Cytoskeleton Volume 44 issue 2 1999 [doi 10.1002%2F%28sici%291097-0169%28199910%2944%3A2-85%3A%3Aaid-cm1-3.0.co%3B2-%23] Sumio Ishijima; Sanae A. Ishijima; Björn A. Afzelius -- Movement of Turrite

Cell Motility and the Cytoskeleton 44:85–95 (1999)
Movement of Turritella Spermatozoa:
Direction of Propagation and Chirality
of Flagellar Bends
Sumio Ishijima,1* Sanae A. Ishijima,2 and Björn A. Afzelius3
1Biological
Laboratory, Faculty of Bioscience and Biotechnology, Tokyo Institute
of Technology, O-okayama, Meguro-ku, Tokyo, Japan
2Department of Biology, Japan Women’s University, Mejirodai, Bunkyo-ku,
Tokyo, Japan
3Department of Ultrastructure Research, Wenner-Gren Institute,
University of Stockholm, Stockholm, Sweden
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
INTRODUCTION
Turritella spermatozoa are unusual in that two types
exist, apyrene and eupyrene spermatozoa, and that the
eupyrene ones, which are usually paired, can swim
backward as well as forward [Afzelius and Dallai, 1983].
These peculiarities are useful for an understanding of the
mechanism generating flagellar bends and of the meaning
of sperm dimorphism and sperm conjugation.
A change in the swimming direction has been
reported in several protozoa and spermatozoa [Holwill
and McGregor, 1976; Afzelius, 1983; Baccetti et al.,
1989; Curtis and Benner, 1991; Ishida et al., 1991;
Ishijima et al., 1994]. A reversal of the swimming
direction of Crithidia cells and spermatozoa of tephritid
flies and of Myzostomum has been shown to be due to a
change in the direction of bend propagation and to be
r 1999 Wiley-Liss, Inc.
regulated by intracellular Ca2⫹ concentrations [Holwill
and McGregor, 1976; Baccetti et al., 1989; Ishijima et al.,
1994]. Furthermore, a reversal of the swimming direction
of the tephritid spermatozoa was accompanied by a
reversal of rotation of the spermatozoa around the long
axis [Baccetti et al., 1989]; the significance of these
phenomena, however, remained to be studied.
It has long been known that two types of spermatozoa are produced in several phyla (sperm dimorphism),
especially Lepidoptera and Gastropoda [Wilson, 1925].
*Correspondence to: Dr. Sumio Ishijima, Biological Laboratory, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
12-1, O-okayama 2-chome, Meguro-ku, Tokyo 152-8551, Japan.
E-mail: sishijim@bio.titech.ac.jp
Received 30 November 1998; accepted 14 July 1999
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Ishijima et al.
Fig. 1. Phase-contrast video micrographs of unpaired eupyrene Turritella spermatozoa propagating
bending waves toward the base of the flagellum (A) and toward the tip (B). The time interval between
successive images is 1/30 s (A) and 1/60 s (B). Arrowheads indicate the corresponding bends. Bar ⫽ 30 µm.
TABLE I. Movement Characteristics of Eupyrene Turritella Spermatozoa
Free-swimming
spermb
Sperm attached to coverslipa
Direction of bend
propagation
Base to tip
Unpaired spermatozoa
Paired spermatozoa
Tip to base
Unpaired spermatozoa
Amplitude
(µm)
Wavelength
(µm)
Beat frequency
(Hz)
Swimming
speed (µm/s)
7.2 ⫾ 1.7
7.3 ⫾ 2.2
52.9 ⫾ 4.2
51.2 ⫾ 2.3
30.0 ⫾ 10.0
30.0 ⫾ 14.1
144.0 ⫾ 27.2
169.0 ⫾ 33.4
3.6 ⫾ 1.5
25.6 ⫾ 2.0
8.1 ⫾ 1.7
11.8 ⫾ 5.1
⫾ S. D. for samples of 23 spermatozoa propagating bends from base to tip from four experiments and of 20
spermatozoa propagating bends from tip to base from five experiments.
bMean ⫾ S. D. for samples of 15 spermatozoa propagating bends from base to tip from four experiments and of 18
spermatozoa propagating bends from tip to base from five experiments.
aMean
This phenomenon has attracted many investigators, and
light and electron microscopic studies have been carried
out on the morphology of the apyrene and the eupyrene
spermatozoa and their spermatogenesis [Koike and Nishiwaki, 1980; Braidotti and Ferraguti, 1982]. The eupyrene
spermatozoa are known to be essential for fertilization
[Kennedy and Keegan, 1992], but the function of the
apyrene spermatozoa remains obscure.
