Carbon Dioxide Uptake During Anaerobic Metabolism of Bovine Spermatozoa1

Carbon Dioxide Uptake During Anaerobic Metabolism of Bovine Spermatozoa1 L. A. KRAFT2 and J. R. LODGE

Department of Dairy Science, University of Illinois Urbana 61801 Abstract

Introduction

Carbon dioxide markedly influences metabolism of spermatozoa. The successful room temperature storage of bull sperm in a C02 containing diluent by VanDemark and Sharma (29) indicated that C02 could help control metabolism and extend liveability and fertility of bull spermatozoa. Medium and high pCOu inhibit both aerobic and anaerobic spermatozoan metabolism (7, 14, 19, 21-23, 25, 26) whereas low levels, i.e. under 5% C02, are stimulatory and under certain conditions necessary for survival (6, 8, 9, 11-13). Bicarbonate, as a component of oviduct fluid (5) or when added exogenously (4), stimulated spermatozoan metabolism by 10 to 29%. Despite all these studies and except for the knowledge that more is involved than pl=I changes due to carbonic acid formation, mechanisms underlying effects of C02 on spermatozoan metabolism remain obscure. Recently an NADtt-dependent uptake of CO 2 by bovine spermatozoa was shown and a fixation with pyruvate suggested (11). Studies involving 14C02 have indicated that fixation occurred in spermatozoa of the ram (18), codfish (17), and bull (24, 30). These labeling experiments have indicated that carbon obtained by CO, fixation can be incorporated into a variety of metabolites. Thus, CO: fixation may be involved in important biosynthetic pathways of sperm. The metabolic pathway(s) responsible for this CO 2 uptake and fixation has not been elucidated nor is its physiological role known. Consequently, the phenomenon of NADH-dependent C02 uptake was studied with the hope of obtaining information useful in establishing its role in spermatozoan metabolism.

Anaerobic CO~ uptake by bovine spermatozoa has been studied by direct manometric measurements with the W a r b u r g respirometer. This uptake phenomenon requires spermatozoa or their components, pyruvate as substrate, N A D H as a cofactor, and a C02 containing gas phase. The magnitude of the uptake was directly associated with available N A D H in the incubation system since increasing concentrations of N A D H between .0-.032 increased the quantity and duration of CO 2 uptake. Maximum dose-response, however, was not obtained. Mild soniflcation, which disrupted the spermatozoa into head and midpiece-tail components, caused a much more rapid and somewhat greater C02 uptake than whole spermatozoa. Separation of sonified spermatozoa into head-rich and midpiece-tail-rich fractions with a sucrose gradient showed that the majority of uptake activity was located in the midpieee-tail portion of the sperm cell. Whole spermatozoa subjected to a sucrose gradient showed marked uptake activity. Fractionations indicated that the responsible cellular materials had leached into the supernatant forming a cell-free extract with uptake activity characteristic of whole cells. Activity of this supernatant could be preserved at low temperatures and was reduced but not completely eliminated by Millipore filtration (.22 ~). Addition of avidin and malonate as specific enzymatic inhibitors was ineffective in reducing uptake activity of any cell fractions studied.

Methods

Received for publication July 21, 1971. 1 This work was supported in part by U.S. Public Health Grant GM-1380 awarded to L. A. Kraft and the Illinois Agricultural Experiment Station. 2 Present address: Department of Pharmacology, Ortho Research Foundation, Raritan, New Jersey 08869.

Spermatozoa were collected with an artificial vagina from dairy bulls either as standard semen or epididymal-like spermatozoa (ELC) (20). The ELC were washed free from the inhibitory diluent by centrifuging 15 min at 1,000 × g and suspended in .9% NaC1 so that .2 ml in each incubation flask contained 2.40 × 10 S to 2.60 × 10 s cells. Sperm samples

