w. T. Pommerenke, Ph.D., M.D.

Transcription

1 Analysis of Carbohydrates in Human Cervical Mucus* Mary Alice B. Breckenridge, M.S., and w. T. Pommerenke, Ph.D., M.D. SPERM ACTIVITY, in common with that of all living cells, is dependent upon the expenditure of energy derived from foodstuffs. If sugar is essential for the motility of human spermatozoa, and the available evidence indicates that it is, then a functional explanation of the presence of carbohydrates in the genital secretions of both the male and female is on hand. MacLeod 1, 11, 13 has demonstrated that only glucose, maltose, fructose, mannose, or glycogen can serve as an energy source for human sperm motility. Because glycolysis, rather than respiration, is characteristic of the metabolism of these spermatozoa, they are well adapted for the environment of the cervical canal where it would seem likely that the supply of oxygen is lowp The semen is well supplied with a utilizable sugar, fructose.14 It is doubtful, however, that spermatozoa transport with them any appreciable quantity of this extracellular sugar from their native environment when they infiltrate the cervical secretion. Their intracellular stores of carbohydrate are also apparently low since motility is markedly impaired when human spermatozoa are washed and resuspended in glucose-free Ringer's solution,u Aided by a grant from the Ortho Research Foundation, Raritan, New Jersey. Data presented in part before the American Physiological Society, April 21, 195, Atlantic City, New Jersey. From the Department of Obstetrics and Gynecology, University of Rochester, Rochester, N. Y. Read at the Annual Meeting of The American Society for the Study of Sterility, San Francisco, June 25,

2 3 BRECKENRIDGE & POMMERENKE [Fertility & Sterility However, washing the spermatozoa and resuspending them in glucose-free Ringer's solution does not impair their ability to penetrate cervical mucus in vitro.is Pommerenke and ViergiverI7. 29 have reported that cervical mucus contains reducing substances, some free and others that are liberated on hydrolysis, in amounts comparable to the reducing substances in semen. Furthermore, incubation of spermatozoa with mucus results in a significant decrement in these reducing substances beyond that for incubated mucus alone. 9 If the reducing substances in cervical mucus were utilizable carbohydrates, the cervical mucus would present spermatozoa with an environment favorable for their nutrition and migration, and one could thus explain the sperm longevity of seventy-two hours in this new medium.26 The present study is concerned with the identification and quantitative estimation by chromatographic methods of individual sugars present in the cervical mucus during various phases of the menstrual cycle. Cervical mucus is defined as that material which can be aspirated from the cervical canal at the os. METHODS Mucus was obtained by aspiration from cervices of young healthy women having normal pelvic structures and normal menstrual cycles of from days. Samples were collected daily, beginning shortly after the end of the menstrual period, through the phase of increased secretion, and as long as samples large enough for analysis could be obtained. Specimens weighing less than 25 mg. proved to be too small for most analyses. Precautions were taken to avoid vaginal contamination of the specimens during collection, and specimens contaminated with blood were not employed. Free simple sugars were extracted from the whole mucus specimen by 95 per cent ethyl alcohol at a final concentration of 8 per cent. Loss of these sugars by adsorption on the protein-polysaccharide residue was negligible, as evidenced by the recovery of sugars added to the mucus in known amounts before the alcohol extraction. The extract from each specimen was concentrated and applied in toto to the chromatogram which was developed in butanol-acetic acid and sprayed with a benzidine-acetic acid reagent6 for location of the sugar spots. Estimation of the amount of each sugar was made by comparison with a series of known sugars chromatographed simultaneously. Therefore, all statements of amount are approximations. The. lower