Conjugated spermatozoa have been described from
four or five animal groups; American marsupials [Biggers and
Creed, 1962; Biggers and DeLamater, 1965; Phillips, 1972;
Temple-Smith and Bedford, 1980; Taggart et al., 1993], the
snail family Turritella [Afzelius and Dallai, 1983], the primitive apterygote insect group Thysanura [Dallai and Afzelius,
1984], dytiscid water beetles [Dallai and Afzelius, 1985,
1987], and the diplopod myriapods [Reger and Fitzgerald,
1979]. These studies suggest the pairing is an adaptation for a
more effective swimming capacity, although many other
suggestions as to the function of the conjugated spermatozoa
have been offered [Afzelius and Dallai, 1987].
Movement of Turritella Spermatozoa
87
Fig. 3. A eupyrene Turritella spermatozoon swimming in a relatively
deep suspension. Micrographs of a spermatozoon swimming in a
circular path close to a coverslip when an observer views the cell from
above. Because the beating plane is parallel to the optical axis,
segments of the sperm flagellum are in focus. The time interval
between successive images is 1/30 s. Arrowheads indicate the corresponding bends. Bar ⫽ 30 µm.
TABLE II. Percentage of Spermatozoa With Flagellar Waves
Propagating Proximally*
Medium
Fig. 2. The time-course of flagellar movement of an unpaired eupyrene
Turritella spermatozoon. Bar ⫽ 30 µm. See text for details.
The purpose of this study was to investigate in detail the
observation by one of us (B.A.A.). The movements of
Turritella spermatozoa, not only the eupyrene spermatozoa
but also the apyrene spermatozoa, were recorded by means of
a charged-coupled device (CCD) video camera with a highspeed shutter. Movement characteristics of conjugated eupyrene spermatozoa were compared with those of unpaired
spermatozoa. Effects of free calcium ions and of viscosity
variations on these movements were also examined. The
results obtained suggest that changes in the calcium concentration affect the direction of bend propagation, which in turn
determines the swimming direction of the spermatozoa. From
Standard solution ⫹ 0.3 mM EGTA
Standard solution ⫹ 0.1 mM EGTA
Standard solution ⫹ 1 mM CaCl2
Standard solution ⫹ 3 mM CaCl2
%
n
0
16
88
100a
30
37
32
29
*Standard solution consists of Ca-free seawater (470 mM NaCl, 10
mM KCl, 54 mM MgSO4 , and 10 mM Tri-HCl, pH 8.2) and 20 µM
calcium ionophore A23187.
aSpermatozoa ceased their movement within one minute.
these results, it is argued that the rotation of spermatozoa
around their long axis is related to the chirality of the bending
waves.
MATERIALS AND METHODS
Turritella communis Lamarck snails were collected
near the Kristineberg Marine Biological Station, Fiske-
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Ishijima et al.
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.
Snails having a total length of 2.3–3.0 cm were used.
Spermatozoa were obtained by micropipettes from the
gonad after removing the shell. To prepare the unpaired
eupyrene spermatozoa, the conjugated spermatozoa were
separated by pipetting several times. The movements of
the sperm and their flagella were observed in natural
seawater, artificial seawater consisting of 470 mM NaCl,
10 mM KCl, 10 mM CaCl2, 54 mM MgSO4, and 10 mM
Tris-HCl, pH 8.2, and artificial seawater with the viscosities of 110, 230, 780, and 3,000 cP, which were increased
with methylcellulose (viscosity of 2% aqueous solution:
4,000 cP, Tokyo Kasei Kogyo Co., Ltd., Tokyo). The
viscosity of artificial seawater with methylcellulose was
measured by a falling ball viscometer [Ishijima et al.,
1986] at 18°C. In experiments examining the effect of
Ca2⫹ on the sperm movement, CaCl2 or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid [EGTA] was added
to a standard solution, which consists of Ca-free artificial
seawater and 20 µM calcium ionophore A23187 (ICN
Biomedicals, Inc., Aurora, OH). A stock solution of 10
mM calcium ionophore A23187 was prepared in dimethyl
sulfoxide (E. Merck, Darmstadt, Germany) and stored at
4°C. The presence of dimethyl sulfoxide did not change
the movement characteristics of the spermatozoa.
