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Effect of Egg yolk and its Isolated Constituents Upon the Dehydrogenase Activity of Bovine Spermatozoa1
EFFECT OF EGG YOLK AND ITS ISOLATED CONSTITUENTS UPON THE DEHYDROGENASE ACTIVITY OF BOVINE SPERMATOZOA ~ J. T. S M I T H , D. T. MAYER, ~ AND C. P. MERILAN Agricult~ral Che~nistry,~Animal Husbandry, and Dairy H~¢sbandry Departments University of Missouri, Columbia
An active succinic dehydrogenase was first demonstrated in the semen of man and bull b y L a r d y and Phillips (16) and MacLeod (22) by use of the Thunberg technique. Subsequently, Zittle and Zitin (~1) demonstrated the presence of cytochrome oxidase in bull spermatozoa, and M a n n (24, 25) observed the complete eytochrome spectrum in the spermatozoa of man, ram, bull, and boar. However, MacLeod was unable to demonstrate a n y connection between succinate oxidation and the persistence of spermatozoan motility. R y a n (33) observed an increase in respiration of cold-shocked spermatozoa in the presence of succinate, which a p p e a r e d to be associated with an increase in spermatozoan motility. Although the necessity for an active succinic dehydrogenasc in the maintenance of spermatozoan viability has not been established, the importance of dehydrogenases in general to spermatozoa has been confirmed by the relationships which have been observed between " r e d u c t i o n t i m e " and motility, longevity, and per cent n o n r e t u r n s by Beck and Salisbury (4) and E'rb and Ehlers (11). Since the introduction of egg yolk as a desirable constituent of a semen diluent by Phillips (29) and Phillips and L a r d y (30), Tosic and Walton (37, 38, 39) and L aslcy and Mayer (21) have described its effect upon spermatozoan respiration. Tosic and Walton obtained an initial stimulation of respiration followed b y a decrease when egg yolk was added to ejaculated bull spermatozoa and explained the observed decrease as caused by the presence of a dialyzable organic peroxide in egg yolk. Lasley and Mayer, however, observed no respirat o r y effect following the addition of egg yolk to ejaculated r a m spermatozoa but found an increase in the respiration of epididymal spermatozoa. Lasley et al. (19), Easley et al. (9), Lasley and Bogart (18), Lasley and Mayer (20), and Bogart and M a y e r (6) have suggested that egg yolk is of importance in protecting spermatozoa against adverse environmental conditions. Fractionation of egg yolk has yielded fractions valuable in the preservation of spermatozoa. L a r d y and Phillips (17), K a m p s c h m i d t ctal. ( l i ) , and Ryan (.?3) have discussed the beneficial effects of egg yolk phospholipids and lipoproteins upon spermatozoan longevity. K a m p s c h m i d t et al. and Bogart and Mayer recognized two distinct egg yolk factors, one of which protects sperlnatozoa against the h a r m f u l effects of cold shock and a second which contributed to survival (a storage factor). E g g yolk phospholipids, p a r t i c u l a r l y lecithin, have Received for publication September 16, 1955. This investigation was supported ill part by a research grant from the National Institutes of I][e.qlth, Public Health Service. Missouri Agricultural Experiment Station, Journal Series No. 1560. 552
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been identified as resistance factors b y R y a n and K a m p s c h m i d t et aL, whereas L a r d y and Phillips demonstrated the oxidative metabolism of egg yolk phospholipids whenever spermatozoa were deprived of a metabolizable hexose. I n recognition of the importance of dehydrogenases and the m a n y beneficial effects of the addition of egg yolk to spermatozoa, as well as its effect upon spermatozoan respiration in general, a series of experiments was conducted to determine the effect of egg yolk in the medium upon the dehydrogenases concerned with c a r b o h y d r a t e metabolism of spermatozoa. MATERIALS AND METHODS
The semen was collected f r o m healthy dairy bulls m a i n t a i n e d as a regular p a r t of the University of Missouri E x p e r i m e n t Station d a i r y herd. A n artificial vagina was used to collect the semen, which was immediately placed in a Thermos bottle of water at a t e m p e r a t u r e no lower t h a n 15 ° C. for t r a n s p o r t to the laboratory. W h e n received at the laboratory, the vial of semen was placed in a 400-ml. beaker of water and stored in the refrigerator. The large water volmne insured gradual cooling to a storage t e m p e r a t u r e of 4 ° C., thereby p r e v e n t i n g cold shock. Generally, the semen specimens f r o m two or more bulls were pooled. I f the spermatozoa were to be washed, they were mixed with about five volumes of 0.9% saline. The wash solution and spermatozoa were separated by centrifugation at 950 G or 2,200 r.p.nl, in a Servall model G centrifuge for about 10 minutes. The seminal plasma saline m i x t u r e was removed b y use of a h a n d controlled automatic pipette. The washing process was repeated four times, a f t e r which the spermatozoa were resuspended in sufficient isotonic saline to attain the original semen volume. Spermatozoan concentration counts were made b y using an eosin counting fluid as described by Smith and Mayer (35). Five counts were made on each sample, a n d their average was used to calculate the cubic millimeters of CO: liberated by 10 s spermatozoa per hour. The W a r b u r g flasks were cleaned by the potassium p e r m a n g a n a t e - s o d i m n hydroxide method and calibrated by fillling them with mercury, according to the method of Umbreit et al. (40). Since spermatozoan suspensions are difficult to p r e p a r e with sufficient optical ~ransparency to facilitate application of spectrophotometric techniques for the determination of enzyme activity, the manometric method of Quastel and Wheatley (31) was employed to estimate the dehydrogenase activity of the spermatozoa in the different suspending media. This method, used more recently b y Rubinstein and Denstedt (32) in an investigation of the metabolism of the avian erythroeyte, uses ferricyanide as an artificial hydrogen carrier to liberate COe f r o m a bicarbonate medimn. The f e r r i e y a n i d e was placed in a side a r m of the ~ a r b u r g flasks and tipped in a f t e r the equilibration period at zero time to initiate the reaction, and the m a n o m e t r i c readings were recorded at 5-minute intervals for a period of ] hour. E g g yolk was added to the flasks as a 1 : 1 m i x t u r e of egg yolk a n d 1.3% N a H C O , . The volume of the m i x t u r e added to each flask was 0.6 ml., equivalent
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to 0.3 ml. of egg yolk per flask. E g g yolk lecithin was p r e p a r e d by the method of P a n g b o r n (28). Twelve rag. of lecithin p r e p a r e d in this m a n n e r was added to each flask as an enmlsion. The cholesterol, a commercial preparation, "~ was incorporated into a water emulsion b y dissolving 275 mg. in 15 ml. of hot methanol and adding the hot solution to 10 ml. of water. A volume of 0.6 nil. of this emulsion, containing 6.6 rag. of cholesterol, was added to each reaction flask. The fl-carotene emulsion was p r e p a r e d by nfixing 3 ml. of a synthetic fl-carotene solution in petroleum ether (50 × 10 " g. per milliliter) with 3 ml. of mineral oil and 3 ml. of d.d. II.,O and the petroleum ether, removed in vacua. Then 1 ml. of the lecithin p r e p a r a t i o n in absolute alcohol, in a concentration of 300 rag. of lecithin per milliliter, was added as an enmlsifying agent. E x a c t l y 0.3 nil. of this emulsion was added to each flask to give 4 × 10 -G g. of carotene per flask, or about the same amount as is contained in 0.3 nil. of egg yolk. To rule out the effect of either lecithin or mineral oil, 0.3 nil. of a solution containing the above ingredients with the exception of carotene was also added to the control flasks. A erude flavine-adenine-dinucleotide concentrate was p r e p a r e d by the method of D i m a n t et al. (8), with the exception that egg yolks were used instead of pig liver or yeast. The egg yolks were fraetionated into aeetolm sohlble and insoluble fractions b y the method of P a n g b o r n (28). Skinnnilk, obtained from the University of Missom'i D a i r y l ) e p a r t m e n t , was used unheated in some experiments and for others was heated in a water bath at 92 ° C. for lO minutes. RESUI,TS
The data showing the effect of whole egg yolk upon the dehydrogenase activity are presented ill Table 1. These data demonstrate all a p p a r e n t stimulation t)y egg yolk of all of the dehydrogenases studied in this investigation. F u r t h e r , they show a highly significant stimulation of the suceinic dehydrogenase activity of both washed and unwashed bovine spermatozoa following addition of the egg yolk mixture. The effect of egg yolk upon the snecinic dehydrogcnase aetivity of washed and unwashed spermatozoa is a p p r o x i m a t e l y the same, au increase of about 25 cu. mm. COo. Since fewer samples of the unwashed spermatozoa were available for study, the results a p p e a r to be less significant. Although the data (Table 1) indicate a slight s t i m u l a t o r y effect of egg yolk upon glyceraldehyde-3-phosph~te and malie dehydrogenases, lhese data were not statistically significant. The value of lecithin to spermatozoan viability has been established by K a m p sehmidt et al. (1.1), L a r d y and Phillips (17), and R y a n (33), and its importance to the succinic oxidase system in homogenized muscle cell p r e p a r a t i o n s was reported b y N y g a a r d and Stunner (27) and E d w a r d s and Ball (10). IIenee. it seemed logical t h a t lecithin m i g h t be responsible for the dehydrogenase stinlula"'Cholesterol preparation obtained from the American Lecithin Co.
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TABLE 1 Effect of egg yolk upon bovine spermatozoan dehyd.roge~ase activity Cu. ram. CO~ liberated per l0 s spermatozoa per hour
Dehydrogenase system
No egg yolk added
Succinic a~ Succinic b b Glyceraldehyde-3-phosphate ~-Vlalic P = ~
32.0 30.6 6.8 10.8
Added 0.6 nil. of ] : 1 egg yolk: 1.3% NaItCO~
(8) ¢ (4) (9) (3)
57.7 55.6 8.6 11.1
0.01.
b P - - ~ 0.05. ¢ Values in parentheses indicate number of runs which were averaged. Conditions: all spermatozoa except those used in succinate b, which were unwashed, were washed four times with isotonic saline and resuspended in sufficient saline to r e a t t a i n the original seminal volume. Flask contents: for all systems, NaIICO3, 1.86 × 10 -~ M, K3Fe(CN)6 0.2 ml. of an 11% solution neutralized with NatICOs, 0.3 and 0.5 nil. of spermatozoan suspension, sufficient saline to make the total flask volume 2.5 ml., added when measuring (succinic dehydrogenase) Nasuccinate, 0.08 M, glyceraldehyde-3-phosphate dehydrogenase, D P N 3 × 10 -4 M, nicotinamide 0.03 M, malic dehydrogenase, D P N 3 × 10 -~ M, nicotinaniide 0.3 M, Na-l-malate 0.8 M, NaCN 0.2 nil. of a solution of 0.65 M neutralized with tIC1. Gas phase: 5% CO: 95% N.~, incubation temperature 38 ° C., time 1 hour; values are expressed as eu. ram. CO: liberated per 10 ~ spermatozoa per hour, a f t e r correction for a blank cont a i n i n g no substrate. t i o n e f f e c t o f e g g y o l k . l l o w e v e r , t h e r e s u l t s ( T a b l e 2) s h o w a s l i g h t b f l f i b i t i o n of succinic dehydrogenase activity in the presence of lecithin. The failure to find a stimulation of suceinic dehydrogenase in the presence of lecithin suggested that another egg yolk constituent was enhancing the enzyme activity. Succinic dehydrogeuase has been shown to be dependent upon riboflaviu a n d f l a v i n e - a d e n i n e - d i n u c l e o t i d e s f o r m a x i m a l a c t i v i t y b y A x e l r o d e l al. (2) a n d B a l l a n d C o o p e r (3). T h e r e f o r e , a n e g g y o l k c o n c e n t r a t e o f f l a v i n e w a s t e s t e d for its effect upon succinic dehydrogenase a c t i v i t y w i t h o u t s u c c e s s , as s h o w n by the limited data presented
i n T a b l e 2.
B e c a u s e o f a n i n a b i l i t y t o a s s o c i a t e t h e s e t w o e g g y o l k c o n s t i t u e n t s , know~l to affect succinie oxidase systems, with spermatozoan succinic dehydrogenase stimulation, isolation of some egg yolk fractions with organic solvents was performed in an attempt to associate the active substance with a particular fraction TABLE 2 Effect of egg yolk fractions and ,milk upon the s~weiJ~ic dehydrogenase activity of bovine spermatozoa Cu. ram. CO.oliberated per 10 ~ spermatozoa per hour Factor added 12 rag. lecithin 0.6 nil. FAD concentrate 0.6 ml. acetone soluble 0.6 nil. acetone insoluble 0.6 nil. heated milk 0.6 ml. unheated milk
No addition 37.1 45.5 45.8 42.8 50.9 50.9
(6) '~ (2) (4) (6) (2) (2)
Factor added 20.9 37.2 64.7 48.2 54.1 47.4
Values in parentheses indicate number of samples averaged. Conditions arc the same as given for succinic dehydrogenase estimation in Table 1 except t h a t all spermatozoa were washed.
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of egg yolk. The d a t a ( T a b l e 2) s u g g e s t t h a t t h e a c t i v e s u b s t a n c e was p r e s e n t in t h e a c e t o n e soluble f r a c t i o n of e g g yolk. The i m p o r t a n c e of c h o l e s t e r o l to c e l l u l a r m e t a b o l i s m , its effect on t h e diffusion of a c i d s a n d a l k a l i s i n t o g e l a t i n a n d a g a r , a n d its beneficial effect of p r e v e n t i n g h e m o l y s i s b y m u m p s v i r u s h e m o l y s i n h a v e been r e p o r t e d b y A f f o n s k y (1), M a g i s t r i s (23), a n d M o r i m o t o a n d M o r g a n (26). S i n c e cholesterol c o m p r i s e d a l a r g e p e r c e n t a g e of t h e a c e t o n e s o l u b l e f r a c t i o n of egg y o l k a n d possessed t h e p r o p e r t i e s cited, its effect on b o v i n e s p e r m a t o z o a n d e h y d r o g e n a s e s was m e a s u r e d . A h i g h l y s i g n i f i c a n t a c t i v a t i o n of s u c c i n i c d e h y d r o g e n a s e b y c h o l e s t e r o l (P---< 0.01) was o b s e r v e d ( T a b l e 3). H o w e v e r , a s i g n i f i c a n t i n h i b i t i o n of g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e a n d a n a p p a r e n t i n h i b i t i o n of m a l i c d e h y d r o genase were o b s e r v e d ( T a b l e 3). TABLE 3 Effect of cholesterol upon bovine spermatozoan dehydroge~tase activity Cu. mm. CO: liberated per 10~ spermatozoa per hour Dehydrogenase system
No cholesterol added
6.6 mg. cholesterol added to each flask
Succinic :' Glyceraldehyde-3-phosphatet' Malic
49.5 (15) 6.3 (10) 10.3 (4)
61.9 3.6 S.1
" P ~ 0.01. b p = 0.05. Conditions are the same as in Table 1 except that all spcrnmtozoa were washed. S i n c e t h e e n t i r e s t i m u l a t i n g effect of egg y o l k c o u l d n o t be a t t r i b u t e d to cholesterol, one o r m o r e a d d i t i o n a l e g g y o l k c o n s t i t u e n t s w e r e p o s s i b l y involved. H e r i s s e t (12) f o u n d t h a t , in t h e p r e s e n c e of m e t h y l e n e b l u e as a h y d r o g e n a c c e p t o r , t h e a d d i t i o n of a s m a l l a m o u n t of c a r o t e n e - i n - o i l e m u l s i o n a c c e l e r a t e d t h e a c t i o n of S c h a r d i n g e r enzyme, s u c c i n i c d e h y d r o g e n a s e , a n d g l u t a m i c a c i d d e h y d r o g e n a s e . O n t h e basis of these r e s u l t s , t h e effect of c a r o t e n e e n m l s i o n s u p o n m a l i c a n d s u c c i n i c d e h y d r o g e n a s e s of b o v i n e s p e r m a t o z o a was i n v e s t i g a t e d . The effect of c a r o t e n e , a n o t h e r c o m p o n e n t of t h e a c e t o n e soluble f r a c t i o n of egg yolk, u p o n b o v i n e s p e r m a t o z o a n s u c c i n i c a n d m a l i c d e h y d r o g e n a s e s is p r e s e n t e d in T a b l e 4. These d a t a show t h a t c a r o t e n e s t i m u l a t e s t h e a c t i v i t y of b o t h succinic and malic dehydrogenases. I n o r d e r to d e t e r m i n e i f h e a t e d m i l k , w h i c h has been shown to be a s a t i s f a c t o r y s e m e n e x t e n d e r , w o u l d also a c t i v a t e s u c c i n i c d e h y d r o g e n a s e , a c o m p a r a t i v e TABLE 4 Effect of carotene ~pon the succinic and malic dehydrogenase activity of bovine spermatozoa Cu. mm. CO-,liberated per 10s spermatozoa per hour Dehydrogenase system
No carotene added
4 × 10-0 g. carotene added per flask
Succinic 1V[alic
25.8 (6) 10.0 (3)
34.6 20.1
Conditions are the same as in Table 1 except that all spermatozoa were washed.
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deternfination with unheated milk was made. I t was f o u n d (Table 2) that heated milk has a slight a p p a r e n t s t i m u l a t o r y effect upon suecinic dehydrogenase, whereas unheated milk, which is spermicidal, was slightly inhibitory in its effect upon bovine spermatozoan succinic dehydrogenases. DISC U S S I O N
Activation of the suecinic a n d malic dehydrogenase systems of bovine spermatozoa by egg yolk or certain of its constituents a p p e a r s to be compatible with previous evidence obtained in this and the Wisconsin laboratories. Smith et al. (3.t) demonstrated that, in an egg yolk diluent, maintenance of an osmotic pressure within certain optinlal limits was dependent upon the addition of a metabolyzable hexose. Unpublished observations in this l a b o r a t o r y indicate that tile buffering capacity of the egg yolk-glucosc-NaIICO:~ diluent (13), though low, is sufficient to p r e v e n t a dangerous shift towards an acid p H t h r o u g h o u t a ]0-day storage period. L a r d y and Phillips (17) obtained evidence suggesting that spermatozoa metabolized egg yolk phospholipids in the absence of a nletabolizable hexose. I t is possible, then, that the egg yolk activated dehydrogenases f u n c t i o n to remove the acid products of glyeolysis in a diluent, thereby m a i n t a i n i n g both a physiological p H and osmotic pressure. The a p p a r e n t failure of egg yolk lecithin to stimulate the sueeinic dehydrogenase activity of bovine spermatozoa is interesting in view of the excellent results obtained with lecithin in protecting spernmtozoa against adverse environmental conditions. However, it m a y be recalled that two egg yolk factors, one a storage and the other a resistance factor, were recognized in egg yolk and that lecithin was identified as a resistance factor. Could it be t h a t lecithin a n d / o r leeitho-protein complexes act as resistance factors whereas cholesterol a n d / o r carotene, or complexes of these compounds, m a y be classified as storage factors ? Cholesterol, on the other hand, has not been closely associated with cellular metabolic activities by investigations in this field. The literature has cited the effects of cholesterol upon m e m b r a n e permeability and the m a r k e d ability of this hydrophobie compound to f o r m water-in-oil emulsions, thus holding w a t e r in the tissues. Cholesterol, also, counteracts the effects of Ca + + upon lecithin when the latter is in contact with w a t e r and tends to reduce the surface occupied by the lecithin, according to Bloor (5). The only a p p a r e n t explanation of the stinmlation of suecinic dehydrogenase activity by cholesterol would seem to be its effect upon cellular permeability. This effect m a y be a direct one or it m a y be an indirect effect in which it prevents Ca + + inhibition of lecithin. The indirect effect is valid if it be assumed, as it was for the e r y t h r o c y t e b y Morimoto and Morgan (26), that cholesterol and lecithin are components of the spermatozoan cellular membrane. Investigation of the chemistry of m a m m a l i a n spermatozoa now in progress in this l a b o r a t o r y suggests the validity of this assumption. The results also show that cholesterol inhibits glyceraldehyde-3-phosphate and malic dehydrogenase activity of bovine spermatozoa. This finding suggests another possible explanation of the mechanism whereby cholesterol stimulates succinie dehydrogenase activity. The two enzynie systems, which are inhibited
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by cholesterol, have at least one common p r o p e r t y that may influence suecinie dehydrogenase a c t i v i t y - - t h e y both require the participation of D P N for activity. D P N has been shown by Keilin and H a r t r e e (15) to inhibit sueeinic dehydrogenase activity of nmscle preparations and by Smith and Mayer (36) to inhibit suecinic dehydrogenase activity of bovine spermatozoa. This inhibition by DPN is effeeted by the catalysis of the reaction malate ---> oxalaeetate, oxalaeetate being inhibitor.v in concentrations as low as 10 4M. Therefore, it may be that the stinmlatory effect of cholesterol upon bovine spermatozoan sueeinie dehydrogeuase and its inhibitory effect upon glyceraldehyde-3-phosphate dehydrogenase may be the result of its inhibition in some maimer of I)PN catalysis. Some reservation is placed upon this explanation, however, since the stimulation of succinic dehydrogenase by cholesterol occurred in washed spermatozoa, which have been shown by Smith and Mayer to be deficient in DPN. However, since it is the D P N catalysis of malate ---> oxalaeetate which is inhibitory, any inhibition of malie dehydroo'enase would result in an apparent suceinie dehydrogenase stinmlation. Explanation of the mechanism of stinmlation of bovine spermatozoan dehydrogenase activity by carotene stinmlation is difficult. The only known function of caroteue in enzyme systems is its postulated role in photosynthesis by Davis (7), who assumes that carotene functions in photosynthesis as an electron transport system by virtue of its large number of resonating double bonds. It does not seem unreasonable that it might function in the bovine spermatozoan suceinic dehydrogenase system in a similar lnanner, but there is no affirmative evidence. SUMMARY
The effect of whole egg yolk and its constituents upon the dehydrogenase activity of bovine spermatozoa was determined. Whole egg yolk was found to stimulate the succinic, malic, and glyceraldehyde-3-phosphate dehydrogenase activity of these cells. The dehydrogenase-stimulating effect of egg yolk was found to be associated with the acetone-soluble fraction of the yolk and appeared to be the result of the stimulatory effect of cholesterol and of carotene upon succinic dehydrogenase activity. Cholesterol was observed to inhibit both glyceraldehyde-3-phosphate and malic dehydrogenase activity of bovine spermatozoa. The importance and possible mechanisms of dehydrogenase stimulation and inhibition by egg yolk, cholesterol, and carotene have been discussed, as well as the a p p a r e n t failure of lecithin to stimulate succinie dehydrogenase activity. ACKNOWLEDGMENTS T h e a u t h o r s wish to a c k n o w l e d g e t h e g e n e r o u s d o n a t i o n of fl-carotene by L a u r a M. F l y n n a n d t h e service of K . W. Bower, who collected t h e semen. R E F E I{ENCES (1) AI~PONSKY, S. I. ~ b e r des E i n f l u e s s e s der L i p o i d e a u f die D i f f u s i o n der S a u r e n u n d A l k a l e i n in Gallerten. Biochem. Z., 195: 387. 1928. (2) AXELROD, A. E., SWINGLE~ K . F., AND ELVEItJEM, C. A. S t u d i e s on t h e Succinoxidase S y s t e m of R a t L i v e r in Riboflavine Deficiency. J. Biol. Chem., 145: 297. 1942.
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(3) BALL, E. G., AND COOPER~ O. The Activity of Succinate Oxidase in Relation to P h o s p h a t e and P h o s p h o r u s Compounds. J. Biol. Chem., 180: 113. 1949. (4) BECK, G. H., AND SALISBURY, G. W. l~apid Methods for E s t i m a t i n g tile Quality of Bull Semen. J. Dairy Sci., 26: 483. 1943. (5) BLOOR, W. R. Bioche~istry o f tl~e F a t t y Acids. Reinhold P u b l i s h i n g Corp., New York. 1943. (6) BOGART, R., AND MAYER, D. T. Tile Effects of E g g Yolk on the Various Physical and Chemical Factors Detrimental to Spermatozoan Viability. J. A n i ~ a l Sci., 9: 143. 1950. (7) DAVIS, C. New I n t e r p r e t a t i o n of ]Role of Carotenoids in Photosynthesis. _Federation Prec., 14: 200. 1955. (8) DI~ANT, E., SANADI, D. ]~., AND tIt:NNE~=INS, F. M. The Isolation of Flavin Nucleotides. J. Am. Cicero. Soc., 74: 5440. 1952. (9) EASLE¥, G. T., MAYER, D. T., AND BOGAICT, R. Influence of Diluters. Rates of Cooling and Storage Temperature on Survival of Bull Sperm. Am. J. Vet. Researcl~, 3: 358. 1942. (10) EDWARDS, S. W., AND BALL, E. G. The Action of Phospholipases oa Succinate Oxidase and Cytochrome Oxidase. J. Biol. Chem., 209: 619. 1954. (11) ERB, R. E., AND EtILERS, M. It. Resazurin Reducing Time as an Indicator of Bovine Semen Fertilizing Capacity. J'. Dairy Sci., 33: 853. 1950. (12) HERISSET, A. Influence of Carotene on Reactions Catalyzed in Vitro by Some Dehydrogenases. B~dl. Soc. Chem. Biol., 36: 809. 1954. ( F r o m Chem. Abstr. 49: 1832d) 1955. (13) KAMPSCHJ~IDT, R. F., MAYER, D. T., HERMAN, H. A., AND DICKE~SON, G. E. Viability of Bull Spernmtozoa ~s Influenced by Electrolyte Concentration, Buffer Efficiency, and Added Glucose in Storage Media. J. Dairy Sci., 33: 45. 1951. (14) KAMPSCHi~IIDT, R. F., MAYER, D. T., AND HERMAN, H. A. Lipid and Lipoprotein Cons t i t u e n t s of E g g Yolk in Resistance and Storage of Bull Spermatozoa. J. Dairy Sci., 36: 733. 1953. (15) KEILIN, D., AND ttAR~REE, E. F. Succinic Dehydrogenase Cytochrome System of Cells, Intracellular ]~espiratory System Catalyzing Aerobic Oxidation of Succinic Acid. Prec. Roy. Soc., B., 129: 277. 1940. (16) LARDY, H. A., AND PHILLIPS, P. H. The Effect of Certain Inhibitors a n d Activators on Sperm Metabolism. J. Biol. Cl~em., 138: 195. 1941. (17) LARDY, H. A., AND PHILLIPS, P. It. Phospholipids as a Source of E n e r g y for Motility of Bull Spernmtozoa. Am. J. Pl~ysiol., 134: 542. 1941. (18) LASLEY, ,.T.F., AND BOGART, R. Some F a c t o r s Affecting the Resistance of E j a c u l a t e d a n d Epididymal Spermatozoa of the Boar to Different Environmental Conditions. Am. J . Physiol., 141: 619. 1944. (19) LASLEY, J. F., EASLI~Y, G. T., AND BOGART, R. Some Factors Influencing the Resistance of Bull Sperm to Unfavorable E n v i r o n m e n t a l Condition. J. A~dnlal Sci., 1: 79. 1942. (20) LASLEY, J. F., AND MAYER, D. T. A Variable Physiological Factor Necessary for the Survival of Bull Spermatozoa. J. Animal Sci., 3: 121. 1944. (21) LASLEY, J. F., AND MAYEI~, D. T. The Effect of Glucose, E g g Yolk and Seminal P l a s m a on the Comparative Metabolic Activities of Epididymal a n d E j a c u l a t e d Spermatozoa of the Ram. J. Animal Sci., 10: 1027. 1951. (22) MAcL~OD, J. The Role of Oxygen in the Metabolism a n d Motility of H u m a n Si~ermatozoa. Am. J. Physiol., 138: 512. 1943. (23) MAGISTRIS, H. Zur Biochemie und Physiologic Oranischer P h o s p h o r v e r b i n d u n g e n in Pflanze und Tier. Ein Beitr~g zur F r a g e des Einflusses van Tierisehen u n d flanzlichena Lecithin zur die Diffusion von Sauren u n d Alkalien in Gallerten. Biochem. Z., 2 5 3 : 81. 1932. (24) MANN, T. Studies on the Metabolism of Semen. I. General Aspects, Occurrence a n d Distribution of Cytochrome, Certain E n z y m e s and Co-enzymes. Biochem. J., 39: 451. 1945.
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(25) MAN~-, T. Studies on thc Metabolism of Semen. 7. Cytochrome in Hmnan Spermatozoa. Biochem. J., 48: 386. 1951. (26) M0zIMOT0, M., AND MORG.~N, H. l]. Effect of Lecithin, Cholesterol and Cardiolipin on Mumps Virus Hemolysin. Proc. Soc. Exptl. Biol. Med., 86: 795. 1954. (27) NYOAARI), A. P., AXD SUMNEa, J. B. The Effect of Lecithinase A. ml the Succinoxidase System. J. Biol. Chem., 200: 723. 1953. (28) PA~CGBOR~¢,M. C. A Simplified Purification of Lecithin. J. Biol. Chem., 188: 471. 1951. (29) PHILLIPS, P. t{. Preservation of Bull Semen. J. Biol. Chem., 130: 415. 1939. (30) PHILLIPS, P. It., AND LARDY, I-I. A. A Yolk Buffer Pabulum for the Preservation of Bull Semen. J. Dairy Sci., 23: 399. 1940. (31) QUAST~L, J. H., AND WHEATLEY, A. I'i. M. On Ferrieyanide as a Reagent for the Manometric Investigation of Dehydrogenase Systems. Biochem. J., 32: 936. 1938. (32) RUBINSTEIN, D., AND D~NSTEDT, O. F. The Metabolism of the Erythrocyte. I I I . The Tricarboxylic Acid Cycle in the Avian Erythrocyte. J. Biol. Chem., 204: 623. 1953. (33) RYA~, W. L. A Metabolic Study of Cold Shock in Mammalian Spermatozoa. Ph.D. thesis, Univ. of Missouri. 1953. (34) SMITH, ft. T., i~AYER, 1). W., AND I'IERSIAN, ~[{. A. A Comparison of the Ability of .Certain E g g Yolk Diluents to Maintain Optinlum Osmotic Conditions During the Storage of Bull Semen. J. Dairy Sci., 37: 684. 1954. (35) SMITH, J. T., AND MAYEa, D. T. Evaluation of Sperm Concentration by the Hemacytometer Method: A Comparison of Four Comtting Fluids. Fertility and Sterility, 6: 271. 1955. (36) SMITH, J. T., AND MAYFA~, D. T. (Unpublished lab. data) 1955. (37) TOSlC, J., AND WALTON, A. Respiration of Sperm~tozoa in Egg Yolk Medium. Nat~tre, 156: 507. 1945. (38) TosIc, J., ANn WALTON, A. Effect of Egg Yolk and Its Constituents on the Respiration and Fertilizing Capacity of Sl)ermatozoa. J. Agr. Sei., 37: 69. lq46. (39) TosIc, J., AND WALTON, A. Metabolism of Spermatozoa. The Formation and Elimination of Hydrogen Peroxide by Spermatozoa and Effects on Motility and Survival. BiDchem. J., 47: 199. 1950. (40) U~IBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F. Manometric Teelodq~ws and Tiss~lte Metabolism. Burgess Publ. Co., Minneapolis. 1949. (41) ZITTLE, C. A., ANI) ZITIN, B. The Amount ar.d Distribution of Cytochrome Oxidase in Bull Spermatozoa. J. Biol. Chem., 144: 99. 1942.
An active succinic dehydrogenase was first demonstrated in the semen of man and bull b y L a r d y and Phillips (16) and MacLeod (22) by use of the Thunberg technique. Subsequently, Zittle and Zitin (~1) demonstrated the presence of cytochrome oxidase in bull spermatozoa, and M a n n (24, 25) observed the complete eytochrome spectrum in the spermatozoa of man, ram, bull, and boar. However, MacLeod was unable to demonstrate a n y connection between succinate oxidation and the persistence of spermatozoan motility. R y a n (33) observed an increase in respiration of cold-shocked spermatozoa in the presence of succinate, which a p p e a r e d to be associated with an increase in spermatozoan motility. Although the necessity for an active succinic dehydrogenasc in the maintenance of spermatozoan viability has not been established, the importance of dehydrogenases in general to spermatozoa has been confirmed by the relationships which have been observed between " r e d u c t i o n t i m e " and motility, longevity, and per cent n o n r e t u r n s by Beck and Salisbury (4) and E'rb and Ehlers (11). Since the introduction of egg yolk as a desirable constituent of a semen diluent by Phillips (29) and Phillips and L a r d y (30), Tosic and Walton (37, 38, 39) and L aslcy and Mayer (21) have described its effect upon spermatozoan respiration. Tosic and Walton obtained an initial stimulation of respiration followed b y a decrease when egg yolk was added to ejaculated bull spermatozoa and explained the observed decrease as caused by the presence of a dialyzable organic peroxide in egg yolk. Lasley and Mayer, however, observed no respirat o r y effect following the addition of egg yolk to ejaculated r a m spermatozoa but found an increase in the respiration of epididymal spermatozoa. Lasley et al. (19), Easley et al. (9), Lasley and Bogart (18), Lasley and Mayer (20), and Bogart and M a y e r (6) have suggested that egg yolk is of importance in protecting spermatozoa against adverse environmental conditions. Fractionation of egg yolk has yielded fractions valuable in the preservation of spermatozoa. L a r d y and Phillips (17), K a m p s c h m i d t ctal. ( l i ) , and Ryan (.?3) have discussed the beneficial effects of egg yolk phospholipids and lipoproteins upon spermatozoan longevity. K a m p s c h m i d t et al. and Bogart and Mayer recognized two distinct egg yolk factors, one of which protects sperlnatozoa against the h a r m f u l effects of cold shock and a second which contributed to survival (a storage factor). E g g yolk phospholipids, p a r t i c u l a r l y lecithin, have Received for publication September 16, 1955. This investigation was supported ill part by a research grant from the National Institutes of I][e.qlth, Public Health Service. Missouri Agricultural Experiment Station, Journal Series No. 1560. 552
EGG Y O L K AND H Y D R O G E N A S E A C T I V I T Y OF S P E R M A T O Z O A
553
been identified as resistance factors b y R y a n and K a m p s c h m i d t et aL, whereas L a r d y and Phillips demonstrated the oxidative metabolism of egg yolk phospholipids whenever spermatozoa were deprived of a metabolizable hexose. I n recognition of the importance of dehydrogenases and the m a n y beneficial effects of the addition of egg yolk to spermatozoa, as well as its effect upon spermatozoan respiration in general, a series of experiments was conducted to determine the effect of egg yolk in the medium upon the dehydrogenases concerned with c a r b o h y d r a t e metabolism of spermatozoa. MATERIALS AND METHODS
The semen was collected f r o m healthy dairy bulls m a i n t a i n e d as a regular p a r t of the University of Missouri E x p e r i m e n t Station d a i r y herd. A n artificial vagina was used to collect the semen, which was immediately placed in a Thermos bottle of water at a t e m p e r a t u r e no lower t h a n 15 ° C. for t r a n s p o r t to the laboratory. W h e n received at the laboratory, the vial of semen was placed in a 400-ml. beaker of water and stored in the refrigerator. The large water volmne insured gradual cooling to a storage t e m p e r a t u r e of 4 ° C., thereby p r e v e n t i n g cold shock. Generally, the semen specimens f r o m two or more bulls were pooled. I f the spermatozoa were to be washed, they were mixed with about five volumes of 0.9% saline. The wash solution and spermatozoa were separated by centrifugation at 950 G or 2,200 r.p.nl, in a Servall model G centrifuge for about 10 minutes. The seminal plasma saline m i x t u r e was removed b y use of a h a n d controlled automatic pipette. The washing process was repeated four times, a f t e r which the spermatozoa were resuspended in sufficient isotonic saline to attain the original semen volume. Spermatozoan concentration counts were made b y using an eosin counting fluid as described by Smith and Mayer (35). Five counts were made on each sample, a n d their average was used to calculate the cubic millimeters of CO: liberated by 10 s spermatozoa per hour. The W a r b u r g flasks were cleaned by the potassium p e r m a n g a n a t e - s o d i m n hydroxide method and calibrated by fillling them with mercury, according to the method of Umbreit et al. (40). Since spermatozoan suspensions are difficult to p r e p a r e with sufficient optical ~ransparency to facilitate application of spectrophotometric techniques for the determination of enzyme activity, the manometric method of Quastel and Wheatley (31) was employed to estimate the dehydrogenase activity of the spermatozoa in the different suspending media. This method, used more recently b y Rubinstein and Denstedt (32) in an investigation of the metabolism of the avian erythroeyte, uses ferricyanide as an artificial hydrogen carrier to liberate COe f r o m a bicarbonate medimn. The f e r r i e y a n i d e was placed in a side a r m of the ~ a r b u r g flasks and tipped in a f t e r the equilibration period at zero time to initiate the reaction, and the m a n o m e t r i c readings were recorded at 5-minute intervals for a period of ] hour. E g g yolk was added to the flasks as a 1 : 1 m i x t u r e of egg yolk a n d 1.3% N a H C O , . The volume of the m i x t u r e added to each flask was 0.6 ml., equivalent
554
~. T. S ~ I I T H ET AL
to 0.3 ml. of egg yolk per flask. E g g yolk lecithin was p r e p a r e d by the method of P a n g b o r n (28). Twelve rag. of lecithin p r e p a r e d in this m a n n e r was added to each flask as an enmlsion. The cholesterol, a commercial preparation, "~ was incorporated into a water emulsion b y dissolving 275 mg. in 15 ml. of hot methanol and adding the hot solution to 10 ml. of water. A volume of 0.6 nil. of this emulsion, containing 6.6 rag. of cholesterol, was added to each reaction flask. The fl-carotene emulsion was p r e p a r e d by nfixing 3 ml. of a synthetic fl-carotene solution in petroleum ether (50 × 10 " g. per milliliter) with 3 ml. of mineral oil and 3 ml. of d.d. II.,O and the petroleum ether, removed in vacua. Then 1 ml. of the lecithin p r e p a r a t i o n in absolute alcohol, in a concentration of 300 rag. of lecithin per milliliter, was added as an enmlsifying agent. E x a c t l y 0.3 nil. of this emulsion was added to each flask to give 4 × 10 -G g. of carotene per flask, or about the same amount as is contained in 0.3 nil. of egg yolk. To rule out the effect of either lecithin or mineral oil, 0.3 nil. of a solution containing the above ingredients with the exception of carotene was also added to the control flasks. A erude flavine-adenine-dinucleotide concentrate was p r e p a r e d by the method of D i m a n t et al. (8), with the exception that egg yolks were used instead of pig liver or yeast. The egg yolks were fraetionated into aeetolm sohlble and insoluble fractions b y the method of P a n g b o r n (28). Skinnnilk, obtained from the University of Missom'i D a i r y l ) e p a r t m e n t , was used unheated in some experiments and for others was heated in a water bath at 92 ° C. for lO minutes. RESUI,TS
The data showing the effect of whole egg yolk upon the dehydrogenase activity are presented ill Table 1. These data demonstrate all a p p a r e n t stimulation t)y egg yolk of all of the dehydrogenases studied in this investigation. F u r t h e r , they show a highly significant stimulation of the suceinic dehydrogenase activity of both washed and unwashed bovine spermatozoa following addition of the egg yolk mixture. The effect of egg yolk upon the snecinic dehydrogcnase aetivity of washed and unwashed spermatozoa is a p p r o x i m a t e l y the same, au increase of about 25 cu. mm. COo. Since fewer samples of the unwashed spermatozoa were available for study, the results a p p e a r to be less significant. Although the data (Table 1) indicate a slight s t i m u l a t o r y effect of egg yolk upon glyceraldehyde-3-phosph~te and malie dehydrogenases, lhese data were not statistically significant. The value of lecithin to spermatozoan viability has been established by K a m p sehmidt et al. (1.1), L a r d y and Phillips (17), and R y a n (33), and its importance to the succinic oxidase system in homogenized muscle cell p r e p a r a t i o n s was reported b y N y g a a r d and Stunner (27) and E d w a r d s and Ball (10). IIenee. it seemed logical t h a t lecithin m i g h t be responsible for the dehydrogenase stinlula"'Cholesterol preparation obtained from the American Lecithin Co.
EGG Y O L K A N D t I Y D R O G E N A S E
ACTIVITY OF SPER-~c[ATOZOA
555
TABLE 1 Effect of egg yolk upon bovine spermatozoan dehyd.roge~ase activity Cu. ram. CO~ liberated per l0 s spermatozoa per hour
Dehydrogenase system
No egg yolk added
Succinic a~ Succinic b b Glyceraldehyde-3-phosphate ~-Vlalic P = ~
32.0 30.6 6.8 10.8
Added 0.6 nil. of ] : 1 egg yolk: 1.3% NaItCO~
(8) ¢ (4) (9) (3)
57.7 55.6 8.6 11.1
0.01.
b P - - ~ 0.05. ¢ Values in parentheses indicate number of runs which were averaged. Conditions: all spermatozoa except those used in succinate b, which were unwashed, were washed four times with isotonic saline and resuspended in sufficient saline to r e a t t a i n the original seminal volume. Flask contents: for all systems, NaIICO3, 1.86 × 10 -~ M, K3Fe(CN)6 0.2 ml. of an 11% solution neutralized with NatICOs, 0.3 and 0.5 nil. of spermatozoan suspension, sufficient saline to make the total flask volume 2.5 ml., added when measuring (succinic dehydrogenase) Nasuccinate, 0.08 M, glyceraldehyde-3-phosphate dehydrogenase, D P N 3 × 10 -4 M, nicotinamide 0.03 M, malic dehydrogenase, D P N 3 × 10 -~ M, nicotinaniide 0.3 M, Na-l-malate 0.8 M, NaCN 0.2 nil. of a solution of 0.65 M neutralized with tIC1. Gas phase: 5% CO: 95% N.~, incubation temperature 38 ° C., time 1 hour; values are expressed as eu. ram. CO: liberated per 10 ~ spermatozoa per hour, a f t e r correction for a blank cont a i n i n g no substrate. t i o n e f f e c t o f e g g y o l k . l l o w e v e r , t h e r e s u l t s ( T a b l e 2) s h o w a s l i g h t b f l f i b i t i o n of succinic dehydrogenase activity in the presence of lecithin. The failure to find a stimulation of suceinic dehydrogenase in the presence of lecithin suggested that another egg yolk constituent was enhancing the enzyme activity. Succinic dehydrogeuase has been shown to be dependent upon riboflaviu a n d f l a v i n e - a d e n i n e - d i n u c l e o t i d e s f o r m a x i m a l a c t i v i t y b y A x e l r o d e l al. (2) a n d B a l l a n d C o o p e r (3). T h e r e f o r e , a n e g g y o l k c o n c e n t r a t e o f f l a v i n e w a s t e s t e d for its effect upon succinic dehydrogenase a c t i v i t y w i t h o u t s u c c e s s , as s h o w n by the limited data presented
i n T a b l e 2.
B e c a u s e o f a n i n a b i l i t y t o a s s o c i a t e t h e s e t w o e g g y o l k c o n s t i t u e n t s , know~l to affect succinie oxidase systems, with spermatozoan succinic dehydrogenase stimulation, isolation of some egg yolk fractions with organic solvents was performed in an attempt to associate the active substance with a particular fraction TABLE 2 Effect of egg yolk fractions and ,milk upon the s~weiJ~ic dehydrogenase activity of bovine spermatozoa Cu. ram. CO.oliberated per 10 ~ spermatozoa per hour Factor added 12 rag. lecithin 0.6 nil. FAD concentrate 0.6 ml. acetone soluble 0.6 nil. acetone insoluble 0.6 nil. heated milk 0.6 ml. unheated milk
No addition 37.1 45.5 45.8 42.8 50.9 50.9
(6) '~ (2) (4) (6) (2) (2)
Factor added 20.9 37.2 64.7 48.2 54.1 47.4
Values in parentheses indicate number of samples averaged. Conditions arc the same as given for succinic dehydrogenase estimation in Table 1 except t h a t all spermatozoa were washed.
556
J . T . S S [ I T H ET AL
of egg yolk. The d a t a ( T a b l e 2) s u g g e s t t h a t t h e a c t i v e s u b s t a n c e was p r e s e n t in t h e a c e t o n e soluble f r a c t i o n of e g g yolk. The i m p o r t a n c e of c h o l e s t e r o l to c e l l u l a r m e t a b o l i s m , its effect on t h e diffusion of a c i d s a n d a l k a l i s i n t o g e l a t i n a n d a g a r , a n d its beneficial effect of p r e v e n t i n g h e m o l y s i s b y m u m p s v i r u s h e m o l y s i n h a v e been r e p o r t e d b y A f f o n s k y (1), M a g i s t r i s (23), a n d M o r i m o t o a n d M o r g a n (26). S i n c e cholesterol c o m p r i s e d a l a r g e p e r c e n t a g e of t h e a c e t o n e s o l u b l e f r a c t i o n of egg y o l k a n d possessed t h e p r o p e r t i e s cited, its effect on b o v i n e s p e r m a t o z o a n d e h y d r o g e n a s e s was m e a s u r e d . A h i g h l y s i g n i f i c a n t a c t i v a t i o n of s u c c i n i c d e h y d r o g e n a s e b y c h o l e s t e r o l (P---< 0.01) was o b s e r v e d ( T a b l e 3). H o w e v e r , a s i g n i f i c a n t i n h i b i t i o n of g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e a n d a n a p p a r e n t i n h i b i t i o n of m a l i c d e h y d r o genase were o b s e r v e d ( T a b l e 3). TABLE 3 Effect of cholesterol upon bovine spermatozoan dehydroge~tase activity Cu. mm. CO: liberated per 10~ spermatozoa per hour Dehydrogenase system
No cholesterol added
6.6 mg. cholesterol added to each flask
Succinic :' Glyceraldehyde-3-phosphatet' Malic
49.5 (15) 6.3 (10) 10.3 (4)
61.9 3.6 S.1
" P ~ 0.01. b p = 0.05. Conditions are the same as in Table 1 except that all spcrnmtozoa were washed. S i n c e t h e e n t i r e s t i m u l a t i n g effect of egg y o l k c o u l d n o t be a t t r i b u t e d to cholesterol, one o r m o r e a d d i t i o n a l e g g y o l k c o n s t i t u e n t s w e r e p o s s i b l y involved. H e r i s s e t (12) f o u n d t h a t , in t h e p r e s e n c e of m e t h y l e n e b l u e as a h y d r o g e n a c c e p t o r , t h e a d d i t i o n of a s m a l l a m o u n t of c a r o t e n e - i n - o i l e m u l s i o n a c c e l e r a t e d t h e a c t i o n of S c h a r d i n g e r enzyme, s u c c i n i c d e h y d r o g e n a s e , a n d g l u t a m i c a c i d d e h y d r o g e n a s e . O n t h e basis of these r e s u l t s , t h e effect of c a r o t e n e e n m l s i o n s u p o n m a l i c a n d s u c c i n i c d e h y d r o g e n a s e s of b o v i n e s p e r m a t o z o a was i n v e s t i g a t e d . The effect of c a r o t e n e , a n o t h e r c o m p o n e n t of t h e a c e t o n e soluble f r a c t i o n of egg yolk, u p o n b o v i n e s p e r m a t o z o a n s u c c i n i c a n d m a l i c d e h y d r o g e n a s e s is p r e s e n t e d in T a b l e 4. These d a t a show t h a t c a r o t e n e s t i m u l a t e s t h e a c t i v i t y of b o t h succinic and malic dehydrogenases. I n o r d e r to d e t e r m i n e i f h e a t e d m i l k , w h i c h has been shown to be a s a t i s f a c t o r y s e m e n e x t e n d e r , w o u l d also a c t i v a t e s u c c i n i c d e h y d r o g e n a s e , a c o m p a r a t i v e TABLE 4 Effect of carotene ~pon the succinic and malic dehydrogenase activity of bovine spermatozoa Cu. mm. CO-,liberated per 10s spermatozoa per hour Dehydrogenase system
No carotene added
4 × 10-0 g. carotene added per flask
Succinic 1V[alic
25.8 (6) 10.0 (3)
34.6 20.1
Conditions are the same as in Table 1 except that all spermatozoa were washed.
EGG Y O L K AND H Y D R O G E N A S E A C T I V I T Y OF S P E R M A T O Z O A
557
deternfination with unheated milk was made. I t was f o u n d (Table 2) that heated milk has a slight a p p a r e n t s t i m u l a t o r y effect upon suecinic dehydrogenase, whereas unheated milk, which is spermicidal, was slightly inhibitory in its effect upon bovine spermatozoan succinic dehydrogenases. DISC U S S I O N
Activation of the suecinic a n d malic dehydrogenase systems of bovine spermatozoa by egg yolk or certain of its constituents a p p e a r s to be compatible with previous evidence obtained in this and the Wisconsin laboratories. Smith et al. (3.t) demonstrated that, in an egg yolk diluent, maintenance of an osmotic pressure within certain optinlal limits was dependent upon the addition of a metabolyzable hexose. Unpublished observations in this l a b o r a t o r y indicate that tile buffering capacity of the egg yolk-glucosc-NaIICO:~ diluent (13), though low, is sufficient to p r e v e n t a dangerous shift towards an acid p H t h r o u g h o u t a ]0-day storage period. L a r d y and Phillips (17) obtained evidence suggesting that spermatozoa metabolized egg yolk phospholipids in the absence of a nletabolizable hexose. I t is possible, then, that the egg yolk activated dehydrogenases f u n c t i o n to remove the acid products of glyeolysis in a diluent, thereby m a i n t a i n i n g both a physiological p H and osmotic pressure. The a p p a r e n t failure of egg yolk lecithin to stimulate the sueeinic dehydrogenase activity of bovine spermatozoa is interesting in view of the excellent results obtained with lecithin in protecting spernmtozoa against adverse environmental conditions. However, it m a y be recalled that two egg yolk factors, one a storage and the other a resistance factor, were recognized in egg yolk and that lecithin was identified as a resistance factor. Could it be t h a t lecithin a n d / o r leeitho-protein complexes act as resistance factors whereas cholesterol a n d / o r carotene, or complexes of these compounds, m a y be classified as storage factors ? Cholesterol, on the other hand, has not been closely associated with cellular metabolic activities by investigations in this field. The literature has cited the effects of cholesterol upon m e m b r a n e permeability and the m a r k e d ability of this hydrophobie compound to f o r m water-in-oil emulsions, thus holding w a t e r in the tissues. Cholesterol, also, counteracts the effects of Ca + + upon lecithin when the latter is in contact with w a t e r and tends to reduce the surface occupied by the lecithin, according to Bloor (5). The only a p p a r e n t explanation of the stinmlation of suecinic dehydrogenase activity by cholesterol would seem to be its effect upon cellular permeability. This effect m a y be a direct one or it m a y be an indirect effect in which it prevents Ca + + inhibition of lecithin. The indirect effect is valid if it be assumed, as it was for the e r y t h r o c y t e b y Morimoto and Morgan (26), that cholesterol and lecithin are components of the spermatozoan cellular membrane. Investigation of the chemistry of m a m m a l i a n spermatozoa now in progress in this l a b o r a t o r y suggests the validity of this assumption. The results also show that cholesterol inhibits glyceraldehyde-3-phosphate and malic dehydrogenase activity of bovine spermatozoa. This finding suggests another possible explanation of the mechanism whereby cholesterol stimulates succinie dehydrogenase activity. The two enzynie systems, which are inhibited
558
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by cholesterol, have at least one common p r o p e r t y that may influence suecinie dehydrogenase a c t i v i t y - - t h e y both require the participation of D P N for activity. D P N has been shown by Keilin and H a r t r e e (15) to inhibit sueeinic dehydrogenase activity of nmscle preparations and by Smith and Mayer (36) to inhibit suecinic dehydrogenase activity of bovine spermatozoa. This inhibition by DPN is effeeted by the catalysis of the reaction malate ---> oxalaeetate, oxalaeetate being inhibitor.v in concentrations as low as 10 4M. Therefore, it may be that the stinmlatory effect of cholesterol upon bovine spermatozoan sueeinie dehydrogeuase and its inhibitory effect upon glyceraldehyde-3-phosphate dehydrogenase may be the result of its inhibition in some maimer of I)PN catalysis. Some reservation is placed upon this explanation, however, since the stimulation of succinic dehydrogenase by cholesterol occurred in washed spermatozoa, which have been shown by Smith and Mayer to be deficient in DPN. However, since it is the D P N catalysis of malate ---> oxalaeetate which is inhibitory, any inhibition of malie dehydroo'enase would result in an apparent suceinie dehydrogenase stinmlation. Explanation of the mechanism of stinmlation of bovine spermatozoan dehydrogenase activity by carotene stinmlation is difficult. The only known function of caroteue in enzyme systems is its postulated role in photosynthesis by Davis (7), who assumes that carotene functions in photosynthesis as an electron transport system by virtue of its large number of resonating double bonds. It does not seem unreasonable that it might function in the bovine spermatozoan suceinic dehydrogenase system in a similar lnanner, but there is no affirmative evidence. SUMMARY
The effect of whole egg yolk and its constituents upon the dehydrogenase activity of bovine spermatozoa was determined. Whole egg yolk was found to stimulate the succinic, malic, and glyceraldehyde-3-phosphate dehydrogenase activity of these cells. The dehydrogenase-stimulating effect of egg yolk was found to be associated with the acetone-soluble fraction of the yolk and appeared to be the result of the stimulatory effect of cholesterol and of carotene upon succinic dehydrogenase activity. Cholesterol was observed to inhibit both glyceraldehyde-3-phosphate and malic dehydrogenase activity of bovine spermatozoa. The importance and possible mechanisms of dehydrogenase stimulation and inhibition by egg yolk, cholesterol, and carotene have been discussed, as well as the a p p a r e n t failure of lecithin to stimulate succinie dehydrogenase activity. ACKNOWLEDGMENTS T h e a u t h o r s wish to a c k n o w l e d g e t h e g e n e r o u s d o n a t i o n of fl-carotene by L a u r a M. F l y n n a n d t h e service of K . W. Bower, who collected t h e semen. R E F E I{ENCES (1) AI~PONSKY, S. I. ~ b e r des E i n f l u e s s e s der L i p o i d e a u f die D i f f u s i o n der S a u r e n u n d A l k a l e i n in Gallerten. Biochem. Z., 195: 387. 1928. (2) AXELROD, A. E., SWINGLE~ K . F., AND ELVEItJEM, C. A. S t u d i e s on t h e Succinoxidase S y s t e m of R a t L i v e r in Riboflavine Deficiency. J. Biol. Chem., 145: 297. 1942.
