• Energy Research

The effects of exposure to low pH on platelets isolated from plasma by saline-EDTA washing or by gel filtration were compared. Platelets isolated by the two methods responded similarly with respect to nucleotide metabolism, secretion and leakage. Both showed decrease in adenylate energy charge and metabolic ATP level after exposure to pH 5.3 and reversal to almost normal levels after return to pH 7.6, while the pH 5.3-induced secretion (about 50% of preabsorbed serotonin) was continued after the return to higher pH and reached about 80%. The response of the cytoskeleton of EDTA-washed and gel-filtered platelets to low pH differed quantitatively. Analysis of transmission electron micrographs (TEM):TEM of Triton-insoluble residue showed similar structures and SDS-PAGE showed primarily actin (42,000d) and likely myosin (200,000d). Conclusion: The method of isolation did not affect the response of platelets to low pH when studied by platelet nucleotide metabolism, secretion or leakage but did affect the response when studied by the organization of the cytoskeleton (actin filaments).

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to twenty-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane intact spermatozoa supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.

Mammalian spermatozoa utilize ATP as the energy source for key functions on the route to fertilization. ATP and its precursor nucleotides ADP and AMP are determined in many sperm physiology studies, mostly by bioluminescence assays. Assay results vary widely, mainly due to different efficiency in nucleotide extraction and prevention of their enzymatic degradation. Here, we describe a revised, validated protocol for efficient phosphatase inhibition and adenine nucleotide extraction resulting in consistently high ATP concentrations exceeding previously reported values for boar spermatozoa up to twenty-fold. The revised assay is applicable for determining ATP concentrations and adenylate energy charge in extracts from fresh and frozen samples, thereby allowing simultaneous assessment of semen samples from long-term storage experiments. After validation, the assay was applied to liquid preserved boar spermatozoa stored at 17°C and 5°C for 24 and 72h. Cooling to 5°C, but not storage duration, reduced ATP concentration in spermatozoa (P<0.05), which was accompanied by the appearance of AMP and ADP in the preservation medium. ATP and energy charge were highly correlated to the proportion of membrane intact spermatozoa supporting the idea of nucleotides leaking through disrupted membranes in cold-shocked cells. The present assay allows highly standardized studies of energy metabolism in spermatozoa.

(1981). Phosphoadenylate concentrations and energy charge in two freshwater crustaceans: Responses to physical and chemical stressors. SIL Proceedings, 1922-2010: Vol. 21, No. 1, pp. 205-220.