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Metabolism and motility of turkey (Meleagris gallopavo) spermatozoa in the presence or absence of oxygen, glucose and fructose
Camp. Biochem.Physrol. Vol.
SOA.No.
$3.00+0.00 0300-9629/85
3, pp. 325-327,1985
0 1985Pergamon Press Ltd
Printed in Great Bntrun
METABOLISM AND MOTILITY OF TURKEY (MELEAGIUS GALL0PAV0) SPERMATOZOA IN THE PRESENCE OR ABSENCE OF OXYGEN, GLUCOSE AND FRUCTOSE D. AMIR, ORA PINTO, H. SWINDLER and S. HURWITZ Institute of Animal Science, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel (Received 23 May 1984) Abstract-l. Sugar consumption by turkey spermatozoa for 1 hr at 37°C was similar in the presence or absence of oxygen, and with glucose or fructose in the medium. 2. Motility of the spermatozoa at the end of the above incubation period was lower under anaerobic than aerobic conditions. 3. Fructose enhanced the oxygen uptake of spermatozoa in comparison with that in the glucosecontaining medium. Omission of sugar from the medium depressed respiration but not motility of the spermatozoa. 4. The rate of oxygen uptake by the spermatozoa during a 3-hr incubation period was higher in a 3.3-mM than a 15.0-mM glucose medium. 5. Fructose was formed from glucose under aerobic but not under anaerobic conditions. Fructose originating from glucose was used for fructolysis, when the glucose reserve in the medium was almost exhausted.
INTRODUCTION Oxygen uptake by turkey spermatozoa was found to increase with increasing pH of the medium from 6.3 to 8.8 (Bade et al., 1956; Sexton and Giesen, 1982; Pinto et al., 1984). The 0s uptake was also higher in the presence of glucose or fructose than in the absence of sugar in the medium (Sexton, 1974). Turkey spermatozoa oxidized more [U-‘4C]glucose than [U-‘4C]fructose (Kraft et al., 19%). In a previous study (Pinto et al., 1984), we found
that under aerobic conditions, fructose was formed by turkey spermatozoa from glucose and accumulated in the medium, similar to fowl spermatozoa (Lorenz, 1958). The reason for this conversion remains obscure. Furthermore, the occurrence of this process under anaerobic conditions, and the possibility of the utilization of the formed fructose by the spermatozoa, have not been investigated. In the present work, the use by turkey spermatozoa of fructose, added to the medium or generated from glucose, was studied under both aerobic and anaerobic conditions, and the results were compared with those obtained under the same conditions and in the same medium containing glucose.
10 pg neomycin/ml and long kanamycin/ml. Sugar content of the medium varied from 0 to 15 mM and the osmolarity of the medium (330 mosmol) was maintained at the expense of osmotically equivalent concentrations of NaCl. The medium was buffered at 7.3 with 15 mM Hepes (N-2-hydroxyethyl piperazine-N-2-etbane sulphonic acid). Anaerobic conditions were obtained by saturating the manometer flask assemblies used for the oxygen uptake measurements, with nitrogen bubbled through water. The results were subjected to analyses of variance and to Duncan’s multiple range test, or to the multiple comparison test according to Scheffe (1953).
