I N T E R N A T I O N A L J O U R N A L O F A N D R O L O G Y

8 (1985) 119-127

Department of Biochemistly, The University ofSydney, Sydney, Australia

The anti-glycolytic action of (S)-achlorohydrin

on epididymal bovine spermatozoa in vitro

BY

A. R. Jones and J. I. du Toit

(S)-a-Chlorohydrin interferes with glycolysis in bovine spermatozoa whereas the (R)-isomer is ineffective. The action of the (S)-isomer, which involves inhibition of the reaction catalysed by glyceraldehyde 3-phosphate dehydro- genase, is not immediate but is evident only after a brief period of incubation with the spermatozoa. This inhibitory action is prevented when glycerol is present suggesting that the mechanism of action of (S)-a-chlorohydrin requires its oxidation to (S)-3-~hlorolactaldehyde which is the active metabolite. Addition of racemic 3-chlorolactaldehyde to bovine spermatozoa caused immediate inhibition of glycolysis. It is proposed that the action of (S)-a- chlorohydrin in bovine spermatozoa is similar to that observed in the sperma- tozoa of other species in being a two-stage process; first, its oxidation to (S)-3-~hlorolactaldehyde, and then inhibition of the glycolytic enzyme by this metabolite.

Key wmds: (S)-a-chlorohydrin - (R,S)-3-~hlorolactaldehyde - metabolic inhibi- tion - bovine spermatozoa.

(S)-a-Chlorohydrin (I, Fig. I), the active isomer of (R,S)-a-chlorohydrin (Stevenson &Jones 1982), has an antifertility action which involves the inhibition of glycolysis in mature spermatozoa of the boar (Hutton et al. 1980), ram (Brown-Woodman et d. 1978), rhesus monkey, rat, hamster and man (Ford et al. 1979). This decreases the glycolytic flux by affecting the activity of glyceraldehyde 3-phosphate dehydro- genase (EC 1.2.1.12) (see Jones 1983), and the spermatozoa are, therefore, unable to synthesize the ATP necessary to maintain motility. The mechanism of this action

Received on November 16th, 1984.

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CH20H CHO I I

CHO I

Fig. 1. The structures of (S)-a-chlorohydrin (I), its active metabolite (S)-3-~hlorolactaldehyde (11)

and the substrate for the inhibited enzyme (R)-glyceraldehyde-3-phosphate (111).

involves a specific metabolite of (S)-a-chlorohydrin which has been identified in boar and rat spermatozoa (Jones et al. 1981) as (S)-3-~hlorolactaldehyde (11, Fig. 1). A detailed study of the production of this metabolite together with a proposal for its inhibitory effect on glycolysis has been made using boar spermatozoa in vitro (Stevenson &Jones 1982; Jones & Stevenson 1983). This has been shown to involve, firstly, production of the metabolite within the spermatozoa by an NADPC- dependent dehydrogenase and, secondly, its inhibition of glyceraldehyde 3-phos- phate dehydrogenase due to its stereochemical similarity to the substrate for the enzyme, (R)-glyceraldehyde-3-phosphate (111, Fig. 1). Further work with rabbit (Ford & Jones 1983) and guinea pig spermatozoa (Jones & Ford 1984) had indicated that this two-stage process could apply to spermatozoa in general.

It has recently been reported that (R,S)-a-chlorohydrin inhibits the oxidative metabolism of glucose by bovine spermatozoa (Ford & Waites 1982). This brief study, which did not investigate the mechanism of the inhibitory action by the racemic mixture, was performed on samples of ejaculated bovine spermatozoa which had narrowly failed to meet the UK standards imposed for freezing for commercial use in artificial insemination. In th is paper we describe the action of (S)-a-chlorohydrin on epididymal bovine spermatozoa.

