Most of the energy that sperm need to maintain its characteristic motility pattern is obtained through glycolysis, also known as the Embden-Meyerhof pathway, which is highly active under both aerobic and anaerobic conditions. The effects of substrate, substrate concentration and changes in medium composition on the rate of glycolysis in human sperm were first reported by Peterson and Freund [1]. In this study, the conditions under which fructose and glucose levels limit glycolysis were described. They also found that the transport of carbohydrates into sperm was not the rate-limiting step of glycolysis and that the sites of glycolytic control are downstream hexokinase. These sites are related to the regulation of the activity of glycolytic enzymes by a key cofactor: inorganic phosphate. Phosphofructokinase is involved in this critical control given its low intracellular concentration in sperm and its sensitivity to adenine nucleotides. Inorganic phosphate stimulates phos-phofructokinase activity in cell-free extracts and glycolysis in intact sperm [2, 3], However, this stimulation by inorganic phosphate only accounts for a 30% increase in the rate of glycolysis, even at phosphate concentrations up to 40 mM, thus suggesting that phosphate control of glycolysis is perhaps less important than control by other cofactors.

The enzymatic profile of human spermatozoa is not typical of cells that exhibit a high rate of aerobic glycolysis. This implies that certain metabolic changes must take place after ejaculation. This hypothesis was postulated based on the similarities in the enzymatic profile between human and bull spermatozoa. Early work by Lardy and Parks indicated that glycolysis in epididymal bull sperm sharply decreased when sperm were exposed to oxygen and increased after ejaculation, thus suggesting that a metabolic regulator may be released at the time of ejaculation that uncouples oxidative phosphorylation which, in turn, decreases the respiratory inhibition of glycolysis [3] .

Human sperm contain glucose-6-phosphate dehydrogenase and 6-phosphoglu-conate dehydrogenase, the entry enzymes into the hexose monophosphate shunt. The shunt pathway has four major functions: (1) to provide reducing power in the form of NADPH required to drive biosynthetic reactions; (2) to provide the pentose needed for nucleic acid biosynthesis; (3) to provide an additional mechanism for the oxidative generation of ATP; and (4) to provide NADPH to maintain operative the glutathione reductase/glutathione peroxidise protective system for reduction of hydrogen peroxide and lipid hydroperoxides [4] . However, based on the fact that there is no biosynthetic activity in human sperm after their release into the seminiferous tubules, it is highly unlikely that the first two functions be of any relevance on sperm function. In addition, sperm are unable to metabolize ribose-5-phosphate, adenosine, or uridine, which are pentose shunt metabolites usually converted to lactic acid at rapid rates in cells where the shunt pathway supports biosynthesis. However, the physiological relevance of the third function indicated above is supported by the work of Sarkar et al. [5], who found that sperm incubated in the presence of NADP and glucose-6-phosphate or 6-phosphogluconate produce high levels of NADPH.

At this point is worth mentioning the significance of the low levels of a-glycer-ophosphate dehydrogenase in human sperm. This enzyme plays an important role in oxidative metabolism because it catalyzes the conversion of dehydroxyacetone phosphate to a-glycerophosphate which can be directly oxidized by mitochondria. Furthermore, this enzyme competes with lactate dehydrogenase for NADH, the reduced form of NAD and, therefore, can provide additional NAD for the reduction of pyruvate to lactate, a key step in sperm's metabolic strategy. Therefore, differences in the activity a-glycerophosphate dehydrogenase could explain, at least in part, some of the differences observed in oxidative metabolism between human sperm and other mammalian species. For example, higher levels of this enzyme in ram spermatozoa account for the higher rates of glucose oxidation observed in this species and also for the higher rates of sorbitol and glycerol oxidation.

The catalytic activity of glycolytic enzymes in mammalian spermatozoa exceeds the rate of glycolysis, thus indicating that glycolysis is not limited by the concentration of any particular enzyme. In sharp contrast, phosphofructokinase is found in very low concentration in sperm, suggesting a potential regulatory role for this enzyme in sperm metabolism. Furthermore, the activity of phosphofructokinase in sperm homogenates is significantly affected by cofactors known to be involved in other cell types. High concentrations of ATP inhibit phosphofructokinase activity, while AMP and inorganic phosphate stimulate its activity. These observations imply that phosphafructokinase plays an important role as a rate-limiting step in glycoly-sis control.

The overall enzymatic profile of epididymal sperm is characteristic of cells that exhibit a pronounced Pasteur effect. That is, oxygen inhibition of glycolysis. An important feature of ejaculated human sperm, however, is the absence of any detectable Pasteur effect. Addition of dinitrophenol, known to release the inhibitory effect of oxidative metabolism on glycolysis, to sperm suspensions barely stimulates the rate of aerobic glycolysis. The absence of a significant Pasteur effect in human sperm also provides an explanation for the relatively small effect of inorganic phosphate on the rate of glycolysis.

The rate of conversion of glucose to lactate by motile sperm suspended in seminal plasma-free medium is constant over a 30-fold range in glucose concentration (1-30 mM), whereas lactate conversion from fructose increases less than twofold over the same concentration range. The observation that glucose-6-phosphate enters the sperm cell without prior dephosphorylation and is metabolized to lactate at the same rate as fructose and glucose provides further evidence that glycolysis in human sperm is not substrate-limited. This also indicates that the fructose levels usually present in semen are more than enough to sustain a high rate of glycolysis, even at very low concentrations, as is the case in the female reproductive tract.

Rabbit spermatozoa convert more than 70% of the glucose consumed to lactate under aerobic conditions through the Embden-Meyerhof pathway, as shown by Murdoch and White [6]. These investigators used both the measurement of glucose uptake and lactate production of 14CO2 produced from glucose labeled at the 1- and 6-positions. One of the two enzymes in the Embden-Meyerhof pathway that produce net ATP is pyruvate kinase [7, 8], which can be readily measured in hypotonically treated epididymal spermatozoa [9] . Based on kinetic studies on their substrates, cofactors, and effectors [10, 11], two major isozymes of pyruvate kinase have been identified. One is the type showing allosteric control by fruc-tose-1,6-biphosphate. An isoenzyme of this type, designated L [12], is found in yeast [7] and hepatocytes [13]. Other isozymes of this type are M2 from liver nonparenchymal cells [13-15] and A from kidney [16]. The control characteristics of this type of enzyme and, in particular its allosteric activation by fructose-1,6-biphosphate, are those expected for an enzyme operating in a pathway in which the flux of substrate is closely controlled [8]. The biosynthetic pathways functioning in liver parenchymal cells require maximal utilization of ATP with minimal utilization of glucose [17]. That is, the glucose used is effectively converted to either H2O and CO2 or to precursors to be used in anabolic pathways. The same consideration applies to kidney cells that have the gluconeogenic pathway and the A isozyme of pyruvate kinase.

The second type of pyruvate kinase, characteristic of the muscle isozyme M [18], does not have this kind of allosteric control. It is, therefore, well suited for handling higher fluxes of glucose for conversion into lactate through the Embden-Meyerhof pathway [8]. The efficiency of ATP production, taken as moles of ATP produced per mole of glucose, is lower, but it can support a high rate of ATP production. These control characteristics of the muscle enzyme are those required for conversion of metabolic energy into mechanical work in which efficiency of glucose utilization is subordinated to maximal rate of ATP production [17]. The flux of glucose through the Embden-Meyerhof pathway is usually high in order to maintain speed and force of muscle contraction. Therefore, the kinetic properties of the pyruvate kinase in any given cell type are useful in terms of inferring the regulatory characteristics of glycolysis in any particular cell type.

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