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Biology of Reproduction 64, 1165-1175 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Expression of Heparin/Heparan Sulfate Interacting Protein/Ribosomal Protein L29 During the Estrous Cycle and Early Pregnancy in the Mouse1

JoAnne Juliana, Sanjoy K. Dasb, Sudhansu K. Deyb, Doug Baraniaka, Van-Thanh Taa, and Daniel D. Carson2,a

a Department of Biological Sciences, University of Delaware, Newark, Delaware 19707 b Department of Obstetrics/Gynecology & Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7338

ABSTRACT

Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.

decidua, early development, female reproductive tract, implantation, uterus

INTRODUCTION

Interactions between heparin/heparan sulfate proteoglycans (HSPGs), located on mammalian cell surfaces or in extracellular matrices, and their respective binding proteins, contribute to a variety of biological processes, including cell adhesion, cytokine/growth factor action, viral pathogenicity, and modulation of blood coagulation [1]. In addition, multiple lines of evidence suggest that HSPGs and HSPG-binding proteins participate in one or more aspects of embryo implantation. These include the increased expression of HSPGs and the mRNA encoding their core proteins during the acquisition of attachment competence by embryos [2–5], the inhibition of blastocyst attachment in vitro by heparin (Hp)- or heparan sulfate (HS)-degrading enzymes or by inhibition of HS synthesis [2, 3]. Acquisition of attachment competence by murine blastocysts in vitro is accompanied by expression of the HSPG, perlecan, at the external surface of trophectoderm [5], and HSPGs and certain HSPG-binding proteins display a complementary pattern of expression at implantation sites and the fetal-maternal interface [6–8]. Nonetheless, recent studies of perlecan-null mice do not indicate an implantation defect, suggesting functional redundancy in the HS core proteins in this regard [9, 10]. In order for HSPGs to interact with uterine epithelia during the initial stage of implantation, the epithelia must express HSPG binding sites. Consistent with this are the observations that heparin-binding epidermal growth factor (EGF)-like growth factor is induced at murine implantation sites, supports embryo attachment, and promotes embryonic development in vitro [11]. Other HS-binding proteins also may be involved in aspects of embryo-epithelial interactions.

Both primary cultures of mouse uterine epithelial cells and human uterine cell lines display specific, saturable cell surface Hp/HS-binding sites [12, 13]. Examination of heparin-binding tryptic peptides released from the cell surface of a human uterine adenocarcinoma cell line led to the identification of heparin/HS interacting protein/ribosomal protein L29 (HIP/RPL29) [14]. Both the intact protein and a peptide sequence within HIP/RPL29 recognize cell surface HS expressed by human choriocarcinoma and breast cancer cell lines [15, 16]. HIP/RPL29 or HIP/RPL29 peptides also support HS-dependent attachment of a variety of human cell lines [15–17]. Furthermore, cell surface expression of HIP/RPL29 has been demonstrated for several human cell lines [7, 15, 18].

HIP/RPL29 is expressed by luminal and glandular epithelium of the normal cycling human uterus [18]; however, examination of the potential role for HIP/RPL29 in embryo attachment requires the use of an animal model. The murine orthologue to human HIP/RPL29 was recently identified and characterized [19]. Murine HIP/RPL29 is differentially expressed in a variety of adult tissues and cell types, including uterine epithelium and, similar to its human orthologue, murine HIP/RPL29 binds heparin and HS with high affinity and selectivity [19]. Nevertheless, no information is available regarding the expression of HIP/RPL29 during the estrous cycle or early pregnancy in mice, nor is it clear whether HIP/RPL29 can interact directly with peri-implantation-stage mouse blastocysts. The present study examines the expression of HIP/RPL29 in the mouse uterus during the estrous cycle and early pregnancy. We demonstrate that mouse blastocysts adhere strongly to HIP/RPL29. This interaction is, surprisingly, not inhibited by soluble glycosaminoglycans, suggesting that cell surface components in addition to proteoglycans can bind to HIP/RPL29.

MATERIALS AND METHODS

Peptide Synthesis and Antibody Generation

A synthetic peptide corresponding to murine HIP/RPL29 amino acids 117–131 (CQPKPKVQTKAGAKA) was synthesized, conjugated to maleimide-activated keyhole limpet hemocyanin, and used for rabbit immunization as described previously [19]. Polyclonal antibodies were purified on affinity resin prepared using the same synthetic peptide linked to maleimide-activated BSA and conjugated to cyanogen bromide-activated Sepharose (Sigma, St. Louis, MO) using a modification of the manufacturer's protocol (Imject kit; Pierce, Rockford, IL). In order to protect the heparin-binding site during the conjugation step, the peptide-BSA (1 mg relative to the peptide) was preincubated with acetylated heparin (1 mg). Both bound and unbound acetylated heparin were removed by a 1 M NaCl rinse after conjugation.

Immunohistochemistry

CF-1 virgin female mice were staged by vaginal smear or mated and killed on the indicated day of the estrous cycle or gestation, respectively. Day 1 of gestation was determined by the presence of a vaginal plug. Day 4.5 (day of implantation) was confirmed by examining flushings of one uterine horn for the presence of hatched blastocysts. Uterine horns were frozen in OCT (Miles, Elkhart, IN) and 6-µm sections prepared on a Reichert Jung cryostat. Sections were fixed in 100% methanol for 10 min at room temperature, rehydrated in PBS for 15 min at room temperature, and incubated with primary antibody for 1 h at 37°C. After three rinses in PBS, sections were incubated for 40 min at 37°C with fluorescein isothiocyanate-conjugated donkey anti-rabbit immunoglobulin G (IgG; Amersham Pharmacia Biotech, Piscataway, NJ) diluted 1:10 in PBS, followed by three rinses in PBS. Affinity-purified antibody generated against peptide sequence CQPKPKVQTKAGAKA was used at a concentration of 0.05 mg/ml. Staining controls included deletion of primary antibody and a peptide preadsorption of primary antibody. In the latter case, the reactive sulfhydral group on the terminal cysteine of the peptide was quenched with 5 mM N-ethylmaleimide prior to incubation with the antibody.

