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Movement of Spermatozoa:: Direction of Propagation and Chirality of Flagellar Bends
Movement of Spermatozoa:: Direction of Propagation and Chirality of Flagellar Bends
Tokyo, Japan
3Department of Ultrastructure Research, Wenner-Gren Institute,
The marine snail, Turritella communis, produces two types of spermatozoa, named
apyrene and eupyrene. Eupyrene spermatozoa are usually paired, but unpaired
ones are involved in fertilization. Movements of these spermatozoa were analyzed
using a video camera with a high-speed shutter. The eupyrene spermatozoa usually
swim with the head foremost but are able to swim flagellum foremost. A reversal of
the direction of their swimming was found to be the result of a change in the
direction of flagellar bend propagation, which changed with calcium concentration.
Reversal of the direction of bend propagation was accompanied by a reversal of
direction of the rotational movement of the spermatozoa around their long axis,
suggesting that the bending waves keep the sense of their three-dimensional form.
The swimming speed of apyrene spermatozoa in natural seawater was about
one-eighth of that of the eupyrene ones and remained almost constant in highly
viscous medium.The swimming speed of conjugated eupyrene spermatozoa was
the same as that of unpaired spermatozoa over a wide viscosity range (⬍3,000 cP).
No advantage of swimming by two spermatozoa could be detected in Turritella
spermatozoa. Cell Motil. Cytoskeleton 44:85–95, 1999. r 1999 Wiley-Liss, Inc.
Key words: bidirectional swimming; conjugated spermatozoa; marine snail; 9 ⴙ 2 axoneme; rotational
movement; sperm dimorphism
This phenomenon has attracted many investigators, and Creed, 1962; Biggers and DeLamater, 1965; Phillips, 1972;
light and electron microscopic studies have been carried Temple-Smith and Bedford, 1980; Taggart et al., 1993], the
out on the morphology of the apyrene and the eupyrene snail family Turritella [Afzelius and Dallai, 1983], the primi-
spermatozoa and their spermatogenesis [Koike and Nishi- tive apterygote insect group Thysanura [Dallai and Afzelius,
waki, 1980; Braidotti and Ferraguti, 1982]. The eupyrene 1984], dytiscid water beetles [Dallai and Afzelius, 1985,
spermatozoa are known to be essential for fertilization 1987], and the diplopod myriapods [Reger and Fitzgerald,
[Kennedy and Keegan, 1992], but the function of the 1979]. These studies suggest the pairing is an adaptation for a
apyrene spermatozoa remains obscure. more effective swimming capacity, although many other
Conjugated spermatozoa have been described from suggestions as to the function of the conjugated spermatozoa
four or five animal groups; American marsupials [Biggers and have been offered [Afzelius and Dallai, 1987].
Movement of Turritella Spermatozoa 87
Fig. 4. Phase-contrast video micrographs of the rotational movement of Turritella spermatozoa observed
along the sperm axis. Spermatozoa propagating bending waves distally roll counterclockwise when viewed
from the head (A), whereas spermatozoa propagating bending waves proximally roll clockwise (B). The
time interval between successive images is 1/60 s (A) and 1/2 s (B). Arrows indicate the direction of the
beating planes determined from the parts of the flagellum in focus. Bar ⫽ 10 µm.
bäckskil, Sweden, in the summers of 1990 and 1996. Ca2⫹ on the sperm movement, CaCl2 or [ethylene-
Snails having a total length of 2.3–3.0 cm were used. bis(oxyethylenenitrilo)]tetraacetic acid [EGTA] was added
Spermatozoa were obtained by micropipettes from the to a standard solution, which consists of Ca-free artificial
gonad after removing the shell. To prepare the unpaired seawater and 20 µM calcium ionophore A23187 (ICN
eupyrene spermatozoa, the conjugated spermatozoa were Biomedicals, Inc., Aurora, OH). A stock solution of 10
separated by pipetting several times. The movements of mM calcium ionophore A23187 was prepared in dimethyl
the sperm and their flagella were observed in natural sulfoxide (E. Merck, Darmstadt, Germany) and stored at
seawater, artificial seawater consisting of 470 mM NaCl, 4°C. The presence of dimethyl sulfoxide did not change
10 mM KCl, 10 mM CaCl2, 54 mM MgSO4, and 10 mM the movement characteristics of the spermatozoa.