RESULTS
MATERIALS AND METHODS The experimental birds and the methods of collection, evaluation and dilution of semen and measurements of oxygen uptake and glycolysis have been described previously (Pinto et al., 1984). The composition of the medium for the sperm suspension was (in mM): NaCl, 118.5; KCl, 3.0; CaCl,, 0.8; MgCl,, 0.5; NaH,PO, , 2.0. To this medium were added 7.Opg vitamins/100 ml and 1.0 mg amino acids/100 ml (Eagle’s Basal Medium without L-glutamine: Bio-Lab Ltd., Jerusalem, Israel), 200 U penicillin/ml, 200 pg streptomycin/ml, 325
The metabolism of the spermatozoa under aerobic or anaerobic conditions and with glucose or fructose in the medium, was evaluated (Table 1). The oxygen uptake of the spermatozoa was significantly higher in the fructose-containing than Likewise, a the glucose-containing medium. significantly greater accumulation of lactic acid was found in the former than in the latter under aerobic conditions, although the hexose consumption was similar in the two media. Under anaerobic conditions no formation of fructose from glucose was detected. Hexose consumption in both media tended to be lower (P > 0.05) than under aerobic conditions. The lactic acid accumulation, on the other hand, was significantly higher than that in the glucose-containing medium, under aerobic conditions. The motility of the spermatozoa at the end of the 1 hr incubation period was adversely affected by the presence of fructose in the medium, under aerobic conditions. Under anaerobic conditions, the sperm motility was significantly lower than under aerobic conditions. Table 2 presents the effect of different concen-
326
D. AMIR et al. Table 1. Metabolism of turkey spermatozoa during 1 hr incubation at 37°C III the presence of glucose or fructose (15 mM) m the medmm, and under aerobic or anaerobxc conditions (~molj109 cells)* Aerobic Oxygen uptake Hexose disappearance Fructose formatIon Hexose consumption Lactic acid accumulation Sperm motdity after incubation (% mot&e cells)
Anaerobic
Glucose
Fructose
3.71 i 0.28t 2.50 ct 0.28 1.17 + 0.12 1.33 * o.K?p
4.67 + 0.28$ 1.43*01s 1.43*015t
0.38 * 0.06t 86ct 1.91
Fructose
CihXBe
1.08f 0.22
1 17 + 0.29
0 1.08 & 0.22t
1.17 i 0.29t
0.57 + 0.07$
0 64 -t_0.06$
0 62 * 0 07f
69 + 6.8t
30 i: 6.38
26 + 8.9§**
*Five replicates; means + SEM. Numbers followed by d&rent superscripts represent si~l~~ntly d&rent results. Comoarisons within rows according to Duncan’s multiple-range test. **Four rephcates.
trations of glucose in the medium on the respiratory and survival rates of turkey spermatozoa during a 3-hr incubation period. Oxygen uptake was significantly higher in the presence of 15 mM glucose than in the absence of sugar (Experiment 1). In 15 mM glucose, the O2 uptake decreased with time, whereas no such decrease was observed in 3.3 mM glucose (Experiment 2). The motility of the spermatozoa at the end of the incubation period in both experiments was not significantly affected by the presence of sugar in the medium nor by glucose concentration. The changes in the glucose concentrations and in the formed fructose in the medium during incubation (Experiment 2) are shown in Fig. 1. In the highglucose medium, the progressive decrease in the glucose concentration during incubation was accompanied by a continuous increase in the concentration of fructose. In the low-glucose medium, part of the glucose was consumed by the spermatozoa during the first hour of incubation; most of the remaining glucose was converted to fructose, which was in turn used during the following period of incubation. l?ISCUSSION
Formation of fructose from glucose by turkey spermatozoa was reported by Pinto et at. (1984). In the present study, it was found that the fo~ation occurs only under aerobic conditions. Lorenz (1958) also reported that the rate of fructose formation by cock sperm, under semi-anaerobic conditions, was approx. half of that under aerobic conditions. The similar hexose ~nsumption by turkey spermatozoa under aerobic and anaerobic conditions found in the present study, shows that no Pasteur effect (Mann and Lutwak-Mann, 1981) occurs in these cells. consequently, the total amount of energy generated per time unit in the sperm suspensions was lower in a N, atmosphere than under aerobic conditions, and this was reflected in a significantly lower sperm motility. These results are not in accordance with those of Wishart (1982), who reported a higher glucose utilization by turkey spermatozoa in the presence than in the absence of 0,. The disagreement may be due to the fact that Wishart measured glucose disappearance, whereas in the present work glucose consumption was also measured. In this study too, glucose disappearance was greater under aerobic than anaerobic conditions (Table 1). Oxygen uptake was significantly higher in the
(P i 0.05)
fructose- than in the glucose-containing medium, a result differing from that of Sexton (19’74), who reported a similar 0, uptake by turkey spermatozoa in fructose- and glucose-containing media. The higher lactic acid accumulation in the former medium under aerobic conditions, together with the same sugar consumption, suggests that less fructose than glucose is oxidized. Kraft et al. (1978) also reported that turkey spermatozoa oxidize more glucose than fructose. The present study gives no clue as to the substances which may have been oxidized instead of part of the available fructose. The fructose formed by the spermatozoa from glucose is used by them when the glucose in the medium is almost exhausted. At this stage, i.e. when fructose became the prevailing sugar in the medium, there was a significant increase in the 0, uptake of the spermatozoa during the following hours of incubation, as occurred during the first hour of incubation in the medium which contained fructose as the only hexose right from the start of incubation. In the high-glucose-containing medium, fructose was also formed from glucose but, since sufficient glucose was available, the spermatozoa did not use fructose and accordingly did not increase their 0, uptake. On the other hand, omission of glucose from the medium resulted in a depression of 0, uptake by the spermatozoa, as reported in previous studies (Sexton, 1974; Wishart, 1982).
