Comparison of Glycolysis and Oxidative Phosphorylation as Energy Sources for Mammalian Sperm Motility, Using the Combination of Fluorescence Imaging, Laser Tweezers, and Real-Time Automated Tracking and Trapping

Quantitative measurements of sperm are critical for assessing their overall quality and viability. Several systems for sperm analysis have been developed. For example, Computer Assisted Sperm Analysis (CASA) systems measure motility parameters of sperm populations (Amann and Katz, 2004). Flow cytometry systems in combination with fluorescent probes that monitor sperm mitochondrial membrane potential (MMP) have been used to demonstrate that high MMP correlates with increased motility (Marchetti et al., 2002) as well as high fertility (Kasai et al., 2002; Marchetti et al., 2004; Gallon et al., 2006). Laser tweezers have been used to measure sperm swimming force, a measurement that positively correlates with sperm swimming speed (Tadir et al., 1990; Nascimento et al., 2006, 2007). The recent combination of laser tweezers, fluorescent imaging, and the real-time automated tracking and trapping system (RATTS) (Shi et al., 2006) has provided a system that can measure sperm motility parameters, such as curvilinear velocity (VCL) and swimming force (in terms of the laser power at which a sperm can escape the optical trap, Pesc), simultaneously with MMP (Shi et al., 2008). This system’s capabilities have been described in detail in Nascimento et al. (2008). Specifically, the system can monitor changes in MMP over time, study the effects of prolonged exposed to the laser tweezers on VCL and MMP, and quantify VCL, Pesc and MMP in real-time for hundreds of sperm. The study also found that when sperm were exposed to a mitochondrial uncoupling agent (carbonyl cyanide 3-chlorophenylhydrazone, CCCP), a significant decrease in MMP was observed yet VCL appeared to be unaffected. As a result of these findings, it was suggested that perhaps a pathway other than oxidative phosphorylation, such as glycolysis which is known to occur along the sperm tail (Turner, 2003; Mukai and Okuno, 2004; Ford, 2006), may be providing the ATP (energy) needed to support motility (Nascimento et al., 2008). Oxidative phosphorylation is, indeed, a more efficient means of generating ATP than glycolysis (the metabolism of sugars in mitochondria can produce fifteen times more ATP than glycolysis) (Alberts et al., 2002). However, there is some doubt that ATP can sufficiently diffuse from the midpiece down to the distal segments of the tail (Turner, 2003; Ford, 2006). Recent studies suggest glycolysis is an important pathway to generate ATP in sperm cells. For example, analysis of the flagellum has shown that several glycolytic enzymes are localized to the fibrous sheath in mouse and human sperm (Krisfalusi et al., 2006; Kim et al., 2007) as well as an ADP/ATP carrier protein in human sperm (Kim et al., 2007). Having energy production and translocation mechanisms localized to the sperm tail suggests that sperm may rely upon glycolysis for the generation of ATP that is used for motility (Kim et al., 2007). Other studies have demonstrated the importance of glucose for sperm motility. Williams and Ford (2001) showed that the percent of motile human sperm as well as their smoothed path velocity was statistically greater when incubated in media containing glucose. One of the findings in Mukai and Okuno (2004), who measured the beat frequency of mouse sperm flagella, was that the beat frequency was significantly reduced for sperm in media without substrate and that exposure to an electron transport chain inhibitor (antimycin A) did not affect flagellum beat frequency so long as the media contained glucose. They also found that sperm were motile with the mitochondrial substrate, pyruvate, in the absence of glucose, and that the beat frequency was the same in this medium as in the medium containing glucose. Sperm motility was significantly reduced in this medium in the presence of the uncoupler, CCCP, as would be expected if the ATP for motility were being supplied by oxidative phosphorylation with pyruvate as substrate. Krzyzosiak et al. (1999) found that bull sperm were motile in the presence of both antimycin A and rotenone (another electron transport chain inhibitor) only when suspended in media that contained glucose. Miki et al. (2004) found that motility (measured by CASA) was reduced in sperm from mice lacking a sperm-specific glycolytic enzyme (glyceraldehyde 3-phosphate dehydrogenase-S, GAPDS) compared to the wild-type with glucose as substrate. In this article, laser tweezers, fluorescent imaging, and RATTS (Nascimento et al., 2008; Shi et al., 2008) are combined to study the roles of two sources of ATP: oxidative phosphorylation, which occurs in the mitochondria located in the sperm midpiece, and glycolysis, which occurs along the sperm tail (flagellum). Since previous studies showed that there is a relationship between VCL and Pesc (Nascimento et al., 2006) and that high MMP correlated with increased motility (Marchetti et al., 2002), the custom system is first used to analyze the relationships between MMP (i.e., oxidative phosphorylation) and sperm swimming force and swimming speed in domestic dog and human species. Second, the effects of different media, the presence/absence of glucose in the media, and both oxidative phosphorylation and glycolytic inhibitors on human sperm motility (VCL, Pesc) and MMP are studied. The results provide quantitative evidence supporting the theory that glycolysis is a critical pathway for the generation of energy (ATP) for human sperm motility.