Metabolism of motile zebrafish sperm

Comparative Biochemistry and Physiology, Part A 158 (2011) 461–467

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Comparative Biochemistry and Physiology, Part A j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p a

Metabolism of motile zebrafish sperm R.L. Ingermann ⁎, C.L.F. Schultz, M.K. Kanuga, J.G. Wilson-Leedy 1 Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, ID 83844-3051, USA

a r t i c l e

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Article history: Received 11 August 2010 Received in revised form 1 December 2010 Accepted 2 December 2010 Available online 13 December 2010 Keywords: Creatine kinase Danio rerio Fish Energetics Motility Oxidative phosphorylation

a b s t r a c t As metabolism of motile fish sperm is not well understood, the current study examined the metabolism of saline-activated zebrafish (Danio rerio) sperm. Activation of sperm with inhibitors of oxidative phosphorylation (potassium cyanide, 2,4 dinitrophenol or carbonyl cyanide 3-cholorophenylhydrazone) negatively impacted sperm motility by 60–90 s postactivation. Incubation of quiescent sperm with 2,4 dinitrophenol prior to activation resulted in a 67% decrease in the percent motile sperm assessed 15 s postactivation. Thus, production of ATP in quiescent sperm is important for motility upon activation and nascent ATP production via oxidative phosphorylation by motile sperm appears important at 60–90 s postactivation. Exposure of sperm to iodoacetamide, an inhibitor of creatine kinase, at activation was without effect. However, incubation of quiescent sperm with iodoacetamide prior to activation resulted in a 77% reduction in percent motile sperm and decreased velocity and wobble at 15 s postactivation. These results suggest that creatine kinase and phosphocreatine shuttle are physiologically important at, or shortly after the initiation of motility. Finally, sperm were exposed to lactate, pyruvate, or acetate as well as to several monosaccharides upon activation. The results provided no evidence supporting any metabolic role of exogenous organics (potentially from the female via ovarian fluid) in sperm once motility has begun. © 2010 Elsevier Inc. All rights reserved.

1. Introduction The fertility of fish sperm is largely based on sperm motility (Rurangwa et al., 2004) and the motility of sperm of many externally fertilizing fishes appears based primarily on the ATP stored prior to the onset of motility rather than upon ATP generated once motility is initiated (Christen et al., 1987; Perchec et al., 1995). The extent to which nascent ATP production occurs while the sperm is motile may be limited as the duration of sperm motility in water tends to be very short, often less than 60 s in many species (e.g., Huxley, 1930; Billard, 1978; Scott and Bayness, 1980; Cosson et al., 1985; Lahnsteiner et al., 1997; He and Woods, 2003). In addition to limitations in ATP storage and production, the brevity of motility appears associated with the deleterious effect of a hyposmotic medium on sperm structure and function (Schlenk and Kahmann, 1938; Billard, 1978; Benau and Terner, 1980; Morisawa et al., 1983; Christen et al., 1987). For example, activation of carp (Cyprinus carpio) sperm with a hyposmotic medium is associated with a coiling of the flagellum and swelling of the head with a 3-fold increase of cell volume (Marian et al., 1993; Perchec et al., 1996). However, the motility of sperm of numerous teleosts can be reinitiated upon activation following incubation with

⁎ Corresponding author. Tel.: +208 885 6280; fax: + 208 885 7905. E-mail address: rolfi@uidaho.edu (R.L. Ingermann). 1 Current address: School of Medicine & Dentistry, University of Rochester, Rochester, NY 14627, USA. 1095-6433/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2010.12.008