For double staining, sections were coincubated with anti-HIP/RPL29 (peptide) and rat monoclonal antibody, MAB 1948, recognizing perlecan core protein (Chemicon, Temecula, CA) used at a 1:20 dilution. The procedure was as described above. Subsequently, sections were incubated with a combination of fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and rhodamine conjugated affinipure F(abN)2 fragment donkey anti-rat IgG (Jackson Immunoresearch Lab, Inc., West Grove, PA) diluted 1:10 and 1:20, respectively. Desmin was detected with a polyclonal rabbit antibody (Sigma) used at a 1–50 working dilution.

SDS-PAGE and Western Blotting

Uterine epithelium and stroma were isolated as previously described [20]. Where indicated, whole endometrium was separated from myometrium by scraping the longitudinally opened uterine horn with a scalpel blade. Cell pellets and endometrial scrapings were solubilized in sample extraction buffer: 8 M urea, 1% (w/v) SDS, 50 mM Tris pH 7.0, 1% (v/v) ß-mercaptoethanol, and 0.01% (w/v) phenylmethylsulfonyl fluoride. Solubilized samples were concentrated by precipitation in 10% (w/v) trichoracetic acid at 4°C, rinsed with 10% (w/v) trichloracetic acid and 100% acetone, and air-dried. Samples redissolved in equal volumes of sample extraction buffer and sample buffer [21] were heated for 2 min at 90°C and resolved by SDS-PAGE on a 15% (w/v) resolving gel as described [19]. Electrophoresis-grade reagents were obtained from Bio-Rad (Hercules, CA). Gels were transferred to nitrocellulose membrane at 4°C for 5 h at 40 V in a Transblot apparatus (Bio-Rad) in 100 mM Tris base and 100 mM glycine pH 9.2. The transferred blot was processed as described previously [19]. Alternately, the blot was blocked at least 6 h at 4°C in 3% (w/v) BSA in PBS/0.1% (v/v) Tween 20, incubated overnight at 4°C in primary antibody diluted in the same solution, rinsed three times with 0.1% (v/v) Tween 20 in PBS, incubated with horseradish peroxidase conjugated anti-rabbit IgG (1:200 000 dilution) in 3% (w/v) BSA in PBS/0.1% (v/v) Tween 20 for 2 h at 4°C, rinsed three times with 0.1% Tween 20 in PBS, and visualized with enhanced chemiluminescence reagent (Pierce Chemical, Rockford, IL). Preincubation of the HIP/RPL29-directed antibody with its corresponding peptide antigen completely blocked detection on these blots and demonstrated specificity.

In Situ Hybridization

In situ hybridization was performed as described previously [6, 22] using the full-length murine HIP/RPL29 coding sequence [19]. Sense and antisense 35S-labeled cRNA probes were generated using appropriate polymerases from cDNAs. Small pieces of tissues were flash-frozen in liquid Histo-Freeze (Fisher Scientific, St. Louis, MO). Frozen sections (11 µm) were mounted onto poly-L-lysine-coated slides, fixed in cold 4% paraformaldehyde solution in PBS, acetylated, and hybridized at 45°C for 4 h in hybridization buffer containing the 35S-labeled antisense cRNA probes. After hybridization, the sections were incubated with ribonuclease A (RNAase A, 20 µg/ml) at 37°C for 20 min. RNAase A-resistant hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Sections hybridized with sense probes served as negative controls.

Northern Blotting

Total RNA was extracted from whole uteri pooled from 7 to 10 mice on the indicated days of pregnancy by a modified guanidine thiocyanate procedure [6]. RNA (7 µg) was denatured, separated by formaldehyde-agarose gel electrophoresis, and transferred to nylon membranes. RNA was cross-linked to the membranes by UV irradiation (Spectrolinker, XL-1500; Spectronics Corp., Westbury, NY) and the blots were prehybridized, hybridized, and washed as previously described [6]. 32P-Labeled cRNA probes specific for murine HIP/RPL29 and rpL7 transcripts as described [23] were generated. The hybrids were detected by autoradiography.

Blastocyst Binding to Recombinant Mouse HIP

Recombinant murine HIP/RPL29 with an added N-terminal 27 amino acid sequence containing an oligo-histidine motif was expressed and purified on Talon affinity resin (Clontech, Palo Alto, CA) as described previously [19] with the following modifications to improve yield and recovery: addition of four freeze/thaw cycles at -80°C prior to sonication and preblocking of both the cobalt resin and heparin agarose with nontransformed bacterial extract lacking HIP/RPL29.

For blastocyst-binding assays, virgin 6-wk-old CF-1 females were superovulated. Blastocysts were flushed on Day 4.5 of pregnancy as previously described [5]. Hatched, expanded blastocysts were maintained in 0.1% (w/v) BSA in CMRL 1066, 100 U/ml penicillin, 100 µg streptomycin, 0.25 µg/ml fungizone (binding medium; components from Gibco BRL, Grand Island, NY) on CM culture dish inserts (Millipore; Bedford, MA) at 37°C in a humidified atmosphere of 5% (v/v) CO2:air while the wells were being prepared for the binding assay. All procedures using mice were approved by the Institutional Animal Care and Use Committee.

