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Summary. Ram cauda epididymal spermatozoa were incubated for 10 min at 34°C with or without 1·0 mm-RS-α-chlorohydrin before (1) 5 mm-d-glucose or (2) 10 mm-l-lactate plus 1 mm pyruvate or (3) 5 mm-d-glucose plus 10 mm-l-lactate plus 1 mm-pyruvate or (4) no substrate was added. Without α-chlorohydrin, the motility, the ATP concentration and the energy charge of the spermatozoa were maintained for 240 min by substrate combinations 1–3 but with no added substrate (4) the motility declined after 60 min. All the values decreased dramatically after 10 min in spermatozoa exposed to α-chlorohydrin in substrate conditions 1 and 3 (glucose present) but α-chlorohydrin had no significant effect in conditions 2 and 4 (no glucose) except after prolonged incubation. In a dose—response experiment glucose-dependent ATP dissipation began to occur with 0·025 mm-rs-α-chlorohydrin. A similar effect was seen in boar spermatozoa exposed to 0·1–5·0 mm-α-chlorohydrin and 5 mm-d-glucose. With boar spermatozoa the presence of 10 mm-l-lactate and 1 mm-pyruvate as well as glucose prevented the loss of ATP.

We conclude that this concerted action of α-chlorohydrin and glucose is probably responsible for the contraceptive action of α-chlorohydrin and propose that it may depend on 'futile substrate cycling' in the glycolytic pathway.

Summary. Washed human spermatozoa had an endogenous oxygen uptake of 2·14 ± 0·17 nmol O₂/10⁸ spermatozoa/min (mean ± s.e.m., n = 35) which was stimulated by succinate (V _max = 9·64 ± 0·44 nmol O₂/10⁸ spermatozoa/min) but not by other substrates. The ATP concentration in freshly washed spermatozoa was 12·18 ± 0·54 (s.e.m.) nmol/10⁸ spermatozoa (n = 26) and was maintained for 2 h in the presence of 2 mm-d-glucose but fell to 9·56 ± 0·73 (s.e.m.) nmol/10⁸ spermatozoa (n = 13) in its absence. The presence of 2 μm-antimycin A, 2 μm-rotenone, 0·4 μm-carbonyl cyanide m-chlorophenyl hydrazone or 8 μm-oligomycin caused the ATP concentration to fall to <2 nmol/10⁸ spermatozoa but their effect was partly alleviated by 2 mm-glucose. Sodium malonate (5 mm) prevented the stimulation of respiration by succinate but had no effect on the ATP concentration of the spermatozoa or their ability to produce ¹⁴CO₂ from [U-¹⁴C]glucose.

The least active of the tricarboxylic acid cycle enzymes was 2-oxoglutarate dehydrogenase (EC 1.2.4.2) (3·1 ± 0·6 (s.e.m.) nmol substrate transformed/10⁸ spermatozoa/h (n = 4). Cytochrome c oxidase (EC 1.9.3.1) was much less active than in rat spermatozoa (22·3 ± 6·0 (s.e.m., n = 4) and 615 ± 87 (n = 4) nmol transformed/10⁸ spermatozoa/min).

It is concluded that human spermatozoa can obtain ATP by the respiration of endogenous substrate but the substrates and metabolic pathways involved remain obscure.

Summary. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (mUnits/10⁶ spermatozoa: mean ± s.e.m., N = 12) in spermatozoa from the rat epididymis declined from 22·0 ± 1·4 in the caput to 14·1 ± 1·3 in the corpus region but there was no further decrease in the cauda region. In hamsters (N = 4), GAPDH activity in spermatozoa declined from 24·8 ± 2·2 in the caput to 15·2 ± 1 ·2 in the distal cauda epididymidis with the greatest decrease between the corpus and proximal cauda regions. In guinea-pigs (N = 4) GAPDH activity in spermatozoa increased from 11·4 ± 0·79 in the caput to 18·0 ± 0·7 in the corpus and cauda regions of the epididymis. The activity of GAPDH in spermatozoa therefore changes during maturation in a species dependent manner.

GAPDH in spermatozoa from the distal cauda epididymidis of rats given α-chlorohydrin (4, 8 or 25 mg/kg/day by mouth) or 6-chloro-6-deoxyglucose (24 or 96 mg/kg/day by mouth) for 10 days was inhibited by > 80% but was only inhibited by 25– 45% in spermatozoa from the caput epididymidis. The enzyme was inhibited to an intermediate and dose-dependent extent in spermatozoa from the corpus region. A similar pattern of inhibition was seen in spermatozoa from hamsters given α-chlorohydrin (50 or 100 mg/kg/day) for 10 days. α-Chlorohydrin (66 mg/kg/day s.c.) for 10 days inhibited GAPDH in spermatozoa from the caput or corpus epididymidis of the guinea-pig by <20% but decreased GAPDH activity by almost 90% in the cauda region. In rats the greater effect of α-chlorohydrin on spermatozoa from the cauda region of the epididymis occurred even after short periods of treatment or when the passage of spermatozoa through the duct was interrupted by a ligature around the corpus region, indicating that the effect is not simply a reflection of the length of time the spermatozoa have spent in the epididymis. It is concluded that either spermatozoa undergo a maturational change which increases their sensitivity to α-chlorohydrin or that α-chlorohydrin (or an active metabolite) is concentrated in the lumen of the cauda epididymidis.