33r
Incubationtime (mm) Fig. 1. changes in the glucose (A----& and fructose concentration in sperm suspensions (O-O) (1 x 109cells/ml) containing 15.0mM (a) or 3.3 mM (b) glucose during incubation at 37°C (means of three replicates).
321
Metabolism ana mOUllLy of turkey spermatozoa
The physiological significance of the conversion of glucose to the less metabolically efficient fructose remains unclear. It could be speculated that under aerobic conditions the “on-demand’ regulation, which controls the penetration rate of the actively transported glucose (Mann and Lutwak-Mann, 1981), does not apply to turkey spermatozoa, thus permitting an over-influx of glucose into the cell. A resulting excess could have been prevented by the conversion of glucose to fructose and the passive transport of the latter out of the cell (Boszormenyi et al., 1972). Under a N2 atmosphere, active transport of glucose is suppressed (Crane, 1960), and its transport into turkey sperm cells may not have exceeded the latter’s glycolytic capacity. The formation of fructose as a regulating mechanism was therefore unnecessary. No consistent results as to the effect of fructose on the motility of spermatozoa were obtained. Whereas an adverse effect was obtained when the spermatozoa were suspended in a medium with added fructose, sperm motility was not affected when the fructose in the medium was formed from glucose. Acknowledgements-This paper is contribution No. 878-E, 1983 series from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. The study was supported by the U.S.-Israel Binational Agricultural Research and Development Fund (BARD) under contract No. I-79-85. The authors thank Drs. A. Genizi and Ruth Marcus for guidance with the statistical analysis and Miss C. Bechar and Mr. H. Gassitua for assistance.
REFERENCES Bade
M. L., Wiegers H. and Nelson L. (1956) Oxygen uptake, motility and fructolysis of turkey spermatozoa. J. appl. Physiol. 9, 91-96. Boszormenyi Z., Cseh E., Gardos G. and Kertai P. (1972) Transport Processes in Living Organisms. Akademiai Kiado, Budapest. Crane R. K. (1960) Intestinal absorption of sugars. Physiol. Rev. 40, 789-825.
Kraft L. A., Foley C. W., Howarth B. Jr., Blum M. S. and Johnson A. D. (1978) Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species. Comp. Biochem. Physiol. 60B, 233-238. Lorenz F. W. (1958) Carbohydrate metabolism of cock spermatozoa. Nature, Lond. 182, 397-398. Mann T. and Lutwak-Mann C. (1981) Male Reproductive Function and Semen. Springer, Berlin. Pinto O., Amir D., Schindler H. and Hurwitz S. (1984) Effect of pH on the metabolism and fertility of turkey spermatozoa. J. Reprod. Fert. 70, 437-442. Scheffe H. (1953) Judging all contrasts in the analysis of variance. Biometrika 40, 87-104. Sexton T. J. (1974) Oxidative and glycolytic activity of chicken and turkey spermatozoa. Comp. Biochem. Physiol. 48B, 59-65.
Sexton T. J. and Giesen A. F. (1982) Beltsville poultry semen extender. 6. Holding turkey semen for six hours at 15°C. Poult. Sci. 61, 1202-1208. Wishart G. J. (1982) Maintenance of ATP concentrations in and of fertilizing ability of fowl and turkey spermatozoa in vitro. J. Reprod. Fert. 66, 457-462.