3 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 31 limits of detection under the conditions used were 3 micrograms for glucose and 5 micrograms for maltose. The protein-polysaccharide residue from the alcohol extraction was incubated with.5 ml. of filtered saliva for two hours at 37 C. for digestion of glycogen. The glucose and maltose thus produced from the glycogen were extracted from the remaining proteins and polysaccharides by 95 per cent alcohol at a final concentration of 8 per cent. This extract was concentrated and applied to the chromatogram. An alcohol extract of.5 ml. of saliva was run simultaneously as a control, and chromatograms were developed as with free simple sugars. An estimation of the amount of glucose and maltose on the chromatogram was taken as a measure of the glycogen originally present. Recovery as determined from known amounts of pure glycogen carried through the same digestion and chromatographic procedure was at least 9 per cent. Other polysaccharides present in the mucus were determined on the protein-polysaccharide precipitate remaining after digestion of the glycogen. This precipitate was hydrolyzed with 1 ml. of various concentrations of hydrochloric acid (.1 N,.5 N, 1 N, 4 N, and 6 N) at 1 C. for various periods of time from one to twenty-four hours. Humin formed during the hydrolysis was removed by centrifugation and the hydrochloric acid removed as completely as possible from the supernatant by evaporation in a vacuum desiccator. The residue was taken up in water for application to the chromatogram. Of the pairs of solvents tried for the two-dimensional chromatograms, phenol and butanol-acetic acid appeared to give the best separation of all the sugars present. Three reagents were of particular usefulness in location and identification of the sugar spots: benzidine-acetic acid, silver nitrate followed by sodium thiosulfate,21 and acetylacetone followed by p-dimethylaminobenzaldehyde. Diastase activity of the mucus was determined by the Somogyi method employing incubation of the mucus sample with added starch, reaction of the reducing sugars produced with an alkaline copper reagent, and iodometric titration.23 Two modifications of the method were employed, however: (1) For determination of reducing substances originally present before diastase breakdown of the starch began, a control containing' the complete reaction mixture, including half of each mucus sample, and deproteinized at zero time was used instead of the Somogyi control lacking starch. (2) A titration blank carried through the same procedure as the experimental

4 32 BRECKENRIDGE & POMMERENKE [Fertility & Sterility samples, but without added starch, was used in place of a water blank. Diastase activity is expressed as mg. glucose produced from the added starch by 1 ml. of mucus in thirty minutes. RESULTS Ninety-nine specimens from 6 subjects followed through a total of fourteen menstrual cycles showed up to 65 mg. per cent glucose per specimen in the alcohol extract (Fig. 1). The total amount of glucose per specimen is 7 6 r- 5 r- \oj II). u :::>..J.,e " 3 :IE 2. I. FIGURE 1. 1 I : to, I I I ; I I 16 I t BEFORE MAXIMUM SECRETION AFTER DAYS Concentration of glucose per specimen in relation to phase of increased mucus secretion. given in Figure 2. The variations in glucose concentration in individual cycles are illustrated in Figure 3 by three typical curves. The only other free simple sugar present in the mucus in amounts detectable on the chromatogram was maltose, which occurred at concentrations up to 8 mg. per cent in approximately half of the specimens examined (Fig. 4). The total amount of maltose per specimen is given in Figure 5. Final identification of the maltose followed elution of the spot from the chromatogram, hydrolysis, and rechromatography-a procedure which yielded only glucose. Fructose was absent from the mucus as evidenced by spraying free sugar chromatograms with a phloroglucinol rea gene which reacts with keto sugars.

5 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 33 Individual menstrual cycles show little consistent phasic tendency either in absolute amount or in concentration of the free sugars glucose and mal :::;; Ul 3 a: \ll2 8 ::i ' Cl 'l OL FIGURE 2. BEFORE DAYS MAXIMUM SECRETION AFTER Total amount of glucose per mucus specimen. 2Or , ' M " DAYS BEFORE NEXT MENSES FIGURE 3. Glucose concentration in three typical cycles. The day of maximum secretion is circled in each graph. In the top graph, samples on days 2 and 19 showed no glucose on the chromatogram. The samples were so small that the absolute amount of glucose present may have been below the sensitivity of the method. The broken line indicates the maximum concentration which could have been present without showing on the chromatogram. tose. However, the over-all picture as shown in Figures 1 and 4 suggests some decrease in concentration in the phase of increased mucus secretion. Any tendency toward a cyclic variation in concentration is apparent only in M