saline (Schlenk and Kahmann, 1938; Sneed and Clemens, 1956; Christen et al., 1987; Redondo-Müller et al., 1991; Perchec et al., 1995) indicating that the brief duration of motility is not solely the result of irreversible osmotic damage. The short duration of sperm motility in these fishes is associated with a large, but not complete, depletion of ATP levels (Christen et al., 1987; Billard and Cosson, 1992; PerchecPoupard et al., 1998; Dreanno et al., 1999a; Fauvel et al., 1999; Cosson, 2010). These findings suggest that the brief motility of teleost sperm is primarily due to limited quantities of stored ATP prior to the onset of motility and to the limited capacity of the metabolic machinery to support the flagellar apparatus as well as other ATP dependent processes with sufficient nascent ATP. As isotonic ovarian fluid is released with eggs (Ellis and Jones, 1939; Czihak et al., 1979; Lahnsteiner et al., 1997), sperm are exposed to this fluid immediately prior to fertilization. The presence of ovarian fluid has been shown to have positive effects on sperm motility in several freshwater teleosts (e.g., Rucker et al., 1960; Litvak and Trippel, 1998; Turner and Montgomerie, 2002, Woolsey et al., 2006). For example, ovarian fluid prolongs the motility of trout (Salmo trutta) sperm to over 5 min (Lahnsteiner, 2002) and those of the threespined stickleback (Gasterosteus aculeatus) from less than 60 s to 7 h (Elofsson et al., 2003, 2006) and its presence increases sperm velocity in the Arctic charr (Salvelinus alpinus) (Turner and Montgomerie, 2002; Urbach et al., 2005). In at least some of these fishes, the observed effects of ovarian fluid can be mimicked by the saline component of this fluid (Lahnsteiner, 2002; Elofsson et al., 2006). It is conceivable that ovarian fluid may provide organic nutrients that can

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provision the sperm in addition to providing a more favorable osmotic environment (Ingermann, 2008). Despite the use of the zebrafish as a model organism in biological research, little is known about the basis for sperm motility in this species. A decrease in intracellular K+ is known to be associated with the onset of motility in zebrafish sperm (Takai and Morisawa, 1995). Furthermore, zebrafish sperm motility is appreciably prolonged in a buffered saline as opposed to tap water (Wilson-Leedy et al., 2009). In the current study, we utilized this observation and specific inhibitors to probe the metabolic basis of motile, as opposed to quiescent, sperm. Sperm were exposed, either prior to or upon activation, to an inhibitor of cytochrome oxidase (potassium cyanide [KCN]), or to uncouplers of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone [CCCP] and 2,4 dinitrophenol [2,4 DNP]) to assess the importance of oxidative phosphorylation to motile sperm. (These inhibitors have been used in similar studies; e.g., Inoda et al., 1988; Lahnsteiner et al., 1999). To establish whether creatine kinase, and the reversible reaction it catalyzes (phosphocreatine [PCr] + MgADP− + H+ ↔ creatine [Cr] + MgATP2−), are important to actively swimming sperm, sperm were exposed, either prior to or upon activation to the inhibitor of creatine kinase, iodoacetamide (Kindig et al., 2005). Finally, we investigated whether the presence of exogenous nutrients (including monosaccharides, pyruvate, acetate and lactate), as potentially supplied by ovarian fluid, has positive effects on sperm as assessed by the percent of sperm that become motile upon activation and their velocity, straightness of swimming and wobble (an indicator of forward swimming efficiency). The study predicted that 1) inhibitors of important metabolic processes would reduce the percent of motile sperm upon activation and 2) that exogenous nutrients would increase the percent of motile sperm if sperm have the capability to take up, metabolize and generate ATP from those nutrients. 2. Materials and methods 2.1. Collection and handling of biological samples The original stock of zebrafish (Danio rerio) was obtained from Scientific Hatcheries (Scientific Hatcheries, Huntington Beach, CA, USA) and had been housed and bred for more than 30 generations in the University of Idaho recirculating zebrafish facility built by Aquaneering Inc. (San Diego, CA, USA). Fish were fed twice daily with flake food and live brine shrimp and maintained, year round, at 28.5 °C on a 14 h light:10 h dark photoperiod. All animals were used and cared for in accordance with guidelines of the University of Idaho Animal Care and Use Committee (protocol #2009-24). Fish were anaesthetized with 170 mg/L of NaOH-neutralized tricaine followed by decapitation. Fish were blotted dry and the testes were removed and immediately placed in 50 μL of ice-cold sperm immobilizing solution (SIS; in mM: 140 NaCl, 10 KCl, 2 CaCl2, 20 HEPES titrated to pH 8.5 with NaOH). The testes were minced and gently mixed by hand as described (Jing et al., 2009). With the exception of the sperm suspension which was kept on wet ice, all solutions were maintained and data were generated at room temperature (20–23 °C) and within 8 h of sperm collection. Activating solution osmolalities (Table 1) were determined using a VAPRO 5520 vapor pressure osmometer (Wescor Inc., Logan, UT, USA); pH was determined with a model 815 MP Accumet pH meter with an Accu pHast electrode (Fisher Scientific, Pittsburgh, PA, USA) calibrated at 20 °C. Reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Activation of sperm for analysis To establish the influence of water versus saline as an activation medium, sperm were activated in deionized water or in sperm activating saline (AS; in mM: 40 NaCl, 20 HEPES, titrated to pH 8.5 with NaOH).

