Transient Ca²⁺ mobilization caused by osmotic shock initiates salmonid fish sperm motility

Sperm motility is indispensable for fertilization success. Fish sperm are known to be motile for very short periods, usually limited to minutes (Cosson, 2010). Thus, they must be kept immotile until they come into contact with female gametes. Therefore, initiation and activation of sperm motility must be precisely controlled in order to prevent exhaustion prior to ejaculation.

The sperm of teleosts are kept immotile in the testis. Upon spawning, their motility is triggered by exposure to an environment different from seminal plasma (Alavi and Cosson, 2006). Teleosts inhabit, and reproduce in, various environments. Therefore, initiation and activation of teleost sperm motility have adapted to these various environments. For example, sperm of freshwater teleosts, such as common carp and zebrafish, are immotile in an isotonic environment (∼300 mosmol kg^–1). When these sperm were suspended into a hypotonic condition (<300 mosmol kg^–1, such as fresh water), they initiate motility in a Ca²⁺-dependent and cAMP-independent manner (Krasznai et al., 2000; Takai and Morisawa, 1995; Morisawa and Suzuki, 1980). By contrast, sperm of marine teleosts, such as flounder and puffer fish, become motile in a Ca²⁺-dependent and cAMP-independent manner (Oda and Morisawa, 1993; Takai and Morisawa, 1995) when released into hypertonic conditions (>300 mosmol kg^–1, such as sea water). Moreover, sperm of tilapia, a euryhalin teleost, activate their motility by a change in osmolarity between the seminal plasma and the external medium. Tilapia sperm, which have acclimated to fresh water, activate their motility by hypo-osmotic shock, whereas tilapia sperm, which have acclimated to seawater, activate motility by hyper-osmotic shock. The signaling pathway of tilapia sperm is Ca²⁺ dependent and cAMP independent (Morita et al., 2003). These facts suggest that osmotic shock and a Ca²⁺ change commonly play an important role in sperm motility activation in various teleosts.

By contrast, sperm motility of salmonid fishes is suppressed by the millimolar concentrations of extracellular K⁺ in seminal plasma, and a decrease in extracellular K⁺, upon spawning, triggers the signaling for initiation of sperm motility (Morisawa and Suzuki, 1980). The decrease in K⁺ causes efflux of K⁺ through certain K⁺ channels and is followed by hyperpolarization of the plasma membrane (Tanimoto et al., 1994; Boitano and Omoto, 1991). Subsequently, the intracellular Ca²⁺ concentration increases (Cosson et al., 1989; Boitano and Omoto, 1992; Tanimoto et al., 1994), stimulating synthesis of cAMP (Morisawa and Ishida, 1987). The increase in cAMP leads to the initiation of sperm motility through cAMP-dependent protein phosphorylation of the axonemal components, including the 15-kDa protein and 22-kDa dynein light chain (Morisawa and Okuno, 1982; Morisawa and Hayashi, 1985; Hayashi et al., 1987; Inaba et al., 1998).

However, Morita and colleagues (Morita el al., 2005) reported that treatment of sperm with a 10% glycerol solution, before subsequent dilution into a low-osmotic solution, induced the initiation of motility even though a high concentration of extracellular K⁺ – for example, 100 mmol l^–1 – was present. It was also reported that some axonemal components, including the 22-kDa dynein light chain, were phosphorylated in the glycerol-treated sperm in the absence of cAMP synthesis. The detected pattern of phosphorylation was different from that formed with accompanying cAMP synthesis. This result indicates the presence of a novel salvaging pathway for salmonid fish sperm motility initiation that bypasses K⁺ efflux and the synthesis of cAMP. However, a detailed mechanism of motility initiation due to glycerol treatment remains unclear, and certain questions arise concerning which effect of glycerol treatment induces sperm motility and what is happening downstream from the glycerol treatment.

Although this glycerol-treated initiation of sperm motility is an artificial, yet specific, means of initiation, elucidation of this mechanism and comparison with conventional signaling pathways are expected to give us valuable information for understanding the signaling mechanism for salmonid fish sperm motility. Thus, in the present study, we investigated the motility initiation mechanism by glycerol treatment, specifically focusing on the triggering factor for initiation and the involvement of Ca²⁺, using the fluorescent dye Fluo-4. Finally, we revealed that the motility of salmonid fish sperm could be initiated in an osmolarity- and Ca²⁺-dependent manner similar to that of the sperm of other teleosts.

Chelerythrine, calphostin C, H-89, H-85, BAPTA-AM, U 73122 and U 73343 were purchased from Merck KGaA (Darmstad, Germany). Dithiothreitol (DTT) was purchased from Sigma (St Louis, MO, USA). EGTA and Fluo-4 AM were purchased from Dojindo (Kumamoto, Japan). All other chemicals were reagent grade and were purchased from Wako Pure chemicals (Osaka, Japan).

