Glycolysis by Human Spermatozoa: Levels of Glycolytic Intermediates

BIOLOGY OF REPRODUCrION 5, 221-227 (1971) Glycolysis by Human Spermatozoa: Levels of Glycolytic Intermediates R. N. PETERSON AND M. FREUND Laboratory of Reproductive Pharmacology, Departments of Pharmacology and of Obstetrics aiid Gynecology, New York Medical College, Flower and Fifth Avenue Hospitals, New York, New York Received April 12, 1971 The steady-state levels of glycolytic intermediates of washed sperm suspensions containing glucose under aerobic and anaerobic conditions were determined from perchloric acid extracts. Under both conditions, the products of the phosphofructokinase and aldolase steps accumulate and substantially exceed the level of hexose monophosphates. These high levels of triose phosphate and fructose diphosphate do not appear to be the result of an unfavorable NAD/NADH equilibrium since added pyruvate does not substantially reduce their accumulation in washed cells and since the same intermediates accumulate in semen where pyruvate concentrations are normally high. The aerobic metabolite levels apparently reflect the inability of oxidative metabolism in human sperm to develop high levels of ATP and also suggest possible sitesof glycolytic control at either glyceraldehyde phosphate dehydrogenase or phosphoglycerate kinase. Concentrations of a-glycerophosphate in sperm were found to be low; the reasons for this and itspossible significance for glycolysis are discussed In a previous report (Peterson and Freund, 1970b) a possible role for phosphofructokinase in the control of human sperm glycolysis was suggested on the basis of the comparatively low amount of this enzyme in cells compared to other glycolytic enzymes. In addition, phosphofructokinase activity in sperm extracts was shown to be sensitive to several cofactors known to be involved in control in other cells. However, the absence of a significant Pasteur effect in human sperm together with the rather small stimulatory effect of inorganic phosphate on glycolysis in intact cells (Peterson and Freund, 1969) make uncertain the extent to which phosphofructokinase participates in control. We have examined this further by determining the steady-state levels of certain key glycolytic intermediates in washed sperm suspensions supplemented with glucose and in sperm suspended in seminal plasma. Our results suggest that enzymatic sites in addition to phosphofructokinase are involved in control. METHODS AND MATERIALS Semen specimens obtained from normal donors were pooled before use. The work up of these Specimens and the procedure used for preparing plasmafree cell suspensions have been previously described (Peterson and Freund, 1968). A Tris-buffered salts medium similar to that used in earlier studies but at a phosphate concentration closer to that in semen was used in experiments with washed cells.this medium had the following composition: sodium chloride, 0.1 l8m; tris(hydroxymethyl)aminomethane (Tris)- chloride, 0.03M; dipotassium hydrogen phosphate, 0.003M; magnesium chloride, 0.005M; and disodium EDTA, 0.0001M. The ph was 7.4 at 37 C. Ext,action and Assay of Metabolites. Washed sperm were resuspended in 1-2 ml buffered salts medium in 25-mi flasks to give a sperm concentration of at least 2 x 108 cells/mi. When seminal plasma was used in the suspending medium, the pooled specimens were centrifuged once (300g, 10 mm) to remove any remaining cells. This preparation was used in controls. Experiments were carried out in a Dubnoff shaker at 37 C. When anaerobic conditions were required, flasks were stoppered with rubber septa and gassed through syringe needles with purified nitrogen (99.99%) for 10 mm prior to the addition of glucose. The sperm suspension was shaken vigorously for an additional 20 mm. Metabolism was stopped by the rapid injection of an equal volume of ice-cold 20% 221