Table 1 Mean osmotic concentrations of saline activating solutions: controls (AS and AS + DMSO) and AS containing uncouplers of oxidative phosphorylation (CCCP + DMSO or 2,4 DNP), an inhibitor of oxidative phosphorylation (KCN), an inhibitor of creatine kinase (iodoacetamide) and potential nutrients (sodium acetate, pyruvate, lactate and monosaccharides). N ≥ 3; SEM b 3% of the mean. Activating solution

mmol/kg

Activating solution

mmol/kg

AS AS + DMSO (0.1%, v/v) 50 μM CCCP 5 mM 2,4 DNP 10 mM KCN 2 mM iodoacetamide 5 mM sodium acetate 5 mM pyruvate

102 119 120 111 113 103 114 114

5 mM lactate 5 mM D-mannitol 5 mM 3-O-methyl-D-glucose 5 mM D-glucose 5 mM D-fructose 5 mM D-xylose 5 mM D-galactose

113 109 109 111 108 106 108

To determine whether oxidative phosphorylation contributed to the metabolism of motile sperm, two experiments were conducted. First, sperm were activated in the presence of 5 mM 2,4 DNP or 10 mM KCN, each dissolved in AS, relative to the AS control. Sperm were also activated in the presence of 50 μM CCCP dissolved in dimethyl sulfoxide (DMSO; final concentration in activating medium: 0.1%, v/v) relative to the AS + DMSO control. AS containing 2,4 DNP was titrated to pH 8.50 with NaOH; AS containing KCN was titrated to pH 8.50 with HCl. Second, to determine whether exposure of quiescent sperm to these inhibitors impacted subsequent motility, sperm suspended in SIS were mixed 2:1 with SIS containing 15 mM 2,4 DNP or 30 mM KCN, incubated at room temperature for 30 min then activated with AS containing 5 mM 2,4 DNP or 10 mM KCN and motility assessed. To determine the effect of creatine kinase inhibition on sperm motility, sperm were exposed to iodoacetamide upon activation or prior to and during motility. Sperm were activated with AS containing 2 mM iodoacetamide previously titrated to pH 8.50 with NaOH. Also, to establish whether exposure of quiescent sperm to this inhibitor had subsequent effects on motile sperm, sperm in SIS were mixed 2:1 with SIS containing 6 mM iodoacetamide, pH 8.50, incubated at room temperature for 30 min then activated with AS containing 2 mM iodoacetamide and motility assessed. To test the hypothesis that the presence of exogenous nutrients can impact sperm motility, sperm were activated in AS containing 5.0 mM sodium acetate, pyruvate or lactate (titrated to pH 8.50). Sperm were also activated in AS containing 5.0 mM D-glucose, D-fructose, D-xylose, D-galactose, 3-O-methyl-D-glucose or D-mannitol. Sperm motility parameters were obtained by computer assisted sperm analysis (CASA) as previously described (Wilson-Leedy and Ingermann, 2007; Wilson-Leedy et al., 2009). Glass slides were coated with 1% (w/v) polyvinyl alcohol (Sigma Aldrich) to reduce the sperm sticking. An 8-well, 0.5 mm deep CoverWell perfusion chamber gasket (Invitrogen, Carlsbad, CA, USA) was attached to the coated slide and activations were conducted within this chamber. Sperm were activated by diluting 1 μL of sperm suspension into 20 μL of deionized water, AS or all combinations of AS plus other reagents. Motility data were collected at 97 fps and recorded for 300 s after activation. 2.3. Statistical analysis Each experiment was conducted with sperm from 5 fish. For percent motile sperm N = 5. Determinations of velocity (curvilinear velocity, μ/s), straightness and wobble were of motile sperm in a treatment; samples with less than 25 motile sperm were not analyzed and therefore for these motility parameters N ≤ 5. (Straightness is determined as straight line velocity divided by velocity average path; wobble is determined as velocity average path divided by curvilinear velocity; Wilson-Leedy et al., 2009. Straightness represents the trajectory of swimming; wobble represents the average distance traveled by the sperm versus the distance traveled by the sperm head