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SPERI~IATOZOA

were examined for motility before and after incubation. Some samples were subjected to low intensity sonic oscillations from a Model $75 Bronson sonifier set at 2 to 4 amperes. Exposure for approximately 30 sec broke the sperm into head and midpiece-tail components (sonified cells). The sonified sperm cells were centrifuged at 1,500 X g for 15 rain at room temperature. The pellet was washed twice with .9% NaC1 and resuspended to the original volmne (resuspended pellet) for incubation. The supernatant was vacuum filtered through a sintered glass filter to remove larger cellular structures and where indicated was again filtered through a .22 g Millipore filter before incubation. Sonified spermatozoa were separated into head-rich and midpiece-tail-rieh fractions by a layered density gradient of sucrose. Three milliters of 76%, 1.5 ml of 70%, and 3 ml of 65% sucrose were layered from the bottom upwards in 12 ml conical pyrex centrifuge tubes. Two milliters of sonified cells were placed above the top layer and stirred in with the upper 2 ml of this layer. After eentri_fugation (60 rain, 1,500 × g, 4 C), the upper layer was removed and used as a midpiece-tail-rich fraction. The intermediate layers, containing both heads and tails, were removed and discarded and the remaining pellet was the head-rich fraction. Both fractions were washed twice with .9% NaC1 to remove the sucrose. Whole cell controls were subjected to the same sucrose gradient procedure with intact spermatozoa recovered from the sperm pellet. Carbon dioxide gaseous exchange was measured manometrically at 37 C by the direct method of Warburg (27). Incubations were for 2 or 4 hr in 15 ml single-sidearm Warburg flasks at a shaking rate of approximately 120 strokes per minute. The diluting medium was IVT-pyruvate (Illini Variable Temperature Diluter containing 35 m ~ sodium pyruvate) (29) to which NADH and metabolic inhibitors were added as indicated. The total volume per flask (.8 ml diluent and .2 ml cell suspension or fraction) was 1.0 ml. Each flask was gassed individually with a 5% Co2-95 % Nu gas mixture by at least 10 alternating evacuations with vacuum and gassing sequences followed by immediate attachment to the manometer and placement into the water bath. Spermatozoa from the same sample were in control and treated groups. Results

Results from incubations of bovine ejaculate and its components, illustrated in Figure 1,

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Fro. 1. Carbon dioxide exchange of bovine semen, seminal plasma, and washed cells during anaerobic incubation in I1]ini variable temperature diluter-pyravate containing .016 ~ NADH. (1Vumbets below zero represent uptake and above zero evolution.) indicate that an uptake of C02 occurred only with washed spermatozoa and that the presence of seminal plasma resulted in an evolution of COe. Figure 2 shows the effect of .00 to .032 NADH on C02 uptake. A dose-response relationship existed between NADH and amount and duration of CO 2 uptake. Maximum doseresponse, however, was not obtained. With no NADH a progressive evolution of C02 always occurred, whereas all amounts of N A D t t stimulated an uptake of C02 which eventually was followed by an evolution of C02. When incubation was extended to 8 hr, a continuous evolution of CO 2 occurred after the maximum uptake was reached suggesting that with prolonged incubations a net evolution of CO2 would result even with the higher NADH. Final measured p H was higher and generally no motile sperm were found after either 4 or 8 hr incubation when the amount of N A D t t exceeded .02 ~. Spermatozoa were sonified to alter cell membrane permeability and facilitate passage of JOURNAL 0~" DAIRY SCIENCE ~¢~OL. 55~ NO, 4

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were approximately 90% pure, is shown in Figure 4. The midpiece-tail-rich fraction had over four times the CO 2 uptake activity of the head-rich fraction. Since a major portion of the uptake of the head-rich fraction was possibly due to midpiece-tail contamination, C02 uptake activity is largely, if not entirely, located in the midpiece-tail portion of the spermatozoa. An attempt was made to determine if a biotin enzyme and possible C02 fixation were involved in this uptake reaction. Avidin which inhibits any biotin dependent reaction (15) was added to the incubation medium. Results showed that up to 10 mg of avidin per milliliter of IVT-pyruvate did not change COe uptake of any cellular fraction. The effect of TCA cycle inhibition on the CO 2 uptake activity of spermatozoa was studied with malonate which is a competitive inhibitor of succinie dehydrogenase (15). There was no inhibition of the C02 uptake activity of any of the cellular fractions.

