COMPARATIVE GLYCOLYTIC METABOLISM OF SPERM FROM NORMAL, ASTHENOSPERMIC, AND OLIGOASTHENOSPERMIC MEN*

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1 FRTLTY AND STRLTY Copyright " 1975 The American Fertility Society Vol. 6, No. 4, April1975 Printed in U.SA. COMPARATV GLYCOLYTC MTABOLSM OF SPRM FROM NORMAL, ASTHNOSPRMC, AND OLGOASTHNOSPRMC MN* NVS PDRON, M.D., JUAN GNR, M.D., JUAN J. HCKS, M.D., PH.D., AND ADOLFO ROSADO, M.D., PH.D. Division de Biologia de la Reproducci6n, Departmento de lnvestigacion Cientifica, Centro Medico Nacional, lnstituto Mexicano del Segura Social, Mexico 1, DJi'. The diagnostic procedures presently used to assess the causes of male sterility are based on morphologic and functional studies. 1 n the clinic these procedures are limited to the study of seminal fluid for number, motility, and morphology of the spermatozoa, to the functional evaluation of the hypothalamic-pituitary-testicular axis, and to the histopathologic analysis of the testis. 3 Despite the availability of modern biochemical techniques and a knowledge of spermatozoal metabolism in some mammalian species,4.5 studies designed to uncover differences in the glycolytic metabolism of spermatozoa from fertile and infertile human subjects have not been reported. For this reason we undertook a comparative study of glycolysis in spermatozoa from normal, asthenospermic, and oligoasthenospermic men. MATRALS AND MTHODS Forty men, 0 to 35 years of age, were selected and classified as normal, asthenospermic, or oligoasthenospermic according to clinical findings and semen analysis. The normal group consisted of 15 men with normal clinical findings, spermatozoa concentrations of more than 40 million/ml of semen, normal sperm motility according to Kremer's capillary Received June 13, *Supported in part by a research grant from The Ford Foundation. 309 method, 6 and normal sperm morphology in at least 80% of the cells. The asthenospermic group consisted of ten men with decreased sperm motility (one standard deviation below the average for the normal group at 30 and 60 minutes), but with normal concentrations of spermatozoa and normal sperm morphology. The oligoasthenospermic group consisted of 15 men with spermatozoa concentrations of less than 40 million/ml, decreased sperm motility, and higher percentages of abnormal forms than those of the normal group. Three samples of semen were taken from each subject at seven-day intervals. They were obtained by masturbation between 8 and 9 AM, collected in glass beakers, and allowed to liquefy at room temperature for 60 minutes. Spermatozoa were obtained from these fresh ejaculates by centrifugation of the liquefied semen for ten minutes at 3,000 g. The pellets were resuspended in enough phosphate buffer (ph 7.4, 0.085M) to restore the original volume of semen and were washed once. The spermatozoa were finally resuspended in the same phosphate buffer. 7 Pilot experiments, which were conducted with 0.085M Hepes (N--hydroxyethylpiperazine-N --ethanesulfonic acid) buffer to discount the possible effect of high phosphate concentrations, failed to show any significant difference in spermatozoal metabolism with either buffer; this