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per unit time and thus, is an indicator of forward movement efficiency.) Percent motile sperm, straightness and wobble data were subjected to arcsine square root transformation before statistical analysis. Data comparing percent motility of sperm activated in deionized water versus AS (Fig. 1) were analyzed by Student's t-test as were the data comparing sperm motility parameters generated in the presence of inhibitors of oxidative phosphorylation to the AS control at 60 and 90 s in Fig. 3. Of particular interest were the comparisons between treatment and control at specific times rather than as changes over time. Data in Figs. 2, 4–6 were analyzed by one-way analysis of variance (ANOVA). To isolate significant treatments, Fisher LSD method was used to conduct pairwise multiple comparisons for Figs. 4 and 5. The statistical package used for these analyses was SAS version 9.1 (SAS Institute, Cary, NC, USA). Alpha (α) was set at 0.05 and significant P values b 0.05. 3. Results Motility of sperm activated by dilution in deionized water decreased to about 3% at 120 s; sperm activated in AS demonstrated about 55% and 35% motility at 120 s and 300 s, respectively (Fig. 1). Subsequent experiments evaluated the motility of sperm following activation with AS with or without various compounds. 3.1. Inhibition of oxidative phosphorylation To determine whether oxidative phosphorylation contributed to the metabolism of motile sperm, sperm were activated in the presence of 50 μM CCCP, 5 mM 2,4 DNP or 10 mM KCN relative to the AS (for 2,4 DNP and KCN) or AS + DMSO (for CCCP) controls (Fig. 2). At 15 s postactivation, there was no discernible effect of these agents on percent motile sperm. There was also no effect on the velocity (curvilinear velocity), straightness or wobble of those sperm that were motile (data not shown). At 300 s, the percent of sperm that were motile was about 2% for each treatment; too few sperm were motile to allow for the other motility characteristics to be assessed. (By comparison, the percent motile sperm of the AS and AS + DMSO controls at 300 s was about 30%.) To better assess the time course of inhibition via interference with oxidative phosphorylation, sperm were activated in the presence of 5 mM 2,4 DNP or 10 mM KCN and the decline in motility assessed over a period of 120 s (Fig. 3). There was no impact of these agents on percent motile sperm or straightness at 60 s; there were significant decreases in percent motility by 2,4 DNP and KCN and in straightness by 2,4 DNP at 90 s. However, a significant effect of both agents was discernible at 60 s postactivation for velocity and for 2,4 DNP on wobble. Incubation of

Fig. 2. Percent motile sperm at 15 s (white) and 300 s (black) after activation with AS or AS containing 0.1% DMSO (v/v; AS DMSO), 50 μM CCCP (+0.1% DMSO), 5 mM 2,4 DNP or 10 mM KCN. N = 5. Groups with different letters are significantly different.

