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P. J. QUINN

Summary.

The methyl green assay method has been used to determine the activity of DNase I and DNase II in ram, bull, human, dog and rabbit seminal plasma. The activity of DNase II was higher except in the ram. The DNase I activity of ram seminal plasma was inhibited by low concentrations of citrate, zinc and cetyltrimethylammonium bromide ; this detergent also caused a reduction of DNase II activity. There was a stimulation of DNase I activity by 10 mm-citrate but not by concentrations less than 2 mm. Addition of antibiotics or shaking of ram seminal plasma with toluene did not affect the activity of either DNase. After deep freezing of ram semen there was a decrease of DNase I activity in the plasma and an increase of DNase I and II activity in extracts obtained from disintegrated spermatozoa. No significant change in DNase activity could be detected in the plasma of ram semen which had previously been subjected to cold shock.

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P. J. QUINN and I. G. WHITE

The active transport of sodium and potassium has been demonstrated in dog spermatozoa (Quinn & White, 1967) and the existence of a cation pump, involving a sodium—potassium activated ATPase has been postulated to account for the gradients of alkali metal ions between the cell and seminal plasma (Quinn, White & Wirrick, 1965). Recently, Uesugi & Yamazoe (1966) have reported the occurrence of a sodium—potassium activated ATPase in boar epididymal spermatozoa, and the present experiments were undertaken to determine the activity and distribution of ATPases in ram and bull spermatozoa.

Semen was diluted with 2·5 vol of 250 mm-sucrose, centrifuged, and the spermatozoa washed twice with 5-ml aliquots of sucrose. Washed spermatozoa, resuspended in sucrose buffered to pH 7·4 (37° C) with 50 mm-tris (tris [hydroxylmethyl] amino methane) and 40 mm

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Lynn R. Fraser and P. J. Quinn

Summary. The substrate requirements for capacitation of epididymal mouse spermatozoa, initiation of the acrosome reaction and support of fertilization of mouse eggs in vitro were examined by assessing not only fertilization rates, but also the stages of egg activation and sperm head decondensation in fertilized eggs to monitor the temporal aspects of these processes. Although early events of capacitation did not require exogenous substrates, the fertilization process was effectively blocked at the terminal stages of capacitation in the absence of a glycolysable sugar, and addition of glucose was obligatory to initiate both the acrosome reaction and the whiplash motility associated with fertilizing ability. Once the spermatozoa had been primed by glucose, however, the removal of exogenous glucose did not block fertilization. The need for oxidative metabolism was excluded because successful fertilization could be achieved with glucose as the sole exogenous substrate under strictly anaerobic conditions or in the presence of 2·5 μm-oligomycin. We suggest, therefore, that epididymal mouse spermatozoa can be almost completely capacitated in the absence of metabolic processes simply by release into substrate-free medium. They require exposure to glucose to permit induction of the acrosome reaction and motility changes, necessary prerequisites for fertilization; such exposure is followed by rapid and synchronous penetration of eggs.

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P. J. QUINN and I. G. WHITE

Since mammalian spermatozoa are generally considered to be less affected by hypertonic than by hypotonic solutions (see Mann, 1964) it was surprising to find that dialysis of ram semen for 6 hr in the continuous flow dialysis apparatus (CFDA) of Dott & Walton (1960) against a hypotonic diluent, did not affect the impedance change frequency (ICF) whereas a hypertonic diluent abolished the ICF within 2 hr (Quinn, White & Voglmayr, unpublished).

The most obvious difference between the CFDA and diluting semen directly in tubes is that high molecular weight substances present in the seminal plasma remain within the dialysis sac and are not further diluted. To test whether the non-dialysable components of the seminal plasma were responsible for the effect, ram semen was diluted with 10 vol. of Krebs-Ringer buffered to pH 7·4 with 50 mm sodium

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P. J. QUINN and I. G. WHITE

Summary.

Cold-shocking and deep-freezing bull and ram semen caused an influx of sodium to and an efflux of potassium and magnesium from the spermatozoa. The movement of these cations was accentuated when the semen was diluted 1 : 8 in sodium phosphate buffers. Calcium is actively accumulated in the spermatozoa of these two species after cold-shocking.

No significant changes in the cation content of human, dog, rabbit or fowl spermatozoa were found after cold-shocking. There was no great disturbance of the cation status of human and fowl spermatozoa, even after deep-freezing.

Lecithin prevented calcium accumulation in bull and ram spermatozoa on cold-shocking, and glycerol also had this effect on cold-shocked bull spermatozoa. An egg-yolk-citrate/glycerol-fructose diluent protected bull and ram spermatozoa to a considerable extent from the influx of sodium and the efflux of potassium that occurs after deep-freezing to −79°C.