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Yle. 2. Carbo~ dioxide exchange by bovine epididymal-like spermatozoa during anaerobic incubation in Illini variable temperature diluterpyruvate containing varying NADH. (Numbers below zero represent uptake and above zero evolution) compounds into cellular fractions. Sonified suspension was separated into cell-free supernatant and pellet fractions by eentrifugation. The CO 2 uptake of these spermatozoan fractions is in Figure 3. The marked, rapid, and greater total uptake by the sonified cell and cell-free supernatant fractions over the whole cell control was evident. The resuspended pellet fraction had a somewhat slower, although an almost equal, total uptake as compared to other sperm fractions. Uptake activity was completely destroyed when any sperm fraction was heated prior to incubation or if either pyruvate, N A D H , C02 or spermatozoa was removed from the incubation system. The uptake activity of the cell free supernatant could be preserved for several days at low temperatures and was reduced, but not completely eliminated, by Millipore filtration (.22 /~). The sperm midpiece is rich in enzymes and is the site of most of the metabolic activity (16). Therefore, it seemed reasonable that the midpiece portion would also be responsible for most C02 uptake. The uptake activity of headrich and mid-piece-tail-rich fractions, which JOURIqAL O~ DAIRY SCIENCE VOL. 55, 1~0. 4

Discussion

The results reported here confirm previous reports (10, 11) that washed bovine spermatozoa in the presence of pyruvate, .02 ~[ NADH, and 5% C02-95% N2 demonstrate a marked, rapid uptake of C02 which is reversed to an evolution with continued incubation. The response to increased concentrations of N A D H and the results from the sonified fractions suggest that N A D H limited the magnitude of C02 uptake in earlier studies. Ejaculated spermatozoa collected as standard semen required removal of seminal plasma to demonstrate CO 2 uptake activity. Similar findings have been reported for epididymal spermatozoa (10). Uptake always occurred with ELC since seminal plasma is removed during collection and preparation. Bistocchi et al. (2) recently reported that seminal plasma and epididymal fluids rapidly destroyed all p y r idine-nucleotide coenzymes in the spermatozoa; therefore, it is possible that C02 uptake was inhibited by these fluids due to their destruction of exogenous N A D H . Since uptake values per l 0 s cells are comparable only when the total cells per flask are approximately equal, results from the headrich and midpiece-tail rich fractions are comparable with each other but not with the other fractions included in Figure 4 because inefficient separation necessitated using fewer total heads and midpiece-tails per flask. The large increase in C02 uptake by whole cells

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FIG. 3. Effect of sonification and fractionation of bovine epididymal-like spermatozoa on carbon dioxide exchange during anaerobic incubation in IVTopyruvato containing .016 ~ I~ADH. Resuspended pellet and cell free supernatant are fractions from centrifugation of sonified cell suspension. (Numbers below zero represent uptake and above zero evolution) exposed to sucrose gradient was unexpected and though larger, appears similar to that caused by sonification. This result is presumably due to the high osmotic pressure of the sucrose gradient increasing cell permeability to N A D H . Beatty (1) reported that spermatozoa are heavier than most cells so that with conventional media as sucrose or other low molecular weight substances, the osmotic pressure is lethal at the main isopycnie point. The osmotic pressure of the sucrose gradient in these studies exceeded 5 osmolar. The C02 uptake period is associated with concentration of spermatozoa in the incubation (30). Our results show that the uptake period is associated also with the concentration of N A D H in the incubation medium as higher N A D H extended the period of CO s uptake. Uptake period and magnitude varied among ejaculates. This variability among ejaculates was less with cell fractions than with whole cells suggesting that much of the variability was due to differences in membrane permeability. Mann (16) reports that fragmentation of middle-pieces and tails probably release the sperm cytoplasm because enzymatic behavior is more easily demonstrated in disrupted than intact sperm. Flipse (3) reported that metabolic activity of certain TCA metabolites was increased by sonification of bovine sper-