Tris-HCl, pH 8.2, and artificial seawater with the viscosi- For examination and recording of sperm movement,
ties of 110, 230, 780, and 3,000 cP, which were increased samples were transferred onto a glass slide beneath a
with methylcellulose (viscosity of 2% aqueous solution: raised coverslip (0.18 mm depth). In the experiments
4,000 cP, Tokyo Kasei Kogyo Co., Ltd., Tokyo). The examining the rotational movement of the spermatozoa
viscosity of artificial seawater with methylcellulose was around their long axis, samples were placed into a
measured by a falling ball viscometer [Ishijima et al., 0.30-mm-deep observation chamber and covered with a
1986] at 18°C. In experiments examining the effect of coverslip precoated with 0.5% agar and dried [Ishijima et
Movement of Turritella Spermatozoa 89
flagellar waves and swimming speed of spermatozoa) of their flagellar waves [Taylor, 1952; Gray, 1962;
could be detected between paired and unpaired spermato- Ishijima et al., 1992]. Therefore, valuable information on
zoa (Table I). the three-dimensional geometry of flagellar waves will be
obtained from the parameters of the rotational movement
Effects of Calcium Ion on the Propagating of spermatozoa; for example, knowledge of the direction
Direction of Flagellar Waves of sperm rotation gives us the sense of the three-
To elucidate the factor influencing the direction of dimensional geometry in flagellar waves. To examine
flagellar wave propagation, the effects of calcium ion on whether or not the conformational change of flagellar
the propagation direction of flagellar waves were exam- waves of spermatozoa is caused by changing the propaga-
ined by altering the concentration of CaCl2 or EGTA in tion direction of bending waves, the rotational movement
the presence of 20 µM calcium ionophore A23187. A of spermatozoa around their axis was examined using
decrease in EGTA and an increase in CaCl2 in the unpaired eupyrene spermatozoa in standard solution
standard solution increased the percentage of the sperma- containing either 0.3 mM EGTA or 1 mM CaCl2. In the
tozoa propagating flagellar waves proximally (Table II). former solution, all spermatozoa had distally propagating
The parameters of flagellar waves propagating proxi- flagellar waves, whereas in the latter solution almost all
mally in the standard solution did not differ essentially spermatozoa had proximally propagating flagellar waves
from that in natural seawater (data not shown) except for (Table II).
the duration of motility, which rapidly decreased with All spermatozoa (n ⫽ 58) propagating bending
high calcium concentrations. waves from the base of the flagellum to the tip rotated
counterclockwise, as viewed from the head to the tail
Rotational Movement of an Unpaired Eupyrene (Fig. 4A). The average rotation frequency was 10.1 ⫾ 3.1
Spermatozoon Around Its Axis Hz (n ⫽ 15). On the other hand, all spermatozoa (n ⫽ 22)
The rotational movement of spermatozoa around propagating bending waves from the tip to the base
their long axis is due to the three-dimensional geometry rotated clockwise with the rotation frequency of 1.2 ⫾
Movement of Turritella Spermatozoa 91
Fig. 7. Phase-contrast video micrographs of paired eupyrene spermatozoa beating in natural seawater (A)
and in viscous seawater of 110 cP (B). The time interval between successive images is 1/60 s. Arrowheads
indicate the corresponding bends. Bar ⫽ 30 µm.
0.49 Hz (n ⫽ 12) (Fig. 4B). These results show that the to the one-third power of the viscosity (Fig. 5). No
chirality of flagellar waves does not change when the significant difference in the swimming speed could be
direction of wave propagation along the sperm flagella detected between paired and unpaired spermatozoa in
changes. seawater or in the four viscous seawaters. Typical flagel-
lar waves of unpaired spermatozoa in viscous seawaters
Effects of Viscosity on Eupyrene Sperm and Their are shown in Figure 6.
Flagellar Movements It is well known that two individual flagella beat
Turritella communis has internal fertilization; there- synchronously with one another even though they do not
fore, their spermatozoa will have to swim in fairly have physical contact with one another, as spirochetes
viscous medium in situ. To investigate the meaning of also do [Gray, 1928; Taylor, 1952]. To test the synchroni-
pairing of spermatozoa and the change in the movements zation in the paired eupyrene Turritella spermatozoa, the
of spermatozoa and their flagella in viscous medium, the flagellar movements were examined in seawater and the
sperm and their flagellar movements were examined in four viscous seawaters. In natural seawater, the two
viscous seawaters of 110, 230, 500, and 3,000 cP. The spermatozoa in a pair beat with different phases (Fig. 7A),
swimming speed was found to be inversely proportional whereas in the seawater with viscosities over 110 cP,
