Vol. 26, No. 2, February 1975 Printed in U.S.A.
FERTILITY AND STERILITY Copyright c 1975 The American Fertility Society
OXIDATIVE AND GLYCOLYTIC METABOLISM OF SEMEN COMPONENTS BY WASHED GUINEA PIG SPERMATOZOA GEDALIA FRENKEL, PH.D.,* RUDOLPH N. PETERSON, PH.D.,t
AND
MATTHEW FREUND, PH.D.
Laboratory of Reproductive Pharmacology, Department of Pharmacology, New York Medical College, Valhalla, New York 10595
,. The metabolism of washed guinea pig epididymal and ejaculated spermatozoa is characterized by high rates of aerobic glycolysis and oxygen consumption. Anaerobically, glycolysis is low and motility is not maintained. 1-3 This behavior is in marked contrast to that of spermatozoa of several other species, especially human sperm, where metabolism is predominantly glycolytic and where glycolysis supports motility under both aerobic and anaerobic conditions. We analyzed the mechanisms of metabolic control in both species (human and guinea pig) with the view that comparison of particular metabolic features will contribute to an understanding of the basic causes of metabolic differences. In this report, we present an analysis of guinea pig semen for potentially metabolizable substances and also report on the comparative ability of these substances to support sperm ATP levels and motility. MATERIALS AND METHODS
Adult guinea pigs (Carworth, New City, NY), weighing approximately 1 kg, were used in all the experiments. Semen was obtained by electroejaculation as described by Freund. 4 Semen was collected in centrifuge tubes containing tris buffer, pH 7.4. 5 The coagulated Received April 3,.1974. *Permanent address: Institute for Study and Treatment of Fertility Di~turbances, MunicipalGovernment Medical Center, Tel-Aviv, Israel. t Address inquiries: Dr. Peterson.
ejaculate was minced and dispersed in the buffer. Tubes were then centrifuged for 20 minutes at 100 g. The supernatant was boiled for ten minutes, was centrifuged, and the clear solution was analyzed or frozen until assay. The treatment and isolation of sperm from caudal segments of the epididymis and vas deferens has been described elsewhere. 2 A tris-based salts medium, buffered at pH 7.45 was used in all experiments. Experiments were performed in stoppered 25-ml Erlenmeyer flasks. Incubations were carried out with shaking in a thermostated Dubnoff bath at 37°. Substrates were added at zero time to final concentration of 2.5 millimol. Motility was determined with small portions of the suspensions taken at various times. Aliquots of the sperm suspensions were removed from the flasks at zero time and after two hours of incubation. They were deproteinized immediately by boiling for ten minutes and then centrifuged at 25,000 g. The clear solution was then analyzed or frozen until assay. Oxygen uptake was measured polarographically (Beckman, Model160, oxygen analyzer). After determining the endogenous respiration, the substrate was added with a microsyringe (Hamilton) through a hole in the Teflon sleeve jacketing the electrode. Assays. All assays were determined fluorometrically in a Turner Model 111.
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METABOLISM OF SEMEN COMPONENTS
Vol. 26, No.2
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Fructose, glucose, pyruvate, and lactate were analyzed by the methods described earlier. 7 Glycerol and a-glycerophosphate were analyzed as described elsewhere. 8 For the determination of sorbitol, the following substances were added to fluorometric tubes: triethanolamine (0.1M, pH 7 .5), magnesium chloride (0.003 M), NAD (0.000002 M), ATP (0.0005 M), hexokinase (0.005 mg). Aliquots (25 p.l) of the extract were added to the tubes. Finally, sorbitol dehydrogenase (0.25 mg) was added and the increase in fluorescence was recorded. ATP and ADP were also determined by standard fluorometric procenures.9 Materials. Bakers reagent grade chemicals were used. Fructose was purchased from the Pfanstiehl Company. Highly purified enzymes were purchased from the Boehringer-Mannheim Company. RESULTS AND DISCUSSION
Ejaculated guinea pig semen was assayed for the glycolyzable and oxidizable substrates that have been found in the semen of other mammalian species (Table 1). The rates of utilization of these substrates and their ability to support ATP synthesis and motility are shown in Table 2. Both glucose and fructose were present at high concentrations in guinea pig semen. The average fructose concentration was similar to that reported earlier by Freund and Borelli. 10 No glucose was TABLE !.-Concentrations of Metabolizable Substrates in Guinea Pig Semen Substance
No. of estimations
Concentration•
Glucose Fructose Sorbitol Glycerol a-glycerophosphate Pyruvate Lactate
33 31 8 8 8 34 31
9.81±0.61 6.67±0.45 0.26±0.02 0.77±0.16 0.13±0.04 0.06±0.01 0.53±0.04
8
Mean ± SE.