Glycerol solution consisted of 0.5–2 mol l^–1 glycerol (or other substances), 150 mmol l^–1 KCl, 0.5 mmol l^–1 DTT, 0.5 mmol l^–1 EDTA and 20 mmol l^–1 Hepes–NaOH, pH 8.0. Sodium test solution (STS) contained 100 mmol l^–1 NaCl, 10 mmol l^–1 Hepes–NaOH, pH 8.0, and potassium test solution (PTS) contained 100 mmol l^–1 KCl and 10 mmol l^–1 Hepes–NaOH, pH 8.0. Artificial seminal plasma (ASP) consisted of 130 mmol l^–1 NaCl, 40 mmol l^–1 KCl, 2.5 mmol l^–1 CaCl₂, 1.5 mmol l^–1 MgCl₂ and 10 mmol l^–1 Hepes–NaOH, pH 8.3. The composition of ASP was based on the data of Kho and colleagues (Kho et al., 2001), with slight modification.

BAPTA-AM and inhibitors were dissolved in dimethylsulfoxide (DMSO), and the final concentration of DMSO in all experimental solutions was set below 0.5% (v/v). For control experiments, the same concentration of DMSO was added to each experimental solution.

In the present study, the sperm of the rainbow trout Oncorhynchus mykiss Walbaum 1792 and chum salmon Oncorhynchus keta (Walbaum 1792) were used as salmonid fish sperm. Chum salmon returning to the Ohtsuchi River in Iwate prefecture, Japan, from late November to December were kindly provided by the Ohtsuchi fisherman’s union. Rainbow trout were kindly provided by the Oshino Branch of Yamanashi Prefectural Fisheries Experiment Station. Two strains of rainbow trout were used: a ‘fall strain’, which gave sperm from November to January and a ‘summer strain’ that matured from June to July. No significant difference in sperm behavior between the two strains was detected. Semen was collected by inserting a pipette directly into the sperm duct and stored on ice until use.

In the present study, sperm motility was activated by two methods as follows. Semen was diluted directly into 2000 volumes of potassium-free STS (intact sperm). Semen was diluted 100-fold with ice-cold hypertonic solution for 15 s; and then 2 μl of the sperm suspension was resuspended in 40 μl of PTS on a glass slide (glycerol-treated sperm). Sperm movements were recorded using a video recorder (HR-G11, Victor JVC, Kanagawa, Japan) and a CCD camera (WAT-902H Ultimate, Watec, Yamagata, Japan) mounted on a phase-contrast microscope (Optiphoto; Nikon, Tokyo, Japan). In order to elucidate which factor in the glycerol treatment is important for motility initiation, glycerol was substituted for other substances at various concentrations.

The effect of extracellular Ca²⁺ on motility activation was examined using the Ca²⁺ chelator EGTA. 1 mmol l^–1 EGTA was added to the STS in intact sperm or PTS in glycerol-treated sperm to remove extracellular Ca²⁺. The resultant concentrations of free Ca²⁺ in those solutions were calculated to be less than 10^–10 mol l^–1, including Ca²⁺ contamination from semen. The concentration of free Ca²⁺ was calculated by the algorithms published by Goldstein (Goldstein, 1979).

To investigate the effect of intracellular Ca²⁺ on motility activation, intracellular Ca²⁺ was eliminated by using BAPTA-AM. To load BAPTA into sperm cells, semen was diluted twofold in ASP containing 200 or 1000 μmol l^–1 of BAPTA-AM (final concentration 100 and 500 μmol l^–1, respectively) and incubated for 30 min at room temperature. After incubation, the sperm suspension was treated by the two methods of activation previously described.

Intracellular Ca²⁺ was monitored by a Ca²⁺-fluorescent dye, Fluo-4 (excitation 495 nm; emission 518 nm). Semen was suspended in the same volume of ASP, containing 150 μmol^–1 of Fluo-4 AM (final concentration 75 μmol l^–1) and 0.6% (v/v) Powerload (final concentration 0.3%) and incubated at 10°C for 2 h for dye loading. After dye loading, semen was placed at a window of the perfusion chamber. The perfusion chamber was made by longitudinally putting two strips of plastic tape at 10 mm intervals on glass slides and then fixing a coverslip to the strips with additional plastic tapes. ASP without Fluo-4 was applied to one side the chamber window and absorbed by filter paper at the other window. Additionally, various experimental solutions (glycerol solution, STS, PTS) were perfused using the same method. Fluorescent images emitted from sperm were captured using fluorescence microscopy (BX51, Olympus, Tokyo, Japan) and an Olympus filter set (excitation filter, BP470–490; dichromatic mirror, DM 505; emission filter, BA 510–550) and recorded digitally using an EMCCD camera (Luca S, Andor Technology, Belfast, Northern Ireland). Recorded images were analyzed with Andor Solis software (Andor Technology, Belfast, Northern Ireland). The fluorescence intensity (FI) of the sperm head was determined by selecting a square region surrounding the sperm head and then calculating the mean FI value of this region using Andor Solis. Calculated values were multiplied by the area of the square and the background FI value was subtracted, which was determined in the same manner as that for the sperm head. The FI value of the tail region was determined similarly, except for the size of the square, using the center of the sperm tail.