6 34,BRECKENRIDGE & POMMERENKE [Fertility & Sterility relation to this phase of increased mucus secretion and not in relation to the number of days before or after a menstrual period. The middle of the phase + 8 t- 7t- 6 t- 3-2 lot-. t. 1..l 1 1. i t BEFORE MAXIMUM SECRETION AFTER FIGURE 4. DAYS Concentration of maltose per mucus specimen z u on It:... on g o16l , t BEFORE MAXIMUM SECRETION AFTER FIGURE 5. DAYS Total quantity of maltose per mucus specimen. of increased secretion occurred between 16 and 9 days before the beginning of the next menstrual period in this series.

7 Vol. 2, No.1, ANALYSIS OF CARBOHYDRATES 35 Up to 157 mg. per cent glycogen was found in 63 specimens examined from eight cycles on 5 subjects (Fig. 6). Total amount of glycogen per specimen is shown in Figure 7. In most individual cycles the pattern of glucose, maltose, and glycogen variations followed each other rather closely. Figure 8 illustrates a typical cycle. The co-existence of glycogen, maltose, and glucose in the mucus suggested 9 t l7 }1I8 till z '" <.> 4 '" 3 '" " : l' BEFORE MAXIMUM SECRETION AFTER FIGURE 6. Concentration of glycogen per mucus specimen. Results from three cycles on one subject, who almost consistently showed higher absolute amounts and higher concentrations of glycogen and greater variation in these between phases of the cycle than did the other subjects, are shown by a vertical line through the dot. DAYS the presence of a diastase. Nineteen specimens from 3 subjects, representing various phases of the menstrual cycle, were found to have a diastase activity up to 5 mg. glucose/loo m!. mucus/3 minutes without dependence on phase of the cycle (Table 1). This is up to 4 times the diastase activity of normal blood. 25 Figure 9 illustrates a typical chromatogram of the hydrolyzed nonglycogen polysaccharide fraction of cervical mucus. By addition of known sugars to the hydrolyzed polysaccharide mixture before chromatographing,

8 36 BRECKENRIDGE & POMMERENKE [Fertility & Sterility TABLE 1. Number of specimens Average diastase activity (mg. glucose/ioo ml. mucus/3 min.) Range of activity Diastase Activity of Cervical Mucus Preovulatory Ovulatory Postovulatory or Il. '" Z 4 '".., o <> 3 C> ;,! 2 1 to It BEFORE MAXIMUM SECRETION AFTER DAYS FIGURE 7. Total amount of glycogen per mucus specimen. (See notation under Fig. 6.) 8 spot B was identified as glucosamine, spot C as galactose, and spot E as fucose. Confirmatory evidence for these identifications was obtained by migration in additional solvents and by the differences in reaction of the various sugars with the spraying reagents. Hexuronic acids were absent. Spots A and D could not be identified as any of the known sugars. Spot D migrates to the position of mannose in butanol-acetic acid and in collidinelutidine, but not as far as mannose in phenol. Elution of the spot from the chromatogram and rechromatographing one-dimensionally in phenol with known mannose showed this difference still more precisely.

9 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 37 It was noted that browning of the hydrolysis mixture and humin formation was evident even after the first twenty minutes of hydrolysis. Reaction of sugars with certain amino acids (lysine, glycine, arginine) has been reported to be an intermediate step in this humin formation. 3 These amino acid-sugar reaction products have been shown to react with acetylacetone followed by p-dimethylaminobenzaldehyde, with the p-dimethylaminobenzaldehyde alone, and with silver nitrate, as well as with ninhydrin. 3,5 In the present experiments some amino acids as well as sugars were shown chromatograph- 6 5 It I( A------f, GLU CO SE MALTOSE GLYCOGEN ", DAYS BEFORE NEXT MENSES FIGURE 8. Glycogen, maltose, and glucose concentrations in a typical cycle. The circle indicates the day of maximum secretion. The broken lines for day 8 show the maximum concentrations of glucose and maltose which could have been present since none appeared on the chromatogram of this small sample. ically to be released even on short hydrolysis of the protein-polysaccharide fraction of the cervical mucus. Hydrolysis products on the chromatogram, in addition to glucosamine, were found to react with acetylacetone followed by p-dimethylaminobenzaldehyde and with the p-dimethylaminobenzaldehyde alone. These facts suggested that the two unidentified spots on the hydrolyzed polysaccharide chromatogram might be due to such amino acidsugar reaction products. To test this hypothesis, polysaccharide hydrolysis conditions were simulated but pure samples of galactose, fucose, glucosamine, or glucose with the reactable amino acids, all of which are liberated from the mucus protein on hydrolysis,16 were substituted for the protein-polysaccharide substrate. Each yielded amino acid-sugar reaction products detectable on the chromatogram with the benzidine and the silver nitrate

10 38 BRECKENRIDGE & POMMERENKE [Fertility & Sterility reagents, but the reaction appeared to proceed at different rates for the different sugars. A single two-dimensional chromatogram of all four mixtures was essentially identical with the experimental chromatograms from the hydrolyzed cervical mucus polysaccharides, showing all five spots and several additional faint ones which appear occasionally on the hydrolyzed r.w-.. \) \) <C( t..i e B Z 4: 5 CD A FIGURE 9. Chromatogram of the hydrolyzed non-glycogen polysaccharide of cervical mucus. A, amino acid-sugar reaction product formed during hydrolysis; B, glucosamine; C, galactose; D, another amino acid-sugar reaction product formed during hydrolysis; E, fucose. polysaccharide chromatograms. Therefore it seems likely that A and Dare amino acid-sugar reaction products rather than additional free sugars. The five spots shown in Figure 9 were present after hydrolyses with concentrations of hydrochloric acid from.1 N to 1 N and with all hydrolysis times from one to twenty-four hours. The intensity of spots increased as length of hydrolysis increased. After hydrolysis with 4 N Hel for eight hours only spot B, glucosamine, remained, as might be expected. Hydrolysis

11 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 39 of the total protein-polysaccharide fraction without previous digestion of the glycogen showed only one difference in the picture-a glucose spot between spots C and D and shifted slightly to the left. On cycles chroma tographed one-dimensionally before glycogen removal, the galactose to fucose ratio was consistently about 2:1 while the glucose to fucose ratio varied from 4:1 to 1:3 in individual specimens. 2C FIGURE 1. w <Jl 14 ::J lj <9 1.. ::;: C C t.. r t t BEFORE MAXIMUM SECRETION AFTER DAYS Concentration of fucose per hydrolyzed mucus specimen (giving estimate of relative concentration of mucopolysaccharide). The only polysaccharide other than glycogen present in the cervical mucus in amounts detectable by present methods is probably, then, a mucopolysaccharide consisting of galactose, fucose, and glucosamine. That this is a neutral polysaccharide was further evidenced by its failure to precipitate with protein in dilute acetic acid. It is also alkali-labile, releasing marked amounts of sugar in twenty minutes at 1 C. with 2 per cent

12 4 BRECKENRIDGE & POMMERENKE [Fertility & Sterility NaHC3-Na2C3. The viscosity of an alkaline solution of cervical mucus rapidly decreases even at room temperature, indicating degradation of the mucopolysaccharide. An estimate of the relative concentration of this mucopolysaccharide in the cervical mucus throughout the intermenstrual cycle was obtained from the amount of fucose produced on hydrolysis with 1 ml. of.5 N HCI at 1 C. for twenty-four hours without prior glycogen digestion. Sixty-four specimens from nine cycles on 7 subjects showed a definite decrease in concentration of the mucopolysaccharide as the amount of secretion increased and the viscosity decreased (Fig. 1). DISCUSSION The trend reported here for a decrease in concentration of the individual free simple sugars in the mucus in the phase of increased secretion, regarded as the ovulatory phase, follows the finding of Viergiver and Pommerenke 29 that the total free reducing substances in the mucus as measured by the Somogyi copper reagent, show a decrease in this phase. However, the sum of the free sugars as determined by chromatography is considerably less than the total free reducing substances determined by the Somogyi method. This is apparently due in part at least to the fact that the mucopolysaccharide in the mucus is alkali-labile and releases reducing sugar during the heating period with the alkaline copper reagent. That the protein of the mucus may also contribute to the total copper reducing power, as previously suggested by Pommerenke,17 is also a strong possibility. The over-all picture in the total amount of glucose and maltose per specimen collected at one aspiration shows no marked cyclic change (Figs. 3 and 6). In evaluating these results, several factors must be considered, however. During the pre- and post-ovulatory phases when the mucus is highly viscid, a greater per cent of the total amount produced in twenty-four hours can be aspirated at one time than during the ovulatory phase when much of the fluid mucus escapes spontaneously into the vagina. Also the longer the mucus has remained in the cervical canal before aspiration, the greater has been its exposure to bacterial and enzyme action. Both of these factors may affect the absolute amount of sugar in the specimen as collected. Therefore little can be concluded about the absolute amounts of sugars produced throughout various phases of the menstrual cycle unless the entire twenty-four-hour production of mucus can be collected at one time. For sperm motility, concentration of utilizable sugars present at a given time is the important factor, not the total amount of the sugars produced. Atkinson et al. have reported the absence of glycogen in the cervical mucus of the lumen, as determined histologically, and conclude that the glucose produced on hydrolysis of aspirated mucus comes from the mucopolysaccharide. However, the following considerations confirm previous evidence17 that the glucose-containing polysaccharide in the mucus must be glycogen or a glycogen-like molecule:

13 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 41 (1) When polysaccharides of the mucus are followed through various phases of the menstrual cycle by hydrolysis and chromatography, the ratio of galactose to fucose is constant while the ratio of glucose to fucose is highly variable. (2) The glucose is entirely removed from the combined proteins and polysaccharides of the mucus with filtered saliva, with no other change in the sugars of this polysaccharide fraction. (3) Salivary amylase is specific for 1,4- (and, controversially, 1,6-) a-glucosidic linkages as in glycogen and starch. The source of the glycogen in the cervical mucus remains a question. The endocervical epithelium, to even a greater degree than the isthmus epithelium,22 appears to contain no appreciable glycogen.1,2 The squamous epithelium of the ectocervix normally contains glycogen,19 but whether this may contaminate the cervical mucus within the canal is undetermined. The extent to which glycogen from the uterus could appear in the aspirated cervical mucus would depend on a number of factors: the cyclic variation in glycogen in the endometrium with the peak sometime after ovulation;2,3 the production of uterine fluid, which experimentally appears to vary with hyperemia of the uterus;27 the amount of uterine fluid which descends to the cervix in various phases of the menstrual cycle; the amount of glycogen which this uterine fluid carries to the cervix; and what per. centage of this uterine fluid and glycogen is collected with the mucus obtained at a single aspiration. Determinations on specimens derived from subjects on whom supracervical hysterectomy has been performed should clarify some of these points. Whatever the source of the glycogen in the cervical mucus, it does seem likely that a number of factors must be operative in determining the concentration since glycogen, maltose, and glucose concentrations usually show a somewhat erratic pattern in any single cycle instead of a rather smooth decrease and subsequent increase like the mucopolysaccharide. All three of these sugars-glucose, maltose, and glycogen-are utilizable by once-washed spermatozoa for energy for motility. MacLeod reports that further washing destroys the ability to utilize glycogen. He has shown that the range of effective sugar concentrations for spermatozoa is 2-2 mg. per cent.l The concentration of total utilizable sugars in cervical mucus is within this range, usually in the lower part of it, and shows, if anything, a tendency to be slightly lower during the phase of increased secretion and decreased viscosity of the mucus. Penetrability of the mucus by spermatozoa has been noted to be maximal at this time.28 One may conclude, then, that concentration of nutritive sugars is not a limiting factor in this cyclic penetrability. However, concentrations of utilizable sugars below the effective range noted by MacLeod may well contribute to some cases of sterility. The diastase in the mucus probably controls the relative concentrations of the three carbohydrates glycogen, maltose, and glucose, depending on accessibility of the substrate glycogen to the enzyme and the factor of time. Substrate and enzyme concentrations may also have an effect on the proportions of the three sugars, as with salivary, pancreatic, and urine amylases.4,24 Breakdown of the glycogen by this diastase would leave unaccounted by the methods of estimation used here, one small fraction of carbohydrates which would be potentially sperm-utilizable. Dextrins too small to be precipitated with the