Materials and Methods

(R)- and (S)-a-Chlorohydrin (Porter & Jones 1982) and (R,S)-3-~hlorolactaldehyde (Williams et al. 1960) were synthesized by established procedures. The compounds were homogenous according to TLC analysis and had melting and boiling points that corresponded to values in the literature. Enzymes, substrates and co-factors were purchased from Sigha Chemical Co. (St. Louis Missouri USA), and all other chemicals were of analytical grade. Radioactive substrates were obtained from Amersham International plc (Amersham, UK).

For each experiment, entire testis-epididymis complexes were obtained from

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groups of 6-8 mature bulls (Bos taurus, Hereford Crossbreeds) within 2 h of slaughter at Riverstone abattoir, near Sydney. Spermatozoa were flushed from the cauda epididymides with modified phosphate-buffered saline (PBS) (Stevenson & Jones 1982) at 34°C. The suspension of spermatozoa was washed twice with PBS as previously described (Hutton et al. 1980) to lower the content of endogenous substrates, and finally suspended in PBS to give a concentration of about 0.5 ml packed spermatozoa in 10 ml suspension; when examined under the microscope the spermatozoa were seen to be motile. Aliquots (1.0 ml), containing approxi- mately 8-15 mg protein/ml (about lo7 spermatozoa/ml), were added to conven- tional Warburg flasks containing PBS (0.9 ml). To each flask was added 50 nCi (50 pl) of radioactive substrate {D-[U-14C]fructose (1 mM), [U-14C]pyruvate (2 mM),

L-[U-14C]lactate (2 m ~ ) , [U-14C]glycerol (2 a) or [U-14C]glycerol-3-phosphate (2 mM)} and 50 pl of the test compound {(R)- or (S)-a-chlorohydrin or (R,S)-3- chlorolactaldehyde, as solutions in distilled water and at concentrations indicated in the text}, to give a final volume of 2.0 ml. The sealed flasks were incubated for 1 h at 34°C at 120 oscillations/min. The centre well of each Warburg flask contained 0.1 m12 M NaOH to trap metabolic l4CO2. Incubations were terminated by the addition of 0.2 ml 3 M HC104 to each flask and the l4CO2, trapped in the centre well as Naz I4CO3, was estimated by liquid scintillation counting as described by Dawson (1977). The degrees of inhibition were determined by comparing the amounts of I4CO2 produced in the presence and absence of the various inhibitors. The absolute rates of metabolism of D-[U-14C]fructose, [U-14C]pyruvate, L-[LV4C]lactate, [U-14C]- glycerol and [U-14C]glycerol-3-phosphate by bovine spermatozoa were determined to be 10.5 k 2.1, 2.9 f 0.09, 13.5 k 2.9, 3.8 f 0.06 and 41.7 f 3.1 nmoles/h/mg protein, respectively (mean +_ SEM).

Deproteinized and neutralized supernatant solutions, derived from the incuba- tions by the method of Stevenson & Jones (1982), were used for the assay of metabolic intermediates. Established procedures were employed for the assay of fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde 3- phosphate (Michael & Beutler 1974) and lactate (Hohorst 1963). Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard, and the results calculated with the linear transform equation of CoaMey & James (1978). Results are given as the mean f SEM of 4 separate experiments, each experiment having its own zero time and control values.

Results

In the presence of 50 m~ (S)-a-chlorohydnn, the oxidation of [U-14C]pyruvate (2 mM) or L-[U-14C]lactate ( 2 m ) to 14C02 by bovine spermatozoa was not inhibited (Table 1). In fact there were increases in their degrees of oxidation

12 1

Concentrations of (S)-a-chlorohydrin (mM)

Substrate

Pyruvate Lactate Fructose Fructose + glycerol* (10 mM) Glycerol . Glycerol-3-phosphate Glycerol-3-phosphate +

glycerol* (10 mM)

COZ produced (% of control)

50 50

0.2 0.2 0.2 0.2

0.2

214 i 39 129 ? 5.8 50 ? 5.2

101 k 16 87 +_ 6.5 57 f 5.5

99 f 2.1 ~ ~-

Values are mean f SEM of 4 separate experiments. * = Non radioactive.

