Distinct Ca²⁺ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt, Cynops pyrrhogaster

Abstract

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca²⁺ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca²⁺ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca²⁺ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca²⁺ channels mediate the intracellular Ca²⁺ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.

Introduction

The hyperactivation of sperm, with a specific flagellar beating, high amplitude and asymmetry at the midpiece, propels the sperm through the oviduct in the internal fertilization of mammals (Yanagimachi 1994; Mortimer 1997). It is critical for the success of mammalian fertilization that sperm swim up the oviduct and fertilize the egg in the ampulla. The oviduct‐dependent fertilization mode is also observed in aves (Bakst 1998) and reptiles (Blackburn 1998), although it is unknown whether a similar state of motility is induced in the internal fertilization of non‐mammalian vertebrates.

Amphibian internal fertilization is a primitive, oviduct‐dependent mode of vertebrates that is observed in a few species of anurans, most urodeles and all caecillians among more than 4000 species (Duellman & Trueb 1994; Wake & Dickie 1998). In the red‐bellied newts, Cynops pyrrhogaster, although sperm do not swim up the oviduct, they penetrate through the oviduct‐secreted matrix called the egg jelly. At the onset of fertilization, Cynops sperm that have been quiescently stored in a sperm reservoir are mechanically pushed onto the surface of the egg jelly surrounding an egg. Sperm motility is initiated by sperm motility‐initiating substance (SMIS), a 34‐kDa protein included in the egg jelly (Watanabe et al. 2010) in association with an another factor, acrosome reaction‐inducing substance (Watanabe et al. 2011). Forward movement of the sperm is provided by a wave of the undulating membrane (UM), which is located from the anterior midpiece to the posterior principal piece (Fig. 1). A long axoneme is situated through the lateral tip of the UM (Scheltinga & Jamieson 2003a) and produces wave movement. Previously, we found that Cynops sperm time‐dependently received a faster UM wave after motility initiation in the jelly substances (Watanabe & Onitake 2003; Watanabe et al. 2003). The fast wave may be accompanied with a specific wave pattern such as seen in the beating flagellum of the hyperactivated mammalian sperm, although the character of the UM wave has not been qualitatively analyzed.

Figure 1

Scanning electron microscope image of Cynops pyrrhogaster sperm. Single, double and triple arrowheads indicate the acrosome at the tip of the sperm head, the border between the head and midpiece, and the border between the midpiece and the principal piece, respectively. The undulating membrane (UM) extends from the anterior end of the midpiece to the posterior end of the principal piece. The inset shows a high magnification view of the principal piece. The UM was alternately situated on the axial rod. Arrows and dashed arrows indicate the sites in which the UM is at the front side and far side, respectively. Bar: 50 μm.

In the present study, we analyzed the UM waves of Cynops sperm and found that the fast wave was constructed with different wave velocities in the anterior and posterior midpiece. The high motility state was maintained by distinct Ca²⁺ channels that mediated intracellular Ca²⁺ levels in the anterior and posterior ends of the midpiece.

Materials and methods

Gametes

Experimental animals were treated according to the guidelines for proper conduct of animal experiments in Japan. Ovulation was induced in sexually mature C. pyrrhogaster females by two injections of gonadotropin (150 IU) (Asuka Pharmacy Inc, Tokyo, Japan) at 24 h intervals. The eggs were obtained from the posterior‐most portion of the oviduct termed the uterus 2–3 days following the last injection. Sperm were obtained from the vas deferentia of males.

Scanning electron microscopy

Cynops sperm were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.0) at 4°C overnight. The sperm were then rinsed with phosphate buffer, dehydrated and dried in a critical point apparatus (HCP‐1; Hitachi). They were coated with platinum using a magnetron sputter (JUC‐5000; JEOL) and observed with a scanning electron microscope (JSM‐6510LV, JEOL).

