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Metabolism of Human Spermatozoa in Semen
Metabolism of Human Spermatozoa in Semen M. Edward Davis, M.D., and William W. McCune, M.D.
of mammalian spermatozoa have been largely confined to the last two decades. There are two factors responsible for this recent interest; problems arising from artificial insemination of livestock and, in the case of human spermatozoa, the realization that in 40 to 50 per cent of all barren marriages, the husband is the responsible party. Spermatozoa are readily accessible for study in their own physiologic environment, where their energy exchange can be correlated with their motility. This property adapts them well to metabolic studies. Probably the first report on the metabolism of human spermatozoa was that of McCarthy et al. They determined changes in lactic acid and glucose levels in semen after incubation at 38° C., and concluded that lactic acid production was not sufficient to account for all of the glucose utilization. Their findings were confirmed by Goldblatt. These authors minimized the significance of bacterial contamination, which undoubtedly introduced a large error in their experiments. 0 In 1939, MacLeod, 11 using the Warburg manometric technic, concluded that the metabolism of spermatozoa suspended in Ringer solution is preSTUDIES OF THE PHYSIOLOGY
From the Chicago Lying-In Hospital and the Division of Chemistry, Department of Medicine, University of Chicago. The authors gratefully acknowledge the valuable assistance and advice of Dr. E. S. G. Barron and his co-workers, of the Department of Medicine, Division of Chemistry, University of Chicago, who made available the facilities of their laboratory for this work. This work was done under a grant from the Douglas Smith Foundation for Medical Research and the Joseph Bolivar DeLee Research Fund, the University of Chicago. • During the course of the present work, we noted that semen without penicillin, incubated at 38° C., shows a logarithmic acceleration in respiration corresponding with bacterial growth, which starts after two or three hours of incubation. 158
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HUMAN SPERMATOZOA IN SEMEN
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dominantly glycolytic, and that the rate of anaerobic glycolysis is higher and more sustained than aerobic glycolysis. In a more recent report, 15 he gave evidence suggesting that the apparent inhibition of glycolysis by 02 might be due to the formation of H202. He found 13 that when glycolysis is inhibited by monoiodoacetate and sodium fluoride, motility is correspondingly inhibited. Both motility and the rate of glycolysis were accelerated by the addition of glucose to an exhausted specimen. He concluded from these observations that the energy for motility is the result of a glycolytic process. He later demonstrated15 that, in spite of the low rate of respiration of spermatozoa under normal conditions, they possess a complete cytochrome system. Opinion concerning the degree of respiration and its role in the physiology of spermatozoa is varied. Shettles reported a relatively high rate of respiration of human spermatozoa in semen, and concluded that the rate of respiration varied inversely with the age of the specimen. It has been demonstrated that semen devoid of spermatozoa takes up 02, and MacLeod12 has suggested that this oxygen uptake is simply an absorption of 02 by seminal fluid itself. However, Ross et al., after correcting the total oxygen uptake for respiration of the seminal fluid, were able to demonstrate a small, but significant oxygen uptake by spermatozoa. They studied the spermatozoa from three groups of fertile, probably fertile,· and subfertile men, and concluded that there was no significant difference in the aerobic glycolysis of spermatozoa from each of these groups, and that although the oxygen uptake of spermatozoa in semen was higher in the fertile group, the number of experiments was too small to enable them to draw any definite conclusions. Walton and Edwards, 25 and Walton, 24 showed that in sheep, cattle, and horses, respiration of spermatozoa can be used as a criterion of sperm motility, and that motility is closely related to fertility in these animals. T. Mann17 recently proved that the reducing substance in the seminal fluid of various mammals, including man, is fructose, that the origin of fructose is in the seminal vesicles, and that its formation is controlled by the testicular hormone, testosterone. 18 The importance of sulfhydryl groups in cellular metabolism has been demonstrated in a large number of investigations. Barron and Singer3· 22 •23 found that the presence of -SH groups was essential for the activity of many
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enzymes concerned with the metabolism of cells. Barron et aU also demonstrated that sulfhydryl reagents in small concentrations increased respiration. They concluded that the soluble sulfhydryl groups must regulate respiration by inhibiting the rate of reoxidation of the cytochrome system, and that in small concentration the sulfhydryl reagents combined with these groups abolishing this regulating mechanism of respiration. In higher concentrations, the sulfhydryl reagents combined with fixed sulfhydryl groups in the side chains of the protein moiety of enzymes, thus inactivating these enzymes, which are essential to respiration. There are presented in this paper data on the 02 uptake and fructolysis of human spermatozoa, and the effect of small concentrations of sulfhydryl reagents.
..
METHODS Semen specimens were obtained from husbands of sterility patients being studied at Chicago Lying-In Hospital. Husbands were instructed in the care to be exercised in collecting specimens at home, by coitus interruptus. The age of the specimens at the beginning of experiments ranged from one and one-haH to four hours. All specimens were examined routinely as soon as they arrived in the laboratory, and count, volume, motility, and morphology were determined. They were classified as normal, subfertile, probably sterile, or sterile. Motility was recorded in terms of per cent motile and quality of motility ( 1 plus to 4 plus), according to the method described by Hotchkiss. It was found that the percentage of motile forms could most accurately and simply be determined by employing a cross-hatched micrometer disc in the microscope ocular. Approximately 5 to 20 nonmotile cells in a given number of squares (depending on the density of cells in the field) are counted, then the number of motile cells in the same number of squares is counted. This procedure is repeated three times, using different fields, and the results averaged. Counts were determined and morphology studied according to the methods described by Hotchkiss, except that the triple-stain technic of Keaty and Hamblen was used to stain smears for morphological study. Oxygen Uptake
Semen was buffered to pH 7.4 with phosphate buffer ( 0.1 cc., one-tenthmolar buffer to 1 cc. semen), 1 cc. portions of the sample were placed in
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small ( 5 cc.) Warburg vessels, with 0.1 cc. of KOH in the center cup. The vessels were equilibrated at 38 ° C. for 10 minutes. Experiments were run in duplicate and triplicate, depending on the volume of the specimen, and readings taken at 15-minute intervals.
