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A. R. Jones and L. Gillan

Boar spermatozoa incubated with glycerol 3-phosphate as substrate produced CO2 and an accumulation of dihydroxyacetone phosphate and fructose-1,6-bisphosphate. The rate of oxidation of glycerol 3-phosphate was decreased, as was the production of CO2 and the two glycolytic intermediates, in the presence of (R,S)-α-bromohydrin phosphate. In the presence of inhibitors of stage two of the glycolytic pathway, CO2 production was prevented, there was a marked increase in the concentration of the glycolytic intermediates but the rate of metabolism of the substrate was unaffected. Oxygen consumption by spermatozoa incubated with glycerol 3-phosphate was unaffected in the presence of rotenone, whereas it was decreased when lactate was offered as the substrate. The results reported here confirm that in boar spermatozoa glycerol 3-phosphate dehydrogenase is an FAD-linked enzyme that is inhibited by (R,S)-α-bromohydrin phosphate in, possibly, a competitive manner.

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D. Stevenson and A. R. Jones

Summary. The (S)-isomer of the male antifertility agent α-chlorohydrin was metabolized by mature boar spermatozoa in vitro to (S)-3-chlorolactaldehyde. This oxidative process, which did not occur when (R)-α-chlorohydrin was offered as a substrate, was catalysed by an NADP+-dependent dehydrogenase that converts glycerol to glyceraldehyde. (S)-3-chlorolactaldehyde, produced by this metabolic reaction or when added to suspensions of boar spermatozoa, was a specific inhibitor of glyceraldehyde 3-phosphate dehydrogenase as assessed by the accumulation of fructose 1,6-bisphosphate and the triosephosphates. When glycerol and (S)-α-chlorohydrin were added concomitantly to boar spermatozoa in vitro, the presence of glycerol decreased the degree of inhibition of glyceraldehyde 3-phosphate dehydrogenase. Extracts of glyceraldehyde 3-phosphate dehydrogenase that were obtained from boar spermatozoa incubated with (S)-α-chlorohydrin or (R,S)-3-chlorolactaldehyde showed significant reductions in their enzymic activity.

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A. R. Jones and L. Gillan

Under anaerobic conditions boar spermatozoa metabolized fructose and glucose to lactate but did not produce ATP to the extent of that produced under aerobic conditions; the ketogenic amino acids leucine, tryptophan, phenylalanine and tyrosine were not oxidatively metabolized. Glycerol 3-phosphate was metabolized rapidly in the presence or absence of the glycolytic inhibitor, 3-chloro-1-hydroxypropanone (CHOP). In the absence of CHOP, glycerol 3-phosphate was converted to CO2, lactate, glucose 6-phosphate and fructose 6-phosphate, and ATP was produced. In the presence of CHOP, glycerol 3-phosphate did not produce CO2, lactate or ATP, but formed fructose 1,6-bisphosphate and dihydroxyacetone phosphate. With dihydroxyacetone phosphate as substrate, fructose 1,6-bisphosphate, lactate, glucose 6-phosphate, fructose 6-phosphate and ATP were produced. Accumulation of glucose 6-phosphate and fructose 6-phosphate from glycerol 3-phosphate appeared to depend on the production of ATP; if ATP was not produced, dihydroxyacetone phosphate and fructose 1,6-bisphosphate accumulated. The conversion of glycerol 3-phosphate to glycolytic intermediates appeared to be a mechanism for the conversion of substrates for the ultimate production of lactate.

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A. R. Jones and D. Milmlow

When incubated in the absence of exogenous substrates, washed boar spermatozoa maintained a high energy charge potential (ECP) for at least 5 h. Addition of 3-chloro-1-hydroxypropanone, an inhibitor of triosephosphate isomerase and glyceraldehyde 3-phosphate dehydrogenase, at any time caused the ECP to decline and fructose-1,6-bisphosphate, dihydroxyacetone phosphate and glycerol to accumulate. There appear to be two endogenous substrates that are degraded ultimately to produce the triosephosphates which allow the cells to produce lactate for the mitochondrial synthesis of ATP. One substrate generates minor amounts of glycerol 3-phosphate whereas the other substrate degrades to glycerol and may be di-glycerides, or tri-glycerides, or both.

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R. C. JONES and I. C. A. MARTIN

Summary.

In a factorial experiment skim milk, egg-yolk-citrate and synthetic diluents composed of fructose or lactose, sodium chloride and phosphate buffer containing 3·0% w/v of a lyophilized preparation of non-dialysable solids from (cow) milk were used as diluents for deepfreezing ram spermatozoa and for incubating spermatozoa at 37° C after thawing. All samples of semen were diluted forty-fold before freezing. Egg-yolk-citrate was inferior to skim milk as a diluent for freezing spermatozoa and for incubating spermatozoa after thawing. Of the two synthetic diluents, lactose synthetic was better for freezing spermatozoa whilst fructose synthetic was better for incubating spermatozoa after thawing. Only spermatozoa frozen in the lactose synthetic diluent and resuspended after thawing in the fructose synthetic survived incubation at 37° C for 2 hr as well as spermatozoa frozen and incubated in skim milk diluents.