Egg jelly extract

Mature eggs were immersed in modified Steinberg's salt solution (ST: 58.2 mmol/L NaCl, 0.67 mmol/L KCl, 6 mmol/L CaNO₃, 0.83 mmol/L MgSO₄, 10 mmol/L Tris‐HCl; pH 8.5) with a volume of 50 μL per egg. Following agitation of the egg suspension at 4°C for 1 h, ST was collected and centrifuged at 16 000 × g at 4°C for 30 min. The supernatant was the egg jelly extract (JE), which was stored at −30°C.

Motility state analysis

Sperm were suspended in the JE or 10 mmol/L Hepes‐NaOH (pH 6.9), and motility was observed under a phase‐contrast microscope (BX51, Olympus) equipped with a 20× objective at room temperature. The UM wave was recorded on a personal computer with a digital high‐speed camera (HAS‐220; DITECT). The motility rate was estimated by the percentages of sperm with the UM moving in most, a part, or none of the midpiece and principal piece. The wave velocity was measured with cell movement analyzing software, Bohboh (Bohboh Soft) by estimating the time that a wave phase moved through a distance of 20 μm at two different portions, 40–50 μm and 140–180 μm from the anterior end as the representatives of the anterior and posterior midpiece, respectively. The significant differences in wave velocity were evaluated by Student's t‐test. In some experiments, the sperm were treated with: verapamil (50 or 500 μmol/L), an L‐type voltage‐dependent Ca²⁺ channels (VDCCs) blocker that also blocks T‐type VDCC with a low affinity (Bezprozvanny & Tsien 1995; Arnoult et al. 1998); mibefradil (100 μmol/L), a VDCC blocker with a dominant effect on T‐type VDCC (Bezprozvanny & Tsien 1995); or nitrendipine (50 μmol/L), an L‐type VDCC blocker (McCarthy & Fry 1988), 5 min after suspension in the JE. More than three independent experiments were performed under each condition.

Insemination assay

Sperm suspension (1 μL), containing approximately 6 × 10² sperm, was put on the surface of the egg jelly of each mature egg in a moist chamber. In 0–1, 2–3, 4–5, 9–10, or more than 60 min, the egg was immersed into a tap of distilled water, and its development was observed. For the positive control, naturally spawned eggs were observed in distilled water. The eggs developing into two‐cell or gastrula stage embryos were evaluated as fertilized and normally developing. More than three independent experiments were performed under each condition.

Ca²⁺ imaging

Sperm were incubated with 5 μmol/L Fluo8H‐AM (AAT Bioquest) for 2 h. They were suspended in JE; after 5 min, verapamil (50 or 500 μmol/L), mibefradil (100 μmol/L) or nitrendipine (50 μmol/L) was added to the suspension. Images were captured with an inverted fluorescence microscope (IX71, Olympus) equipped with a 20× objective and a cooled EM‐charge‐coupled device (CCD) camera (ImagEM, C9100‐13, Hamamatsu Photonics). Filter sets of BP490‐500 and BA510‐550 were used for excitation and emission, respectively. Fluorescence was processed and analyzed with AQUACOSMOS software (Hamamatsu Photonics). For the evaluation of the differences in the intracellular Ca²⁺ levels of the midpiece, the ratio of the fluorescence intensity to the mean fluorescence intensity of the entire midpiece was calculated, and the mean ratios of 6 sperm in the JE, 16 and 10 sperm treated with 50 and 500 μmol/L verapamil, respectively, in the JE, 11 sperm treated with 100 μmol/L mibefradil in the JE, or 12 sperm treated with 50 μmol/L nitrendipine in the JE were expressed throughout the entire midpiece.

Results

Characteristics of Cynops sperm

Cynops pyrrhogaster sperm were morphologically typical in urodele amphibians (Scheltinga & Jamieson 2003a). From the anterior to posterior, the sperm were approximately 400 μm in length, with a head of approximately 100 μm, a midpiece of approximately 200 μm and a principal piece of approximately 100 μm (Fig. 1). A long UM was located from the anterior tip of the midpiece to the posterior tip of the principal piece. Sperm in the JE moved forward exhibiting a continuous UM wave from the anterior to the posterior (Movie S1).