...
Glycolysis One cubic-centimeter samples of the specimen were placed in small Warburg vessels without center cup, and the system saturated with a mixture of 95 per cent N2 and 5 per cent C02 for 10 minutes. Because of the poor gas exchange in a viscous medium, those specimens in which complete liquefaction had not taken place were not used. In almost all of the experiments, it was found that saturation of the semen with N2-C02 had taken place within 10 minutes, if the vessels were agitated constantly during the passage of the gas mixture through the system. In all of the experiments manometric measurements were pedormed on spermatozoa in whole semen. C02 Retention by Seminal Fluid The figures given for the anaerobic glycolysis have not been corrected for the C02 bound to semen protein. To measure this C02 retention, 1 cc. of cell-free seminal fluid was added to the small Warburg vessel, and 0.05 to 0.1 cc. of a lactic acid solution of known molarity was added to the sidearm. Mter saturation with N2-C02 mixture, the vessels were equilibrated in the bath at 38 o C. for 10 minutes. After equilibration the lactic acid was added to the seminal fluid. The difference between the actual and theoretical liberation of C02, divided by the theoretical figure, gives the per cent of C02 retained by the seminal fluid. The results were confirmed by chemical analysis, in which fructose was determined immediately before and after glycolysis. Bicarbonate in Seminal Fluid Bicarbonate was measured manometrically by a method similar to that described above for C02 retention. To the unaltered, cell-free seminal fluid 0.3 cc. of one-tenth normal HCl was added from the side-arm. The C02 given off represents an equal molar concentration of HCOa- ions in the seminal fluid.
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Fructose
Fructose was determined according to the method of Roe with the Coleman spectrophotometer at 490 mp.. Protein-free filtrates of the semen were obtained by diluting 0.1 cc. semen to 4 cc. with water, adding 2 cc. 10 per cent ZnS04 and 2 cc., one-tenth normal NaOH, and heating in boiling water for two minutes. In some cases, where a turbid supernatant indicated incomplete precipitation of the protein, an additional 1 cc. of ZnS04 and 1 cc. of NaOH were added. The solution was then filtered through No. 40 Whatman filter paper. TABLE 1.
Experiment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
P'/2 3 4 2 3"/2 P'/2 2 H4
m
2 21'/4 P'/2 2 4 1 3 2 1 3* 1* 3* 21'/4 3 2 2* 3 4 3* 2
Million Cells per cc. Semen
98 90 111 122 83 82 62 136 60 216 265 73 55 251 78 76 66 44 323 68 210 64 124 64 82 47 73 144 240
•
Respiration of Normal Spermatozoa
Motility Age (hours)
..
Motile (per cent)
50 75 85
75 90 60 65 80 75 50 60 70 60 55 90 70 75 90 80 90 75 90 80 80 80 70
0 2 Uptake per Hr. per 108 cells Quality
(c.mm.)
++ +++ ++ +++ ++++ ++++ ++ ++++ ++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++++ ++ +++ ++++ ++++ ++++ +++ ++++ ++ ++++ +++ ++
26.5 10.5 4.5 9.4 11.0 17.8 16.9 5.9 14.7 5.7 4.0 8.5 8.9 8.0 11.0 17.8 19.7 26.8 4.2 19.0 6.7 11.3 1.8 6.3 9.0 18.5 9.3 13.2 5.4
..
..
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..
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•
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HUMAN SPERMATOZOA IN SEMEN
RESULTS
02 Uptake Table 1 gives the results of individual experiments in which the oxygen utilization of 29 normal semen specimens was determined by the method described. It will be noted that the motility or cell concentrations of several of the specimens listed is lower than the value usually considered as normal, but in those instances the over-all evaluation of the specimens (age,
~
'
TABLE 2.
Effect of Cell Concentration, Age, and Motility on Respiration and Glycolysis of Spermatozoa
Glycolysis Respiration Oz per Hr. per C02 per Hr. per 108 Cells 108 Cells Number of Number of Experiments (c.mm.) Experiments (c.mm.)
!-. .,.
'
~
i
+
I
.
'
I ~
•
Count per cc. X 10-o Less than 100 Greater than 100 Age, hours 1 to Hf 2 to 2~f 3 to 4 Motility I 90%++++ II 75%++++ to 85%++++ III 50%++++ to 70%++++ 75%+++ to 90%+++ IV 40%+++ to70%+++ to 90%++ 75%++ v 10%++ to 70++ Classification of Specimen Normal Subfertile
18 11
14.1 6.3
7 9
23.5 23.6
8 10 11
16.3 9.7 9.6
7 6 3
30.1 18.3 19.2
5 4 5
10.0 8.3 11.6
0 3 3
24.3 18.5
9
14.7
4
20.5
3
8.1
6
27.9
29 0
11.1
8 8
24.1 23.1
morphology, volume, etc.), placed them within normal limits. The results are not corrected for respiration of the cell-free seminal fluid, the volume of most of the semen samples being too small to permit the determination of this error in each experiment. The average 02 uptake of 2 cell-free seminal fluid specimens was found to be 3.4 c.mm. 02, + 0.4. The mean, uncorrected oxygen uptake, with mean deviation, of the 29 experiments listed in Table 1 is 11.1 c.mm. + 5.2, 02 per 108 cells per hour (range, 1.8 to 26.8). The wide variation in the oxygen uptake of individual specimens cannot be explained
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TABLE 3.
