. Magnification x10. D) Region below the primary decidual zone; Bar = 500 µm. The mesometrial aspect is oriented to the top of each panel. e, embryo.


Figure 04
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  		FIG. 4 HPSE localization in Day 8 implantation sites. Frozen sections of Days 5 and 8 pregnant mouse uterus were stained by indirect immunofluorescence as described in Materials and Methods with anti-HPSE (red), antiperlecan (green), and Draq5 (blue), a nuclear stain. A and B) Opposing sides of an implantation chamber. Note the yellow staining highlighted by the arrows indicating colocalization of perlecan and HPSE. C) Decidua just below, i.e., antimesometrial to, the implantation chamber. D) Deep decidua in the antimesometrial aspect. Bar = 200 µm for all panels.

Uterine HPSE Activity Assay

To determine if HPSE from mouse endometria was enzymatically active, we established a HPSE activity assay. 35SO4-labeled products released by this reaction were partially included on a molecular exclusion column and displayed a similar size distribution when uterine extracts or B16BL6 mouse melanoma positive control extracts were used as a source of enzyme (Fig. 5A). Much more 35SO4-labeled material was released by Day 8 endometrial extracts than Day 5 extracts. ß-Elimination verified that the fragments released in the peak fraction were not attached to a protein core, and nitrous acid degradation confirmed that all the 35SO4-labeled material in these fractions was HS (Fig. 5B). These data identified the 35SO4-labeled material definitively to be HS fragments and, therefore, the products of HPSE action. Day 8 endometrial extracts were used to characterize this activity further. 35S-HS release was linear throughout the 24-h time course routinely used and was protein-concentration dependent (Fig. 6, A and B). Furthermore, the reaction displayed a pH optimum of approximately pH 5 and was inhibited significantly by the inclusion of heparin, properties consistent with those of HPSE (Fig. 6, C and D; [28, 29]).


Figure 05
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  		FIG. 5 HPSE activity assay. ECM-H[35S]PGs extracted from WiDr cells were used as a substrate to test for HPSE activity as described in Materials and Methods. A) Day 1, Day 5, and Day 8 endometria were used to test for HPSE enzymatic activity. 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. Reaction products were analyzed by molecular exclusion chromatography as described in Materials and Methods. B) Validation of the assay was performed using the peak fraction obtained from Day 8 reaction. This material was subjected to either ß-elimination or nitrous acid degradation. Total represents the undigested peak fraction.


Figure 06
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  		FIG. 6 Characterization of endometrial HPSE activity. ECM-H[35S]PGs extracted from WiDr cells were used as a substrate to characterize HPSE activity as described in Materials and Methods. Activity was examined as a function of time (A), protein concentration (B), or pH (C). Day 8 endometrial protein extract (50 µg) was used in each case except as indicated in B. (D) Inhibition by the inclusion of low-molecular-weight heparin with a mean molecular weight of 3000. Points indicate the average ± range of values obtained for duplicate determinations in each case.

As suggested in Figure 5A, HPSE activity was much greater in Day 8 endometrial extracts than Day 1 or Day 5 extracts. Quantification of the change in activity is shown in Table 1. These observations indicate that endometrial HPSE activity increased 18-fold and 44-fold in Day 5 and Day 8 extracts relative to Day 1 extracts, respectively. Moreover, by Day 8 the activity was comparable to that observed for B16BL6 mouse melanoma cell extracts, a robust source of HPSE activity [16]. Mixing experiments determined that the difference in activity among these extracts was not due to the presence of diffusible activators or inhibitors (data not shown). Thus, in addition to the increases in Hpse mRNA and protein expression described above, these studies demonstrate that HPSE activity also increased.


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  		TABLE 1 Endometrial heparanase activity increases during early pregnancy.