Motility of Cynops sperm in the JE

To characterize the motility state of the sperm in the jelly substances, sperm were suspended in the JE and their motility was observed. After 5 min, we recorded the UM wave of 73 sperm for 2 min. Most sperm in the JE moved forward with a circular trajectory. All sperm maintained the continuous UM wave throughout the midpiece and principal piece during the observed time period in contrast with only 12 ± 4.3% of the sperm in the ST as the control (Fig. 2). The sperm in the JE showed a variety of UM wave velocities in both anterior and posterior midpieces (Fig. 3A,B). They were divided into three groups according to the wave velocity at the posterior midpiece as group I (more than 200 μm/s), group II (100–200 μm/s) and group III (<100 μm/s), and their wave was characterized using the wave velocity of the UM at the anterior and the posterior midpiece (Fig. 4A). The group I sperm showed a high wave velocity: 266 ± 9.16 μm/s at the anterior midpiece and 294 ± 9.98 μm/s at the posterior. The group II sperm showed medium wave velocity: 115 ± 11.7 μm/s at the anterior midpiece and 140 ± 5.58 μm/s at the posterior. The group III sperm showed low wave velocity: 24.7 ± 4.68 μm/s at the anterior midpiece and 34.8 ± 7.30 μm/s at the posterior. The group I sperm was 64 ± 6.4% of total sperm (Fig. 4G), suggesting that the majority of sperm in the JE was categorized in group I during the observed time period. The UM wave velocities in the group I sperm were significantly higher at the posterior midpiece (P < 0.05) compared with the anterior, whereas no difference was observed in the UM wave velocity in the midpiece of the group III sperm. Wavelength of the UM at the anterior midpiece did not differ from that at the posterior in both group I and group III sperm (Fig. 5). This indicates that UM wave frequency is higher at the posterior midpiece than at the anterior of group I sperm.

Figure 2

Percentages of motile sperm in suspension. Sperm were suspended in jelly extract (JE), 10 mmol/L Hepes‐NaOH (pH 6.9) as a hyposmotic solution or modified Steinberg's salt solution (ST). Voltage‐dependent Ca²⁺ channel (VDCCs) blockers were added to the sperm suspension 5 min after suspending sperm in the JE. The motility of the undulating membrane (UM) was captured at the midpiece with a high‐speed video camera. Sperm with the UM fully and partially waving were expressed as “whole” and “partial”, respectively. Hypo: 10 mmol/L Hepes‐NaOH (pH6.9), vera‐50 and 500: 50 and 500 μmol/L verapamil, mib: 100 μmol/L mibefradil, nit: 50 μmol/L nitrendipine.

Figure 3

Distributions of wave velocity of the undulating membrane (UM) in the anterior and posterior midpiece. The motility of the UM was captured as described in Fig. 2. The UM wave was estimated at the anterior and posterior portions of the midpiece of the sperm in each condition. Hypo: 10 mmol/L Hepes‐NaOH (pH6.9), vera‐50 and 500: 50 and 500 μmol/L verapamil, mib: 100 μmol/L mibefradil, nit: 50 μmol/L nitrendipine.

Figure 4

Mean wave velocity of the undulating membrane (UM) at the anterior and posterior midpiece. (A–F): The motility of the UM was captured as described in Fig. 2. The sperm with the UM waving in the midpiece and principal piece was divided into three groups: sperm with more than 200 μm/s, 100–200 μm/s, and < 100 μm/s of the UM wave velocities at the posterior midpiece were included in groups I, II, and III, respectively. The values represent the mean ± SE. The wave velocities that significantly increased or decreased at the posterior midpiece against the anterior are indicated by single (P < 0.05) and double (P < 0.01) asterisks or crosses, respectively. (G): Percentages of groups I‐III sperm to total sperm with the UM waving in the midpiece and principal piece. Roman numeral indicates each group. Hypo: 10 mmol/L Hepes‐NaOH (pH6.9), vera‐50 and ‐500: 50 and 500 μmol/L verapamil, mib: 100 μmol/L mibefradil, nit: 50 μmol/L nitrendipine, a: anterior, p: posterior.