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Effect of Sulfhydryl Reagents in Small Concentration on the Respiration of Human Sperm
02 Uptake per Hr. per lQH Cells Sulfhydryl Concentration Control Reagent (Molar) (c.mm.) (c.mm.)
Sulfhydryl Reagent
5X 5 X 5X 5 X 5X
Iodoacetamide p-chloromercuribenzoate p-chloromercuribenzoate Arsenite Cadmium chloride
I0-5 I0-5
10-4 I0- 5
10-5
16.1 10.5 4.5 17.1 8.1
Increase (per cent)
26.5 12.0 1.8 17.8 11.6
39.3 12.5 -82.0 3.9 30.1
on the basis of differences in motility, but it is apparent that the rate of oxygen uptake is much higher in specimens having relatively low counts, and in fresh specimens (Table 2). It must be recalled that Barron and Harrop found that overcrowding diminished the respiration of leukocytes. In agreement with the findings of Barron et aP on sea urchin sperm, small concentrations of four sulfhydryl reagents increased the respiration of spermatozoa, as shown in Table 3. One of the reagents, p-chloromercuribenzoate, caused an inhibition of respiration in higher concentration. TABLE 4.
Classification Subfertile Fertile
Glycolysis of Normal and Subfertile Spermatozoa
Age (hours)
X
3
X
2~ X X
X X
X X X X X X
P'/2 P'/4 3 2 3 2 2 21'/4
m B4
X
1 lJf 2
X
P'/2
X X
Motility Million Cells Motile per cc. Semen (per cent) Quality
86 196 99 55 119 77 219 102 88 150 72 67 185 234 286 222
80 80 25 33 36 55 75 40 48 46 9 66 80 75 78 60
+++ ++++ ++ ++ ++ +++ ++++ +++ +++ ++ ++ +++ ++++ +++ +++ ++
C02 per Hr. per 108 Cells (c.mm.)
17.4 16.3 26.2 36.4 25.9 15.2 14.2 20.6 20.5 16.8 23.6 25.8 42.4 17.9 20.2 38.6
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Anaerobic Glycolysis The anaerobic glycolysis of 8 normal and 8 subfertile semen specimens was determined, and the results are summarized in Table 4. Determinations of C02 gave mean values, with mean deviation, of 23.6 c.mm. ± 6. 7 C02 per 108 cells per hour (range 14.2 to 42.4), for the entire group, and there is no significant difference in the glycolysis of the normal and subfertile groups (Table 2). It is also apparent (Table 2) that cell concentration has no significant effect on the rate of fructolysis, and that the rate of fructolysis is much higher in fresh specimens. TABLE 5.
Experiment
1 2 3
Manometric Determination of C0 2 Retention by Semen
Found ( c.mm.)
C02 Formation Theoretical ( c.mm.)
95.8 50.1 18.3
Bound C02 (per cent)
194.2 97.1 37.9
50.7 48.4 51.7
C02 Retention by Seminal Fluid Table 5 gives the results of 3 experiments in which it was found that the average C02 bound to seminal fluid was 50.3 per cent, ± 0.9. This large retention was confirmed by chemical analysis of fructose utilization (Table 6) by which it was found that 48.5 per cent of the C02 was bound. TABLE 6.
Chemical Determination of C0 2 Retention by Semen
C02 Formation
Experiment
Found (c.mm.)
Fructose Utilization (c.mm. C02)
Bound C02 (per cent)
1 2
109.5 43.6
238 143
53.8 43.3
Bicarbonate An excess of HCl added to cell-free seminal fluid liberated 177.5 c.mm. C02 per cc. of semen. This quantity is equivalent to 7.92 micromols of bicarbonate, and is in excess of the highest values recorded for glycolysis of spermatozoa in seminal fluid (Table 1).