PI-88, a HPSE Inhibitor, Inhibits Implantation In Vivo

To determine the role of HPSE in embryo implantation we used PI-88, a HPSE inhibitor [45]. PI-88 is a heterogenous mixture of sulfonated oligosaccharides consisting mainly of sulfated phosphomannopentose and phosphomannoteraose, which has been used to inhibit HPSE activity in vitro and in vivo [49–51]. Initially, we confirmed that PI-88 inhibited endometrial HPSE activity in vitro (Fig. 7). We observed a 50% inhibition at approximately 40 µg/ml of PI-88 and almost complete inhibition of HPSE activity at the highest PI-88 concentration tested (1000 µg/ml). These studies confirmed that PI-88 was able to inhibit endometrial HPSE activity effectively. For in vivo experiments, we used a dose of PI-88 previously shown to be effective in vivo in studies of tumor progression in mice [45]. A spectrum of effects on implantation were observed in PI-88-injected mice (Fig. 8, B–D). In some cases, no implantation sites (decidual swellings) were observed, whereas in others implantation sites of relatively normal size and morphology were seen. At a low dose of PI-88 (10 mg/kg per day), we observed a reduction (~37%) in the number of implantation sites (Fig. 8, B and E). Nonetheless, most of the mice injected with 30 mg/kg per day of PI-88 showed a marked decrease in the number of implantation sites compared to PBS-injected controls (P < 0.001; Fig. 8E). Histological examination of Day 8 implantation sites revealed that the morphology of the decidua appeared different compared to the PBS-injected controls (Fig. 9, compare A and B). H&E staining suggested that the normal orientation of the embryo in the mice injected with 30 mg/kg per day of PI-88 was compromised. To confirm this, we stained implantation sites from our control and PI-88-injected (30 mg/kg per day) mice for cytokeratin, a trophoblast marker. These studies revealed that trophoblasts invaded the endometrium poorly in PI-88-injected mice (30 mg/kg per day) compared to the controls (Fig. 9, C and D). In addition, the nuclear stain used as a contrast revealed the absence of well-organized embryonic tissues, suggesting that embryonic development was impacted as well. Moreover, in many of the cases in which implantation occurred, both trophoblast invasion and embryonic development were abnormal. Studies also were performed to determine if HPSE was involved in the adhesion of the blastocyst to the luminal epithelia. In this case, mice were injected with 30 µg/kg body weight of PI-88 on Day 4, and implantation sites were assessed 24 h later. There were no significant differences in the number of implantation sites at Day 5 between these groups (data not shown). Collectively, these studies indicate that PI-88 severely inhibits embryo implantation in a dose-dependent manner; however, PI-88 did not impact the initial events of embryo attachment.


Figure 07
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  		FIG. 7 PI-88 inhibits endometrial HPSE activity. Endometrial extracts from Day 8 mice were collected and assayed for HPSE activity in the presence of the indicated concentrations of PI-88 as described in Materials and Methods. Points indicate the average ± range of values obtained for duplicate determinations in each case.


Figure 08
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  		FIG. 8 PI-88 inhibits embryo implantation. PI-88 (10 or 30 mg/kg per day) was injected on Days 5, 6, and 7 of pregnancy. Mice were killed on Day 8 and the number of implantation sites were scored as described in Materials and Methods. Photographs of uterine horns from (A) PBS injected, (B) 10 mg/kg per day and (C and D) 30 mg/kg per day PI-88-injected mice are shown. E) The number of implantation sites in control and PI-88 injected were determined and the data presented as the means ± SD (P < 0.001). Bar (pin head) = 1.3 cm (A–D).


Figure 09
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  		FIG. 9 Implantation sites are abnormal in PI-88-injected mice. PI-88 (30 mg/kg per day) was injected on Days 5, 6, and 7 of pregnancy. Mice were killed on Day 8 and implantation sites were studied morphologically as described in Materials and Methods. H&E-stained cross sections of the implantation sites of (A) PBS-injected or (B) PI-88-injected mice are shown. Magnification x10. Arrows in B indicate regions at edge of trophoblast invasion suggesting an abnormal morphology. The arrowhead indicates residual uterine lumen. Frozen sections of implantation sites of (C) PBS-injected or (D) PI-88-injected mice were stained with cytokeratin (red) and Draq5 (blue). Arrows in D point to the edges of the trophoblast indicating a highly irregular pattern of invasion. Also note the poorly organized tissues within the embryo suggested by the lack of Draq5 staining. Bar = 500 µm (C and D). meso, mesometrial end; am, antimesometrial end; emb, embryo.