Figure 5

Wavelength of the waving undulating membrane (UM) of the sperm in jelly extract (JE). Sperm were suspended in JE and the motility of the UM was captured at the midpiece with a high‐speed video camera. Wavelength of the waving UM was measured at the anterior and posterior portions of the midpiece of groups I and III sperm.

Motility of Cynops sperm in hyposmotic solution

Low osmolality in freshwater is a trigger for motility initiation in the sperm of many externally fertilizing anurans (Inoda & Morisawa 1987) and can immediately induce sperm motility of internally fertilizing urodeles (Hardy & Dent 1986; Ukita et al. 1999). To evaluate the specific feature of the sperm motility state in the jelly substances included in JE, we characterized the motility state of sperm in a hyposmotic solution. Sperm were suspended in 10 mmol/L Hepes‐NaOH (pH6.9) and, after 1 min, motility was recorded in 83 sperm for 9 min. Only 35 ± 7.7% of the total sperm maintained a continuous UM wave throughout the midpiece and principal piece during the observed time period (Fig. 2). Although these sperm often exhibited no forward movement, they showed a variety of UM wave velocities both in the anterior and posterior midpiece (Fig. 3C,D). The group I sperm showed wave velocities of 274 ± 16.0 μm/s at the anterior midpiece and 329 ± 21.2 μm/s at the posterior (Fig. 4B). The group II sperm showed wave velocities of 158 ± 49.5 μm/s at the anterior midpiece and 141 ± 9.03 μm/s at the posterior. The group III sperm showed wave velocities of 169 ± 45.9 μm/s at the anterior midpiece and 71.2 ± 9.37 μm/s at the posterior. The wave velocities of the UM at the posterior midpiece of the group I sperm were significantly higher (P < 0.05) compared with the anterior, whereas those of the group III sperm were significantly lower (P < 0.01). The group I sperm in the hypotonic solution were only 22 ± 4.0% (Fig. 4) whereas they were 64 ± 6.4% in JE, suggesting that the high motility state is caused predominantly in the 34 kDa jelly substances.

Significance of fast wave of the UM in fertilization

In C. pyrrhogaster sperm, the UM waves are sporadically initiated in the jelly substances within 4 min (Watanabe et al. 2003, 2011) and gradually become faster during approximately 10 min (Watanabe & Onitake 2003), whereas egg jelly swells and hardens, which prevents the sperm from penetrating it within 5 min of immersion in distilled water (Matsuda & Onitake 1984). Based on these features of the sperm and egg jelly, we elucidated the significance of the high motility state of the sperm for the success of fertilization. Mature eggs were inseminated with 6 × 10² sperm per egg without contact with any solution, which is a condition similar to fertilization in vivo (Takahashi et al. 2006), followed by immersion in distilled water. Four percent and 27% of the eggs were fertilized when the inseminated eggs were immersed in distilled water within 1 min and in 2–3 min, respectively (Table 1). The fertilization rate rose to 43% in the eggs immersed in distilled water 4–5 min after insemination and to 68% in eggs immersed 9–10 min after insemination. All eggs were fertilized when they were immersed in distilled water more than 1 h after insemination. Naturally spawned eggs had a fertilization rate of 91%. Most of the fertilized eggs developed normally to gastrula stage embryos. These results suggest that the fast wave of the UM such as seen in group I sperm is needed for the inseminated sperm to contribute to fertilization.

Table 1. Effect of spawned timing on fertilization and development after insemination

	Total	Fertilized	%	Developed* *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	%† *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.
0–1 min‡ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	24	1	4	1	100
2–3 min‡ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	30	8	27	6	75
4–5 min‡ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	28	12	43	11	92
9–10 min‡ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	28	20	71	17	85
1 h<‡ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	17	17	100	12	71
Spawned§ *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10^2 sperm. ^§Naturally spawned by females.	30	27	90	24	90

• *Developed normally to the tailbud stage embryos. ^†Percentage of normally developed embryo in fertilized eggs. ^‡Time of immersing eggs into distilled water after artificial insemination with 6 × 10² sperm. ^§Naturally spawned by females.