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DISCUSSION Data concerning the rate of respiration and glycolysis of human spermatozoa in seminal fluid are limited. This may be due, in part, to the belief of many investigators that the sole function of seminal plasma is as a medium for transporting the spermatozoa to the female genital tract. The analysis of human seminal plasma by Goldblatt and by Huggins et aU· 7 • 8 has demonstrated the chemical complexity of seminal plasma, and has cast doubt on the above concept. Evidence indicates that spermatozoa are in a dormant metabolic state until they reach the vicinity of the prostate gland. 7 The comparison of the reducing sugar and lactic acid contents of fractionated ejaculates of normal semen and of semen with azoospermia16 indicates that when the spermatozoa come in contact with the secretions of certain accessory glands of reproduction, prior to ejaculation, glycolysis is initiated. The high buffering capacity of the seminal plasma 5 protects the spermatozoa against the low pH of the vaginal secretions, and the high fructose content of the seminal vesicle component of the seminal plasma17 supplies the spermatozoa with a readily utilizable substrate until they are able to reach the more hospitable environment of the cervical canal. In view of the relatively high incidence of disease of the male accessory glands of reproduction, without involvement of the testes, it is evident that the pathology in many subfertile or sterile semen specimens may be due to the harmful effect of abnormal seminal plasma on normal spermatozoa. Shettles' figures of approximately 14 c.mm. 02 uptake per 108 cells per hour compare favorably with our results. Shettles reported no respiration in seminal fluid, and his results are uncorrected for that error as are those of the present report. He concluded that the rate of respiration varies inversely with the age of the specimen and directly with the cell concentration. The data presented in this paper do not agree with his conclusion regarding cell concentration. Although there was a higher rate of respiration in fresh specimens, no direct ratio between age and respiratory rate was found (Table 2). Ross et al. reported lower values (3 to 9 c.mm. 02 per hour with 2 values of 1 c.mm. 02 and 1 of 13 c.mm. 02). Three of their experiments were corrected for blanks made on seminal fluid of the same specimens, and the remainder for the average value of blanks made on several seminal fluids. The striking difference in the rate of respiration between specimens of less than 100 million per cc. cell concentration and specimens of greater than 100 million per cc. cell concentration (Table 2) may be explained on the
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basis of the seminal fluid respiration error. This error is increased or de· creased, depending on whether one multiplies or divides in converting the observed oxygen uptake to the oxygen uptake per 108 cells. Thus, in making this conversion it is found that the error is inversely proportional to the cell concentration of the specimen. The lack of correlation between the degree of motility and the rate of respiration or glycolysis is apparent from Table 2. MacLeod 14 has demonstrated a definite relationship, in individual specimens, between changes in the degree of motility and the rate of glycolysis. In a larger series of experiments than have been undertaken in this work such a relationship would probably be found between the initial motility of a specimen and its rate of glycolysis. Search of the literature revealed only one other report on the glycolysis of human spermatozoa in seminal fluid. Ross et al. reported the results of 10 such experiments in which they found an average C02 liberation of 8.2 c.mm. C02 per 108 cells per hour, considerably lower values than ours ( 23.6). However, the ages of their specimens varied from 1 to 12 hours, and the average age was greater than ours. The slightly higher rate of glycolysis in the fertile group ( 24.1 c.mm. C02) over the subfertile group ( 23.1 c.mm. C02) undoubtedly falls within the limits of error of the method. Ross et al. were, likewise, unable to determine a correlation between glycolysis and the degree of fertility of the specimen.
SUMMARY Respiration and glycolysis of human spermatozoa in unaltered semen were measured independently by the direct method of W arburg. The mean rate of respiration of 29 normal specimens was found to be 11.1 c.mm. 02 per 108 cells per hour, and certain sulfhydryl reagents, in low concentrations, increased respiration. Fresh specimens respired at a higher rate, but no correlation between the degree of motility and the rate of respiration could be demonstrated. It was determined manometrically, and confirmed chemically, that seminal plasma retains approximately 50 per cent of the C02 liberated during glycolysis. The rate of glycolysis of spermatozoa is more nearly proportional to cell concentration than is the rate of respiration. No evidence was found, as has been reported in the case of some lower mammals, that either the degree of motility or the potential fertility of the specimen determines the rate of glycolysis. For references see p. 168.
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REFERENCES I. Barron, E. S. G., and Harrop, G. A., Jr.: J. Bioi. Chern. 84:89, 1929. 2. Barron, E. S. G., and Nelson, L., and Ardao, M. 1.: J. Gen. Physiol. 32:179, 1948. 3. Barron, E. S. G., and Singer, T. P.: J. Bioi. Chem.157:221, 1945. 4. Berg, 0. C., Huggins, C., and Hodges, C. V.: Am. J. Physiol. 133:82, 1941. 5. Goldblatt, M. W.: Biochem. J. 29:1346, 1935. 6. Hotchkiss, R. S.: Fertility in Men. Philadelphia, Lippincott, 1944. 7. Huggins, C. B., and Johnson, A. A.: Am. J. Physiol. 103:574, 1933. 8. Huggins, C. B., Scott, W. W., and Heinen, J. H.: Am. J. Physiol. 136:467, 1942. 9. Keaty, E. S., and Hamblen, E. C.: J. Clin. Endocrinol. 5:286, 1945. 10. McCarthy, J. F., Stepita, C. T., Johnson, W. B., and Killian, J. F.: J. Urol. 19:43, 1928. 11. MacLeod, J.: Proc. Soc. Exper. Bioi. & Med. 42:153, 1939. 12. MacLeod, J.: Am. J. Physiol. 132:193, 1941. 13. MacLeod, J.: Endocrinology 29:583, 1941. 14. MacLeod, J.: Human Fertil. 7:129, 1942. 15. MacLeod, J.: Am. J. Physiol. 138:512, 1943. 16. MacLeod, J., and Hotchkiss, R. S.: J. Urol. 48:225, 1942. 17. Mann, T.: Biochem. J. 40:481, 1946. 18. Mann, T., and Parsons, V.: Nature 160:294, 1947. 19. Ross, V., Miller, E. G., Jr., and Kurzrok, R.: Endocrinology 28:885, 1941. 20. Roe, J. H.: J. Bioi. Chern. 107:15, 1934. 21. Shettles, L. B.: Am. J. Physiol. 128:408, 1940. 22. Singer, T. P., and Barron, E. S. G.: Proc. Soc. Exper. Bioi. & Med. 56:120, 1944. 23. Singer, T. P., and Barron, E. S. G.: J. Bioi. Chern. 157:241, 1945. 24. Walton, A.: Commem. Spallanzaniane 4:36, 1939. 25. Walton, A., and Edwards, J.: Proc. Am. Soc. Anim. Prod. 31:254, 1938.