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(3) BALL, E. G., AND COOPER~ O. The Activity of Succinate Oxidase in Relation to P h o s p h a t e and P h o s p h o r u s Compounds. J. Biol. Chem., 180: 113. 1949. (4) BECK, G. H., AND SALISBURY, G. W. l~apid Methods for E s t i m a t i n g tile Quality of Bull Semen. J. Dairy Sci., 26: 483. 1943. (5) BLOOR, W. R. Bioche~istry o f tl~e F a t t y Acids. Reinhold P u b l i s h i n g Corp., New York. 1943. (6) BOGART, R., AND MAYER, D. T. Tile Effects of E g g Yolk on the Various Physical and Chemical Factors Detrimental to Spermatozoan Viability. J. A n i ~ a l Sci., 9: 143. 1950. (7) DAVIS, C. New I n t e r p r e t a t i o n of ]Role of Carotenoids in Photosynthesis. _Federation Prec., 14: 200. 1955. (8) DI~ANT, E., SANADI, D. ]~., AND tIt:NNE~=INS, F. M. The Isolation of Flavin Nucleotides. J. Am. Cicero. Soc., 74: 5440. 1952. (9) EASLE¥, G. T., MAYER, D. T., AND BOGAICT, R. Influence of Diluters. Rates of Cooling and Storage Temperature on Survival of Bull Sperm. Am. J. Vet. Researcl~, 3: 358. 1942. (10) EDWARDS, S. W., AND BALL, E. G. The Action of Phospholipases oa Succinate Oxidase and Cytochrome Oxidase. J. Biol. Chem., 209: 619. 1954. (11) ERB, R. E., AND EtILERS, M. It. Resazurin Reducing Time as an Indicator of Bovine Semen Fertilizing Capacity. J'. Dairy Sci., 33: 853. 1950. (12) HERISSET, A. Influence of Carotene on Reactions Catalyzed in Vitro by Some Dehydrogenases. B~dl. Soc. Chem. Biol., 36: 809. 1954. ( F r o m Chem. Abstr. 49: 1832d) 1955. (13) KAMPSCHJ~IDT, R. F., MAYER, D. T., HERMAN, H. A., AND DICKE~SON, G. E. Viability of Bull Spernmtozoa ~s Influenced by Electrolyte Concentration, Buffer Efficiency, and Added Glucose in Storage Media. J. Dairy Sci., 33: 45. 1951. (14) KAMPSCHi~IIDT, R. F., MAYER, D. T., AND HERMAN, H. A. Lipid and Lipoprotein Cons t i t u e n t s of E g g Yolk in Resistance and Storage of Bull Spermatozoa. J. Dairy Sci., 36: 733. 1953. (15) KEILIN, D., AND ttAR~REE, E. F. Succinic Dehydrogenase Cytochrome System of Cells, Intracellular ]~espiratory System Catalyzing Aerobic Oxidation of Succinic Acid. Prec. Roy. Soc., B., 129: 277. 1940. (16) LARDY, H. A., AND PHILLIPS, P. H. The Effect of Certain Inhibitors a n d Activators on Sperm Metabolism. J. Biol. Cl~em., 138: 195. 1941. (17) LARDY, H. A., AND PHILLIPS, P. It. Phospholipids as a Source of E n e r g y for Motility of Bull Spernmtozoa. Am. J. Pl~ysiol., 134: 542. 1941. (18) LASLEY, ,.T.F., AND BOGART, R. Some F a c t o r s Affecting the Resistance of E j a c u l a t e d a n d Epididymal Spermatozoa of the Boar to Different Environmental Conditions. Am. J . Physiol., 141: 619. 1944. (19) LASLEY, J. F., EASLI~Y, G. T., AND BOGART, R. Some Factors Influencing the Resistance of Bull Sperm to Unfavorable E n v i r o n m e n t a l Condition. J. A~dnlal Sci., 1: 79. 1942. (20) LASLEY, J. F., AND MAYER, D. T. A Variable Physiological Factor Necessary for the Survival of Bull Spermatozoa. J. Animal Sci., 3: 121. 1944. (21) LASLEY, J. F., AND MAYEI~, D. T. The Effect of Glucose, E g g Yolk and Seminal P l a s m a on the Comparative Metabolic Activities of Epididymal a n d E j a c u l a t e d Spermatozoa of the Ram. J. Animal Sci., 10: 1027. 1951. (22) MAcL~OD, J. The Role of Oxygen in the Metabolism a n d Motility of H u m a n Si~ermatozoa. Am. J. Physiol., 138: 512. 1943. (23) MAGISTRIS, H. Zur Biochemie und Physiologic Oranischer P h o s p h o r v e r b i n d u n g e n in Pflanze und Tier. Ein Beitr~g zur F r a g e des Einflusses van Tierisehen u n d flanzlichena Lecithin zur die Diffusion von Sauren u n d Alkalien in Gallerten. Biochem. Z., 2 5 3 : 81. 1932. (24) MANN, T. Studies on the Metabolism of Semen. I. General Aspects, Occurrence a n d Distribution of Cytochrome, Certain E n z y m e s and Co-enzymes. Biochem. J., 39: 451. 1945.
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(25) MAN~-, T. Studies on thc Metabolism of Semen. 7. Cytochrome in Hmnan Spermatozoa. Biochem. J., 48: 386. 1951. (26) M0zIMOT0, M., AND MORG.~N, H. l]. Effect of Lecithin, Cholesterol and Cardiolipin on Mumps Virus Hemolysin. Proc. Soc. Exptl. Biol. Med., 86: 795. 1954. (27) NYOAARI), A. P., AXD SUMNEa, J. B. The Effect of Lecithinase A. ml the Succinoxidase System. J. Biol. Chem., 200: 723. 1953. (28) PA~CGBOR~¢,M. C. A Simplified Purification of Lecithin. J. Biol. Chem., 188: 471. 1951. (29) PHILLIPS, P. t{. Preservation of Bull Semen. J. Biol. Chem., 130: 415. 1939. (30) PHILLIPS, P. It., AND LARDY, I-I. A. A Yolk Buffer Pabulum for the Preservation of Bull Semen. J. Dairy Sci., 23: 399. 1940. (31) QUAST~L, J. H., AND WHEATLEY, A. I'i. M. On Ferrieyanide as a Reagent for the Manometric Investigation of Dehydrogenase Systems. Biochem. J., 32: 936. 1938. (32) RUBINSTEIN, D., AND D~NSTEDT, O. F. The Metabolism of the Erythrocyte. I I I . The Tricarboxylic Acid Cycle in the Avian Erythrocyte. J. Biol. Chem., 204: 623. 1953. (33) RYA~, W. L. A Metabolic Study of Cold Shock in Mammalian Spermatozoa. Ph.D. thesis, Univ. of Missouri. 1953. (34) SMITH, ft. T., i~AYER, 1). W., AND I'IERSIAN, ~[{. A. A Comparison of the Ability of .Certain E g g Yolk Diluents to Maintain Optinlum Osmotic Conditions During the Storage of Bull Semen. J. Dairy Sci., 37: 684. 1954. (35) SMITH, J. T., AND MAYEa, D. T. Evaluation of Sperm Concentration by the Hemacytometer Method: A Comparison of Four Comtting Fluids. Fertility and Sterility, 6: 271. 1955. (36) SMITH, J. T., AND MAYFA~, D. T. (Unpublished lab. data) 1955. (37) TOSlC, J., AND WALTON, A. Respiration of Sperm~tozoa in Egg Yolk Medium. Nat~tre, 156: 507. 1945. (38) TosIc, J., ANn WALTON, A. Effect of Egg Yolk and Its Constituents on the Respiration and Fertilizing Capacity of Sl)ermatozoa. J. Agr. Sei., 37: 69. lq46. (39) TosIc, J., AND WALTON, A. Metabolism of Spermatozoa. The Formation and Elimination of Hydrogen Peroxide by Spermatozoa and Effects on Motility and Survival. BiDchem. J., 47: 199. 1950. (40) U~IBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F. Manometric Teelodq~ws and Tiss~lte Metabolism. Burgess Publ. Co., Minneapolis. 1949. (41) ZITTLE, C. A., ANI) ZITIN, B. The Amount ar.d Distribution of Cytochrome Oxidase in Bull Spermatozoa. J. Biol. Chem., 144: 99. 1942.