Nickel chelate-coated Reacti Strips (Pierce) in a 96-well format that had been blocked by the manufacturer were coated for 3 h at 4°C on a shaker with 50 µl of the indicated solution per well. Wells were then rinsed three times with 200 µl of unsupplemented medium per well. Each well was filled with 200 µl of binding medium and 2–3 blastocysts were added per well. During incubation at 37°C in 5% (vv) CO2:air, adhesion was monitored by gently blowing a stream of medium against the blastocyst using a mouth-controlled pipette with a bore slightly larger than the blastocyst while observing under phase contrast at low magnification [24]. Attachment was scored as no movement in response to this procedure. Control coatings included polyhistidine (1 mg/ml in PBS) and bacterial extract lacking HIP/RPL29. Recombinant HIP/RPL29 was bound to well surfaces either from bacterial extract or after purification. HIP/RPL29 binding to Reacti Strip wells was monitored by ELISA using antibody recognizing HIP/RPL29 peptide.

In competition assays, coated wells were preincubated with either heparin (1 mg/ml) or chondroitin-6-sulfate (l mg/ml) for 30 min at 37°C in medium before addition of embryos. Competitors remained during the period of blastocyst adhesion.

RESULTS

HIP/RPL29 Expression During the Estrous Cycle

An affinity-purified antibody generated against a synthetic peptide sequence designed from the predicted amino acid sequence of murine HIP/RPL29 [19] was used to detect HIP/RPL29 in frozen sections of mouse uteri obtained at various stages of the estrous cycle. As shown in Figure 1, this antibody detected expression throughout the cell in epithelia as well as what appeared to be nuclear staining in the underlying stroma (Fig. 1a). Preabsorption of the antibody with the peptide antigen reduced staining to a similar background level as observed when primary antibody was omitted (Fig. 1, b and c, respectively). Western blotting was performed as a further test of antibody specificity. Although the predicted molecular weight based on amino acid sequence is 17 600, both mouse and human HIP/RPL29 consistently migrate at 24 000 Mr in SDS gel electrophoresis as has been observed for other highly basic proteins [18, 19]. In isolated uterine epithelia, obtained at either estrus or diestrus (Fig. 2, lanes 1 and 3) the antibody displayed specific reactivity with a doublet at 24 000 and 22 000 Mr; however, even though the antibody appeared to react specifically with nuclear components in the stroma, minimal reactivity was detected in stroma isolated from estrus (Fig. 2, lane 2). Increased stromal reactivity was observed in extracts from diestrus, Day 4.5 of pregnancy, and Day 10 decidua. Thus, HIP/RPL29 appeared to be constitutively expressed in epithelia, but varied in stroma during the estrous cycle. Further immunohistological examination of HIP/RPL29 expression during the estrous cycle revealed that this protein was expressed in epithelia at all times (Fig. 3). When isolated epithelia from each stage of the cycle were analyzed by Western blotting, densitometric analysis of HIP/RPL29 expression relative to a constant amount of total cell protein supported the immunohistological results with an average decrease of 35% from estrus to diestrus epithelium (data not shown). In contrast, densitometric analysis of HIP/RPL29 expression in isolated stroma showed an average 7.8-fold increase from estrus to diestrus for a constant amount of total cell protein.



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  		FIG. 1. Specificity of HIP/RPL29 protein detection by anti-HIP/RPL29. Serial sections of estrous stage mouse uteri were stained by indirect immunofluorescence with antibody directed against HIP/RPL29 peptide 3 as described in Materials and Methods. The primary antibody solutions used in each panel were a) anti-HIP/RPL29, b) anti-HIP/RPL29 preadsorbed with the peptide used to raise the antibody, or c) PBS only. The bar in c represents 60 µm. Location of the luminal epithelium (le) and stroma (s) are indicated.



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  		FIG. 2. Western blot analyses of HIP/RPL29 in total protein extracts of isolated uterine cells. Western blotting was performed with antibody to HIP/RPL29 peptide 3 as described in Materials and Methods. Each lane contained 50 µg total protein from isolated epithelia (lanes 1, 3, and 5) or stroma (lanes 2, 4, and 6) from estrous (lanes 1 and 2), diestrous stages (lanes 3 and 4), or Day 4.5 of pregnancy (lanes 5 and 6) or decidua of Day 10 of pregnancy (lane 7). In most cases a doublet of 24 000 and 22 000 Mr was detected.



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  		FIG. 3. Spatial pattern of HIP/L29 protein expression in the cycling mouse uterus. Sections of proestrous (a), estrous (b), metestrous (c), and diestrous (d) stage mouse uteri were stained by indirect immunofluorescence using anti-HIP/RPL29 as described in Materials and Methods. Strong staining is detected in both luminal (le) and glandular (ge) epithelium in all cases. Reduced staining is observed in underlying stroma (s) and myometrium. Bar = 93 µm

