# Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies.

IntroductionOptimal motility of spermatozoa is essential for fertility; in particular, swimming speed is the most important parameter for successful fertilization (Rothschild and Swann, 1951; Levitan, 2000; Gage et at, 2004). However, the questions of how the motility and morphological parameters of the spermatozoa relate to each other and what is the most important motility parameter affecting the swimming speed of the spermatozoa remain unanswered. For investigating these questions, fish spermatozoa are extremely suitable specimens because of their variations in sperm motility and morphology. However, detailed information about the sperm and flagellar movements of fish spermatozoa has been provided in only a few cases, and the reported data still vary and disagree (Terner, 1986). The discrepancies arise in part from the short duration of the active motility of the fish spermatozoa (Ishijima a al., 1998), but are mainly due to differences in the method used to evaluate motility (Scott and Baynes, 1980; Stoss, 1983; Terner, 1986). To dispel these uncertainties and clarify the important invariant feature of the sperm motility, the sperm and their flagellar movements were quantitatively described in the lancelet and fish spermatozoa by using high-speed video microscopy (Ishijima a al., 1998).

An analysis of mammalian spermatozoa revealed two modes of flagellar movement (Ohmuro and Ishijima, 2006; Ishijima, 2007): a nearly constant-curvature mode that was mainly observed in normal spermatozoa and a nearly constant-frequency mode that was mainly observed in hyper-activated spermatozoa. The constant-frequency mode, the so-called isochronisms of oscillations (the period of oscillation is independent of the amplitude), is the essential feature of hyperactivated mammalian spermatozoa, and the maintenance of a low oscillation frequency seems to be essential for the functions of these spermatozoa (Ishijima, 2011). On the other hand, the constant-curvature mode is likely to be found in almost all normal spermatozoa, in invertebrates as well as in mammals, because the same flagellar axoneme functions in both the mammalian and echinoderm spermatozoa (Kaneko et al., 2007; Ishijima, 2007). However, its detailed features are still not clarified. To verify the characteristics of this mode and the mechanism underlying it, the wide variations in the motility of fish spermatozoa are extremely useful; namely, in determining whether the maximum amount of microtubule sliding as well as the maximum curvature of the flagellar waves are consistently maintained while the beat frequency significantly changes.

To understand the basic mechanisms underlying flagellar movement and sperm function, it is necessary to quantitatively estimate the hydrodynamic characteristics--for example, the flagellar force generated by a flagellum, the power output dissipated by a flagellum, and the bending moment acting on a flagellum, as well as the swimming speed of a spermatozoon (Gray and Hancock, 1955; Brokaw, 1970; Hiramoto and Baba, 1978; Baltz et al., 1988; Ishijima and Hiramoto, 1994; Ishijima, 2011). For this purpose, the resistive-force theory introduced by Gray and Hancock (1955) is extremely useful because these characteristic values can be easily estimated from the measured values of the motility parameters of the sperm and their flagellar movements even if modification of the drag coefficients is necessary (Lighthill, 1975, 1976; Ramia et al., 1993). Various methods of estimating these data have been reported (Lauga and Powers, 2009), but the investigations involving the hydrodynamic analysis of the sperm and their flagellar movements were mainly carried out by theoretical analysis (Higdon, 1979; Dresdner et al., 1980, Evans and Lauga, 2010); thus, the validity of these methods has to be fully confirmed by experimental results.

In the present study, the relationships between the movement and morphological parameters of a lancelet and fish spermatozoa and their effects on sperm swimming speed were examined using the measured values obtained from spermatozoa having different morphologies. The beat frequency of the flagellum correlated well with the sperm swimming speed and the power flagellar waveform varied only slightly and, therefore, were remotely related to the motility parameters. The applicability of the resistive-force theory to the lancelet and fish spermatozoa was also examined, and a correction of the drag coefficients used in the resistive-force theory is suggested to explain the experimental results.

