The Polysaccharide Composition of Human Cervical Mucus

Transcription

1 The Polysaccharide Composition of Human Cervical Mucus landrum B. Shettles, M.D., Ph.D. THE VISCOSITY of the cervical mucus seems to be one of the factors which determine the ability of spermatozoa to penetrate into the uterine cavity.lo Proteins are present in the cervical mucus in a concentration of about 1 per cent and can therefore barely account for its viscosity. It could, however, be due to highly polymerized carbohydrates. The presence of this type of sugars in the cervical mucus has been repeatedly observed and reported, but their nature has not been definitely established. Boyland found glucosamine in bovine cervical mucus, which suggested the presence of a mucopolysaccharide. Viergiver and Pommerenke, on the other hand, found in human cervical mucus an increase in reducing power of the mucus after acid hydrolysis. The reducing substance was removed by yeast. As alcohol precipitation of the cervical mucus yielded a precipitate which reacted with iodine, like glycogen, these authors assumed that the mucus contains glycogen. New sensitive spectrophotometric micromethods of considerable specificity for the identification and quantitative determination of various classes of sugars and individual sugars in polysaccharides were recently developed. These methods have been used to identify and quantitatively determine the constituents of the polysaccharides of the cervical mucus. The present paper deals with these experiments. From the Department of Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University; and the Sloane Hospital for Women, New York City. 361

2 362 SHETTLES [Fertility & Sterility EXPERIMENTAL Material Translucent, acellular cervical mucus can be obtained only around the middle of the menstrual cycle. This investigation was limited to the clear, limpid, midcycle mucus in women with regular menstrual periods and of proven fertility. It was obtained by aspiration with a pip~tte provided with a rubber bulb. The pipette was weighed before and after collecting the sample, the difference of the two weights representing the weight of the sample. The sample was collected under guidance of a speculum and great care was taken not to contaminate it with the contents of the vagina. Other sources of contamination were also ruled out. The mucus was then thoroughly mixed with a nine-fold amount of 0.05 N NaOH, in which it dissolved readily. The identification and quantitative determination of all sugars present in the mucus was carried out in samples pooled from two to three individuals. As the cervical mucus may have contained a significant amount of free glucose, the identification and determination of sugars was carried out on the polysaccharide fraction precipitated from the mucus by 90 per cent ethanol after acidification with glacial acetic acid. The precipitate was centrifuged off, separated by decantation, and redissolved in 0.05 N NaOH. Analytic Methods The total carbohydrates in the mucous solution were determined by the indole reaction described by Dische and Popper and the amount calculated as galactose. This allowed an appropriate dilution of the mucus for the following analytic procedures. The method of Dische and Popper is a color reaction with indole and H2S04 which furnishes a basis for the determination of carbohydrate in animal organs, secretions, and blood. The color produced by boiling a carbohydrate solution with indole and H2S04 is proportional to the carbohydrate concentration. The important feature of this method is that it depends upon a true color reaction and not upon the reducing power of the sugar, and the determination is practicable with a concentration ranging from 0.1 to per cent. In absolute terms, by this procedure 0.05 mg. of carbohydrate can still be determined quantitatively. Methylpentose was detected and determined by the cysteine reaction

3 Vol. 2, No.4, 1951] POLYSACCHARIDES IN CERVICAL MUCUS 363 of Dische and Shettles,4 in which the unknown solution is treated with H2S04, heated in boiling water, cooled, and treated with a solution of cysteine hydrochloride. The color of the reaction product with various sugars depends on the time of heating. The difference between the extinction coefficients at 3960 A. and 4300 A. is used for the quantitative determination of methylpentoses in amounts from 2 to 10 micrograms and in the presence of an excess of other sugars. This difference for a methylpentose is strictly proportional to the concentration of the sugar. The identification of methylpentose was confirmed repeatedly by measuring the rate of destruction of the reaction product of methylpentose with cysteine in two hours after the addition of a certain amount of water.s The presence of keto sugars was then excluded by the reaction with carbazole and cysteine of Dische and Borenfreund. 7 The identification and determination of hexoses was carried out by three modifications of the cysteine reaction of Dische and Shettles. 5 The procedures differ in the concentration of H2S04 used, the time of addition of cysteine, and the heating time. The methods are characterized by a rapid primary reaction and a slow secondary reaction that appears to reach a maximum in forty-eight hours at room temperature. Density measurements at selected times and wave lengths permit the detection and determination of hexoses in mixtures with each other and in the presence of other sugars. This identification of hexoses was based on the assumption that only the three usual aldohexoses are present in the cervical mucus. The recently described reactions with indole in Hel of hexosamines was employed for the determination of hexosamine. This is a new, characteristic, color reaction of hexosamines based on their deamination to 2,5-anhydrohexoses, in which as little as 5 gamma of hexosamine can be quantitatively ascertained. The method can also be used for the quantitative determination of hexosamine in polysaccharides. The mucous solution was hydrolized for two hours with 2 N Hel under reflux, neutralized, and hexosamine was determined after deamination by the reaction with indole and Hel. Longer hydrolysis did not change the values for the concentration of hexosamine. In all these determinations internal standards of methylpentose, hexose, and hexosamine were run. The carbazole reaction of hexuronic acids 3 was used as a test for these compounds. This is a color reaction with H2S04 and carbazole for the microdetermination of hexuronic acids, and it is positive with a solution of glucuronic acid containing 5 gamma per cc. This test was

