Functional Relationships between Capacitation-dependent Cell Signaling and Compartmentalized Metabolic Pathways in Murine Spermatozoa

Abstract

Spermatozoa are highly polarized cells with specific metabolic pathways compartmentalized in different regions. Previously, we hypothesized that glycolysis is organized in the fibrous sheath of the flagellum to provide ATP to dynein ATPases that generate motility and to protein kinases that regulate motility. Although a recent report suggested that glucose is not essential for murine sperm capacitation, we demonstrated that glucose (but not lactate or pyruvate) was necessary and sufficient to support the protein tyrosine phosphorylation events associated with capacitation. The effect of glucose on this signaling pathway was downstream of cAMP, and appeared to arise indirectly as a consequence of metabolism as opposed to a direct signaling effect. Moreover, the phosphorylation events were not affected by uncouplers of oxidative respiration, inhibitors of electron transfer, or by a lack of substrates for oxidative respiration in the medium. Further experiments aimed at identifying potential regulators of sperm glycolysis focused on a germ cell-specific isoform of hexokinase, HK1-SC, which localizes to the fibrous sheath. HK1-SC activity and biochemical localization did not change during sperm capacitation, suggesting that glycolysis in sperm is regulated either at the level of substrate availability or by downstream enzymes. These data support the hypothesis that ATP specifically produced by a compartmentalized glycolytic pathway in the principal piece of the flagellum, as opposed to ATP generated by mitochondria in the mid-piece, is strictly required for protein tyrosine phosphorylation events that take place during sperm capacitation. The relationship between these pathways suggests that spermatozoa offer a model system for the study of integration of compartmentalized metabolic and signaling pathways.Mammalian spermatozoa are highly differentiated cells that display extreme polarization of cellular architecture and function. For example, the sperm head has evolved to interact with the egg's extracellular matrix and plasma membrane, and contains the paternal genetic material, whereas the sperm flagellum acts to provide motility for these cells. In regard to this polarization of function, sperm have two major constraints. First, they have little cytoplasm, and therefore have a reduced ability to translocate metabolic intermediates or substrates from one region to another. In addition, they are transcriptionally inactive, and therefore cannot make new proteins in response to changing needs. To overcome these constraints, we, along with others, have hypothesized that sperm possess compartmentalized metabolic and signaling pathways in specific regions of the cell poised to function in a localized fashion (1-4).The most obvious example of metabolic compartmentalization in spermatozoa is that of oxidative respiration. This pathway is restricted to the mid-piece of the flagellum, because mitochondria are located solely in this region. Oxidative respiration provides the most efficient generation of ATP, yet the major si...

The facilitative glucose transporter GLUT3: 20 years of distinction

“…Of note, glucose concentrations as low as 10 -100 M are all that are necessary to support the tyrosine phosphorylation signaling in murine sperm (116). This low concentration is consistent with a primary role for GLUT3, which is the predominant family member in murine sperm (122).…”

“…However, the ATP produced from glycolysis is essential for normal flagellar motility even in the presence of otherwise functional mitochondria (86,91). In addition, ATP produced by glycolysis is essential for the protein tyrosine phosphorylation events associated with capacitation (116). Thus the glycolytic machinery compartmentalized to the principal piece of the flagellum powers both the dynein ATPases that provide motility, as well as the kinases that regulate it.…”

Mammalian sperm metabolism: oxygen and sugar, friend and foe

“…The mouse sperm principal piece is an ideal compartment for high glycolytic flux: it is long and thin, with a high surface-tovolume ratio, favoring unimpeded transport of glucose into and lactate out of the compartment, and with the glycolytic pathway enzymes arranged as a large unit favoring maximal flux on the fibrous sheath scaffold. Indication that mitochondrial oxidative phosphorylation might not be necessary for flagellar function came from the observation of Travis et al (2001) that sperm maintained normal motility in the presence of uncouplers of oxidative phosphorylation, suggesting that ATP derived from oxidative phosphorylation contributed little if any energy to the flagellum. Impressive evidence for glycolysis as sole driving force for mouse sperm motility was provided by generation of mice in which the gapds gene had been "knocked out"; these were designated gapds -/- (Miki et al, 2004).…”

Novelty of the Pyruvate Metabolic Enzyme Dihydrolipoamide Dehydrogenasein Spermatozoa

“…The two ideal examples of noncanonical localization of metabolic enzymes in spermatozoa are the sperm-specific isoforms of hexokinase (localized in the mid piece and principal piece of the flagella and in the sperm head (25)) and lactate dehydrogenase (localized in the mitochondrial matrix (26)). The non-canonical localizations of metabolic enzymes indicate there is an extra mitochondrial energy production center; the ATP generated after glycolysis has been shown to be the source of tyrosine phosphorylation during capacitation of mouse spermatozoa (3). In our previous report we have established the importance of a post pyruvate metabolic enzyme, dihydrolipoamide dehydrogenase, in hamster sperm hyperactivation and acrosome reaction (27).…”