HIP/RPL29 Expression During Early Pregnancy

Several approaches were used to examine uterine HIP/RPL29 expression during early pregnancy. Western blot analyses of total protein extracted from scraped total endometrium from Days 1 through 7 of pregnancy revealed specific reactivity with a single band of the expected size (i.e., 24 000 Mr, in all cases; Fig. 4). Densitometric analysis of the Western blot in Figure 4 revealed no change in relative HIP/RPL29 expression greater than a 20% decrease on Day 5 and a twofold increase by Day 7. Immunohistology was used to localize HIP/RPL29 protein in frozen sections. HIP/RPL29 was detected in both epithelium and proximal stroma from Days 1–4, similar to the pattern described at Day 4.5 (data not shown). Inspection of implantation sites revealed persistent epithelial expression of HIP/L29 as well as elevated expression in primary and secondary decidua through Day 7.5 (Fig. 5). In addition, trophectoderm of peri-implantation-stage blastocysts displayed comparable HIP/RPL29 expression as surrounding cells at Day 4.5 (Fig. 5b). Embryonic HIP/RPL29 expression extended throughout the embryo at Day 7.5 (Fig. 5e). Nevertheless, we were unable to demonstrate cell surface expression of HIP/RPL29 in attachment competent blastocysts, suggesting that this protein predominantly participates in intracellular functions at this stage (data not shown). A more intense HIP/RPL29 accumulation surrounded the implanted embryo at day 7.5 (Fig. 5e). An accumulation of mRNA in a similar region of the embryo was detected by in situ hybridization (see Fig. 9h). Cross-reactive staining was found in stromal nuclei during the peri-implantation period, similar to that seen in cycling stroma. Staining in the postimplantation period revealed enhanced reactivity with HIP/RPL29 antibodies throughout cytoplasm and nuclei in the decidua of both primary and secondary zones (Fig. 5). Examination of antimesometrial decidua stained with antibodies to both HIP/RPL29 (green) and a potential HIP/RPL29 ligand, perlecan ([25]; red) revealed a strong cellular expression of HIP/RPL29 and complementary pericellular staining of perlecan at both Days 5.5 and 7.5 (Fig. 6, a and b, respectively). Thus, HIP/RPL29 and a potential ligand were expressed at appropriate sites for interaction in vivo if a portion of HIP/RPL29 expressed in decidua is located at the cell surface. Through Day 8 of pregnancy, expression of HIP/L29 was of equal intensity in antimesometrial and mesometrial decidua. By Day 10, the developing differences in cellular morphology, physiological characterization, and protein production [26] were reflected by HIP/RPL29 expression in the two decidual regions (Fig. 7). Expression of HIP/RPL29 in antimesometrial decidua (Fig. 7, c and e) appeared more intense and extended throughout the cell while expression in mesometrial decidua (Fig. 7, d and f) appeared restricted to the nucleus. A similar difference in relative intensity was evident for desmin (Fig. 7, a and b), the intermediate filament protein that distinguishes decidua from nondecidualized stroma in rodent [27]. In contrast, perlecan expression was most intense in the basal lamina associated with vascular elements of mesometrial decidua (Fig. 7f), although a region of intense expression was associated with decidual cells immediately proximal to antimesometrial trophoblast giant cells (Fig. 7e).



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  		FIG. 4. Western blotting of HIP/RPL29 in total protein extracts of uterine endometrium during pregnancy. Western blotting was performed using antibody to HIP/RPL29 as described in Materials and Methods. Each lane contained 100 µg total protein solubilized from whole endometrium scraped from uteri of Day 1 (lanes 1 and 2), Day 3 (lane 3), Day 4.5 (lane 4), Day 5 (lanes 5 and 6), Day 6 (lanes 7 and 8), and Day 7 of pregnancy (lanes 9 and 10). Duplicate samples on Days 1, 5, 6, and 7 represent independent endometrial preparations. No significant changes were detected during this period



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  		FIG. 5. Spatial patterns of HIP/RPL29 protein expression during decidualization. Frozen sections of Day 4.5 (a and b), Day 6.5 (c), and Day 7.5 (d and e) pregnant mouse uteri were stained by indirect immunofluorescence with anti-HIP/RPL29 as described in Materials and Methods. In a, an implantation site is shown demonstrating HIP/RPL29 expression in the blastocyst (bl), epithelium (e), primary decidual zone (pdz), and associated stroma (s). A higher magnification of the implantation site is shown in b. Panel c shows HIP/RPL29 expression in the antimesometrial secondary decidual zone (sdz) at Day 6.5. Panels d and e show HIP/RPL29 expression through the decidua and embryo (emb) and minimal expression in the myometrium (myo). Bars = 70 µm for a, c, d, and e and 14 µm for b



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  		FIG. 9. Distribution of HIP/RPL29 mRNA during the periimplantation period. In situ hybridization was performed on longitudinal sections of Day 1 (a), Day 2 (b), Day 3 (c), Day 4 (d), and Day 5 (e) pregnant uteri or cross-sections through implantation sites of Day 6 (f), Day 7 (g), and Day 8 (h) pregnant uteri. Red signals are sites of mRNA and green signal is due to hematoxylin and eosin counterstaining. In c, stroma (s), luminal epithelium (le), glandular epithelium (ge), and myometrium (myo) are designated. Panels e–h indicate locations of the blastocyst (bl), embryo (em), primary decidual zone (pdz), secondary decidual zone (sdz), and the mesometrial (m) and antimesometrial (am) aspects. Original magnification x40 (a–e) and x20 (f–h)



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  		FIG. 6. Complementary pattern of HIP/RPL29 and perlecan in decidua. Frozen sections of Day 5.5 (a) and Day 7.5 (b) were doubly stained by indirect immunofluorescence with anti-HIP/RPL29 (green) and anti-perlecan (red) as described in Materials and Methods. Note the intense pericellular accumulation of perlecan in close apposition to HIP/RPL29-positive decidual cells. Bar = 14 µm