SOA.No.
$3.00+0.00 0300-9629/85
3, pp. 325-327,1985
0 1985Pergamon Press Ltd
Printed in Great Bntrun
METABOLISM AND MOTILITY OF TURKEY (MELEAGIUS GALL0PAV0) SPERMATOZOA IN THE PRESENCE OR ABSENCE OF OXYGEN, GLUCOSE AND FRUCTOSE D. AMIR, ORA PINTO, H. SWINDLER and S. HURWITZ Institute of Animal Science, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel (Received 23 May 1984) Abstract-l. Sugar consumption by turkey spermatozoa for 1 hr at 37°C was similar in the presence or absence of oxygen, and with glucose or fructose in the medium. 2. Motility of the spermatozoa at the end of the above incubation period was lower under anaerobic than aerobic conditions. 3. Fructose enhanced the oxygen uptake of spermatozoa in comparison with that in the glucosecontaining medium. Omission of sugar from the medium depressed respiration but not motility of the spermatozoa. 4. The rate of oxygen uptake by the spermatozoa during a 3-hr incubation period was higher in a 3.3-mM than a 15.0-mM glucose medium. 5. Fructose was formed from glucose under aerobic but not under anaerobic conditions. Fructose originating from glucose was used for fructolysis, when the glucose reserve in the medium was almost exhausted.
INTRODUCTION Oxygen uptake by turkey spermatozoa was found to increase with increasing pH of the medium from 6.3 to 8.8 (Bade et al., 1956; Sexton and Giesen, 1982; Pinto et al., 1984). The 0s uptake was also higher in the presence of glucose or fructose than in the absence of sugar in the medium (Sexton, 1974). Turkey spermatozoa oxidized more [U-‘4C]glucose than [U-‘4C]fructose (Kraft et al., 19%). In a previous study (Pinto et al., 1984), we found
that under aerobic conditions, fructose was formed by turkey spermatozoa from glucose and accumulated in the medium, similar to fowl spermatozoa (Lorenz, 1958). The reason for this conversion remains obscure. Furthermore, the occurrence of this process under anaerobic conditions, and the possibility of the utilization of the formed fructose by the spermatozoa, have not been investigated. In the present work, the use by turkey spermatozoa of fructose, added to the medium or generated from glucose, was studied under both aerobic and anaerobic conditions, and the results were compared with those obtained under the same conditions and in the same medium containing glucose.
10 pg neomycin/ml and long kanamycin/ml. Sugar content of the medium varied from 0 to 15 mM and the osmolarity of the medium (330 mosmol) was maintained at the expense of osmotically equivalent concentrations of NaCl. The medium was buffered at 7.3 with 15 mM Hepes (N-2-hydroxyethyl piperazine-N-2-etbane sulphonic acid). Anaerobic conditions were obtained by saturating the manometer flask assemblies used for the oxygen uptake measurements, with nitrogen bubbled through water. The results were subjected to analyses of variance and to Duncan’s multiple range test, or to the multiple comparison test according to Scheffe (1953).