matozoa apparently due to increased permeability. Some metabolic reactions were greatly increased and others only slightly suggesting that mitochondrial permeability was probably only slightly affected and still remained a barrier to compounds such a s citrate. I n our studies sonification of spermatozoa appeared to increase permeability to N A D H as well as to cause a leaching from cells of components responsible for uptake. Perhaps cell components responsible for C02 uptake are primarily extra-mitochondrial since they appeared to be easily removed from the cell. Activity after Millipore filtration Suggests that these components are somewhat soluble in physiological saline. Results from addition of avidin agree with those previously reported (11) for incubations with whole cells. I n addition, present results suggest that impermeability of the cell membrane, as previously suggested (11), is not the reason for lack of inhibition by avidin since no inhibition of CO 2 uptake was observed by cell free extracts of bovine spermatozoa. Thus, biotin containing enzymes do not a p p e a r to be involved in this uptake phenomenon. No effect on the CO 2 uptake by any cell fractions was observed when malonate was added during incubation. Since two other steps are present between oxalaeetate and the succinic JOURNAL OF DAIRY S~JlE~CE VOL, 55, NO. 4

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with pyruvate. Malic enzyme and pyruvate carboxylase catalyzed reactions are the only two known to involve a direct C02 fixation with pyruvate to yield dicarboxylic acids (28). Malic enzyme activity was in acetone powder extracts of bovine (24) and cod sperm (17), but its role in C02 fixation by these cells is still unknown. Pyruvate carboxylase activity has not been demonstrated in sperm; however, it can not yet be eliminated as a possibility since this enzyme favors oxalacetate production (15, 28) while malic enzyme is generally believed to function primarily in pyruvate production. The physiological importance of these C02 uptake and fixation reactions during normal metabolism of spermatozoa is unknown.

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FIG. 4. Carbon dioxide uptake by bovine epididymal-like spermatozoa (ELC) and cell fractions by sonification and sucrose gradient separation incubated anaerobically in IYT-pyruvate containing .016 ~ NADtt. Whole cells represent ELC without treatment, whole cells from sucrose gradient are ELC recovered after passing through a sucrose gradient, sonified cells are sonified ELC in suspension, head-rich and tail-rich fractions are fractions of sonified ELC separated by a sucrose gradient. (Numbers below 0 represent uptake) deoxyhydrogenase reaction, it might be that even with malonate inhibition an initial C02 uptake and fixation with pyruvate would not be inhibited. Mounib and Eisan (17), have reported that .074 ~ malonate depressed C0~ fixation by cod sperm, and the pyruvate additions counteracted this depression. Carbon dioxide fixation by spermatozoa during incubations without exogenous N A D H have been reported (17, 18, 24), but no gas exchange values were given to indicate whether there was a net evolution or uptake of CO 2. In the absence of exogenous N A D H , uptake would be less than evolution and, therefore, would only be detectable with labeled C02. The initial C02 fixation reaction(s) with pyruvate has not been elucidated in any of these studies with spermatozoa. Direct reversal of the pyruvate kinase reaction to produce phosphoenolpyruvate ( P E P ) is physiologically unfavorable (15) suggesting that P E P - C 0 o fixation reactions are not involved in initial fixation J O U R N A L OF D A I R Y SCIEI~CE ~ O L .

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(1) Beatty, 1~. S. 1964. Density gradient media for mammalian spermatozoa. Prec. 5th Int. Congr. Animal Reprod. Trento, 3: 276. (2) Bistocchi, M., G. D'Alessio, and E. Leone. 1968. Pyridine-nucleotide coenzymes in bull and rabbit spermatozoa. J. Reprod. Fert., 16 : 223. (3) Flipse, R. J. ]967. Metabolism of bovine semen. XV. Rate-limiting reactions in the citric acid cycle. J. Dairy Sci., 50: 281. (4) Foley, C. W., and W. L. Williams. 1967. Effect of bicarbonate and oviduct fluid on respiration of spermatozoa. Prec. Soc. Exp. Biol. Meal., 126: 634. (5) Hamner, C. E., and W. L. Williams. 1964. Identifcation of sperm stimulating facto1 of rabbit oviduct fluid. Prec. Soc. Exp. Biol. Med., 117: 240. (6) Humphrey, G. F., and T. Mann. 1949. Studies on the metabolism of semen. 5, Citric acid in semen. Biochem. J., 44: 97. (7) Jones, E. E., and G. W. Salisbury. 1962. Action of carbon dioxide as a reversible inhibitor of mammalian spermatozoan respiration. Abstr. Federation Prec., 21: 86. (8) Lodge, E. R., C. N. Graves, and G. W. Salisbury. 1963. Carbon dioxide in pre. vention of the deleterious dilution effect on metabolism and motility of mammalian spermatozoa. Prec. Soc. Exp. Biol. Med.~ 113 : 824. (9) Lodge, J. R., C. N. Graves, and G. W. Salisbury. 1963. Effect of CO~ on anaerobic glycolysis of unwashed, once- and twicewashed spermatozoa from the same semen samples. Prec. See. Exp. Biol. Med., 113: 827. (10) Lodge, J. R., C. N . Graves, and G. W. Salisbury. 1964. COs in anaerobic epididymaI spermatozoan metabolism. Prec. 5th Int. Congr. Animal Reprod. Trento, 2: 152. (11) Lodge, J. R., C. N. Graves, and G. W. Salisbury. 1968. Carbon dioxide in anaerobic spermatozoan metabolism. J. Dairy S~i., 51 : 96.