92 Ishijima et al.
DISCUSSION
Bidirectional Swimming of Eupyrene Spermatozoa
Turritella spermatozoa can move forward and back-
ward. The bidirectional swimming seems to be a general
characteristic of the snail spermatozoa because the eupy-
rene spermatozoa of the mud creeper (Terebralia palus-
tris) and the black snail (Semisulcospira bensoni) also
showed bidirectional swimming (Ishijima and Furukawa,
unpublished observations). There are basic differences in
the movement parameters between waves propagating
toward the tip of the flagella and those moving toward the
base, as shown in Table I and Figure 1. The distally
propagating waves were more effective than the proxi-
mally propagating ones: the swimming speed of sperma-
tozoa with distally propagating waves was more than 12
times that of spermatozoa with proximally propagating
Fig. 8. Phase-contrast video micrographs of the flagellar movement of waves, and the distally propagating waves generally
apyrene spermatozoa. A spermatozoon swims in natural seawater (A) preceded the proximally propagating ones in the time
and in viscous seawater of 3,000 cP (B). Bar ⫽ 30 µm. course of the flagellar movement of eupyrene spermato-
zoa, even though the reverse transition occasionally
paired spermatozoa were aggregated and beat in union happened. Thus, it is unlikely that backward swimming in
(Fig. 7B). Turritella spermatozoa plays an important role. The
presence of calcium ion increased the percentage of
Movements of Apyrene Spermatozoa spermatozoa with the proximally propagating waves and
It has been suggested that the apyrene spermatozoa seemed to inhibit the distally propagating waves; thereby,
may aid in the transport of the eupyrene spermatozoa due the proximally propagating waves were able to appear on
to their greater motility [Friedländer and Gitay, 1972]. the sperm flagella. This interpretation seems to be sup-
Apyrene spermatozoa from Turritella were hence re- ported by several findings. Caffeine or 8-bromoadenosine
corded with respect to their movement in natural seawater 3’:5’-cyclic monophosphate, both of which activate the
or the seawaters with different viscosities. An apyrene sperm motility [Mann and Lutwak-Mann, 1981], pro-
Turritella spermatozoon consists of a head (without a longed the duration of flagellar movement of distally
nucleus) and eight or more flagella (8.6 ⫾ 1.7, propagating waves and restored the active movement of
mean ⫾ S.D., n ⫽ 12). One of these flagella is longer sperm flagella that had proximally propagated bending
(0.23 ⫾ 0.11 mm, n ⫽ 11) than the others (0.04 ⫾ 0.02 waves. Only at unusually high concentrations of calcium
mm, n ⫽ 11). The average swimming speed of the did all the spermatozoa exhibit the proximally propagat-
apyrene spermatozoa was 19.3 µm/s (S.D. ⫽ 12.0, n ⫽ 18) ing waves. Turritella spermatozoa can move backward as
in natural seawater and 11.2 µm/s (S.D. ⫽ 4.1, n ⫽ 20) in well as forward like some other spermatozoa and many
the 3,000 cP seawater. The swimming speed of the protozoa [Holwill and McGregor, 1976; Baccetti et al.,
apyrene spermatozoa in natural seawater was rather 1989; Ishijima et al., 1994]. Crithidia cells and tephritid
slower than that of the eupyrene spermatozoa but was and Myzostomum spermatozoa, in which the backward
almost the same as that in the 3,000 cP seawater. Many swimming is usual, abruptly change their swimming
flagella beat independently in natural seawater (Fig. 8A), direction, but such a rapid change in swimming direction
whereas they beat in union in the 3,000-cP seawater (Fig. 8B). could not be detected in Turritella spermatozoa, although
spermatozoa with proximally propagating waves restored
Movements of Distal Tail Fragments of Eupyrene the flagellar waves propagating distally within a short
Spermatozoa time. Thus, the role of a change in the swimming
An unexpected finding in this study was that of the direction of Turritella spermatozoa seems to differ from
distal tail fragments of the eupyrene sperm flagella that of these cells.
Movement of Turritella Spermatozoa 93
Eupyrene Turritella spermatozoa swam almost in beating plane has also been described for the spermatozoa
circles when moving freely near a coverslip, as sea urchin of the humpbacked fly [Curtis and Benner, 1991].
spermatozoa also do [Ishijima and Hamaguchi, 1992].
However, the circular rotation of Turritella spermatozoa Direction of Wave Propagation and Chirality of
is due to the bending of the beating plane of flagellar Flagellar Waves
waves (Fig. 3), rather than to an asymmetry of the The eupyrene Turritella spermatozoa reversed the
flagellar waves (a difference in curvature between the direction of their swimming as a result of a change in the
principal and reverse bends) as seen in other spermato- direction of bend propagation, this direction apparently
zoa, including those of sea urchins and mammals. Be- being regulated by calcium ions. When the spermatozoa
cause the beating plane of Turritella spermatozoa is in the were viewed from head to tail, a reversal of direction of
direction perpendicular to the line connecting the central the bend propagation accompanied the reversal of the
microtubules [Afzelius and Dallai, 1983] as the others do, sperm rotating around their long axis. Baccetti et al.
the bending of the beating plane in the Turritella sperma- [1989] reported the same findings in spermatozoa of
tozoa is perhaps caused by a mechanism that is different tephritid flies; thus their flagellar waves, which propagate
from that of other spermatozoa. The bending of the from the tip of the flagellum toward the head, induce the
94 Ishijima et al.
Moore and Taggart, 1995]. The difference in orientation Gray J. 1962. Flagellar propulsion. In Bishop DW, editor. Spermato-
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ACKNOWLEDGMENTS axis. J Exp Biol 163:15–31.
Ishijima S, Ishijima SA, Afzelius B. 1994. Movement of Myzostomum
We thank the director, Prof. J.-O. Strömberg, and spermatozoa: calcium ion regulation of swimming direction.
the staff of the Kristineberg Marine Biological Station, Cell Motil. Cytoskeleton 28:135–142.
Fiskebäckskil, Sweden, where this work was carried out, Kaneda Y. 1965. Movement of sperm tail of frog. J Fac Sci Univ Tokyo,
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for their generous hospitality. We also thank Dr. Y. Kennedy JJ, Keegan BF. 1992. The encapsular developmental se-
Hamaguchi for his active interest and advice. quence of the mesogastropod Turritella communis (Gastropoda:
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