Materials and Methods

Materials

Concentrated spermatozoa were obtained from a lancelet (Branchiostoma belcheri) and 35 fish species (Deania calcea, Chimaera phantasma, Sardinops melanostictus, Engraulis japonica, Osmerus eperlanus mordax, Spirinchus lanceolatus, Hypomesus pretiosus japonicus, Hypomesus transpacificus nipponensis, Plecoglossus altivelis altivelis, Salangichthys microdon, Oncorhynchus mykiss, Hypoptychus dybowskii, Aulichthys japonicus, Gasterosteus aculeatus, Syngnathus schlegeli, Hexagrammos agrammus, Hexagrammos octogrammus, Hexagrammos otakii, Pseudoblennius cottoides, Rhinogobius sp. CB, Rhinogobius sp. DA, Tridentiger kuroiwae, Eviota prasina, Entomacrodus striatus, Istiblennius bilitonesis, Lateolabrax japonicus, Apogon semilineatus, Pagrus major, Zoarces elongates, Ammodytes personatus, Trichiurus japonicus, Arctoscopus japonicus, Takifugu niphobles, Rudarius ercodes, and Paralichthys olivaceus) (Hara and Okiyama, 1998; Ishijima et al, 1998).

Observations and recording

The spermatozoa of most fish species have a short period of progressive motility after release into a medium (Ishijima et al., 1998); accordingly, the following procedures were used for observation and recording of the sperm and their flagellar movements. Approximately 25 [micro]1 of the concentrated spermatozoa was placed on a glass slide on the stage of a Nikon phase-contrast microscope (Nikon Corp., Tokyo, Japan), and 50 [micro]l of egg seawater, physiological solutions, or seawater (depending on the species, Ishijima et al., 1998) was placed next to the drop. To initiate sperm motility, the concentrated spermatozoa were diluted by placing a cover slip (22 mm x 22 mm, coated with 1% bovine serum albumen) over them.

To record these movements, the microscope objective (plan 40 x BM, Nikon Corp.) was focused on an area about 3 [micro]m from the lower surface of the coverslip, and then the specimen was moved to the center of the visual field. At this distance, it is easier to record many spermatozoa and reduce the effect of the coverslip surface. The recording usually began within 5 s after dilution. The sperm movements were recorded on a VHS 1/2-inch cassette videotape at 200 fields per second using a Nac microscopic high-speed video system (MHS-200, Nac, Inc., Tokyo, Japan). The observations and recording were made at 23 [degrees]C.

To examine the effect of the coverslip surface on the sperm motility, the freely swimming spermatozoa of the green bubble goby (Eviota prasina) were recorded at approximately 1 [micro]m, 2 [micro]m, and 3 [micro]m from the lower surface of the coverslip. When the flagella of the spermatozoa swimming at 1 [micro]m from the lower surface of the coverslip were in focus, the beating flagella of the spermatozoa attached to the coverslip by their heads were also sharply in focus; hence, the free-swimming sperm flagella probably beat at 0.80-1.13 [micro]m from the lower surface of the coverslip because the width of the sperm head was 1.6 [micro]m (Hara and Okiyama, 1998) and the depth of focus of the 40x objective is about 0.65 [micro]m.

Analysis of sperm and flagellar movements.

The spermatozoa swim by wave-like movements--that is, flagellar waves--that usually propagate backward along the flagellum, pushing the water backward and propelling the sperm forward. To define the flagellar movements of the spermatozoa in quantitative terms, the beat frequency, the wavelength, and the amplitude of the flagellar waves are usually used as parameters, analogous to the descriptions of wave motion in physics.

To obtain these parameter values from the images of the sperm and their flagella, the images recorded on the videotape were traced from a video monitor (TN-96, Panasonic, Osaka, Japan) onto transparent plastic sheets using a finepoint marker; the final magnification was 1580 times. Measurements of the flagellar and head lengths were directly made on these traces. The amplitude of the flagellar waves was defined as the maximum transverse displacement of the flagellum from the wave axis, which was drawn through the center of the beat envelope. The wavelength and the number of waves contained in a flagellum were determined by reference to the wave axis. The beat frequency was calculated from the period required for one complete beat. The swimming speed was determined by measuring the distance between the positions of the sperm head at the beginning and the end of 0.5-s intervals.