Effect of Ca²⁺ channel blockers in jelly substance‐induced motility

Calcium‐ion channels participate in controlling sperm motility in many animal species (Yoshida et al. 1994; Ren et al. 2001; Darszon et al. 2008), including C. pyrrhogaster (Watanabe et al. 2003). To estimate the mechanism for controlling the high motility state of Cynops sperm, we used an L‐type and T‐type VDCC blocker, verapamil, to treat moving sperm in JE. Ninety‐five ± 5.1% of the sperm treated with 50 μmol/L verapamil maintained the continuous UM wave throughout the midpiece and principal piece during the observed time period (Fig. 2). Most of the sperm exhibited forward movement although they showed a weakened wave velocity of the UM in both the anterior and posterior midpiece (Fig. 3E,F). The wave velocity was notably low at the anterior midpiece of the group I sperm compared to the non‐treated, group I sperm despite it being similar at the posterior (Fig. 4A,C). Unlike the non‐treated group III sperm, the wave velocity was significantly lower in the posterior midpiece than the anterior of the verapamil‐treated, group III sperm.

When sperm were treated with 500 μmol/L verapamil, all maintained the UM wave in both the midpiece and principal piece (Fig. 2), although they showed no forward movement. The wave velocity was severely weakened in both the anterior and posterior midpiece (Fig. 3G,H), and no group I sperm and a few group II sperm were observed (Fig. 4D,G). These results suggest that the high motility state was completely suppressed by the high concentration of verapamil. In addition, 86 ± 14% of them stopped the wave movement at a limited point of the posterior midpiece (Movie S2). In these sperm, the forward wave propagation was maintained from the anterior tip of the midpiece to the posterior, whereas the reverse wave occurred to propagate from the posterior tip of the principal piece to the posterior midpiece. This indicates that the UM wave is independently controlled at the anterior midpiece, posterior midpiece and the posterior principal piece, and the wave at the posterior midpiece was especially suppressed by the high concentration of verapamil.

Verapamil blocks T‐type VDCC, with a lower affinity than L‐type VDCC (Arnoult et al. 1998). Thus, we next examined the effects of mibefradil, which blocks T‐type VDCCs with a higher affinity than L‐type VDCCs, and nitrendipine, which dominantly blocks L‐type VDCC. When sperm moving in the JE were treated with mibefradil, 97 ± 3.3% of them maintained the UM wave in both the midpiece and principal piece (Fig. 2). The UM wave velocity was severely weakened in the posterior midpiece (Fig. 3I,J) and the sperm exhibited no forward movement. No group I sperm were observed in the sperm treated with mibefradil (Fig. 4G). Unlike the non‐treated sperm in the JE, the UM wave velocity was significantly lower at the posterior midpiece of the mibefradil‐treated group II (P < 0.05) and group III sperm (P < 0.01) compared with the anterior (Fig. 4E). In addition, the wave velocities at the anterior midpiece of the mibefradil‐treated sperm of groups II and III displayed similar levels to the non‐treated sperm of groups I and II, respectively, in the JE (Fig. 4A,E). In 77 ± 13% of the sperm, the UM wave was stopped in a limited point of the posterior midpiece shortly after treatment with mibefradil (Fig. 6, Movie S3), similar to many sperm treated with 500 μmol/L verapamil. These indicate that the inhibitory effect of mibefradil predominantly expressed in the posterior midpiece.

Figure 6

Suppression of undulating membrane (UM) wave at the limited point of the posterior midpiece of the mibefradil‐treated sperm. (A) Moving sperm in jelly extract (JE). Continuous UM wave was maintained from the anterior midpiece to the posterior end of the principal piece. (B) Moving sperm treated with mibefradil in JE. The UM wave was completely suppressed at a limited point of the posterior midpiece (arrowhead), and the reverse wave was observed from the posterior end of the principal piece. The arrows in (A) and (B) indicate the direction of the UM wave. Bar: 50 μm.

