Mode of action of α-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by α-chlorohydrin and location of block in glycolysis

Abstract

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.

Glycolysis plays an important role in energy transfer from the base to the distal end of the flagellum in mouse sperm

“…In contrast, ACH, which inhibits GAPDH through oxidation in cytoplasm (Mohri et al, 1975;Brown-Woodman et al, 1978;Stevenson and Jones, 1985), caused a significant decrease in the bend angle and sliding velocity even in the presence of respiratory substrates such as pyruvate or BHB (Fig. 1).…”

Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility

“…It remains to be determined whether the germ cell-specific glycolytic enzymes have lower K m values for substrates and͞or less sensitivity to feedback regulation by ATP, leading to higher local ATP concentrations for the sperm dynein ATPases. Another difference between the GAPDH isozymes is that GAPDS appears to be more sensitive than the somatic isozyme to substrate inhibition by the active metabolite of ␣-chlorohydrin and related toxicants (39)(40)(41). Based on its unique features and restricted expression during spermatogenesis, as well as the infertility, lack of progressive sperm motility and low sperm ATP levels of Gapds Ϫ/Ϫ males, GAPDS may be an excellent and highly specific target for male contraceptive strategies.…”

The interaction of -chlorohydrin with glycerol kinase

“…In fact, synthetic a-chlorohydrin phosphate has been shown to be an inhibitor of both sperm glyceraldehyde phosphate dehydrogenase and trióse phosphate isomerase (Mohri et al, 1975;Mashford & Jones, 1978). Moreover, high concentrations of glycerol can overcome the inhibitory effect of a-chlorohydrin on ram spermatozoa (Edwards et al, 1976;Brown-Woodman et al, 1978), suggesting competition between the two compounds for the same pathway. Such competition is supported by the report that a-chlorohydrin is a competitive inhibitor of glycerol kinase prepared from Escherichia coli (Thorner, 1972; see Brooks, Hamilton & Mallek, 1974 (Blackshaw, 1954) and the spermatozoa were separated from seminal plasma by the procedure of Harrison (1976).…”

“…For instance, a period of preincubation of ram spermatozoa with 0-1-1 mM-a-chlorohydrin is required before an inhibition of glycerol metabolism is noted (Edwards, Dacheux & Waites, 1976). Furthermore, the activity of a number of glycolytic enzymes is substantially reduced when measured in ram spermatozoa which have been incubated with 0-03-300 mM-a-chlorohydrin before extraction, yet these same enzymes are not inhibited by the direct addition of a-chlorohydrin to extracts of unincubated spermatozoa (Mohri, Suter, Brown-Woodman, White & Ridley, 1975; Brown-Woodman, Mohri, Mohri, Suter & White, 1978). A number of acrosomal enzymes is also inhibited after, but not before, incubation with 50-120 mM-a-chlorohydrin (Yang & Srivastava, 1976).…”

“…That the inhibition in the spectrophotometric assay was due to an interaction of achlorohydrin with glycerol kinase rather than with the coupling enzyme, glycerolphosphate dehydrogenase, as has been suggested by Brown-Woodman et al (1978), was checked by adding different amounts of the latter enzyme. This resulted in no change in reaction rate, The degree of inhibition of glycerol kinase by a-chlorohydrin (AT, of 30 mM) is considerably less than that noted previously (Brooks, 1979).…”