(mM)
145
detected in the earlier study; however, because our new observations showed substantial amounts of glucose in all specimens examined, we attribute the earlier finding to some defect in assay. The high levels of glucose in guinea pig semen are in contrast to the very low levels of this carbohydrate that are ordinarily found in the seminal plasma of human, ram, boar, and bull. Both glucose and fructose were used by guinea pig sperm at high rates (Table 2), and both compounds sustained a high level of cellular ATP. In view of the fact, however, that hexokinase in nearly all tissue has a much higher affinity for glucose than for fructose, glucose probably is the main energy source for sperm metabolism in the guinea pig. Glucose and fructose are apparently also oxidized by guinea pig sperm since a substantial fraction of the carbohydrate utilized did not appear in the medium as lactic acid. Glucose oxidation probably leads to a compensatory decrease in the oxidation of endogenous substrate since neither glucose nor fructose increased sperm respiration above the endogenous rate. Guinea pig semen contained appreciable amounts of sorbitol; the semen of many other mammalian species also contain sorbitol. However, unlike the case in some of these other species (eg, the ram), sorbitol utilization proceeded very slowly and did not support a high motility or a high rate of ATP synthesis. Glycerol, which is present at about 7 mg % in guinea pig semen (Table 1), was not used by sperm nor did it maintain sperm motility. This fact is attributed to an absence of glycerokinase in guinea pig sperm since the phosphorylated derivative, a-glycerophosphate, was used at a high rate. a-glycerophosphate was also oxidized at a high rate, as indicated by its stimulation of sperm respiration to a level more than double the
't
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February 1975
FRENKEL ET AL
TABLE 2.-Metabolism Rate, a ATP Level, a Percentage Motility of Washed Guinea Pig Sperm ZO,
Substrate (2.5 mM)
Glucose Fructose Sorbitol a-glycerophosphate Pyruvate Lactate Glycerol ~-hydroxybutyrate
Succinate
No. of assays
4 4 4 4 4 4 4 1 1
Substrate utilized (p.mol/10' sperm/hr)
Lactate produced (p.mol/10' sperm/hr)
1.44±0.12 1.37±0.04 0.15±0.02 1.09±0.01 0.64±0.03 0.51 ±0.01 0
1.70±0.09 1.71 ±0.09 0 0 0.22±0.01 0
Endogenous (p.l/10' sperm/hr)
Substrate (.ul/10' sperm!hr)
ATP (p.mol/10' sperm)
36.0±3.8 32.6±6.1 37.2±4.9 28.6±3.8 37.4±2.8 36.0±3.8 36.8±3.7 33.2 33.2
42.2±8.7 32.6±6.1 38.5±5.1 62.1b±5.8 69.5°±10.8 68.8d±4.8 35.9±2.8 53.0 85.0
80.4±2.1 89.8±19.0 8.2±2.3 28.1±3.3 9.7±1.2 21.9±0.9
Motility (%)
50 50 20 60 50 60 10
8 Mean ± SE of at least three separate experiments. bSignificantly different from endogenous rate (P
FERTILITY AND STERILITY Copyright c 1975 The American Fertility Society
OXIDATIVE AND GLYCOLYTIC METABOLISM OF SEMEN COMPONENTS BY WASHED GUINEA PIG SPERMATOZOA GEDALIA FRENKEL, PH.D.,* RUDOLPH N. PETERSON, PH.D.,t
AND
MATTHEW FREUND, PH.D.