14 42 BRECKENRIDGE & POMMERENKE [Fertility & Sterility glycogen by alcohol would be present in the alcohol extract of the mucus. However, these dextrins may be too large to migrate to any appreciable degree on the chromatogram or may have too few reducing groups in the total amount to react to a detectable degree with the spraying reagent. Occasionally a spot of low migration, possibly a trisaccharide, was seen on the chromatogram of free sugars, along with the glucose and maltose. In the action of diastase on glycogen and starch, glucose is produced not by splitting of the maltose but by removal of glucose units from certain dextrins. The concentration of glucose among the resulting products of diastase action is always considerably below that of maltose, regardless of time of enzyme action.24 If diastase alone were active in this breakdown of glycogen in the cervical mucus, one would expect to find maltose as the predominant free simple sugar. However, glucose is present in higher concentrations than maltose in the majority of cases, while following the same variations. Therefore, it is likely that a maltase is also present in the mucus. The observation that the relative concentration of mucopolysaccharide, as estimated from the fucose liberated on hydrolysis, decreases markedly in the phase of increased mucus secretion and decreased viscosity would be anticipated from the fact that the percentage water content increases during this ovulatory phase, and also from the fact that viscosity of mucus secretions appears to be associated principally with the mucopolysaccharide. Since such a polysaccharide is undoubtedly not sperm-utilizable, its chief ehect on sperm migration through the cervical canal probably results from its relationship to viscosity. On the basis of fermentation studies, Viergiver and Pommerenke first reported evidence for this polysaccharide other than glycogen in the cervical mucus.29 Shettles and Dische, using a spectrophotometric method following hydrolysis of whole specimens of mid-cycle cervical mucus, reported that up to 2 per cent of the carbohydrate consisted of methyl pentose and up to 55 per cent of galactose, the remainder being glucose. Certain obvious difficulties are inherent in the determination of the exact percentage composition of the mucopolysaccharide. The reaction between the sugars and amino acids liberated during hydrolysis, the fact that these reactions proceed at diherent rates for the diherent sugars, and the reaction of liberated sugars with the protein itself8, 15 would alter the relative as well as the absolute amounts of the sugars remaining to be measured at the termination of hydrolysis. The fact that the amino acid-sugar reaction products supplement the actual glucosamine in the standard quantitative method for glucosamine5 must also enter into consideration. SUMMARY Free simple sugars in human cervical mucus have been identified as glucose and maltose and have been quantitatively estimated through various phases of the menstrual cycle by means of paper partition chromatography. The only cyclic variation noted was some tendency for a decrease in