sperm for 30 min at room temperature in SIS with 5 mM 2,4 DNP followed by activation with AS containing 5 mM 2,4 DNP was associated with a 67% decrease in percent motile sperm at 15 s postactivation; 29% motile sperm in the treatment group versus 88% motile sperm in the control (Fig. 4A). At 30 s postactivation, the results were statistically indistinguishable from the 15 s data and by 300 s, the 2,4 DNP-incubated and activated sperm showed no motility (Fig. 4A). 3.2. Inhibition of creatine kinase To assess the role of creatine kinase in sperm motility, sperm were incubated for 30 min at room temperature with or without 2 mM iodoacetamide then activated with AS with or without 2 mM iodoacetamide. Exposure of sperm to iodoacetamide only at activation was without effect relative to AS control at 15 s, 30 s and 300 s postactivation (Fig. 4B). In contrast, incubation of sperm with iodoacetamide, coupled with continued exposure after activation, resulted in a reduced number of motile sperm after activation (Fig. 4B). At 15 s, 20% of sperm exposed to iodoacetamide before and after activation were motile; 88% of controls were motile. Sperm exposed pre- and postactivation to iodoacetamide showed reduced motility at 30 s and 300 s as well (Fig. 4B). Not only did pre- and postactivation exposure of sperm to iodoacetamide reduce the percent of motile sperm, such exposure also resulted in a significant reduction in velocity and wobble, but not straightness within 15 s postactivation (Fig. 5). 3.3. Influence of exogenous potential nutrients The possibility that motile sperm can take up and metabolize exogenous nutrients was assessed by activating sperm in the presence of 5 mM acetate or pyruvate or lactate. The presence of these potential nutrients at activation had no effect on the percent motile sperm at 15 s or 300 s postactivation (Fig. 6A) or on the motility characteristics of those sperm that were motile (data not shown). Similar negative results were obtained when sperm were activated in AS containing, 5 mM D-glucose, D-fructose, D-xylose, D-galactose or the controls, D-mannitol or 3-O-methyl-D-glucose (Fig. 6B). 4. Discussion

Fig. 1. Percent motile sperm versus time after activation with AS or deionized water (DI). Mean ± SEM (all figures); N = 5. Percent motility of sperm activated in AS versus DI differed statistically at all time points at and after 60 s.

Zebrafish sperm activated in saline (AS) demonstrated motility for a greater duration than did those activated in water. This finding is consistent with that found for zebrafish sperm in a previous study (Wilson-Leedy et al., 2009) as well as results with sperm from other

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Fig. 3. Sperm motility parameters after activation with AS, 5 mM 2,4 DNP or 10 mM KCN: A) percent motile sperm, B) velocity (curvilinear, μ/s), C) straightness and D) wobble. N = 5 except for KCN at 90 s (N = 4) and for 2,4 DNP at 120 s (N = 3). Data generated with inhibitors were compared to control at each time point; statistical difference between 2,4 DNP and control are denoted with an “a” and a difference between KCN and control are denoted with a “b”.

teleosts (e.g., Lahnsteiner et al., 1997; Elofsson et al., 2006). The increased duration of motility provided the experimental opportunity to address questions concerning the metabolism of motile zebrafish sperm. Exposure of zebrafish sperm to inhibitors of oxidative phosphorylation at activation resulted in a marked decrease of percent motile sperm by 300 s but not at 15 s postactivation. This suggests that stored ATP is the basis for motility soon after initiation of motility but that prolonged motility relies on oxidative phosphorylation and nascent ATP generation. The effect of the uncoupler, 2,4 DNP, and the inhibitor of cytochrome oxidase, KCN, on percent motile sperm were not apparent until 60–90 s after the onset of motility. Thus, the time required for these agents to penetrate the cell and exert their biological effects is not clear but the results indicate that stored ATP becomes limiting in supporting motility within 60–90 s postactivation. Of note, the inhibition of oxidative phosphorylation impacted not only the percent motile sperm but reduced the velocity of swimming, the trajectory of swimming (straightness) and wobble. The effect on wobble suggests a decrease in the efficiency of the sperm's average forward movement. Therefore, ATP generation and availability are linked to the characteristics of sperm swimming in addition to the number of motile sperm. Zebrafish sperm incubated with 2,4 DNP to inhibit ATP synthesis in quiescent sperm for 30 min prior to activation demonstrated a 67% decrease in the percent of motile sperm at 15 s postactivation. These results are consistent with observations that maintaining trout and carp sperm in a quiescent state with cyanide or under nitrogen to block ATP production results in a decrease in motility (and fertility) upon activation (Christen et al., 1987; Perchec et al., 1995; Bencic et al., 1999). Similar results have been reported for the sperm of the turbot, a marine teleost (Dreanno et al., 1999b). Overall, the current results are consistent with the hypothesis that ATP production is maintained in quiescent fish sperm and is necessary for a) maintenance of ionic balance and b) to ensure sufficient ATP stores are available to support initial phases of motility.

