Composition and Properties of Human Cervical Mucus

Composition and Properties of Human Cervical Mucus III. A Preliminary Study of the Mucoid Component OTTO W. NEUHAUS, Ph.D., and KAMRAN S. MOGHISSI, M.D.

functions in some manner in sperm migration, abnormalities in its composition could easily lead to a state of biolOgical infertility. Studies of its composition are, therefore, of fundamental importance. In previous reports the proteins of cervical mucus were studied by dilute agar gel electrophoresis9and by immunoelectrophoresis. 10 The former showed the presence of albumin, gamma globulin, and a nonmigrating fraction amounting to 43 per cent of the total protein-staining material (Fig. 1, Fraction C). Immunoelectrophoresis showed the presence of albumin, 2 alpha2 globulins, 3 beta1 globulins, 2 beta2 globulinS, and gamma globulins. There was, however, no high concentration of an immunologically active material coinciding with the so-called
SINCE CERVICAL MUCUS

From the Departments of Physiological Chemistry, Anatomy, and Obstetrics and Gynecology, Wayne State University College of Medicine, and from the Detroit Receiving Hospital and the Grace Hospital, Detroit. Mich. This study was aided by Grant RG-7031 from the United States Public Health Service. Samples of Mylar film were generously contributed by the DuPont Company, Wilmington, Del. Gratitude is expressed to Drs. M. Schubert, W. Pigman, and G. Biserte, for samples of some materials used. We wish to thank Dr. James W. Holleman for performing the amino acid analysis. The participation of Doctor Michael Iacobellis, and the technical assistance of Miss Virginia Sogoian in this study are also gratefully acknowledged.

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Shettles found a 1: 1 ratio for galactose and hexosamine in cervical mucus and consequently suggested the presence of a heteropolysaccharide composed of repeating disaccharide units (galactose plus hexosamine) combined with some fucose-containing components. Both human and rabbit cervical

Fig. 1. Electrophoretic separation of proteins of cervical mucus in an agar gel medium. Top: normal human serum. Bottom: cervical mucus.

gland and secretions were observed to react metachromatically with toluidine blue, indicating the presence of an acid mucopolysaccharide.13' 19, 20, 21 Recent experiments with S35-labeled sulfate have led Zacchariae to believe that cervical mucus contains a sulfated acid mucopolysaccharide.20 This polysaccharide in his opinion is a keratosulfate composed of galactose and glucosamine.21 Werner suggested that cervical mucus contains a glycoprotein which forms the bulk of the solid material and is responsible for its specific characteristicS. 1S ,17 This glycoprotein is, in his opinion, of the fucose (fucomucin) and sialic acid (sialomucin) type. Likewise, Gibbons, using bovine cervical mucus in pregnancy and during estrus, found in either case that this secretion contains a mucoid (by definition a carbohydrate-rich glycoprotein) consisting of 75-80 per cent carbohydrate and 20-25 per cent amino acid residues. Thus the possibilities fall into two general categories: (a) mucoprotein: a mucopolysaccharide-protein complex perhaps resembling the chondroitin sulfate-protein combination of connective tissues,s,l1 and (b) mucoid: a carbohydrate-rich glycoprotein resembling salivary mucin6 or the orosomucoid of bl()od serum. 1S It is the purpose of this report to determine which of these two types of substances the active material of human cervical mucus most nearly resembles.

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MATERIAL AND METHODS Collection of Specimens

Samples of midcycle cervical mucus were obtained from selected patients attending the gynecologic outpatient clinic at Detroit Receiving Hospital and from the admitting clinic and planned parenthood clinic at the Grace Hospital, Detroit. Care was taken to exclude all cases of menstrual disorder, vaginitis, and cervicitis as well as other pelvic abnormalities. The majority of patients were healthy young women of proved fertility. After cleaning the cervix gently with a cotton ball, the cervical mucus was aspirated from the cervical canal with a tuberculin syringe without needle. All samples for immediate use were kept refrigerated while those for later use were stored in the frozen state. Specimens contaminated with blood were discarded. Electrophoresis

The dilute agar gel electrophoretic technique described by Zak and coworkers was used throughout. To visualize the separation of proteins, the electrophoretic process was performed on vegetable parchment paper and then stained with bromphenol blue. 9 To demonstrate the carbohydrate containing substances, the separation was performed on Mylar film and then stained using the periodic acid Schiff reagents as described by Uriel and Grabar. RESULTS