222 PETERSON AND FREUND perchloric acid. The sperm suspension was then placed in ice for 60 mm before being centrifuged (15,000g, 10 mm, OC) to remove precipitated materials. The supernatant was neutralized to ph 7 by the slow addition, with constant swirling, of solid potassium carbonate. After neutralization, the suspension was kept in ice an additional 30 mm before again being centrifuged. The supernatant from this last step was used for the assay of intermediates. The fluorometric assays used were similar to those originally described by Lowry et al. (1964). Assay conditions were as follows: G/ucose-6 -phosphate, Fructose-6-phosphaie. Tnethanolamine buffer (ph 7.5, 0.IM); NADP (12.8 MM); glucose-6-phosphate dehydrogenase (0.002 mg); and phosphohexose isomerase (0.005 mg). Total volume 4.0 ml. Both metabolites were usually assayed from the same aliquot of extract. In some instances, however, when total sperm numbers were low, both metabolites were assayed simultaneously and reported as total hexose monophosphate. Dihydroxyacetone Phospha:e, Glyceraidehyde-3- phosphate, Fructose-i, 6-diphosphate. Triethanolamine buffer (0.IM, ph 7.5); NADH (O.8-4.OpM);a-glycerophosphate dehydrogenase (0.050 mg); triosephosphate isomerase (0.005 mg); and aldolase (0.005 mg). All three metabolites were assayed from the same aliquot of extract. 3-Phosphoglycerate. Triethanolamine buffer (0.1M, ph 7.5); ATP (0.4 mm); MgCI, (6 mm); mercaptoethanol (5 mm); NADH (1 MM); muscle glyceraldehyde phosphate dehydrogenase (0.100 mg); phosphoglycerate kinase (0.005 mg); phosphoglycerate mutase (0.060 mg). This assay was also used in an attempt to detect 1, 3-diphosphoglyceric acid and 2-phosphoglyceric acid. However, the levels of these metabolites were too low for accurate assay. Phosphoenolpyruvale, Pyruvate. Phosphate buffer (0.lM, ph 7.0); NADH (1.0-5.0 MM as needed); lactic dehydrogenase (beef heart, 0.005 mg); ADP (0.02 mm); MgCl, (0.005M); and pyruvate kinase (0.015 mg). Both metabolites were assayed from the same aliquot of extract; ADP, MgCI,, and pyruvate kinase were added after the completion of the fluorescent change due to pyruvate reduction. a-glycerophosphate. Hydrazine (350 mm); hydrazine hydrochloride (50 mm); NAD (70 MM); and a-glycerophosphate dehydrogenase (0.075 mg). Lactic acid. Lactic acid was determined enzymatically as previously described (Peterson and Freund, 1969). NAD. The procedure is essentially that of Estabrook and Maitra (1962). An aliquot of the neutralized extract (0.3-0.9 ml) was diluted to 4.0 ml with buffer at ph 10 containing 0.2M glycine, 0.075M semicarbazide, and 0.15 mg alcohol dehydrogenase. ATP, ADP. ATP was measured fluorometrically by the fire-flyassay described previously (Peterson and Freund, 1970b). ADP was measured as the increase in ATP after the addition of 3 pmoles phosphoenolpyruvate and 0.020 mg pyruvate kinase. Creatine phosphate. Tniethanolamine buffer (ph 7.5, 0.IM); NADP (12.8 MM); MgCl, (0.003M); glucose (0.OIM); ADP (0.25mM); hexokinase (0.010 mg); glucose-6-phosphate dehydrogenase (0.010 mg); and creatine phosphokinase (0.010 mg). After the fluorescent change due to ATP was complete, ADP was added. Small amounts of contaminating ATP gave rise to a further increase in fluorescence. When the fluorescence reading became constant, creatine phosphokinase was added and the fluorescent change due to creatine phosphate was recorded. All measurements were made in optically matched cuvettes at room temperature in a Turner Model 111 recording fluorometer. Internal standards were run with all assays. The total volume in all assays was 4.0 ml. Materials. Chemicals used in these experiments were either Sigma grade or Baker reagent grade. All enzymes were highest purity and purchased from the Boehringer-Mannheim Co. RESULTS Table 1 shows the intermediate profiles obtained with washed cells incubated aerobically and anaerobically with glucose as substrate. Results are given as the average value and standard error from all experiments in which the particular intermediate was assayed. The sperm numbers available did not permit a determination of all components in a single assay. First, as regards the aerobic profile, the most striking feature is the comparatively high levels of triose phosphate and fructose diphosphate. On average, the sum of the concentrations of these metabolites exceeds by about fivefold the level of total hexose monophosphate which ordinarily might be expected to accumulate under aerobic conditions. This aspect of the aerobic profile is not substantially different from that obtained under anaerobic conditions where there is about a 40% further increase in the levels of triose phosphate and fructose diphosphate. The change to anaerobic conditions which is also accompanied by about a