,
of mammalian spermatozoa have been largely confined to the last two decades. There are two factors responsible for this recent interest; problems arising from artificial insemination of livestock and, in the case of human spermatozoa, the realization that in 40 to 50 per cent of all barren marriages, the husband is the responsible party. Spermatozoa are readily accessible for study in their own physiologic environment, where their energy exchange can be correlated with their motility. This property adapts them well to metabolic studies. Probably the first report on the metabolism of human spermatozoa was that of McCarthy et al. They determined changes in lactic acid and glucose levels in semen after incubation at 38° C., and concluded that lactic acid production was not sufficient to account for all of the glucose utilization. Their findings were confirmed by Goldblatt. These authors minimized the significance of bacterial contamination, which undoubtedly introduced a large error in their experiments. 0 In 1939, MacLeod, 11 using the Warburg manometric technic, concluded that the metabolism of spermatozoa suspended in Ringer solution is preSTUDIES OF THE PHYSIOLOGY
From the Chicago Lying-In Hospital and the Division of Chemistry, Department of Medicine, University of Chicago. The authors gratefully acknowledge the valuable assistance and advice of Dr. E. S. G. Barron and his co-workers, of the Department of Medicine, Division of Chemistry, University of Chicago, who made available the facilities of their laboratory for this work. This work was done under a grant from the Douglas Smith Foundation for Medical Research and the Joseph Bolivar DeLee Research Fund, the University of Chicago. • During the course of the present work, we noted that semen without penicillin, incubated at 38° C., shows a logarithmic acceleration in respiration corresponding with bacterial growth, which starts after two or three hours of incubation. 158
Vol. 1, No. 2, 1950]
HUMAN SPERMATOZOA IN SEMEN
159
dominantly glycolytic, and that the rate of anaerobic glycolysis is higher and more sustained than aerobic glycolysis. In a more recent report, 15 he gave evidence suggesting that the apparent inhibition of glycolysis by 02 might be due to the formation of H202. He found 13 that when glycolysis is inhibited by monoiodoacetate and sodium fluoride, motility is correspondingly inhibited. Both motility and the rate of glycolysis were accelerated by the addition of glucose to an exhausted specimen. He concluded from these observations that the energy for motility is the result of a glycolytic process. He later demonstrated15 that, in spite of the low rate of respiration of spermatozoa under normal conditions, they possess a complete cytochrome system. Opinion concerning the degree of respiration and its role in the physiology of spermatozoa is varied. Shettles reported a relatively high rate of respiration of human spermatozoa in semen, and concluded that the rate of respiration varied inversely with the age of the specimen. It has been demonstrated that semen devoid of spermatozoa takes up 02, and MacLeod12 has suggested that this oxygen uptake is simply an absorption of 02 by seminal fluid itself. However, Ross et al., after correcting the total oxygen uptake for respiration of the seminal fluid, were able to demonstrate a small, but significant oxygen uptake by spermatozoa. They studied the spermatozoa from three groups of fertile, probably fertile,· and subfertile men, and concluded that there was no significant difference in the aerobic glycolysis of spermatozoa from each of these groups, and that although the oxygen uptake of spermatozoa in semen was higher in the fertile group, the number of experiments was too small to enable them to draw any definite conclusions. Walton and Edwards, 25 and Walton, 24 showed that in sheep, cattle, and horses, respiration of spermatozoa can be used as a criterion of sperm motility, and that motility is closely related to fertility in these animals. T. Mann17 recently proved that the reducing substance in the seminal fluid of various mammals, including man, is fructose, that the origin of fructose is in the seminal vesicles, and that its formation is controlled by the testicular hormone, testosterone. 18 The importance of sulfhydryl groups in cellular metabolism has been demonstrated in a large number of investigations. Barron and Singer3· 22 •23 found that the presence of -SH groups was essential for the activity of many
160
DAVIS & McCUNE
[Fertility & Sterility
enzymes concerned with the metabolism of cells. Barron et aU also demonstrated that sulfhydryl reagents in small concentrations increased respiration. They concluded that the soluble sulfhydryl groups must regulate respiration by inhibiting the rate of reoxidation of the cytochrome system, and that in small concentration the sulfhydryl reagents combined with these groups abolishing this regulating mechanism of respiration. In higher concentrations, the sulfhydryl reagents combined with fixed sulfhydryl groups in the side chains of the protein moiety of enzymes, thus inactivating these enzymes, which are essential to respiration. There are presented in this paper data on the 02 uptake and fructolysis of human spermatozoa, and the effect of small concentrations of sulfhydryl reagents.
..
METHODS Semen specimens were obtained from husbands of sterility patients being studied at Chicago Lying-In Hospital. Husbands were instructed in the care to be exercised in collecting specimens at home, by coitus interruptus. The age of the specimens at the beginning of experiments ranged from one and one-haH to four hours. All specimens were examined routinely as soon as they arrived in the laboratory, and count, volume, motility, and morphology were determined. They were classified as normal, subfertile, probably sterile, or sterile. Motility was recorded in terms of per cent motile and quality of motility ( 1 plus to 4 plus), according to the method described by Hotchkiss. It was found that the percentage of motile forms could most accurately and simply be determined by employing a cross-hatched micrometer disc in the microscope ocular. Approximately 5 to 20 nonmotile cells in a given number of squares (depending on the density of cells in the field) are counted, then the number of motile cells in the same number of squares is counted. This procedure is repeated three times, using different fields, and the results averaged. Counts were determined and morphology studied according to the methods described by Hotchkiss, except that the triple-stain technic of Keaty and Hamblen was used to stain smears for morphological study. Oxygen Uptake
Semen was buffered to pH 7.4 with phosphate buffer ( 0.1 cc., one-tenthmolar buffer to 1 cc. semen), 1 cc. portions of the sample were placed in
'
Vol. 1, No. 2, 1950]
HUMAN SPERMATOZOA IN SEMEN
161
small ( 5 cc.) Warburg vessels, with 0.1 cc. of KOH in the center cup. The vessels were equilibrated at 38 ° C. for 10 minutes. Experiments were run in duplicate and triplicate, depending on the volume of the specimen, and readings taken at 15-minute intervals.