0 1 0 0.1 0.2 0.3 0.4 0.5

Concentrotion of (S)-a-chlorohydrin (mM)

I 4

0 30 60 90

Incubation time (min)

Fig. 2. The effect of @)-a-chlorohydrin on the oxidation of D-[U-14C]fructose to 14C02 by bovine spermatozoa during incubation at 34°C. A: The response to increasing concentrations of

(S)-a-chlorohydrin over 1 h. B: The effect of 0.2 mM(S)-a-chlorohydrin over 1.5 h.

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100, A

' Concentrotion of (S)-a-chlorohydrin (mM)

Fig. 3 . The effect of various concentrations of (S)-a-chlorohydrin on the amounts of glycolytic intermediates and lactate in bovine spermatozoa incubated for 1 h at 34°C. A: The decrease in concentration of endogenous lactate. B : The increase in concentrations of fructose-1,6- bisphosphate (-0-0-), dihydroxyacetone phosphate (-A-A-) and glyceraldehyde-3-

phosphate (-B-B-).

showing that the presence of (S)-a-chlorohydrin increased their value as metabolic fuels. With D-[U-14C]fructose as the substrate, the presence of low concentrations of (S)-a-chlorohydrin strongly inhibited its oxidation to 14C02, a concentration of 0.2 m~ causing almost 50% inhibition in 1 h (Fig. 2A). When the effect of this concentration of (S)-a-chlorohydrin on the oxidation of D-[U-14C]fructose to I4CO2 was examined over a period of 1.5 h, the degree of inhibition increased as the incubation time was increased (Fig. IB). After the shortest period of incubation that was practicable (2.5 min), (S)-a-chlorohydrin had no effect on the oxidation of D-[U-14C]fructose. When spermatozoa were incubated in the presence of increasing amounts of (S)-a-chlorohydrin and with D-[U-14C]fructose (1 m ~ ) as substrate, there was a decrease in the concentrations of endogenous lactate (Fig. 3A), with a concomitant increase in the amounts of fructose-l,6-bisphosphatc, dihydroxy- acetone phosphate and glyceraldehyde-3-phosphate (Fig. 3B). These results are compatible with the inhibition by (S)-a-chlorohydrin of glyceraldehyde 3-phosphate dehydrogenase, as has been observed in other species (Jones 1983). At a concentra- tion of l o w , (R)-a-chlorohydrin had no significant effect on any of these parameters (data not shown).

The effect of (S)-a-chlorohydrin (0.2 a) on the oxidation of several [U- 14C]labelled substrates to 14C02 is shown in Table 1. Addition of unlabelled glycerol

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100

80 - 0

: 't; 60

c

20 -0 0

0

0

1 40

0" " 20

0 0 10 20 30

Incubation time (rnin)

Fig. 4. The effect of (R,S)-3-~hlorolactaldehyde (5 m M) on the oxidation of D-[U-14C]fructose to I4CO2 by bovine spermatozoa incubated 34°C. Values are the mean of two experiments only.

(10 111~) to incubations in which D-[U-'4C]fructose (1 rrii~). or [U-14C]glycerol-3- phosphate (2 111~) were the substrates prevented the inhibitory action of 6)-a- chlorohydrin. Over 1 h the oxidation of [U-14C]glycerol (2 m ~ ) to 14COz was inhibited by only about 13% when (S)-a-chlorohydrin and the substrate were both added at zero time. However, when spermatozoa were pre-incubated with @)-a- chlorohydrin (0.2 m) for 5 rnin before addition of the [U-14C]glycerol, the production of 14C02 over the following hour was decreased from 87.6 f 6.5 to 54 k 13% of control values (n = 4). Similarly, incubation of spermatozoa with (S)-a-chlorohydrin (0.2 mbf) for 5 min before addition of [U-14C]glycerol-3-phos- phate (2 m ~ ) as the substrate, caused the production of 14C02 to be decreased from 57 k 5.5% (concomitant addition) to 22 f 16% (pre-incubation) (n = 4).