Reactivation of sperm flagellar movement was performed based on the methods of Morisawa and Okuno (Morisawa and Okuno, 1982) and Cosson and colleagues (Cosson et al., 1995), with slight modifications. Briefly, dry sperm was diluted in ASP (or STS, glycerol solution in other experiments) by a factor of 100. After the appropriate incubation time, 5 μl of the sperm suspension was mixed with 200 μl of demembranating solution, containing 150 mmol l^–1 K-acetate, 0.5 mmol l^–1 EDTA, 20 mmol l^–1 Hepes–KOH (pH 8.2), 2 mmol l^–1 DTT and 0.04% Triton-X100. After a 30 s incubation on ice, 2.5 μl of the demembranated sperm suspension was again diluted with 50 μl of reactivation solution, containing 150 mmol l^–1 K-acetate, 1 mmol l^–1 EGTA, 20 mmol l^–1 Hepes–KOH (pH 8.2), 2 mmol l^–1 DTT and 0.2 mmol l^–1 ATP, with or without 10 μmol l^–1 cAMP. After gentle mixing, an aliquot of reactivated sperm suspension was placed onto glass slides, covered with a coverslip and observed using a phase-contrast microscope.

The semen was diluted twofold in ASP containing various concentrations of inhibitors, as illustrated in the figures, and incubated for an appropriate time (30–60 min). After incubation, the sperm motility was examined by the two methods described previously using solutions containing the same concentrations of inhibitors. The effects of inhibitors on sperm motility were expressed in terms of the percentage of motile spermatozoa. The incubation time and temperature were noted during individual experiments. U 73122 [an inhibitor of phospholipase C (PLC)], H-89 [an inhibitor of protein kinase A (PKA)] and chelerythrine and calphostin C [both inhibitors of protein kinase C (PKC)] were used in this study.

Morita and colleagues (Morita et al., 2005) reported that, when chum salmon sperm were treated once with 1.3 mol l^–1 glycerol or an organic alcohol solution followed by dilution with a hypotonic solution, motility initiation was observed even in motility-inhibiting PTS. In rainbow trout sperm, treatment with 1.3 mol l^–1 glycerol and an organic alcohol, such as erythritol, also provided high sperm motility in PTS (data not shown). To elucidate the motility-triggering factor of treatment with glycerol, we first substituted several compounds for glycerol and looked at the effect of osmolarity on rainbow trout sperm motility. Fig. 1 shows the result of using glycerol, NaCl and KCl. No sperm motility was observed when sperm were treated with a 0.5 osmol l^–1 glycerol solution. Treatment with a glycerol solution of more than 0.75 osmol l^–1 caused sperm motility, and they exhibited the highest motility at 1.25 osmol l^–1. Treatment with a glycerol solution of more than 1.5 osmol l^–1 caused a gradual decrease in sperm motility. Similar results were obtained with electrolyte solutions (NaCl or KCl). However, motility was considerably lower in high-osmotic (>1.25 osmol l^–1) electrolyte solutions owing to sperm aggregation.

To investigate the effect of the chemical properties of solutes, such as molecular weight and charge, on sperm motility initiation, we treated chum salmon sperm with various compounds, including organic alcohol (glycerol, ethylene glycol, polyethylene glycol 200), sugar (sucrose), organic ion (choline chloride) and inorganic ions (NaCl, KCl and sodium acetate). The results are shown in Table 1. As previously shown by Morita and colleagues (Morita et al., 2005) and Fig. 1, organic alcohol and inorganic ions, with the exception of ethylene glycol, induced sperm motility. In addition, treatment with organic ions, such as choline chloride, and sugars, such as sucrose, also induced motility. Furthermore, osmolarity greater than 0.75 osmol l^–1 was required for motility initiation in all solutes. These results indicate that treatment with a hyper-osmotic solution and the subsequent dilution into hypo-osmotic solution induces salmonid fish sperm motility, independent of solute molecular weight or charge.

The percentage of motility-initiated sperm reached a plateau at 1–1.125 osmol l^–1 of a glycerol–NaCl–KCl solution. Therefore, hypertonic solutions of 1.125 osmol l^–1 were used in the following experiments.