Nitrendipine weakened the UM wave (Figs 3K,L, 4G) but did not stop it at any point. Most of the nitrendipine‐treated sperm exhibited forward movement. Compared with the posterior midpiece, the UM wave velocity at the anterior was significantly lower in the sperm of groups II and III (Fig. 4F), and the sperm of group I showed significantly lower wave velocity (P < 0.01) at the anterior midpiece than the non‐treated, group I sperm in the JE (Fig. 4A,F). These indicate that nitrendipine affects predominantly in the anterior midpiece.

Changes of intracellular Ca²⁺ levels in the midpiece of moving sperm by VDCC blockers

To elucidate whether intracellular Ca²⁺ level was changed by VDCC blockers, free Ca²⁺ was visualized throughout the midpiece cytoplasm by Fluo8H fluorescence intensities. The sperm in the JE showed a typical distribution pattern of fluorescence intensities, which is relatively high in the anterior end of the midpiece (Fig. 7A,D). After treatment with verapamil or mibefradil, the fluorescence intensities of the sperm changed to a lower level in the anterior end of the midpiece and a higher level in the posterior (Fig. 7B,D,F). Whereas after sperm were treated with nitrendipine, the fluorescence intensities changed to a lower level in the anterior end of the midpiece, although no change was detected in the other portions (Fig. 7C,F). These results suggest that intracellular Ca²⁺ levels are predominantly changed in the anterior and posterior ends of the midpiece by the VDCC blockers.

Figure 7

Distributions of intracellular Ca²⁺ in the midpiece of moving sperm in jelly extract (JE). Fluo8H‐loaded sperm were suspended in JE. After 5 min, verapamil (vera; 50 μmol/L or 500 μmol/L), mibefradil (mib; 100 μmol/L) or nitrendipine (nit; 50 μmol/L) were added to the suspension. Fluorescence from the loaded Fluo8H was captured with a fluorescence microscope and a cooled charge‐coupled device camera. (A–C) The pseudocolored images of fluorescence intensities prepared by ImageJ that show the relative Ca²⁺ levels along the midpiece of a sperm in JE (A), in JE containing mibefradil (B) or in JE containing nitrendipine (C). The arrows and arrowheads indicate the anterior and posterior ends of the midpiece, respectively. (D) and (E) The mean ratios of fluorescence intensities to the mean fluorescence intensity of the midpiece, which was continuously expressed from the anterior end to the posterior of the midpiece of moving sperm in JE (n = 6), JE + verapamil (n = 16; 50 μmol/L, or n = 10; 500 μmol/L), JE + 100 μmol/L mibefradil (n = 11), or JE + 50 μmol/L nitrendipine (n = 12).

Discussion

Sperm motility is initiated in C. pyrrhogaster on the surface of the egg jelly at the onset of fertilization. In this study, we found that moving Cynops sperm showed two types of motility states with different UM wave velocities (Fig. 4A). The high motility state, which was predominantly induced in the jelly substances, was characterized by a significantly higher wave velocity at the posterior midpiece and was different from the low motility state, which had no significant difference in wave velocities throughout the midpiece. It is known that many Cynops sperm in JE show motility with a slow UM wave at the beginning of motility initiation and then develop vigorous motility with a faster UM wave (Watanabe & Onitake 2003; Watanabe et al. 2003). This suggests that the motility state of moving Cynops sperm changes to the high motility state in the jelly substances in JE.