In the presence of (R,S)-3-~hlorolactaldehyde (5 m) the production of 14C02 from D-[U-14C]fmctose was severely inhibited (Fig. 4). After the shortest practic- able period of incubation (2.5 rnin), this inhibition was 60% of control values which contrasts with the ineffectiveness of (S)-a-chlorohydrin on this process after the same period of time (see Fig. 2).

Discussion

Low concentrations of (S)-a-chlorohydrin inhibited the oxidative metabolism of fructose to COz by bovine spermatozoa. As there was no inhibitory effect on the oxidative metabolism of either lactate or pyruvate, the site of inhibition is within the

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glycolytic pathway beyond the entry of fructose and before the production of pyruvate. Accumulation of the glycolytic intermediates fructose- 1,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate is compatible with the inhibition of glyceraldehyde 3-phosphate dehydrogenase. The bull, therefore, becomes the fourth species to be identified in which the (S)-isomer of a-chloro- hydrin exerts a specific antiglycolytic action on mature spermatozoa in vitro by inhibition of this particular enzyme, the other susceptible species being the ram (Ford et al. 1977), boar (Stevenson &Jones 1982) and guinea pig (Jones & Ford 1984). Furthermore, the metabolic activity of bovine spermatozoa is unaffected by (R)-a-chlorohydrin which agrees with results obtained with spermatozoa from the boar (Stevenson & Jones 1982), rabbit (Ford &Jones 1983) and guinea pig (Jones & Ford 1984).

The effect of (S)-a-chlorohydrin on boar spermatozoa is not immediate, an incubation period of 20 min being required before an inhibitory action was noticeable (Stevenson &Jones 1982). This pre-incubation time was shown to be due to the conversion of (S)-a-chlorohydrin within the spermatozoa to the active inhibitory metabolite, (S)-3-~hlorolactaldehyde (11, Fig. 1). In the present study bovine spermatozoa have also been shown to require a period of pre-incubation before a response to the action of (S)-a-chlorohydrin was observed, although this time was much shorter than for the boar, being somewhat less than 5 min. While this indicates that bovine spermatozoa are more responsive than boar spermatozoa to the action of (S)-a-chlorohydrin, it does not prove that the metabolite is being produced. Direct evidence for its formation could not be obtained due to the supply of bovine spermatozoa becoming unavailable. However, the results of several experiments suggest that the aldehyde is the inhibitory metabolite.

Addition of (R,S)-3-~hlorolactaldehyde (a synthesis of the (S)-isomer has not been achieved) to incubations of bovine spermatozoa caused an immediate inhibition of the oxidation of fructose to COz. There was no evidence of a delay in this inhibitory activity, and the compound had no effect on the oxidation of either pyruvate or lactate. Further evidence for the involvement of (S)-3-~hlorolactaldehyde as the active metabolite was derived from a series of metabolic studies. The formation of the aldehyde by boar spermatozoa involves an enzyme that converts glycerol to glyceraldehyde (Jones & Stevenson 1983). The production of the aldehyde from (S)-a-chlorohydrin, therefore, was prevented when glycerol was added to incubates in which fructose or glycerol-3-phosphate were substrates or when glycerol itself was the substrate. A similar sequence of events is presumed to occur within bovine spermatozoa (Table 1) which leads us to propose that a similar metabolic trans- formation is necessary and that (S)-3-chlorolactaldehyde is the inhibitory meta- bolite.

The proposal for the mechanism of action of (S)-a-chlorohydrin in bovine spermatozoa is shown in Fig. 5. (S)-a-Chlorohydrin is converted into (S)-3-chloro- lactaldehyde by an enzyme which converts glycerol into glyceraldehyde. This would

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glycerol ~ di hydroxyacetone fructose 3-phosphate phosphate

glyceraldehyde - glyceraldehyde 1 A frurtose

t

3-phosphate 1.6-bisphosphate glycerol

/I u n

S-3-chloro- INHIWION S-a-chlorohydrin lactaldeh yde T

1,3-bisphospho- - - - Co2 glycerate

Fig. 5. Proposed pathway for the production of (S)-3-chlorolactaldehyde from (S)-a-chlorohydnn by

bovine spermatozoa and its effect upon the glycolytic pathway.

explain the apparent ‘protective’ effect exhibited by the presence of glycerol in the metabolic studies. The active metabolite then inhibits glyceraldehyde 3-phosphate dehydrogenase resulting in the accumulation of fructose- 1,6-bisphosphate and the triose phosphates.

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia. The co-operation of the Commonwealth Meat Inspectors, especially Des Clegg, at Riverstone abattoir was much appreciated.

References

Brown-Woodman P D C, Mohri H, Mohri T , Suter D & White D (1978): Mode of action of a-chlorohydrin as a male antifertility agent. Inhibition of the metabolism of ram spermato- zoa by a-chlorohydrin and location of block in glycolysis. Biochem J 170: 23.

Coakley W T &James C J (1978): A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mgiml. Anal Biochem 85: 90.

Dawson A G (1977): Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules. Biochim Biophys Acta 499: 85.

Ford S A &Jones A R (1983): The effect of a-chlorohydrin on the oxidation of fructose by rabbit spermatozoa. Contraception 28: 565.

Ford W C L, Harrison A & Waites G M H (1977): Effects of the optical isomers of a-chlorohydrin on glycolysis by ram testicular spermatozoa and the fertility of male rats. J Reprod Fertil5 1 : 105.

Ford W C L, Harrison A, Takkar G L & Waites G M H (1979): Inhibition of glucose catabolism in rat, hamster, rhesus monkey and human spermatozoa by a-chlorohydrin. Int J Androl2: 275.

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Ford W C L & Waites G M H (1982): Activities of various 6-chloro-6-deoxysugars and (S)-a-chlorohydrin in producing spermatocoeles in rats and paralysis in mice and in inhibiting glucose metabolism in bull spermatozoa in vitro. J Reprod Fertil65: 177.

Hohorst H-J (1963): L(+)Lactate. Determination with lactate dehydrogenase and DPN. In: Bergmeyer H U (ed). Methods of Enzymatic Analysis, p 266. Academic Press, New York.

Hutton P, Dawson A G & Jones A R (1980): Inhibition of glycolysis in boar sperm by a-chlorohydrin. Contraception 22: 505.

Jones A R (1983): Antifertility actions of a-chlorohydrin in the male. Aust J Biol Sci 36: 333. Jones A R & Ford S A (1984): The action of (S)-a-chlorohydrin and 6-chloro-6-deoxyglucose

on the metabolism of guinea pig spermatozoa. Contraception 30: 261. Jones A R & Stevenson D (1983): Formation of the active antifertility metabolite of

(S)-a-chlorohydrin in boar sperm. Experientia 39: 784. Jones A R, Stevenson D, Hutton P & Dawson A G (1981): The antifertility action of

a-chlorohydrin: metabolism by rat and boar sperm. Experientia 37: 340. Lowry 0 H, Rosebrough N J, Farr A L & Randall R J (1951): Protein measurement with the

Folin reagent. J Biol Chem 193: 265. Michal G & Beutler H-0 (1974) : D-Fructose- 1,6-diphosphate, dihydroxyacetone phosphate

and D-glyceraldehyde-3-phosphate. In: Bergmeyer H U (ed). Methods of Enzymatic Analysis. Vol3: 13 14. Academic Press, New York.

Porter K E &Jones A R (1982): The effect of the isomers of a-chlorohydrin and racemic P-chlorolactate on the rat kidney. Chem-Biol Interact 41 : 95.

Stevenson D &Jones A R (1982): Inhibition of fructolysis in boar spermatozoa by the male antifertility agent (S)-a-chlorohydrin. Aust J Biol Sci 35: 595.

Williams P H, Payne G B, Sullivan W J & Van Ess P R (1960): Chemistry of glycidaldehyde. J Am Chem SOC 82: 4883.

Author’s address: A. R. Jones, Department of Biochemistry, The University of Sydney, Sydney, NSW 2006, Australia.

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