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Gossypol uncoupling of respiratory chain and oxidative phosphorylation in ejaculated boar spermatozoa
CONTRACEPTION
GOSSYPOL UNCOUPLING OF RESPIRATORY CHAIN AND OXIDATIVE PHOSPHORYLATION
IN EJACULATED BOAR SPERMATOZOA
Wung-Wai Tso* and Chi-Sing Lee Department of Biochemistry The Chinese University of Hong Kong Shatin, N.T., Hong Kong
ABSTRACT Gossypol, a promising male contraceptive isolated from This anticottonseed oil, inhibits spermatozoa1 motility. motility effect accompanies a biphasic change in the spermatozoal respiratory rate: a stimulation at low concentrations With various and an inhibition at high concentrations. respiratory inhibitors, gossypol was shown to take effect by the uncoupling of the respiratory chain and the oxidative phosphorylation. The implication of this uncoupling action in the strategy of producing an ideal gossypol-like male contraceptive is discussed.
Submitted for publication March 31, 1982 Accepted for publication May 27, 1982
*
To whom correspondence should be addressed.
JUNE
1982 VOL. 25 NO. 6
649
CONTRACEPTION
INTRODUCTION Laboratory mammals forced-fed with gossypol, a promising male contraceptive isolated from cottonseed oil (1,2), produced dead spermatozoa in the caudal epididymis and abnormal spermatozoa in the testes(3). This compound has also been tested clinically with higher than ninetynine percent success and exhibited no obvious adverse effect to the treated individual (4). In vitro studies with mature boar spermatozoa showed that gossypol abolished spermatozoa1 forward motility. This action was accompanied by a sizable respiratory enhancement at low concentrations and an inhibition at high concentrations of gossypol (5). Although this antimotility effect has been suggested to be a possible contributing factor for the gossypol antifertility action, the mechanism of the gossypol effect on spermatozoa1 respiration is not understood. In rat liver mitochondria, gossypol has been shown to be an uncoupler of respiratory chain and oxidative phosphorylation (6). Since artificial electron acceptors such as methylene blue may stimulate respiration by acting as an alternative pathway for the rate limiting electron transport system, gossypol with a structure similar to hydroquinone, may possess a redox potential which enables it to function as an artificial electron acceptor. Recently, lactate dehydrogenase-X, has been shown to be highly susceptible to gossypol inhibition (7). This enzyme, being located only in the gonad, would seem to be one of the most favorable targets on which a male contraceptive works (8,9). It is the aim of this study to characterize the nature of the gossypol effect on spermatozoa1 respiration and to discuss the possible role of this effect in contraception.
MATERIALS
AND METHODS
Fresh gossypol solution was prepared by dissolving gossypol acetic acid (Sigma) in 0.5 M NaOH immediately before use. Where pH adjustment Oligomycin was dissolved was needed, a 0.5 M HCl solution was employed. in absolute ethanol and added when tested. Fresh boar semen, collected by an artificial vagina, was filtered through sterilized gauge and then centrifuged at 700 g to remove gelatinous particles. Only those batches with sperm count between 150 and 300 millions/ml and not less than 70% progressive motility were used for experiments (6). The respiratory assay medium contained 4 ml of 10% (v/v) seminal plasma in isotonic Tris-Cl buffer (0.172 M Tris), pH 7.0 (6). The spermatozoa1 respiratory rate was followed with a YSI Model 53 Biological Oxygen Monitor (Yellow Springs Instrument, Yellow Springs, Ohio, U.S.A.) with temperature maintained either at 25' ? 0.5OC (for intact spermatozoa) or at 30" 2 0.5"C (for hypotonically-treated spermatozoa). Hypotonically-treated spermatozoa were prepared by the method slightly modified from Calvin and Tubbs (10). Spermatozoa were washed twice by centrifugation at 18" + 2°C with 60 mM morpholinopropane sulfonic acid (MOPS, pH 7.4 by KOH) containing 0.2 M sucrose. The spermatozoa1 pellet was then suspended in 5 M MOPS solution (pH 7.4) and kept in an icebath for 10 min, followed by centrifugation at 1,300 g for 5 min at 4°C. The hypotonically-treated spermatozoa so obtained were resuspended in an assay medium containing 0.2 M sucrose, 60 mM MOPS, 10 mM KH2PO4 and 5 mM MgC12 (pH 7.4) and studied for respiration at a fixed cell concentration in the presence of substrate.
