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Gamete Biology |
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ATP, glycolysis, sperm, motility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There are two pathways for ATP production in mammalian sperm, glycolysis and mitochondrial respiration. In several mammals, glycolysable substrates are present in seminal fluid (human [7], bull [8], guinea pig [9]) and female reproductive fluid (pig [10], sheep [11], cattle [12], mouse [13]), and enzymatic activity of glycolysis is detected in mammalian sperm [14, 15]. Glycolysis-related enzymes localize in the tail of boar and murine sperm [16– 19]. In addition, several studies have documented the relationship between glycolysis and capacitation-dependent cell signaling [4]. It was also demonstrated that the glycolysable substrate played significant roles in capacitation of sperm [20, 21] and penetration of zona pellucida [22– 24]. These reports suggest that glucose and/or glycolysis is one of the factors involved in signal transduction via tyrosine phosphorylation.
Although the importance of glycolysis for protein phosphorylation and fertilization has received recognition in recent years, it has not been noticed in the matter of energy production for flagellar movement. In the present study, we focused on the relationship between flagellar movement and energy metabolism following activation. Flagellar movement is the product of dynein ATPase activity that is localized along the entire length of a flagellum and depends on the supply of ATP. Seminal fluid and female reproductive fluid in mammals contain metabolic substrates such as fructose and glucose [8]. Therefore, it is generally assumed that ATP, the substrate of dynein, is supplied via glycolysis and respiration, which metabolize these extracellular substrates. Because respiration at mitochondria is superior to glycolysis in the efficiency of ATP synthesis, it has been widely supposed that, under normal conditions, the ATP required for sperm motility is produced by mitochondrial respiration.
Because a sperm flagellum is very long and thin (mouse sperm is about 150 µm long), diffusion may not be sufficient to transport ATP to the tip of the tail at the rate required for sperm motility [25]. To overcome the difficulty, Tombes and Shapiro suggested that a phosphorylcreatine (PCr) shuttle proposed in muscle mediated the energy transport in sperm [26]. In sea urchin sperm, the enzymes associating with the PCr shuttle were detected [27]. Therefore, PCr shuttle can function successfully as an energy transport system from the mitochondria to the tip of the tail in sea urchin. On the other hand, the enzymes related to the PCr shuttle system are not present in boar and bull sperm [28] and probably have low capacity in other mammalian species [29], including mouse sperm [30]. If this is the case, another mechanism is required to ensure the supply of ATP throughout the length of the sperm tail.
In this article, we demonstrate that glycolysis is very important for mouse sperm motility. We reveal a previously unsuspected role for glycolysis in sperm energy metabolism that may resolve the problem of supplying ATP throughout the length of the mammalian sperm tail.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sperm were obtained from the cauda epididymidis of 10- to 14-wk-old ICR male mice (from Nihon SLC, Shizuoka, Japan) in accordance with the guideline of the University of Tokyo. The epididymis was removed and the dry sperm was gently squeezed out with forceps. For observation before and after the beginning of sperm activation, the dry sperm was first diluted into 100 µl of sucrose solution (300 mM sucrose, 10 mM Hepes-NaOH, pH 7.4), in which sperm exhibited very low activity (about 1 Hz of beat frequency), referred to as the initiated sperm by Fujinoki et al. [31]. The sperm suspension was diluted with the test solution for the following experiments. The test solution contained 150 mM NaCl, 5.5 mM KCl, 0.4 mM MgSO4, 1 mM CaCl2, 10 mM NaHCO3, 10 mM Hepes-NaOH, pH 7.4, and metabolic substrates such as glucose. One volume of sucrose solution containing sperm was diluted into 10 volumes of each test solution, and this suspension was incubated at 37°C for observation.
For inhibition experiments, sperm were incubated in the sucrose solution containing carbonyl cyanide m-chlorophenylhydrazone (CCCP) or antimycin A for 10 min, followed by mixing with the test solution containing the inhibitors for observation. A glucose analogue, 2-deoxy-D-glucose (DOG), was added to the test solution along with other metabolic substrates.
High-quality sperm samples (motility > 70–80%, beat frequency > 15 Hz), assessed as described previously [32], were used within 3 h for experiments.
