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Variation in the Amount of T Antigen and N-Acetyllactosamine Oligosaccharides in Human Cervical Mucus Secretions with the Menstrual Cycle

Variation in the Amount of T Antigen and N-Acetyllactosamine Oligosaccharides in Human Cervical Mucus Secretions with the Menstrual Cycle

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 Variation in the Amount of T Antigen and
-Acetyllactosamine Oligosaccharides in HumanCervical Mucus Secretions with the Menstrual Cycle
 Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114
It has been hypothesized that the carbohydrate portion of mucins present in the endocervical canal plays an importantrole in conferring specific physicochemical properties (
viscosity and hydration) to the mucus gel through the men-strual cycle. Our recent finding showing an increase in theamount of MUC5B mucin protein at midcycle has raised thequestionofwhetherthemucin
-glycancontentalsovariestoconfer specific hydrodynamic properties to secreted mucinsduring ovulation. Using lectins as carbohydrate probes, wehave identified two common mucin oligosaccharide struc-tures, T antigen and
acetyllactosamine, within secretorygranules in human endocervical glands during the prolifer-ative phase of the menstrual cycle. Analysis of endocervicalsecretions by enzyme-linked lectin assay revealed that theamountsofTantigenand
acetyllactosaminearemaximalatmidcycle. Lectin blot assay of immunoprecipitated MUC5Bdemonstrated that the mucin is a carrier of the T antigen and
acetyllactosamine oligosaccharides in cervical mucus se-cretions. The amounts of T antigen and
acetyllactosamineoligosaccharides on MUC5B increased during the first half of the cycle, peaked at midcycle, and dramatically dropped atthe end of the cycle. The peak in MUC5B mucin protein andcarbohydrate content coincides with the change in mucuscharacter that occurs at midcycle. The role of 
-glycans onmucins may be to hold water within the endocervical canalduring ovulation to facilitate sperm migration. (
 J Clin Endo-crinol Metab
87: 5641–5648, 2002)
UMANCERVICALMUCUSplaysanimportantroleinthe protection of the endocervical epithelium against bacterial invasion, fluid loss, and, most importantly, in theregulationofspermtransporttotheupperreproductivetract(1).Changesinthephysicochemicalpropertiesofthecervicalmucus during the different phases of the menstrual cycle areknown to correlate directly with receptivity to sperm, thuscontributing to reproductive success (2, 3).The cervical mucus is a viscoelastic gel composed primar-ily of water, ions, various secreted substances, and cells. Themajor structural components (
85% of the total macromol-ecules) of the mucus gel in the cervix are a group of extraor-dinarily long and heavily
-glycosylated proteins, termedmucins (4). To date, 16 mucin genes have been described inhumans(5–7).Theendocervicalepitheliumexpressesatleast5differentmucingenes,thesecretedMUC5AC,MUC5B,andMUC6 mucins and the membrane-spanning MUC1 andMUC4 mucins (8, 9). Of these, the most predominant mucintranscripts are MUC5B and MUC4 mucins (10).The specific rheological and hydrodynamic properties of mucins can be ascribed to their extensive
-linked glycosyl-ation (11, 12). Most of the carbohydrate chains on cervicalmucins, which represent up to 65% of the mass by weight,occur as a microheterogeneous population of neutral, sialy-lated,andsulfatedoligosaccharidesvaryingfromtwotoninesugar residues in length (13, 14). Compositional analysis of the
-linked carbohydrate chains in preparations of purifiedhuman cervical secretions collected at midcycle has revealedhigh levels of galactose,
acetylgalactosamine (GalNAc),
acetylglucosamine, fucose, and neuraminic acid (15).Structural studies of cervical mucins as well as in other ep-ithelial mucins have demonstrated the common presence of the tetrasaccharide core structure Gal
1 linked to serine or threonine, suggest-ing that this is an integral structural unit of human epithelialmucins (14, 16). The Thomsen-Friedenreich antigen or T an-tigen (Gal
1Ser/Thr) constitutes the inner coreof this structure and is recognized by the
Arachis hypogaea
orpeanut agglutinin lectin (PNA).