4 364 SHETTlES [Fertility & Sterility carried out on the twenty-fold diluted mucus which was shown not to depress the reaction of hexuronic acids. To account for the absorption due to methylpentose and hexoses present in the mucus a standard containing the same amount of these sugars as that present in the mucus was run simultaneously with the mucous sample. RESULTS Composition of the Mucous Polysaccharide Fraction Precipitated with Ethanol Two series of experiments were carried out. In the first series the identification and quantitative determination of sugars was performed on the precipitated polysaccharide of pooled mucous samples collected from two or three individuals and kept overnight at 4 0 C. in alkaline solutions. The results of these determinations are listed in Table 1. To ascertain the extent TABLE 1. Composition of the polysaccharide fraction precipitated by 90 per cent ethanol from pooled samples of cervical mucus (Mg. per 100 cc. of original mucus volume) Ratio Ratio '".: '" 0.~ ~ '" 0 0 1:1 ti ti..s ~..s ~ '" ;::., Total <:l <:l~ " Methyl- b.o'" ~ mucopoly- Sample No. pentose Galactose Glucose Hexosamine b.ol ~ saccharide I II III to which the precipitation of the polysaccharides had been complete and whether the mucus contains mono- and oligosaccharides the cysteine reaction for methylpentoses and for hexoses was also carried out in the original mucous solution, and the results compared with those obtained with corresponding ethanol precipitates. As can be seen from Table 1 the polysaccharide fraction precipitated by 90 per cent ethanol contains a methylpentose, an hexosamine, and two hexoses (galactose and glucose). The amount calculated for mannose was zero in mucus II and in mucus I the value was no more than 8 per cent of the total hexose and not significant. The values for the two other hexoses

5 Vol. 2, No.4, 1951] POLYSACCHARIDES IN CERVICAL MU:US 365 in mucus III were calculated, under the assumption that no mannose is present in the mucus. The ratio between glucose and galactose varies from one mucus to another between 1 and 5. This indicates that the two hexoses are constituents of two different polysaccharides. The ratio galactose:hexosamine is very nearly 1, the deviation in both directions not exceeding 13 per cent. These deviations cannot be regarded as significant. The ratio galactose: methylpentose varies between the narrow limits of 2.5 and 3.1. Comparison of the Sugar Content in the Original Mucus and in the Precipitate The ethanol precipitation of mucus I and III was carried out on the ten-fold dilution of the original sample, containing 400 and 140 gamma per cc. of polysaccharide respectively. Mucus II, which originally did not differ essentially in its sugar content from mucus I, was diluted thirty-fold instead of ten-fold for the ethanol precipitation. The ethanol precipitation of polysaccharides was complete in mucus I (Table 2). In the two other more TABLE 2. Comparison of the composition of the polysaccharides in the original pooled mucous samples and in the corresponding alcohol precipitate (Mg. per 100 cc. of original mucus volume) Sample No. I II III M ethylpentose Original sample Precipitate Total hexose as galactose Original sample Precipitate diluted mucous samples 15 to 20 per cent of the methylpentm:e remained in solution. The values for hexose remaining unprecipitated in these cases obtained by the cysteine reaction and calculated as galactose correspond to those for methylpentose. It is clear, therefore, that the cervical mucus does not contain any significant amounts of mono- or oligo saccharides. The carbazole reaction for hexuronic acids carried out on the twenty-fold diluted mucus I, using an internal standard of glucuronic acid, failed to reveal any significant amounts of hexuronic acids. As the carbazole test permits the detection of 5 gamma per cc. of glucuronic acid, this negative result indicates that the amount of polyuronides possibly present in the mucus cannot exceed 5 per cent of the total polysaccharide.

6 366 SHETTLES [Fertility & Sterility Methylpentose and Galactose in Individual Mucous Samples In addition to the analysis of pooled samples of mucus, quantitative determinations of methylpentose, galactose, and glucose were carried out on three individual samples of cervical mucus without any alcohol precipitation. The results of this series of determinations are presented in Table 3. TABLE 3. Content in methylpentose, galactose, and glucose in individual mucous samples (Mg. per 100 cc. of original mucus volume) Ratio ~ ~ ~ ] ~ ~~ Sample No. Methylpentose Galactose Glucose 1: I II III Finally, quantitative determinations of methylpentose alone and a qualitative test for galactose were performed on nine individual samples of cervical mucus. Methylpentose and galactose were found in everyone of these samples. DISCUSSION Our analytic data show that the content in methylpentose, galactose, and hexosamine varies very nearly to the same extent from one mucous sample to another. As the ratio of galactose:hexosamine does not differ significantly from unity, we can assume the presence of a heteropolysaccharide consisting of a chain of disaccharide units in which one galactose is attached to one hexosamine molecule. Some of these disaccharide units are combined with a methylpentose. Mucopolysaccharides of this type are constituents of blood group substances. The content in glucose on the other hand changes to a very different extent as compared with the three other sugars. This fact and the finding of a polysaccharide giving the iodine reaction of glycogen by Vier giver and Pommerenke suggest that glucose is present in the mucus in the form of glycogen. The amount of glucose drops in some samples of cervical mucus to less than one-sixth of the total contents in sugars which sug-

7 Vol. 2, No.4, 1951] POLYSACCHARIDES IN CERVICAL MUCUS 367 gests that glucose is an accidental constituent Of the cervical mucus, and its presence may be due to contamination with contents of the uterine cavity. The heteropolysaccharide, on the other hand, seems to be an essential constituent, combined probably with proteins to a mucoprotein of considerable viscosity. The great variations in the concentration of the mucopolysaccharide in individual mucous samples may be due either to constitutional factors or to the fact that the day on which our material was collected did not correspond in every case to the same point in the menstrual cycle. Blood group substances with identical immunologic reactivity as those in blood cells are found in the body fluids of 75 to 85 per cent of individuals of a population. Mucopolysaccharides of the type found in blood group substances have been prepared from saliva, linings of gastric mucosa, and the fluid of ovarian cysts. It is not known, however, whether these mucopolysaccharides represent the main constituent of the polysaccharide fraction of these fluids. There can be little doubt that a specific mucopolysaccharide of an identical or nearly identical composition will be found in every individual sample of cervical mucus, even in those without immunologic activity. This mucopolysaccharide depends on finer details of the structure of the polymer, or its combination with a polypeptide. Polysaccharides of identical composition may represent different immunologic types or be devoid of any immunologic activity. If the viscosity of the cervical mucus depends on the degree of polymerization of the neutral mucopolysaccharide or its combination with proteins, the activity of the liquefying enzyme in the seminal fluid described by Kurzrok and Miller should involve either the hydrolysis of the mucopolysaccharide or the rupture of the corresponding mucoprotein into carbohydrate and protein. The presence of enzymes splitting blood-group polysaccharides in some tissues and body fluids have been reported sporadically by various authors. This type of enzymatic activity in semen may playa role in the mechanism of fertilization and the lack of such activity may be related to certain cases of sterility. SUMMARY 1. In the human mid cycle cervical mucus 75 to 80 per cent of the polysaccharides consists of a neutral mucopolysaccharide containing as constituents methylpentose, galactose, and hexosamine.

8 368 SHETTLES [Fertility & Sterility 2. The ratio of galactose to hexosamine is 1: 1 and the content of methylpentose varies between 15 and 20 per cent of the total mucopolysaccharide. 3. The composition of the mucopolysaccharide is very similar to that of human blood group substances. 4. The viscosity of the cervical mucus is related to the ability of spermatozoa to penetrate into the uterine cavity, and it may depend on the degree of polymerization of the neutral mucopolysaccharide resulting from the activity of a liquefying enzyme in semen. Accordingly, certain cases of sterility may result from failure of this type of enzymatic activity. REFERENCES 1. Boyland, E.: Biochern. J. 40: , Dische, Z., and Popper, H.: Biochern. Ztschr. 175: , Dische, Z.: J. BioI. Chern. 167: , Dische, Z., and Shettles, L. B.: J. BioI. Chern. 175: , Dische, Z., Shettles, L. B., and Osnos, M.: Arch. Biochern. 22: , Dische, Z., and Shettles, L. B.: (In press) J. BioI. Chern. 7. Dische, Z., and Borenfreund, E.: (In press) J. BioI Chern. 8. Dische, Z., and Borenfreund, E.: J. BioI. Chern. 184: , Kurzrok, R., and Miller, E. G.: Am. J. Obst. & Gynec. 15:56-72, Shettles, L. B.: Obst. & Gynec. Surv. 4: , Viergiver, E., and Pornrnerenke, W. T.: Am. J. Obst. & Gynec. 54: , 1947.