The effects of Ca²⁺ on sperm motility initiation were examined. To test the effect of Ca²⁺ on motility activation of glycerol-treated and intact sperm, extracellular and intracellular Ca²⁺ ions were removed using EGTA and BAPTA-AM, respectively.

In both intact and glycerol-treated sperm from rainbow trout, the elimination of extracellular Ca²⁺ by 1 mmol l^–1 EGTA had no effect on sperm motility initiation (Fig. 2A), although the swimming paths of intact sperm were altered considerably. This effect occurred at a Ca²⁺ concentration calculated to be <10^–10 mol l^–1. By contrast, when sperm were preloaded with BAPTA-AM to eliminate intracellular Ca²⁺, the motility of both intact and glycerol-treated sperm was significantly inhibited, in a dose-dependent manner (Fig. 3A).

In the case of chum salmon sperm, motility initiation of intact sperm was significantly inhibited by 1 mmol l^–1 EGTA, unlike rainbow trout sperm, whereas glycerol-treated chum salmon sperm were not inhibited, similar to rainbow trout sperm (Fig. 2B). As in the case of rainbow trout sperm, BAPTA-loaded chum salmon sperm motility was severely inhibited in both intact and glycerol-treated sperm (Fig. 3B). These results indicate that Ca²⁺ plays an important role in both intact and glycerol-treated sperm; intact sperm are more dependent on extracellular Ca²⁺, contrary to glycerol-treated sperm, which showed no dependency on extracellular Ca²⁺.

In glycerol-treated sperm, high motility was observed even when extracellular Ca²⁺ had been depleted with EGTA. However, when sperm were loaded with BAPTA-AM, glycerol-treated sperm showed decreased motility. This result suggests that mobilization of Ca²⁺ from intracellular Ca²⁺ stores might be involved in the regulatory mechanism of glycerol-treated sperm. In order to examine the involvement of Ca²⁺ during motility activation, intracellular Ca²⁺ was monitored with the fluorescent dye Fluo-4, using a perfusion technique. Fig. 4 shows the change in the level of intracellular Ca²⁺ in rainbow trout sperm during motility activation.

When sperm were perfused with a hyper-osmotic glycerol solution, a slight decrease in FI was observed following the onset of perfusion, and a subsequent rapid increase of FI, mainly in the head-to-neck region, was also observed (Fig. 4A,B,F,G,K). The increased FI was followed by a gradual decrease over time (Fig. 4B,G). By contrast, PTS perfusion following glycerol solution perfusion (glycerol treatment) caused a rapid decrease in FI in the head region (Fig. 4A,F). The FI at the center of the tail region did not change in glycerol-treated sperm (Fig. 4K). Perfusion with other hypertonic solutions, which consisted of either ionic or nonionic substances, also caused an increase in FI, whereas perfusion of hypotonic STS, following hypertonic solution perfusion, caused a decrease in FI, as shown in Fig. 4A,F (data not shown). These results demonstrate that hypertonic treatment causes a rapid increase of intracellular Ca²⁺, and the subsequent hypotonic shock causes a rapid decrease in intracellular Ca²⁺.

It was very hard to determine the absolute value of the intracellular Ca²⁺ concentration owing to individual variance and the technical difficulty of these experiments. However, the maximum value of intracellular Ca²⁺ in the head region during perfusion with a glycerol solution was assumed to be approximately 10^–6 mol l^–1, as derived from the similar value obtained when sperm were perfused with a Ca²⁺ ionophore (data not shown). When sperm were preloaded with BAPTA, almost no change of FI during perfusion was observed, suggesting the removal of intracellular Ca²⁺ by BAPTA (Fig. 4E,J).

Intact sperm exhibited a different pattern of FI. Perfusion of STS, using intact sperm, also caused a rapid increase of FI, following a slight decrease of FI at the beginning of perfusion. However, in contrast to glycerol-treated sperm, the fluorescence of intact sperm was observed mainly in the tail region, and fluorescence of the head region was less bright (Fig. 4C,H,L). In fact, the maximum FI value of intact sperm in the head region was only 1.5-fold higher when compared with the value before perfusion, whereas glycerol-treated sperm showed more than a fourfold increase (Fig. 5A). In addition, the time required for the onset of the increase in FI of the head in intact sperm was significantly shorter than that of glycerol-perfused sperm (Fig. 5B). Moreover, the fluorescence emission pattern was quite different between intact and glycerol-perfused sperm. In glycerol-perfused sperm, emission started from the specific region of the head around the basal body and mid-piece (Fig. 6A). By contrast, no such specific region was observed in intact sperm (Fig. 6B). These results show that the change in intracellular Ca²⁺ during motility activation is different between intact and glycerol-treated sperm. There was no significant change when sperm were perfused with PTS (Fig. 4D,I).