DISCUSSION

In situ hybridization and real-time RT-PCR demonstrated that endometrial Hpse mRNA expression increased during the peri-implantation period. On Day 5, during the initial stages of implantation, Hpse expression was highest in the region surrounding the implanting embryo. A number of processes occurred in the implantation site. Attachment of the blastocyst to the uterine luminal epithelia occurred on the evening of Day 4 [3]. Previous studies have shown that HPSE can mediate cell adhesion, an activity that does not require proteolytic conversion to an enzymatically active state activation [30, 31]. Therefore, it is possible that HPSE participates in blastocyst attachment to the uterine luminal epithelia, although it seems likely that this would involve the enzymatically inactive precursor form. Subsequently, the basement membrane underlying the uterine epithelia at the attachment site, must be breached so that the growing embryo can invade the uterus. Perlecan is an HSPG that is a major component of basal lamina. In addition, perlecan is found on the external surface of the attachment competent blastocyst as well as in the interstitial matrix of decidua [52–54]. Thus, it is possible that HPSE can participate in HS-dependent interactions occurring between the blastocyst and uterine epithelia.

In the present study, we confirmed that endometrial HPSE was enzymatically active by an in vitro enzyme assay. Thus, decidual HPSE is likely to cleave the HS components of the basal laminae as well as decidual HSPGs, facilitating embryo implantation by partially degrading or remodeling the extracellular matrix. In addition, HPSE is likely to release HS-binding growth factors involved in promoting embryo development or in the decidualization process [6, 46, 55–57]. The activity assay indicated that the increase in HPSE activity greatly exceeded the increase in mRNA. Mixing experiments revealed that this was not due to the presence of diffusible activators or inhibitors. It is possible that nondiffusable activators or inhibitors are present that account for the disparity between changes in mRNA levels and activity. It also is possible that other heparanases other than HPSE may be expressed. Hpse-related mRNAs have been identified in human uteri, though it is not clear if these proteins are enzymatically active [44]. It also is possible that structurally nonrelated proteins could have heparanase activity that has not yet been reported.

On Day 6, Hpse mRNA expression was intense in the primary decidual zone. HPSE protein expression was also intense in the apical surface of the luminal epithelia. At this stage the lumen is closed and the epithelial cells of the lumen are in contact with one another. On Day 8, the expression of Hpse mRNA and protein is intense in the decidual compartments of the uterus. In contrast, the myometrium appeared to have very low levels of Hpse. We examined HPSE protein localization in Day 8 implantation sites. HPSE staining was most intense in the decidua. At Day 8, this staining persisted in regions where Hpse mRNA expression was no longer robust. It has been reported that HPSE is localized on the cell surface and ECM as well as in intracellular compartments [17]. Inside the cell, it is primarily located in the lysosomes [58], though HPSE can be associated with the nucleus [59, 60]. Intracellularly, it has been suggested that the primary function of HPSE is HS degradation [17, 34]. On the cell surface, HPSE may be associated with HSPGs or other cell-surface components [61]. Our data suggests that decidual HPSE is present in both intracellular and extracellular compartments and persists in decidual regions after a transient rise in mRNA expression detected by in situ hybridization. Taken together, these observations suggest that: 1) HPSE protein is relatively stable and 2) the increase in steady state levels of mRNA encoding Hpse is both transient and proceeds in a wave emanating from the embryo attachment site.