Observation and Analysis of Sperm Motility
In the present experiments, we focused on the activity parameters of sperm movement rather than percent motility because sperm were activated already by the presence of Ca2+ and bicarbonate [32]. We analyzed the beat frequency and wave form of the flagella.
After dilution to the test solution, an aliquot (40 µl) of sperm suspension was placed on a prewarmed glass slide and immediately covered with a coverslip. We could observe the two types of motile sperm, one swimming freely and the other attaching the head to the glass surface with moving flagellum. For the analysis of beat frequency and wave form, the attached sperm were recorded using a phase-contrast microscope (Optiphoto; Nikon, Tokyo, Japan) and a video system (CCD camera; Hitachi, Tokyo, Japan; VTR; Mitsubishi, Tokyo, Japan). Observations and recording were made at 37°C using a warm plate placed on the stage. Most of the epididymal sperm beating stably exhibited similar activity. We chose at least 15 head-attached sperm randomly in each experimental condition for the beat frequency measurement. The measurements were repeated three times, each with sperm from a different mouse and averaged.
The analyses were performed from video recording frame by frame. Images of the attaching sperm flagellum were traced from a video monitor onto transparent plastic sheets using a fine-point marker. Beat frequency and amplitude were calculated from the period required for one complete beat cycle. Wave form was determined from the traces of one complete beat.
Evaluation of Mitochondrial Activity
Sperm mitochondrial activity was evaluated by staining with JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide). JC-1 was dispensed from 1 mM stocks in DMSO and diluted to 10 µM in sucrose solution and then mixed with one-tenth volume of the sperm suspension. After loading for 20 min at room temperature, one volume of sample was diluted to 10 volumes of the test solution. The diluted sperm solution was transferred into the quartz cuvette, and the fluorescence of JC-1 (excitation, 490 nm; emission, 530, 590 nm) was measured by fluorescent spectrophotometer (F-4500; Hitachi). The quantitative data were displayed using the fluorescent intensity (FI) ratio of 530 nm to 590 nm. Moreover, phase-contrast fluorescent microscopy was used to visualize the sperm, and images were recorded on a digital camera (Cool Pix 950; Nikon). We recorded the sperm attached to the slide glass or cover glass.
Measurement of ATP by Reversed-Phase HPLC
We measured the amount of ATP contained in mouse sperm by reversed-phase HPLC (LC10VP series; Shimadzu, Kyoto, Japan) [33]. Sperm suspended in the test solution were incubated at 37°C for 20 min, quickly mixed with one-tenth volume of ice-chilled 3% perchloric acid (PCA) to remove proteins, and incubated for an additional 10 min on ice. The solution was centrifuged and the supernatant was filtered with a membrane of 0.22-µm pore size. Fifty microliters of filtered supernatant was applied to a reversed-phase HPLC column (Phenomenex Luna 5µC18, 4.6 x 150 mm; Shimadzu GLC, Tokyo, Japan). The eluent was prepared with 100 mM potassium phosphate (pH 6.8) and 1 mM tetrabutyl-ammonium hydroxide, and 7.5% methanol. The number of sperm cells was counted in each sample, and the content of ATP was calibrated per 106 sperm.
Statistical Analysis
Values for beat frequency, ATP content, and fluorescent intensity ratio were expressed as the mean and standard deviation (SD). Statistical tests were performed using one-way analysis of variance followed by least squares differences as multiple range test.
Reagents
CCCP and antimycin A were purchased from Sigma Chemical Co. (St. Louis, MO), JC-1 was from Molecular Probe (Eugene, OR), and other chemicals were reagent grade from Wako Pure Chemicals Co. Ltd. (Osaka, Japan).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We first confirmed the support of sperm flagellar movement by exogenous substrates. Cauda epididymal sperm involve mature flagellar apparatus so that they exhibit approximately 20 Hz of beat frequency in the test solution containing calcium and bicarbonate in addition to glucose (see Materials and Methods). The beat frequency was higher than that reported previously [32] because the solutions for the previous experiment contained only the minimal ingredients such as sucrose, CaCl2, and Hepes buffer. The present study revealed that the flagellar beat was maintained during the period of observation, from immediately after dilution to 30 min of incubation.