For examination and recording of sperm movement,
samples were transferred onto a glass slide beneath a
raised coverslip (0.18 mm depth). In the experiments
examining the rotational movement of the spermatozoa
around their long axis, samples were placed into a
0.30-mm-deep observation chamber and covered with a
coverslip precoated with 0.5% agar and dried [Ishijima et
Movement of Turritella Spermatozoa
al., 1992]. This treatment with agar increased the number
of spermatozoa attached vertically to the coverslip and
did not change the rolling direction and the rotation
frequency of the spermatozoa (data not shown). The
movements of Turritella sperm and their flagella were
recorded using a Leitz Dialux 22 microscope equipped
with a phase-contrast condenser, objectives (Neofluor
40/0.75), and 12.5⫻ eyepieces, or a Leitz Laborlux S
microscope with a phase-contrast condenser, objectives
(EF 40/0.65 and 25/0.50), and 10⫻ eyepieces. Images
were recorded on VHS 1⁄2-inch cassette videotape with a
Panasonic CCD video camera with a shutter of 1/500 s
(WV-BD 400, Matsushita Communication Industrial Co.,
Ltd., Yokohama). For preparation of the figures, images
on the video monitor were photographed with a frozen
field using Fuji Neopan F 35-mm film. Observations and
recording were made at 17–20°C.
The movements of the sperm and their flagella were
analyzed by methods previously described [Ishijima et
al., 1994]. The direction of sperm rotation around its
longitudinal axis was determined from the direction of
rotation of flagellar images [Ishijima et al., 1992].
RESULTS
Bidirectional Propagation of Flagellar Waves of
Eupyrene Spermatozoa in Natural Seawater
In natural seawater, both forward and backward
swimming spermatozoa were seen in a microscopic field.
All spermatozoa (more than 100 spermatozoa) that propagated the flagellar waves from the base of the flagellum to
the tip swam with the head foremost, whereas all
spermatozoa (more than 100 spermatozoa) that propagated the waves from the tip to the base swam with the
flagellum foremost. These spermatozoa exhibited one
characteristic feature: the distal end of the flagella hardly
exhibited transverse movement while the sperm flagella
beat (Fig. 1). Movement characteristics of these two types
were, however, quite different; spermatozoa swimming
with the head foremost moved fast and had flagellar
waves of large amplitude and high beat frequency,
compared with spermatozoa swimming with the flagellum foremost (Table I and Fig. 1).
Turritella spermatozoa changed their swimming
direction by a spontaneous reversal of bend propagation.
Generally, the spermatozoa kept propagating distally the
flagellar waves for approximately 10 min (Fig. 2A). The
proximally propagating flagellar waves appeared first in
the tip region of the flagella, then gradually extended
toward the base of the flagellum (Fig. 2B and C), and
finally covered the whole flagellum (Fig. 2D) by approximately 5 min. Usually the proximally propagating waves
kept beating for more than 10 min. The sequence of these
changes in flagellar waves was not so exact; the spermato-
89
Fig. 5. Relationship between the swimming speed and the viscosity of
the medium. Each point represents the average of 11 to 27 spermatozoa. The vertical bars are standard deviations. The lines are weighted
least square regression lines with a slope of ⫺0.35 for paired (䊉) and
⫺0.33 for unpaired (䊊) spermatozoa.
zoa that had flagellar waves propagating proximally often
recovered the flagellar waves of large amplitude and of
high beat frequency and changed the direction of wave
propagation. Addition of 10 mM caffeine or 8-bromoadenosine 3’:5’-cyclic monophosphate (Sigma Chemical
Co., St. Louis, MO) to the sperm suspensions prolonged
the movement of the distally propagating waves and
restored the active movement of sperm flagella that had
propagated proximally. Therefore, three types of sperm
flagella could be seen in the field of viewing: (1) flagella
propagating large bending waves distally; (2) flagella
propagating short bending waves proximally; and (3)
flagella propagating waves simultaneously in two directions with the distally propagating waves in the proximal
region of a flagellum and the proximally propagating
ones in the distal region. The propagation velocity of the
bending waves was not always the same in a flagellum;
the propagation velocity of distally propagating waves in
the distal region was faster than that in the proximal
region.