Previous reports have suggested that HPSE may be involved in the induction of PTGS2 in breast cancer progression and therefore regulates the expression of PTGS2 [62, 63]. The mechanism of such action is not known. Interestingly, both Ptgs2 and Hbegf mRNA are induced rapidly and locally at implantation sites in the mouse uterus [46, 64]. Therefore, it is possible that HPSE induction is related to Hbegf or Ptgs2 induction, at least during the attachment stage. We did not find that PI-88 inhibited the increase in vascular permeability triggered by embryo attachment (Pontamine Blue reaction), a process that is dependent on PTGS2 induction [1]. Assuming PI-88 efficiently diffuses to the uterine lumen, these observations suggest that heparanase activity is not required either for initial embryo attachment or induction of PTGS2. Numerous reports have shown that HPSE is involved in angiogenesis either by releasing angiogenic factors or by degrading the subendothelial basement membrane to support endothelial cell migration [32, 33, 65–67]. Angiogenesis is essential for the formation of the maternal-fetal blood supply and to ensure proper growth of the developing embryo. HPSE activity increased greatly in Day 5 endometria compared to Day 1 and even further by Day 8. This suggests that endometrial HPSE is enzymatically active. Endometrial HPSE activity is highest at pH 5.0 (Fig. 5C), consistent with previous reports [28, 29]; however, a substantial amount (33%) of activity remained at pH 7 indicating that this enzyme is very likely to function in HS hydrolysis at most sites of deposition in this tissue. HPSE degrades HS at discrete sites, releasing HS fragments rather than small oligosaccharides [66]. Thus, HPSE can release angiogenic growth factors, such as FGF2, in association with HS fragments as well as degrading the subendothelial basement membrane to facilitate angiogenesis or tissue invasion.

To define HPSE function in the context of embryo implantation, we used an HPSE inhibitor, PI-88 [45]. We confirmed the efficacy of PI-88 in inhibiting endometrial HPSE using our in vitro enzyme activity assay. PI-88 injection significantly (P < 0.001) reduced the number of implantation sites in vivo. In addition, the size of the decidual capsule appeared to be smaller in PI-88 injected mice. We noted the variability among the implantation sites from PI-88 injected mice. We speculate that this variability may be due to differential accessibility. PI-88 can exert its action in two different ways: 1) by inhibiting HPSE action and 2) by competing with HS-binding growth factors. Nonetheless, the effect of PI-88 is on HS-dependent processes. PI-88 may exert biological effects either by directly inhibiting HPSE or by competing for HS growth-factor binding [45]. Previous reports have shown that MMP inhibitors reduce decidual growth and cause embryo misorientation [7]. In the mice injected with a high dose of PI-88, the implantation chamber was not normal. HPSE has been shown to promote cell invasion; therefore, it is possible that inhibition of HPSE caused trophoblasts to invade the uterus in an inappropriate manner. Although our study verifies that PI-88 should significantly inhibit uterine HPSE activity under these conditions, we cannot exclude effects on HS/growth factor interactions as well. We attempted to knock down HPSE expression via a virally delivered ribozyme, but observed no consistent effect on implantation (data not shown). It is unclear if this is due to inefficient delivery of the virus to implantation sites or poor expression of the ribozyme. Nonetheless, since reduced HPSE expression is likely also to affect HS-binding growth-factor delivery as well as ECM remodeling, HPSE knockdown or knockout would not discriminate between these two actions. The PI-88 results are informative in demonstrating that at least one of these HPSE-dependent processes is critical.

In summary, we show that HPSE expression and activity increases markedly during early pregnancy in the mouse. In addition, inhibition of HPSE activity in vivo severely impairs the implantation process. Thus, these data indicate that HPSE plays an important role in the normal program of events occurring during implantation, most likely by controlling HS-dependent processes. Additional studies should examine both the expression and function of HPSE during the later stages of pregnancy as well as placentation to determine if this HS-modifying enzyme plays additional roles in the reproductive process.

ACKNOWLEDGMENTS

The authors wish to thank Progen Industries, Ltd., Australia, for providing us with PI-88. The authors wish to thank all the staff working in the Animal Facility at the University of Delaware. The authors also wish to thank Dr. Catherine Kirn-Safran, JoAnne Julian, and Benjamin Rohe for critically reading the manuscript. The authors wish to thank Anissa J. Brown, Rick Focht, Daniel Oristian, and all members of Dr. Carson's and Dr. Farach-Carson's laboratories for their discussions and insightful suggestions. We are grateful for the excellent secretarial assistance of Ms. Sharron Kingston.

FOOTNOTES

1This work was supported by the NIH grant HD25235 (D.D.C.) and Gynecologic Cancer Foundation Award (T.D.). Back

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

Received: 7 March 2007.

First decision: 25 March 2007.

Accepted: 8 May 2007.

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