RESULTS
MATERIALS AND METHODS The experimental birds and the methods of collection, evaluation and dilution of semen and measurements of oxygen uptake and glycolysis have been described previously (Pinto et al., 1984). The composition of the medium for the sperm suspension was (in mM): NaCl, 118.5; KCl, 3.0; CaCl,, 0.8; MgCl,, 0.5; NaH,PO, , 2.0. To this medium were added 7.Opg vitamins/100 ml and 1.0 mg amino acids/100 ml (Eagle’s Basal Medium without L-glutamine: Bio-Lab Ltd., Jerusalem, Israel), 200 U penicillin/ml, 200 pg streptomycin/ml, 325
The metabolism of the spermatozoa under aerobic or anaerobic conditions and with glucose or fructose in the medium, was evaluated (Table 1). The oxygen uptake of the spermatozoa was significantly higher in the fructose-containing than Likewise, a the glucose-containing medium. significantly greater accumulation of lactic acid was found in the former than in the latter under aerobic conditions, although the hexose consumption was similar in the two media. Under anaerobic conditions no formation of fructose from glucose was detected. Hexose consumption in both media tended to be lower (P > 0.05) than under aerobic conditions. The lactic acid accumulation, on the other hand, was significantly higher than that in the glucose-containing medium, under aerobic conditions. The motility of the spermatozoa at the end of the 1 hr incubation period was adversely affected by the presence of fructose in the medium, under aerobic conditions. Under anaerobic conditions, the sperm motility was significantly lower than under aerobic conditions. Table 2 presents the effect of different concen-
326
D. AMIR et al. Table 1. Metabolism of turkey spermatozoa during 1 hr incubation at 37°C III the presence of glucose or fructose (15 mM) m the medmm, and under aerobic or anaerobxc conditions (~molj109 cells)* Aerobic Oxygen uptake Hexose disappearance Fructose formatIon Hexose consumption Lactic acid accumulation Sperm motdity after incubation (% mot&e cells)
Anaerobic
Glucose
Fructose
3.71 i 0.28t 2.50 ct 0.28 1.17 + 0.12 1.33 * o.K?p
4.67 + 0.28$ 1.43*01s 1.43*015t
0.38 * 0.06t 86ct 1.91
Fructose
CihXBe
1.08f 0.22
1 17 + 0.29
0 1.08 & 0.22t
1.17 i 0.29t
0.57 + 0.07$
0 64 -t_0.06$
0 62 * 0 07f
69 + 6.8t
30 i: 6.38
26 + 8.9§**
*Five replicates; means + SEM. Numbers followed by d&rent superscripts represent si~l~~ntly d&rent results. Comoarisons within rows according to Duncan’s multiple-range test. **Four rephcates.
trations of glucose in the medium on the respiratory and survival rates of turkey spermatozoa during a 3-hr incubation period. Oxygen uptake was significantly higher in the presence of 15 mM glucose than in the absence of sugar (Experiment 1). In 15 mM glucose, the O2 uptake decreased with time, whereas no such decrease was observed in 3.3 mM glucose (Experiment 2). The motility of the spermatozoa at the end of the incubation period in both experiments was not significantly affected by the presence of sugar in the medium nor by glucose concentration. The changes in the glucose concentrations and in the formed fructose in the medium during incubation (Experiment 2) are shown in Fig. 1. In the highglucose medium, the progressive decrease in the glucose concentration during incubation was accompanied by a continuous increase in the concentration of fructose. In the low-glucose medium, part of the glucose was consumed by the spermatozoa during the first hour of incubation; most of the remaining glucose was converted to fructose, which was in turn used during the following period of incubation. l?ISCUSSION
Formation of fructose from glucose by turkey spermatozoa was reported by Pinto et at. (1984). In the present study, it was found that the fo~ation occurs only under aerobic conditions. Lorenz (1958) also reported that the rate of fructose formation by cock sperm, under semi-anaerobic conditions, was approx. half of that under aerobic conditions. The similar hexose ~nsumption by turkey spermatozoa under aerobic and anaerobic conditions found in the present study, shows that no Pasteur effect (Mann and Lutwak-Mann, 1981) occurs in these cells. consequently, the total amount of energy generated per time unit in the sperm suspensions was lower in a N, atmosphere than under aerobic conditions, and this was reflected in a significantly lower sperm motility. These results are not in accordance with those of Wishart (1982), who reported a higher glucose utilization by turkey spermatozoa in the presence than in the absence of 0,. The disagreement may be due to the fact that Wishart measured glucose disappearance, whereas in the present work glucose consumption was also measured. In this study too, glucose disappearance was greater under aerobic than anaerobic conditions (Table 1). Oxygen uptake was significantly higher in the
(P i 0.05)
fructose- than in the glucose-containing medium, a result differing from that of Sexton (19’74), who reported a similar 0, uptake by turkey spermatozoa in fructose- and glucose-containing media. The higher lactic acid accumulation in the former medium under aerobic conditions, together with the same sugar consumption, suggests that less fructose than glucose is oxidized. Kraft et al. (1978) also reported that turkey spermatozoa oxidize more glucose than fructose. The present study gives no clue as to the substances which may have been oxidized instead of part of the available fructose. The fructose formed by the spermatozoa from glucose is used by them when the glucose in the medium is almost exhausted. At this stage, i.e. when fructose became the prevailing sugar in the medium, there was a significant increase in the 0, uptake of the spermatozoa during the following hours of incubation, as occurred during the first hour of incubation in the medium which contained fructose as the only hexose right from the start of incubation. In the high-glucose-containing medium, fructose was also formed from glucose but, since sufficient glucose was available, the spermatozoa did not use fructose and accordingly did not increase their 0, uptake. On the other hand, omission of glucose from the medium resulted in a depression of 0, uptake by the spermatozoa, as reported in previous studies (Sexton, 1974; Wishart, 1982).
33r
Incubationtime (mm) Fig. 1. changes in the glucose (A----& and fructose concentration in sperm suspensions (O-O) (1 x 109cells/ml) containing 15.0mM (a) or 3.3 mM (b) glucose during incubation at 37°C (means of three replicates).
321
Metabolism ana mOUllLy of turkey spermatozoa
The physiological significance of the conversion of glucose to the less metabolically efficient fructose remains unclear. It could be speculated that under aerobic conditions the “on-demand’ regulation, which controls the penetration rate of the actively transported glucose (Mann and Lutwak-Mann, 1981), does not apply to turkey spermatozoa, thus permitting an over-influx of glucose into the cell. A resulting excess could have been prevented by the conversion of glucose to fructose and the passive transport of the latter out of the cell (Boszormenyi et al., 1972). Under a N2 atmosphere, active transport of glucose is suppressed (Crane, 1960), and its transport into turkey sperm cells may not have exceeded the latter’s glycolytic capacity. The formation of fructose as a regulating mechanism was therefore unnecessary. No consistent results as to the effect of fructose on the motility of spermatozoa were obtained. Whereas an adverse effect was obtained when the spermatozoa were suspended in a medium with added fructose, sperm motility was not affected when the fructose in the medium was formed from glucose. Acknowledgements-This paper is contribution No. 878-E, 1983 series from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. The study was supported by the U.S.-Israel Binational Agricultural Research and Development Fund (BARD) under contract No. I-79-85. The authors thank Drs. A. Genizi and Ruth Marcus for guidance with the statistical analysis and Miss C. Bechar and Mr. H. Gassitua for assistance.
REFERENCES Bade
M. L., Wiegers H. and Nelson L. (1956) Oxygen uptake, motility and fructolysis of turkey spermatozoa. J. appl. Physiol. 9, 91-96. Boszormenyi Z., Cseh E., Gardos G. and Kertai P. (1972) Transport Processes in Living Organisms. Akademiai Kiado, Budapest. Crane R. K. (1960) Intestinal absorption of sugars. Physiol. Rev. 40, 789-825.
Kraft L. A., Foley C. W., Howarth B. Jr., Blum M. S. and Johnson A. D. (1978) Radiorespirometric studies of carbohydrate metabolism by washed spermatozoa of various species. Comp. Biochem. Physiol. 60B, 233-238. Lorenz F. W. (1958) Carbohydrate metabolism of cock spermatozoa. Nature, Lond. 182, 397-398. Mann T. and Lutwak-Mann C. (1981) Male Reproductive Function and Semen. Springer, Berlin. Pinto O., Amir D., Schindler H. and Hurwitz S. (1984) Effect of pH on the metabolism and fertility of turkey spermatozoa. J. Reprod. Fert. 70, 437-442. Scheffe H. (1953) Judging all contrasts in the analysis of variance. Biometrika 40, 87-104. Sexton T. J. (1974) Oxidative and glycolytic activity of chicken and turkey spermatozoa. Comp. Biochem. Physiol. 48B, 59-65.
Sexton T. J. and Giesen A. F. (1982) Beltsville poultry semen extender. 6. Holding turkey semen for six hours at 15°C. Poult. Sci. 61, 1202-1208. Wishart G. J. (1982) Maintenance of ATP concentrations in and of fertilizing ability of fowl and turkey spermatozoa in vitro. J. Reprod. Fert. 66, 457-462.