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R. N. PETERSON and M. FREUND

Summary.

In the presence of glucose, respiration and the levels of citric acid cycle intermediates in washed human spermatozoa are low. Citrate levels are not markedly increased by pyruvate, lactate or acetate, nor do these compounds increase respiration. Succinate, which is oxidized at high rates, increases citrate levels about fourfold; however, the increased respiration is attributed to the succinate—fumarate conversion alone and not to a general stimulation of the citric acid cycle. The addition of succinate, malate, or fumarate to sperm suspensions containing pyruvate increases citrate levels 50- to 100-fold, markedly stimulates α-ketoglutarate formation, decreases glycolysis and almost doubles the rate of pyruvate utilization. The increased rates of the synthesis of citrate and other intermediates of the citric acid cycle are not accompanied by increased rates of respiration. These data argue for the presence of a powerful pyruvate dismutation pathway in human spermatozoa in which lactic dehydrogenase competes successfully for the reducing equivalents generated by pyruvate oxidation and argues against the idea that oxidation in spermatozoa is limited by substrate entry into the citric acid cycle.

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R. N. Peterson and M. Freund

Summary.

The motility of washed suspensions of human spermatozoa was completely inhibited by tetraphenylboron at concentrations that had little effect on sperm energy metabolism. The inhibition of motility was reversed by quaternary ammonium salts, albumin, caffeine, dibutyryl cyclic AMP and potassium ions. The addition of ouabain to cells rendered immotile by tetraphenylboron prevented reinitiation of motility by potassium but not by the other compounds. These observations, together with the effect of tetraphenylboron on the fluorescence of sperm suspensions treated with 1-anilinonaphthalene 8-sulphonic acid, suggest that the binding of tetraphenylboron to sites on the sperm plasma membrane is involved in the inhibition of sperm motility and that cyclic AMP may be involved in the regulation of ion transport across the plasma membrane.

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R. N. PETERSON and M. FREUND

Summary.

The level of human sperm respiration in semen and in artificial media has been estimated by manometric and radiochemical techniques. Our results show that the respiration is small, exhibiting rates that rarely exceed 1 to 2 μl/108 cells/hr. These low Zo2 values and the limited sensitivity of the manometric apparatus stress the point that pooled specimens with high cell concentrations must be used in order to obtain reliable estimates of oxygen consumption. Attention is also drawn to the fact that measurements of oxygen uptake by cells in whole semen are further complicated by the high rates and variability of oxygen absorption by seminal plasma and that small errors in the estimation of this absorption can lead to substantial errors in estimates of cellular oxygen uptake. Although isotopic measurements of respiration are more sensitive than manometric techniques, some reserve is also required when interpreting results based on 14CO2 evolution alone. Experiments with [U-14C]glucose and [6-14C]glucose indicate that the carbon atoms of glucose are not converted to CO2 in an identical manner. The higher rates of 14CO2 produced from the metabolism of uniformly labelled glucose suggest that a portion of the glucose molecule may be converted to CO2 through non-oxidative pathways. In view of such a possibility, the ability of washed cells and whole semen to convert [6-14C]glucose into labelled CO2 provides less equivocal radiochemical evidence than that previously reported for true respiratory activity in human sperm cells. High phosphate concentrations inhibit the conversion of glucose to carbon dioxide. It is suggested that this may represent the presence of competing reactions for limiting intermediates involved in glycolytic and respiratory pathways. We have also described the use of the rapid Millipore filtration technique to follow glucose uptakes and incorporation into acid-insoluble (lipid) components of sperm cells. Uptake into acid-insoluble compounds is rapid and represents a substantial portion of the radio-active glucose that is retained by cells. The potential usefulness of this method to measure endogenous lipid turnover and the role of cellular lipid in endogenous respiration is also considered.

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L. D. Russell and R. N. Peterson

Summary. Criteria were devised for determining the elongate spermatid—Sertoli cell ratio in various mammalian species at the electron microscope level. When data from particular species were pooled, the values were: rabbit, 12·17:1, hamster, 10·75:1; gerbil, 10·64:1; rat, 10·32:1; guinea-pig, 10·10:1; vole, 9·75:1; and monkey, 5·94:1. The elongate spermatid—Sertoli cell ratio is a measure of the workload of the Sertoli cell and is a prime factor determining their efficiency. The higher the ratio, the higher the sperm output is likely to be per given weight of seminiferous tubule parenchyma for a particular species.

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R. N. PETERSON, K. SILVERSTEIN and M. FREUND

Laboratory of Reproductive Pharmacology, Department of Pharmacology, New York Medical College, Valhalla, New York 10595, U.S.A.

(Received 3rd June 1974)

The dye, ethidium bromide, markedly increases its fluorescence quantum efficiency when bound to double-stranded undenatured nucleic acid (Le Pecq, Yat & Paoletti, 1964; Le Pecq & Paoletti, 1966). When added to sperm suspensions, ethidium bromide binds almost exclusively to the sperm head and induces an intense fluorescence (Edelman & Millette, 1971).

In this report, a method for DNA assay in human semen and washed sperm suspensions is described. The method takes advantage of these earlier findings and permits quantitative estimates of DNA and, indirectly, of sperm counts to be made in less than 15 min.

Semen specimens were obtained from medical students. Sperm counts were made in quadruplicate using a haemocytometer, as described by Freund & Carol (1964). The procedure for preparing washed sperm suspensions and the composition of the

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R. N. Peterson, D. Seyler, Donna Bundman and M. Freund

Summary. Radioactive calcium uptake by suspensions of washed boar and human spermatozoa was inhibited by the mitochondrial uncoupling agent carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Theophylline + dibutyryl cyclic AMP also inhibited calcium uptake in the presence or absence of FCCP. Uptake of low concentrations of calcium (0·1 mm) was inhibited by the calcium ionophore A23187, but at high calcium concentrations the ionophore stimulated calcium uptake. These observations are explained in terms of a mechanism for the regulation of calcium uptake in spermatozoa based on competing mitochondrial and plasma membrane pumps. Uptake of 32P was also inhibited. These effects provide evidence that cyclic AMP plays a role in the transport of ions across the plasma membrane of spermatozoa.

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N. Saxena, R. N. Peterson, S. Sharif, N. K. Saxena and L. D. Russell

Summary. Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2·0, 4·85 and 5·0, and one specific for an unreferenced PM protein (M r 80 000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5·0 and M r 80 000) or a delocalization of fluorescence on the head PM (PM proteins 2·0 and 4·85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5·0 and that of M r 80 000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5·0 and that of M r 80 000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4·85 and 5·0, a much smaller percentage of caput spermatozoa (∼20%) showed specific localization changes compared to those of the cauda (∼ 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.