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P. J. Quinn, P. Y. W. Chow and I. G. White

Summary. Adding phospholipid dispersions to washed ram spermatozoa provided immediate protection against cold shock as judged by the effect on motility. Re-washing the spermatozoa free of dispersed lipid rendered then susceptible to cold shock. Analysis of the spermatozoa showed that exogenous phospholipid had not become interpolated into the cell membranes. The ratio of phospholipid to cholesterol in the spermatozoon was not changed when dispersed phospholipid was added and stayed the same during incubation for 3 h at 37°C. It is concluded that the protective effect of phospholipid against cold shock in ram spermatozoa is due to a 'loose' interaction of the lipid structures with the plasma membrane of the cells.

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P. J. QUINN, I. G. WHITE and J. K. VOGLMAYR

Summary.

When ram semen was dialysed against phosphate-glucose diluents with a cation composition similar to rete testis fluid, epididymal and ejaculated seminal plasma and ejaculated ram spermatozoa, the impedance change frequency (ICF) was maintained best over 6 hr in the latter diluent. Krebs-Ringer-glucose buffered with sodium phosphate or trisodium citrate maintained the ICF at a high level, whereas tris-HCl and veronal-HCl buffers caused a rapid decline in ICF. Hypertonic dialysis media severely depressed the ICF within 1 hr, whereas hypotonic diluents gave results similar to Krebs-Ringer-phosphate.

Adding 1 mm-calcium or 5 to 51 mm-potassium to an isotonic dialysis fluid was beneficial and the effects on ICF were additive. The omission of potassium from the hypotonic diluent caused no significant reduction in ICF.

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P. J. QUINN, I. G. WHITE and B. R. WIRRICK

Summary.

The atomic absorption spectrophotometer provides a simple and easy method for determining sodium, potassium, calcium and magnesium in whole semen, spermatozoa and seminal plasma. Analyses of ram, bull, human, dog, rabbit and fowl semen all reveal a reciprocal relation of potassium to sodium in the spermatozoa and seminal plasma. The high potassium and low sodium concentration of the spermatozoa, coupled with a low potassium and high sodium concentration in the seminal plasma, suggest the operation of a sodium pump mechanism in spermatozoa similar to that postulated for erythrocytes. The magnesium concentration of the spermatozoa always exceeded that of the seminal plasma, whilst the same is true for calcium, except in the bull where the seminal plasma has an exceptionally high calcium content. The distribution of cations between the epididymal spermatozoa and epididymal plasma in the ram and bull is, in general, similar to that between ejaculated spermatozoa and seminal plasma. However, there were substantial differences in absolute values: the sodium concentration of epididymal ram spermatozoa was half that of the ejaculated cells and the potassium concentration about double.

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P. J. QUINN, I. G. WHITE and K. W. CLELAND

Summary.

There was little or no loss of DNA or phospholipid from ram spermatozoa after washing three times with an equal volume of isotonic tris buffer. There was substantial loss of non-dialysable orcinoland biuret-reactive substances and also acid soluble phosphorus and phospholipids after cold-shocking and freezing ram spermatozoa. These components were also rendered more extractable with 10% NaCl after cold treatment. DNA was lost from cold-shocked and frozen ram spermatozoa after incubation at 37° C for 6 hr and the rate of degradation of DNA into dialysable components was initially higher in coldshocked or frozen spermatozoa.

Electron microscopy showed that cold shock and freezing caused profound changes in the appearance of the corrugated and terminal parts of the acrosome in the majority of spermatozoa but there were no apparent changes in the smooth region. There was considerable swelling of the acrosome with reduction in the density of the material contained within the acrosomal membranes. The effects were more pronounced in frozen than in cold-shocked spermatozoa.

In the middle pieces, changes occurred in the matrix of the mitochondria making up the mitochondrial sheath. The matrix in cold-shocked and frozen spermatozoa appeared lighter than in the controls and loss of protein from the middle piece was confirmed histochemically.

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P. J. QUINN, I. G. WHITE and B. R. WIRRICK

Summary.

Mild dilution (<1·8) of ram and bull semen in isotonic sodium phosphate diluent of pH 7·0 caused an influx of sodium and an efflux of potassium, calcium and magnesium from the spermatozoa. Equilibrium was reached soon after dilution and little further change in the concentration of these cations was observed over 3 hr.

Similar changes occurred when ram and bull semen was diluted in tris and veronal diluents of pH 7; however, calcium was found to accumulate in ram spermatozoa diluted in tris. Diluting semen in buffers of pH range 8·5 to 5·5 indicated that potassium loss from the spermatozoa increased with increasing pH.

Greater dilution (1:100) and exhaustive washing (four times) had a similar but more severe effect on the cation concentration of ram and bull spermatozoa. A large influx of sodium was accompanied by a marked depletion of potassium which tended to be further depleted over 3 hr.