When the eupyrene spermatozoa were swimming in
a deep observation chamber, they moved in spiral paths.
Spermatozoa that came in contact with the coverslip or
the slide glass moved almost in circles. Their beating
plane was curved and perpendicular to the glass surface
(Fig. 3). A curved beating plane was also noticed in
spermatozoa sticking to the glass surface by the head; the
entire length of a sperm flagellum was not completely in
focus (Fig. 1). The flagellar waves were fairly symmetrical with a constant amplitude over the entire flagellum
when viewed from the direction perpendicular to the
beating plane.
No significant difference in the movement characteristics (amplitude, wavelength, and beat frequency of
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Ishijima et al.
Fig. 6. Typical bending waves of unpaired
eupyrene Turritella spermatozoa swimming in
media of increased viscosity. A: 110 cP. B:
230 cP. C: 3,000 cP. Bar ⫽ 30 µm.
flagellar waves and swimming speed of spermatozoa)
could be detected between paired and unpaired spermatozoa (Table I).
Effects of Calcium Ion on the Propagating
Direction of Flagellar Waves
To elucidate the factor influencing the direction of
flagellar wave propagation, the effects of calcium ion on
the propagation direction of flagellar waves were examined by altering the concentration of CaCl2 or EGTA in
the presence of 20 µM calcium ionophore A23187. A
decrease in EGTA and an increase in CaCl2 in the
standard solution increased the percentage of the spermatozoa propagating flagellar waves proximally (Table II).
The parameters of flagellar waves propagating proximally in the standard solution did not differ essentially
from that in natural seawater (data not shown) except for
the duration of motility, which rapidly decreased with
high calcium concentrations.
Rotational Movement of an Unpaired Eupyrene
Spermatozoon Around Its Axis
The rotational movement of spermatozoa around
their long axis is due to the three-dimensional geometry
of their flagellar waves [Taylor, 1952; Gray, 1962;
Ishijima et al., 1992]. Therefore, valuable information on
the three-dimensional geometry of flagellar waves will be
obtained from the parameters of the rotational movement
of spermatozoa; for example, knowledge of the direction
of sperm rotation gives us the sense of the threedimensional geometry in flagellar waves. To examine
whether or not the conformational change of flagellar
waves of spermatozoa is caused by changing the propagation direction of bending waves, the rotational movement
of spermatozoa around their axis was examined using
unpaired eupyrene spermatozoa in standard solution
containing either 0.3 mM EGTA or 1 mM CaCl2. In the
former solution, all spermatozoa had distally propagating
flagellar waves, whereas in the latter solution almost all
spermatozoa had proximally propagating flagellar waves
(Table II).
All spermatozoa (n ⫽ 58) propagating bending
waves from the base of the flagellum to the tip rotated
counterclockwise, as viewed from the head to the tail
(Fig. 4A). The average rotation frequency was 10.1 ⫾ 3.1
Hz (n ⫽ 15). On the other hand, all spermatozoa (n ⫽ 22)
propagating bending waves from the tip to the base
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
chirality of flagellar waves does not change when the
direction of wave propagation along the sperm flagella
changes.
Effects of Viscosity on Eupyrene Sperm and Their
Flagellar Movements
Turritella communis has internal fertilization; therefore, their spermatozoa will have to swim in fairly
viscous medium in situ. To investigate the meaning of
pairing of spermatozoa and the change in the movements
of spermatozoa and their flagella in viscous medium, the
sperm and their flagellar movements were examined in
viscous seawaters of 110, 230, 500, and 3,000 cP. The
swimming speed was found to be inversely proportional
to the one-third power of the viscosity (Fig. 5). No
significant difference in the swimming speed could be
detected between paired and unpaired spermatozoa in
seawater or in the four viscous seawaters. Typical flagellar waves of unpaired spermatozoa in viscous seawaters
are shown in Figure 6.
It is well known that two individual flagella beat
synchronously with one another even though they do not
have physical contact with one another, as spirochetes
also do [Gray, 1928; Taylor, 1952]. To test the synchronization in the paired eupyrene Turritella spermatozoa, the
flagellar movements were examined in seawater and the
four viscous seawaters. In natural seawater, the two
spermatozoa in a pair beat with different phases (Fig. 7A),
whereas in the seawater with viscosities over 110 cP,
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Ishijima et al.
swimming in natural seawater. Some fragments moved
proximally (Fig. 9A), but most moved distally (Fig. 9B).
