0013-7227/92/1301-0240$03.00/0 Endocrinology Copyright
0 1992 by The Endocrine
Vol. 130,No. 1 Printed
Society
Steroid Hormones Differentially Modulate Glycoconjugate Synthesis and Vectorial Secretion Polarized Uterine Epithelial Cells in Vitro* SHAILAJA
K. MANI,
DANIEL
D. CARSON,
AND
STANLEY
in U.S.A.
by
R. GLASSER
Department of Cell Biology and the Center for Population Research and Studies in Reproductive Biology, Baylor College of Medicine, and the Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center (D.D.C.), Houston, Texas 77030
one caused a 4- to 5-fold accumulation of mucosialoglycoproteins in the cell-associated fraction, suggesting regulation by the hormone at the level of secretion, rather than synthesis. Estrogen and progesterone both stimulated the synthesis and secretion of hyaluronate by the polarized UE cells. Neither hormone substantially altered the synthesis or secretory pattern of heparan sulfate proteoglycans. Collectively, these studies provide the first comprehensive characterization of the major glycoconjugates synthesized and secreted by rabbit UE cells. Furthermore, these observations demonstrate marked differential direct influences of steroid hormones on the production of distinct classes of UE cell glycoconjugates. (Endocrinology 130: 240-248,1992)
ABSTRACT. Characterization of glycoconjugates synthesized by polarized immature rabbit uterine epithelial (UE) cells in vitro, their vectorial patterns of secretion, and regulation by ovarian steroid hormones are reported. Large (mol wt, >230 kDa) sialomucoglycoproteins and hyaluronate were primarily (86-96%) secreted from the apical cell surface domain, while heparan sulfate proteoglycans were predominantly secreted from the basal cell surface of the polarized UE cells. The polarized UE cells responded to estrogen and progesterone in vitro and exhibited distinct profiles in their synthesis and secretion of different glycoconjugates. Progesterone and/or estrogen reduced the secretion of the mucosialoglycoproteins; however, progester-
B
ment. Mouse UE cells in uiuo and in uitro express Nlinked lactosaminoglycan (LAGS) glycoproteins (13). Moreover, LAG synthesis is stimulated by estrogen (14), and uterine LAGS have been shown to participate in cellcell and cell-substratum adhesion in vitro (15). Furthermore, the LAG-related oligosaccharide, lacto-l\r-fucopentanose-I, is expressed at the apical cell surface domain of mouse UE in uiuo during implantation and inhibits attachment of blastocysts to UE cell monolayers (16). Heparan sulfate proteoglycans (HSPGs) also have been identified at the cell surfaces of both UE (17) and periimplantation stage blastocysts in the mouse and probably participate in embryo-UE cell attachment (18). Keratan sulfate proteoglycans, structurally related to LAGS, as well as HSPGs are produced by polarized rat UE cells and immature mouse UE cells in vitro (13, 19). Collectively, these studies suggest that glycoproteins bearing LAG-related stuctures as well as HSPGs are produced by rodent UE cells and may participate in multiple aspects of adhesive interactions occurring between UE cells and trophoblast. Most of the information on the modification of UE cell glycoconjugates during pre- and periimplantation stages is based on the studies in mammals, in which implantation is interstitial, i.e. the rat and mouse. In this
LASTOCYST implantation involves adhesion of the trophoblast to the luminal epithelium of the transiently receptive uterus conditioned by the ovarian steroid hormones estrogen and progesterone (1, 2). The molecular basis for the cell-cell interactions resulting in adhesion remains unknown. Based on the recent evidence for the involvement of glycoproteins in various cell-cell interactions (3, 4), including fertilization (5), glycoproteins and/or glycolipid oligosaccharides can be assumed to play an important role in trophoblast-endometrial interactions. Recent data indicate that uterine epithelium produces both secreted and membrane-bound glycoconjugates (6). Furthermore, it is clear that the mechanisms that regulate uterine glycoprotein biosynthesis are under the control of estrogen and progesterone (7,W Both morphological (9, 10) and biochemical (11, 12) studies describe changes in glycoconjugate expression on the surfaces of the trophoblast and uterine epithelium (UE) during the initial phase of embryo-UE cell attachReceived July 25, 1991. Address requests for reprints to: Stanley R. Glasser, Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030. * This work was supported by NIH Grants HD-13663 and HD-07485 (to S.R.G.) and HD-25235 (to D.D.C.). 240 Downloaded from https://academic.oup.com/endo/article-abstract/130/1/240/3034250 by Adelaide University user on 07 March 2018
