Metabolism of Pyruvate by Epididymal-Like Bovine Spermatozoa

METABOLISM

OF PYI~UVATE BY EPIDIDYMAL-LIKE BOVINE SPERMATOZOA

C. 1'~. GRAVES, J. R. LODGE, AND G. W. SALISBURY Department of Dairy Science, University of Illinois, Urbana SUMIq[ARY

Various factors affecting aerobic and anaerobic pyruvate metabolism by bovine spermatozoa were studied. With pyruvate as the sole exogenous substrate, DNP ( 1 0 ' ~ ) stimulated the respiration of ELC (epididymal-like sperm cells) of some bulls but had no effect on the respiration of others. DNP also depressed the aerobic pyruvate utilization and lactate accumulation by ELC. The dismutation reaction, in which two molecules of pyruvate undergo dismutatlon to one molecule each of lactate, CO~, and acetate, was studied and was found to be stimulated anaerobically by both phosphate and sulfite. Phosphate, NAD, lipoic acid, and A T P stimulated the respiratory activity of ELC in the presence of pyruvate. The pivotal position occupied by pyruvate during the metabolic breakdown of the hexoses and other carbohydrates makes this compound an important area of study. After its formation by glycolytic systems, its subsequent transamination to alanine, reduction to lactate, or its decarboxylation followed by oxidation in the nfitochondria are but a few examples of the metabolic pathways utilized during the metabolism of the compound (17). The rapid oxidation of the pyruvic acid in semen (12) and its preferential utilization as compared to the other carbohydrates (18) attest to the activity of these various consuming reactions as they occur within motile mammalian spermatozoa. The differential rate of oxidative metabolism of pyruvate in the presence and absence of 2,4-dinitrophenol (DNP) as a measure of the fertilizing ability of bovine spermatozoa was proposed by Melrose and Terner (13) and confirmed by Glew (6). Groups of semen samples reportedly could be distinguished which differed in their ability to oxidize pyruvate with high, intermediate, or low oxygen/pyruvate ratios and which varied characteristically in. their respiratory response to DNP. Fluoride was found to be essential in these studies to abolish the utilization of hexoses by the twice-washed cells. Melrose and Terner (13) first suggested the anaerobic dismutation by bovine spermatozoa of two moles of pyruvate to one mole each of carbon dioxide (CO~), acetate, and lactate. Its presence during both the aerobic and anaerobic utilization of pyruvate was later demonstrated by Terner (19) using isotope techniques. Recvived for publication July 31, 1964.

The recent availability of substrate-free epididymal-like spermatozoa (ELC) (15), i.e., sperm cells subjected to the substrates of the seminal plasma only momentarily and at low concentration during collection stimulated reexamination of both the aerobic and anaerobic utilization of pyruvate by bovine spermatozoa. The effect of DNP, sulfite, and phosphate on respiratory activity, as well as a verification of the dismutation reaction, also was included. ~tATERIAnS AND ~ET~ODS The method for the collection and preparation of ELC has been reported (15), as have the methods used for estimation of both sperm concentrations and sperm motility (2). Following the 4-hr, 37 C incubation period, fructose (11), pyruvate (5), and lactic acid (1) analyses were performed to determine substrafe utilization or accumulation. The diluents used were isotonic (0.154 ~ ) sodium chloride or an isosmotic phosphate buffer (16) containing 777 mg % phosphate. When pyruvate or acetate were added as substrates, they were added at a level of 1.0 mg per milliliter of diluent. Sodium fluoride, 0.01 ~ ; DNP, 10.4 ~ ; fumarate, 2 × 10-' ~ ; coenzyme A, 2 X 10 ' ~ ; and sodium sulfite, 30 7 sulfur per milliliter of diluent, were added to the incubation diluents where indicated. I n the experiment designed to study the effects of the various cofactors on pyruvate metabolism (Table 4), pyruvate was present at 0.02 ~, phosphate at 90 mg per 100 ml diluent, and each cofactor present at 2 × 10 -~ ~. I n all studies the ELC suspensions were diluted one to four with the respective diluents and incubated at 37 C for 4 hr in 17 ml single sidearm flasks in a Warburg respirometer, with either air or 100% N~ as the

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C . N . GRAVES, J. ~. LODGE, AND G. W. S A L I S B U R Y

gas phase. I n the aerobic studies 2 0 % p o t a s sium h y d r o x i d e was added in the center well to a b s o r b the CO~. I n i t i a l a n d final ( a f t e r incub a t i o n ) aliquots were used f o r nmtility estim a t e s a n d chemical analysis. I~ESULTS

Effect of DNP.

The metabolic r e s p o n s e of the s p e r m a t o z o a ( E L C ) of f o u r r a n d o m l y selected bulls to D N P a n d fluo,~ide is shown in Table 1. I n the first f o u r lines of the table are p r e s e n t e d the responses of the cells receiving no exogenous substrate. Since fluoride, a n inh i b i t o r of enolase, w h e n added to these cells caused no decrease in oxygen uptake, one can conclude t h a t t h e r e were no glycolytic endp r o d u c t s u b s t r a t e s seeping into the T C A cycle f r o m endogenous sources. Because of this the oxygen u p t a k e is similar, t h o u g h not identical in the presence or absence of fluoride. F l u o ride was, however, toxic to the m o t i l i t y of the cells a n d in the presence of p y r u v a t e increased lactate accumulation. D N P depressed endogenous r e s p i r a t i o n b y a b o u t a b o u t 30%, but stimulated r e s p i r a t i o n in the presence of added

p y r u v a t e f r o m 13 to 26%. A c c o m p a n y i n g this s t i m u l a t e d r e s p i r a t i o n , D N P also m a r k e d l y decreased the r e d u c t i o n of p y r u v a t e to lactate a n d depressed motility. A b r e a k d o w n of the combined r e s p i r a t o r y d a t a ( s u m m a r i z e d in Table 1) by bulls is shown in Table 2. As i n d i c a t e d in this table, the a d d i t i o n of p y r u v a t e to the s p e r m a t o z o a of one of the bulls (no. 39) caused a large increase in the r e s p i r a t o r y activity, which was not f u r t h e r increased b y the a d d i t i o n of D N P . The metabolic b e h a v i o r of E L C of the o t h e r three bulls (no. 40, 41, a n d 840) differed, however, since p y r u v a t e caused o n l y a medium increase in r e s p i r a t o r y rate, which was f u r t h e r increased a p p r o x i n m t e l y 2 5 % by D N P . A differential r e s p i r a t o r y q u o t i e n t d u r i n g the metabolism of p y r u v a t e has p r e v i o u s l y been rep o r t e d for these two g r o u p s of bulls (10). Dismutation reaction. Table 3 shows the results of a n e x p e r i m e n t designed t o examine the presence of the d i s m u t a t i o n reaction in aerobic s p e r m a t o z o a n metabolisnl. I n this exp e r i m e n t , in which 20 samples were involved, 5.1 t t ~ p y r u v a t e a n d 4.8 t~I 0~ were converted

TABLE l Effect of D N P and fluoride on the oxygen uptake, pyruvate utilization, lactate accumulatiou, and livability of ELC incubated 4 hr a t 37 C (n = 16)

Saline Saline-DNP Saline-F1 Saline-F1-DNP Saline-Pyr Saline-Pyr-DNP Saline-Pyr-F1 Saline-Pyr-F1-DNP " ~M/10s/4 hr.

O2 uptake "

% Motile

Pyruvate utilization "

Lactate accumulation a

0.57 0.40 0.56 0.33 2.27 2.60 1.61 2.02

24.7 5.3 8.1 0.0 7.9 7.6 0.3 0.0

0.0 0.0 0.0 0.0 2.76 2.56 2.72 2.23

0.0 0.0 0.0 0.0 1.61 0.43 1.93 0.66

TABLE 2 Effect of D N P on the oxygen uptake (t~M/10s/4 hr) of ELC of different bulls (n = 8) Bull no. Diluent Saline Saline-Pyruvate Saline-DNP Saline-Pyruvate-DNP

39

40

41

840

0.43 2.54 0.17 2.58

0.59 ].38 0.36 2.05

0.70 1.92 0.55 2.42

0.54 1.91 0.38 2.28

TABLE 3 Presence of tile dismutation reaction in aerobic metabolism of pyruvate by ELC ( n - 2 0 ) Divalent Saline Saline-Pyruvate (0.02 •) ~ / 1 0 s / 4 hr.

O_, uptake ~

CO~ production "

R.Q.

0.5 4.8

0.7 6.7

1.40 1.39

Pyruvate Lactate utilized a accumulation ~ 0.0 5.1

0.0 2.2

METABOLISM OF P¥]4UVATE BY SPEIC~Y[ATOZOA to 2.2 FM lactate a n d 6.7 gM C02. h i this conversion 2.2 tLzl p y r u v a t e were accounted f o r by the lactate accunmlation, a n d ( a s s m n i n g t h a t 2.5 /x~ of oxygen are r e q u i r e d to completely oxidize one t~[ of p y r u v a t e ) a n a d d i t i o n a l 1.9 Fh~ of p y r u v a t e was accounted f o r b y the 0.: consumed, leaving 1 t ~ p y r u v a t e u n a c c o u n t e d for. Since 5.7 t ~ COz would be f o r m e d f r o m the complete oxidation of 1.9 /x~ p y r u v a t e (3 t~M CO~ p e r t ~ p y r u v a t e oxidized) t h e r e would be 1 F~r CO~ p r o d u c e d d u r i n g the i n c u b a t i o n p e r i o d b u t still u n a c c o u n t e d for. I f this CO~ arose f r o m the d e c a r b o x y l a t i o n of the 1 ~5I p y r u v a t e u n a c c o u n t e d for, one t ~ of acetate would be f o r m e d ; however, no assay was m a d e in this s t u d y f o r acetate or o t h e r metabolic end p r o d u c t s . Effect of various co factors. The effect of various nmtabolic eofactors in the p r e s e n c e a n d absence of a low level of p h o s p h a t e (90 m g % ) on the r e s p i r a t o r y activity of bovine spermatozoa is seen i n Table 4. The r e s p i r a t i o n in all diluents c o n t a i n i n g p h o s p h a t e in this series was h i g h e r t h a n the n o n p h o s p h a t e controls. A n analysis of v a r i a n c e showed this difference, as well as the difference i n r e s p i r a t i o n in r e s p o n s e to the different cofactors, to be

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significant. The lactate a n d p y r u v a t e data did n o t show the same u n i f o r m effect of p h o s p h a t e as was s h o w n b y the r e s p i r a t i o n values. T h e r e was, however, a s t i m u l a t i o n o f p y r u v a t e utilization a n d lactate a c c u m u l a t i o n in the p r e s ence of p h o s p h a t e f o r all cofactors used except f o r lipoie acid. I n the presence of lipoic acid, p h o s p h a t e depressed t h e p y r u v a t e utilization a n d lactate a c c u m u l a t i o n b u t s t i m u l a t e d the oxygen uptake. Of the various cofaetors used, lipoic acid, N A D , a n d A T P s t i m u l a t e d the s p e r m a t o z o a n r e s p i r a t o r y activity b o t h in the p r e s e n c e a n d absence of the a d d e d p h o s p h a t e b u t decreased the p y r a v a t e utilization. Anaerobic conversion to lactate. I n a n att e m p t to d e m o n s t r a t e a n a n a e r o b i c d i s m u t a t i o n reaction, as well as to s t u d y the effect of phosp h a t e a n d sulfite on the a n a e r o b i c conversion of p y r u v a t e to lactate, E L C were i n c u b a t e d u n d e r N~ in b o t h saline a n d p h o s p h a t e (777 m g % P ) diluents. Sodium sulfite was added at a c o n c e n t r a t i o n of 30 t~g s u l f u r / m i l l i l i t e r . The 4-hr values, as shown in Table 5, indicated t h a t p h o s p h a t e s t i m u l a t e d t h e CO~ p r o d u c t i o n b u t i n h i b i t e d the conversion of p y r u v a t e to lactate, whereas sulfite depressed the accumulation of b o t h end products. The f o r n m t i o n of

TABLE 4 Effect of various cofactors in the presence of 0.02 ~ pyruvate with and without 90 mg % PO~ on tim metabolism of ELC during a 4-hr incubation period (n = 6) Cofactor None (0.02 ~t pyruvate) None -4- phosphate (90 mg % ) Lipoic acid Lipoic acid ÷ phosphate NAD NAD -4- phosphate ATP A T P + phosphate Ace±ate Acetate + phosphate Oxa]acetate Oxalacetate 4- phosphate Fructose Fructose ÷ phosphate Thiamine Thiamine + phosphate " ~/lOS/4 hr.

O~ uptake ~

Pyruvatc disappearance "

Lactate accumulation "

1.33 1.85 1.79 2.21 1.79 2.21 2.11 2.29 1.31 2.04 1.51 1.95 1.52 1.89 1.23 1.83

3.04 4.03 2.84 2.47 2.89 3.30 2.83 3.53 2.38 3.01 2.28 3.24 1.72 3.40 3.21 3.83

1.26 1.87 1.43 0.84 1.16 1.76 1.43 1.82 1.04 1.78 1.50 2.27 2.10 2.86 ] .38 2.20

TABLE 5 Effect of sulfltc on the anaerobic metabolism of pyruvate by ELC incubated in saline and phosphate diluenCs (n = 11) Diluen± Saline-Pyruvate Saline Pyruvate-SO~ Phosphate-Pyruvate Phos~)h~ te-Pyruvate- SO,~ " ~ / 2 0 s / 4 hr.

CO_-prod. ~

Pyruvate disappearance "~

Lactate accumulatioll ~

1.41 1.16 1.93 1.24

7.99 6.05 7.95 8.17

5.32 3.89 4.61 3.67

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c. N. GRAVES, J. R. LODGE, AND G. W. SALISBURY

CO~ as in this experiment at least indicates the presence of the dismutation reaction in anaerobic metabolism, but by no nieans does it account for the pyruvate which did not occur as lactate. No analyses were made in this study for other reaction products. DISCUSS/ON The stimulatory effect of DNP on spermatozoan respiration as shown in Table 1 agrees with results of previously reported studies (13). A similar increase in the respiratory activity of liver tissue in response to the DNP has been attributed to a stimulation of the initochondrial adenosine triphosphotase (ATPase) (4), with the degree of stimulation, however, decreasing as the tissue ages. A loss in capacity for stimulation by D N P during aging also has been reported for spermatozoa (6), as has a decline in their fertility (14). Whether the decline of the two is parallel has not been tested. The decreased utilization of pyruvate by ELC in the presence of DNP (Table 1) confirms previous observations with washed bovine spermatozoa (19). Terner (19) found that low levels of D N P stinmlated the utilization of glucose, decreased the uptake of pyruvate, but had little effect on the metabolism of acetate, with the lactate values being increased from the breakdown of glucose but much diniinished during the metabolism of pyruvate. Huggins and Smith (8), working with rat diaphragms, also reported a stinmlation of glucose uptake and subsequent lactate accunmlation when D N P was added to the incubation niedia. These diverse effects on the rate of utilization of the various carbohydrates suggests that D N P affects the oxidation of carbohydrates in a twofold manner: first, by stimulating a DNP-induced ATPase; secondly, by influencing the pyruvate to acetyl Coenzyme A conversion before entrance of' the two-carbon fragment into the TCA cycle. The importance of the physiological role of the pyruvate dismutation reaction in spermatozoa is unknown. Pyruvate has been reported present in bovine senien at ejaculation, but is rapidly utilized by the spermatozoa (12). Recent studies (7) showing the production of forniie, acetic, and glycolic acids as well as lactic acid during the anaerobic metabolism of fructose indicate that the dismutation reaction may be a normal metabolic process occurring in the utilization of hexoses. The stimulatory effect of low levels of phosphate on pyruvate oxidation, as well as on the conversion of pyruvate to lactate (Table 4),

indicates that phosphate is limiting in the ELC and is required for optinmm enzymatic activity for pyruvate utilization. Similar studies using fructose (unpublished) showed 90 mg % phosphate depressed respiratory activity but was somewhat stilnulatory to glycolysis. The stfinulatory effects of lipoic acid, N A D H , and A T P on metabolism appear to be independent of the stimulatory effect of phosphate, since the metabolic activity in the presence of each of these compounds was increased both in the presence and absence of phosphate. The increased C02 production but decreased lactate accumulation in the phosphate diluent (Table 5), as compared to the saline controls, suggests that phosphate stimulated the dismutation reaction~ with acetate or some other metabolic acids being produced. The decreased CO~ and lactate occurring in the presence of sulrite reportedly is due partially to inhibition of lactic dehydrogenase (3); however, sulfite apparently inhibits also the oxidation of the pyruvate to acetate, CO.~, and other compounds. REFERE)TCES (1) BARKER, S. B., AND SU~IMERSON,W. H. The Colorimetric Determination of Lactic Acid in Biological Material. J. Biol. Chem., 138: 535. 1941. (2) BISHOP, M. W. H., AND SALISBURY, G. W. Effect of Dilution with Saline and Phosphate Solutions oll Oxygen Uptake of Bull Semen. Am. J. Physiol., 181: 114. ]955. (3) ]~ROQUIST, H. P., AND LODGE, J. R. Lactic Dehydrogenase Studies with Cell-Free Extracts of Bovine Semen. J. Dairy Sci., 43: 881. 1960. (4) CHE:~URKA, F. Some Observations on the Effect of a Naturally Occurring Agent(s) on the 2,4-Dinltrophenol-Induced ATPase of Liver Mitoehondria. Canadian J. BiGchem. Physiol., 39: 1941. 1961. (5) PKIED~AN, T. E., AND HAUGEN, G. E. Pyruvic Acid. II. The Determination of Keto Acids in Blood and Urine. J. Biol. Chem., 147: 415. 1943. (0) GEEW, G. An Investigation into tile Relationship Between the Metabolism of Pyruvate in Bull Spermatozoa and Fertility. J. Agr. Sci., 48:153. 1953. (7) GRAVES,C. N., LODGE, J. R., AbTDSALISBURY, G.W. Organic Acids Produced by Bovine Spermatozoa During Anaerobic Metabolism. J. Dairy Sci., 47: 705. 1964. (8) ttUGGIRTS,A. K., AND SI~IITH, M. J. H. Uncoupling Re~gents and Metabolism. 5. Effects of Salicylate and 2,4-Dinitrophenol on the Metabolism of Isolated Rat Diaphragm. Biochem. J., 85: 394. 1962. (9) LARDY, H. A., AND PItlLLIPS, P. H. The Effect of Thyroxine and Dinitrophenol on

METABOLIS~ OF PY~UYATE BY SPERMATOZOA

(10)

(11)

(12)

(13)

(14)

Sperm Metabolism. J. Biol. Chem., 149: 177. 1943. LODGe, J. R., AND SALISBUaY, G. W. Respiratory Quotients of Bovine Spermatozoa. Proc. 4th Intern. Congr. Animal Reprod., The Hague, 2:263. 1961. MAI~N, T. Fructose Content and Fructolysis in Semen. Practical Application in the Evaluation of Semen Quality. J. Agr. Sci., 38: 323. 1948. MARDEN, W. G. R. Source of Endogenous Pyruvie Acid in Bovine Seminal Fluid and Utilization. J. Dairy Sei., 44: 1688. 1961. MELI~OSE, D. IR., AND TERNER, C. The Metabolism of Pyruvate by Bull Spermatozoa. Biochem. J., 53: 296. 1953. SALISBVI~Y, G. W., AND FLEI~CHINGEI~ G. H. In ¥ i t r o Aging of Spermatozoa and Evidence for Embryonic or Early Fetal Mot-

(15)

(16)

(17)

(18)

(19)

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tality in Cattle. Proc. 4th Intern. Congr. Animal Reprod., The Hague, 3:501. 1961. SAliSBUrY, G. W., .~N~) GRAVES, C. N. Substrate-free Epididymal-like Bovine Spermatozoa. J. Reprod. Fertil., 6" 351. 1963. SALISBURY, G. W., AN]) KINNEY, W. C., J~. Factors Influencing Metabolic Activity of Bull Spermatozoa. III. pH. J. Dairy Sci., 40: 1343. 1957. SALmBUaY, G. W., AND LODGE, J. R. Metabolism of Spermatozoa. Adv. in Enz., 24: 35. 1962. TERNER, C. Effect of Nitrophenols on the Metabolism of Spermatozoa. Federation Proc., 16: 261. 1957. TERNEI~,C. The Effect of 2,4-Dinitrophenol and p-Nitropheno] on the Aerobic and Anaerobic Metabolism of Bull Spermatozoa. Biochlm. et Biophys. Acta, 36:479. 1959.