ENERGY METABOLISM IN HUMAN SPERMATOZOA 223 TABLE 1 LEVELS OF GLYCOLYTIC INTERMEDIATES IN SPERM SUSPENDED IN GLUCOSE-SALTS MEDIUM Intermediate Air amoles/cell Nitrogen Glucose-6-phosphate 15.4 ± 1.7 (18) 16.8 ± 1.6 (3) Fructose-6-phosphate 5.4 ± 0.7 (18) 4.9 ± 1.8 (1) Fructose-l,6-diphosphate 50.1 ± 5.9 (9) 63.8 ± 12.3 (5) Dihydroxyacetone-phosphate 51.2 ± 6.1 (9) 81.5 ± 6.1 (5) Glyceraldehyde-3-phosphate 3.1 ± 1.6 (9) - a-glycerophosphate 43.9 ± 7.7 (16) 75.2 ± 7.0 (4) 3-Phosphoglycerate 11.2 ± 2.4 (6) 12.2 ± 1.3 (3) Phosphoenolpyruvate 6.0 ± 0.9 (4) 9.06 (2) Pyruvate 163.9 ± 53.7 (3) 91.9 ± 21.9 (3) Lactate 4,405 ± 395 (3) 4,896 ± 681 (3) ADP 88.9 ± 11.3 (4) 91.0 ± 2.8 (3) ATP 145.3 ± 9.6 (4) 125.6 ± 28.2 (3) Creatine phosphate 6.3 ± 2.6 (3) - NAD 8.3 ± 0.9 (5) - ATP/ADP 1.63 1.38 Lactate/pyruvate 26.8 53.3 a-glycero-p/dihydroxyacetone-p 0.86 I.29 Sperm were incubated for 20 mm at 37 C in Tnis-buffered medium containing 0.OlM glucose. number of experiments involved in a particular assay is given in parentheses. 1 attomole 1 amole = 10-18 mole. The 10% increase in glucose utilization and lactic acid production did not markedly affect the levels of the other intermediates assayed. A second feature of the aerobic profile, and one that is probably related to the accumulation of the products of the phosphofructokinase step, is the rather low ATP/ADP ratio. This ratio, which was less than 2 in all experiments, was only slightly higher than that observed under anaerobic conditions. Similar low ratios were also obtained with unwashed cells (see Table 3). In view of the known sensitivity of phosphofructokinase activity to high levels of ATP, a low ATP/ADP ratio together with the accumulation of triose phosphate and fructose diphosphate suggest that phosphofructokinase, under both aerobic and anaerobic conditions, is ordinarily not under appreciable inhibition. Table I also shows that significant amounts of pyruvic acid accumulate under both aerobic and anaerobic conditions. Most of the pyruvate accumulates in the medium and, like lactic acid, the amount of pyruvate formed increases with time. A smaller amount of pyruvate is formed under anaerobic conditions, although the amount of lactic acid formed is slightly increased. These differences are probably related to the equilibrium at lactic dehydrogenase and the sensitivity of this equilibrium to the NADH/NAD+ ratio in the cytoplasm. The lactate/pyruvate ratio, which should be proportional to the NADH/NAD+ ratio, increased twofold from 26.8 to 53.3 when cells were shifted from aerobic to anaerobic conditions. A similar effect might also be expected to occur at a-glycerophosphate dehydrogenase since the same pyridine nucleotide cofactor is required by this enzyme. Further, if it is assumed that a common pool of pyridine nucleotides supplies both lactic dehydrogenase and a-glycerophosphate dehydrogenase, one might expect

224 PETERSON AND FREUND the dihydroxyacetone phosphate/a-glycerophosphate and lactate/pyruvate ratios to be similar in view of the similarity of the equilibrium constants for both reactions. However, as can be seen from the table, there is considerable difference between the ratios. The much lower ratio at a-glycerophosphate dehydrogenase may be the result of a rate limitation due to the small amounts of the enzyme known to be present in the cell (Peterson and Freund, l970a). Such a situation can have a marked effect on the glycolytic rate as will be discussed below. Since glyceraldehyde-phosphate dehydrogenase activity also depends on pyridine nucleotide levels, it was possible that the cytoplasmic NADH/NAD+ ratio was also affecting the accumulation of triose phosphate. If this were true, a potential oxidant such as pyruvate could be expected to reduce substantially the triose phosphate level. This was tested and the results are shown in Table 2. As can be seen 0.OlM pyruvate had little effect on the level of hexose monophosphate but lowered the combined level of triose phosphate and fructose diphosphate by about 35 % in the three experiments. However, even under these conditions, the combined level of the latter intermediates still substantially exceeded the total concentration of hexose monophosphates. It was also of interest to determine the level of these metabolites in semen where cells are buffered at much lower lactate/ pyruvate