Figure 1 shows the changes in beat frequency (A) and the waveform (B–E) of flagella with or without metabolic substrates. When substrate was absent, the beat frequency gradually decreased with time and, after 10 min, sperm no longer exhibited vigorous motility. After 20 min, the beat frequency was about 1 Hz and both the wavelength and the wave amplitude, especially in the distal part of flagella, increased (Fig. 1E). On the other hand, sperm kept vigorous motility both in beat frequency and waveform for more than 30 min in the solutions containing metabolic substrates such as glucose, fructose, pyruvate, and lactate. Pyruvate and lactate, substrates for respiration, could support movement for more than 30 min as well as glucose and fructose. The beat frequency was not significantly different among them.
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Sperm Motility Can Be Maintained by Glycolysis
CCCP [34], an uncoupler, was used to inhibit mitochondrial ATP production. As shown in Figure 2A, sperm motility supported by substrates of respiration (pyruvate or lactate) was significantly inhibited by CCCP. The beat frequency decreased down to the same level observed in the substrate-free solution (Fig. 2A). When glucose was present in addition to CCCP, however, sperm maintained high beat frequency (about 20 Hz) for more than 30 min (Fig. 2B). The waveform was similar to those observed without CCCP (data not shown). Sperm motility in the presence of fructose was the same as that of glucose (data not shown). Antimycin A, another respiratory inhibitor [35], inhibited sperm motility like CCCP (Fig. 2, A and B).
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We assessed the activity of mitochondria by means of fluorescence of JC-1. When the membrane potential of inner mitochondrial membrane is high, indicating the active state, JC-1 emits orange (590 nm) light, while green (530 nm) light is emitted at low membrane potential [36]. As shown in Figure 3A, mitochondria exhibited orange emission in the presence of glucose representing the active state. Addition of CCCP reduced the mitochondrial inner membrane potential as an uncoupler (Fig. 3B) because of green emission from mitochondria. When glucose was present in addition to CCCP, mitochondria showed the green fluorescence; however, it exhibited high beat frequency. Furthermore, we employed the FI ratios (595 nm/535 nm) for the mitochondrial activity. The low FI ratio (0.351, P < 0.05) implied that mitochondrial activity was suppressed (Fig. 3D).
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For the next step, the amount of ATP was directly measured by HPLC. Figure 4 showed the ATP content 20 min after the incubation. There was about 200 pmol ATP per 106 sperm in the absence of substrate and about 1000 pmol ATP per 106 sperm in the presence of glucose. Even when sperm were treated with CCCP, there was about 900 pmol ATP per 106 sperm in the presence of glucose. This amount of ATP was almost equivalent to the level observed without CCCP. When glucose was substituted for pyruvate, the amount of ATP was also about 900 pmol per 106 sperm unless CCCP was present. However, the amount of ATP drastically decreased down to that observed without substrate when CCCP was added. This result indicates that ATP production from pyruvate results from mitochondrial activity.
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Inhibition of Glycolysis by a Glucose Analogue, DOG
Although it appeared that the glycolysis was sufficient for sperm motility, the possibility was not eliminated that glycolysis might be activated to compensate for suppression of respiration. Therefore, DOG was used to inhibit glycolysis. DOG can enter into the cell and is phosphorylated to DOG-6-phosphate by hexokinase. DOG-6-phosphate, however, cannot be metabolized further [37, 38]. DOG was found to be a competitive inhibitor to glucose with respect to beat frequency (Fig. 5, A and B). DOG did not completely inhibit motility in the presence of glucose because the apparent Km for glucose (0.027 mM) was much smaller than Ki for DOG (0.752 mM). Then we examined the effect of DOG more closely. We confirmed that DOG had no effect on mitochondrial respiration by means of fluorescence of JC-1 (Fig. 3). When DOG and either glucose or pyruvate were present, mitochondria emitted orange light, suggesting that DOG did not affect the mitochondrial activity (Fig. 3C) and the FI ratios of pyruvate with DOG and glucose with DOG were not significantly different from the ratio of glucose or pyruvate only (Fig. 3D).
