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
. (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
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,
(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
Oligosaccharides in Cervical Mucus during Menstrual Cycle J Clin Endocrinol Metab, December 2002, 87(12):5641