The flagellar wave of the spermatozoa is therefore approximately described by the sinusoidal shape given by

y-b sin 2[pi](x/[lambda]-ft),

where b is the amplitude, [lambda] is the wavelength, and f is the beat frequency (Hiramoto and Baba, 1978; Ohmuro and Ishijima, 2006). The maximum tangential angle (shear angle) was obtained when the flagellum intersects the x-axis; that is, arctan(27[pi]b/[lambda]).

Calculations of swimming speed and power output by the resistive-force theory

The swimming speed was also calculated using the resis-tive-force theory based on the following equation (Gray and Hancock, 1955):

V=2(c-1)[[pi].sup.2]f[b.sup.2][[lambda].sup.-1][[1+2C[[pi].sup.2][b.sup.2]/[[lambda].sup.2]-[(1+2[[pi].sup.2][b.sup.2]/[[lambda].sup.2]).sup.1/2](3a)(n[lambda]){ln(d/2[lambda])+1/2}].sup.-1], (Equation 1)

where V is the desired swimming speed, C is the ratio of the normal to the tangential drag coefficient, f is the beat frequency, b is the amplitude, [lambda] is the wavelength, a is the half length of the head, n is the number of waves, and d is the radius of the flagellum. The dimensionless swimming speed was calculated by V/(f[lambda]). In the original resistive-force theory by Gray and Hancock (1955), C = 2. In the present study, the value of C was determined in order to explain the measured values of the dimensionless swimming speed of the spermatozoa swimming at different distances from the surface of the coverslip. The hydrodynamic power output, P, dissipated by a single flagellum against the medium (Rikmenspoel, 1984) and its non-dimensional form (inverse efficiency, [[eta].sup.-1]; Higdon, 1979) were calculated by

P=4[[pi].sup.3] [micro] [f.sup.2][b.sup.2]L/{0.62-ln(2[pi]d/[lambda])}

[[eta].sup.-1]=P/[{6[pi] [micro]a+2[pi] [micro]L/ln(2L/d)}[v.sup.2]],

respectively, where yr is the viscosity of the medium, L is the total length of the flagellum, and v is the measured swimming speed.

Statistical analysis

All data are expressed as the mean [+ or -] s.d. Linear and nonlinear regression analyses were carried out using SPSS 11.0.1 J and SPSS Regression Models 9.0J (SPSS Japan Inc., Tokyo). The most appropriate regression models for a given data set were determined using the curve estimation procedure. The significant level was considered to be P < 0.05. The correlation coefficient (r) and the r-squared ([R.sup.2]) value were used to understand the relationships between the motility and morphological parameters. The groups in Table 2 were compared by one-way ANOVA followed by Scheffe post hoc test using SPSS 11.0.1J.

Results

Movement characteristics of the lancelet and fish spermatozoa

The flagellar movements of the spermatozoa of several fishes are shown in Figure 1. Two features were quickly realized: (1) the longer the sperm flagellum, the greater the number of waves on the flagellum; and (2) the flagellar waveform remained almost constant. The movement characteristics and the head and flagellar lengths of the lancelet and fish spermatozoa are summarized in Table 1. The parameters defining the flagellar waveform, the wavelength, and the amplitude varied only slightly in the spermatozoa of different species, although the flagellar length varied from 13.7 [micro]m (half-lined cardinal, Apogon semilineatus) to 163 (Birdbeak dogfish, Deania calcea). Furthermore, the number of waves contained in a flagellum increased with the flagellar length (Fig. 2A). These results suggest that the flagellar waveform remains almost constant in the spermatozoa having different morphologies. On the other hand, the beat frequency and the swimming speed varied significantly (Table 1). Furthermore, since the swimming speed was proportional to the beat frequency (Fig. 2B), these results suggest that the swimming speed is mainly determined by the beat frequency and only slightly affected by the parameters defining the flagellar waveform. There was a weak relationship between the number of waves and the head length (r = 0.64), although a slight relationship was found between the head and flagellar lengths (r = 0.34). The amplitude of the flagellar waves was insensitive to changes in the flagellar length (r = 0.13). The hydrodynamic power output dissipated by a single flagellum was estimated to be 1.83 [+ or -] 1.42 x [10.sup.-14] W, similar to that reported for sea urchin spermatozoa (Brokaw, 1966). The power output also did not correlate with the parameters defining the flagellar waveform, but strongly correlated with the approximate square of the beat frequency (Fig. 3) or the swimming speed.

Table 1 Movement characteristics and head and flagellar lengths of the lance let and fish spermatozoa Statistic Wavelength Amplitude Head Number Flagellar ([mu]m) ([mu]m) length of length ([mu]m) waves ([mu]m) Mean [+ or 17.1 [+ or 3.1 [+ or 0.88 [+ 1.7 [+ 39.1 [+ or -] s.d. -] 3.1 -]0.7 or -] or -] -] 28.3 0.41 0.9 Minimum 11.2 1.3 0.45 0.8 13.7 (a) Max:Min 2.4 3.3 5.9 6.4 11.9 (b) Statistic Beat Swimming frequency speed (Hz) ([mu]m/s) Mean [+ or 40 [+ or -] 102 [+ or -] s.d. 14.9 -] 54.7 Minimum 3.8 13.1 (a) Max:Min 19.2 20.2 (b) Data were collected from at least ten spermatozoa from each species of a lancelet and 35 fishes, (a.)The minimum value of the motility and morphological parameters. (b.) The ratio of the maximum to minimum values of the motility and morphological parameters. It represents the variation in each parameter.

To clarify the relationship between the waveform and the beat frequency, the beat frequency was plotted versus the maximum shear angle of the flagellar wave (Materials and Methods) in Figure 4. The beat frequency varied widely without any significant change in the maximum shear angle (0.85 [+ or -] 0.08 rad). Since the shear angle of a flagellar wave is directly related to the amount of sliding between the doublet microtubules in the sperm flagellum (Ohmuro and Ishijima, 2006), this result proved that the spermatozoa beat in the constant sliding displacement mode; namely, the maximum amount of microtubule sliding remained almost constant while the beat frequency was able to change widely.

The effect of the flagellar length on the swimming speed was not simple (Fig. 5). However, the least squares regression analysis revealed two things. First, the short flagella were at a disadvantage in developing the swimming speed; however, so were the extra-long flagella. Second, the dimensionless swimming speed, which was the measured swimming speed divided by the beat frequency and the wavelength, had a maximum value (0.155). Therefore, most species had spermatozoa with moderately short tails (Fig. 5).

The inverse efficiency varied widely and did not reach a specific value (Fig. 6). This parameter was well correlated with the dimensionless swimming speed (Fig. 6); therefore, either of these parameters can be used to estimate the efficiency of the sperm motility.

Effect of boundary on the sperm motility

The swimming speeds calculated by the original resistive-force theory (Gray and Hancock, 1955) using the measured motility and morphological parameters (Eq. 1, Materials and Methods) were plotted versus the measured swimming speeds (Fig. 7). The calculated data correlated well with the experimental ones; however, the calculated swimming speed was 42.7% on average greater than the swimming speed measured at approximately 3 [micro]m from the coverslip in the present study.