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  		FIG. 7. Regional HIP/RPL29 protein expression in mid-gestation decidua. Frozen sections of Day 10 pregnant mouse uteri were stained by indirect immunofluorescence as described in Materials and Methods with anti-desmin (a and b), anti-HIP/RPL29 (c and d), or a combination of rabbit anti-HIP/RPL29 (green) with rat monoclonal anti-perlecan (red; e and f). Anti-mesometrial (a, c, and e) and mesometrial (b, d, and f) decidua, defined by expression of desmin, exhibit a marked difference in relative expression of HIP/RPL29. Trophoblast giant cells (T), decidua (D), myometrium (M), and Reichert's membrane (R) are indicated. Bar = 117 µm

HIP/RPL29 expression in uterine tissue compartments during early pregnancy also was confirmed by Northern blotting and in situ hybridization. Northern blot analyses indicated that HIP/RPL29 mRNA with an approximate size of 1.2 kilobase was detected in total uterine RNA extracts throughout pregnancy and at similar relative levels when compared with mRNA encoding ribosomal protein, rpL7, used as a loading control (Fig. 8). As shown in Figure 9, in situ hybridization of uterine longitudinal sections revealed specific hybridization primarily with epithelial components from Days 1–3 with some expression found in stroma in association with epithelia (Fig. 9, a–c). Similar to the immunohistochemical results, little or no signal was detected in myometrium at any point. At Day 4, hybridization began to be detected in stroma at a level comparable to the epithelium (Fig. 9d). On Day 5, strong hybridization was detected in the developing decidua surrounding the implantation site; however, stroma away from the implantation site displayed hybridization close to background levels, while epithelia at these distal sites again displayed strong hybridization (Fig. 9e). By Day 6 of pregnancy (Fig. 9f), strong hybridization was detected in both primary and secondary decidual zones at both the mesometrial and antimesometrial aspects of the endometrium. The decidual signal persisted through Days 7 and 8 of pregnancy (Fig. 9, g and h, respectively). Furthermore, a strong signal was detected in embryonic tissues at Day 7 and 8, similar to the pattern of accumulation of HIP/RPL29 protein observed in embryos at this time (Fig. 5e).



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  		FIG. 8. Northern blotting of HIP/RPL29 and rpL7 mRNAs in total RNA from whole uterus during the periimplantation period. Northern blot analysis was performed on 6 µg total RNA isolated from whole uterus as described in Materials and Methods. 32P-Labeled cRNA probes hybridized to transcripts of HIP/L29 and rpL7 (upper and lower panels, respectively). No significant changes were detected in the relative level of HIP/RPL29 mRNA expression throughout pregnancy. Lanes 1–8, RNA from Days 1–8 of pregnancy, respectively

Blastocyst Adhesion to Recombinant Murine HIP/RPL29

The ability of recombinant murine HIP/RPL29 to support blastocyst attachment was examined by a solid phase assay in which recombinant murine HIP/RPL29 containing an N-terminal oligohistidine sequence was anchored to a nickel chelate-coated solid support by the N-terminal oligohistidine sequence. Previous studies demonstrate that this recombinant protein retains the properties of native HIP/RPL29 and that the oligohistidine sequence does not participate in heparin/HS-binding [19, 28]. Blastocysts were harvested at Day 4.5 of gestation when approximately 80% were hatched from the zona pellucida. In vivo the blastocysts acquire attachment competence shortly after hatching and attach within a few hours. These blastocysts were used to determine whether HIP/RPL29 could support embryo attachment in vitro.

Attachment-competent blastocysts incubated on recombinant HIP/RPL29 attached within 6–12 h. As shown in Figure 10, blastocysts attached to HIP/RPL29-coated surfaces did so by assuming a position where the inner cell mass was oriented to one end, indicating an attachment via the mural trophectoderm. Tight adhesion, characterized by no movement when subjected to a gentle stream of medium, followed within 24 h. During the transition from loose to tight adhesion, the blastocyst usually collapsed. During the subsequent 48 h, the trophectoderm dissociated, with individual cells migrating away from the inner cell mass, which remained intact (Fig. 10b). Cells remained rounded during the attachment and dissociation phases. Characteristic outgrowth (see Fig. 10c, for example) accompanied by cell flattening and spreading was not observed in these cultures. On control surfaces a transient loose adhesion was observed for 20%–30% of embryos. By 24 h of culture, all controls were again unattached. Their retention of attachment competence and viability was tested at 72 h of culture when all unattached embryos were transferred to uncoated tissue culture plastic in the presence of serum. One hundred percent of unattached embryos attached within 2 h and displayed characteristic spreading and outgrowth within 6 h.