The role of CK was investigated. The creatine kinase/phosphocreatine (CK/PCr) system represents a “temporal energy buffer” regenerating ATP as ADP is produced by ATP hydrolysis (Wallimann et al., 1992). The CK/PCr system also functions in some cell types as a “phosphate energy shuttle” linking the sites of ATP formation/storage and its usage (e.g., Bessman and Carpenter, 1985; Tombes and Shapiro, 1985, 1989; Wallimann et al., 1992; van Dorsten et al., 1997). Salmonid and cyprinid sperm possess CK and PCr and levels of PCr decrease after the initiation of motility (Robitaille et al., 1987; Saudrais et al., 1998; Tombes and Shapiro, 1989; Kamp et al., 1996; Lahnsteiner et al., 1996; Woolsey and Ingermann, 2003). To determine whether the CK/PCr system plays a physiological role in the motility of the zebrafish sperm, sperm were treated with the CK inhibitor, iodoacetamide. Exposure of sperm to iodoacetamide upon activation had no demonstrable effect relative to the control suggesting either that the CK/PCr system has a negligible role in motility or that iodoacetamide does not penetrate the cell quickly enough to yield a discernible effect in this experimental design. That the latter explanation is probably correct was supported by the results obtained after activating sperm (in the presence of iodoacetamide) previously incubated with iodoacetamide for 30 min. Sperm exposed to iodoacetamide prior to activation demonstrated a 77% decrease in the percent of motile sperm at the earliest time point analyzed following activation, 15 s. Thus, while the use of newly generated ATP via oxidative phosphorylation became important after 60 s, the CK/ PCr system appears important within 15 s postactivation. We cannot eliminate the possibility that the CK/PCr system is important only at the initiation of motility. Nonetheless, the current data are consistent with the reported role of this system as an energy shuttle in sea urchin sperm (Tombes and Shapiro, 1985, 1989; van Dorsten et al., 1997) and the interpretation that the shuttle links mitochondrial ATP production and/or ATP stores with the dynein ATPase activity of the zebrafish sperm flagellum. Furthermore, consistent with the results of the inhibition of oxidative phosphorylation (Fig. 3), sperm incubated with the CK inhibitor prior to activation demonstrated reduced velocity

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Fig. 4. Percent motile sperm following exposure to 2,4 DNP or iodoacetamide either at activation or prior to as well as following activation. A) Sperm were incubated for 30 min at room temperature in SIS with (DNP) or without (SIS) 5 mM 2,4 DNP, then activated with AS with (DNP) or without (AS) 5 mM 2,4 DNP and motility assessed at 15 s (white bar), 30 s (gray bar) and 300 s (black bar). N = 5 except for DNP/DNP at 30 s where N = 4 (as one sample had zero motility). B) Sperm were incubated for 30 min at room temperature in SIS with (IA) or without (SIS) 2 mM iodoacetamide, then activated with AS with (IA) or without (AS) 2 mM iodoacetamide and motility assessed at 15, 30 and 300 s. N = 5. Within each treatment group and within each duration postactivation, columns with different letters are significantly different.