The movements of flagellar fragments were less active
than that of whole flagella.
DISCUSSION
Bidirectional Swimming of Eupyrene Spermatozoa
Fig. 8. Phase-contrast video micrographs of the flagellar movement of
apyrene spermatozoa. A spermatozoon swims in natural seawater (A)
and in viscous seawater of 3,000 cP (B). Bar ⫽ 30 µm.
paired spermatozoa were aggregated and beat in union
(Fig. 7B).
Movements of Apyrene Spermatozoa
It has been suggested that the apyrene spermatozoa
may aid in the transport of the eupyrene spermatozoa due
to their greater motility [Friedländer and Gitay, 1972].
Apyrene spermatozoa from Turritella were hence recorded with respect to their movement in natural seawater
or the seawaters with different viscosities. An apyrene
Turritella spermatozoon consists of a head (without a
nucleus) and eight or more flagella (8.6 ⫾ 1.7,
mean ⫾ S.D., n ⫽ 12). One of these flagella is longer
(0.23 ⫾ 0.11 mm, n ⫽ 11) than the others (0.04 ⫾ 0.02
mm, n ⫽ 11). The average swimming speed of the
apyrene spermatozoa was 19.3 µm/s (S.D. ⫽ 12.0, n ⫽ 18)
in natural seawater and 11.2 µm/s (S.D. ⫽ 4.1, n ⫽ 20) in
the 3,000 cP seawater. The swimming speed of the
apyrene spermatozoa in natural seawater was rather
slower than that of the eupyrene spermatozoa but was
almost the same as that in the 3,000 cP seawater. Many
flagella beat independently in natural seawater (Fig. 8A),
whereas they beat in union in the 3,000-cP seawater (Fig. 8B).
Movements of Distal Tail Fragments of Eupyrene
Spermatozoa
An unexpected finding in this study was that of the
distal tail fragments of the eupyrene sperm flagella
Turritella spermatozoa can move forward and backward. The bidirectional swimming seems to be a general
characteristic of the snail spermatozoa because the eupyrene spermatozoa of the mud creeper (Terebralia palustris) 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 proximally propagating ones: the swimming speed of spermatozoa with distally propagating waves was more than 12
times that of spermatozoa with proximally propagating
waves, and the distally propagating waves generally
preceded the proximally propagating ones in the time
course of the flagellar movement of eupyrene spermatozoa, even though the reverse transition occasionally
happened. Thus, it is unlikely that backward swimming in
Turritella spermatozoa plays an important role. The
presence of calcium ion increased the percentage of
spermatozoa with the proximally propagating waves and
seemed to inhibit the distally propagating waves; thereby,
the proximally propagating waves were able to appear on
the sperm flagella. This interpretation seems to be supported by several findings. Caffeine or 8-bromoadenosine
3’:5’-cyclic monophosphate, both of which activate the
sperm motility [Mann and Lutwak-Mann, 1981], prolonged the duration of flagellar movement of distally
propagating waves and restored the active movement of
sperm flagella that had proximally propagated bending
waves. Only at unusually high concentrations of calcium
did all the spermatozoa exhibit the proximally propagating waves. Turritella spermatozoa can move backward as
well as forward like some other spermatozoa and many
protozoa [Holwill and McGregor, 1976; Baccetti et al.,
1989; Ishijima et al., 1994]. Crithidia cells and tephritid
and Myzostomum spermatozoa, in which the backward
swimming is usual, abruptly change their swimming
direction, but such a rapid change in swimming direction
could not be detected in Turritella spermatozoa, although
spermatozoa with proximally propagating waves restored
the flagellar waves propagating distally within a short
time. Thus, the role of a change in the swimming
direction of Turritella spermatozoa seems to differ from
that of these cells.
Movement of Turritella Spermatozoa
93
Fig. 9. Phase-contrast video micrographs of distal fragments of unpaired eupyrene spermatozoa. A
fragment that moves toward the base of the flagellum (A) and one toward the tip (B). The swimming speed
of the flagellar fragment (with a total length of 51.4 µm) was 6.6 µm/s in A and that of the flagellar fragment
(the total length of 67.5 µm) was 3.9 µm/s in B. Amplitude of the flagellar fragment in B was 2.5 µm; beat
frequency, 4.6 Hz; and wavelength, 25.2 µm. The time interval between successive images is 2 s. Bar ⫽ 30 µm.