15 Vol. 2, No.1, 1951] ANALYSIS OF CARBOHYDRATES 43 concentration of these sugars in the phase of increased mucus secretion, regarded as the ovulatory phase. The glycogen content of the mucus has been followed through various phases of the menstrual cycle by salivary amylase digestion and subsequent chromatography of the glucose and maltose produced. Total concentration of nutritive sugars appears not to be a limiting factor in the cyclic penetrability of cervical mucus by spermatozoa. The mucus was found to have significant diastase activity without dependence on phase of the menstrual cycle. An alkali-labile, neutral mucopolysaccharide containing galactose, fucose, and glucosamine was found to be the remaining detectable carbohydrate constituent of the cervical mucus. Concentration of this polysaccharide shows a marked decrease with the increased mucus secretion of the ovulatory phase. The significance of these results, especially as they relate to sperm migration through the cervix, is discussed. During the final preparation of this manuscript, an advance reprint of a paper by Bergman and Weiner in Acta obstet. gynec. Scandinav. 195, has come to the attention of the authors. In this they report the presence of galactose, mannose, fucose, hexosamine, and glucose in the polysaccharide fraction of human cervical mucus. The glucose was believed to come from a glycogenic admixture. REFERENCES 1. Atkinson, W. B., ShettIes, L. B., and Engle, E. T.: Am. J. Obst. & Gynec. 56:712, Driessen, L. F.: Zentralbl. f. Gynak. 35:138, Gottschalk, A., and Partridge, S. M.: Nature, London 165:684, Hanes, C. S., and Cattle, M.: Proc. Roy. Soc., London, s. B. 125:387, Horowitz, H. N., Ikawa, M., and Fling, M.: Arch. Biochem. 25:226, Horrocks, R. H.: Nature, London 164:444, Horrocks, R. H., and Manning, G. B.: Lancet 256:142, Lea, C. H., and Hannan, R. S.: Nature, London 165:438, Lipphardt, E. M., and Pommerenke, W. T.: Am. J. Obst. & Gynec. 59:918, MacLeod, J.: Am. J. Physiol. 132:193, MacLeod, J.: Endocrinology 29:583, MacLeod, J.: Human Fertil. 7:129, MacLeod, J.: Am. J. Physiol. 138:512, Mann, T.: Biochem. J. 4:481, Mohammad, A., Fraenkel-Conrat, H., and Olcott, H. S.: Arch. Biochem. 24:157, Pederson, D. P.: Unpublished.

16 44 BRECKENRIDGE & POMMERENKE [Fertility & Sterility 17. Pommerenke, W. T.: Am. J. Obst. & Gynec. 52:123, Pommerenke, W. T., and Viergiver, E.: Proc. Soc. Exper. BioI. & Med. 66:161, Schiller, W.: Lancet 23:1228, Shettles, L. B., and Dische, Z.: Federation Proc. 7:114, Simmons, N. S.: Unpublished. 22. Simon, H. J.: Am. J. Obst. & Gynec. 27:284, Somogyi, M.: J. BioI. Chern. 125:399, Somogyi, M.: J. BioI. Chern. 134:31, Somogyi, M.: J. BioI. Chern. 134:315, Stein, I. F., and Cohen, M. R.: Fertility and Sterility 1:169, Sturgis, S. H.: Endocrinology 31:664, Viergiver, E., and Pommerenke, W. T.: Am. J. Obst. & Gynec. 51:192, Viergiver, E., and Pommerenke, W. T.: Am. J. Obst. & Gynec. 54:459, Wegelin, c.: ZentralbI. all. Path. u. path. Anat. 22:1, DISCUSSION DR. CARL HARTMAN, Raritan, New Jersey: I was interested to learn that there is glycogen in the cervical mucus and also amylases to digest them. Grafenberg years ago commented on this. Perhaps these amylases are able to digest the polysaccharides of exfoliated vaginal cells. These polysaccharides are not glycogen and are not digestible with saliva nor malt diastase but are with Taka-diastase. We shall see if they dissolve with amylases of cervical mucus. DR. JOHN MACLEOD, New York City: First of all, I shall say that I have a great admiration for Dr. Pommerenke and his persistence in going after the cervical mucus problem. We accept the fact (and I will dismiss it rather briefly) that the glucose concentration in cervical mucus probably acts as a substrate for the spermatozoa so that motility can be maintained for long period. I am much more interested in this acid problem, but have a rather different point of view. First of all, while amino acids are not necessary in providing energy for fertility, certain amino acids may be highly important in another way, namely, the protection of motility in the spermatozoa. The amino acids Dr. Pommerenke has described this afternoon are all right; they are good things to have, but there are two or three others that I wish Dr. Pommerenke and his colleagues would look for because they may have a very definite function in protecting the sperma-. tozoa. I refer to the sulfhydril amino acids, such as cysteine and glutamine. Dr. Pommerenke has described methionene which is -SH containing acid, but he describes it as being in rather negligibly small quantities. I wish that he would look further because I believe it will eventually be shown that such amino acids in the mucus may act as highly protective substances against toxic substances, substances which may affect the spermatozoa. They are very important from my point of view.