Demembranated and reactivated trout sperm motility experiments were performed to examine whether glycerol treatment induces axonemal maturation (Fig. 7). As reported previously by Morisawa and Okuno (Morisawa and Okuno, 1982) and Cosson and colleagues (Cosson et al., 1995), demembranated trout sperm were almost completely immotile in the reactivation solution that contained 200 μmol l^–1 ATP without cAMP. Supplementation of cAMP to the reactivation solution caused reactivated motility in more than 70% of sperm. By contrast, when trout sperm were diluted in STS, in which sperm exhibited motility, for 30 s before demembranation, flagellar movement occurred without cAMP, as reported by Morisawa and Okuno (Morisawa and Okuno, 1982). This result indicates that reduction of external K⁺ induces maturation of the sperm motility apparatus, including protein phosphorylation for motility initiation. Similarly, when sperm were diluted in a glycerol solution for 15 s before demembranation, >50% of the sperm were reactivated when cAMP was not supplemented in the reactivation solution. Demembranation of sperm after glycerol treatment also resulted in reactivated motility in the absence of cAMP; however, the percentage of motile sperm was slightly lower than the preceding three preparations. These results indicate that hypertonic shock induces maturation of axonemal components, similar to the effects of external K⁺ reduction.

When U 73122, an inhibitor of PLC, was applied to experimental solutions, motility activation of both intact and glycerol-treated sperm was significantly inhibited at 20 μmol l^–1 and was completely inhibited at 50 μmol l^–1 (Fig. 8). By contrast, the same concentrations of U 73343, an inactive analog of U 73122, did not inhibit sperm motility (Fig. 8).

Fig. 9 shows the effect of H-89, a potent inhibitor of PKA. In rainbow trout, H-89 significantly inhibited activation of intact sperm motility in a dose-dependent manner. Only 12% of intact sperm were activated in the presence of 30 μmol l^–1 H-89. In contrast, 50% of glycerol-treated sperm activated motility at the same concentrations of H-89. H-85, an inactive analog of H-89, did not significantly inhibit either intact or glycerol-treated sperm motility at a concentration of 50 μmol l^–1 or less. Chum salmon sperm exhibited results similar to those of the rainbow trout sperm (data not shown).

The effect of the PKC inhibitors chelerythrine and calphostin C on both intact and glycerol-treated sperm were examined (Fig. 10). Applying 15 μmol l^–1 chelerythrine significantly inhibited glycerol-treated sperm motility in both the rainbow trout and chum salmon (Fig. 10A,B). By contrast, the same concentrations of chelerythrine showed weaker motility inhibition effects on intact sperm (Fig. 10A,B). Similar results were obtained with calphostin C. Calphostin C inhibited glycerol-treated sperm motility in a dose-dependent manner, whereas calphostin C did not affect intact sperm motility at these concentrations (Fig. 10C,D).

It has been known that activation of salmonid fish sperm motility is triggered by a decrease of extracellular K⁺ ions (Morisawa and Suzuki, 1980). However, our previous study demonstrated that, when salmonid fish sperm were treated with a 10% glycerol solution and subsequently diluted into the low-osmotic solution, sperm initiate motility even in the presence of a high concentration of extracellular K⁺ (Morita et al., 2005). It was also reported that not only glycerol treatment but also treatment with erythritol, an organic alcohol, induced initiation of sperm motility contrary to ethylene glycol treatment, which did not induce sperm motility initiation. Based on these results, we previously concluded that the chemical properties and molecular weight of organic alcohol might be important factors for initiation of motility. In the present study, however, we found that treatment of sperm with compounds other than organic alcohol (NaCl, KCl, etc.) also induced sperm motility, and motility initiation was dependent on the osmolarity of the solution (Fig. 1 and Table 1). These results indicate that motility initiation by glycerol is dependent on the osmotic shock caused by glycerol treatment, but less dependent on the molecular weight and electric charge of the substances.

The effects of intracellular Ca²⁺ increase in rainbow trout sperm motility have been revealed by many authors, but their source and function are still obscure (Tanimoto and Morisawa, 1988; Cosson et al., 1989; Boitano and Omoto, 1992). Several reports suggested that the increase in intracellular Ca²⁺, by the entry of external Ca²⁺, is important for the initiation of sperm motility (Tanimoto and Morisawa, 1988; Cosson et al., 1989). In addition, Kho and colleagues (Kho et al., 2001) reported that the influx of Ca²⁺, through dihydropyridine-sensitive T- and/or L-type Ca²⁺ channels, caused the changes in membrane potential, resulting in the activation of adenylyl cyclase to produce cAMP. Boitano and Omoto (Boitano and Omoto, 1992) reported that, in addition to activation of motility, Ca²⁺ release from intracellular stores also regulates the swimming pattern in trout sperm in a manner similar to that of sea urchin sperm (Brokaw, 1979; Okuno and Brokaw, 1981). By contrast, Okuno and Morisawa (Okuno and Morisawa, 1989) have shown an inhibitory effect of relatively low Ca²⁺ concentrations on the reactivation of the demembranated trout sperm.