To explain this difference between the calculated and measured swimming speeds, we examined the effect of the glass surface on the swimming speed of spermatozoa of the green bubble goby because these spermatozoa had almost the same parameter values of the flagellar waveform as the average values of the lancelet and fish spermatozoa. The swimming speed and the wavelength and amplitude of the flagellar waves of the spermatozoa swimming at about 1 [micro]m from the glass surface were almost identical to those of the spermatozoa swimming at about 2 [micro]m and 3 [micro]m from the glass surface (Table 2), whereas the beat frequency significantly decreased when the spermatozoa approached the glass surface. Therefore, the dimensionless swimming speed calculated from these parameter values also significantly increased with decreasing distance from the glass surface (Table 2). The ratios of the normal to tangential drag coefficients for these dimensionless swimming speeds evaluated by Eq. 1 were determined and compared (Table 2) to those from the original resistive-force theory by Gray and Hancock (1955). At about 1 pm from the glass surface, the ratio between the normal and tangential drag coefficients was 1.99, identical to that used by Gray and Hancock. This result suggests that the original resistive-force theory by Gray and Hancock (1955) gives the swimming speed of the spermatozoa swimming at about 1 [micro]m from the glass surface. On the other hand, the ratios of the normal-to-tangential drag coefficients also increased with decreasing distance from the glass surface (Table 2). The ratio of the coefficients calculated from the swimming speed of the spermatozoa of the lancelet and fishes was 1.63 at about 3 [micro]m from the glass surface. Consequently, for correctly estimating the swimming speed using the resistive-force theory it is necessary to use a drag coefficient ratio of less than 2, corresponding to the distance from the glass surface.

Table 2 Effect of distance from the coverslip surface on the swimming speed Parameter 1 (a) 2 (a) 3 (a) 3 (a) ([mu]m) ([mu]m) ([mu]m) ([mu]m) Swimming speed 141 [+ or 142 [+ or 148 [+ or 102 [+ or (v, [mu]m/s) -] 28 -] 16 -] 24 -] 54.7 Wavelength 17.5 [+ or 16.9 [+ or 16.7 [+ or 17.1 [+ or ([lambda], -] 1.6 -] 1.3 -] 1.3 -] 3.1 [mu]m) Beat frequency 39.1 (b) [+ 46.3 (b) [+ 51.2 (b) [+ 40 [+ or -] (f, Hz) or -]6.4 or -] 6.0 or -]5.5 14.9 v/(f[lambda]) 0.207 (c) 0.180 (c) 0.169 (c) 0.152 [+ or [+ or -] [+ or -] [+ or -] -]0.047 0.039 0.031 0.036 C (d) 1.99 1.75 1.82 1.63 Parameter Gray and Hancock, 1955 Swimming speed (v, [mu]m/s) Wavelength ([lambda], [mu]m) Beat frequency (f, Hz) v/(f[lambda]) 0.23 C (d) 2.00 The first three columns were obtained from the green bubble goby (Eviota prasina) and the fourth was from a lancelet and 35 fishes. The values of 1 [micro]m, 2 [micro]m, and 3 [micro]m were obtained from 21, 20, and 16 spermatozoa, respectively. (a.) The value has an error of approximately 0.65 [micro]m due to the depth of focus of the 40X objective (Materials and Methods). (b.) These values are significantly different (P < 0.05). (c.) These values are significantly different except between 2 [micro]m and 3 [micro]m (P < 0.05). (d.) The ratio between the normal and the tangential drag coefficients (C) was evaluated by Eq. 1 (Materials and Methods).

Discussion

Constant sliding displacement mode in sperm motility

The motility and morphological parameters of the spermatozoa obtained from a lancelet and 35 fishes were determined using high-speed video microscopy because these spermatozoa had wide variations in their motility and morphology. The motility parameters describing the flagellar wave, the wavelength, and the amplitude remained almost constant while the beat frequency and the swimming speed varied widely. These movement characteristics suggested that the lancelet and fish spermatozoa beat with flagellar waves of constant curvature, which was expected from the analysis of the flagellar movement of mammalian spermatozoa (Ohmuro and Ishijima, 2006; Ishijima, 2007, 2011). Because the parameters describing the flagellar waveform were not strongly determined by the hydrodynamic calculation (Brokaw, 1975; Higdon, 1979; Dresdner et al., 1980; Ishijima, 2011) and the number of waves on a flagellum increased with the flagellar length (Fig. 2A), the flagellar waves of a nearly constant amplitude and wavelength observed in the present study seem to be determined by the basic mechanisms for generating flagellar movement (Ohmuro and Ishijima, 2006). Furthermore, the relationship between the beat frequency and the maximum shear angle obtained in the present study (Fig. 4) revealed that the nearly constant waves were generated by the constant sliding displacement between the doublet microtubules of the axoneme. Since the curvature of the flagellar waves of the mammalian spermatozoa also was proportional to the amount of microtubule sliding between the doublets (Ohmuro and Ishijima, 2006; Ishijima, 2007), the flagellar movement of the constant sliding displacement mode found in the present study must generally apply in both invertebrate and mammalian spermatozoa.