...
Glycolysis One cubic-centimeter samples of the specimen were placed in small Warburg vessels without center cup, and the system saturated with a mixture of 95 per cent N2 and 5 per cent C02 for 10 minutes. Because of the poor gas exchange in a viscous medium, those specimens in which complete liquefaction had not taken place were not used. In almost all of the experiments, it was found that saturation of the semen with N2-C02 had taken place within 10 minutes, if the vessels were agitated constantly during the passage of the gas mixture through the system. In all of the experiments manometric measurements were pedormed on spermatozoa in whole semen. C02 Retention by Seminal Fluid The figures given for the anaerobic glycolysis have not been corrected for the C02 bound to semen protein. To measure this C02 retention, 1 cc. of cell-free seminal fluid was added to the small Warburg vessel, and 0.05 to 0.1 cc. of a lactic acid solution of known molarity was added to the sidearm. Mter saturation with N2-C02 mixture, the vessels were equilibrated in the bath at 38 o C. for 10 minutes. After equilibration the lactic acid was added to the seminal fluid. The difference between the actual and theoretical liberation of C02, divided by the theoretical figure, gives the per cent of C02 retained by the seminal fluid. The results were confirmed by chemical analysis, in which fructose was determined immediately before and after glycolysis. Bicarbonate in Seminal Fluid Bicarbonate was measured manometrically by a method similar to that described above for C02 retention. To the unaltered, cell-free seminal fluid 0.3 cc. of one-tenth normal HCl was added from the side-arm. The C02 given off represents an equal molar concentration of HCOa- ions in the seminal fluid.
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Fructose
Fructose was determined according to the method of Roe with the Coleman spectrophotometer at 490 mp.. Protein-free filtrates of the semen were obtained by diluting 0.1 cc. semen to 4 cc. with water, adding 2 cc. 10 per cent ZnS04 and 2 cc., one-tenth normal NaOH, and heating in boiling water for two minutes. In some cases, where a turbid supernatant indicated incomplete precipitation of the protein, an additional 1 cc. of ZnS04 and 1 cc. of NaOH were added. The solution was then filtered through No. 40 Whatman filter paper. TABLE 1.
Experiment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
P'/2 3 4 2 3"/2 P'/2 2 H4
m
2 21'/4 P'/2 2 4 1 3 2 1 3* 1* 3* 21'/4 3 2 2* 3 4 3* 2
Million Cells per cc. Semen
98 90 111 122 83 82 62 136 60 216 265 73 55 251 78 76 66 44 323 68 210 64 124 64 82 47 73 144 240
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Respiration of Normal Spermatozoa
Motility Age (hours)
..
Motile (per cent)
50 75 85
75 90 60 65 80 75 50 60 70 60 55 90 70 75 90 80 90 75 90 80 80 80 70
0 2 Uptake per Hr. per 108 cells Quality
(c.mm.)
++ +++ ++ +++ ++++ ++++ ++ ++++ ++ ++++ ++++ ++++ ++++ +++ +++ +++ +++ ++++ ++ +++ ++++ ++++ ++++ +++ ++++ ++ ++++ +++ ++
26.5 10.5 4.5 9.4 11.0 17.8 16.9 5.9 14.7 5.7 4.0 8.5 8.9 8.0 11.0 17.8 19.7 26.8 4.2 19.0 6.7 11.3 1.8 6.3 9.0 18.5 9.3 13.2 5.4
..
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RESULTS
02 Uptake Table 1 gives the results of individual experiments in which the oxygen utilization of 29 normal semen specimens was determined by the method described. It will be noted that the motility or cell concentrations of several of the specimens listed is lower than the value usually considered as normal, but in those instances the over-all evaluation of the specimens (age,
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TABLE 2.
Effect of Cell Concentration, Age, and Motility on Respiration and Glycolysis of Spermatozoa
Glycolysis Respiration Oz per Hr. per C02 per Hr. per 108 Cells 108 Cells Number of Number of Experiments (c.mm.) Experiments (c.mm.)
!-. .,.
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Count per cc. X 10-o Less than 100 Greater than 100 Age, hours 1 to Hf 2 to 2~f 3 to 4 Motility I 90%++++ II 75%++++ to 85%++++ III 50%++++ to 70%++++ 75%+++ to 90%+++ IV 40%+++ to70%+++ to 90%++ 75%++ v 10%++ to 70++ Classification of Specimen Normal Subfertile
18 11
14.1 6.3
7 9
23.5 23.6
8 10 11
16.3 9.7 9.6
7 6 3
30.1 18.3 19.2
5 4 5
10.0 8.3 11.6
0 3 3
24.3 18.5
9
14.7
4
20.5
3
8.1
6
27.9
29 0
11.1
8 8
24.1 23.1
morphology, volume, etc.), placed them within normal limits. The results are not corrected for respiration of the cell-free seminal fluid, the volume of most of the semen samples being too small to permit the determination of this error in each experiment. The average 02 uptake of 2 cell-free seminal fluid specimens was found to be 3.4 c.mm. 02, + 0.4. The mean, uncorrected oxygen uptake, with mean deviation, of the 29 experiments listed in Table 1 is 11.1 c.mm. + 5.2, 02 per 108 cells per hour (range, 1.8 to 26.8). The wide variation in the oxygen uptake of individual specimens cannot be explained
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TABLE 3.