UE GLYCOCONJUGATES
regard, much less is known in animals exhibiting syncytial implantation, e.g. the rabbit. Morphological studies of rabbit luminal UE cells demonstrate extensive differentiation accompanied by a loss of surface negativity and alterations in the luminal glycocalyx (20, 21, 22). Biochemical studies on stage-specific alterations in protein and saccharide composition demonstrate a reduction of sialic acid residues and the increased expression of Nacetylglucosamine residues on the apical surface of UE cells during the periimplantation period (11). Lectin binding studies demonstrate that the accumulation of terminal galactosyl groups at the apical cell membrane of UE cells has been implicated in the acquisition of receptivity, and a role for the galactosyl moieties has been proposed in trophoblast adhesion to the UE cells (11). Earlier studies on hormonal effects on the in vivo metabolism of glycoproteins and glycosaminoglycans in the rabbit uterus reported stimulated synthesis of sulfated glycosaminoglycans in the presence of estrogen; progesterone treatment antagonized the estrogen stimulation (23). It remains unclear whether glycoconjugates detected in the thick apical glycocalyx are integral to the plasma membrane or are secreted and trapped at the apical surface. There is evidence in rodents suggesting intimate association between secretory sustances and the apical cell surface of UE (24, 25), and identical glycoproteins can be detected in both the cell-associated and secreted fractions (13, 19, 25). Extremely important in the blastocyst-endometrial relationship are the cell-cell and cellmatrix interactions at the apical and basolateral surfaces of the UE cell, respectively. These interactions are likely to be modulated by the steroid hormones (1,2). We have recently developed a cell culture system for immature rabbit UE cells that allows the cells to retain their polarized state and respond to steroid hormones in vitro (26). In the present study we have used this culture system to characterize the glycoproteins and proteoglycans secreted from the apical and basolateral domains of polarized UE cells and demonstrate hormonally induced changes in glycoprotein expression in vitro. Materials
and Methods
241
glucoside, Sephadex G-50 (fine), pokeweed mitogen agarose (PWM), Datura stramonium agglutinin-agarose (DSA), wheat germ agglutinin (WGA)-agarose, pronase, heparin, 3-[(3-cholamido-propyl)dimethyl ammonioll-propane sulfonate (CHAPS), Triton X-100, leupeptin, benzamidine, aprotinin, chymostatin, pepstatin, 17@estradiol, and progesterone were obtained from Sigma Chemical Co. (St. Louis, MO). Protein mol wt (Mr) calibration standards were purchased from Bethesda Research Laboratories (Gaithersburg, MD). Endo+ galactosidase (Eschrichia freundii) was purchased from VLabs (Covington, LA). Peptide-N-glycanase and Streptomyces hyaluronidase were obtained from Genzyme (Boston, MA), and neuraminidase (Clostridium perfringens) was purchased from Boehringer Mannheim (Indianapolis, IN). Urea and guanidine hydrochloride were obtained from Schwarz/Mann Biotec (Cleveland, OH). All chemicals used were reagent grade or better. of polarized
Preparation
cell cultures
The isolation of epithelial cells from immature rabbit uteri and their culture on matrix-coated Millicell HA inserts in a serum-free, phenol red-free, defined medium were performed as described previously (26). The extent of confluency and the development of cell polarity were monitored by morphological examination. Functional polarity was confirmed by previously established criteria (26). The cells were cultured for 7 days in four groups of four filters each in defined medium containing 1) no hormone addition, 2) progesterone (1 X 10m7M), 3) 17/3estradiol (1 X lo-’ M), and 4) progesterone and estradiol. Polarized UE cells maintained a confluent density in all groups at the time the cells were metabolically labeled, as described below. Metabolic
labeling
Media from the cultured polarized cells were removed, the cells were rinsed with Hanks’ Balanced Salt Solution, and the basal surface of the filter inserts was blocked using 1% (wt/ vol) BSA in Dulbeccos’ Modified Eagle’s Medium-Ham’s F-12 for 1 h at 37 C. After rinsing with Hanks’ Balanced Salt Solution, the polarized cells were labeled with [3H]glucosamine (0.1 mCi/ml) and H2[35S]0, in RPMI-1640 (sulfate free) supplemented with 3.3 mM MgC&, 1.2 g/liter NaCO,, 15 mM HEPES (pH 7.2), 2.5 U penicillin/ml, and 2.5 rg/ml streptomycin sulfate. The incubations were performed overnight (1618 h) in a humidified atmosphere of 95% air-5% CO, (vol/vol) at 37 C. Isotope incorporation into macromolecules was linear over this time interval (19, 26).
Materials
New Zealand White immature female rabbits (4 weeks old) were purchased from Ray Nichols Rabbitry (Lumberton, TX). Millipore HA filter inserts were obtained from Millipore Continental Water Systems (Bedford, MA). Tissue culture media and supplies were obtained from Gibco (Grand Island, NY). Mito plus serum extender and Matrigel were procured from Collaborative Research (Lexington, MA). Tissue culture plates and supplies were obtained from Corning (Corning, NY). H,35S04 (carrier-free) and [6-3H]glucosamine (25 Ci/mmol) were purchased from ICN Radiochemicals (Irvine, CA). Octyl-
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Glycoconjugate
extraction
Media from the apical and basal secretory compartments were individually collected, and the macromolecular material was precipitated with ice-cold 20% (wt/vol) trichloroacetic acid-6% (wt/vol) phosphotungstic acid. The fractions obtained by centrifugation at 5000 x g for 10 min were precipitated twice with 10% (wt/vol) trichloroacetic acid, followed by rinsing with ice-cold 95% (vol/vol) ethanol. The efficiency of precipitation of total glucoconjugates by this procedure has been addressed previously (18). The precipitates were dissolved in solution
UE GLYCOCONJUGATES
242
Endo. 1992 Voll30. No 1
containing 4 M urea, 0.1% (wt/vol) octylglucoside, 25 mM EDTA (pH 7.0), 20 mM Tris acetate (pH 7.0), and 0.02% (wt/ vol) sodium azide. Cell/filter-associated material was extracted by incubation with a solution of 4 M guanidine hydrochloride, 1% (wt/vol) CHAPS, 25 mM EDTA (pH 7.0), and a mixture of protease inhibitors (14) at room temperature with continuous agitation overnight. Glycoconjugates from this extraction solution were precipitated and processed in the same manner as the secretions. Liquid
chromatography
of glycoconjugates
Macromolecules from the apical and basal secretory compartments and from cell/filter-associated material were initially fractionated by anion exchange liquid chromatography. The chromatographic system, obtained from Beckman Instruments (Palo Alto, CA), consisted of two model 100 A pumps controlled by a model 421 A controller. Chromatography was performed on a 0.5 x 5-cm Mono Q column (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated with 0.5 M urea, 20 mM Trisacetate (pH 7.0), 0.01% (wt/vol) octylglucoside, and 0.02% (wt/ vol) sodium azide. The gradient was developed with O-4 M sodium chloride. The buffer was pumped at a flow rate of 1 ml/ min at room temperature, with a back pressure of -400 psi. Fractions were collected every 0.5 min. Recoveries from these analyses ranged from 85-95%. Analyses for LAGS The radioactive macromolecules eluting in the void volume (peak A; Fig. 1) and 0.8-1.0 M NaCl gradient (peak B: Fig. 1) on the Mono Q column from the secreted and cell-associated fractions were pooled, dialyzed against water, and lyophilized. The products were analyzed for the presence of LAGS by lectin affinity chromatography and endo+galactosidase sensitivity (13,14,25). Lectin binding studies were performed as described previously (13). The lyophilized products from peak fractions A and B were dissolved in PBS containing 0.1% (wt/vol) octylglucoside and 0.1% (wt/vol) hemoglobin and incubated with PWM, DSA, or WGA-agarose for 1 h with continuous agitation at room temperature. The lectin resins were eluted with 4 M guanidine hydrochloride, 1% (wt/vol) CHAPS, and 20 mM Tris-acetate (pH 7.0) to obtain the bound material. Digestions with E. freundii endo+galactosidase were performed at 37 C in 50 mM sodium acetate (pH 5.8) containing 0.3 U enzyme/ml. After 24 h, an additional 0.3 U enzyme/ml was added, and the incubations were carried on for a further 24 h. The products of this digestion were analyzed by molecular exclusion chromatography, using a 1 X 30-cm column of Superose 12 (Pharmacia). The column was eqilibrated with 2 M guanidine hydrochloride, 20 mM Tris-acetate (pH 7.0), 0.01% (wt/vol) octylglucoside, and 0.02% (wt/vol) sodium azide and eluted at a flow rate of 0.7 ml/min. The fractions were collected every 0.5 min, with greater than 85% recovery. The sensitivity of the glycoconjugate to the enzyme was monitored by a shift in the elution pattern relative to that of the undigested controls. Enzymatic
and chemical
degradation
of glycoconjugates
Pronase digestions were performed, as previously described (13), by incubating the material with predigested pronase so-
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4
6
12 MirluBttes 20
24
28
32
1. Anion exchange liquid chromatography of polarized UE cell glycoconjugates. Confluent cultures of polarized UE cells were metabolically labeled for 16-18 h with [3H]glucosamine and H;SO,. The secretions from the apical basal compartments and cell-associated fractions were extracted and processed as described in Materials ana’ Methods. The extracted glycoconjugates were chromatographed on a Mono Q column, eluted at a flow rate of 1 ml/min at room temperature with 20 mM Tris acetate (pH 7.0) containing 0.5 M urea, 0.01% (wt/ vol) octylglucoside and 0.02% (wt/vol) sodium azide. The NaCl gradient (O-4 M) used is indicated (- - -). The elution profiles of both [sH] glucosamine-labeled (0) and ?‘3-labeled (0) material are shown. FIG.
lution at 50 C for 24 h. Fresh pronase was added after 24 h, and the digestion was continued for a further 24 h. Peptide-Nglycanase digestions (10 U/ml) of peak B were performed at 25 C for 16 h in 150 mM sodium phosphate (pH 8.5), 60 mM EDTA, 1% (vol/vol) ,&mercaptoethanol, 1% (vol/vol) Triton X-100, and 2% (wt/vol) sodium dodecyl sulfate. Mild alkaline hydrolysis (p-elimination) was performed for 48 h at 37 C in 0.1 M sodium hydroxide and 0.25 M sodium borohydride. Digestion of glycoconjugate material in peak B fraction with 3.3 U of Streptomyces hyaluronidase in 1 ml 20 mM sodium acetate (pH 5.0) was carried out for 24 h at 37 C. Fresh enzyme was added after 12 h. Parallel control incubations were performed, in which 1 mg commercial hyaluronate was digested under identical conditions in the presence and absence of enzyme. Hyaluronate degradation was monitored in these controls by cetyl pyridinium chloride precipitation, as previously described (15). Degradation of the radioactive material from all of the above digestions was monitored by Superose-12 column chromatography. The material was considered to have been degraded by the treatment when it migrated further into the included volume compared to the undigested controls. The radioactive material migrating in the unbound fraction of the anion exchange column (peak A) was subjected to neuraminidase digestions, as previously described (13). This digested material, products from peptide-N-glycanase digestion or from mild alkaline hydrolysis of components of peak A, as
UE GLYCOCONJUGATES well as the intact material were subjected to molecular exclusion chromatography on a 1.5 X 84-cm Sephadex G-50 column equilibrated with 0.1 M sodium bicarbonate containing 5% (vol/ voi) ethanol and 0.03% (wt/vol) sodium azide. Radioactive samples eluting in the high salt gradient between 2.5-4.0 M NaCl (peak C) were subjected to mild alkaline hydrolysis and nitrous acid degradation. Nitrous acid degradation was performed for 4 h at room temperature in 1 ml aqueous solution of 0.25 N HCl containing 5% (vol/vol) n-butyl nitrite and 25% (vol/vol) ethanol. The products were analyzed by Superose-I2 chromatography. Intact material also was chromatographed in parallel samples to monitor the extent of degradation. Other procedures
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the material from peak A was carried out under reducing conditions, according to the procedure of Porzio and Pearson (27). Fluorography was performed after the fixed gel was incubated in the fluorographic enhancer, En3Hance (New England Nuclear Research Products, Boston, MA).
Results Synthesis and secretion of glycoconjugates Primary cultures of immature rabbit UE cells grown on matrix-coated semipermeable filter inserts demonstrated morphological and functional polarity. This polarized UE cell culture system was used to analyze the glycoconjugates in the cell/filter-associated fraction and in the apical and basal secretory compartments of the UE cells receiving different steroid hormone regimens in vitro. Although the number of cells per filter varied with each hormone treatment, the cells were confluent and displayed a high degree of polarity in all cases. Consequently, the data were normalized to represent glycoconjugates in both the secretions and cell-associated fractions on the basis of lo5 cells/filter insert. Polarized UE cells were labeled metabolically with [3H]glucosamine and H235S04 for 16-18 h. The radioactive macromolecules in the cell-associated fraction and those secreted into the apical and basal compartments were acid precip-
itated and fractionated by anion exchange liquid chromatography. Three major charge classes were obtained, as shown in Fig. 1. These were components that did not bind to the resin (peak A), components that bound and eluted with 0.8-1.0 M NaCl (peak B), and components that bound and eluted with 2.5-4.0 M NaCl (peak C). Most of the sulfate-labeled components in the apical secretions and 3H-labeled molecules in the basal secretions (Fig. 1) were lost upon dialysis using 3500 Mr cutoff dialysis tubing (data not shown). These observations
indicated that most of the radioactivity in these fractions was in the form of low Mr components and/or unincorporated precursor. The macromolecular components in
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243
the unbound fraction (peak A) and the 0.8-1.0 M NaCl eluate (peak B) from the anion exchange resin were labeled primarily with 3H and exhibited preferential apical secretion. These labeled macromolecules were also observed in the cell-associated fraction. Sulfate-labeled components eluting from the resin in the high salt eluate (peak C) were predominantly found in the basal secretions and the cell-associated fraction. This pattern of vectorial expression of the labeled glycoconjugates was similar under all hormonal treatments examined, however, the relative amounts produced varied in response to steroid hormones (see below). The components of these three charge classes in each compartment were analyzed further by subjecting them to a series of specific enzymatic and chemical digestions, followed by molecular exclusion chromatographic analyses. Characterization of glycoconjugates fraction (peak A)
in the unbound
The components in the unbound fraction from the anion exchange resin were labeled primarily with 3H (as shown in Fig. 1). In all cases, the behavior of coeluting sulfated molecules
(if any) was the same as that of the
3H-labeled com p onents (data not shown). As shown in Fig. 2, the intact components
of this fraction
migrated
as a single major peak of radioactivity, exhibiting a median hydrodynamic radius greater than that of a 230kDa protein. Pronase digestion of this material completely shifted the distribution of the radioactivity in this
peak to that of the hydrodynamic radius of a 5kDa protein, indicating that the radiolabeled components
0 Minutes
FIG. 2. Identification of glycoconjugatesin the unbound fraction (peak A) from the anion exchangeresin. Secreted and cell-associated glycoconjugates eluting in the unbound fraction of the anion exchange column were pooled, subjected to enzyme and chemical degradation, and chromatographed on a Superose-12 column, as described in Materials and Methods. Representative profiles of the intact material from the apical secretions (A), pronase-digested (0) material, product obtained after alkaline hydrolysis (A), and endo-j3-galactosidase-digested (0) materials are shown. Peak A materials in the cell-associated fractions exhibit similar behavior. The elution positions of blue dextran [void volume (V,); Mr, 2 X 106], catalase (230K), immunoglobulin G (150K), BSA (68K), carbonic anhydrase (30K), cytochrome-c (12.5K), and potassium dichromate [total volume (VJ] are indicated at the top of the figure.
UE GLYCOCONJUGATES
244
were protein linked. Mild alkaline hydrolysis of this radiolabeled fraction (peak A) completely released the radioactivity to a form with a similar Mr as the pronasedigested material. This observation indicated that the oligosaccharides were primarily O-linked to protein. The radioactive components in this peak A fraction were insensitive to endo-/3-galactosidase, indicating the absence of polysaccharides containing GlcNac (pl-3) Gal sequences (data not shown). Very little material in this fraction (peak A) bound to PWM and DSA, while approximately 3040% bound to WGA; however, estrogen treatment increased the proportion of radioactivity bound to PWM or DSA in the cell-associated fraction and basal secretions (Table 1). Digestion with peptide-N-glycanase followed by molecular exclusion chromatography on Sephadex G-50 demonstrated no significant shift in the mobility patterns (Fig. 3). Chromatography of the products obtained after mild alkaline hydrolysis displayed a median size distribution of 3.5 kDa relative to glycopeptide standards. Digestion of mild alkali-released oligosaccharides with neuraminidase generated two distinct peaks of radioactivity with lower Mr. The lower Mr products migrating near the total included volume were assumed to contain the released sialic acids, while the larger Mr products were assumed to contain the residual core oligosaccharides. It was apparent that the glycoproteins in the unbound fraction from the anion exchange column (peak 1. Lectin affinity chromatography fraction of Mono Q eluate
TABLE
of glycoproteins
in peak A
Treatment
Lectin
Apical secretion (% bound)
Basal secretion (% bound)
Cellassociated (W bound)
No hormone
PWM DSA WGA
9.3 f 1.00 6.6 f 0.50 50.0 + 2.00
1.5 + 0.10 2.6 + 0.05 10.0 f 2.00
1.4 f 0.20 3.2 + 0.50 28.5 + 1.20
+E
PWM DSA WGA
5.6 f 1 7.8 + 1 27.9 f 2
13.8 + 0.1 16.1 f 1 31.7 f 1
6.2 f 0.1 9.9 f 0.25 27.5 + 1
+E+P
PWM DSA WGA
11.2 f 2 9.1 + 1 47 f 1
10.8 f 1 15.0 + 1 30 f 1
5.6 k 2 4.2 f 1 37.5 f 4
PWM 5.7 f 1 1.7 * 0.2 2.8 k 0.2 DSA 3.4 + 1 15 f 0.5 2.9 f 0.1 WGA 28.7 + 0.5 42.6 + 2 36.7 f 0.5 For the lectin-binding studies, the 3H-labeled fractions (50,000 dpm) obtained on anion exchange chromatography, were incubated with PWM, DSA, or WGA, as described previously (13, 14, 25). The percentage of radioactivity remaining bound to the resin after exhaustive elution with PBS is presented. The values represent the average + SE of triplicate determinations per sample. The treatments were 1 X 10m9 M estrogen (E), 1 X 10e7 M progesterone (P), and estrogen plus progesterone (E+P). +P
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Endo. 1992 Vol130.No1
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Fraction
130
knber
FIG. 3. Sephadex G-50 chromatography of 3H-iabeled glycoconjugates in the unbound fraction (peak A) from the anion exchange resin. Material from the unbound fraction from the anion exchange resin was analyzed by Sephadex G-50 chromatography before (0) and after flelimination (O), peptide-N-glycanase digestion (A), or neuraminidase digestion (- - -) The 1.5 X 84-cm column was equilibrated with 0.1 M sodium bicarbonate containing 5% (vol/vol) ethanol and 0.03% (wt/ vol) sodium azide. The elution positions of blue dextran (V,), potassium dichromate (V,), and fetuin glycopeptides 1 (Mr, 3300) and 2 (Mr, 1500) are indicated.
A) were primarily composed of sialic acid-containing Olinked glycoproteins, i.e. mucin glycoproteins. The large Mr of the intact mucin glycoproteins also was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where their mobility into the resolving gel was restricted, with the greater amount retained in the stacking gel (data not shown). The components in this fraction (peak A) from the apical and basal secretions and the cell-associated material behaved in an identical fashion (data not shown). Quantitative differences were observed in the synthesis and secretion of mucinglycoproteins by UE cells in response to the hormones examined. As shown in Table 2, in most cases, mucin secretion was preferentially apical (86%) and was greater in the absence of hormonal treatment. Estrogen alone decreased mucin production; however, the pattern of secretion appeared to be similar to that in nonhormone-treated controls. Progesterone alone caused a marked accumulation of mucin glycoproteins in the cell-associated fraction. Interestingly, combined treatment with estrogen and progesterone not only stimulated mucin accumulation in the cell-associated fraction, but also shifted the vectorial preference for secretion from predominantly apical to nearly equal in either compartment. Characterization of glycoconjugates in the 0.8- to 1.0-M NaCl elude (peak B)
These components (peak B from the anion exchange column) migrated near the fully excluded column volume of Superose-12, indicating that they had hydrodynamic
UE TABLE 2. Sialomucoglycoproteins distribution cells in response to steroid hormones dpm X lo-‘/lo’ Fraction Apical secretion Basal secretion Cell-associated Total
A/B
No treatment 32.7 3.8 6.0 42.5
f + + +
8.6
2 0.1 1 3.1
+E 13.7 2.4 1.2 17.3
+ + + + 5.7
GLYCOCONJUGATES
in polarized rabbit UE cells +E+P
+P
1 9.8 f 1.7 12.6 + 0.5 0.2 16.5 zk 3 3.3 + 0.1 0.05 22.7 & 2 29.0 + 1 1.25 49.0 f 6.7 44.9 + 1.6 0.6
3.8
Polarized cultures of rabbit UE cells grown in the presence and absence of estrogen (E; 1 x 10e9 M) and/or progesterone (P; 1 X lo-’ M) were metabolically labeled overnight with [3H]glucosamine and Hz%O,, as described in Materials and Methods. The secreted and cellassociated glycoconjugates were extracted and processed for anion exchange chromatography, as described in Materials and Methods. The 3H-labeled material eluting in the run-through fraction (peak A) was further analyzed. The data presented are the averages f SE of values obtained from triplicate determinations in representative experiments. The ratio of the average values obtained for the sialomucoglycoprotein apical and basal secretions (A/B) is indicated.
Minutes
FIG. 4. Identification of glycoconjugates in the 0.8- to 1.0 M-NaCl eluate (peak B) from the anion exchange resin. 3H-Labeled material from the 0.8- to l.O-M NaCl eluate from the anion exchange column was analyzed as described in Materiuk and Methods. The elution profiles of intact (0) and hyaluronidase-treated samples (A) are depicted. The Mr markers indicated at the top of the figure are the same as those described in Fig. 2.
radii greater than that of 230-kDa protein standards. When subjected to enzymatic and chemical digestion, these macromolecules were found to be resistant to pronase and peptide-N-glycanase (data not shown). This material also was stable to P-elimination, indicating that that it was not in 0-glycosidic linkage. The components did not bind to PWM, DSA, or WGA and were insensitive to endo-@galactosidase digestion, suggesting the absence of LAGS. However, the radioactive components in this fraction were nearly completely digested by Streptornyces hyaluronidase (Fig. 4). Collectively, these data indicated that radioactive components in peak B were
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245
predominantly composed of hyaluronate. The distribution of hyaluronate among the secreted and cell-associated fractions under different hormonal conditions is presented in Table 3. Hyaluronate synthesized by UE cells was secreted primarily to the apical compartment under all conditions. Estrogen and progesterone, either alone or in combination, stimulated the synthesis and apical secretion of this glycosaminoglycan. Characterization of glycoconjugates NaCl eluate (peak C)
in the 2.5- to
4.0-M
In contrast to the other two peak fractions, the intact components of the high salt gradient eluate were primarily labeled with 35S04, rather than [3H]glucosamine. The intact material migrated on molecular exclusion chromatography with hydrodynamic radii greater than that of a 230-kDa protein (Fig. 5). Mild alkaline hydrolysis generated one major peak of radioactivity, eluting close to the total included volume in the apical and basal secretions (Fig. 5). However, in the cell-associated fraction, P-elimination yielded two additional, quantitatively minor, size classes of sulfated components (Fig. 5). The smaller of these had a median hydrodynamic radius similar to that of a 25-kDa protein standard, while the larger Mr class overlapped with the size of the intact material. Most of the labeled macromolecules in peak C were degraded by nitrous acid, indicating the presence of heparin/heparan sulfate. Pronase digestion also shifted the size distribution of the radioactive peak closer to the included volume (data not shown). Collectively, these data suggest that the constituents in this fraction were comprised predominantly of HSPGs. Most of the HSPGs were secreted basally (Table 4). Neither estrogen nor progesterone had any substantial effect on the synthesis or secretion of this glycoconjugate. TABLE 3. Hyaluronate
distribution
in response to steroid hormones dpm x lo-‘/lo’
Fraction Apical secretion Basal secretion Cell-associated Total
A/B
cells
No +E +E+P +P treatment 8.6 f 1 40.9 f 5 33.7 + 2 44.9 f 1 3.13 + 0.5 1.6 + 0.1 4.95 f 1 2.15 + 0.05 22.6 f 2 16.2 + 1 12.03 _+0.5 7.3 + 1 34.3 + 3.5 58.7 + 6.1 50.6 + 3.5 54.4 + 2.05 2.7
25.6 6.95 20.8 Glycoconjugates from the secreted and cell-associated fractions were processed, as described in Materials and Methods. The material in the 0.8- to l.O-M NaCl eluate (peak B) from the anion exchange resin was further analyzed. The data presented show the average + SE of triplicate determinations in each case. The notations and the concentrations of hormones used are the same as those described in Table 1. The ratio of the average values obtained for the apical and basal secretions (A/ B) is indicated.