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Glycolysis Has a Major Role for Sperm Motility
Although DOG did not suppress the activity of mitochondria, DOG inhibited the motility induced by pyruvate or lactate (Fig. 6A). Figure 6B showed also that the suppression of motility was rapidly reversed by addition of glucose (up to 12 Hz of beat frequency within 1 min), while addition of other substrates failed to recover motility. The recovery attained by glucose was dose dependent (data not shown).
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When sperm activated with pyruvate or lactate were exposed to DOG, the wave amplitude and beat frequency decreased drastically (Fig. 6B). Flagella only exhibited twitching in the proximal region, with almost straight distal region (left of Fig. 6B). The recovery with glucose induced large amplitude of bend and full propagation to the distal end. Therefore, the effect of DOG is reversible.
Then we examined ATP contents in the presence of DOG. As shown in Figure 7, ATP was about 200 pmol per 106 sperm in the pyruvate solution with DOG. This amount was equivalent to that measured in sperm in substrate-free solution. Because DOG does not inhibit ATP synthesis in mitochondria, these observations indicate that normal mitochondrial respiration is not sufficient to maintain the concentration of ATP that is required for sperm flagellar motility.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been demonstrated that the beat frequency of flagella is proportional to the rate of ATP hydrolysis by dynein when the waveform is kept constant [40]. Therefore, we measured the beat frequency of flagella as the parameter of motility. Because the mouse sperm rotate during free swimming [32], we chose the sperm with their heads attached to the glass surface and recorded these on videotape for further analysis. Although the beat frequency is a little bit lower in head-attached sperm compared with that of freely swimming ones, it shows the same linear relationship on double reciprocal plots against the concentration of ATP [40].
In substrate-free solution, sperm were activated with high beat frequency, about 20 Hz, and the motility rapidly decreased down to about 1 Hz. In the present experiment, cauda epididymal sperm were directly diluted in the experimental solution without washing. Therefore, the residual motility might be due to substrates that remained endogenously and/or in epididymal fluid. It was demonstrated that sperm kept the motility for a long time in the presence of substrate such as glucose (Fig. 1).
For assessing metabolic activities, we examined the amount of ATP and the activity of mitochondria by means of fluorescent probe, JC-1. The JC-1 probe provided an advantage for the vital staining of sperm maintaining the motility. Measurement of ATP revealed that the high concentration of ATP, approximately 1000 pmol/106 sperm, corresponded to the high beat frequency at 20 Hz and the low concentration of ATP, 200 pmol/106 sperm did to the low beat frequency, less than 1 Hz. The beat frequency of the intact sperm seems to be dependent on the ATP concentration, as observed in the demembranated sperm flagella [35].
We detected a considerable amount of ATP in the immotile sperm in substrate-free solution (Fig. 4). If sufficient ATP were present in the principal piece of flagella, sperm should move because flagella in the present experiment were functionally activated by the presence of HCO3– and Ca2+ [32]. It might be expected, therefore, that the concentration of ATP should be very low in the principal piece of flagella when motility is very low. However, this must be modified by recognition that reduction in ATP concentration will be accompanied by an increase in ADP concentration, which acts as a competitive inhibitor of flagellar motility [40]. It has been reported that the sperm cell is compartmentalized into head, midpiece, and principal piece [41]. Thus, we postulated that the ATP detected with sperm suspended in substrate-free solution was localized in the midpiece, where mitochondria exist, and head of sperm. It is likely that ATP in the midpiece and head remains there and is not transported throughout the tail at a rate sufficient to support normal flagellar movement. The hypothesis is supported by the evidence that flagella inhibited by DOG exhibited vibration with small amplitude in the midpiece region, which did not propagate in the principal piece. In addition, we could assume that the ability of ATP synthesis by respiration was much lower than that by glycolysis in respect to the results of a series of inhibitor experiments (Figs. 4 and 7).
