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Original Studies |
Schepens Eye Research Institute and Department of Ophthalmology (I.K.G., R.M., S.S.-M., P.A.), Harvard Medical School, Boston, Massachusetts 02114; Brigham and Women’s Hospital and Harvard Medical School (A.R.G., J.A.H.), Boston, Massachusetts 02115; Boston University Medical Center (G.D.O.), Boston, Massachusetts 02118; and Massachusetts General Hospital and Harvard Medical School (H.T.K.), Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Ilene K. Gipson, Ph.D., Schepens Eye Research Institute, 20 Staniford Street, Boston, Massachusetts 02114. E-mail: gipson{at}vision.eri.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The four large gel-forming mucin genes, all of which are clustered on chromosome 11.p15.5, share several structural features (15, 16, 17, 18, 19, 20). Each has a large central tandem repeat region and, 5' to the tandem repeat region, three regions that encode cysteine-rich domains designated D1, D2, and D3, which share homology with the D domains of von Willebrand Factor (21). MUCs 2, 5AC, and 5B also have a region that encodes a fourth D domain, 3' to the central tandem repeat.
Epithelia expressing and secreting mucins often express several of the genes, giving rise to a heterogeneous mucus secretion. All of the cloned mucins, with the exception of MUCs 3 and 7, have been reported to be expressed by the endocervical epithelium, although MUC2 message seems to be sporadic (22, 23, 24). We have previously demonstrated that MUC4 and MUC5B are the predominant mucin messenger RNA (mRNA) transcripts present in the human endocervix through the menstrual cycle and that levels of both correlate inversely with serum progesterone level. Smaller amounts of mRNA for MUCs 5AC and 6 are also expressed (25). Although this study provided insight into the expression levels of mucin genes during the menstrual cycle, little is known about specific mucin protein levels in cervical mucus within the cycle. Price-Schiavi et al. (26) have demonstrated that the membrane-spanning mucin rat Muc4 is posttranscriptionally regulated in mammary epithelium, because protein and mRNA levels do not correlate under certain experimental conditions. Thus, it is of importance to assay MUC5B protein levels to correlate to the demonstrated cyclic changes in MUC5B mRNA (25) and to begin to characterize the molecular basis for the dramatic changes in the physical properties of the mucin gel that occur at midcycle to facilitate sperm migration (27, 28, 29).
Measuring mucin protein levels of a specific mucin in samples containing multiple mucin gene products has proven difficult. This is partly attributable to difficulty in identifying specific gene products, because there have been relatively few well-characterized specific antibodies based on molecular characterization of the molecules, and because individual mucins have not been purified. Thornton et al. (30, 31) have proposed an extensive CsCl density centrifugation purification of the mucins followed by direct measurement of the protein in the isolated fractions by mucin-specific antibodies. This technique is useful for identifying glycoforms (32) but is labor-intensive and requires a large amount of starting material, rendering it impractical for studies of small amounts of mucus secretion. Measurement of mucins within mucus secretions has also been done using antibodies to isolates of mucin in combination with enzyme-linked immunosorbent assay (ELISA) (33). While this technique proves feasibility for direct measurement of mucins within native secretions without mucin purification, levels of a specific mucin gene product are not obtained. An alternative method reported here is the use of antibodies specific to a synthetic peptide that mimics a unique nonglycosylated region of the D4 domain of MUC5B in an ELISA-based assay, to determine relative amounts of a specific mucin protein in a mixed sample. Such assay procedures allow quantitative measures of variance of specific mucins under changing physiologic conditions.
The goal of this study was to develop a quantitative assay for a specific mucin present in a heterogeneous cervical mucin secretion and to expand the previous investigation of mucin RNA levels by determining relative amounts of MUC5B mucin protein in the cervical mucus through the menstrual cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies were made to a synthetic peptide from the deduced amino acid sequence (17) of a unique region of the D4 domain (nontandem repeat region) of MUC5B (VTFNGQVFQARLPYSLFHN). Searching for homology, using BLASTP 2.0.8, results in matches to MUC5B only, indicating that this region lacks identity to other gel-forming mucins. Peptides synthesized by solid-phase procedure, using F moc chemistry, were conjugated to keyhole limpet hemocyanin with glutaraldehyde in the Peptide Synthesis Core of the Reproductive Endocrine Sciences Center, Massachusetts General Hospital. Antibodies against the keyhole limpet hemocyanin peptides were produced in chickens by Avian Antibodies, Inc. (Carlisle, MA).
