Oxidative Metabolism

10.1.3.1 Krebs Cycle

The existence of oxidative metabolism in human sperm has long been established [2, 19], It has also been shown that oxidative phosphorylation can support sperm motility in the absence of glycolysis [20]. However, given the idiosyncrasy of sperm, oxidative metabolism in human spermatozoa is less effective than glycolysis in maintaining intracellular levels of ATP along the length of the flagellum and in supporting sperm motility. The inability of oxidative metabolism to generate high levels of ATP may explain, at least in part, the absence of a significant Pasteur effect in human sperm.

The finding by Peterson and Freund [21] that phosphofructokinase activity in human sperm homogenates is markedly inhibited by ATP and that this inhibition can be prevented or reversed by the addition of inorganic phosphate is consistent with this notion. However, the addition of inorganic phosphate to intact spermatozoa results in a discrete increase in aerobic glycolysis which appears to reflect more a simple ionic requirement than glycolytic control [1]. This suggests that ATP generated aerobically in intact spermatozoa is insufficient to cause significant inhibition of enzymatic activity at the phosphofructokinase level.

With the exception of succinate, Krebs cycle intermediates, glucose and pyruvate, do not significantly increase respiration over the endogenous rate. Possible causes for this low oxidation rates include: (1) the pathway to oxygen is common at coenzyme Q for the oxidation of succinate and pyridine nucleotide-linked substrates. The high rate of succinate oxidation, therefore, eliminates this portion of the electron-transfer chain by limiting the oxidation of other substrates. This assumes that succinate-stimulated respiration is not caused by exposure and activation of succinate dehydrogenase after sperm washing. Hamner and Williams [22] reported that the level of glucose oxidation by human sperm could be increased to the level of succinate oxidation following the addition of bicarbonate. This could be explained by the fact that bicarbonate speeds the turnover rate of the Krebs cycle and of acetyl CoA oxidation by providing increasing amounts of a rate-limiting component: oxaloacetate via CoA fixation to pyruvate. Murdoch and White reported that addition of 6 mM bicarbonate slightly stimulated sperm respiration. These observations were also confirmed by Peterson and Freund using polarographic measurements. When tested in the presence of 1-10 mM sodium bicarbonate, the rate of oxidation of succinate was consistently two to fourfold higher than the rate of glucose oxidation [2, 23]. Furthermore, the respiration rates in semen, where bicarbonate levels are relatively high, are relatively low but can be stimulated by succinate. This indicates that factors other than bicarbonate limit the oxidation of pyridine nucleotide-linked substrates. On this regard, it should be pointed out that the concentration of phosphate acceptors, such as ADP and AMP, do not usually limit the oxidation of pyridine nucleotide-linked substrates. The increased rate of oxidation in the presence of succinate alone suggests that this is so because the succinate pathway bypasses two of the three potential rate-limiting steps involved in ATP biosynthesis.

Citrate and other Krebs cycle intermediates are readily formed from pyruvate in human sperm, provided that a source of oxaloacetate is present. Activation of the Krebs cycle in mammalian spermatozoa appears to be limited by oxaloacetate. The conversion of acetate to citrate, however, is not increased in the presence of substrates that increase oxaloacetate levels. This is in agreement with the report by Terner [19] who found that in human spermatozoa acetate was oxidized at much lower rates than glucose or pyruvate. Although an increased rate of citrate formation may increase the rate of oxidation of a particular substrate, the overall rate of respiration is not increased by stimulating citrate synthesis and, therefore, other substrate must regulate the control of oxidation of pyridine nucleotide-linked substrates. The high rate of succinate oxidation indicates that most Krebs cycle enzymes are present in more than adequate concentrations to handle the substrate load that sperm may usually encounter. Therefore, oxidation may be limited by either the catalytic capacity of mitochondrial pyridine nucleotide dehydrogenase or some form of stringent cofactor control. Several metabolic peculiarities of human sperm are known to contribute to the near absence of a Pasteur effect. First, a-glycerophosphate dehydrogenase activity is very low [24]. This would tend to increase the NADH available to lactate dehydrogenase for pyruvate reduction and, therefore, lower the acetyl CoA available for mitochondrial oxidation through the Krebs cycle. Second, high ATP/ ADP ratios are not found in human spermatozoa perhaps due to the weak coupling of respiration to phosphorylation. This would tend to lower feedback inhibition of glycolysis at several points in the glycolytic pathway.

10.1.3.2 Fatty Acid Oxidation

Long-chain fatty acids must first be converted to the acyl esters of CoA in the cyto-solic compartment before they can be oxidized by mitochondria. Acyl esters of CoA are then converted to the carnitine esters by the corresponding transferase located on the outside of the inner mitochondrial membrane [25-28]. In addition to oxidation of the aliphatic chain, human spermatozoa have been also shown to produce ATP through the conversion of the glycerol moiety of the lipid molecule to l-3-glycerophosphate [29, 30] .

The mitochondrial oxidative activity profile of mammalian spermatozoa differs significantly between the different mammalian species. The profile of human spermatozoa falls between the full set of enzyme activity typical somatic cell mitochondria, like that of bull sperm mitochondria, and the limited set displayed by rabbit spermatozoa [31]. For example, mouse sperm lack the necessary enzymes needed to utilize the malate/aspartate shuttle [32] for transfer of these equivalents, as shown by their inability to oxidize glutamate in the presence of malate [33] . It is likely that the lactate/pyruvate shuttle, common to most mammalian spermatozoa, plays a major role in transferring NADH equivalents from the cytosol to the mitochondria in mouse sperm. On the other hand, mouse sperm can oxidize fatty acids directly from the CoA esters. These CoA esters provide mouse sperm with a supply of endogenous substrate which would enable them to remain motile for more than 4 h in a simple solution containing NaCl, tris-HCl, and CaCl2 [34, 35], Sperm from BL/6 mice maintain their high initial motility for more than 6 h in a defined saline medium buffered with phosphate and bicarbonate and lacking oxi-dizable substrates [36]. The differences in mitochondrial activities of human, bovine, mouse, and rabbit spermatozoa suggest that bovine gametes are more self-sufficient with regard to the ample set of substrates that they could possibly utilize, in addition to their endogenous reserves of fatty acids. If the hypothesis postulated by Storey that sperm mitochondrial activities can be used to predict the concentration of nutrients (from which energy can be derived) in the oviduct is correct, then the bovine oviductal lumen should have low concentrations of these nutrients. Data regarding substrate concentration in the lumen of bovine oviduct support this hypothesis [37-39],

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