Glycolysis by Washed Suspensions of Human Spermatozoa. Effect of Substrate, Substrate Concentration, and Changes in Medium Composition on the Rate of Glycolysis

Abstract

The rate of formation of lactic acid from glucose by motile human spermatozoa suspended in plasma-free medium is constant over a 30-fold change in substrate concentration (.001-03 M) while lactic acid formation from fructose increases less than 2-fold over the same concentration range. The additional observation that glucose-6-phosphate enter cells without prior dephosphorylation and is metabolized to lactic acid at the same maximum rate as fructose and glucose indicates that glycolysis in human sperm is ordinarily not substrate limited. These results also indicate that the fructose levels ordinarily present in semen are sufficient to sustain a high rate of glycolysis even when considerably diluted such as may occur in the female reproductive tract. A transport barrier to sugar influx also does not limit glycolysis since the initial rate of uptake of radioactive glucose into cells continues to increase over a concentration range in which the rate of lactic acid formation is constant. Further, from the observation that the activity of hexokinase in sperm homogenates exceeds the rate of glucose utilization by intact cells, it is possible to conclude that the catalytic capacity of intracellular hexokinase does not limit glycolysis. The sum of these observations indicates that a major rate limiting step (or steps) functions beyond hexokinase. In other experiments aerobic glycolysis was found to be only slightly stimulated by dinitrophenol. Inorganic phosphate also stimulates glycolysis but the magnitude of this effect and the concentration required to produce it are small. The significance of these observations are discussed in terms of the small Pasteur effect exhibited by these cells. A sensitive fluorometric method for determining lactic acid in washed sperm suspensions is also described.