Laboratory of Reproductive Pharmacology, Department of Pharmacology, New York Medical College, Valhalla, New York 10595
,. The metabolism of washed guinea pig epididymal and ejaculated spermatozoa is characterized by high rates of aerobic glycolysis and oxygen consumption. Anaerobically, glycolysis is low and motility is not maintained. 1-3 This behavior is in marked contrast to that of spermatozoa of several other species, especially human sperm, where metabolism is predominantly glycolytic and where glycolysis supports motility under both aerobic and anaerobic conditions. We analyzed the mechanisms of metabolic control in both species (human and guinea pig) with the view that comparison of particular metabolic features will contribute to an understanding of the basic causes of metabolic differences. In this report, we present an analysis of guinea pig semen for potentially metabolizable substances and also report on the comparative ability of these substances to support sperm ATP levels and motility. MATERIALS AND METHODS
Adult guinea pigs (Carworth, New City, NY), weighing approximately 1 kg, were used in all the experiments. Semen was obtained by electroejaculation as described by Freund. 4 Semen was collected in centrifuge tubes containing tris buffer, pH 7.4. 5 The coagulated Received April 3,.1974. *Permanent address: Institute for Study and Treatment of Fertility Di~turbances, MunicipalGovernment Medical Center, Tel-Aviv, Israel. t Address inquiries: Dr. Peterson.
ejaculate was minced and dispersed in the buffer. Tubes were then centrifuged for 20 minutes at 100 g. The supernatant was boiled for ten minutes, was centrifuged, and the clear solution was analyzed or frozen until assay. The treatment and isolation of sperm from caudal segments of the epididymis and vas deferens has been described elsewhere. 2 A tris-based salts medium, buffered at pH 7.45 was used in all experiments. Experiments were performed in stoppered 25-ml Erlenmeyer flasks. Incubations were carried out with shaking in a thermostated Dubnoff bath at 37°. Substrates were added at zero time to final concentration of 2.5 millimol. Motility was determined with small portions of the suspensions taken at various times. Aliquots of the sperm suspensions were removed from the flasks at zero time and after two hours of incubation. They were deproteinized immediately by boiling for ten minutes and then centrifuged at 25,000 g. The clear solution was then analyzed or frozen until assay. Oxygen uptake was measured polarographically (Beckman, Model160, oxygen analyzer). After determining the endogenous respiration, the substrate was added with a microsyringe (Hamilton) through a hole in the Teflon sleeve jacketing the electrode. Assays. All assays were determined fluorometrically in a Turner Model 111.
144
11·
11
METABOLISM OF SEMEN COMPONENTS
Vol. 26, No.2
...