In the present study, we demonstrated that intracellular Ca²⁺ increased upon motility activation, especially in the tail region (Fig. 4C,H,L) and the motility of intact sperm was inhibited by the elimination of intracellular Ca²⁺ by BAPTA-AM (Fig. 3). These results are consistent with previous studies that discussed the effects of Ca²⁺ on trout sperm motility (Tanimoto et al., 1994; Cosson et al., 1989; Boitano and Omoto, 1992). When extracellular Ca²⁺ was eliminated by EGTA, the motility of chum salmon intact sperm was inhibited (Fig. 2B). By contrast, in rainbow trout, EGTA did not severely affect the percentage of intact sperm that initiated motility (Fig. 2A). However, their swimming path was altered from straight to circular (data not shown). Because we frequently found that sperm near the end of the breeding season or poor-quality sperm generally show such a circular swimming path, it is assumed that the intact sperm of rainbow trout were also impaired by EGTA. These results indicate that, in intact sperm of both species, the Ca²⁺ increase is induced more or less by the entry of external Ca²⁺ and that sperm of chum salmon are more dependent on extracellular Ca²⁺ than the rainbow trout. This difference might come from the difference in the habitats of these species – rainbow trout inhabit inland waters, whereas chum salmon migrate.

In glycerol-treated sperm, intracellular Ca²⁺ increased when sperm were exposed to a hypertonic solution (Fig. 4A,F), and the removal of intracellular Ca²⁺ by BAPTA-AM inhibited both sperm motility and the increase of intracellular Ca²⁺ (Fig. 3, Fig. 4E,J). These results indicate that intracellular Ca²⁺ is a prerequisite for the initiation of salmonid fish sperm motility in glycerol-treated sperm, as well as in intact sperm. By contrast, elimination of extracellular Ca²⁺ by EGTA did not influence glycerol-treated sperm motility (Fig. 2). In addition, U 73122, an inhibitor of a PLC, inhibited initiation of glycerol-treated sperm motility (Fig. 8). Therefore, unlike intact sperm, it is likely that the mobilization of Ca²⁺ is from the intracellular Ca²⁺ store by means of the PLC–inositol (1,4,5)-trisphosphate (IP₃) pathways, not through the influx from the extracellular medium, and this plays an important role in the motility initiation of glycerol-treated sperm. This idea is supported by the observation that the intracellular Ca²⁺ concentration increased when sperm were suspended in a hypertonic glycerol solution where most of the extracellular Ca²⁺ had been removed by 0.5 mmol l^–1 EDTA (Fig. 4A,F). The difference in time required for the onset of the increase in intracellular Ca²⁺ between intact and glycerol-perfused sperm might be reflective of the difference in Ca²⁺ mobilization (Fig. 5B). Collectively, the data indicate that hypertonic-shock-induced mobilization of Ca²⁺ from intracellular Ca²⁺ stores by means of the PLC–IP₃ pathway has an important role in glycerol-treated sperm motility initiation. Interestingly, this result coincides with a previous report of puffer fish sperm in which the release of Ca²⁺ from an intracellular store, in response to hypertonic shock, has a key role in motility initiation (Oda and Morisawa, 1993). This idea is very suggestive of evolutionary adaptation in response to reproductive background.

In glycerol-treated sperm, the Ca²⁺ increase occurred from a specific region of the head close to the basal body and mid-piece (Fig. 6A), whereas such a specific point of Ca²⁺ burst could be identified neither in intact sperm (Fig. 6B) nor in sperm treated with A 23187, a Ca²⁺ ionophore (data not shown). These results suggest the existence of a Ca²⁺ store near the basal body that releases Ca²⁺ in response to hypertonic shock. Ho and Suarez (Ho and Suarez, 2001) reported that an IP₃ receptor-gated Ca²⁺ store exists at a redundant nuclear envelope in the neck region of bovine sperm. A similar membrane structure was observed between the basal body and mitochondria of trout sperm with an osmiophilic mass (Billard, 1983). It is plausible that this structure is the IP₃ receptor-gated Ca²⁺ store in trout sperm.