2 310 PDRON TAL April1975 ( confirms the report of Peterson and Freund. 8 Sperm counts were made with a Neubauer hemocytometer. A Gilson differential respirometer adjusted to 37" ± 0.1 C was used for radiorespirometry. The following materials were added to 5-ml Warburg flasks: 0.1 ml of M potassium hydroxide to the center well containing filter paper, 0.1 ml of 0.5M perchloric acid to the side arm, and 0.5 ml of the resuspended spermatozoa to the principal chamber. Measurements were carried out in air at a shaking rate of 70 oscillations/minute. Glucose 14C (U-L) and fructose 14 C (U-L) (New ngland Nuclear Co) were used as substrates in the different experiments. They were adjusted to a specific activity of 33.3 nci/jlmole and added to the flasks at a final concentration of 5 JLmoles/ml. Finally the volume was adjusted to 1.0 ml in all analytical -procedures by adding the same buffer as that used to resuspend the spermatozoa. After ten minutes for temperature equilibration, the flasks were closed and oxygen uptake was measured manometrically at 15-minute intervals in the Gilson differential respirometer. After the chosen time elapsed, the reaction was stopped by tipping in the perchloric acid solution. After 30 minutes, (to allow complete trapping of the 14C0 ), the flasks were opened. Substrate oxidation was calculated by counting the 14C0 trapped in the potassium hydroxide in the center well filter paper with a liquid scintillation spectrophotometer and by correcting counts per minute to disintegrations per minute using the channels ratio method. The contents of the flasks were homogenized in an all glass, Potter-lvejhem homogenizer and the homogenate was centrifuged at 3,000 rpm for ten minutes. Proteins and DNA were measured in the precipitate Pyruvate, lactate, and residual glucose were measured in the supernatant by usual analytical procedures.11 L-+l-""i p. mol ---4 Glucose 14 C {Ll) - Fructose 14 C {Ll). so FG. 1. Comparative utilization of glucose 14 C (U-L) and fructose 14 C (U-L) by human spermatozoa. The 14 C0 (ordinate) produced at the hexose concentrations indicated on the abscissa were plotted for glucose and for fructose. ach point represents the mean of three to four determinations. All the differences between the two curves are statistically significant (P<O.Ol) except that obtained when 10 p.moles of substrate was used. Before this study there was still some question about which hexose was preferentially used by human spermatozoa. Therefore, we decided to study initially whether glucose or fructose was selectively converted to 14 C0 Two approaches were selected. First, utilization of each of the two sugars was measured at several final concentrations by the method described above. n all cases the specific activity was 33.3 nci/jlmole (Fig. 1). Second, utilization of radioactive glucose of higher specific activity (16.6 JLCi/ JLmole) was measured when the system contained various concentrations of nonradioactive fructose. The ratio of radioactive glucose to nonradioactive fructose was 0.1 uci of glucose 14 C/3 JLmoles of fructose in each case. This type of experiment was then repeated using fructose 14 C (1 uciijlmole) and nonradioactive glucose (Figs. and 3). Significance of differences between means <P value) was determined by the use of the Student's nonpaired t test.

3 Vol. 6, No. 4 GLYCOLYTC MTABOLSM 311 c( z Q i;- ~.. Q. 3! 10 p. mol -- Labeled Glucase + Fructose -- Labeled Glucose + Glucose so FG.. Competitive utilization of radioactive glucose in the presence of a large excess of nonradioactive fructose. The 14 C0 produced from radioactive glucose (ordinate) was plotted against the concentration of nonradioactive fructose (abscissa). The ratio of radioactive glucose to nonradioactive fructose was kept constant. For comparison, the curve showing 14 C0 production from glucose 14 C in the presence of nonradioactive glucose is duplicated from Figure 1. RSULTS Hexose utilization by the spermatozoa as measured by the yield of 14 C0 from labeled sugars in a radiorespirometry system showed that glucose is more c( z Q.. "' Q.. Q. 3! ~tmol J LABLD FRUCTOS + GLUCOS -- LABLD FRUCTOS + FRUCTOS. S.D. FG. 3. Competitive utilization of radioactive fructose in the presence of a large excess of nonradioactive glucose. The 14 C0 produced from radioactive fructose (ordinate) was plotted against the concentration of nonradioactive glucose (abscissa). For comparison the curve showing ' 4 C0 production from fructose 14 C in the presence of excess nonradioactive fructose is duplicated from Figure 1. readily oxidized than fructose when the substrate concentration did not exceed the normal concentration in blood ( J.tmoles/100 ml) (Fig. 1). When the concentration of hexose exceeded these values, the difference in utilization tended to disappear. This absence of difference indicates that the hexokinase systems lose their substrate selectivity when substrate concentration is high and it may explain in part why fructose concentration in seminal plasma is almost twice as high as the serum concentration of glucose. n some experiments glucose 14 C (U-L) was added to the oxidation system with a large excess of nonradioactive fructose, and in others fructose 14 C (U-L) was added with a large excess of nonradioactive glucose. The results of both types of experiments clearly indicate that human spermatozoa preferentially utilized glucose as a glycolytic substrate (Figs. and 3). n view of these results, glucose was chosen as substrate to measure glycolysis in sperm from normal and infertile men. The production of 14 C0 from labeled glucose (5 J.tmoles/ml) by spermatozoa from normal, asthenospermic, and oligoasthenospermic men was measured. No significant difference in glucose utilization between spermatozoa from normal and asthenospermic men was found; however, a 3.5-fold increase in glucose consumption by the spermatozoa from oligoasthenospermic men compared to that from normal men was found (Table 1). Table shows that spermatozoa from asthenospermic men produced about the TABL 1. Production of'"co zfrom Glucose '"C (U-L) by Human Spermatozoa (5 p.moles) Group No. of samples Normal 45 Asthenospermic 30 Oligoasthenospermic 45 1 "C0 produceda (dpmlmg DNNhrl 96,595± ,837 ± ,457±50lb Mean±SD. bsignificantly different from the normal group (P<O.OOl).