Fructose, glucose, pyruvate, and lactate were analyzed by the methods described earlier. 7 Glycerol and a-glycerophosphate were analyzed as described elsewhere. 8 For the determination of sorbitol, the following substances were added to fluorometric tubes: triethanolamine (0.1M, pH 7 .5), magnesium chloride (0.003 M), NAD (0.000002 M), ATP (0.0005 M), hexokinase (0.005 mg). Aliquots (25 p.l) of the extract were added to the tubes. Finally, sorbitol dehydrogenase (0.25 mg) was added and the increase in fluorescence was recorded. ATP and ADP were also determined by standard fluorometric procenures.9 Materials. Bakers reagent grade chemicals were used. Fructose was purchased from the Pfanstiehl Company. Highly purified enzymes were purchased from the Boehringer-Mannheim Company. RESULTS AND DISCUSSION
Ejaculated guinea pig semen was assayed for the glycolyzable and oxidizable substrates that have been found in the semen of other mammalian species (Table 1). The rates of utilization of these substrates and their ability to support ATP synthesis and motility are shown in Table 2. Both glucose and fructose were present at high concentrations in guinea pig semen. The average fructose concentration was similar to that reported earlier by Freund and Borelli. 10 No glucose was TABLE !.-Concentrations of Metabolizable Substrates in Guinea Pig Semen Substance
No. of estimations
Concentration•
Glucose Fructose Sorbitol Glycerol a-glycerophosphate Pyruvate Lactate
33 31 8 8 8 34 31
9.81±0.61 6.67±0.45 0.26±0.02 0.77±0.16 0.13±0.04 0.06±0.01 0.53±0.04
8
Mean ± SE.
(mM)
145
detected in the earlier study; however, because our new observations showed substantial amounts of glucose in all specimens examined, we attribute the earlier finding to some defect in assay. The high levels of glucose in guinea pig semen are in contrast to the very low levels of this carbohydrate that are ordinarily found in the seminal plasma of human, ram, boar, and bull. Both glucose and fructose were used by guinea pig sperm at high rates (Table 2), and both compounds sustained a high level of cellular ATP. In view of the fact, however, that hexokinase in nearly all tissue has a much higher affinity for glucose than for fructose, glucose probably is the main energy source for sperm metabolism in the guinea pig. Glucose and fructose are apparently also oxidized by guinea pig sperm since a substantial fraction of the carbohydrate utilized did not appear in the medium as lactic acid. Glucose oxidation probably leads to a compensatory decrease in the oxidation of endogenous substrate since neither glucose nor fructose increased sperm respiration above the endogenous rate. Guinea pig semen contained appreciable amounts of sorbitol; the semen of many other mammalian species also contain sorbitol. However, unlike the case in some of these other species (eg, the ram), sorbitol utilization proceeded very slowly and did not support a high motility or a high rate of ATP synthesis. Glycerol, which is present at about 7 mg % in guinea pig semen (Table 1), was not used by sperm nor did it maintain sperm motility. This fact is attributed to an absence of glycerokinase in guinea pig sperm since the phosphorylated derivative, a-glycerophosphate, was used at a high rate. a-glycerophosphate was also oxidized at a high rate, as indicated by its stimulation of sperm respiration to a level more than double the
't
146
February 1975
FRENKEL ET AL
TABLE 2.-Metabolism Rate, a ATP Level, a Percentage Motility of Washed Guinea Pig Sperm ZO,
Substrate (2.5 mM)
Glucose Fructose Sorbitol a-glycerophosphate Pyruvate Lactate Glycerol ~-hydroxybutyrate
Succinate
No. of assays
4 4 4 4 4 4 4 1 1
Substrate utilized (p.mol/10' sperm/hr)
Lactate produced (p.mol/10' sperm/hr)
1.44±0.12 1.37±0.04 0.15±0.02 1.09±0.01 0.64±0.03 0.51 ±0.01 0
1.70±0.09 1.71 ±0.09 0 0 0.22±0.01 0
Endogenous (p.l/10' sperm/hr)
Substrate (.ul/10' sperm!hr)
ATP (p.mol/10' sperm)
36.0±3.8 32.6±6.1 37.2±4.9 28.6±3.8 37.4±2.8 36.0±3.8 36.8±3.7 33.2 33.2
42.2±8.7 32.6±6.1 38.5±5.1 62.1b±5.8 69.5°±10.8 68.8d±4.8 35.9±2.8 53.0 85.0
80.4±2.1 89.8±19.0 8.2±2.3 28.1±3.3 9.7±1.2 21.9±0.9
Motility (%)
50 50 20 60 50 60 10
8 Mean ± SE of at least three separate experiments. bSignificantly different from endogenous rate (P