650
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION RESULTS The spermatozoa1 respiratory rate measurements for each study were done three times with independently prepared samples (only twice in hypotonically-treated spermatozoa) and essentially similar respiratory patterns were observed. The figures represent typical oxygen electrode traces to illustrate the effects of various treatments on the spermatozoa1 oxygen uptake. The cyanide-inhibited spermatozoa1 respiration was not relieved by 3.2 x 10m5 M gossypol (Fig. 1-A). A very slight increase took place at low gossypol concentrations presumably due to incomplete cyanide blockage. Another experiment with the chemicals added in a reverse sequence showed that the gossypol-stimulated respiration was essentially abolished by the presence of cyanide (Fig. 1-B). In Fig. Z-A, (from 220.3 nmole was then relieved tory rate changed hour/lo* sperm).
Fj-g. 1.
JUNE
an addition of oligomycin caused respiratory arrest 02/hour/108 sperm to 48.1 nmole 02/hour/108 sperm) which by gossypol at low concentration (3.2 x 10W5 M, respirafrom 48.1 nmole 02/hour/108 sperm to 396.6 nmole 02/ On the other hand, oligomycin exhibited insignificant
Effect of gossypol on spermatozoa1 respiration in the presence of Unwashed spermatozoa were diluted with Krebspotassium cyanide. Ringer phosphate solution, pH 7.4, containing 10% (v/v) seminal Spermatozoa (Sp), potassium plasma and 2 mg/ml D-glucose. cyanide (Cy) and gossypol (Go), each to a final concentration of 80 x lo6 cell/ml, 0.9 mM and 3.2 x 10-5 M,correspondingly, The values against the were added in the designated sequence. slope represent the rate of respiration in nmole 02/hour/10* sperm.
1982 VOL. 25 NO. 6
651
CONTRACEPTION
C
700
“rnOlL¶~ 02
Fi4 . 2.
I
Effect of gossypol on spermatozoa1 respiration in the presence Unwashed spermatozoa were diluted with isotonic of oligomycin. Tris-Cl buffer, pH 7.0, containing 10% (v/v) seminal plasma. Assay conditions were identical to that described in Fig. 1, except oligomycin (01) at 1 n /ml and gossypol (Go) at 3.2 x 10V5 M and (Go') at 3.2 x lo-& M,correspondingly,were used.
inhibition on 3.2 x 10m5 M ossypol-stimulated respiration ( nly slightly 8 from 423.0 nmole 02/hour/lO g sperm to 372.1 nmole 02/hour/lO sperm). Furthermore, gossypol at a higher concentration, 3.2 x low4 M (ten-fold higher), completely abolished the oligomycin-insensitive respiration This is a feature commonly observed for (probably state 4 respiration). a common uncoupler of respiratory chain and oxidative phosphorylation (11). In intact spermatozoa, as the midpiece mitochondria are well protected by a plasma membrane, the exogenous substrates added to the assay medium are not freely accessible to the mitochondria (12). The availability of the substrate thus depends heavily on the permeability Any reagent that can alter of the substrate through the plasma membrane. the membrane permeability may subsequently lead to a change in the
652
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION In addition to this, the seminal plasma, which is respiratory rate. supplemented to the assay medium for studies, contains undefined amounts of various substrates such as fructose, lactate and citrate (13). The respiratory stimulation brought about by gossypol additions might even The be a specific effect on the membrane permeability of the substrate. uncoupling effect of gossypol was further examined in the hypotonicallytreated spermatozoa in which the mitochondria are exposed to the assay medium (10,14). The assay medium contained 2 mM sodium arsenite, a system where pyruvate oxidation has been intentionally inhibited (10). Extramitochondrial reducing equivalent (15), in the form of NADH, was supplied to the mitochondria to enable either the lactate-pyruvate shuttle or the malate-aspartate shuttle to operate (Fig. 3, A and B). The respiration of the hypotonically-treated spermatozoa in the presence of various substrates from these two shuttles was inhibited by oligomycin and was relieved by 3.2 x low5 M gossypol.