31 PDRONTAL April1975 TABL. Production of Lactate and Pyruvate by Human Spermatozoa from Normal, Asthenospermic, and Oligoasthenospermic Men Group No.

4 31 PDRONTAL April1975 TABL. Production of Lactate and Pyruvate by Human Spermatozoa from Normal, Asthenospermic, and Oligoasthenospermic Men Group No. of samples Lactate" Pyruvate" Ratio of lactate/pyruvate Normal Asthenospermic Oligoasthenospermic ± ± ±7.19b Mean±SD {J.tmol/mg DNA!hr). bsignificantly different from the normal group (P<O.Ol). same amount of pyruvate and lactate as those from normal men, but spermatozoa from oligoasthenospermic men produced 6.4 times more pyruvate and four times more lactate than those from normal men. DSCUSSON Mann's observation1 that fructose is a source of energy for human spermatozoa and other metabolic studies on the utilization of this hexose led to the proposal that the index of fructolysis is a clinical indicator of the metabolic capacity of spermatozoa. However, it is important to consider that, in the microenvironment of the female genitalia where fertilization takes place, glucose is the main carbohydrate present Furthermore, since the results of the present investigation clearly show that in human spermatozoa, competitive experiments use glucose rather than fructose, we believe that metabolic studies directed to a better knowledge of the oxidative pathways should use glucose as substrate. t was surprising to find that sperm cells from oligoasthenospermic men used much more glucose both by the mbden Meyerhoff pathway (accumulation of lactate and pyruvate) and the Krebs cycle (increase in 14C0 production) than those from normal men. t has been postulated15 16 that a metabolic regulator, released at the time of ejaculation, in some manner uncouples oxidative phosphorylation, decreasing in turn the respiratory inhibition of glycolysis. f this is true, the greater 14C0 production by spermatozoa from oligoasthenospermic men will reflect only an acceleration of glucose 0.45± ± ±.65b utilization due perhaps to the presence of a less sensitive inhibition of glycolysis by oxygen (Pasteur effect) in these cells. However, the activation of the anaerobic glycolysis will be accompanied by an increase in ATP production, which may be used for spermatozoa motility. Because sperm from oligoasthenospermic men are much slower than those from normal men, we may suppose, the main cause of the pathologic problem is either that ATP is quickly hydrolyzed in these cells or that the locomotor machinery rather than the metabolism is defective. This last suggestion is in accord with the fact that sperm with abnormal morphology occur more frequently in oligoasthenospermic men than in normal men. t is interesting to observe that the metabolism of spermatozoa from asthenospermic men is very similar to that of spermatozoa from normal men and different from spermatozoa from oligoasthenospermic men. This observation reflects, perhaps, the fact that these two pathologic conditions result from different pathogenic mechanisms. Artificial insemination with spermatozoa from asthenospermic men produces better results when the first fraction of a split ejaculate is used for the insemination. 16 n accord with the postulated secretion of an uncoupling agent at the moment of ejaculation, concentration of this agent possibly is lower in the first fraction of the split ejaculate and inadequate motility in spermatozoa from asthenospermic men may be due to an increased susceptibility of these cells towards this agent.

