Washed human spermatozoa had an endogenous oxygen uptake of 2.14 +/- 0.17 nmol O2/10(8) spermatozoa/min (mean +/- s.e..m., n = 35) which was stimulated by succinate (Vmax = 9.64 +/- 0.44 nmol O2/10(8) spermatozoa/min) but not by other substrates. The ATP concentration in freshly washed spermatozoa was 12.18 +/- 0.54 (s.e.m.) nmol/10(8) 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/10(8) spermatozoa (n = 13) in its absence. The presence of 2 microM-antimycin A, 2 microM-rotenone, 0.4 microM-carbonyl cyanide m-chlorophenyl hydrazone or 8 microM-oligomycin caused the ATP concentration to fall to less than 2 nmol/10(8) 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/10(8) 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/10(8) 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.
Spermatozoa from the cauda epididymidis of rats treated with RS‐α‐chlorohydrin (10 mg/kg/day p. o. × 7) had the same ATP content as control spermatozoa immediately after collection, but were unable to metabolize glucose in vitro and so their ATP content declined more rapidly than that of control spermatozoa. The in vitro metabolism of glucose by spermatozoa from rat, hamster, rhesus monkey and human was inhibited by 80% or more after 15–30 min preincubation in the presence of RS‐α‐chlorohydrin concentrations of < 1, > 10, 5–10 and > 50 mM, respectively. Inhibition of glucose oxidation was correlated with a reduction in ATP concentration in the spermatozoa. These data support the hypothesis that α‐chlorohydrin acts as an antifer‐tility agent by inhibition of sperm glycolysis but suggest that RS‐α‐chlorohydrin would not be an effective contraceptive in man. However, the glucose metabolism of human spermatozoa was significantly inhibited by < 10 mM of the S‐enantiomer of α‐chlorohydrin.
When 0-1 mM-S alpha-chlorohydrin was present in incubations, glycolysis by ram testicular spermatozoa was almost completely inhibited whereas 10 mM-R alpha-chlorohydrin had no effect. Male rats dosed orally with S alpha-chlorohydrin (3-25 mg/kg/day) became much less fertile than controls but those dosed with R alpha-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 alpha-chlorohydrin.
Ram cauda epididymal spermatozoa were incubated for 10 min at 34 degrees C with or without 1.0 mM-RS-alpha-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 alpha-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 alpha-chlorohydrin in substrate conditions 1 and 3 (glucose present) but alpha-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-alpha-chlorohydrin. A similar effect was seen in boar spermatozoa exposed to 0.1-5.0 mM-alpha-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 alpha-chlorohydrin and glucose is probably responsible for the contraceptive action of alpha-chlorohydrin and propose that it may depend on 'futile substrate cycling' in the glycolytic pathway.
Summary. The activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (mUnits/106 spermatozoa: mean \m=+-\s.e.m., N = 12) in spermatozoa from the rat epididymis declined from 22\m=.\0 \m=+-\ 1\m=.\4in the caput to 14\m=.\1 \m=+-\ 1\m=.\3in the corpus region but there was no further decrease in the cauda region. In hamsters (N = 4), GAPDH activity in spermatozoa declined from 24\m=.\8 \ m=+-\ 2\m=.\2in the caput to 15\m=.\2 \m=+-\1 \ m=. \ 2in 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\m=.\4 \m=+-\0\m=.\79in the caput to 18\m=.\0 \ m=+-\ 0\m=.\7in 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 \ g = a \ \ x = r e q -\ 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\p=n-\ 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 \ g=a\ \ x=r eq-\ chlorohydrin (50 or 100 mg/kg/day) for 10 days. \g=a\-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 \g=a\-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 \g=a\-chlorohydrin or that \g=a\-chlorohydrin(or an active metabolite) is concentrated in the lumen of the cauda epididymidis.
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