Eupyrene Turritella spermatozoa swam almost in
circles when moving freely near a coverslip, as sea urchin
spermatozoa also do [Ishijima and Hamaguchi, 1992].
However, the circular rotation of Turritella spermatozoa
is due to the bending of the beating plane of flagellar
waves (Fig. 3), rather than to an asymmetry of the
flagellar waves (a difference in curvature between the
principal and reverse bends) as seen in other spermatozoa, including those of sea urchins and mammals. Because the beating plane of Turritella spermatozoa is in the
direction perpendicular to the line connecting the central
microtubules [Afzelius and Dallai, 1983] as the others do,
the bending of the beating plane in the Turritella spermatozoa is perhaps caused by a mechanism that is different
from that of other spermatozoa. The bending of the
beating plane has also been described for the spermatozoa
of the humpbacked fly [Curtis and Benner, 1991].
Direction of Wave Propagation and Chirality of
Flagellar Waves
The eupyrene Turritella spermatozoa reversed the
direction of their swimming as a result of a change in the
direction of bend propagation, this direction apparently
being regulated by calcium ions. When the spermatozoa
were viewed from head to tail, a reversal of direction of
the bend propagation accompanied the reversal of the
sperm rotating around their long axis. Baccetti et al.
[1989] reported the same findings in spermatozoa of
tephritid flies; thus their flagellar waves, which propagate
from the tip of the flagellum toward the head, induce the
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Ishijima et al.
sperm to rotate clockwise with respect to the advancement direction, whereas flagellar waves that propagate
from the head toward the tail tip induce the sperm to
rotate clockwise with respect to the advancement direction as well. These findings in the spermatozoa of both
tephritid flies and Turritella snails suggest that the sense
of the three-dimensional flagellar waves of these spermatozoa does not change when the direction of bend
propagation changes (Fig. 10). This response to calcium
ions differs from that of sea urchin spermatozoa; these do
not change the propagation direction along the flagella by
altering the calcium ion concentration, but they change the
sense of flagellar waves [Ishijima and Hamaguchi, 1993].
These differences in the response to calcium ions
between sea urchin and Turritella spermatozoa can be
explained by a different behavior of active sliding between the doublet microtubules. The rotational movement of spermatozoa around their long axis is due to the
three-dimensional movement of their flagella, and the
direction of sperm rotation is determined by the sense of
their three-dimensional geometry [Gray, 1962; Ishijima
and Hamaguchi, 1993]. The three-dimensional flagellar
waves are generated by localized active sliding between
doublet microtubules transmitting around the flagellar
circumference as well as propagating along the flagellum
[Ishijima and Hamaguchi, 1993]. The two propagation
directions, around the flagellum and along it, in response
to Ca2⫹ differ between sea urchin and Turritella spermatozoa. In sea urchin spermatozoa, transmitting the localized active sliding between doublet microtubules around
the flagellum in an alternative direction changes the
chirality of the three-dimensional bending waves. On the
other hand, in Turritella spermatozoa, the propagation of
a localized sliding along the flagella will change the
direction of propagation of the bending waves. In sea
urchin spermatozoa, changing the propagation direction
around the flagella seems to be easier than that along the
flagella, whereas in Turritella spermatozoa, the reverse is
found, possibly because of mechanical conditions at the
distal region of the sperm flagellum. The doublet microtubules may, for instance, attach to the plasma membrane
[Afzelius and Dallai, 1983], maybe as an adaptation to
enable the spermatozoa to change the direction of bend
propagation along rather than around the flagellum
[Machin, 1958; Kaneda, 1965]. The finding that distal fragments of the eupyrene sperm flagella could swim both
forward and backward may support this idea because
such fragments usually cannot move in other spermatozoa [Okuno and Hiramoto, 1976; Woolley and Bozkurt,
1995]. The important feature in the flagellar movement of
Turritella spermatozoa in which the distal end of the
flagella hardly showed transverse movement also implies
that active sliding between the doublet microtubules is
inhibited at the distal end of the Turritella sperm flagella.