Fig. 3.
Effect of gossypol on hypotonically-treated spermatozoa1 Hypotonically-treated respiration using various substrates. spermatozoa (Sp), 180 x lo6 sperm/ml; oligomycin (Ol), 1 pg/ml; gossypol (Go), 3.2 x 10e5 M; lactate (La), 1 mM; malate (Ma), 1 mM; glutamate (Gl), 5 mM; and arsenite (Ar), 2 mM; were added accordingly in the designated sequence.
DISCUSSION Oligomycin is an inhibitor of oxidative phosphorylation. Its presence prevents the ATP-forming mechanism from utilizing the high-energy interGossypol relieves the mediate or state generated by electron transport. oligomycin-inhibited respiration but not the cyanide-inhibited respiration. This observation is consistent with the proven nature of gossypol as a
JUNE
1982 VOL. 25 NO. 6
653
CONTRACEPTION classical uncoupler. In hypotonically-treatedspermatozoa, an essentially similar uncoupling pattern is observed. The present study shows that gossypol uncouples the respiratory chain from oxidative phosphorylation in mature, ejaculated boar spermatozoa. This uncoupling, as compared to other reported observations (6), appears to be neither animal-specific nor tissue-specific. Simultaneously, gossypol also inhibits many other biochemical reactions such as the gonadspecificlactate dehydrogenase isozyme, LDH-X (7) and the testosterone production by Leydig cells (16). This suggests that the uncoupling action of gossypol may not be the primary cause of contraception. It does not rule out, howeverthe possibility that a reduction in energy supply especially in the early developmental process might be vital to spermatogenesis. Moreover, if the antifertility effect of gossypol relies not on its uncoupling action, an ideal gossypol-like male contraceptive canhen be designed which has no uncoupling properties.
ACKNOWLEDGMENT Thanks are due to the Agriculture and Fisheries Department of Hong Kong for supplying boar semen samples and to Mrs. .I.Hill for secretarial assistance. The research was partially supported by a grant from the Chinese Medicinal Material Research Centre of The Chinese University of Hong Kong.
REFERENCES 1. Wang, Y., Luo, Y. and Tang, X. Studies on the antifertility actions of cottonseed meal and gossypol. Acta. Pharm. Sinica -14:662-669 (1979). 2.
Dai, R.X. and Dong, R.H. Studies on antifertility effect of gossypol. I. An experimental analysis by epididymal ligature. Acta Biol. Exp. Sinica -11:15-22 (1978).
3.
Dai, R.X., Pang, S.N., Lin, X.K., Ke, U.B., Lui, Z.L. and Gong, R.H. A study of antifertility of cottonseed. Acta Biol. Exp. Sinica -ll:l-10 (1978).
4.
National coordinating group on male antifertility agents. Gossypol - a new antifertility agent for males. Chinese Med. J. -58:455458 (1978).
5.
Tso, W.-W. and Lee, C.S. Effect of gossypol on boar spermatozoa in vitro. Arch. Androl. 1:85-88 (1981).
6.
Abou-Donia, M.B. and Dieckert, J.W. Gossypol: Uncoupling of respiratory chain and oxidative phosphorylation. Life Sci. -14:19551963 (1974).
7.