[Fertility & Sterility
Effect of Sulfhydryl Reagents in Small Concentration on the Respiration of Human Sperm
02 Uptake per Hr. per lQH Cells Sulfhydryl Concentration Control Reagent (Molar) (c.mm.) (c.mm.)
Sulfhydryl Reagent
5X 5 X 5X 5 X 5X
Iodoacetamide p-chloromercuribenzoate p-chloromercuribenzoate Arsenite Cadmium chloride
I0-5 I0-5
10-4 I0- 5
10-5
16.1 10.5 4.5 17.1 8.1
Increase (per cent)
26.5 12.0 1.8 17.8 11.6
39.3 12.5 -82.0 3.9 30.1
on the basis of differences in motility, but it is apparent that the rate of oxygen uptake is much higher in specimens having relatively low counts, and in fresh specimens (Table 2). It must be recalled that Barron and Harrop found that overcrowding diminished the respiration of leukocytes. In agreement with the findings of Barron et aP on sea urchin sperm, small concentrations of four sulfhydryl reagents increased the respiration of spermatozoa, as shown in Table 3. One of the reagents, p-chloromercuribenzoate, caused an inhibition of respiration in higher concentration. TABLE 4.
Classification Subfertile Fertile
Glycolysis of Normal and Subfertile Spermatozoa
Age (hours)
X
3
X
2~ X X
X X
X X X X X X
P'/2 P'/4 3 2 3 2 2 21'/4
m B4
X
1 lJf 2
X
P'/2
X X
Motility Million Cells Motile per cc. Semen (per cent) Quality
86 196 99 55 119 77 219 102 88 150 72 67 185 234 286 222
80 80 25 33 36 55 75 40 48 46 9 66 80 75 78 60
+++ ++++ ++ ++ ++ +++ ++++ +++ +++ ++ ++ +++ ++++ +++ +++ ++
C02 per Hr. per 108 Cells (c.mm.)
17.4 16.3 26.2 36.4 25.9 15.2 14.2 20.6 20.5 16.8 23.6 25.8 42.4 17.9 20.2 38.6
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Anaerobic Glycolysis The anaerobic glycolysis of 8 normal and 8 subfertile semen specimens was determined, and the results are summarized in Table 4. Determinations of C02 gave mean values, with mean deviation, of 23.6 c.mm. ± 6. 7 C02 per 108 cells per hour (range 14.2 to 42.4), for the entire group, and there is no significant difference in the glycolysis of the normal and subfertile groups (Table 2). It is also apparent (Table 2) that cell concentration has no significant effect on the rate of fructolysis, and that the rate of fructolysis is much higher in fresh specimens. TABLE 5.
Experiment
1 2 3
Manometric Determination of C0 2 Retention by Semen
Found ( c.mm.)
C02 Formation Theoretical ( c.mm.)
95.8 50.1 18.3
Bound C02 (per cent)
194.2 97.1 37.9
50.7 48.4 51.7
C02 Retention by Seminal Fluid Table 5 gives the results of 3 experiments in which it was found that the average C02 bound to seminal fluid was 50.3 per cent, ± 0.9. This large retention was confirmed by chemical analysis of fructose utilization (Table 6) by which it was found that 48.5 per cent of the C02 was bound. TABLE 6.
Chemical Determination of C0 2 Retention by Semen
C02 Formation
Experiment
Found (c.mm.)
Fructose Utilization (c.mm. C02)
Bound C02 (per cent)
1 2
109.5 43.6
238 143
53.8 43.3
Bicarbonate An excess of HCl added to cell-free seminal fluid liberated 177.5 c.mm. C02 per cc. of semen. This quantity is equivalent to 7.92 micromols of bicarbonate, and is in excess of the highest values recorded for glycolysis of spermatozoa in seminal fluid (Table 1).
