Inhibition of Fructolysis in Boar Spermatozoa by the Male Antifertility Agent (S)-a-Chlorohydrin

Abstract

The (S)-isomer of the male antifertility agent IX-chlorohydrin strongly inhibited the oxidative metabolism of fructose by boar spermatozoa in vitro. The result of this action, which has been deduced to be an inhibition of glyceraldehydephosphate dehydrogenase, caused an accumulation of fructose-1,6-bisphosphate and the triosephosphates, and a decrease in substrate-level phosphorylation with a concomitant lowering of the energy charge potential of the spermatozoa. The (R)-isomer of IX-chlorohydrin had no inhibitory activity on fructolysis.A study of the comparative metabolism of (R)-[3-36CI]-IX-chlorohydrin and (R,S)-[3-36Cl]_IX_ chlorohydrin by boar spermatozoa showed that it is the (S)-isomer that specifically undergoes a process of oxidative metabolism to (R)-3-chlorolactaldehyde.It is proposed that this endogenous oxidation product, which has the same absolute configuration as the substrate for glyceraldehydephosphate dehydrogenase, is the active metabolite of (S)-IX-chlorohydrin that inhibits this enzyme. Exogenous (R,S)-3-chlorolactaIdehyde inhibited the oxidative metabolism of fructose by boar spermatozoa, apparently by a mechanism similar to that of (S)-IX-chlorohydrin.

Production of (S)-3-chlorolactaldehyde from(S)- -chlorohydrin by boar spermatozoa and the inhibition of glyceraldehyde 3-phosphate dehydrogenase in vitro

“…The (S')-isomer is not the inhibitory compound per se but it undergoes oxidation within mature spermatozoa to an active metabolite, (5)-3-chlorolactaldehyde (77, fig. 1), which possesses the same stereochemistry as (R)-or (D)-glyceraldehyde-3-phosphate, the substrate for the inhibited enzyme (Stevenson & Jones, 1982).…”

Glycerol 3-phosphate dehydrogenase of boar spermatozoa: inhibition by -bromohydrin phosphate

“…The use of 3-chloro-lhydroxypropanone (Jones el al, 1986) and 3-bromopyruvate (Stevenson and Jones, 1985) at 34°C The sealed flasks were transported to the laboratory where the spermatozoa were flushed from the epididymides with PBS (34°C) from incisions made in the third and fourth segments of the cauda according to the classification of Holtz and Smidt (1976). The spermatozoa were washed and prepared as a 10% (v/v) suspension in PBS at 34°C according to our established procedure (Jones and Chantrill, 1989 (Stevenson and Jones, 1982), fructose-l,6-bisphosphate and dihydroxyacetone phosphate (Michal and Beutler, 1975), glycerol 3-phosphate and lactate (Lang, 1985) were performed according to the literature. Protein was estimated (Lowry et al, 1951) using BSA as a standard.…”

Metabolism of glycerol 3-phosphate by mature boar spermatozoa

“…5b). There was no detectable production of glucose 6-phosphate or fructose 6-phosphate in the presence or absence of CHOP, even though CHOP caused the accumulation of fructose-l,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (Jones et al, 1986 (Stevenson and Jones, 1982) and with glycerol and glycerol 3-phosphate (Jones et al, 1992) (Jones and Chantrill, 1989), suggest that boar sper¬ matozoa are reliant on the glycolytic pathway primarily to produce lactate, rather than ATP, and that it is lactate that is the main metabolic fuel for the mitochondrial synthesis of ATP. A similar conclusion has been reached by Calvin and Tubbs (1978) 6-phosphate and fructose 6-phosphate accumulated in the proportions of 2.3:1 after 2 h. The production of these isomers from fructose 1,6-bisphosphate has been reported when boar spermatozoa were incubated with fructose 1,6-bisphosphate as the substrate in the presence of (S)-a-chlorohydrin (Jones and Montague, 1991).…”

“…Chemicals and substrates (Stevenson and Jones, 1985) (Dawson, 1977) accord¬ ing to established procedures (Stevenson and Jones, 1982 (Stevenson and Jones, 1982) were used for the assays of fructose (Beutler, 1984) fructose 1,6-bisphosphate, dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (Michal, 1984a), lactate and glycerol 3-phosphate (Lang, 1984), ATP (Jaworek and Welsch, 1985a), ADP and AMP (Jaworek and Welsch, 1985b), glucose 6-phosphate and fructose 6-phosphate (Michal, 1984b), using a Shimadzu UV-160A spectrophotometer (Shimadzu, Kyoto). The presence of CHOP in incubation solutions neither interfered with any of the assays nor was it a substrate for any of the enzymes used in the estimations.…”

“…Under anaerobic conditions bull and ram spermatozoa, for example, utilize fructose and main¬ tain high motility, which is accompanied by the production of lactate (Mann and Lutwak-Mann, 1981), whereas boar (Aalbers el al, 1961;Nevo et al, 1970) and guinea-pig (Frenkel et al, 1975) spermatozoa exhibit both decreased motility and fruc¬ tose utilization, and do not produce lactate. Boar spermatozoa are extremely selective in the substrates that they oxidize; these comprise glucose, fructose, glycerol, glycerol 3-phosphate, lactate and, to a limited extent, acetate, suggest¬ ing fhat fhe glycolytic pathway is the major route for fhe production of mitochondrial substrates (Stevenson and Jones, 1982;Jones et al, 1992). The rapid oxidation of endogenous lactate and the extremely low degree of oxidation of pyruvate by these cells indicates that lactate is the major substrate for mitochondrial metabolism (Jones and Chantrill, 1989).…”