Tso, W.-W. and Lee, C.-S. Lactate dehydrogenase-X: an isozyme particularly sensitive to gossypol inhibition. Int. J. Androl. (in press).
6.54
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION
8.
Blanco, A. and Zinkham, W.H. Science -139:601 (1963).
9.
Goldberg, E. Lactate and malic dehydrogenases in human spermatozoa. Science -139:602 (1963).
Lactate dehydrogenase in human testes.
10.
Calvin, .I.and Tubbs, P.K. Mitochondrial transport processes and oxidation of NADH by hypotonically-treated boar spermatozoa. Eur. J. Biochem. -89:315-320 (1978).
11.
Heytler, P.G. Uncouplers of oxidative phosphorylation. In: Methods in Enzymology (Ed.: Fleischer, S. and Packer, L.). Academic Press, New York, pp. 462-472 (1979).
12.
Fawcett, D.W. The structure of the spermatozoa. In: Frontiers in Reproduction and Fertility Control (Ed.: Greep, R.O. and Koblinsky, M.A.). The MIT Press, Cambridge, Massachusetts, pp. 353-378 (1977).
13.
Mann, 'I. Physiology of semen and of the male reproductive tract. In: Reproduction in Domestic Animals, 2nd ed (Ed.: Cole, H.H. and Cupps, P.T.). Academic Press, New York, pp. 277-312 (1969).
14.
Keyhani, E. and Storey, B.T. Energy conservation capacity and morphological integrity of mitochondria in hypotonically-treated rabbit epididymal spermatozoa. Biochim. Biophys. Acta -305:557-569 (1973).
15.
Dawson, A.G. Oxidation of cytosolic NADH formed during aerobic metabolism in mammalian cells. Trends Biochem. Sci. -4:171-176 (1979).
16.
Hadley, M.A., Lin, Y.C. and Dym, M. Effects of gossypol on the reproductive system of male rats. J. Androl. 7:190-199 (1981).
JUNE 1982 VOL. 25 NO. 6
655
GOSSYPOL UNCOUPLING OF RESPIRATORY CHAIN AND OXIDATIVE PHOSPHORYLATION
IN EJACULATED BOAR SPERMATOZOA
Wung-Wai Tso* and Chi-Sing Lee Department of Biochemistry The Chinese University of Hong Kong Shatin, N.T., Hong Kong
ABSTRACT Gossypol, a promising male contraceptive isolated from This anticottonseed oil, inhibits spermatozoa1 motility. motility effect accompanies a biphasic change in the spermatozoal respiratory rate: a stimulation at low concentrations With various and an inhibition at high concentrations. respiratory inhibitors, gossypol was shown to take effect by the uncoupling of the respiratory chain and the oxidative phosphorylation. The implication of this uncoupling action in the strategy of producing an ideal gossypol-like male contraceptive is discussed.
Submitted for publication March 31, 1982 Accepted for publication May 27, 1982
*
To whom correspondence should be addressed.
JUNE
1982 VOL. 25 NO. 6
649
CONTRACEPTION
INTRODUCTION Laboratory mammals forced-fed with gossypol, a promising male contraceptive isolated from cottonseed oil (1,2), produced dead spermatozoa in the caudal epididymis and abnormal spermatozoa in the testes(3). This compound has also been tested clinically with higher than ninetynine percent success and exhibited no obvious adverse effect to the treated individual (4). In vitro studies with mature boar spermatozoa showed that gossypol abolished spermatozoa1 forward motility. This action was accompanied by a sizable respiratory enhancement at low concentrations and an inhibition at high concentrations of gossypol (5). Although this antimotility effect has been suggested to be a possible contributing factor for the gossypol antifertility action, the mechanism of the gossypol effect on spermatozoa1 respiration is not understood. In rat liver mitochondria, gossypol has been shown to be an uncoupler of respiratory chain and oxidative phosphorylation (6). Since artificial electron acceptors such as methylene blue may stimulate respiration by acting as an alternative pathway for the rate limiting electron transport system, gossypol with a structure similar to hydroquinone, may possess a redox potential which enables it to function as an artificial electron acceptor. Recently, lactate dehydrogenase-X, has been shown to be highly susceptible to gossypol inhibition (7). This enzyme, being located only in the gonad, would seem to be one of the most favorable targets on which a male contraceptive works (8,9). It is the aim of this study to characterize the nature of the gossypol effect on spermatozoa1 respiration and to discuss the possible role of this effect in contraception.