ratios. The results of three such experiments are shown in Table 3. In these analyses, corrections had to be made for the presence of hexose monophosphate in the seminal plasma. Although the concentration of these extracellular intermediates was small (ca. lim) compared to other substances in seminal plasma, they were present at concentrations comparable to the levels of intracellular hexose monophosphate. When these corrections are made, the results are quite similar to those obtained with washed cells and show an accumulation of high levels of triose phosphate and fructose diphosphate and comparatively low levels of hexose monophosphates. TABLE 2 EFFECT OF PYRUVATE ON THE ACCUMULATION OF Intermediate Glucose-6-phosphate + Fructose-6-phosphate Mean ± SE Tniose Truosa PHOSPHATE AND FRUCTOSE DIPHOSPHATE in WASHED SPERM phosphate + Fructose diphosphate Mean ± s Lactate Ex- ment No. 2 3 2 3 Control 26.3 20.7 16.9 21.3 ± 111.2 152.1 81.7 amoles/cell 115.0 ± 20.4 4,687 2.7 23. 7 ± 1.8 69.0 89.3 66.9 75.1 ± 7.1 6,561 Experimental conditions were the same as those indicated in Table 1. DISCUSSION It is generally assumed that the Pasteur and Crabtree effects are due to reciprocal interactions between cofactors generated during cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. In 0.01 84 certain cells (e.g., brain) the high levels of pyruvate ATP generated by Krebs cycle oxtdation react with certain glycolytic enzymes reduc- 26.3 ing their activity and, thereby, decrease the 20.0 overall glycolytic rate. Phosphofructokinase 24.5 has been shown to be the key enzyme controlling glycolysis in these cells. This enzyme has also been suggested to be involved in the control of sperm glycolysis in the monkey (Hoskins et a!. (1970)). In human sperm a role for phosphofructokinase is suggested by its comparatively low concentration in cells (Peterson and Freund, 1970a). However, the absence of an appreciable Pasteur effect and the small effects of inorganic

ENERGY METABOLISM IN HUMAN SPERMATOZOA 225 TABLE 3 LEVEL OF GLYCOLYTIC INTERMEDIATES IN HUMAN SEMEN Intermediate Experiment. no. nmoles/ml amoles/cell Semen Seminal plasma Sperm 1 7.45 7.05 5.71 Glucose-6-phosphate 2 6.12 3.54 15.80 + 3 9.55 8.28 2.88 Fructose-6-phosphate 4 3.91 2.88 18.20 Mean ± Se 1.31 10.68 ± 3.76 Tniose-phosphate 1 2 6.76 ± 1.18 5.63 16.30 5.44 ± 0.00 0.00 + 3 4.23 0.00 97.3 Fructose-diphosphate 4 8.63 0.00 81.4 Mean ± sa 8.70 ± 2.70 0.00 89.90 ± 5.3 Pyruvate 1,366 ± 262 (7) Lactate 5,816 ± 551 (7) - - ADP - - 101.9 ± 7.0 (4) ATP - - 153.7 ± 12.0 (4) Lactate/pyruvate 4.3 - - ATP/ADP 1.5 - - Number in parentheses indicates the number of experiments involved in a particular assay. 80.4 100.6 phosphate and AMP, known activators of phosphofructokinase, on aerobic glycolysis indicate that this enzyme is not appreciably inhibited in the intact cell. This conclusion receives further support from the data presented in this report which show that the products of the phosphofructokinase and aldolase steps accumulate at high levels even under aerobic conditions. The accumulation of these intermediates is probably due, in part, to the relatively low ATP/ADP ratio that occurs in human sperm. This ratio is only slightly increased when cells are aerated as compared to anaerobic conditions, and as a result there is only a small change in the relative levels of intermediates associated with phosphofructokinase. Other experiments which also indicate an inability of the oxidative apparatus of human sperm to generate high levels of ATP have been reported earlier (Peterson and Freund, 1970b). It should be stressed that the accumulation of triose phosphate and fructose diphosphate does not preclude an involvement of phosphofructokinase in rate control since steady-state levels of reactants and products are still significantly displaced from the values that would have been expected if this step were at equilibrium. The question is whether the accumulation of triose phosphate and fructose diphosphate is indicative of a significant displacement of control to sites further along the glycolytic chain. In view of the comparatively low levels of 3-phosphoglycerate and phosphoenolpyruvate, it is possible that a rate limitation exists at either glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate kinase. Added pyruvate reduces triose phosphate accumulation to some extent. This appears to be related to an increase in the level of oxidized pyridine nucleotide since this potential oxidant has little effect on the level of adenine nucleotides. By increasing the level of NAD+, pyruvate addition may stimulate glyceraldehyde phosphate dehydrogenase and thus lower the triose phosphate level. However, even after the addition of pyruvate to washed cells, the total triose phosphate and fructose diphosphate levels still substantially exceed the level of