Repeated attempts were made to degrade cervical mucus with hyaluronidase but no changes were observed. In the following studies the effect of proteolytic enzymes on cervical mucus was studied both by electrophoresis in dilute agar solution and by precipitation with cetyltrimethylammonium bromide (CTAB ). In addition, the amino acid composition of the electrophoretically nonmigrating fraction was determined. Incubation of 2 mg. of crystalline trypsin or chymotrypSin with 0.2 m!. of human or bovine cervical mucus at 40°C. for 24 hours caused a complete disappearance of the property of spinnbarkeit.8 A curve relating the change of spinnbarkeit with time is shown in Fig. 2. Electrophoretic separation of the products following 30 min. of digestion revealed comparable reductions in the amounts of albumin, total globulins, and the nonmigrating fractions (Table 1). When electropherograms of such digests were stained with periodic acid Schiff (PAS) reagent, the samples showed little or no PAS-positive material at the origin, while a diffuse yet distinctly positive region appeared to migrate toward the anode (Fig. 3). That

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this diffuse PAS-positive material originated from the nonmigrating fraction as a result of proteolysis, and not from the a-/3 globulins present in the mucus, was confirmed as follows. After the electrophoretic separation of human cervical mucus, the nonmigrating fraction was removed and incubated in a minimum amount of a solution of chymotrypsin at 37° C. for 24 hours. Reelectrophoresis of this fraction, using the original paper strip (origin), resulted in the migration of PAS-positive material and a negative reaction for PAS at the origin. In a control fraction, incubated with enzyme but not separated electrophoretically a second time, the paper origin retained its original degree of PAS reactivity. This experiment showed that proteolysis degraded the nonmigrating fraction into PAS-positive fragments which subsequently migrated in the electrophoretic system. The effect of digestion was also studied by precipitation with CTAB. 5

4

c .~

....o

.:.t!

2

.c c c -0. CJ)

10

20

30

40

50

60

Time in minutes

--------

18 hour

Fig. 2. Change in spinnbarkeit of human cervical mucus digested with chymotrypsin. Cervical mucus, 0.2 mI., was incubated at 40° C. with 2 mg. of crystalline chymotrypSin. Spinnbarkeit was measured as described by Cohn et al.

TABLE 1. Reduction of Bromphenol Blue Binding by Electropherograms of Human Cervical Mucus Digested with Chymotrypsin Undigested (OD units)

Digested (OD units)

Reduction

Fraction

Albumin Globulins N omnigrating

0.325 0.460 0.410

0.110 0.170 0.145

66

(%)

68 65

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A few drops of a 1% solution of CTAB when added to 0.2 ml. of human or bovine cervical mucus caused the formation of a thick, rope-like precipitate. Likewise, adjusting the pH to 4 with acetic acid resulted in precipitation. A solution of the latter precipitate in alkali showed the presence of the nonmigrating PAS-positive material as well as the other serum proteins. Digestion of the cervical mucus with trypsin and chymotrypsin for 24 hours resulted in complete elimination of precipitability with acetic acid or CTAB (Table

2). For purposes of comparison, filtered human saliva and bovine synovial

ALB

I

a

a-B

N

b

I c

•

y

BPB d

Fig. 3. Representation of electrophoretic separation of cervical mucus following digestion with chymotrypsin; a: albumin, b: PAS alpha-beta region, c: nonmigratbefore ing fraction, and d: gamma zone. Stains were bromphenol blue ani! periodic acid Schiff reagent. Compare with Fig. 1.

PAS after

TABLE 2. Effect of Digestion with Trypsin and Chymotrypsin on Precipitability withCTAB Dige8tion Sub8tance

Cervical mucus Synovial fluid (bovine) Saliva Submaxillary mucin Orosomucoid Chondroitin sulfate

Before

+ + + + + +

After

+

+

Approximately 5 drops of a 1% solution of CTAB were added to 0.2 ml. of each sample. Where dried samples were tested, a 1% solution was used. A positive reaction was recorded either with a rope-like clot or a heavy precipitate. A negative reaction was recorded when no precipitate, was observed.

