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Biology of Reproduction 62, 277-284 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Expression of Carbohydrate Antigens in the Goat Uterus During Early Pregnancy and on Steroid-Treated Polarized Uterine Epithelial Cells In Vitro1

J.K. Powella, S.R. Glasserb,c, S. Woldesenbeta, R.C. Burghardtc, and G.R. Newton2,a,c

a Cooperative Agricultural Research Center, Prairie View A&M University, Prairie View, Texas 77446 b Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 c Center for Animal Biotechnology and Genomics, Texas A&M University Health Sciences Center, College Station,Texas 77843


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our objectives were to determine whether specific fucosylated carbohydrate antigens, associated with uterine receptivity in rodents, are expressed in pregnant caprine uterine tissues and polarized uterine luminal epithelial (ULE) cells in culture. Immunofluorescence microscopy on frozen endometrium revealed that expression of the H-type 1 antigen, confined to epithelial cells, was regulated during early pregnancy. Staining was high on Day 5 and low on Days 11 and 13. Strong, uniform apical staining was characteristic of ULE cells between Days 15 and 19 but declined markedly by Day 25. Immunofluorescence analysis of the apical surface of polarized ULE cells cultured in steroid-free medium revealed weak and diffuse staining for the H-type 1 antigen, while progesterone (P4) treatment resulted in the formation of aggregates of punctate staining along the apical surface. Domain-specific biotinylation of polarized ULE cells, coupled with streptavidin precipitation and Western blotting, revealed that six apical surface proteins (31, 33, 42, 55, 60, and 70 kDa) carry the H-type 1 antigen. Therefore, H-type 1 antigen expression is up-regulated in vivo during the periimplantation period, stimulated by P4 on polarized ULE cells in culture, and may be a useful marker for uterine receptivity in this species.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In ruminants, maintenance of progesterone secretion by the corpus luteum (CL) is essential if pregnancy is to be established. Secretion of interferon tau (IFN-{tau}) by the conceptus during the critical period of maternal recognition of pregnancy is responsible for maintenance of CL function [1,2]. Intrauterine infusion of conceptus-derived proteins or recombinant IFN-{tau} from Days 14 to 18 of the estrous cycle results in an extension of luteal life span until approximately Day 28 in sheep [3,4], cattle [5,6], and goats [7]. However, a large number of embryos are lost even though luteal function has been maintained [8]. These losses occur during a stage of pregnancy when embryonic and uterine tissues are closely apposed, cellular adhesions are forming, and definitive attachment is achieved through microvillous interdigitations between trophoblast and uterine epithelium, culminating in placentation [9].

Cellular interactions leading to attachment and initiation of placentation are likely mediated by developmentally regulated changes in the apical plasma membranes of uterine luminal epithelial (ULE) and trophoblast cells. Ultrastructural studies in the rodent indicate a thinning of the glycocalyx, around the time of implantation [10,11], that coincides with a decrease in anionic charges on ULE cells [12,13]. Temporal changes in oligosaccharide markers exposed at both embryonic and uterine cell surfaces during the periimplantation period have also been reported [14,15]. Carbohydrate recognition systems appear to mediate cell-cell interactions in periimplantation mouse embryos [16] and adhesion between cells of the uterine epithelium [17] as well as initiate adhesion between the blastocyst and ULE cells [18]. These lectin-carbohydrate interactions are likely components of a highly regulated sequence of events that includes acquisition of stage-specific membrane proteins [19,20] and receptors for matrix/cell surface molecules [21–23], and they may prepare the uterine epithelium for embryo receptivity.