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DISCUSSION Data concerning the rate of respiration and glycolysis of human spermatozoa in seminal fluid are limited. This may be due, in part, to the belief of many investigators that the sole function of seminal plasma is as a medium for transporting the spermatozoa to the female genital tract. The analysis of human seminal plasma by Goldblatt and by Huggins et aU· 7 • 8 has demonstrated the chemical complexity of seminal plasma, and has cast doubt on the above concept. Evidence indicates that spermatozoa are in a dormant metabolic state until they reach the vicinity of the prostate gland. 7 The comparison of the reducing sugar and lactic acid contents of fractionated ejaculates of normal semen and of semen with azoospermia16 indicates that when the spermatozoa come in contact with the secretions of certain accessory glands of reproduction, prior to ejaculation, glycolysis is initiated. The high buffering capacity of the seminal plasma 5 protects the spermatozoa against the low pH of the vaginal secretions, and the high fructose content of the seminal vesicle component of the seminal plasma17 supplies the spermatozoa with a readily utilizable substrate until they are able to reach the more hospitable environment of the cervical canal. In view of the relatively high incidence of disease of the male accessory glands of reproduction, without involvement of the testes, it is evident that the pathology in many subfertile or sterile semen specimens may be due to the harmful effect of abnormal seminal plasma on normal spermatozoa. Shettles' figures of approximately 14 c.mm. 02 uptake per 108 cells per hour compare favorably with our results. Shettles reported no respiration in seminal fluid, and his results are uncorrected for that error as are those of the present report. He concluded that the rate of respiration varies inversely with the age of the specimen and directly with the cell concentration. The data presented in this paper do not agree with his conclusion regarding cell concentration. Although there was a higher rate of respiration in fresh specimens, no direct ratio between age and respiratory rate was found (Table 2). Ross et al. reported lower values (3 to 9 c.mm. 02 per hour with 2 values of 1 c.mm. 02 and 1 of 13 c.mm. 02). Three of their experiments were corrected for blanks made on seminal fluid of the same specimens, and the remainder for the average value of blanks made on several seminal fluids. The striking difference in the rate of respiration between specimens of less than 100 million per cc. cell concentration and specimens of greater than 100 million per cc. cell concentration (Table 2) may be explained on the
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basis of the seminal fluid respiration error. This error is increased or de· creased, depending on whether one multiplies or divides in converting the observed oxygen uptake to the oxygen uptake per 108 cells. Thus, in making this conversion it is found that the error is inversely proportional to the cell concentration of the specimen. The lack of correlation between the degree of motility and the rate of respiration or glycolysis is apparent from Table 2. MacLeod 14 has demonstrated a definite relationship, in individual specimens, between changes in the degree of motility and the rate of glycolysis. In a larger series of experiments than have been undertaken in this work such a relationship would probably be found between the initial motility of a specimen and its rate of glycolysis. Search of the literature revealed only one other report on the glycolysis of human spermatozoa in seminal fluid. Ross et al. reported the results of 10 such experiments in which they found an average C02 liberation of 8.2 c.mm. C02 per 108 cells per hour, considerably lower values than ours ( 23.6). However, the ages of their specimens varied from 1 to 12 hours, and the average age was greater than ours. The slightly higher rate of glycolysis in the fertile group ( 24.1 c.mm. C02) over the subfertile group ( 23.1 c.mm. C02) undoubtedly falls within the limits of error of the method. Ross et al. were, likewise, unable to determine a correlation between glycolysis and the degree of fertility of the specimen.
SUMMARY Respiration and glycolysis of human spermatozoa in unaltered semen were measured independently by the direct method of W arburg. The mean rate of respiration of 29 normal specimens was found to be 11.1 c.mm. 02 per 108 cells per hour, and certain sulfhydryl reagents, in low concentrations, increased respiration. Fresh specimens respired at a higher rate, but no correlation between the degree of motility and the rate of respiration could be demonstrated. It was determined manometrically, and confirmed chemically, that seminal plasma retains approximately 50 per cent of the C02 liberated during glycolysis. The rate of glycolysis of spermatozoa is more nearly proportional to cell concentration than is the rate of respiration. No evidence was found, as has been reported in the case of some lower mammals, that either the degree of motility or the potential fertility of the specimen determines the rate of glycolysis. For references see p. 168.
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REFERENCES I. Barron, E. S. G., and Harrop, G. A., Jr.: J. Bioi. Chern. 84:89, 1929. 2. Barron, E. S. G., and Nelson, L., and Ardao, M. 1.: J. Gen. Physiol. 32:179, 1948. 3. Barron, E. S. G., and Singer, T. P.: J. Bioi. Chem.157:221, 1945. 4. Berg, 0. C., Huggins, C., and Hodges, C. V.: Am. J. Physiol. 133:82, 1941. 5. Goldblatt, M. W.: Biochem. J. 29:1346, 1935. 6. Hotchkiss, R. S.: Fertility in Men. Philadelphia, Lippincott, 1944. 7. Huggins, C. B., and Johnson, A. A.: Am. J. Physiol. 103:574, 1933. 8. Huggins, C. B., Scott, W. W., and Heinen, J. H.: Am. J. Physiol. 136:467, 1942. 9. Keaty, E. S., and Hamblen, E. C.: J. Clin. Endocrinol. 5:286, 1945. 10. McCarthy, J. F., Stepita, C. T., Johnson, W. B., and Killian, J. F.: J. Urol. 19:43, 1928. 11. MacLeod, J.: Proc. Soc. Exper. Bioi. & Med. 42:153, 1939. 12. MacLeod, J.: Am. J. Physiol. 132:193, 1941. 13. MacLeod, J.: Endocrinology 29:583, 1941. 14. MacLeod, J.: Human Fertil. 7:129, 1942. 15. MacLeod, J.: Am. J. Physiol. 138:512, 1943. 16. MacLeod, J., and Hotchkiss, R. S.: J. Urol. 48:225, 1942. 17. Mann, T.: Biochem. J. 40:481, 1946. 18. Mann, T., and Parsons, V.: Nature 160:294, 1947. 19. Ross, V., Miller, E. G., Jr., and Kurzrok, R.: Endocrinology 28:885, 1941. 20. Roe, J. H.: J. Bioi. Chern. 107:15, 1934. 21. Shettles, L. B.: Am. J. Physiol. 128:408, 1940. 22. Singer, T. P., and Barron, E. S. G.: Proc. Soc. Exper. Bioi. & Med. 56:120, 1944. 23. Singer, T. P., and Barron, E. S. G.: J. Bioi. Chern. 157:241, 1945. 24. Walton, A.: Commem. Spallanzaniane 4:36, 1939. 25. Walton, A., and Edwards, J.: Proc. Am. Soc. Anim. Prod. 31:254, 1938.
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