Two factorial experiments compared egg-yolk-citrate and skim milk as diluents for freezing semen diluted from ten- to forty-fold. At ten-fold dilution, revival was much the same after freezing in milk or egg-yolk-citrate. A twenty- or forty-fold dilution was better than a tenfold dilution in milk, but revival was depressed at these higher dilution rates after freezing in egg-yolk-citrate. When semen was frozen at a tenfold dilution it was advantageous to resuspend spermatozoa in a diluent free of glycerol after thawing. A diluent based on Krebs—Henseleit— Ringer solution containing 0·5% w/v of non-dialysable milk solids was better for incubating spermatozoa after thawing than egg-yolk-citrate or milk.

A period of 5 hr equilibration at 5° C before freezing was better than 30 min equilibration.

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S. J. Cooney and A. R. Jones

Summary. (S)-α-Chlorohydrin and 3-chloro-1-hydroxyacetone inhibited the oxidative metabolism of fructose by boar spermatozoa only after a period of incubation in which they presumably underwent conversion to (S)-3-chlorolactaldehyde, an inhibitor of sperm glyceraldehyde 3-phosphate dehydrogenase. With glycerol as substrate, 3-chloro-1-hydroxyacetone had a similar effect, (S)-α-chlorohydrin was ineffective while (R,S)-3-chlorolactaldehyde was immediately effective with either substrate. All three compounds caused the accumulation of fructose 1,6-bisphosphate and dihydroxyacetone phosphate from fructose but not from glycerol which led to the conclusion that inhibition of triosephosphate isomerase was also associated with the anti-glycolytic action of (S)-3-chlorolactaldehyde. (S)-3-Chlorolactaldehyde caused the depletion of ATP in incubates of boar spermatozoa metabolizing fructose but not glycerol. This suggests that futile substrate cycling may play an important function in the antiglycolytic action of (S)-3-chlorolactaldehyde and/or that boar spermatozoa can synthesize ATP from glycerol by a mechanism not involving the glycolytic pathway.

Keywords: (S)-α-chlorohydrin; (S)-3-chlorolactaldehyde; 3-chloro-1-hydroxyacetone; metabolic inhibition; spermatozoa (boar)

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A Trounson, C Anderiesz and G Jones

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

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R. C. JONES and I. C. A. MARTIN

Summary.

A transmission electron microscope was used to examine samples of ram semen after 30-fold dilution in a buffered glucose solution and storage at either 35° C or 5° C. The appearance of sections of heads and mid-pieces of spermatozoa was systematically scored so that the frequency of occurrence of several changes in ultrastructure in specimens could be compared between treatments.

Incubation of diluted semen at 35° C for 1 or 2 hr did not cause significant changes in acrosomes but a significant proportion of spermatozoa showed condensation of mitochondria or loss of material. Although the acrosomes of spermatozoa in undiluted semen were unaffected by incubation at 35° C for 2 hr, mitochondria had lost material and were scored as 'pale' in 80·5% of spermatozoa; only 13·5% of spermatozoa in the control samples were scored in this category.

Acrosomes and mid-pieces were affected by cooling to 5° C and storage at this temperature for up to 72 hr. The acrosome changes involved swelling and vacuolation or, in a small proportion of spermatozoa, swelling and vesiculation of the outer acrosomal membrane. Mid-pieces showed condensation of mitochondria and loss of material. The inclusion of 3% (v/v) egg yolk in the diluent reduced the frequency of these changes.

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A. R. Jones, L. A. Chantrill and A. Cokinakis

Summary. Mature boar spermatozoa oxidized glycerol to carbon dioxide in the absence of any detectable activity of glycerol kinase. With triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase inhibited by the presence of 3-chloro-1-hydroxypropanone (CHOP), dihydroxyacetone phosphate accumulated in incubates when glycerol-3-phosphate was the substrate, but not when it was glycerol. Both dihydroxyacetone and glyceraldehyde could be used as substrates; in the presence of CHOP, dihydroxyacetone phosphate and fructose-1,6-bisphosphate accumulated when dihydroxyacetone was the substrate, but not when it was glyceraldehyde. The metabolic pathways glycerol→glyceraldehyde→glyceraldehyde 3-phosphate and dihydroxyacetone→dihydroxyacetone phosphate have been shown to operate in these cells.

Keywords: glycerol; glycerol kinase; glyceraldehyde; dihydroxyacetone; spermatozoa; boar

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H. JACKSON, A. R. JONES and E. R. A. COOPER

Summary.

Hexamethylphosphoramide, an important industrial solvent, has been investigated because of its damage potential to reproductive cells. Male rats of proven fertility were given consecutive daily doses at prescribed levels and fertility data were obtained by serial mating. Aspermia in rats followed oral treatment with twenty-one doses of 100 mg/kg and five doses of 500 mg/kg. The former dose induced sterility between the 6th and 12th weeks and fertility did not return in a majority of animals. The histological results at both dose levels were consistent with an antispermatogenic action and correlated with the effects observed on fertility. In the pituitary glands of the sterilized males, considerable enlargement of basophil cells occurred. Simple modifications in the chemical structure of HMPA resulted in loss of the sterilizing action.