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M. FREUND

Summary.

Significant among-donor differences in mean values for semen-specimen volume and sperm concentration, motility, forward progression, and morphology were demonstrated, but no correlation among these characteristics on a specimens-within-donor basis was demonstrable. When emission frequency was increased from 3·5 to 8·6 times per week, there was a decrease in specimen volume (—33%), in sperm concentration ( —55%) and motility ( —15%), but no effect on progression or morphology. At the high frequency, there was a decrease in total sperm number (—31%) and in motile sperm number (—41%) produced per week, but an increase in volume of semen per week (+39%). Specimens were rated at hourly intervals for 9 hr after collection; motility and progression declined at a linear rate with time, but no change in morphology was observed. No effect of seminal plasma on sperm motility or forward progression was noted in a complete factorial seminal-plasma-reversal study. No significant differences were observed in mean values for semen characteristics between masturbated and condom specimens.

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M. FREUND

Summary.

The extra-gonadal sperm reserve (the sperm reserve in the vasa deferentia and the epididymides) was depleted in six donors by an average of 2·4 emissions per day during a 10-day depletion period. Daily sperm output (dso) was then estimated from the examination of one specimen a day for 24 days. During the dso period, specimen volume averaged 59% and sperm concentration per specimen 27% of that in the control or pre-depletion period. Sperm output per specimen and per week did not return to the levels of the pre-depletion period during a 156-day period (equal to three complete 52-day cycles of spermatogenesis and movement of spermatozoa through the duct system) after the completion of dso. At a frequency of fewer than five emissions per week, the greatest single influence on sperm numbers in the specimen is the size of the extra-gonadal sperm reserve. Daily sperm output, after the depletion of the extra-gonadal sperm reserve, may be used to estimate daily sperm production (dsp) by the testes. Changes in dso may be used to measure treatment effects on spermatogenesis. A practical experimental design is proposed for the analysis of factors and treatments affecting dsp in man.

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Z. PALTI and M. FREUND

The mechanism controlling the last step in ovulation—ejection of the follicular contents—is not yet fully explained. Several hypotheses have been proposed, for example, increased intrafollicular pressure (Bartelmez, 1912; Smith, 1934; Jensen & Zacharia, 1960), contractions of the follicular wall (Lipner & Maxwell, 1960), and enzymatic breakdown (digestion) of the follicular wall (Espey & Lipner, 1965; Espey, 1967; Espey, Slagter, Weymouth & Rondell, 1965; Espey & Rondell, 1968; Rondell, 1970) but none has been proven to be the mechanism of ovulation. However, it is agreed by all that a fully developed stigma is essential for ovulation. Recently, spontaneous contractions of cat ovaries in vitro were demonstrated and a response to adrenergic drugs was shown (Rocereto, Jacobowitz & Wallach, 1969). These contractions were assumed to be under the control of sympathetic impulses mediated through the adrenergic innervation (Jacobowitz & Wallach, 1967; Rocereto et al., 1969) to the smooth muscle cells found in

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M. FREUND and J. WIEDERMAN

Summary.

The presence of a true `dilution effect', as opposed to the toxic effects of certain diluents, was indicated in experiments where human semen was diluted 1 : 1, 1 : 5, 1 : 25 and 1 : 125 with six different diluents. Norman-Johnson-Solutions 1 and 2 were found to be superior to all other diluents tested and were shown to be equally useful as media in the freezing of diluted human semen. Twenty-one freezing trials were made, using the technique as developed to this point— 1 : 5 dilution with Norman-Johnson-Solutions 1 or 2, the addition of 10% egg yolk, slow cooling from +25 to +5° C over a 4-hr period, 6 hr of equilibration with 7% glycerol at +5° C, slow freezing from +5 to -70° C at the rate of -1° C/min, and storage at temperatures of -76 to -85° C. Motility ratings made on the day after freezing indicated that 75 to 100% of the motile cells frozen were recovered alive and motile, displaying vigorous forward progression. In none of the twenty-one trials was less than 75% of the motile cells frozen, recovered alive and motile. Storage at -76 to - 85° C for periods up to 10 months resulted in spermatozoal motility ratings almost equal to that after one day of storage, although there was a substantial loss in motility upon further storage in this temperature range during months 10 to 24. Long-term storage of diluted human semen without considerable loss of motility would seem to require lower storage temperatures, i.e. that of liquid nitrogen, -196° C.

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M. FREUND and B. CAROL

Summary.

Each of three technicians made duplicate pipettings from forty-six semen specimens of seven donors and filled two haemocytometer chambers from each pipette. The number of spermatozoa in each of the four large corner squares of the chamber were counted and recorded separately for a total of 2208 counts on 552 chambers. An analysis of variance was made and the percentage variances were calculated. Marked variation was found in counts among technicians and in duplicate counts by the same technician. The technician term and the related technician interaction terms accounted for 14·8 of the total study variance. Approximately one-half of the variance of the haemocytometer technique itself was due to the variance among technicians (57·4%) and one-half to the variance among duplicate haemocytometer determinations by the same technician (42 ·6%). The 95% confidence interval for haemocytometer counts, among technicians, on a single specimen which is equal to the mean of this study (28 million/ml), is ±52%. The variance associated with the haemocytometer technique is large and must be considered in the design and analysis of studies of semen.

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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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W. P. VENTURA and M. FREUND

Sperm transport through the female reproductive tract in most mammals occurs within a few minutes of coitus and is rapid and passive, i.e. it is not due to sperm motility but rather to some agent or agents, such as oxytocin and semen, which modify uterine motility (Evans, 1933; Yamanaka & Soderwall, 1960; First, Short, Peters & Stratman, 1968). The uterine contracting and relaxing properties of human and ram semen have been reported to be due to prostaglandins (von Euler, 1936; Eliasson, 1959; Bygdeman, 1964). Von Euler (1936) isolated and named prostaglandin, a weak organic unsaturated carboxylic acid containing hydroxyl groups, from human and ram seminal plasma and noted that it had a marked stimulatory effect on uterine smooth muscle. Subsequently, many compounds of the prostaglandin family have been isolated (Eliasson, 1959; Bygdeman, 1964). The prostaglandin content of rat semen is unknown, but von Euler (1936), Eliasson (1959), Horton & Thompson

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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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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, 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