5 Vol. 6, No.4 GLYCOLYTC MTABOLSM 313 Although it is difficult to propose definitive conclusions, our results show significant metabolic differences between spermatozoa from the oligoasthenospermic and normal men, and open new and interesting fields of study that may help in understanding the normal and pathologic coupling between metabolism and motility of human spermatozoa. SUMMARY The glycolysis of spermatozoa from normal, asthenospermic, and oligoasthenospermic men was studied using a respirometry technique to measure glucose utilization by the production of 14 C0 from glucose 14 C (U-L). Lactate and pyruvate were measured by a spectrophotometric method using DNA as reference. Fluman spermatozoa preferred glucose to fructose as the glycolytic substrate when concentrations of these hexoses did not exceed normal concentrations in the blood. Spermatozoa from oligoasthenospermic men produced an average of 3.5 times more 14C0 from glucose 14 C (345, 457 dpm/ mg DNA/hour) than did spermatozoa from asthenospermic (88,837 dpm/mg DNA/ hour) and normal men (96,595 dpm/mg DNA/hour). They also formed four times more lactate (9.63p.moles/mg DNA/hour) than spermatozoa from normal men (.33 p.moles/mg DNA/hour) and 6.4 times more pyruvate (.90 p.moles/mg DNA/hour compared to 0.45 p.moles/mg DNA/hour). Spermatozoa from asthenospermic men formed amounts of lactate (3.01 p.moles/ mg DNA/hour) and pyruvate (0.63p.moles/ mg DNA/hour) similar to those produced by spermatozoa from normal men. RFRNCS 1. Amelar RD: nfertility in Men. Diagnosis and Treatment. First dition. Philadelphia, FA Davis Company, 1966, p. Odell WD, Moyer DL: Physiology of Reproduction. St. Louis, CV Mosby Company, 1971, p8 3. Mancini R: Testiculo Humano. ditorial Panamericana, Buenos Aires, 1968, p Scott TW, White G, Annison F: Glucose and acetate metabolism by ram, bull, dog and fowl spermatozoa. Biochem J 83:398, O'Shea T, Voglmayr JK: Metabolism of glucose, lactate and acetate by testicular and ejaculated spermatozoa of the ram. Bioi Reprod :36, Kremer JA: A simple sperm penetration test. nt J Fertil 10:09, Hicks JJ, Pedron N, Rosado A: Modifications of human spermatozoa glycolysis by cyclic adenosine monophosphate (camp), estrogens and follicular fluid. Fertil Steril 3:886, Peterson PH,. Freund M: Profile of glycolytic enzyme activation in human spermatozoa. Fertil Steril 1:151, Lowry OH: Methods in nzymology. Third dition. dited by SP Colowick, NO Kaplan. New York, Academic PreBS, 1957, p Giles JW, Myers H: An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (Lond) 06:93, Hohrst HJ: L(+ )-Lactate. Determination with lactic dehydrogenase and DPN: Methods of enzymatic analysis. dited by HV Bergmeyer, Bernt. New York, Academic Press, 1963, p Mann T: The Biochemistry of Semen and of the Male Reproductive Tract. Second dition. London, Methuen and Co. 1964, p Weed JC, Carrera A: Glucose contents of cervical mucus. Fertil Steril 1:866, Hamner C: The oviduct fluid. n Pathways to Conception. dited by AL Sherman. Springfield, lll., Charles C Thomas, 1971, p Lardy HA, Ghosh D, Plaut GW: A metabolic regulator in mammalian spermatozoa. Science 109:365, Behrman SJ: Techniques of artificial insemination. n Progress in nfertility. dited by SJ Behrman, RW Kistner. Boston, Little, Brown and Company, 1968, p 75

glucose as substrate for bull spermatozoa has been measured: fructose Spermatozoa of the higher mammalians (e.g. bull, dog and human) are known

glucose as substrate for bull spermatozoa has been measured: fructose Spermatozoa of the higher mammalians (e.g. bull, dog and human) are known THE MICHAELIS-MENTEN CONSTANT FOR FRUCTOSE AND FOR GLUCOSE OF HEXOKINASE IN BULL SPERMATOZOA ROBERT RIKMENSPOEL AND The Population Council, The Rockefeller University, Mew York [Received 30th October 1965)