Expression of the H-type 1 carbohydrate antigen by endometrial tissues may represent an important, stage-specific recognition molecule involved in placentation. Immunofluorescent microscopy has demonstrated that acquisition of H-type 1 antigen expression by mouse ULE cells is under control of gonadal steroids, as indicated by intense staining that is present until Day 3 of pregnancy, declines between Days 4 and 5, and is barely detectable after implantation [24,25]. A monoclonal antibody that recognizes lacto-N-fucopentaose-1 (LNF-1) epitopes carried by the H-type 1 antigen blocks attachment of mouse blastocysts to ULE cells in vitro [18]. Similarly, milk-derived LNF-1 inhibits attachment of mouse blastocysts to monolayers of ULE cells in a dose-dependent manner, while related sugars have no effect [18]. Such changes in specific carbohydrate antigen expression by ruminant endometrial tissues during the periimplantation period have not been evaluated. Therefore, the goals of this study were to evaluate endometrial expression of the H-type 1 antigen and related Galß1–3(4)GlcNAc oligosaccharide structures between Days 5 and 25 of pregnancy and to determine whether the H-type 1 antigen is expressed by primary cultures of polarized caprine uterine epithelial cells in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Tissue culture supplies were obtained from Gibco Laboratories (Grand Island, NY) or Sigma Chemical Co. (St. Louis, MO). Pancreatin, glycerol, and p-phenylenediamine were also purchased from Sigma. DNase was acquired from Calbiochem (La Jolla, CA). Acrylamide, N,N'-methylene-bis-acrylamide, collagenase (type III), and dispase were obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Tissue-Tek OCT embedding compound was from Miles Inc. (Elkhart, IN). Matrigel was obtained from Collaborative Research (Bedford, MA), and Millicell HA filters were from Millipore (Bedford, MA). Monoclonal antibodies used to characterize carbohydrate antigen expression by frozen uterine tissues were supplied by Bio-Carb AB (Lund, Sweden). Signet Laboratories (Dedham, MA) supplied monoclonal antibodies used to establish the apical surface location and biochemical characteristics of the H-type 1 antigen on polarized cultures of luminal epithelial cells after verification that tissue staining on Day 15 of pregnancy was similar regardless of antibody sources. Specific clone numbers used in each experiment are indicated below. Biotinylated anti-mouse IgG and streptavidin-fluorescein were purchased from Amersham (now Amersham Pharmaacia Biotech, Piscataway, NJ). T-Max 3200 and X-Omat AR film were purchased from Eastman Kodak (Rochester, NY). All other supplies and reagents were obtained from either Fisher Scientific (Houston, TX) or Sigma and were reagent grade or better.

Tissue Collection

Cyclic female goats (predominantly Alpine or Spanish breeds) were bred upon standing estrus. Reproductive tracts were recovered on Days 5, 11, 13, 15, 17, 19, and 25 of pregnancy (n = 3–5 goats per day) after captive bolt stunning and exsanguination. Reproductive tracts obtained on Days 5–17 were flushed with sterile saline to detect the presence of a blastocyst or conceptus tissue and verify pregnancy. Reproductive tracts were not flushed on Days 19 or 25 of pregnancy because conceptus tissue cannot be removed from the uterus by flushing at this stage of gestation but is visible upon dissection. Cross sections of the ipsilateral uterine horns were snap frozen in OCT and subsequently processed for immunocytochemistry.

Immunocytochemical Analysis of Endometrial Sections

Cryosections (6 µm thick) of uterine tissues were cut with a cryostat microtome (Bright Instrument model OTF, Hacker Instruments, Fairfield, NJ) and mounted on 3-aminopropyltriethoxysilane-coated slides. Samples were then rinsed in 0.02 M PBS (pH 7.3), fixed in -20°C methanol, and washed with PBS containing 0.3% Tween 20 (PBS-Tween). Uterine tissue sections were then incubated at 37°C for 1 h with PBS-Tween, pH 8.0, containing 1% (w/v) BSA, 2.5% (v/v) normal rabbit serum, and 2.5% (v/v) normal goat serum. Samples were washed with PBS-Tween and reacted with monoclonal antibodies to specific carbohydrate structures carried by the Lewis a (318/2B3), Lewis b (64/5B9), Lewis x (630/7H9), H-type 1 (667/9E9), H-type 2 (647/9A2), and blood group A (402/3D9) and B (306/4E4) antigens, for 16 h at 4°C. These carbohydrate structures differ based on the degree of fucosylation and linkage to common backbone structure (Galß1–3(4)GlcNAcß1–3Galß1–4Glc; i.e., LNT; Table 1). All antibodies were titered on frozen tissue and were used at a dilution that resulted in maximum specific fluorescence and minimal background. Negative controls included samples incubated with normal mouse serum (NMS) or monoclonal antibodies to the unfucosylated backbone structure LNT (619/IDZ) and processed as for other samples. After several washes with PBS-Tween, all samples were incubated with a biotinylated secondary antibody for 1 h at room temperature in a humidified chamber. Samples were washed in PBS-Tween and then incubated with streptavidin-fluorescein for 1 h at room temperature in a humidified chamber. Slides were washed in PBS-Tween and overlaid with a coverglass and mounting media containing 1% p-phenylenediamine in glycerol and PBS. Staining intensities of ULE cells, uterine glandular epithelial (UGE) cells, and the stromal compartment were scored visually (absent, weak, moderate, or strong) by two independent observers. Slides were viewed with a Zeiss (Carl Zeiss, Thornwood, NY) Photomicroscope III and photographed using T-Max 3200 film.