MATERIALS
AND METHODS
Fresh gossypol solution was prepared by dissolving gossypol acetic acid (Sigma) in 0.5 M NaOH immediately before use. Where pH adjustment Oligomycin was dissolved was needed, a 0.5 M HCl solution was employed. in absolute ethanol and added when tested. Fresh boar semen, collected by an artificial vagina, was filtered through sterilized gauge and then centrifuged at 700 g to remove gelatinous particles. Only those batches with sperm count between 150 and 300 millions/ml and not less than 70% progressive motility were used for experiments (6). The respiratory assay medium contained 4 ml of 10% (v/v) seminal plasma in isotonic Tris-Cl buffer (0.172 M Tris), pH 7.0 (6). The spermatozoa1 respiratory rate was followed with a YSI Model 53 Biological Oxygen Monitor (Yellow Springs Instrument, Yellow Springs, Ohio, U.S.A.) with temperature maintained either at 25' ? 0.5OC (for intact spermatozoa) or at 30" 2 0.5"C (for hypotonically-treated spermatozoa). Hypotonically-treated spermatozoa were prepared by the method slightly modified from Calvin and Tubbs (10). Spermatozoa were washed twice by centrifugation at 18" + 2°C with 60 mM morpholinopropane sulfonic acid (MOPS, pH 7.4 by KOH) containing 0.2 M sucrose. The spermatozoa1 pellet was then suspended in 5 M MOPS solution (pH 7.4) and kept in an icebath for 10 min, followed by centrifugation at 1,300 g for 5 min at 4°C. The hypotonically-treated spermatozoa so obtained were resuspended in an assay medium containing 0.2 M sucrose, 60 mM MOPS, 10 mM KH2PO4 and 5 mM MgC12 (pH 7.4) and studied for respiration at a fixed cell concentration in the presence of substrate.
650
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION RESULTS The spermatozoa1 respiratory rate measurements for each study were done three times with independently prepared samples (only twice in hypotonically-treated spermatozoa) and essentially similar respiratory patterns were observed. The figures represent typical oxygen electrode traces to illustrate the effects of various treatments on the spermatozoa1 oxygen uptake. The cyanide-inhibited spermatozoa1 respiration was not relieved by 3.2 x 10m5 M gossypol (Fig. 1-A). A very slight increase took place at low gossypol concentrations presumably due to incomplete cyanide blockage. Another experiment with the chemicals added in a reverse sequence showed that the gossypol-stimulated respiration was essentially abolished by the presence of cyanide (Fig. 1-B). In Fig. Z-A, (from 220.3 nmole was then relieved tory rate changed hour/lo* sperm).
Fj-g. 1.
JUNE
an addition of oligomycin caused respiratory arrest 02/hour/108 sperm to 48.1 nmole 02/hour/108 sperm) which by gossypol at low concentration (3.2 x 10W5 M, respirafrom 48.1 nmole 02/hour/108 sperm to 396.6 nmole 02/ On the other hand, oligomycin exhibited insignificant
Effect of gossypol on spermatozoa1 respiration in the presence of Unwashed spermatozoa were diluted with Krebspotassium cyanide. Ringer phosphate solution, pH 7.4, containing 10% (v/v) seminal Spermatozoa (Sp), potassium plasma and 2 mg/ml D-glucose. cyanide (Cy) and gossypol (Go), each to a final concentration of 80 x lo6 cell/ml, 0.9 mM and 3.2 x 10-5 M,correspondingly, The values against the were added in the designated sequence. slope represent the rate of respiration in nmole 02/hour/10* sperm.