When sperm were suspended and maintained in a hypertonic solution, intracellular Ca²⁺ increased in response to hypertonic shock and was maintained at a high level (Fig. 4B,G). Although intracellular Ca²⁺ increased under this condition, the sperm did not initiate motility. When sperm were diluted into a hypotonic solution following hypertonic exposure, they initiated motility concomitantly with a decrease in increased intracellular Ca²⁺ (Fig. 4A,F). These results suggest that not only the increase in Ca²⁺ by hyper-osmotic shock but also the subsequent decrease of intracellular Ca²⁺ are required for motility initiation. It is reported that relatively low concentrations of Ca²⁺ inhibit the motility of demembranated trout sperm (Okuno and Morisawa, 1989). In addition, Su and Vacquier (Su and Vacquier, 2002) reported that the intracellular Ca²⁺ of swimming sea urchin sperm is maintained at a low level by a K⁺-dependent Na⁺/Ca²⁺ exchanger, and blockage of this exchanger inhibited sperm motility. In conclusion, we speculate as follows: the increase of Ca²⁺ induced by hypertonic shock leads to the acquisition of the capacity for motility to the motile apparatus; however, the sperm cannot then move at that moment as hypertonic shock increased intracellular Ca²⁺ to an inhibitory level that is much higher than that of intact sperm (Fig. 5A). Then, the subsequent hypo-osmosis-induced removal of Ca²⁺ triggers the motility initiation. In intact sperm, the Ca²⁺ increase appears to be low enough to avoid an inhibitory effect on the axoneme. The mechanism by which hypotonic shock induces a decrease in intracellular Ca²⁺ is still unclear. It is known that mechano-sensitive cation channels are involved in the regulation of the motility of marine and freshwater fish sperm (Krasznai et al., 2003). Therefore, the opening of a mechano-sensitive channel by hypotonic shock might contribute to the decrease of Ca²⁺. It is also probable that some Ca²⁺ pumps are activated in response to hypotonic shock to remove intracellular Ca²⁺. Additional experiments are needed in order to elucidate the mechanism by which hypotonic shock induces a decrease in intracellular Ca²⁺.

In intact sperm, the magnitude of increase in intracellular Ca²⁺ was less than in glycerol-treated sperm (Fig. 4F,H, Fig. 5A). An increase of Ca²⁺ was mainly observed in the tail region of intact sperm, whereas a major increase of Ca²⁺ was observed in the head region of glycerol-treated sperm (Fig. 4A,C,F,H,K,L). These results lead to speculation that the increase of Ca²⁺ necessary for flagellar activation is as minimal as that observed in intact sperm (Fig. 4, Fig. 5A) and that the burst of Ca²⁺ in the head of glycerol-treated sperm might cause the increase of Ca²⁺ in the flagellum, which is sufficient for motility activation. Alternatively, an increase in Ca²⁺ might be necessary only at the base of the flagellum for motility initiation.

cAMP-dependent phosphorylation of intraflagellar proteins has been demonstrated to be indispensable for activation of sperm motility in many species (Brandt and Hoskins, 1980; Tash and Means, 1982; Ishiguro et al., 1982). In salmonid fish sperm, the involvement of cAMP in motility initiation was first reported by Morisawa and Okuno (Morisawa and Okuno, 1982). They demonstrated that cAMP-induced maturation of the non-motile axonemal apparatus is required for demembranated trout sperm motility (see also Fig. 7). In addition, they demonstrated that flagellar movement occurred without cAMP when trout sperm were diluted in potassium-free medium for 30 s prior to demembranation, suggesting that reduction of external K⁺ induces maturation of the sperm motile apparatus (Morisawa and Okuno, 1982) (Fig. 7). The following studies have demonstrated that maturation induced by cAMP includes the phosphorylation of axonemal proteins, such as the 15-kDa protein and 22-kDa dynein light chain (Morisawa and Hayashi, 1985; Hayashi et al., 1987; Inaba et al., 1998). Our previous study reported that the 22-kDa dynein light chain and other axonemal proteins, except for the 15- and 29-kDa proteins, were phosphorylated in glycerol-treated salmonid fish sperm without the synthesis of cAMP. This result indicates that cAMP-independent maturation must occur in glycerol-treated sperm. In some species of sperm, Ca²⁺-dependent phosphorylation is demonstrated to be important in motility regulation (Morita et al., 2006; White et al., 2007). As the increase of intracellular Ca²⁺ was achieved by hypertonic shock in glycerol-treated sperm, we consider that axonemal maturation might have occurred during exposure to a hypertonic environment. Indeed, the present study demonstrated that dilution of sperm into a glycerol solution, as well as STS, induced maturation of the sperm axoneme. A subsequent hypotonic shock was not necessary to induce maturation of the axoneme (Fig. 7). This result supports the idea, mentioned above, that phosphorylation of axonemal proteins is completed during hypertonic dilution and that subsequent hypotonic shock might function as a trigger. Notably, increasing intracellular Ca²⁺ from the intracellular store in response to hypertonic shock induces the maturation of the axoneme necessary for flagellar movement through Ca²⁺-dependent phosphorylation of axonemal proteins, and the subsequent removal of Ca²⁺ by hypotonic shock specifically induces sperm motility by unlocking the Ca²⁺-inhibitory effect.