UE GLYCOCONJUGATES
246
0.6 0.6
Minutes
5. Identification of glycoconjugates in the 2.5 to 4.0-M NaCl eluate (peak C) from the anion exchange resin. %SO,-Labeled material from the 2.5- to 4.0-M NaCl eluate from the anion exchange resin was analyzed as described in Materials and Methods. Profiles of intact material (0) and products after @-elimination (0) or nitrous acid degradation (Cl) are shown for fractions abtained from apical secretions, basal secretions, and cell-associated material. For simplicity, only the profiles of sulfate-labeled components are shown. The Mr markers indicated at the top of the figure are the same as those described in Fig. 2. FIG.
TABLE
4. HSPG distribution
in response to steroid hormones dpm
Fraction Apical secretion Basal secretion Cell-associated Total
A/B
x
lo-‘/lo’
cells
No +E +E+P +P treatment 0.39 f 0.01 0.52 f 0.1 0.97 f 0.02 0.13 + 0.02 4.41 f 1 4.05 + 0.5 3.07 * 1 6.42 f 1 3.6 f 1 5.72 + 0.5 4.95 f 0.1 3.0 + 0.2 6.8 f 2.0 12.5 f 1.5 9.9 + 1.2 8.0 f 0.7 0.06
0.12
0.23
0.04
Polarized UE cells were metabolically labeled with [3H]glucosamine and H&S360,, as described in Materids and Methods. The sulfatelabeled glycoconjugates in the 2.5- to 4.0~M NaCl eluate of anion exchange chromatography were further analyzed by a series of chemical and enzymatic digestions. The values are the average + SE obtained from triplicate determinations in each case. The notations and the hormone concentrations used are the same as those in Table 1. The ratio of the average values obtained for the apical and basal secretions (A/B) is indicated.
Discussion A growing body of evidence suggests that modification of glycoproteins at the apical plasma membrane of endometrial epithelial cells is involved in the process of blastocyst adhesion in the rabbit (11, 20, 21) as well as other mammals mammals (14-16, 19). In some of these
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studies (ll), particular glycoproteins were identified, but the vectorial patterns of their secretion were not examined in any of the studies cited. The composition of uterine glycoproteins and glycosaminoglycans and their regulation by estrogen and progesterone in the rabbit uterus in uiuo have been studied by several investigators (23, 28); however, these studies did not identify the precise cellular (UE or stroma) source of the macromolecules synthesized. Recent studies suggest that the diverse functions of UE cells are dependent upon the polarized distribution of the proteins and glycoproteins between structurally and functionally distinct apical and basolateral domains (19, 26, 28, 29). Intracellular vesicular trafficking in UE cells also may be modulated by steroid hormones (30,31). In light of these observations, the present study focused on characterizing the major classes of glycoconjugates synthesized and secreted by polarized rabbit UE cells in vitro, their vectorial patterns of secretion, and the regulation of their expression by estrogen and/or progesterone in vitro. Our studies demonstrate that polarized immature rabbit UE cells secrete different glycoconjugates in distinct vectorial patterns. Sialomucoglycoproteins and a nonprotein-linked glycosaminoglycan, hyaluronate, were secreted primarily from the apical cell surface. In contrast, HSPGs were secreted primarily from from the basal cell surface domain of polarized rabbit UE cells. In addition, steroid hormones had interesting differential effects on both the overall production and patterns of secretion of each class of glycoconjugates. As with other mucociliary epithelia (32,33), rabbit UE cells secreted high Mr mucin glycoproteins. Unlike immature mouse and rat UE cell secretions (13, 19), these complex polysaccharides did not contain a significant LAG character, as indicated by both their resistance to endo-@-galactosidase digestion and their low degree of binding to PWM and DSA. The neuraminidase sensitivity of these glycoproteins demonstrated that almost all of the constituent oligosaccharides were sialylated. Thus, sialomucoglycoproteins appear to be a major group of UE cell glycoconjugates in the rabbit. These observations are in agreement with earlier studies demonstrating abundant expression of sialic acid on the uterine luminal surface of the rabbit in uiuo (11,22). Terminal sialic acid residues have been implicated as a major source of luminal negativity in uteri from nonpregnant rabbits (21). It is of interest that the reduction in luminal surface negativity that precedes implantation in the rabbit is associated with a progesterone-regulated decrease in uterine epithelial sialic acid (20-22). Collectively, these observations suggest that mucin glycoproteins are major glycoconjugates expressed at the apical UE cell surface. Decreased apical secretion of mucin glycoproteins by UE cells was observed in the current studies in response
UE GLYCOCONJUGATES to either progesterone or estrogen treatment; however, a 4- to &fold accumulation of sialomucoglycoproteins in the cell-associated fraction was seen only in response to progesterone, not estrogen. Increases in certain other sialoproteins were not observed in the detergent extracts of the uterine luminal surface of progesterone-treated rabbits (11). This variance may reflect different experimental approaches. The major sialomucoglycoproteins identified in the present study were much larger than those studied by Anderson et al. (ll), where the glycoconjugates may have been inefficiently extracted by detergents alone. The apical secretion of sialomucoglycoproteins observed in the absence of hormonal treatment in vitro is also in accordance with earlier in uiuo studies (20-22). Progesterone stimulated UE cell secretion of the sialomucoglycoproteins without changing their overall synthesis. Estrogen alone decreased sialomucoglycoprotein synthesis; however, the pattern of secretion remained similar to that observed for UE cells cultured in the absence of steroid hormones. This agrees with the observations of Anderson and Hoffman (22) in so far as the degree of luminal surface negativity was not influenced by estrogen. From these observations, it is suggested that one aspect of the reduction in the apical glycocalyx observed during the periimplantation period in the rabbit reflects the regulation of sialomucoglycoprotein expression. This regulation may be due to reductions or changes in sialomucoglycoprotein glycosylation, metabolism, or core protein mRNA expression in response to progesterone. Another striking observation in this study was the synthesis and secretion of hyaluronate by the polarized rabbit UE cells. Most (73-96%) of the secreted hyaluronate was released into the apical compartment under all conditions. It seems unlikely that the lack of basal secretion is due to trapping of hyaluronate by the filter, because hyaluronate does not accumulate in the filter (cell-associated material). In uiuo studies on rabbit uteri have indicated stimulation of hyaluronate synthesis by estrogen and antagonism of this effect by progesterone (23). In contrast, we found that estrogen and progesterone, alone or in combination, stimulated the synthesis and apical secretion by polarized rabbit cells in vitro. The in uiuo studies dealt with the entire uterine tissue, which may respond differently to hormones than isolated UE cells, and other uterine cell types may synthesize hyaluronate. The responses of the UE cells alone may be obscured in studies of whole uterine tissue. Furthermore, stromal cells have been shown to influence the behavior of epithelial cells in other cell systems (34). The presence of stromal cells may alter the steroid hormone regulation of hyaluronate expression by UE cells, per se and simulate in uiuo expression. In uiuo and in vitro studies in the mouse have indicated
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247
uterine stromal cells as the primary site of hyaluronate synthesis (35,36). In contrast, neither mouse nor rat UE cells appear to produce significant amounts of hyaluronate in uitro. Increased hyaluronate synthesis has been observed in early pregnancy associated with uterine decidual response in species with invasive implantation (35, 37). Based on the studies in various developmental systems, including migration of neural crest cells (38) and cornea1 development (39,40), it has been suggested that hyaluronate acts to produce an open loose extracellular matrix structure by virtue of its water retention capabilities and facilitates cell migration (41,42). Hyaluronate also has been found to support embryo attachment and outgrowth in vitro (35). These observations suggest that hyaluronate may play a facilitator-y role in the uterine lumen during early stages of implantation. It is possible that hyaluronate elaborated by UE cells facilitates migration to or fusion penetration of syncitial trophoblast through the UE cells, providing a favorable medium for migration and growth, much as the stromal cells do in species with invasive implantation. The major sulfated macromolecules synthesized by the polarized rabbit UE cells are HSPGs, predominantly secreted from the basal aspect of these cells @O-96%). Basally secreted proteoglycans are likely to be deposited in the basal lamina in vitro and interact with the subjacent cells to retain tissue architecture (40). Binding sites for the constituent oligosaccharides of these proteoglycans have been demonstrated on various extracellular matrix molecules, e.g. laminin and fibronectin (43, 44). Synthesis and secretion of HSPGs showed no significant variation in response to steroid hormones in this study; however, previous studies have demonstrated steroid hormone responses of sulfotransferase activity in microsomal fractions of rabbit uterine tissue in uiuo (8,28,45). Again, these studies involved analyses of membrane fractions from the entire uterine tissue. Consequently, the behavior of isolated UE cells may be distinct and not reflect that of the whole uterus. In this regard, it is known that the glycoprotein composition of rabbit uterine myometrium treated with estrogen and/or progesterone differs markedly from that of the whole uterus (46, 47). The in vitro culture system used in the current study will permit the study of uterine stromal cell influences on glycoprotein expression by UE cells in response to steroid hormones as well. Developing coculture systems with both uterine cell types as well as embryos should enable us to determine not only the biochemical aspects of receptive UE and UE-stromal interactions, but also define in biochemical terms the similarities/differences in the adhesion systems that exist in species exhibiting varied types of implantation.
UE
248
GLYCOCONJUGATES
Acknowledgment We acknowledge
David Scarff for his excellent
illustrations.
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