and wobble (but not straightness) at 15 s postactivation suggesting that inadequate delivery of ATP to the flagellar dynein ATPase results in changes in sperm motility characteristics. The sperm of externally fertilizing fishes are exposed to eggassociated ovarian fluid at fertilization and this fluid contains organics (Lahnsteiner et al., 1995) which can potentially support the metabolism of motile sperm. Can activated sperm take up exogenous nutrients? Terner and Korsh (1963) demonstrated that trout (Oncorhynchus mykiss) and sunfish (Lepomis sp.) sperm activated by dilution into isotonic saline oxidize labeled, exogenous pyruvate and acetate and to a lesser extent, glucose, to carbon dioxide. Mounib (1967) reported similar findings for sperm from salmon (Salmo salar) and cod (Gadus morhua). Activating sperm of the cyprinid Chalcalburnus chalcoides in saline containing pyruvate results in a marked increase in duration of motility relative to saline controls (Lahnsteiner et al., 1999). Therefore, the sperm of at least some teleosts appear to take up and metabolize exogenous nutrients. Further, that the sperm of numerous teleosts are motile for longer durations in ovarian fluid versus water is consistent with the hypothesis that the ovarian fluid provisions the sperm with nutrients to prolong motility and enhance fertility; conceivably, the female may influence fertility in this way. In the present study, zebrafish sperm were exposed to acetate, pyruvate or lactate at activation; the presence of these organics had no discernible effect on the percent motile sperm at 300 s or their swimming characteristics. Similarly, exposure of sperm to various monosaccharides (as well as nonmetabolizable 3-O-D-methyl glucose and non-transported, D-mannitol)

465

Fig. 5. Sperm were incubated for 30 min at room temperature in SIS with (IA) or without (SIS) 2 mM iodoacetamide, then activated with AS with (IA) or without (AS) 2 mM iodoacetamide and A) velocity (curvilinear, μ/s), B) straightness and C) wobble assessed at 15 s postactivation. N = 5, except for IA/IA incubation where N = 4 (as one sample had zero motility). Within each panel, columns with different letters are significantly different.

had no effects on number of motile sperm or their swimming characteristics. Therefore, the presence of exogenous nutrients was without effect. Consistent with the findings from studies of a freshwater sculpin (Lahnsteiner et al., 1997) and the three-spined stickleback (Elofsson et al., 2006), the findings are consistent with the hypothesis that the ionic composition of the ovarian fluid rather than any nutrient provisioning accounts for the enhanced duration of motility. Hoysak and Liley (2001) and Liley et al. (2002) reported that in salmonids, about 25% of eggs are fertilized within 0.5 s and at least 80% within 5 s after sperm activation in vitro. If similarly rapid fertilization occurs in zebrafish, oxidative phosphorylation and ATP generation after the onset of motility may be inconsequential. However, in contrast to the trout, it appears to take at least 15 min for all eggs of the three-spined stickleback to be fertilized (Bakker et al., 2006; Elofsson et al., 2006). If fertilization takes longer than approximately 2 min in the zebrafish, it is likely that levels of ATP stored prior to motility become depleted and that nascent ATP production is important to maintain motility and complete fertilization. The results of the current study are consistent with the hypothesis that production of ATP in quiescent fish sperm is necessary for adequate motility upon activation and that nascent ATP production via oxidative phosphorylation becomes important at 60–90 s postactivation. The results are also consistent with the interpretation that a functioning creatine kinase and a phosphocreatine shuttle are physiologically important in sperm motility shortly after the initiation of motility. Finally, the results provide no evidence to support any

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Fig. 6. Percent motile sperm at 15 s (white) and 300 s (black) after activation in AS and AS containing potential nutrients. A) Activation in the presence of 5 mM acetate, pyruvate, or lactate. N = 5. B) Activation in the presence of 5 mM D-mannitol, 3-Omethyl-D-glucose, D-glucose, D-fructose, D-xylose or D-galactose. N = 5. There were no differences within the 15 s or 300 s groups in panels A or B. There were no effects of the presence of these potential nutrients at 15 s or 300 s after activation on sperm velocity, straightness or wobble (data not shown).

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