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  		TABLE 1. Summary of expression of Galß1-3(4)GIcNAc-related oligosaccharide antigens in goat luminal (LE) and glandular (GE) epithelial cells and uterine stromal (S) cells during early pregnancy.a

Immunocytochemical Analysis of the H-Type 1 Antigen on Primary Cultures of Polarized ULE Cells

Reproductive tracts were collected from an additional four cyclic female goats on Day 5 of the estrous cycle. Endometrial epithelial cells were enzymatically isolated as previously described [26]. Briefly, the uterine horn received enough sterile Ca2+/Mg2+-free Hanks' Balanced Salt Solution (HBSS-) containing pancreatin (2.5 mg/ml) and dispase (4.8 mg/ml) to cause mild distension (approximately 20 ml) and was incubated at 4°C for 1 h, warmed to 20°C for 20 min, and then warmed to 37°C for 10 min. Enzyme wash was then gently stripped from the uterine horn and discarded. The horn was refilled with HBSS- and incubated in room-temperature HBSS- for 15 min with occasional gentle massage to loosen luminal epithelial sheets. The wash was then gently stripped from the uterine horn. Washes continued until reduced yield of luminal epithelial sheets or contamination with uterine stromal cells was apparent (i.e., cloudy appearance of the wash). These washes were pooled and poured into a sterile filter apparatus fitted with a 20-µm screen to collect and wash the epithelial cells. Epithelial sheets were then subjected to mild vortexing with a reduced-bore pipette. The resulting ULE cell population was centrifuged (500 x g for 10 min) and resuspended in culture medium (phenol red-free Dulbecco's modified Eagle's medium with F12 salts, pH 7.4, containing 5% [v/v] fetal bovine serum [FBS] and 1% [v/v] penicillin/streptomycin solution; DMEM:F12) and expanded in 75-cm2 culture flasks. When flasks of ULE cells were 80% confluent (approximately 5 days), cells were removed from culture flasks with trypsin and placed into Matrigel (Collaborative Biochemicals, Bedford, MA)-coated Millicell HA filters. Isolated ULE cells readily form polarized monolayers on Matrigel-coated filter inserts and display many characteristics of epithelium in utero. These include transepithelial resistance, tight junctions, apical microvilli, and domain-specific trafficking of proteins and prostaglandins. In addition, polarized ULE cells respond to IFN-{tau} and oxytocin, two major regulators of prostaglandin secretion in utero [26]. Polarized caprine (c) ULE cells (i.e., transmembrane resistance of 600 Ohm cm2 or greater) were then cultured in DMEM:F12 containing 5% (v/v) charcoal-stripped (s) FBS for 24 h. Medium was replaced daily and was supplemented with either estradiol-17ß (E2; 10-9 M), progesterone (P4; 10-7 M), E2+P4, or no additional steroids for an additional 5 days (n = 8 filters per treatment using ULE cells from four goats). Polarized cULE cells on filters were then fixed for 3 h with 4% paraformaldehyde, rinsed with HBSS (pH 7.2), and incubated at 37°C for 1 h with PBS-Tween, pH 8.0, containing 1% (w/v) BSA, 2.5% (v/v) normal rabbit serum, and 2.5% (v/v) normal goat serum. Cells were washed with PBS-Tween and reacted with either NMS (negative control) or monoclonal antibodies to the H-type 1 antigen (17–206) for 16 h at 4°C. After several washes with PBS-Tween, all cells were incubated with a biotinylated-secondary antibody for 1 h at room temperature in a humidified chamber. Cells were washed in PBS-Tween and then incubated with streptavidin-fluorescein for 1 h at room temperature in a humidified chamber. Cells were washed with PBS-Tween before cells on Millicell HA filters were excised from the plastic holders, mounted on glass slides, and overlaid with a coverglass and mounting media containing 1% p-phenylenediamine in glycerol and PBS. Fluorescence image capture was performed with a digital fluorescence imaging system consisting of a Zeiss Axiovert 135TV microscope attached to a CCD camera and image-capturing software (CELLscan; Scanalytics, Bedford, MA). Image processing of optical sections was performed with the CELLscan fluorescence deconvolution software algorithms to reassign out-of-focus fluorescence to the point of origin in the specimen image.

Biotinylation of Apical Membrane Proteins and Western Blotting [27]

Additional primary cultures of P4-treated polarized cULE cells (n = 4 filters using cells from two animals) were used to determine the biochemical characteristics of the proteins carrying the H-type 1 carbohydrate antigen. Cell monolayers were rinsed with HBSS+Ca2+ and Mg2+ (HBSS+CM; 4 times over 30 min) to remove serum proteins and then reacted with sulfo-NHS-biotin (0.6 mg/ml in HBSS+CM) for two 30-min intervals at 4°C. Free sulfo-NHS-biotin was quenched by washing cells with HBSS+CM + 0.1% BSA, and cells were scraped into ice-cold lysis buffer (25 mM Tris-HCl, pH 7.5, containing 250 mM NaCl, 2.5 mM magnesium acetate, 5% Nonidet P-40, 5 mM EDTA, 50 µg/ml pepstatin, 50 µg/ml chymostatin, 10 µg/ml antipain, and 0.1 mM PMSF) and centrifuged in a microfuge at 4°C for 2 min. Concentration of DNA in the supernatant was determined [28], and biotinylated proteins were precipitated using streptavidin-coupled beads. The precipitate was boiled in buffer containing 5% (w/v) SDS and 5% (v/v) 2-mercaptoethanol, separated by SDS-PAGE as previously described [26], and transferred electrophoretically to nitrocellulose. Blots were incubated sequentially with blocking solution, anti-H-type 1 antigen (17–206) or NMS, washing solution, and rabbit anti-mouse gamma globulin coupled to horseradish peroxidase. Blots were washed extensively before immunoreactive bands were visualized using chemiluminescence. Blocking solution was 10 mM Tris-HCl, pH 7.4, containing 1% (w/v) BSA, 0.9% (w/v) NaCl, and 0.05% (v/v) Tween 20. Washing buffer and diluent for antisera and NMS was 20 mM Tris-HCl, pH 7.6, containing 137 mM (w/v) NaCl and 0.05% (v/v) Tween 20. Autoradiographs were prepared using Kodak XAR x-ray film with enhancing screens.

Statistical Analysis

Tissue immunofluorescent staining scores were converted to numerical ratings of 0 (absent), 1 (weak), 2 (moderate), or 3 (strong) and subjected to ANOVA. The final model evaluated the effects of antigen, day of pregnancy, cell type, and appropriate interactions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endometrial expression of Galß1–3(4)GlcNAc-related oligosaccharides (Table 1) varied according to the carbohydrate antigen investigated (P < 0.01), cell type (antigen x cell; P < 0.01), and the day of pregnancy examined (P < 0.01). No staining of ULE cells, uterine glandular epithelial (UGE) cells, or uterine stromal cells was observed in the absence of primary antibody (Fig. 1A). Negligible or very light sporadic staining of ULE cells was detected when monoclonal antibodies to LNT (Fig. 1B) were used. Expression of Lewis x antigen (Galß1–4[Fuc{alpha}1–3]GlcNAcß1–3Gal) was not detected on UGE cells or in the stromal compartment on any of the days tested. ULE cells from some animals displayed aggregates of light to moderate Lewis x antigen staining only on Day 5 or Day 15 (Fig. 1C) of pregnancy. Lewis a antigen (Galß1–3[Fuc{alpha}1–3]GlcNAcß1–3Gal) weakly stained ULE and UGE cells on most days tested. However, moderate uniform staining of cells in the stromal compartment was detected on all days examined, except Day 25 of pregnancy (Fig. 1D).



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  		FIG. 1. Representative photomicrographs of caprine endometrial tissues reacted with blood group-related carbohydrate antigens. No staining was present in the absence of primary antibodies (A). Minimal immunofluorescence was detected when uterine (U) luminal (LE) or glandular (GE) epithelial cells were reacted with unfucosylated LNT (B). Lewis x antigen (C) staining pattern typical of Day 15 of pregnancy. Lewis a antigen (D; Day 19 pregnant endometrial tissue) predominantly stained uterine stromal cells throughout early pregnancy. x150 (published at 57%)

Some of the blood group antigens tested were constitutively expressed during early pregnancy, although spatial differences in staining patterns were evident. Antibodies to the blood group A antigen (GalNAc{alpha}1–3[Fuc{alpha}1–2]Galß1–3/4GlcNAcß1–3Gal) reacted strongly with the apical and basolateral surface of ULE and UGE cells (Fig. 2A–D) until approximately Day 25 of pregnancy. Weak subepithelial staining of the stromal compartment was also evident on Days 5, 15, 17, and 19 of pregnancy. The spatial pattern of endometrial reactivity to anti-blood group B (GalNAc{alpha}1–3[Fuc{alpha}1–2]Galß1–3/4GlcNAc) antibodies was similar to that observed for blood group A but slightly less intense on ULE cells between Days 15 and 19 of pregnancy. Blood group B staining of the apical surface of ULE cells was uniform and strong from Day 5 to 13 and moderate between Days 15 and Day 25 of pregnancy (not shown). Reactivity of the apical surface of ULE and UGE cells to Lewis b antigen (Fuc{alpha}1–2Galß1–3[Fuc{alpha}1–4]GlcNAcß1–3Gal) was strong on Day 5 of pregnancy (Fig. 3A) and variable between Days 11 and 19 of pregnancy. Moderate punctate staining of Lewis b antigen was detected on ULE cells between Days 11 and 15 of pregnancy with reduced staining observed on Days 19 and 25 of pregnancy (Fig. 3B–E). Light to moderate staining of the trophectoderm was noted on Day 25 of pregnancy (Fig. 3F).



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  		FIG. 2. Endometrial staining patterns for blood group A carbohydrate antigen. Uniform and strong staining patterns were characteristic of ULE and UGE cells between Days 5 and 19 of pregnancy. Representative immunofluorescent staining of ULE cells on Days 13 (A) and 19 (B) of pregnancy and UGE cells on Days 13 (C) and 17 (D) pregnancy. x150 (published at 57%)



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  		FIG. 3. Endometrial reactivity toward Lewis b antigen was high on Day 5 of pregnancy (A), but then declined with advancing stage of pregnancy. ULE (A,B,C,E) and UGE (D) cell and conceptus (Con; F) staining patterns on Days 15 (B), 17 (C), 19 (D), and 25 (E,F) of pregnancy are shown. x150 (published at 57%)

There was also evidence of regulation of carbohydrate antigen expression on the apical surface of ULE and UGE cells. The H-type 1 antigen was temporally expressed during the critical period for pregnancy recognition (Fig. 4 and Fig. 5A,B). Intense staining of ULE cells was observed on Day 5 of pregnancy (Fig. 4A). Staining was reduced (Fig. 4B) until Day 15 of pregnancy (Fig. 4C) when uniform strong apical staining of ULE cells was apparent. Between Days 17 and 19 of pregnancy, the strong apical staining pattern was slightly reduced and less uniform, with intermittent unstained areas clearly present (Fig. 4D,E). Light basolateral staining was also detected between Days 5 and 19 of pregnancy. Only light apical staining of clusters of ULE cells was detected on Day 25 of pregnancy. Light to moderate H-type 1 antigen staining of the apical surface of UGE cells was also observed between Days 11 and 19 of pregnancy (Fig. 5A,B). The temporal and spatial pattern of H-type 2 antigen expression was similar to the H-type 1 antigen staining pattern, but slightly less intense (Fig. 5C–F).



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  		FIG. 4. Temporal expression of H-type 1 antigen during early pregnancy. Staining of ULE cells was high on Day 5 of pregnancy (A) and low on Days 11 (B) and 13 of pregnancy. Uniform apical immunofluorescence, typical of ULE cells on Day 15 of pregnancy (C), also included areas of aggregate staining on Days 17 (D) and 19 (E) of pregnancy. Only light staining of ULE cells was detected on Day 25 of pregnancy (F). x150 (published at 57%)



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  		FIG. 5. UGE cell staining of H-type 1 antigen was low on Day 11 (A) and moderate on Day 19 (B) of pregnancy. Moderate reactivity toward H-type 2 antigen was detected only on Day 15 of pregnancy (E). Expression was low on all other days examined. C,D,F) Representative H-type 2 antigen staining patterns of Days 5, 11, and 17 of pregnancy. x150 (published at 57%)

Primary cultures of polarized cULE cells were used to verify the apical surface location of the H-type 1 antigen and exclude the possibility that endometrial staining patterns were the result of absorption from serum transudate. The apical surface of polarized cULE cells, cultured in steroid-free media, stained weakly and diffusely for H-type 1 antigen (Fig. 6A). Cells cultured in E2 produced only a slight increase in H-type 1 antigen expression (not shown). However, P4 treatment caused an increase and/or redistribution of H-type 1 antigen expression by polarized cULE cells and resulted in the formation of aggregates of punctate staining at the apical surface (Fig. 6B). Biotinylation of the apical surface of P4-treated polarized cULE cells, coupled with streptavidin precipitation and Western blotting, revealed six apical membrane proteins (31, 33, 42, 55, 60, and 70 kDa) carrying the H-type 1 carbohydrate antigen (Fig. 7).



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  		FIG. 6. Confocal micrographs depicting immunofluorescent staining found on the apical surface of polarized ULE cells reacted with monoclonal antibodies to the H-type 1 antigen. P4 treatment (B) altered the pattern of H-type 1 antigen expression and resulted in the formation of punctate aggregates of staining. Control cultures (A) were maintained in steroid-free media



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  		FIG. 7. Western blot of biotinylated apical membrane proteins from polarized ULE cells. Six glycoproteins carrying the H-type 1 antigen were detected. The broad band centered at 65 kDa could be resolved, with shorter exposure times, into two bands with apparent molecular sizes of 70 and 60 kDa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The critical period for pregnancy recognition in goats occurs between Days 15 and 19 of pregnancy [7,29]. Ultrastructural studies indicate that growth and elongation of the goat conceptus brings the trophoderm into close apposition with the apical surface of ULE cells between Days 14 and 18 of pregnancy. Hormonally regulated adhesion develops between Day 19 and 23 after mating, resulting in the development of definitive attachment, which is characterized by microvillous interdigitation of the apical surfaces of trophectoderm and ULE cells and migration and fusion of trophoblast binucleate cells with caruncular ULE cells [30–32]. These interactions are likely facilitated by an up-regulation of cell adhesion molecules at the luminal epithelial surface of the endometrium [33].

In this study, temporal changes in endometrial expression patterns of specific fucosylated carbohydrate antigens indicate that active remodeling of the apical membrane glycocalyx of cULE cells occurs during the first 25 days of pregnancy. Similar changes in terminal fucosylated carbohydrate epitopes carried on related N-linked blood group glycans have been reported during the periimplantation period in mice [24,25] and rats [34] and in normal cycling human endometrium [35,36]. The reappearance and strong apical expression of the H-type 1 antigen on luminal epithelium between Days 15 and 19 of pregnancy are noteworthy and may represent a useful marker for uterine receptivity in the goat. Attachment of mouse blastocysts to monolayers of epithelial cells is inhibited by monoclonal antibodies that recognize the H-type 1 antigen and free oligosaccharides containing LNF-1 [18]. This suggests that endometrial H-type 1 antigen associates with a cognate receptor found on the abembryonic mural trophectoderm [37,38] and initiates blastocyst-epithelial attachment in mice. Therefore, alterations in the expression pattern of the H-type 1 antigen in the goat may represent hormonally regulated events that are critical to cell recognition and adhesion during the earliest stages of trophectoderm-endometrial interactions.

Apical membrane-associated proteins, ranging in molecular size from 32 to 70 kDa, cross-reacted with antibodies to the H-type 1 antigen. These proteins are smaller than the 120–130-kDa murine endometrial epithelial glycoprotein(s) carrying the H-type 1 antigen [39]. Multiple proteins destined for the apical plasma membrane of ULE and UGE cells may contain glycosylation sites that are modified to carry the H-type 1 antigen. The enzyme that catalyzes the final step in the formation of the H-type 1 antigen in humans is {alpha}(1–2)-fucosyltransferase (FUT1; [40]). E2-stimulated maximal expression of FUT1 mRNA, enzyme activity, and H-type 1 antigen occurs in the mouse uterus on Day 1 of pregnancy, declines until Day 5, and is barely detectable after implantation [41,42]. E2 also regulates H-type 1 antigen expression in the pig [33]. Expression is greatest in cycling gilts from Days 0 to 4 and weak on Days 8–15. In ovariectomized gilts, H-type 1 antigen expression by ULE cells is increased after E2 treatment but not in P4-, E2+P4-, or vehicle-treated animals. Immunostaining of rat endometrium indicated that H-type antigens are expressed under the influence of P4 [34], which is consistent with the staining patterns observed in the goat. Factors controlling the spatial and temporal expression of H-type 1 antigen in the goat uterus are currently not known. However, the cDNA sequence for the mouse FUT1 gene contains two potential progesterone receptor-binding sites upstream of the coding region [42]. Therefore, P4 regulation of FUT1 gene activity may be responsible for H-type 1 antigen expression during pregnancy recognition and promote uterine receptivity in the goat. Alternatively, H-type 1 antigen expression may be masked between Days 5 and 15 of pregnancy by the abundant apical glycocalyx that is present during the prereceptive phase in most mammals [43]. The mucin, Muc1, is present on the apical surface of ULE cells of prereceptive uteri in various species [23,44–46], but expression is reduced during the receptive phase in the rodent [47,48] and pig [23] uterus. The relationship between Muc1 expression in the goat uterus and the staining patterns of the carbohydrate antigens examined in this study remains undefined.