Fig. 10. Hypothetical diagram explaining the rotational direction of
the spermatozoa and the propagation direction of their bending waves.
Two flagella (open and dotted) with a phase difference of about a
quarter cycle are shown. Distally propagated bending waves of a
left-handed helix rotate a spermatozoon counterclockwise (curved
arrow) when viewed from the head because the direction of rotation of
the spermatozoon is opposite to that (arrow) of the angular movement
of segments of the flagellum (A), whereas proximally propagated
bending waves of the same sense rotate a spermatozoon clockwise (B).
The flagellar waves are tentatively illustrated as flattened helices
because there is no detailed information on it.
Conjugated Spermatozoa
There was little difference in the movement characteristics between the paired and the unpaired eupyrene
Turritella spermatozoa in either normal or viscous seawater (Table I and Fig. 5). This finding differs from the
previous reports on cooperative swimming in the American opossum in which two spermatozoa are more forceful
in their swimming than are single spermatozoa [Biggers
and Creed, 1962; Moore and Taggart, 1995]. This may be
due to a difference in the orientation of the two conjugated spermatozoa in Turritella and in the opossum. In
the Turritella spermatozoa, the two spermatozoa in a pair
face the same direction [Afzelius and Dallai, 1983],
whereas in the American opossum the two spermatozoa
face each other [Temple-Smith and Bedford, 1980; Taggart, et al., 1993]. A difference in the sperm alignment is
also observed in the beating pattern of paired spermatozoa; the paired Turritella spermatozoa beat with a phase
difference that is less than a quarter cycle, whereas the
paired opossum spermatozoa beat with a phase difference
of approximately half a cycle [compare Fig. 7 in the
present paper with fig. 12 in Phillips, 1972, or fig. 3 in
Movement of Turritella Spermatozoa
Moore and Taggart, 1995]. The difference in orientation
of the two spermatozoa may induce different responses to
the viscosity; the paired Turritella spermatozoa beat in
union in highly viscous seawaters, whereas the paired
opossum spermatozoa beat independently in a medium of
approximately the same viscosity [Moore and Taggart, 1995].
The two flagella of paired Turritella spermatozoa
did not beat in phase in natural seawater, in contrast to the
situation of closely adjacent sea urchin spermatozoa. This
is probably due to the accessory fibers of Turritella
spermatozoa. In fact, spermatozoa of the American
opossum also did not beat in phase [Phillips, 1972; Moore
and Taggart, 1995] and neither do adjacent mammalian
spermatozoa [Ishijima, 1992], although large-scale wavy
motions can be seen in a dense sperm suspension of bull
or ram spermatozoa [Rothschild, 1949; Mann and LutwakMann, 1981; Ishijima, 1992].
ACKNOWLEDGMENTS
We thank the director, Prof. J.-O. Strömberg, and
the staff of the Kristineberg Marine Biological Station,
Fiskebäckskil, Sweden, where this work was carried out,
for their generous hospitality. We also thank Dr. Y.
Hamaguchi for his active interest and advice.
REFERENCES
Afzelius BA. 1983. The spermatozoon of Myzostomum cirriferum
(Annelida, Myzostomida). J Ultrastruct Res 83:58–68.
Afzelius BA, Dallai R. 1983. The paired spermatozoa of the marine
snail, Turritella communis Lamarck (Mollusca, Mesogastropoda). J Ultrastruct Res 85:311–319.
Afzelius BA, Dallai R. 1987. Conjugated spermatozoa. In: Mohri H,
editor. New horizons in sperm cell research. Tokyo: Japan
Scientific Societies Press/New York: Gordon and Breach Science Publishers, p 349–355.
Baccetti B, Gibbons BH, Gibbons IR. 1989. Bidirectional swimming in
spermatozoa of Tephritid flies. J Submicrosc Cytol Pathol
21:619–625.
Biggers JD, Creed RFS. 1962. Conjugate spermatozoa of the North
American opossum. Nature 196:1112–1113.
Biggers JD, DeLamater ED. 1965. Marsupial spermatozoa pairing in
the epididymis of American forms. Nature 208:402–404.
Braidotti P, Ferraguti M. 1982. Two sperm types in the spermatozeugmata of Tubifex tubifex (Annelida, Oligochaeta). J Morphol
171:123–136.
Curtis SK, