Glucose Effect on Respiration: Possible Mechanism for Capacitation in Guinea Pig Spermatozoa B. JANE ROGERS,'.' LANA CHANG'.2 AND RYUZO YANAGIMACHI' Pacific Biomedical Research Center and 'The Department of Anatomy and Reproductive Biology, University of Hawaii, Honolulu, Hawaii 96826

ABSTRACT Guinea pig sperm respiration was determined in minimal capacitation medium (MCM) with different energy sources. The Z 0 2 observed for spermatozoa suspended in media containing pyruvate and lactate was 35.7 5.9, pyruvate alone, 27.9 3.8 and D-glucose alone 3.4 +- 1.1. When D-glucose was added to spermatozoa rapidly respiring in media containing pyruvate as the only exogenous energy source, an immediate suppression in respiration was observed. Further reduction was caused by continued addition of D-glucose. Fructose and mannose also produced a suppression in respiratory rate. However, lactose, fucose, sucrose, L-glucose, and galactose did not alter the respiratory rate. The suppression of respiration by metabolizable sugars is paralleled by a suppression of acrosome reaction in guinea pig spermatozoa. The possibility that suppression of respiration is the mechanism for retardation of capacitation and the subsequent acrosome reaction by D-glucose and other metabolizable sugars is suggested.

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D-glucose has been shown t o have an inhibitory effect on the acrosome reaction and fertilization in guinea pig spermatozoa (Rogers and Yanagimachi, '75).The mechanism of this effect is unknown. Rogers and Yanagimachi ('75) postulated that the mechanism of Dglucose retardation on acrosome reaction might be metabolic via an effect of D-glucose on respiration. Previously D-glucose was shown to inhibit oxidative processes in bull spermatozoa (Lardy and Phillips, '41). Respiration has been shown to be related to capacitation in rabbit, fowl, and ram (Hamner and Williams, '63; Mounib and Chang, '64; Wales and Restall, '66; Foley and Williams, '67; Murdoch and White, '67, '68; Black et al., '68; Iritani et al., '69; Stone et al., '73). Hamner and Williams ('63) suggested that an increase in oxygen consumption by spermatozoa residing in the rabbit female tract may be an important part of the capacitation process. Wales and Restall ('66) and Iritani et al. ('69) suggested that part of the stimulation they observed by these reproductive tract fluids may be simply due to substrates in the fluids. This study concerns the effect of substrates, specifically sugars, pyruvate and lactate on respiration. The purpose of this study was to determine whether the suppression of J. EXP. ZOOL. (1979)207: 107-112.

the acrosome reaction by D-glucose and other sugars in guinea pig spermatozoa is paralleled by an effect on respiration. MATERIALS AND METHODS

Guinea pig spermatozoa from the distal portion of one or two cauda epididymides were extruded into 4 ml of 0.9%NaC1. After washing twice in saline by centrifugation (600 X g for 5 minutes each) the spermatozoa were resuspended in 1 to 2 ml of minimal culture medium (MCM) (Barros, '74) a t a final concentration of 0.2 to 2 x lo8 spermatozoa/ml. Sperm counts were made for each determination using a Spencer Bright-Line Hemacytometer. The minimal culture medium contained NaCl (102.3 mM), CaCI, (1.71 mM), NaHCO, (25.1 mM), Na pyruvate (0.25 mM), DL-Na lactate (21.58 mM) and D-glucose (5.56 mM). When the concentration of energy sources was varied, the osmolality was maintained a t approximately 306 mOsM by varying the NaCl concentration. When pyruvate or glucose was the only energy source present, each was tested at 5.00 mM and 5.56 mM respectively. Oxygen uptake was measured with a Clark electrode attached to a Gilson Oxygraph (Gilson Medical Electronics, Middleton, Wisconsin). Respiration rates were determined on

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B. JANE ROGERS, LANA CHANG AND RYUZO YANAGIMACYI

spermatozoa contained in a 1.5 ml volume in a water-jacketed chamber a t 37°C. When determining the effect of added sugars, 2 pmoles of sugar contained in a 20-pl aliquot were added to the 1.5-ml chamber using a Hamilton microsyringe a t 1-minute intervals. All respiration rates were determined within 30 minutes after suspension of the spermatozoa. During the incubation periods in the oxygraph chamber no significant reduction of sperm viability or motility was caused by stirring. Respiration rates are expressed as ZO, which is defined as p1 02/108cellshr. RESULTS

Respiration rates for washed guinea pig spermatozoa suspended in media containing different energy sources were determined (table 1). The ZO, for spermatozoa in media with either pyruvate or pyruvate and lactate was much higher than the Z 0 2 of spermatozoa in media with D-glucose as the only energy source. The presence of lactate in addition to pyruvate gave a slightly higher Z 0 2 than did pyruvate alone (table 1).These concentrations of pyruvate (0.25 mM), lactate (21.58 mM) and glucose (5.56 mM) are commonly used in capacitating media. In another type of experiment D-glucose was added to washed guinea pig spermatozoa TABLE 1

Guinea pig sperm respiration (ZOJ in different energy sources Energy source

20, '

Pyruvate (0.25mM) and lactate (21.58mM) Pyruvate (5.00mM)

35.7 t 5.9 (15determinations) 27.9f 3.8 (18determinations) 3.4 2 1.1 (20determinations)

Glucose (5.56mM) ~~~

~

' 20, values are the mean Istandard error of the number of determinations. Each determination was done with spermatozoa from a different guinea pig.

which were rapidly respiring in media containing 5 mM pyruvate. Figure 1shows the result of a typical experiment of this nature. Dglucose (0.1 MI was added, 20 pl (2 pmoles) a t a time, to the incubation flask containing sperm suspended in MCM with 5 mM pyruvate as the only exogenous energy source. The addition of 2 pmoles of D-glucose, represented by the first arrow, reduced the ZO, from 40.7 to 17.8. Further additions of D-glucose continued to reduce the respiration rate. Table 2 shows the data from several experiments in which aliquots of D-glucose were added. The sperm concentration was kept near 1 x 10' (range 9.1 x lo7 to 1.5 x 10'). The initial ZO, was dramatically reduced by the first addition of D-glucose; in each case the Z 0 2 after one Dglucose addition was less than 50%of the original value (table 2). The addition of only 2 pmoles of D-glucose was sufficient to cause this reduction. No change was detected in the respiration rate if no glucose was added or if 20 pl of MCM without energy source was added. Linearity was maintained over the experimental period in these control type experiments. The ability of other sugars to suppress the respiration of spermatozoa was also tested. Lactose, fucose, sucrose, L-glucose and galactose were found to have no effect on respiration when tested under conditions which produced a glucose effect. These sugars when added to the incubation flask while the sperm were rapidly respiring with pyruvate as exogenous energy source produced no change in the respiration rate as measured by the slope. Fructose and mannose did produce a depression in respiration. Typical results are shown in figures 2 and 3. The respiration rate is reduced after addition of each 20 p1 aliquot of 0.1 M fructose. This reduction continues with subsequent additions of fructose (fig. 2) but is not suppressed to zero or below the level of detection. A similar effect is observed upon

TABLE 2

Effect of glucose ' on guinea pig sperm respiration Sperm conc./ml

1.2 x 1.2 x 8.5 x 1.5 X 1.3 X 1.3 X 9.1 x 1

108 108 107 10' 10' 10' 10'

Initial 20,

+ 20 pl

+ 40 p1

+ 60 pl

+ 80 4

44.3 40.6 47.3 40.7 20.1 18.2 22.9

15.1 15.1 15.0 17.8 9.3 3.0 6.0

13.4 14.6 8.7 12.3 0.4 3.0 1.4

6.0 6.6 5.3 9.1

6.4 4.0

1.5

1.5

Glucose (0.1 M) was added 20 pl at a time to sperm suspended in MCM containing 5 m M pyruvate.

8.2

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GLUCOSE AFFECTS SPERM RESPIRATION

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40.7

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Fig. 1 Effect of glucose on guinea pig sperm respiration. Spermatozoa are suspended in MCM media containing 5 mM pyruvate (equivalent to 7.5 &moles in 1.5 ml chamber). The initial slope is modified by the addition of 2 &moles of glucose in 20 r l aliquots a t approximately 1-minute intervals (indicated by the arrows). The calculated 20,and the percentage of the original 20, are both tabulated for each concentration level.

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5mM pyruvate I

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Fig. 2 Effect of fructose on guinea pig sperm respiration. Spermatozoa are suspended in MCM media containing 5 mM pyruvate. The initial slope is modified by the addition of 2 &moles of fructose (indicated by the arrows).

addition of mannose (fig. 3). Fructose suppresses respiration but not to as great a degree as glucose and mannose do. Upon the addition of 20 pl of D-glucose, respiration is reduced to 44% of the initial rate whereas with fructose it

is reduced to 58% and with mannose it is reduced to 39%. DISCUSSION

The data presented here show that D-glu-

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B. JANE ROGERS, LANA CHANG AND RYUZO YANAGIMACHI

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5mM pyrwate

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Fig. 3 Effect of mannose on guinea pig sperm respiration. Spermatozoa are suspended in MCM media containing 5 mM pyruvate. The initial slope is modified by the addition of 2 pmoles of mannose (indicated by the arrows).

cose, fructose, and mannose reduce the respiration rate of guinea pig spermatozoa, thus providing a possible mechanism for the retarding effect of these sugars on the acrosome reaction. A dose response effect of D-glucose on the acrosome reaction which was previously shown by Rogers and Yanagimachi (‘75) appears to be paralleled by an effect of D-glucose and other metabolizable sugars on respiration. Increasing levels of D-glucose cause both decreasing levels of acrosome reaction and decreasing levels of respiration. A suppression of respiration by D-glucose has also been demonstrated in hamster and rabbit spermatozoa (Rogers, unpublished data). Based on the data of Frenkel et al. (‘75: table 21, lactate and pyruvate are consumed in moderate amounts (less than half the consumption of D-glucose) by guinea pig spermatozoa. They reported that D-glucose was utilized at a rate of 1.44 gmoles/108 sperm/hr while pyruvate was utilized at 0.64 gmol/108 spermhr. Although D-glucose was rapidly metabolized, oxygen consumption was only slightly greater than the endogenous rate and the cellular ATP level was high. Oxidation in the presence of exogenous pyruvate or lactate was apparently complete since the substrate consumption accounts for the increased oxygen consumption. Useful energy production

did not result, however since ATP levels were not maintained. Thus i t seems that guinea pig sperm metabolize D-glucose in preference to pyruvate or lactate. This preferential metabolism is reflected in the respiration experiments presented here in that D-glucose addition reduces the respiration rate significantly because the sperm prefer to metabolize Dglucose by glycolysis. The D-glucose metabolism utilizes available ADP and Pi in competition with Krebs cycle metabolism and respiration is suppressed. Lardy and Phillips (‘41) previously found a decrease in oxygen consumption in bull spermatozoa when D-glucose was present and suggested that this indicated that the spermatozoa shifted to glycolytic metabolism for some of their energy. This D-glucose-induced respiratory inhibition is referred to as the Crabtree effect. Its mechanism is not totally clear (Koobs, ’72). Frenkel et al. (‘73) found that addition of D-glucose after addition of lactic acid to guinea pig sperm decreased respiration slightly, while addition of lactic acid after D-glucose addition increased respiration. The increase in respiration of spermatozoa caused by genital tract fluids has been studied extensively. Grotjan et al. (‘74) found that pig spermatozoa showed an increase in respira-

GLUCOSE AFFECTS SPERM RESPIRATION

tion when incubated in porcine follicular fluid. The stimulatory effect of the follicular fluid was found to be removed by dialysis. They concluded, however, that the stimulatory effect did not appear to be due to substrates since the addition of D-glucose (up to 5 mg/ml) to the buffer did not increase sperm respiration when incubated in porcine follicular fluid. Extending the results using guinea pig spermatozoa to porcine spermatozoa a decrease in respiration by the addition of glucose might be anticipated. The small molecules that were lost by dialysis could have been pyruvate or lactate. A comparison of the respiration of spermatozoa in dialyzed follicular fluid with and without pyruvate might be a meaningful comparison to rule out the effect of substrates. The possibility that the effect of genital tract fluids on respiration is due to energy sources or bicarbonate has been pursued. Foley and Williams (’67)suggested that bicarbonate could account for a considerable portion of the increase in O2uptake but that additional stimulants may be present in female tract secretions. Hamner and Williams (‘64) and Foley and Williams (’67) suggested that one possible explanation for an increase in oxygen consumption with added bicarbonate is that bicarbonate may increase the respiration by increasing the level of tricarboxylic acid cycle intermediates. Storey (‘75) found that the presence of 45 mM HC03 in the medium stimulated the respiration rate with pyruvate but not with glucose. He felt that this was in accord with earlier findings that rabbit oviductal fluid does stimulate rabbit sperm respiration and that bicarbonate is a factor essential to this stimulation. Murdoch and White (‘68)found lactate to be more effective than glucose in increasing O2uptake of rabbit spermatozoa. Spilman et al. (‘70) found there was a significant positive correlation between the amount of lactate in the oviduct fluids and the amount of stimulation of oxygen utilization caused by the fluids. Bedford (‘701 stated that the functional significance of the observed increase in the respiration of spermatozoa incubated in reproductive tract fluids is unknown. One possible function of this increase in respiration could be that this is part of the mechanism of capacitation and acrosome reaction. A possible energy increase caused by the stimulation in respiration may be important for the acro-

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some reaction. Stone et al. (‘73) presented results confirming an increase in oxygen uptake by porcine spermatozoa incubated in the presence of estrous oviductal fluids and indicated that increased oxygen uptake is paralleled by a proportional increase in energy production. We are suggesting that the increase in respiration associated with capacitation and acrosome reaction can also be caused by pyruvate or lactate and that this stimulated respiration is associated with spermatozoa that are capable of fertilization. ACKNOWLEDGMENTS

This work was supported by a grant from NIH HD-03402and an NIH Minority Biomedical Support Program Grant RR8125-02. We thank Marilynn Ueno for suggestions and assistance in preparing the manuscript. LITERATURE CITED Barros, C. 1974 Capacitation of mammalian spermatozoa. In: Physiology and Genetics of Reproduction. Part B. E. M. Coutinho and F. Fuchs, ede. Plenum Press, New York, pp. 3-24. Bedford, J. M. 1970 Sperm capacitation and fertilization in mammals. Biol. Reprod. (Suppl.), 2: 128-158. Black, D. L., L. V. Crowley, R. T. Durby and C.H. Spilman 1968 Oviduct secretion in the ewe and the effect of oviduct fluid on oxygen uptake by ram spermatozoa in uitro. J. Reprod. Fert., 15: 127-130. Foley, C. W., and W. L. Williams 1967 Effect of bicarbonate and oviduct fluid on respiration of spermatozoa. Proc. SOC. Exp. Biol. Med., 126: 634-637. Frenkel, G., R. N. Peterson and M. Freund 1973 Changes in the metabolism of guinea pig sperm from different segments of the epididymis. Proc. SOC.Exp. Biol. Med., 143: 1231-1236. 1975 Oxidative and glycolytic metabolism of semen components by washed guinea pig spermatozoa. Fertil. Steril., 26: 144-147. Grotjan, H. E., B. N. Day and D. T. Mayer 1974 Porcine spermatozoan respiration in the presence of porcine follicular fluid. J. h i m . Sci., 38: 1235-1238. Hamner, C. E., and W. L. Williams 1963 Effect of the female reproductive tract on sperm metabolism in the rabbit and fowl. J. Reprod. Fert., 5: 143-150. 1964 Identification of sperm stimulating factor of rabbit oviduct fluid. Proc. Soc. Exp. Biol. Med., 117: 240-243. Iritani, A., W.P. Gomes and N. L. Vandemark 1969 The effect of whole, dialyzed and heated female genital tract fluida on respiration of rabbit and ram spermatozoa. Biol. Reprod., 1: 77-82. Koobs, D. H. 1972 Phosphate mediation of the Crabtree and Pasteur effects. Science, 178: 127-133. Lardy, H., and P. H. Phillips 1941 The interrelation of oxidative and glycolytic processes as sources of energy for bull spermatozoa. Amer. J. Physiol., 133: 602-609. Mounib, S.,and M. C. Chang 1964 Effect ofin utero incubation on the metabolism of rabbit spermatozoa. Nature, 201: 943-944.

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Murdoch, R. N., and I. G. White 1967 The metabolism of labelled glucose by rabbit spermatozoa after incubation in utero. J. Reprod. Fert., 14: 213-223. 1968 The influence of the female genital tract on the metabolism of rabbit spermatozoa. Aust. J. Biol. Sci., 21: 961-972. Rogers, B. J., and R. Yanagimachi 1975 Retardation of guinea pig sperm acrosome reaction by glucose: the possible importance of pyruvate and lactate metabolism in capacitation and acrosome reaction. Biol. Reprod., 13: 568-575. Spilman, C. H., R. T. Duby and D. L. Black 1970 Effect of an

intrauterine device on sheep oviduct fluids: chemical composition and stimulation of spermatozoan respiration in uitro. Biol. Reprod., 3: 76-81. Stone, R. T., C. W. Foley, J. G. Thorne and T. L. Huber 1973 Effect of oviductal fluids on oxidative phosphorylation in spermatozoa. Proc. Soc. Exp. Biol. Med., 143: 64-67. Storey, B. T. 1975 Energy metabolism of spermatozoa. IV. Effect of calcium on respiration of mature epididymal sperm of the rabbit. Biol. Reprod., 13: 1-9. Wales, R. G., and B. J. Restall 1966 The metabolism of ram spermatozoa in the presence of genital fluids of the ewe. Aust. J. Bio. Sci., 19: 199-209.