-Acetyllactosamine andpoly-
-acetyllactosamine [recognized by the
Erythrina cris-tagalli
lectin (ECA)] result from the sequential addition of Gal
1–3 residues to the terminal Gal
1–6 motif of the tetrasaccharide (Fig. 1), althoughthey may also be present in other
-linked structures,
-glycans or glycolipids (17, 18). PNA and ECA have been pre-viously used to detect carbohydrate ligands on mucins (19).Varying levels of estrogen and progesterone are respon-sible for the cyclic changes in the amount of mucus and itsproperties in the endocervix. We have recently reported asharp increase in MUC5B protein in cervical secretions atmidcycle in normal cycling subjects (20). The amount of bothMUC5B mRNA and protein abruptly falls as serum proges-teronelevelsriseduringthesecretoryphaseofthemenstrualcycle, suggesting an inverse correlation between MUC5Bmucin expression in the cervical epithelium and progester-one. Although an increase in MUC5B mucin protein secre-tion by the endocervical epithelial cells at midcycle mayindicate that there is a concomitant increase in the amount of mucin
-linked oligosaccharides, early attempts to quantifythe levels of carbohydrates in endocervical secretions during
Erythrina cristagalli
lectin;ELLA,enzyme-linkedlectin assay; FITC, fluorescein isothiocyanate; GalNAc,
-acetylgalac-tosamine; PNA, peanut agglutinin lectin.
0013-7227/02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(12):56415648
 Printed in U.S.A.
Copyright © 2002 by The Endocrine Societydoi: 10.1210/jc.2002-020766
theovulatorycycleyieldedconflictingresults.Evidencebothfor (21, 22) and against (23, 24) variation in the carbohydratecomposition of cervical mucin through the menstrual cyclehas been presented. In this study using a highly sensitiveenzyme-linked lectin assay (ELLA), we evaluated the con-tents of T antigen and
-acetyllactosamine, two carbohy-drate structures previously demonstrated in endocervicalmucus secretions (14, 16), throughout the menstrual cycle of healthy women. Additionally, using lectin blot assay wecompared the amount of lectin binding to equivalentamounts of MUC5B purified by immunoprecipitation fromthesamemucussamplestakenduringthecycle.Therelativeamounts of T antigen and
-acetyllactosamine were corre-latedwithbloodlevelsofprogesteroneandLHsurgedataforeach subject.
Materials and Methods
Tissues and secretions
Human endocervical tissue and cervical secretions were collected inaccordance with human study guidelines and approval from the Schep-ens Eye Research Institute and Brigham and Women
s Hospital insti-tutionalreviewboards.Informedconsentwasobtainedfromallsubjectsin accordance with the approved protocols. Endocervical tissue wasobtained from four females at the time of hysterectomy and frozenwithin 30 min of surgery for cryostat sectioning as described previously(20). The menstrual cycle phase for each woman (two proliferative andtwo secretory) was estimated on the basis of histological appearance of the endometrium obtained from that patient as described by Noyes
(25). Samples of cervical secretions were obtained from normal cy-cling females who were not using intrauterine devices or oral contra-ceptives, had normal cervical cytology, and were free from infection.These subjects were asked to self-report cycle day and to determine theLH surge with a commercially available urinary LH detection kit (ClearPlan Easy, Unipath Ltd., Bedford, UK). Initially, six subjects were en-rolled in the study, although only three completed the entire protocol.Samples were collected from this population at four time points withinthe menstrual cycle as described previously (20). Collection points werechosen to be on d 4 and 7 after the start of menses and on d 1 and 7 afterthe LH surge at midcycle. When possible, duplicate samples for eachtime point were taken, but only two collections were made in any cycle,so collection proceeded over four cycles. Cervical mucus was obtained byswabbingthecervixwithWilshirefoamswabs(VWRScientificProd-ucts, Bridgeport, NJ), and then the mucus was extracted from the swabas previously described (20). The protein concentration of each samplewas determined using the bicinchoninic acid protein assay (PierceChemical Co., Rockford, IL). Levels of MUC5B protein in the mucussecretions of these three patients throughout the menstrual cycle werepreviously determined (20). Blood levels of progesterone for each col-lection point were determined by the Reproductive Endocrine SciencesCenterAssayCoreLaboratory,MassachusettsGeneralHospital(Boston,MA) as previously described (10).
 Lectin histochemistry
Cellularlocalizationoflectinbindingtosectionsofhumanendocervixwasperformedfollowingmethodspreviouslyreported(26).Fluoresceinisothiocyanate (FITC)-conjugated lectins purified from
Arachis hypogaea
(PNA) and
Erythrina cristagalli
(ECA) were purchased from Vector Lab-oratories,Inc.(Burlingame,CA).PNAisspecificfor
-galactoseresiduesat the nonreducing terminal position of glycoconjugates and stronglyreacts with the T antigen (19). ECA shows strong specificity for
-acetyllactosamine and to a lesser extent for galactose and
-acetylgalac-tosamine (19). Cryostat sections (6
m thick) were placed on gelatin-coatedglassslides,air-driedatroomtemperaturefor1h,andrehydratedinPBS(pH7.2).NonspecificbackgroundstainingwasblockedwithPBScontaining 1% BSA (Sigma, St. Louis, MO). Slides subjected to PNAstaining were previously digested with 5 mU neuraminidase from
Clos-tridium perfringens
(Calbiochem, San Diego, CA) for 60 min at 37 C toenhance epitope disclosure to the lectin as previously observed (27).FITC-conjugated lectins diluted to 10
g/ml in PBS were applied tosections for 20 min at room temperature. After washing with PBS andcoverslipping with Vectashield mounting medium with propidium io-dide (Vector Laboratories, Inc.), sections were viewed on a Photomi-croscope III (Carl Zeiss, New York, NY). As a lectin specificity control,lectins were preincubated with
)-galactose at a concentration of 0.2
for 20 min before application to sections. Immunohistochemical lo-calization of MUC5B was determined as previously described (20).
ELLA was used to determine the relative amounts of T antigen and
-acetyllactosamine in crude preparations of human cervical mucusthroughout the menstrual cycle. Samples tested for PNA binding werepreviously digested with 10 mU neuraminidase from
C. perfringens
for60minat37C.Preliminaryserialdilutionexperimentsusingdecreasingamounts of crude cervical mucus collected at midcycle were carried outto determine the range of linear response of the lectins in the assay.Cervical secretions (7.5
g when testing PNA or 0.4
g when testingECA) corresponding to the proliferative, ovulatory, and secretoryphases of normal cycling females were coated in triplicate onto micro-titer plates (Costar, Cambridge, MA) and kept overnight at 4 C in 0.05
carbonate/bicarbonate buffer (pH 9.6). The plates were washed fourtimes with PBS and blocked for 2 h with PBS containing 1% BSA. Afterthree washes with PBS, the plates were incubated with peroxidase-labeled PNA or ECA (Sigma) diluted to 0.5
g/ml (PNA) or 4.0
g/ml(ECA)inPBSfor60minat37C.Afterwashing,theplateswereincubatedwith the peroxidase substrate, tetramethylbenzidine (Sigma), at roomtemperature. The reaction was stopped after 30 min by the addition of 0.5
. The OD of each well was read at 450 nm in a SpectraMaxmicroplate spectrophotometer system using SoftMax Pro version 2.1
. 1. Diagram depicting the synthesisof the T antigen and
-acetyllactosaminein mucin
-linked oligosaccharidesthrough the core 2 intermediate.
J Clin Endocrinol Metab, December 2002, 87(12):5641
5648 Argu
et al.
Oligosaccharides in Cervical Mucus during Menstrual Cycle
software(MolecularDevices,Sunnyvale,CA).Allsamplesfromasingleindividual were run in the same ELLA plate. Secretions were tested andwere found to be negative for endogenous peroxidase activity by in-cubation with peroxidase substrate.
 Electrophoresis and Western blot
Glycoproteins in human cervical secretions were separated by SDS-PAGE and blotted onto nitrocellulose membranes as described previ-ously (26). Briefly, SDS-PAGE was performed using the method of Laemmli (28). Electrophoresis was carried out under reducing condi-tions on 6% separating and 4% stacking polyacrylamide gels. Proteinsin gels were transferred to nitrocellulose membranes (Millipore Corp.,Bedford, MA) according to the method of Towbin
et al
. (29). After blotting, membranes were incubated with blocking solution containing10% normal horse serum in Tris-buffered saline (pH 7.5) for 30 min andwith peroxidase-labeled PNA or ECA (20
g/ml) for 60 min at roomtemperature. Membranes were then washed well with Tris-bufferedsaline, followed by colorimetric detection of positive binding with dia-minobenzidine peroxidase substrate (Bio-Rad Laboratories, Inc., Rich-mond, CA). MUC5B protein on nitrocellulose membranes was detectedas described previously (20). Prestained molecular mass markers in-cluded myosin (207 kDa),
-galactosidase (120 kDa), and BSA (78 kDa).
 MUC5B mucin immunoprecipitation
Immunoprecipitation of MUC5B mucin from individual samplestakenthroughoutthemenstrualcyclewascarriedoutusingapolyclonalantibodyraisedagainstasyntheticpeptidefromthededucedaminoacidsequence of a unique region of the D4 domain (nontandem repeatregion) of MUC5B (20). The specificity of this antibody toward MUC5Bmucinhasbeenpreviouslydemonstratedbypreadsorptionexperimentson Western blot and ELISA as well as immunohistochemistry on tissuesexpressing other membrane-bound and gel-forming mucins (20). TocompareamountsofTantigenand
-acetyllactosaminepermucinmol-ecule throughout the cycle, identical units of MUC5B protein wereimmunoprecipitated at each time point analyzed in this study. AnMUC5B unit was defined as the picogram amount of purified cervicalmucinstandardthatcorrespondstotheODreadingobtainedpersamplein a quantitative MUC5B ELISA (20).The anti-MUC5B antibody was covalently attached to amine-termi-natedmagneticparticles(Sigma)usingtheglutaraldehydeprocedureasdescribed in the manufacturer
s protocol. Conjugated beads were trans-ferred to a sterile tube and washed five times with a buffer containing10m
EDTA,0.1%TritonX-100,and0.1%SDS(pH7.4).Samples to be assayed for PNA binding were digested with 250 mUneuraminidase from
C. perfringens
for 60 min at 37 C before incubationwithbeads.MUC5Bproteinunits(5.7
)ofcervicalmucusinmucinisolation buffer (0.1
, 2.0 m
)were added to the conjugated beads and incubated for 2 h on a rockeratroomtemperature.Afterthreewashes,thebeadswereboiledfor2minwith30
Laemmlibuffer(28)andcentrifuged,andthesupernatantwas loaded onto a gel for SDS-PAGE and Western blot as describedabove.Asacontrol,cervicalmucuswasimmunoprecipitatedwithmag-netic particles conjugated to an isotype-matched Ig of irrelevantspecificity.
 Lectin binding to proliferative and secretory endocervical tissue
The two lectins used in this study,
Arachis hypogaea
Erythrina cristagalli
(ECA), bound to sections of humanendocervical epithelium (Fig. 2). Strong binding was ob-served in endocervical epithelia from biopsies taken duringthe proliferative phase of the menstrual cycle (Fig. 2, A andB), demonstrating the presence of T antigen and
-acetyllac-tosamine in intracellular compartments in these specimens.Both lectins bound to cytoplasmic granules in the supranu-clear region of the epithelial cells, as visualized with thenuclearstainpropidiumiodide(datanotshown).Theintensereactivity was observed in most, but not all, endocervicalcells. Binding of PNA and ECA to endocervical epithelialcells was less intense or negative in tissue taken from thesecretory phase of the menstrual cycle (Fig. 2, D and E),indicating that the mucus stored in the endocervical glandsduring the proliferative phase has been secreted into thecervical canal during the secretory phase. Similarly, bindingoftheMUC5Bantibodytosectionsofendocervixwasgreatlydiminished in the late secretory phase (Fig. 2F) comparedwith the proliferative phase (Fig. 2C), thus correlating to thereduction in PNA and ECA binding. No binding was ob-servedinsectionsfromtheproliferativephaseinwhichPNAand ECA were previously incubated with
)-galactose ata concentration of 0.2
, indicating the lack of nonspecific binding(Fig.2,GandH).AllsectionsprobedwithPNAwerepretreated with neuraminidase, because initial experimentsshowed increased intensity in lectin binding compared withuntreated sections (data not shown).
 Lectin binding to cervical secretions
The presence of the T antigen and
-acetyllactosaminewas initially demonstrated in midcycle cervical secretionsusing the ELLA method. Peroxidase-labeled PNA and ECAconsistently bound to microtiter plates coated with decreas-ing amounts of cervical mucus, producing linear responsesstarting with nanogram quantities of total protein (Fig. 3A).ECA binding was observed when plates were coated with atleast 6 ng total protein and was maximal when they werecoated with 400 ng. Binding of PNA was only detectable incervical secretions after previous treatment of the sampleswith neuraminidase (Fig. 3A). The linear response of PNA binding to cervical mucus was ranged between 500 ng and7.5
g total protein (Fig. 3A). Nonspecific binding of perox-idase-conjugated lectins to microtiter wells as well as en-dogenousperoxidaseactivityinmucussecretionswastestedand found to be negative.Western blot analysis further confirmed binding of PNAand ECA to human cervical secretions (Fig. 3B). After neur-aminidase treatment of the sample, PNA bound most in-tensely to a band in the upper region (
207 kDa) of theseparating gel (Fig. 3B, lane 1). The electrophoretic mobilityof this band was similar to those usually obtained whenanalyzing secreted mucins (20). Two additional bands of high molecular mass (
100 and 200 kDa) were detected inthe separating gel, indicating the presence of the T antigeninadditionalglycoproteins.ECAexclusivelyboundtoahighmolecular mass band (
207 kDa) within the separating gel(Fig. 3B, lane 2). Western blot analysis of cervical secretionswith the anti-MUC5B antibody (Fig. 3B, lane 3) confirmedpreviousresultsshowingstrongbindingtoahighmolecularmass band (20).
T antigen and N-acetyllactosamine content in cervicalsecretions taken through the menstrual cycle
The relative amounts of the T antigen and
-acetyllac-tosamine in cervical secretions of the three normal cyclingfemales varied among the proliferative, ovulatory, and se-cretoryphases,asdeterminedbyELLA(Fig.4A).Duringthe
et al.
Oligosaccharides in Cervical Mucus during Menstrual Cycle J Clin Endocrinol Metab, December 2002, 87(12):5641

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