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fluid were also treated with chymotrypsin to determine the effects upon electrophoresis and precipitability with CTAB. Prior to digestion, saliva demonstrated an intensely PAS-positive nonmigrating zone, which was eliminated by the action of trypsin and chymotrypsin (at 37° C.) in 1.5 hours. Precipitability with CTAB was eliminated after only 30 min. of digestion by trypsin and chymotrypSin. A control sample maintained at 37° C. for the same time retained its precipitability. Digestion of bovine synovial fluid for 24 hours removed all of the normal electrophoretic protein zones. As was expected, however,'precipitability with CTAB, of hyaluronic acid in this instance, was unaffected. Crystalline orosomucoid, purified salivary mucin,14 and a commercial preparation of chondroitin sulfate, containing 7.5 per cent protein, and a sample containing 15 per cent protein prepared by the ultracentrifugal separation of cartilage extracts4 were also tested. In chondroitin sulfate preCipitability was retained, while after digestion neither orosomucoid nor salivary mucoid were precipitable. Amino acid composition of the nonmigrating fraction was determined so that a comparison could be made with the bovine mucoids of Gibbons. Several electrophoretically nonmigrating fractions were pooled and hydrolyzed with 5.7N HCl for 24 hours. The hydrolysate was then treated with fluorodinitrobenzene and evaluated chromatographically as described by Biserte and co-workers. Table 3 is a summary of these data. There is no correlation TABLE 3. Amino-acid Composition of the Electrophoretically Nonmigrating Fraction of Human Cervical Mucus Concentration Amino acid

Cystine Glutamic and aspartic Serine Threonine Proline Glycine Alanine Histidine Valine Leucines Phenylalanine Lysine Tyrosine Arginine Methionine

Mole 0/0

Gm./%

0.7 24.3 8.1 8.1 3.6 10.6 9.2 2.9 5.4 11.5 2.9 3.8 3.9 2.7

1.4 27.1 6.7 7.7 3.3 6.3 6.5 3.5 5.0 11.9 3.8

2.4

4.4

5.6 3.7 2.8

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with the results of Gibbons. Obviously the nonmigrating fraction was not homogeneous but only an electrophoretically defined mixture. There is also the question of species difference. It is essential to isolate a quantity of human cervical mucus factor before a definitive study is pOSSible. DISCUSSION

The absence of uronic acid and the repeated demonstration of the negative action of hyaluronidase on human cervical mucus should eliminate cbmpletely the possibility that the active material of this biological fluid is hyaluronic acid. The possibility still exists that the carbohydrate material of cervical mucus is a heteropolysaccharide, perhaps sulfated. The studies of Gibbons and of Werner16 show that the active carbohydraterich material of cervical mucus is glycoprotein in nature. Because of its high concentration of carbohydrate it can be specifically classed as a mucoid. The practical difficulty of obtaining sufficient human mucus for an isolation of this material is obvious. Therefore, for this preliminary study we have chosen to determine the nature, mucopolysaccharide or mucoid, of the active material by observing changes in electrophoretic properties and preCipitability with CTAB. These observations were compared with those using known mucopolysaccharides and mucoids. Proteolytic hydrolysis of the mucus rapidly destroyed its physical properties and changed entirely the electrophoretic picture. Partial hydrolysis also eliminated the precipitability with CTAB. Since this reagent precipitates both mucopolysaccharides and mucoids, it serves as a means for observing the degradation of these substances. Comparisons were made with biological fluids containing a mucoid, saliva, and a mucopolysaccharide, synovial fluid. Only the precipitability of the saliva was effected by the action of the enzymes. The precipitability of purified salivary mucin and also crystalline orosomucoid was destroyed but not that of chondroitin sulfate. It can be argued that the properties of the mucus are the result of mucopolysaccharide-protein complexes such as exist in most connective tissues.s,ll Proteolytic enzymes would destroy the protein moiety of the complex but not the polysaccharide units per se. This could explain the changes in properties of mucus and even a change in electrophoretic behavior.2 Precipitability with CTAB, however, would be expected to be retained, as confirmed by our experiments. On the other hand, mucoids owe their structure to a polypeptide backbone,6,ls so that proteolytic hydrolysis would be expected to change all of its properties including precipitability with CTAB. On the basis of the degradation of an electrophoretically nonmigrating substance to PAS-positive migrating fragments, the loss of spinnbarkeit and of

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viscosity of cervical mucus, and the loss of precipitability with CTAB, it is concluded, in agreement with Werner and Gibbons, that the active substance in cervical mucus is a protein rich in carbohydrate, perhaps comparable in structure with salivary mucoid or orosomucoid. The presence of all of the amino acids in the nonmigrating fraction lends some support to his conclusion although it is recognized that this is an electrophoretically defined mixture and in no way a homogeneous component. SUMMARY

The effect of trypsin and chymotrypsin has been studied on whole cervical mucus and on the PAS-positive electrophoretically nonmigrating material. Proteolysis caused the formation of PAS-positive, electrophoretically migrating fragments, a disappearance of spinnbarkeit and of viscosity, and an elimination of precipitability with cetyltrimethylammonium bromide. Comparisons were made with saliva, purified salivary mucoid, orosomucoid, synovial fluid, and chondroitin sulfates. It was concluded that the carbohydraterich component of human cervical mucus is a glycoprotein or mucoid and not a mucopolysaccharide. K. S. M. 1401 Rivard St. Detroit 7, Mich.

REFERENCES 1. BISERTE, G., HOLLEMAN, J. W., HOLLEMAN-DEHOVE, J., and SAUTIERE, P. Paper chromatography of dinitrophenylamino acids. In Chromatographic Reviews, Vol. 2, M. Lederer, ed. Elsevier, Amsterdam, 1960, p. 59. 2. BLUMBERG, B. S., and OGSTEN, A. G. Physiocochemical studies on hyaluronic acid. In Chemistry and Biology of Mucopolysaccharide, G. E. W. Wolstenholme and M. O'Conner, ed. Churchill, London, 1958, p. 22. 3. COHN, M. R, STEIN, I. F., and KAYE, B. M. Spinnbarkeit: A characteristic of cervical mucus. Significance at ovulation time. Fertil. & Steril. 8:201, 1952. 4. GERBER, B. R, FRANKLIN, E. C., and SCHUBERT, M. Ultracentrrrugal fractionation of bovine nasal chondromucoprotein. J. Biol. Chern. 285:2870,1960. 5. GmBoNs, R A. CheInical properties of two mucoids from bovine cervical mucus. Biochem. J. 78:209, 1959. 6. GoTTSCHALK, A. Correlation between composition, structure, shape, and function of a salivary mucoprotein. Nature 186:949, 1960. 7. KURZROK, R, and BRINBERG, C. The presence of glucosamine in cervical mucus as a test for the date of ovulation. Internat. J. Fertil. 8:80, 1958. 8. MATHEWS, M. B., and LOZAITYTE, I. Sodium chondroitin sulphate-protein complexes of cartilage. I. Molecular weight and shape. Arch. Biochem. Biophys. 74:158, 1958. 9. MOGHISSI, K., NEUHAUS, O. W., and STEVENSON, C. S. Composition and properties of human cervical mucus. I. Electrophoretic separation and identification of proteins. J. Clin. Invest. 89: 1358, 1960. 10. MOGHISSI, K., and NEUHAUS, O. W. Composition and properties of human cervical

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mucus. II. Immunoelectrophoretic studies of the proteins. Am. J. Obst. & Gynec. 83:

149,1962. 11. PARTRIDGE, S. M., and DAVIS, H. F. The presence in cartilage of a complex containing chondroitin sulphate combined with a non-collagenous protein. In Chemistry and Biology of Mucopolysaccharides. Ciba Foundation Symposium. G. E. W. Wolstenholme and M. O'Conner, ed. Churchill, London, 1958, p. 93. 12. SHETTLES, L. B. The polysaccharide composition of human cervical mucus. Fertil. & Steril. 2:361, 1951. 13. SYLVEN, B. True and false metachromatic staining reactions of myelin and "epithelial" mucus. Nord. Med. Arkiv. 25:405, 1945. 14. TsUIKI, S., HAslllMOTO, Y., and PIGMAN, W. Comparison of procedures for the isolation of bovine submaxillary mucin. J. Biol. Chem. 236:2172,1961. 15. URIEL, J., and GRABAR, P. Emploi de colorants dans l'analyse electrophoretique et immunoeIectrophoretique en milieu gelifie. Ann. Inst. Pasteur. Lille 90:427, 1956. 16. WERNER, 1. Studies on glycoproteins from mucous epithelium and epithelial secretions. Acta Soc. med. upsal. 58:1,1953. 17. WERNER,1. The chemistry of cervical mucus. Acta obst. et gynec. scandinav. 38:39, Suppl. 1, 1959. 18. WINZLER, R. J. Glycoproteins. In Plasma Proteins, Vol. 1, F. W. Putnam, ed. Acad. Press, New York, 1960, p. 309. 19. WISLOCKI, G. B., BUNTING, H., and DEMPSEY, E. W. The Chemical Histology of the Human Uterine Cervix. E. T. Engle, ed. Thomas, Springfield, Ill., 1950, p. 23. 20. ZACCHARIAE, F. Autoradiographic (S35) and histochemical studies of sulphomucopolysaccharides in the rabbit uterus, oviduct, and vagina. Acta endocnnol. 29:118,

1958. 21. ZACCHARIAE, F. The acid mucopolysaccharides of cervical mucus. Acta obst. et gynec. scand. 38:86, Suppl. 1, 1959. 22. ZAX, B., VOLIN!, F., BRISKI, J., and WILLIAMS, L. A. Combined agar and gel-paper electrophoresis. Am. J. Clin. Path. 33:75, 1960.

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