The results discussed above raise a new question: what is the key signaling element for phosphorylation of axonemal proteins in glycerol-treated sperm? It has been revealed that the catalytic subunit of PKA, which phosphorylates proteins in a cAMP-dependent manner, is present in trout sperm (Itoh et al., 2003), suggesting that this signaling molecule might play an important role in intact sperm. In our previous study, however, cAMP was not synthesized upon motility initiation of glycerol-treated sperm. Then we proposed that PKA is activated without the synthesis of cAMP. In the present study, H-89, a potent inhibitor of PKA, inhibited the motility of intact sperm in a dose-dependent manner (Fig. 9). This result is consistent with previous reports that cAMP-dependent phosphorylation of axonemal proteins by PKA is implicated in the motility of intact sperm. In glycerol-treated sperm, H-89 inhibited motility at 30 μmol l^–1, which was weaker than what was required for inhibiting intact sperm (Fig. 9). This result suggests that the contribution of PKA to the motility of glycerol-treated sperm is less than that to intact sperm. Alternatively, the inhibition of the motility of glycerol-treated sperm by H-89 was caused by nonspecific inhibition because it is reported that H-89 at concentrations of approximately 30 μmol l^–1 acts on other kinases, as well, in vitro (Chijiwa et al., 1990).

Next, we tested the involvement of Ca²⁺-dependent phosphorylation in sperm motility initiation, such as calmodulin kinases and PKC, using inhibitors as these signaling elements are reported to be involved in the regulation of motility in some species (Morita et al., 2006; White et al., 2007). However, W-7, which inhibits calmodulin, did not inhibit the motility of glycerol-treated sperm (data not shown). By contrast, PKC inhibitors (chelerythrine and calphostin C) inhibited the motility of glycerol-treated sperm at a concentration that did not inhibit intact sperm motility, although inhibition was not so severe in rainbow trout sperm (P<0.05; Fig. 10). These results suggest that PKC, which does not affect intact sperm initiation, might function at least partially on phosphorylation of axonemal proteins in glycerol-treated sperm. The result that U 73122 inhibited glycerol-treated sperm motility supports this idea, as PKC is known to phosphorylate proteins in a diacylglycerol-dependent manner (Fig. 8). Further investigations are needed to elucidate the key signaling molecule in glycerol-treated sperm.

The present study proposes a novel mechanism for sperm motility initiation that acts as a salvaging pathway for the well-known K⁺-dependent pathway through phosphorylation of the same axonemal proteins, including the 22-kDa dynein light chain. This means that salmonid fish sperm possess diverse mechanisms for the initiation of motility. One mechanism revealed in the present study shares features common to those of other teleosts that initiate motility in a osmolarity- and Ca²⁺-dependent manner. Further investigations on the mechanism of glycerol-treated sperm motility and actual function of this novel signaling pathway under physiological condition are needed and might lead to the generation of further valuable information.

 
• ADP

  adenosine diphosphate

 
• ASP

  artificial seminal plasma

 
• ATP

  adenosine triphosphate

 
• BAPTA

  O,O′-bis(2-aminophenyl)ethyleneglycol-N,N,N’,N’-tetraacetic acid

 
• cAMP

  3′-5′-cyclic adenosine monophosphate

 
• DMSO

  dimethylsulfoxide

 
• DTT

  dithiothreitol

 
• EGTA

  ethylene glycol tetraacetic acid

 
• Fluo-4

  1-[2-amino-5-(2,7-difluoro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid, pentaacetoxymethyl ester

 
• H-85

  N-[2-(N-formyl-p-chlorocinnamylamino)ethyl]-5-isoquinolinesulfonamide

 
• H-89

  N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide

 
• Hepes

  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

 
• PKA

  protein kinase A

 
• PKC

  protein kinase C

 
• PLC

  phospholipase C

 
• PTS

  potassium test solution

 
• STS

  sodium test solution

 
• U 73122

  1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione

 
• U 73343

  1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione

We thank the Oshino Branch of Yamanashi Prefectural Fisheries Experiment Station for supplying rainbow trout. We also thank the Ohtsuchi Kaiyo center of the Atmosphere and Ocean Research Institute, The University of Tokyo and Ohtsuchi fisherman’s union for provision of facilities and materials, notably chum salmon. We wish for a quick recovery from the enormous damage caused by the huge 2011 Tohoku earthquake and tsunami.