1982 VOL. 25 NO. 6
651
CONTRACEPTION
C
700
“rnOlL¶~ 02
Fi4 . 2.
I
Effect of gossypol on spermatozoa1 respiration in the presence Unwashed spermatozoa were diluted with isotonic of oligomycin. Tris-Cl buffer, pH 7.0, containing 10% (v/v) seminal plasma. Assay conditions were identical to that described in Fig. 1, except oligomycin (01) at 1 n /ml and gossypol (Go) at 3.2 x 10V5 M and (Go') at 3.2 x lo-& M,correspondingly,were used.
inhibition on 3.2 x 10m5 M ossypol-stimulated respiration ( nly slightly 8 from 423.0 nmole 02/hour/lO g sperm to 372.1 nmole 02/hour/lO sperm). Furthermore, gossypol at a higher concentration, 3.2 x low4 M (ten-fold higher), completely abolished the oligomycin-insensitive respiration This is a feature commonly observed for (probably state 4 respiration). a common uncoupler of respiratory chain and oxidative phosphorylation (11). In intact spermatozoa, as the midpiece mitochondria are well protected by a plasma membrane, the exogenous substrates added to the assay medium are not freely accessible to the mitochondria (12). The availability of the substrate thus depends heavily on the permeability Any reagent that can alter of the substrate through the plasma membrane. the membrane permeability may subsequently lead to a change in the
652
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION In addition to this, the seminal plasma, which is respiratory rate. supplemented to the assay medium for studies, contains undefined amounts of various substrates such as fructose, lactate and citrate (13). The respiratory stimulation brought about by gossypol additions might even The be a specific effect on the membrane permeability of the substrate. uncoupling effect of gossypol was further examined in the hypotonicallytreated spermatozoa in which the mitochondria are exposed to the assay medium (10,14). The assay medium contained 2 mM sodium arsenite, a system where pyruvate oxidation has been intentionally inhibited (10). Extramitochondrial reducing equivalent (15), in the form of NADH, was supplied to the mitochondria to enable either the lactate-pyruvate shuttle or the malate-aspartate shuttle to operate (Fig. 3, A and B). The respiration of the hypotonically-treated spermatozoa in the presence of various substrates from these two shuttles was inhibited by oligomycin and was relieved by 3.2 x low5 M gossypol.
Fig. 3.
Effect of gossypol on hypotonically-treated spermatozoa1 Hypotonically-treated respiration using various substrates. spermatozoa (Sp), 180 x lo6 sperm/ml; oligomycin (Ol), 1 pg/ml; gossypol (Go), 3.2 x 10e5 M; lactate (La), 1 mM; malate (Ma), 1 mM; glutamate (Gl), 5 mM; and arsenite (Ar), 2 mM; were added accordingly in the designated sequence.
DISCUSSION Oligomycin is an inhibitor of oxidative phosphorylation. Its presence prevents the ATP-forming mechanism from utilizing the high-energy interGossypol relieves the mediate or state generated by electron transport. oligomycin-inhibited respiration but not the cyanide-inhibited respiration. This observation is consistent with the proven nature of gossypol as a
JUNE
1982 VOL. 25 NO. 6
653
CONTRACEPTION classical uncoupler. In hypotonically-treatedspermatozoa, an essentially similar uncoupling pattern is observed. The present study shows that gossypol uncouples the respiratory chain from oxidative phosphorylation in mature, ejaculated boar spermatozoa. This uncoupling, as compared to other reported observations (6), appears to be neither animal-specific nor tissue-specific. Simultaneously, gossypol also inhibits many other biochemical reactions such as the gonadspecificlactate dehydrogenase isozyme, LDH-X (7) and the testosterone production by Leydig cells (16). This suggests that the uncoupling action of gossypol may not be the primary cause of contraception. It does not rule out, howeverthe possibility that a reduction in energy supply especially in the early developmental process might be vital to spermatogenesis. Moreover, if the antifertility effect of gossypol relies not on its uncoupling action, an ideal gossypol-like male contraceptive canhen be designed which has no uncoupling properties.