Primary cultures of polarized cULE cells expressed H-type 1 antigen in vitro. P4 altered the distribution pattern of the H-type 1 antigen and resulted in large aggregates of staining on the apical surface of ULE cells, a phenomenon also observed in endometrial tissues obtained between Days 17 and 25 of pregnancy. Immunostaining of rat endometrium indicates that the H-type structures are also expressed under the influence of P4, with maximal expression seen on rat luminal epithelium just before the time of implantation [34]. Binding experiments using oligosaccharides on lipid or protein carriers indicate that the mode of presentation is a crucial factor determining whether functional carbohydrate-lectin interactions occur [49]. This is attributed to the low affinities many endogenous mammalian lectins have for their carbohydrate ligands. To obtain high avidity binding and trigger a response in one or both cell types, receptor and carbohydrate ligand need to be in a clustered state [49,50]. Recent data support the idea that selectins and glycoprotein ligands of the selectins may participate in activation of integrins [51,52]. Uterine epithelial integrins have been identified for a variety of species [23,53–55]. Specific integrins are modulated during the reproductive cycle and early pregnancy [21,23] and may play a role in the implantation process [33]. It is not known to what extent the H-type 1 antigen, or other blood group antigens, interact with uterine epithelial integrins. However, aggregation of the H-type 1 antigen within the apical plasma membrane of UE cells may represent an important characteristic that is required for interaction with trophectoderm and/or uterine epithelial integrins.

Other blood group carbohydrate antigens have been implicated in trophectoderm-uterine epithelial cell interactions. Lewis x antigen has been localized by immunostaining on ULE cells of pregnant pigs from Days 14 to 30 of pregnancy [56]. Lewis x antigen is a ligand for E-selectin and has also been shown to play a role in homotypic adhesion (Lewisx-Lewisx) in mouse compaction and F9 embryonal carcinoma cell adhesion [57]. Lewis x staining in goat endometrium was minimal during most days examined. We did not evaluate Lewis y reactivity in goat endometrium. However, monoclonal antibodies directed to the Lewis y carbohydrate antigen recognize numerous endometrial glycoproteins and inhibit implantation in the mouse [58]. Interestingly, specific interactions between Lewis y and H-type I and II antigens indicating carbohydrate-carbohydrate interactions may also contribute to membrane apposition during implantation [58].

In several biological systems, cell adhesion occurs through a highly regulated set of sequential interactions involving lectin-carbohydrate interactions, triggering molecules, and/or integrins. The periimplantation period of the goat is characterized by specific patterns of endometrial protein expression [59] and glycosylation. The H-type 1 antigen may mediate blastocyst attachment to uterine epithelium in rodents [18,39] and is temporally expressed on the apical surface of uterine epithelial cells during the critical period for pregnancy recognition. This suggests that the H-type 1 antigen, either directly or through cooperative interactions with other molecules, may facilitate firm adhesion during the earliest stages of trophectoderm-endometrial interaction during early pregnancy in the goat.


   FOOTNOTES
 
First decision: 16 August 1999.

1 Supported by NIH 2S06GM08094 and USDA 97-38814-4157 to G.R.N. Back

2 Correspondence: G.R. Newton, Prairie View A&M University, P.O. Box 4079, Prairie View, TX 77446-4079. FAX: 409 857 2325; gary_newton{at}pvamu.edu Back

Accepted: September 22, 1999.

Received: July 2, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bazer FW. Mediators of maternal recognition of pregnancy in mammals. Proc Soc Exp Biol Med 1992; 199:373–384.[Medline]
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