Cynops sperm began to initiate motility approximately 1 min after contacting the jelly substances (Watanabe et al. 2011), and approximately 70% of the inseminated sperm initiated motility on the surface of the egg jelly within 3 min (Watanabe et al. 2003). Because < 20 s is the estimated time necessary for moving Cynops sperm to penetrate through an egg jelly of approximately 500 μm thickness (Ukita et al. 1999), a 5‐min time period should be sufficient for the inseminated sperm to fertilize the eggs. However, fertilization rates were still low when the inseminated eggs were immersed in distilled water within 5 min (Table 1). Matsuda & Onitake (1984) reported that immersing the eggs in distilled water swelled and hardened the jelly matrix, which resulted in blocking the sperm from being able to penetrate into the matrix, although the egg cell itself was fertilizable. Therefore, it is suggested that, in the present study, many of the moving inseminated sperm took longer than 5 min to access the vitelline membrane through the egg jelly. This was supported by the result that the fertilization rate increased when the eggs were immersed in distilled water more than 9–10 min after insemination (Table 1). The rising pattern of fertilization rates corresponds well with the increasing instance of sperm with fast UM wave (Watanabe & Onitake 2003), suggesting that the high motility state enables Cynops sperm to penetrate the jelly matrix. The high motility state is suggested to be composed of higher wave velocity at the posterior midpiece (Fig. 3). Because this feature is ascribed to the different UM wave frequencies in the midpiece (Fig. 5), the high wave velocity should not be caused passively by the structural nature of the UM, but produced by the local enhancement of axonemal sliding. Although the exact role of the higher UM wave velocity at the posterior midpiece is unknown, it may be needed for sperm with curved morphology to forwardly progress with line trajectory, which is shown by the sperm moving through jelly matrix (Ukita et al. 1999). Because the jelly matrix is derived from oviductal secretions (Okimura et al. 2001), the high motility state of Cynops sperm is supposed to have a synonymous role with the hyperactivated motility of mammalian sperm in the sense that the high motility state of the sperm are for propelling through the oviductal matrix to fertilize an egg (Yanagimachi 1994).

Intracellular Ca²⁺ is a common mediator for controlling sperm motility among many animals (Morisawa et al. 1999; Darszon et al. 2006) including urodeles (Watanabe et al. 2011). In the present study, the high motility state was completely suppressed by a high concentration of verapamil (Figs. 3G,H,4D), although the UM wave was maintained (Fig. 2). This suggests that the sperm Ca²⁺ channels mediate to produce the characteristics of the high motility state of the Cynops sperm, which include a higher UM wave velocity that is relatively higher in the posterior midpiece (Fig. 4A). T‐type VDCC is a candidate mediator for the UM wave in the posterior midpiece because mibefradil and high concentration of verapamil suppressed the wave at the corresponding site (Fig. 4D,E) and stopped it at a limited point of the posterior midpiece (Fig. 6, Movie S2, S3). Treating sperm with mibefradil or verapamil increased the relative intracellular Ca²⁺ level in the posterior end of the midpiece (Fig. 7), suggesting that the verapamil‐ and mibefradil‐sensitive Ca²⁺ channel affects local control of the intracellular Ca²⁺ level in the posterior midpiece. It is unknown why blockage of the Ca²⁺ channel results in an increase of intracellular Ca²⁺ levels (Fig. 7). In a recent pharmacological study in our lab, an adenylate cyclase inhibitor similarly increased the intracellular Ca²⁺ level in the posterior midpiece (unpublished data). Downstream signaling appears to participate in the control of the Ca²⁺ level in the posterior midpiece.

Verapamil, mibefradil and nitrendipine all weakened the UM wave in the whole midpiece, which was shown in the decrease of group I sperm (Fig. 4G). All the VDCC blockers were as effective as the L‐type VDCC, but with different affinities (Bezprozvanny & Tsien 1995). Among them, nitrendipine has a high specificity to the L‐type VDCC (McCarthy & Fry 1988). In the present study, the effect of nitrendipine was predominantly observed in the anterior midpiece (Figs. 3K,L,4F), which was supported by the Ca²⁺ imaging (Fig. 7), suggesting that the nitrendipine‐sensitive Ca²⁺ channel, unlike the mibefradil‐sensitive one, predominantly affects the UM wave in the anterior midpiece.