ACKNOWLEDGMENT Thanks are due to the Agriculture and Fisheries Department of Hong Kong for supplying boar semen samples and to Mrs. .I.Hill for secretarial assistance. The research was partially supported by a grant from the Chinese Medicinal Material Research Centre of The Chinese University of Hong Kong.
REFERENCES 1. Wang, Y., Luo, Y. and Tang, X. Studies on the antifertility actions of cottonseed meal and gossypol. Acta. Pharm. Sinica -14:662-669 (1979). 2.
Dai, R.X. and Dong, R.H. Studies on antifertility effect of gossypol. I. An experimental analysis by epididymal ligature. Acta Biol. Exp. Sinica -11:15-22 (1978).
3.
Dai, R.X., Pang, S.N., Lin, X.K., Ke, U.B., Lui, Z.L. and Gong, R.H. A study of antifertility of cottonseed. Acta Biol. Exp. Sinica -ll:l-10 (1978).
4.
National coordinating group on male antifertility agents. Gossypol - a new antifertility agent for males. Chinese Med. J. -58:455458 (1978).
5.
Tso, W.-W. and Lee, C.S. Effect of gossypol on boar spermatozoa in vitro. Arch. Androl. 1:85-88 (1981).
6.
Abou-Donia, M.B. and Dieckert, J.W. Gossypol: Uncoupling of respiratory chain and oxidative phosphorylation. Life Sci. -14:19551963 (1974).
7.
Tso, W.-W. and Lee, C.-S. Lactate dehydrogenase-X: an isozyme particularly sensitive to gossypol inhibition. Int. J. Androl. (in press).
6.54
JUNE
1982 VOL. 25 NO. 6
CONTRACEPTION
8.
Blanco, A. and Zinkham, W.H. Science -139:601 (1963).
9.
Goldberg, E. Lactate and malic dehydrogenases in human spermatozoa. Science -139:602 (1963).
Lactate dehydrogenase in human testes.
10.
Calvin, .I.and Tubbs, P.K. Mitochondrial transport processes and oxidation of NADH by hypotonically-treated boar spermatozoa. Eur. J. Biochem. -89:315-320 (1978).
11.
Heytler, P.G. Uncouplers of oxidative phosphorylation. In: Methods in Enzymology (Ed.: Fleischer, S. and Packer, L.). Academic Press, New York, pp. 462-472 (1979).
12.
Fawcett, D.W. The structure of the spermatozoa. In: Frontiers in Reproduction and Fertility Control (Ed.: Greep, R.O. and Koblinsky, M.A.). The MIT Press, Cambridge, Massachusetts, pp. 353-378 (1977).
13.
Mann, 'I. Physiology of semen and of the male reproductive tract. In: Reproduction in Domestic Animals, 2nd ed (Ed.: Cole, H.H. and Cupps, P.T.). Academic Press, New York, pp. 277-312 (1969).
14.
Keyhani, E. and Storey, B.T. Energy conservation capacity and morphological integrity of mitochondria in hypotonically-treated rabbit epididymal spermatozoa. Biochim. Biophys. Acta -305:557-569 (1973).
15.
Dawson, A.G. Oxidation of cytosolic NADH formed during aerobic metabolism in mammalian cells. Trends Biochem. Sci. -4:171-176 (1979).
16.
Hadley, M.A., Lin, Y.C. and Dym, M. Effects of gossypol on the reproductive system of male rats. J. Androl. 7:190-199 (1981).
JUNE 1982 VOL. 25 NO. 6
655