The motility and morphological parameters of the spermatozoa were found to be relatively insensitive to the swimming speed as well as to the beat frequency. In fact, the beat frequency significantly correlated with the swimming speed and the power output (Figs. 2B, 3). A strong correlation between the swimming speed and the beat frequency was also verified by the nearly constant dimensionless swimming speed except in spermatozoa having short flagella (Fig. 5). As previously reported (Gibbons, 1975; Goldstein, 1981; Brokaw, 1996), long spermatozoa showed a marked tendency to beat slowly, but no clear relationship between tlagellar length and beat frequency was found in the present study. The increase in beat frequency seems to be a mechanical effect (Gibbons, 1975; Goldstein, 1981).

Effect of boundary on the sperm motility

It is well known that sperm motility is modified by a solid plane boundary, such as the glass of a microscope slide (Brokaw and Gibbons, 1975; Hiramoto and Baba, 1978). However, there is no clear experimental evidence for this effect. In the present study, the lancelet and fish spermatozoa freely swimming at about 3 p.m from the lower surface of the coverslip were recorded because it is easier at this distance to record many swimming spermatozoa having a short period of motility (Materials and Methods). Furthermore, to quantitatively clarify their sperm motility near the coverslip surface, the sperm motility of the green bubble goby (Eviota prasina) was recorded at 1 [micro]m, 2 [micro]m, and 3 [micro]m from the lower surface of the coverslip. The swimming speed and the flagellar waveform of these spermatozoa did not change at the different distances from the coverslip; however, the beat frequency significantly decreased (Table 2). Furthermore, the dimensionless swimming speed, which was swimming speed divided by beat frequency and wavelength, significantly increased when the spermatozoa approached the coverslip surface, an effect caused by the constancies of the swimming speed and the wavelength and by the decrease in the beat frequency. The decrease in the beat frequency at about 1 [micro]m from the coverslip surface would produce the decrease in the swimming speed because the swimming speed was proportional to the beat frequency (Fig. 2B). Therefore, the constant swimming speed observed in the present study demonstrated the increase in the ratio of the normal-to-tangential drag coefficients. Several hydrodynamic investigations on the effect of a boundary on sperm motility have been reported (Katz and Blake, 1975; Fulford and Blake, 1983; Ramia et at, 1993; Gillies et at, 2009) and predicted that the ratio of the normal to tangential drag coefficients increased near the boundary (Fulford and Blake, 1983; Ramia et at, 1993). For a 38-[micro]m-long slender rod such as the fish sperm flagellum in the present study, the ratios of the normal to tangential drag coefficients are 1.91, 1.87, and 1.84 for the 1 [micro]m, 2 [micro]m, and 3 [micro]m distances from the boundary (fig. 4 (c) in Ramia et at, 1993), respectively.

Acknowledgments

We thank Dr. M. Okiyama and Miss M. Hara for their technical assistance.

Received 25 November 2011; accepted 18 June 2012.

* To whom correspondence should he addressed. E-mail: sishijim@bio.titech.ac.jp

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SUMIO ISHIJIMA *

Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Tokyo, 152-8551, Japan

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Author: | Ishijima, Sumio |
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Publication: | The Biological Bulletin |

Article Type: | Report |

Geographic Code: | 9JAPA |

Date: | Jun 1, 2012 |

Words: | 4795 |

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