Summary. [U-¹⁴C]Sucrose and d-[1-¹⁴C]mannitol were used to determine the tritiated water space of human spermatozoa to validate these compounds as markers for the extracellular space. Calculations based on 0·03 mm-[U-¹⁴C]sucrose gave a negative water space. The water space estimated with 0·03 mm-d-[1-¹⁴C]mannitol was unstable but a stable result was obtained with 0·3 mm-d-[1-¹⁴C]mannitol in incubations up to 2 h. The mean water space was 2·21 ± 0·106 μ1/10⁸ spermatozoa (mean ± s.e.m. for 6 batches of pooled semen). The water space was decreased or abolished by Triton X-100, cold shock, sonication or hypotonic treatment. The water space responded to changes in the osmolarity of the medium by increasing in dilute media. It is concluded that mannitol is an effective extracellular marker for human spermatozoa if concentrations ≥ 0·3 mm are used.

When the kinetics of the uptake of 2-deoxyglucose by the spermatozoa were studied by using mannitol as an extracellular marker, the transport was saturable and was inhibited by cytochalasin B. The K _m was 1·6 ± 0·33 mm and the V _max was 4·2 ± 0·52 nmol/10⁸ spermatozoa/10 sec (mean ± s.e.m., n=4).

Summary. Spermatozoa from the cauda epididymidis of the rhesus monkey had metabolic properties similar to those published for ejaculated spermatozoa. The concentration of glycolytic intermediates was low until 2 mm-glucose was added; glucose 6-phosphate, fructose 1,6-bis-phosphate and the triose phosphates then increased but glycerate 3-phosphate did not. It was concluded that the activity of glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) limited flux through the glycolytic pathway. The production of lactate and CO₂ from glucose was strongly inhibited in the presence of 5 or 10 mm-α-chlorohydrin. The energy charge of the spermatozoa was low before and after 1 h incubation with 2 mM-glucose (0·5 ± 0·05 and 0·05 ±0·06 respectively) and the value after the incubation was decreased in the presence of 5 or 10 mm-α-chlorohydrin (0·17 ± 0·05 and 0·15 ± 0·04 respectively). α-chlorohydrin inhibited glycolysis at the glyceraldehyde 3-phosphate dehydrogenase reaction.

Summary. Spermatozoa from rats treated with 6-chloro-6-deoxyglucose (120 μmol/kg/day) were as motile as those from controls soon after dilution but rapidly became immotile when 2 mm-d-glucose was the only substrate present. Control spermatozoa were more motile when 2 mm-pyruvate + 4 mm-dl-lactate rather than 2 mm-d-glucose was present and the former substrate allowed spermatozoa from treated rats to remain motile.

The concentrations of the adenine nucleotides declined rapidly during 40 min incubations when the substrate was 2 mm-d-glucose. The ATP/ADP ratio and the energy charge (ATP + ½ADP/ATP + ADP + AMP) were maintained in spermatozoa from control rats but not in those from treated rats. When the substrate was 2 mm-pyruvate + 4 mm-dl-lactate adenine nucleotides were not lost as rapidly and there was less difference between spermatozoa from control and treated rats.

Summary. When 0·1 mM-S α-chlorohydrin was present in incubations, glycolysis by ram testicular spermatozoa was almost completely inhibited whereas 10 mM-R α-chlorohydrin had no effect. Male rats dosed orally with S α-chlorohydrin (3·25 mg/kg/day) became much less fertile than controls but those dosed with R α-chlorohydrin (13 mg/kg/day) did not. The loss of fertility was associated with a reduced ability of spermatozoa from the cauda epididymidis of these rats to oxidize glucose. It is concluded that the S enantiomer is responsible for both the inhibition of sperm glycolysis and the reduction in fertility caused by the racemic mixture of α-chlorohydrin.

Summary. 6-Chloro-6-deoxyfructose or 6-chloro-6-deoxyglucitol (>90 μmol/kg/ day), 6-chloro-6-deoxyglucose or 6-chloro-6-deoxymannose (>120 μmol/kg/day) and 6-chloro-6-deoxygalactose (>300 μmol/kg/day) all had an antifertility action in the male rat when given by mouth. Spermatozoa from the infertile rats were unable to oxidize glucose. This effect was always produced by a lower dose than the antifertility effect and the threshold dose for the 2 effects varied in a parallel fashion between the different 6-chloro-6-deoxysugars. Glucose oxidation appeared to be inhibited at the triose phosphate isomerase or glyceraldehyde 3-phosphate dehydrogenase reaction. These effects of 6-chloro-6-deoxysugars are similar to those of α-chlorohydrin.

The activities of the glycolytic enzymes were greatly in excess of the observed flux through the pathway and high concentrations of glucose 6-phosphate and triose phosphate accumulated in rat spermatozoa incubated with 2 mm-d-glucose.