The results of the pharmacological studies using Ca²⁺ channel blockers conclusively indicate that the high motility state of Cynops sperm is produced by the control of local intracellular Ca²⁺ in the anterior and the posterior midpiece. Recent studies demonstrated that the motility state of mouse sperm could be modulated through a local increase of intracellular Ca²⁺ levels around the anterior and/or posterior midpiece (Ho & Suarez 2001; Chang & Suarez 2011). Presumably, the local control of Ca²⁺ levels in the midpiece is a common machinery in vertebrate species to coordinate the movement of the long sperm tail. In the case of mammalians, the local increase of intracellular Ca²⁺ appears to be caused by the release of Ca²⁺ from intracellular stores and the influx through a membrane‐associated cation channel. It is unknown which Ca²⁺ channels are present in the Cynops sperm and further study is needed to elucidate the local control of the intracellular Ca²⁺ to induce and maintain a high motility state.

It is noteworthy that the CatSper channel may be involved in the high motility state of Cynops sperm because mibefradil is known to inhibit the Ca²⁺ current through the CatSper channel in mammalian sperm (Lishko et al. 2011; Strünker et al. 2011). CatSper is a sperm‐specific, plasma membrane‐associated cation channel (Ren et al. 2001; Qi et al. 2007) that is critical in inducing the hyperactivated motility of mouse sperm. The genes for the CatSper subunits are widely found in the genomes of deuterostomes; however, they are partly lost in a lineage‐specific manner, most likely because they were redundant for certain modes of fertilization (Cai & Clapham 2008). Although the genes for the CatSper subunits have been lost in the genomes of Xenopus species (Cai & Clapham 2008), which are amphibian model animals and undergo external fertilization, the CatSper function in mammalian sperm motility appears to be related to the process specific for internal fertilization. We will need to clarify whether they exist in the genome of urodeles.

In the present study, we unexpectedly found that a reverse wave was propagated in the verapamil‐ or mibefradil‐treated sperm (Movie S2, S3). Although it looks very clear that the UM alone waves in the tail of the moving C. pyrrhogaster sperm, Bufo sperm that have thin axial rods (Scheltinga & Jamieson 2003b) exhibit the typical wave movement of the tail itself using the undulating membrane (O'Brien et al. 2011). Supposedly, the substantial axial rod of C. pyrrhogaster sperm produces less interference of the UM wave in between the midpiece and principal piece, which results in the appearance of the reverse wave in the principal piece of the verapamil‐ or mibefradil‐treated sperm. Although how the UM reverse wave is produced and independently maintained is unknown now, a new mechanism for controlling flagellum motility is expected to present in the sperm principal piece of newt sperm.

Amphibian egg jelly has a significant role in the successful fertilization by controlling sperm motility (Campanella & Gabbiani 1979; Olson et al. 2001; Watanabe et al. 2010; O'Brien et al. 2011; Takayama‐Watanabe et al. 2012; Tholl et al. 2011), inducing acrosome reaction (Omata 1993; Campanella et al. 1997; Sasaki et al. 2002; Watanabe et al. 2009) and binding to egg envelope (Hiyoshi et al. 2007); however, many of these roles are not always common among amphibians. For example, jelly substances prolong the lifespan of Xenopus laevis sperm (Tholl et al. 2011) and promote the forward motility of Bufo arenarum sperm (O'Brien et al. 2011). The fertilization modes of amphibians are highly diversified to adapt to their specific environment for fertilization in freshwater or on land (Wake & Dickie 1998), which may be one reason why egg jelly has species‐specific functions. It is unknown whether the high motility state of Cynops sperm is common in amphibians or specific for the mode of internal fertilization. In the present study, although we found that the high motility state was controlled by local Ca²⁺ levels and sperm Ca²⁺ channels were involves, it is difficult to determine the exact channel types by pharmacology alone, fundamentally based on the mammalian cells. Further study about the mechanism which induces and maintains the high motility state of Cynops sperm will be needed to clarify the relationship of the high state of sperm motility with the mode of internal fertilization in amphibians.

Acknowledgments

This work was supported by a Grant‐in‐Aid for Scientific Research (C) 24570246 and (A) 24240062 from the Japan Society for the Promotion of Science and was performed in part as joint research in the Japanese Association for Marine Biology (JAMBIO).