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  		FIG. 10. Mouse blastocysts attach but do not spread on HIP/RPL29-coated surfaces. Mouse blastocysts were incubated with HIP/RPL29-coated surfaces for 11 h (a) or 60 h (b) as described in Materials and Methods. Panel a shows four separate attached blastocysts with inner cell masses (icm) oriented at one pole, indicating that adhesion occurred via the mural trophectoderm. Panel b demonstrates that even after an extended incubation, attached cells fail to spread or demonstrate typical trophoblast (troph) outgrowths such as shown in c. In c, five blastocysts were attached and outgrown on uncoated plastic in the presence of 10% (v/v) FBS for 48 h. Bar = 28 µm

In other instances, cell attachment to HIP/RPL29 and HIP/RPL29 peptides has been shown to be HS-dependent [7, 15, 16]; however, blastocyst attachment to recombinant HIP/RPL29 was HS-independent (Table 1). Blastocysts did not attach to surfaces coated with polyhistidine or extracts of bacteria transformed with an empty histidine-tagged vector. Blastocysts also failed to attach to surfaces coated with HIP/RPL29-expressing bacterial extracts preabsorbed with HIP/RPL29 antibodies (data not shown). Preincubation of HIP/RPL29 with 1 mg/ml of a heparin preparation, which had previously been demonstrated to inhibit binding of 3H-Hp to recombinant murine HIP/RPL29 by >95% in a solid-phase assay, did not inhibit subsequent adhesion of blastocysts. In addition, neither low molecular weight heparin (1 mg/ml) nor chondroitin-6-sulfate (1 mg/ml) were able to inhibit blastocyst attachment to recombinant HIP/RPL29. Embryos failed to attach to surfaces to which only the oligohistidine sequence was attached. Thus, this biological activity was associated with HIP/RPL29 itself rather than the oligohistidine sequence. It was concluded that murine HIP/RPL29 was capable of supporting embryo attachment, apparently via a novel (i.e., HS-independent) interaction with embryonic cell surface components.


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  		TABLE 1. HIP/RPL29 supports mouse blastocyst attachment

DISCUSSION

Similar to the pattern of expression observed in the human uterus during the menstrual cycle [18], murine HIP/RPL29 persisted in uteri throughout the estrous cycle. HIP/RPL29 also is believed to function as a ribosome-associated protein [29]. Nonetheless, HIP/RPL29 is not expressed significantly by all cells of adult tissues, suggesting an accessory, rather than essential, role in ribosomal function [14, 19]. Furthermore, HIP/RPL29 expression appears to be restricted to individual cell types within a tissue, as is the case in the uterus. In various human cell lines, HIP/RPL29 is found at the cell surface [7, 14, 15]. In the present study, the most intense expression of HIP/RPL29 occurred in epithelia during proestrus, the stage of epithelial proliferation. The subcellular distribution of HIP/RPL29 protein at this stage, while not excluding a cell surface expression, would indicate that HIP/RPL29 is predominantly intracellular because intense staining was evident throughout the cell. This distribution pattern and the increased intensity of signal displayed in epithelium at a stage of the cycle requiring increased protein synthesis to support cell growth is consistent with a primary role for HIP/RPL29 in translation. Likewise, increased HIP/RPL29 mRNA and protein were detected in uterine stromal cells undergoing decidualization, a process involving proliferation, cellular growth, and differentiation [30]. HIP/RPL29 also accumulates in predecidual cells of the human uterus during the late stages of the menstrual cycle [18]. In this case, the protein is distributed throughout the cytoplasm and nucleus.

Expression of HIP/RPL29 in adult tissues seems to correlate with a regenerative potential retained beyond embryonic development. The limited and variable distribution of HIP/RPL29 in adult tissues, including the uterus, may be related to properties that distinguish HIP/RPL29 from the general group of constitutively expressed essential ribosomal proteins. HIP/RPL29 was found to be differentially monomethylated at Lys 4 in liver, brain, and thymus, suggesting a distinct regulatory role for HIP/RPL29 methylation [31].

While the expression pattern of HIP/RPL29 in uterine cells was consistent with a primary role in translation, potential participation in the implantation process relates to the secondary function of HIP/RPL29 as an HP/HS-binding protein. For HIP/RPL29 to participate in one or more implantation processes as a binding partner of HSPGs, it would have to be capable of interacting with either HS chains or the protein core. We have previously demonstrated that perlecan is capable of HS-dependent binding to a human HIP/RPL29 peptide [7]. Furthermore, both intact HIP and HIP peptides bind heparin and HS with high affinity and selectivity [13, 16–18]. Nonetheless, antibodies directed at particular HS-binding motifs within HIP/RPL29 compete for no more than 50% of 3H-Hp binding [17]. Additional studies indicate that almost all the HIP/RPL29 protein participates in HS binding [28]. Therefore, multiple motifs within the protein appear to be involved. Although mouse HIP/RPL29 demonstrates selective and high-affinity binding of heparin and HS [19], these proteins diverge in sequence in the C-terminal region, which contains a particularly strong and selective HS-binding motif in the human protein [16, 32]. An Hp/HS-binding function has not been established for the C-terminal region of murine HIP/RPL29. Furthermore, it was predicted that the presence of prolines in the C-terminal is likely to disrupt any alpha-helical structures that bind Hp, and that additional domains within murine HIP/RPL29 were responsible for its Hp binding activity [19].