When glucose was present, sperm showed high beat frequency and produced large amounts of ATP. Therefore, it could be assumed that the concentration of ATP was high in the principal piece. Then how is ATP supplied in the principal piece? It is usually believed that ATP is synthesized in mitochondria by respiration and supplied to the tip of the flagella by diffusion. If this is the case, mitochondria play significant roles in energy production. Present experiments, however, provide evidence against this idea. Even when CCCP or antimycin A was present, flagella exhibited high beat frequency in the presence of glucose, while pyruvate or lactate failed to maintain the motility. In addition, these inhibitors could not suppress ATP synthesis of sperm in the presence of glucose, while they succeeded in decreasing ATP concentration when only pyruvate or lactate was present without glucose. Moreover, if ATP is synthesized at mitochondria, ATP should be supplied to the entire length of flagellum by diffusion. It seems unlikely because the creatine shuttle demonstrated in sea urchin sperm [26] has not been reported in mouse sperm [30] and flagella of mouse sperm are more than three times longer than those of sea urchins.
An alternative explanation is suggested by the present experiments indicating an unexpectedly important role for glycolysis. Sperm continued both high activity of beating and high concentration of ATP in the presence of glucose even when the activity of mitochondria was suppressed by inhibitors such as CCCP and antimycin A. These results showed that ATP produced from glycolysis was sufficient for flagellar movement. Moreover, it has been reported that the several key enzymes of glycolysis are localized in the principal piece of the flagellum [16–19]. Because dynein ATPase localizes along the entire length of flagella for generating active bending force, it seems quite reasonable that glycolysis predominantly provides ATP for dynein ATPase in the entire length of the flagella. In addition, the beat frequency of flagella induced by glucose was inhibited competitively by DOG (Fig. 5). Several observations that support present results have been reported. Recently, it was reported that glycolysis-related enzymes and hexose transporters localized at tail of boar and murine sperm [16–19]. It is known that lactate is produced in motile bull sperm [8]. We detected lactate produced in the presence of glucose in mouse sperm (unpublished observation). In addition, it has been reported that the seminal plasma is rich in lactate, probably due to motile sperm, in several mammals such as bull [8], human [42], and guinea pig [43]. The fluid of the oviduct, where sperm meets ovum for fertilization, also contained large amount of lactate in sheep [44] and pig [45]. Our results suggest that sperm could produce lactate by glycolysis and might consume it by another pathway described below.
A difficulty arises that sperm motility is maintained for a long time with the substrate for respiration, pyruvate, without glucose. Recently, Marin et al. [46] reported that glycolysis played a significant role in energy source in boar sperm. They demonstrated the production of lactate/pyruvate and a little glycogen. However, it looks hard to apply the system to mouse sperm because our observation revealed that pyruvate could keep vigorous motility for a long time as well as glucose in mouse sperm. Then we would like to introduce the hypothesis shown in Figure 8. When there are extracellular substrates for glycolysis, sperm metabolize those substrates by glycolysis to provide energy for flagellar movement. On the other hand, when there is little substrate for glycolysis, sperm metabolize respiratory substrates. We suggest that these respiratory substrates function as substrates for gluconeogenesis in the midpiece, resulting in glucose that can diffuse to other regions of the sperm flagellum. The inhibition of flagellar movement by DOG might be explained as a result of the competition of DOG to glucose and/or glucose-6-phosphate as products of gluconeogenesis [37, 38]. This hypothesis should be proven in further investigations. It is very interesting to examine if this glycolysis model is suitable for other mammalian sperm.
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Our results demonstrate that sperm motility could not be maintained in the presence of respiratory substrates unless glycolysis is functional. Thus, glycolysis should play a major role in supplying ATP for sperm motility that may be at least as significant as the production of substrates for respiration or the support of motility under anaerobic conditions. Sperm must maintain motility to reach the oocyte in the female reproductive tract. Although mitochondrial respiration is highly efficient for using substrates to produce ATP, in the absence of a phosphocreatine shuttle, glycolysis must play a major role in providing energy throughout the entire tail in mouse sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received: 26 December 2003.
First decision: 14 January 2004.
Accepted: 30 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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15. The beat frequency of flagella at 5 min after the onset of incubation in the substrate-free solution is significantly different than others (P < 0.01). B–E) Waveform of flagella with various substrates 20 min after dilution. B) Glucose, (C) lactate, (D) pyruvate, and (E) substrate free. Concentration of substrates is 5 mM. In each figure, several trace lines from one beat cycle are superimposed. Sperm showed 1 Hz of beat frequency in substrate-free solution and about 20 Hz in other conditions. Arrowheads show the tip of the sperm head