SPERMATOZOA METABOLISM (12) Lodge, J. 2 , and G. W. Salisbury. 1962. Initiation of anaerobic metabolism of mammalian spermatozoa by carbon dioxide. Nature, 195: 293. (13) Lodge, J. R., and G. W. Salisbury. 1963. Factors influencing metabolic activity of bull spermatozoa. VI. Metabolic C0~ and fructose. J. Dairy Sci., 46: 140. (14) Lodge, J. R., and G. W. Salisbury. 1965. Control of sulfite release of carbon dioxide inhibition of anaerobic glycolysis by bovine spermatozoa. J . Dairy Sci., 48: 1688. (15) Mahler, H. R., and E. H. Cordes. 1966. Biological Chemistry. Harper and Row, New York City. (16) Mann, T. 1964. The Biochemistry of Semen and of the Male Reproductive Tract. John Wiley and Sons, Inc., New York City. (17) Mounib, M. S., and J. S. Eisan. 1968. Carbon dioxide fixation by spermatozoa of cod. Comp. Biochem. Physiol., 25: 703. (18) O'Shea, T., and R. G. Wales. 1967. Fixation of carbon dioxide by ram spermatozoa. J. Reprod. Fert., 14: 333. (19) Salisbury, G. W. 1959. The reversal of metabolic regulators of CO~-induced inhibition of mammalian spermatozoa. Prec. Soc. Exp. Biol. Meal., 101 : 187. (20) Salisbury, G. W., and C. N. Graves. 1963. Substrate-free epididymal-like bovine spermatozoa. J. Reprod. Fert., 6: 351. (21) Salisbury, G. W., and N. L. VanDemark. 1957. Carbon dioxide as a reversible inhibitor of spermatozoan metabolism, lgature, 180 : 989.

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(22) Salisbury, G. W., and N. L. VanDemark. 1957. Sulfa compounds in reversible inhibition of sperm metabolism by carbon dioxide. Science, 126 : 1118. (23) Salisbury, G. W., N. L. VanDemark, J. R. Lodge, and R. G. Cragle. 1960. The inhibition of spermatozoan metabo]ism by pCO:, pH, k-ion, and antibacterial compounds. Amer. J. Physiol., 198: 659. (24) Sexton, T. J., and R. J. Flipse. 1971. Carbon dioxide fixation by bovine semen, washed spermatozoa, and seminal p]asma. g. Dairy Sci., 54: 417. (25) Shettles, L. B. 1940. The respiration of human spermatozoa and their response to various gasses and low temperature. Amer. J. Physiol., 128: 408. (26) Sherries, L. B. 1940. Carbon dioxide tension and its relation to quiescence of spermatozoa in rive. Prec. See. Exp. Biol. Med., 45:318. (27) Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1957. Manometric Techniques. 3rd ed., Burgess Pub. Co., Minneapolis, Minnesota. (28) Utter, M. F. 1969. Metabolic roles of 0xalacetate. I n Citric Acid Cycle. P. 249. J. M. Lowenstein, ed. Marcel Dekker, New York and London. (29) VanDemark, N. L., and U. D. Sharma. 1957. Preliminary fertility results from the preservation of bovine semen at room temperature. J. Dairy Sci., 49: 438. (30) Yoshida, S. 1966. Unpublished data. University of Illinois. Department of Dairy Science.

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