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...

In somatic cells, caveolin-1 plays several roles in membrane dynamics, including organization of detergent-insoluble lipid rafts, trafficking of cholesterol, and anchoring of signaling molecules. Events in sperm capacitation and fertilization require similar cellular functions, suggesting a possible role for caveolin-1 in spermatozoa. Immunoblot analysis demonstrated that caveolin-1 was indeed present in developing mouse male germ cells and both mouse and guinea pig spermatozoa. In mature spermatozoa, caveolin-1 was enriched in a Triton X-100-insoluble membrane fraction, as well as in membrane subdomains separable by means of their light buoyant densities through sucrose density gradient centrifugation. These data indicated the presence of membrane rafts enriched in caveolin-1 in spermatozoa. Indirect immunofluorescence analysis revealed caveolin-1 in the regions of the acrosome and flagellum in sperm of both species. Confocal immunofluorescence analysis of developing mouse male germ cells demonstrated partial co-localization with a marker for the acrosome. Furthermore, syntaxin-2, a protein involved in acrosomal exocytosis, was present in both raft and nonraft fractions in mature sperm. Together, these data indicated that sperm membranes possess distinct raft subdomains, and that caveolin-1 localized to regions appropriate for involvement with acrosomal biogenesis and exocytosis, as well as signaling pathways regulating such processes as capacitation and flagellar motility.

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.

Sperm structure has evolved to be very compact and compartmentalized to enable the motor (the flagellum) to transport the nuclear cargo (the head) to the egg. Furthermore, sperm do not exhibit progressive motility and are not capable of undergoing acrosomal exocytosis immediately following their release into the lumen of the seminiferous tubules, the site of spermatogenesis in the testis. These cells require maturation in the epididymis and female reproductive tract before they become competent for fertilization. Here we review aspects of the structural and molecular mechanisms that promote forward motility, hyperactivated motility, and acrosomal exocytosis. As a result, we favor a model articulated by others that the flagellum senses external signals and communicates with the head by second messengers to affect sperm functions such as acrosomal exocytosis. We hope this conceptual framework will serve to stimulate thinking and experimental investigations concerning the various steps of activating a sperm from a quiescent state to a gamete that is fully competent and committed to fertilization. The three themes of compartmentalization, competence, and commitment are key to an understanding of the molecular mechanisms of sperm activation. Comprehending these processes will have a considerable impact on the management of fertility problems, the development of contraceptive methods, and, potentially, elucidation of analogous processes in other cell systems.

Sperm need to mature in the epididymis to become capable of fertilization. To understand the molecular mechanisms of mouse sperm maturation, we conducted a proteomic analysis using saturation dye labeling to identify proteins of caput and cauda epididymal sperm that exhibited differences in amounts or positions on two-dimensional gels. Of eight caput epididymal sperm-differential proteins, three were molecular chaperones and three were structural proteins. Of nine cauda epididymal sperm-differential proteins, six were enzymes of energy metabolism. To validate these proteins as markers of epididymal maturation, immunoblotting and immunofluorescence analyses were performed. During epididymal transit, heat shock protein 2 was eliminated with the cytoplasmic droplet and smooth muscle γ-actin exhibited reduced fluorescence from the anterior acrosome while the signal intensity of aldolase A increased, especially in the principal piece. Besides these changes, we observed protein spots, such as glutathione S-transferase mu 5 and the E2 component of pyruvate dehydrogenase complex, shifting to more basic isoelectric points, suggesting post-translational changes such dephosphorylation occur during epididymal maturation. We conclude that most caput epididymal sperm-differential proteins contribute to the functional modification of sperm structures and that many cauda epididymal sperm-differential proteins are involved in ATP production that promotes sperm functions such as motility.

Triosephosphate isomerase 1 (TPI1) is a member of the glycolytic pathway, which is a critical source of energy for motility in mouse sperm. By immunoblotting, we detected two male, germ line-specific TPI1 bands (Mr 33,400 and 30,800) as well as the somatic-type band (Mr 27,700). Although all three bands were observed in spermatogenic cells, somatic-type TPI1 disappeared from sperm during epididymal maturation. In vitro dephosphorylation analysis suggested that the two male, germ line-specific TPI1 bands were not the result of phosphorylation of the 27,700 Mr TPI1 band. The Mr 33,400; 30,800; and 27,700 TPI1 bands corresponded to the respective sizes of the proteins predicted to use the first, second, and third possible initiation codons of the Tpi1 cDNA. We performed immunofluorescence on epididymal sperm and determined that TPI1 specifically localized in the principal piece. The antibody staining was stronger in cauda epididymal sperm than in caput epididymal sperm, a finding consistent with the identification of TPI1 as a cauda epididymal sperm-enriched protein. Immunofluorescence with sodium dodecyl sulfate (SDS)-insoluble flagellar accessory structures showed a strong TPI1 signal only in the principal piece, indicating that TPI1 is a component of the fibrous sheath. Northern blot hybridization detected longer Tpi1 transcripts (1.56 kb) in mouse testis, whereas somatic tissues had shorter transcripts (1.32 kb). As there is only one triosephosphate isomerase gene in the mouse genome, we conclude that the three variants we see in sperm result from the use of alternative translation start codons in spermatogenic cells. Mol. Reprod. Dev. 80: 862–870, 2013. © 2013 The Authors. Published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.