Tissues and secretions
Human cervical mucus, endocervical epithelium, cervical tissue, conjunctival tissue, intestine, salivary (submandibular) gland, blood, urine, tears, and saliva were collected in accordance with human study guidelines and approval. Informed consent was obtained from all subjects in accordance with protocols approved by the Schepens Eye Research Institute and Brigham and Women’s Hospital Institutional Review Boards. Human cervical mucus was obtained by swabbing the cervix with Wilshire foam swabs (VWR Scientific Products, Bridgeport, NJ). Swabs were used, rather than aspiration, to maximize harvest of mucus from cell surfaces, because mucus is adherent to cell surfaces. Human endocervical epithelium was obtained by cytobrush collection as previously described (25). Serum, urine, tears, and saliva from volunteers were used for characterization of the MUC5B antibody. Endocervix was obtained at the time of hysterectomy, conjunctival tissue was obtained at the time of surgery from subjects undergoing cataract extraction, and salivary gland (submandibular) tissue was obtained through the National Disease Research Interchange (Philadelphia, PA). Tissue used for immunohistochemistry was frozen, within 30 min of surgery, in Tissue Tek II OCT compound (Lab Tek Products, Naperville, IL) for cryostat sectioning.
Two populations of subjects were used to obtain cervical mucus. The first population consisted of patients at the intrauterine insemination clinic and was used to obtain cervical mucus samples for mucin purification. The second population consisted of normal cycling females who were not using intrauterine devices or oral contraceptives, had normal cervical cytology, and were free of infection. Subjects in the second population were asked to self-report cycle day and to determine the LH surge with a commercially available urinary LH detection kit (Clear Plan Easy, Unipath Ltd., Bedford, UK). Samples were collected on days 4 and 7 after the start of menses and on days 1 and 7 after the LH surge at midcycle. Only samples from cycles with a confirmed LH surge followed by an increase in blood progesterone levels were used. When possible, duplicate samples for each time point were taken, but only two collections were done in any cycle, so collection proceeded over four cycles. Initially, six subjects were enrolled; three did not complete the protocol. Cervical mucus samples were collected just before the cytobrush collection of endocervical epithelium used for RNA isolation. Estradiol and progesterone levels in blood samples taken at each collection point were determined by the Reproductive Endocrine Sciences Center Assay Core Laboratory, MA General Hospital, Boston, MA (funded by NIH Grant HD-28138) as previously described (25).
Purification of cervical mucins for cervical mucin standard
Because there is currently no source of purified MUC5B for the generation of a standard curve for quantitative assays of MUC5B protein, cervical mucins were purified to be used as a cervical mucin standard. Fifty samples of cervical mucus, collected from patients at the intrauterine insemination clinic (patients with both spontaneous and stimulated cycles included), were used to obtain a purified cervical mucin preparation that could be used as a cervical mucin standard for determining relative amounts of MUC5B in crude mucus samples obtained from cycling subjects. Cervical mucin was purified from these samples essentially as previously described (5, 34, 35).
Preparation of cervical mucus/RNA from individual subjects
Cervical mucus was extracted from each individual swab from the normal cycling women (ages 29, 36, and 36) with 1 mL of 0.1 mol/L NH4HCO3, 2.0 mmol/L phenylmethylsulfonylfluoride, 0.5 mol/L NaCl, 5 mmol/L EDTA, 2 mmol/L N-ethylmaleimide, and 0.02% NaN3. Each mucus solution was centrifuged at 16,000 x g for 30 min at 4 C to remove insoluble material.
Total RNA was isolated from cytobrushes with TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s recommended protocol. Yield was measured by optical density using a Shimadzu Spectrophotometer (Shimadzu Scientific Instruments, Braintree, MA).
Immunoblotting and immunohistochemistry
Cervical mucus protein was run under reducing conditions on 6% separating, 4% stacking SDS-PAGE. Protein was transferred to nitrocellulose for immunoblot detection of MUC5B antibody binding as previously described (36). Cellular localization of MUC5B antibody binding was detected by immunofluorescence microscopy on cryostat sections of human endocervix, conjunctiva, intestine, and submandibular (salivary) gland as previously described (36).
ELISA
Indirect ELISAs were used to determine the relative quantity of MUC5B in cervical mucus samples using standard methods (37). Briefly, cervical mucus was coated in triplicate (50–200 ng per well) on EIA microtiter plates (Costar, Cambridge, MA) in 0.05 mol/L carbonate/bicarbonate buffer, pH 9.6. Care was taken to coat equivalent amounts of total protein from each sample. Plates were blocked with PBS containing 3% (wt/vol) Fish Gel (Sigma, St. Louis, MO). Antibodies were diluted in PBS containing 1% (wt/vol) Fish Gel. The plates were washed with PBS containing 0.05% (vol/vol) NP-40 followed by PBS. Secretions were tested and found to be negative for endogenous peroxidase activity by incubation with peroxidase substrate alone. Care was taken to run all samples from a single individual in the same ELISA assay. Aliquots of dilutions of the cervical mucin standard (125–8000 pg) were used with all individuals, to generate the standard curve. To verify the accuracy of the quantitation of MUC5B mucin in crude mucus samples, so-called add-back experiments were performed. Known amounts of purified cervical mucin were added to crude cervical mucus samples and analyzed by ELISA.
Semiquantitative RT-PCR
Total RNA was reverse-transcribed using the Superscript II First Strand cDNA Synthesis Kit (Life Technologies, Inc./BRL, Gaithersburg, MD). MUC5B mRNA levels were assayed using RT-PCR methodology previously described (25).
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Results |
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Recently, we determined that MUC5B transcripts are the predominant
gel-forming mucin mRNAs expressed by the human endocervix, with two
other gel-forming mucins expressed (MUC5AC and MUC6) but at lower
levels (25). To develop methods to assay specific mucin
gene products in the cervical secretion, a polyclonal antibody specific
for MUC5B was made. This antibody, designated no. 799, was to a
synthetic peptide from the deduced amino acid sequence of the D4 domain
of MUC5B, residues 418–436 (17). The sequence was from an
intercysteine-rich region and lacked apparent N or O glycosylation
sites. When used on immunoblots of human cervical mucus run on
denaturing SDS-PAGE (6% separating, 4% stacking), the antibody bound
to a high-molecular-weight band that just entered the separating gel
(Fig. 1A
, lane 1). The specificity of the
antibody to its immunizing peptide was confirmed by loss of binding to
this band after preadsorption of the antibody with 100 µg/mL of the
unconjugated peptide (Fig. 1A, lane 2). By ELISA, preadsorption of the
antibody, with 200 µg of immunizing MUC5B peptide per mL, reduced
binding by 50% to 60%, whereas preadsorption with a MUC4-specific
peptide had no effect. By ELISA (Fig. 1B), we observed strong binding
to secretions known to contain MUC5B [cervical mucus (22, 25), saliva (38)], and no binding to secretions
(tears) known to contain MUC4 and MUC5AC but not MUC5B
(39) or to body fluids lacking mucins (serum and urine).
There was a 2-fold increase in binding of the antibody to the purified
cervical mucin isolate used as the cervical mucin standard, compared
with the crude midcycle cervical mucus used in its preparation,
indicating at least a 2-fold purification (Fig. 1B).
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). The antibody also bound
to submandibular gland (salivary tissue), known to express MUC5B (Fig. 2B) (32). The antibody did not bind to the goblet cells of
human conjunctiva (Fig. 2C), which do not express MUC5B but do express
MUC5AC; nor did the antibody bind to intestinal epithelia known to
express MUC2 (Fig. 2D) (6, 39). These data demonstrate
lack of binding of the antibody to other gel-forming mucins.
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The MUC5B antibody consistently bound by ELISA in a linear fashion
to picogram quantities (250–4000 pg) of the purified cervical mucin
standard (Fig. 3A). Because this cervical
mucin standard is a purified mucin fraction that contains all mucins in
the cervical mucus, we report the amount of MUC5B protein as MUC5B
units per nanogram of total mucus protein. A MUC5B unit is defined as
the picogram amount of cervical mucin standard corresponding to the
optical density reading obtained per sample. The accuracy of using the
ELISA to measure MUC5B mucin in a complex biological fluid (crude
mucus) was further confirmed by doing add-back experiments, in which
known amounts of purified cervical mucin were added to an aliquot of a
crude mucus sample (20 ng). As shown in Fig. 3, the predicted amounts
of MUC5B protein, as determined from the standard curve for this assay
(Fig. 3B), correlated to the actual assay value representing the amount
in crude plus added purified mucin (Fig. 3C). This data supports the
use of this ELISA for the quantitation of MUC5B protein in cervical
mucus samples.
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ELISAs were performed on the individual mucus samples collected
from three subjects, each over four hormone cycles. No more than two
samples were taken per cycle, to prevent inflammation from
oversampling, and ovulation was verified for each cycle by documenting
LH surge followed by increase in blood progesterone levels (see
Materials and Methods). For each subject, cumulatively,
two-to-three samples were taken before the LH surge, one around the LH
surge, and one-to-two after the LH surge. All samples from a single
individual were run in the same ELISA against the same standard curve.
To assure the full range of detectability in all samples, the assays
were performed on a range of concentrations (50, 100, and 200 ng per
well) of the cervical mucus samples taken at the time points in the
menstrual cycle indicated in Fig. 4. At
all concentrations of the cervical mucus tested, there was an obvious
peak in MUC5B antibody binding to samples from midcycle, compared with
those from early or late in the cycle. The assays reported in Fig. 4A
reflect the average of values obtained from 50, 100, and 200 ng
cervical mucus per well, with each assay repeated three times. These
results demonstrate that the amount of MUC5B mucin present per nanogram
of mucus protein displays a dramatic 3- to 7-fold increase at midcycle
(at cycle day LH plus 1). The amount of MUC5B protein from
corresponding cycle time points among the three subjects was
comparable. The amount of MUC5B in the cervical mucus in the luteal
phase dropped dramatically as mRNA levels dropped and as blood
progesterone levels increased (Fig. 4B). There were no apparent
correlations between MUC5B levels and blood estradiol levels (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observed peak in MUC5B protein levels in cervical secretions at midcycle is consistent with early reports of increase in amounts of mucus (as measured by wet weight) in the cervix at midcycle (40) and with a more recent report of maximal glycoprotein content at midcycle (as measured by densitometry of periodic acid Schiff staining of SDS-PAGE gels) (27). Along with the increase in mucin/glycoprotein content at midcycle, there is increased water content in the cervical mucus, with estimates of water content at 97.85–99% in midcycle mucus, compared with 91% in the luteal-phase mucus (27, 41). Morales et al. (27) have quantified the protein content (as estimated by densitometry of Coomassie blue-stained SDS-PAGE gels) of periovulatory and luteal mucus, estimating it to be 2.45 ± 0.36 and 14.95 ± 1.10 mg/mL wet weight, respectively. Thus, at midcycle, cervical mucus is greater in quantity, has more mucin and less protein, and has a higher water content than in the luteal phase. How do these observations fit with the long-observed decrease in viscosity of the mucus at midcycle (40), as well as with the enhanced ability to allow migration of spermatozoa (28, 29)? Increase in amount of mucins in a solution is expected to increase the viscosity of the fluid at neutral pH (42); but, if the amount of water present increases at the same time, such that a dilution occurs, one might anticipate a decrease in mucus viscosity. What role does the increased mucin play? At least two hypotheses may be made. First, increased mucin in the endocervical canal may, because of its hydrophilic character, function to retain or hold water in place at the cell/canal surface to keep the canal patent for sperm motility. Mucins are extraordinarily hydrophilic, and the ability of their surfaces to bind water accounts for a part of the mucins’ gel-forming and space-filling ability (43). A second hypothesis is that increased mucin in the canal, at a period of high water content, is required for protection of the cervix and uterus. Pathogens and other seminal fluid components may be excluded from entering the uterus by mucin trapping. The function of specific mucins in the cervical mucus remains to be determined.
The results presented here also confirm our previous findings of an inverse correlation between MUC5B mRNA levels, compared with serum progesterone levels (25). Other studies have described hormonal regulation of mucin genes, mostly in MUC1. There is little information in the literature regarding hormonal regulation of expression of mucin genes other than the membrane-spanning mucins. Recent studies show that sialomucin complex (SMC) mRNA (the rat homologue of MUC4) is expressed in the uterus of the rat (44). They studied SMC protein level in ovariectomized rats supplemented with estrogen, progesterone, or a combination of the two, by immunoblot of uterine homogenates. They found high levels of the SMC protein in the uterus of the estrogen-supplemented rats, a diminution in estrogen-plus-progesterone-supplemented rats, and no SMC protein with progesterone supplementation alone. These data suggest that progesterone down-regulated the SMC message, as is the case with MUC5B in the human endocervix.
Previous studies to quantify specific gel-forming mucins from native secretions have required extensive purification protocols, including CsCl density centrifugation followed by fractionation (30, 31). These types of protocols, though useful for identification of glycoforms (32), prove impractical for small samples and are labor intensive. Measurement of the amount of mucin-like proteins within airway lavage samples, using a sandwich-type ELISA, has been reported (33). This study demonstrated the feasibility of directly measuring mucins in secretions without mucin purification, but the antibodies used for these assays were not directed toward a specific mucin gene product. Direct measurement by ELISA with specific, characterized peptide antibodies allows a rapid, reproducible, and sensitive assay for these large molecules.
The specificity and binding of antibody no. 799 to native MUC5B protein may relate not only to the lack of glycosylation sites in the antigenic region but also to its position within an intercysteine region of the D4 domain of MUC5B. Such intercysteine sequences typically loop out from associated cysteines, thus providing ready access to antibody binding (45). Besides surface exposure, the relative flexibility of the looped sequences may more readily replicate appropriate components among the ensemble of shapes assumed by small, flexible peptide antigens (46). Examples of looped epitopes among secreted proteins include the gonadotrophins LH/human CG (47, 48) and FSH (49), as well as human GH (50). Unique intercysteine sequences from the D domains in other regions of MUC5B or in other gel-forming mucins may thus also be candidate sites for developing region- or mucin-specific antibodies.
Although this study, for the first time, provides quantitative protein data for a specific mucin component of the cervical mucus through the menstrual cycle, some limitations must be addressed. Because of the difficulty in obtaining timed cervical mucus swabs and cytobrush samples, the sample size in this study was limited. Within the three subjects studied, the number of samples obtained was spread over several cycles to minimize the effects of any potential inflammatory response induced by the sampling technique. The result is a collection of static time points, rather than a fluid description of protein and mRNA levels throughout each day of the cycle.
In summary, the peak levels of MUC5B protein at midcycle are consistent with high mRNA levels present in the proliferative phase (25) and with the change in mucin character that facilitates sperm migration. Availability of the MUC5B-specific antibody will enable future studies of the role of MUC5B in sperm transit as well as other aspects of MUC5B character, including posttranslational modifications such as glycosylation.
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Footnotes |
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Received June 30, 2000.
Revised September 22, 2000.
Accepted October 13, 2000.
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-subunit of human follicle-stimulating hormone using
site-specific antipeptide antisera. Endocrinology. 127:573–579.This article has been cited by other articles:
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F. Ceric, D. Silva, and P. Vigil Ultrastructure of the human periovulatory cervical mucus J. Electron Microsc. (Tokyo), October 1, 2005; 54(5): 479 - 484. [Abstract] [Full Text] [PDF]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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) and blood levels of
progesterone (B; P4 
) are shown for each subject on the collection
day, relative to LH surge.