For HIP/RPL29 to participate in embryo attachment during implantation, HIP/RPL29 expression must occur in the luminal uterine epithelia during the receptive phase of the uterus. This phase occurs shortly after hatching of the blastocyst from the zona pellucida, at which time the surfaces of trophectoderm and uterine epithelium are brought together through closure of the uterine lumen [33]. HIP/RPL29 is expressed by uterine luminal epithelia throughout the preimplantation and peri-implantation stages and is present at blastocyst implantation sites. Loss of apically disposed mucins that occurs during this time may create access to HIP/RPL29 and other HS-binding proteins (e.g., amphiregulin and heparin-binding EGF-like growth factor [6, 23]). Nevertheless, while HIP/RPL29 is expressed by uterine epithelia at the site of embryo attachment and is capable of promoting embryo attachment in vitro, it does not appear that HIP/RPL29 is expressed at the cell surface in the mouse. Using multiple approaches, we have been unable to detect significant cell surface HIP/RPL29 expression on either mouse uterine epithelial cells in primary culture or normal murine mammary gland cells (NMuMG; unpublished results). It is possible that the high level of mucin glycoprotein expression by these cells impairs access to cell surface HIP/RPL29 [34, 35]; however, based on our immunohistochemical results, it seems likely that the vast majority of HIP/RPL29 in mouse uterine epithelia is intracellular. This is in contrast to various human epithelial cells and cell lines that display cell surface HIP/RPL29 [7, 15, 18]. Nevertheless, studies of HIP/RPL29 functions in the context of mouse embryo attachment may provide useful insights regarding this protein's role in human implantation. Moreover, HIP/RPL29 may promote implantation-related events in mice because the embryo may itself trigger HIP/RPL29 release in the locale of the embryo. Triggered or alternate protein export systems have been proposed for another HS-binding protein lacking a signal sequence, fibroblast growth factor-2 [36]. Moreover, there are multiple examples of embryos triggering local responses in uterine tissues [23, 33, 37]. In addition to promoting embryo attachment, it is possible that HIP/RPL29 may have growth-regulating activities either by displacing HS-bound growth factors or by having growth factor activity itself. These possibilities currently are being considered in our laboratory.

The most surprising finding in the current study is that HIP/RPL29 supports embryo attachment in an HS-independent manner. Prior to this report all other extracellular activities associated with HIP/RPL29 have been shown to be HS-dependent. In this regard, it is noteworthy that membrane-associated HIP/RPL29, in both mouse and human cell lines, is not extractable by either soluble heparin or heparinase digestion. In contrast, salt efficiently solubilizes HIP/RPL29 from membrane preparations [18, 19]. Thus, it appears that non-HS-bearing molecules participate in HIP/RPL29 retention on membranes. It will be important to identify these novel HIP/RPL29-binding components to fully understand HIP/RPL29 interactions at cell surfaces and other membrane compartments.

ACKNOWLEDGMENTS

We are grateful to Drs. M.C. Farach-Carson, R. Gomes, C. Kirn-Safran, and A. Liu for their careful reading of the manuscript and many helpful discussions. We are also indebted to Ms. Sharron Kingston for her excellent secretarial assistance.

FOOTNOTES

First decision: 8 August 2000.

1 Supported by National Institutes of Health grants HD 25235 and HD 29963 to D.D.C., HD 29968 to S.K. Dey, and ES 07814 to S.K. Das. This work was performed in part as a component of the National Cooperative Program for Markers of Uterine Receptivity. Back

2 Correspondence: Daniel Carson, Department of Biological Sciences, 117 Wolf Hall, University of Delaware, Newark, DE 19707. FAX: 302 831 2281; dcarson{at}udel.edu Back

Accepted: November 13, 2000.

Received: July 14, 2000.