226 PETERSON AND FREUND hexose monophosphate. This is also the situation in semen where sperm are ordinarily exposed to high levels of pyruvate. Under these conditions it is not clear to what extent glycolysis is controlled by flow through glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase. Further analysis will require an accurate determination of all intermediates associated with these steps, especially 1, 3-diphosphoglycerate and 2, 3-diphosphoglycerate which are present at concentrations too low for analysis with the sperm numbers presently available. We should also bring attention to the rather low levels of a-glycerophosphate that are observed in human sperm especially in view of the high levels of dihydroxyacetone phosphate and the equilibrium constant for the reaction catalyzed by a-glycerophosphate dehydrogenase which lies far in the direction of c-glycerophosphate formation. These low levels do not appear to be due to an unfavorable NADH/NAD+ ratio since the lactate/pyruvate ratio, which is also determined by the redox state of the pyridine nucleotides, is high under the same conditions. This could mean that lactic dehydrogenase and a-glycerophosphate dehydrogenase are in different compartments in the cell and thus are separated from a common pool of pyridine nucleotides. However, it has already been shown that the activity of cs-glycerophosphate dehydrogenase in human sperm is much lower than other glycolytic enzymes (Peterson and Freund, l970a). This fact suggests the alternative that the formation of a-glycerophosphate is limited by the amount of a-glycerophosphate dehydrogenase in the cell. One effect of this situation is that a-glycerophosphate dehydrogenase would be less effective in competing with lactic dehydrogenase for pyridine nucleotide reducing equivalents which, in turn, would lead to a greater rate of conversion of pyruvate to lactate and a higher aerobic glycolysis. The second factor that appears to contribute to the high aerobic glycolysis is the low ATP/ADP ratio and the resultant increased activity of phosphofructokinase as discussed above. Apparently ATP is synthesized by glycolytic and mitochondrial mechanisms in the mature sperm at a rate just sufficient to meet the cells energy demands. Indeed, since the anabolic pathways that ordinarily require larger pools of ATP are absent in these cells, high levels of this compound would serve no function and may even be deleterious to mechanisms involved in ion transport and motility. The presence of a greater biosynthetic capacity in testicular and epididymal spermatozoa and a smaller demand for channeling high energy intermediates into the ion transport mechanisms involved in motility may raise ATP levels considerably in these cells and may explain why these maturing cells exhibit much higher Pasteur effects than do ejaculated sperm. ACKNOWLEDGMENTS This investigation was supported by Grant HD- 00488-12 from the National Institute of Child Health and Human Development, National Institutes of Health, U. S. Public Health Service. The authors gratefully acknowledge the technical assistance of Mrs. Gloria Edwards, Miss Sandra Stoner, and Miss Ollie Brown. Matthew Freund IS a Career Scientist of the Health Research Council of the City of New York (1-218). REFERENCES ESTABROOK, R. W., AND MAITRA, P. J. (1962). A fluorometric method for the quantitative microanalysis of adenine and pyridine nucleotides. A,zab. Biochem. 3, 369-382. HosKiNs, D., STEPHENS, D. T., AND CASILLAS, E. (1970). Regulation of monkey sperm cell fructolysis. Fed. Proc. Fed. Arne,. Soc. Exp. Biol. 29, 888. LowRy, 0. H., PASSONNEAU, J., HASSELBERGER, F., AND SCHULZ, D. (1964). Effect of mschemia on known substrates and cofactors of the glycolytic pathway in brai i. J. Biol. C/ten,. 239, 18-30. PETERSON. R. N., AND FREUND, M. (1968). An evalua-