REFERENCES

  1. Lander AD, Selleck SB. The elusive functions of proteoglycans: in vivo veritas. J Cell Biol 2000; 148:227–232.[Abstract/Free Full Text]
  2. Farach MC, Tang J-P, Decker GL, Carson DD. Differential effects of p-nitrophenyl-D-xylosides on mouse blastocysts and uterine epithelial cells. Biol Reprod 1988; 39:443–455.[Abstract]
  3. Farach MC, Tang J-P, Decker GL, Carson DD. Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro. Dev Biol 1987; 123:401–410.[CrossRef][Medline]
  4. Smith SE, French M, Julian J, Paria BC, Dey SK, Carson DD. Expression of heparan sulfate proteoglycan (perlecan) in the mouse blastocyst is regulated during normal and delayed implantation. Dev Biol 1997; 184:38–47.[CrossRef][Medline]
  5. Carson DD, Tang J-P, Julian J. Heparan sulfate proteoglycan expression by periimplantation stage embryos. Dev Biol 1993; 155:97–106.[CrossRef][Medline]
  6. Das KS, Wang X-N, Paria BC, Damm D, Abraham JA, Klagsbrun M, Andrews GK, Dey SK. Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 1994; 120:1071–1083.[Abstract]
  7. Rohde LH, Janatpore MU, McMaster MT, Fisher S, Zhou Y, Lim KH, French M, Hoke D, Julian J, Carson DD. Complementary expression of HIP, a cell-surface heparan sulfate binding protein, and perlecan at the human fetal-maternal interface. Biol Reprod 1998; 58:1075–1083.[Abstract/Free Full Text]
  8. Kirn-Safran CB, Carson DD. Dynamics of uterine glycoconjugate expression and function. Semin Reprod Endocrinol 1999; 17:217–227.[Medline]
  9. Costell M, Gustafsson E, Aszodi A, Morgelin M, Bloch W, Hunziker E, Addicks K, Timpl R, Fassler R. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol 1999; 147:1109–1122.[Abstract/Free Full Text]
  10. Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y. Perlecan is essential for cartilage and cephalic development. Nat Genet 1999; 23:354–358.[CrossRef][Medline]
  11. Raab G, Kover K, Paria BC, Dey SK, Ezzell RM, Klagsbrun M. Mouse preimplantation blastocysts adhere to cells expressing the transmembrane form of heparin-binding EGF-like growth factor. Development 1996; 122:637–645.[Abstract]
  12. Wilson OF, Jacobs A, Stewart S, Carson DD. Expression of externally disposed heparin/heparan sulfate binding sites by uterine epithelial cells. J Cell Physiol 1990; 143:60–67.[CrossRef][Medline]
  13. Raboudi N, Julian J, Rohde LH, Carson DD. Identification of cell surface heparin/heparan sulfate binding proteins of a human uterine epithelial cell line (RL95). J Biol Chem 1992; 267:11930–11939.[Abstract/Free Full Text]
  14. Liu S, Smith SE, Karin N, Rohde LH, Julian J, Carson DD. cDNA cloning and expression of heparin/heparan sulfate interacting protein (HIP). J Biol Chem 1996; 271:11817–11823.[Abstract/Free Full Text]
  15. Jacobs AL, Julian J, Sahin AA, Carson DD. Heparin/heparan sulfate interacting protein expression and functions in human breast cancer cells and normal breast epithelia. Cancer Res 1997; 57:5148–5154.[Abstract/Free Full Text]
  16. Liu S, Julian J, Carson DD. A peptide sequence of heparin/heparan sulfate (HP/HS)-interacting protein supports selective, high affinity binding of HP/HS and cell attachment. J Biol Chem 1998; 273:9718–9726.[Abstract/Free Full Text]
  17. Liu S, Hoke D, Julian J, Carson DD. Heparin/heparan sulfate (HP/HS) interacting protein (HIP) supports cell attachment and selective, high affinity binding of HP/HS. J Biol Chem 1997; 272:25856–25862.[Abstract/Free Full Text]
  18. Rohde LH, Julian J, Babaknia A, Carson DD. Cell surface expression of HIP, a novel heparin/heparan sulfate binding protein of human uterine epithelial cells and cell lines. J Biol Chem 1996; 271:11824–11830.[Abstract/Free Full Text]
  19. Hoke DE, Regisford EG, Julian J, Amin A, Bégue-Kirn C, Carson DD. Murine HIP/L29 is a heparin-binding protein with a restricted pattern of expression in adult tissues. J Biol Chem 1998; 273:25148–25157.[Abstract/Free Full Text]
  20. Jacobs AL, Sehgal PB, Julian J, Carson DD. Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro. Endocrinology. 1992; 131:1037–1046.
  21. Laemmli UK. Cleavage of structural proteins during assembly of the head of the bacteriophage T4. Nature 1970; 277:680–685.
  22. Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, Dey SK. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997; 91:197–208.[CrossRef][Medline]
  23. Das SK, Chakraborty I, Paria BC, Wang XN, Plowman G, Dey SK. Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus. Mol Endocrinol 1995; 9:691–705.[Abstract]
  24. DeSouza MM, Surveyor GA, Price RE, Julian J, Kardon R, Zhou X, Gendler S, Hilkens J, Carson DD. MUC1/episialin: a critical barrier in the female reproductive tract. J Reprod Immunol 1999; 45:127–158.[Medline]
  25. French MM, Smith SE, Akanbi K, Sanford T, Farach-Carson MC, Carson DD. Expression of the heparan sulfate proteoglycan perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro. J Cell Biol 1999; 145:1103–1115.[Abstract/Free Full Text]
  26. Bell SC. Decidualization: regional differentiation and associated function. Oxf Rev Reprod Biol 1983; 5:220–271.
  27. Glasser SR, Lampelo S, Munir MI, Julian J. Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells. Differentiation 1987; 35:132–142.[CrossRef][Medline]
  28. Hoke DE, LaBrenz SR, Höök M, Carson DD. Multiple domains contribute to heparin/heparan sulfate binding by human HIP/L29. Biochemistry 2001; (in press).
  29. Chan Y-L, Olvera J, Paz V, Wool IG. The primary structure of rat ribosomal protein L29. Biochem Biophys Res Commun 1993; 192:583–589.[CrossRef][Medline]
  30. Abrahamsohn PA, Zorn TM. Implantation and decidualization in rodents. J Exp Zool 1993; 266:603–628.[CrossRef][Medline]
  31. Williamson NA, Raleigh J, Morrice NA, Wettenhall REH. Post-translational processing of rat ribosomal proteins: ubiquitous methylation of Lys 22 within the zinc-finger motif of RL40 (carboxy-terminal extension protein 52) and tissue-specific methylation of RL 29. Eur J Biochem 1997; 246:786–793.[Medline]
  32. Liu S, Zhou F, Höök M, Carson DD. A heparin-binding synthetic peptide of heparin/heparan sulfate interacting protein modulates blood coagulation activities. Proc Natl Acad Sci U S A 1997; 94:1739–1744.[Abstract/Free Full Text]
  33. Parr MB, Parr EL. The implantation reaction. In: Wynn RM, Jollie WP (eds.), The Uterus. New York: Plenum Press; 1989: 233–277.
  34. Valdizan MC, Julian J, Carson DD. WGA-binding, mucin glycoproteins protect the apical cell surface of mouse uterine epithelial cells. J Cell Physiol 1992; 151:451–465.[CrossRef][Medline]
  35. Pimental RA, Julian J, Gendler SJ, Carson DD. Synthesis and intracellular trafficking of Muc. 1 and mucins by polarized mouse uterine epithelial cells. J Biol Chem 1996; 271:28128–28137.[Abstract/Free Full Text]
  36. Florkiewicz RZ, Anchin J, Baird A. The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase. J Biol Chem 1998; 273:544–551.[Abstract/Free Full Text]
  37. Hoffman LS, Olson GE, Carson DD, Chilton BS. Progesterone and implanting blastocysts regulate Muc1 expression in rabbit uterine epithelium. Endocrinology 1998; 139:266–271.[Abstract/Free Full Text]



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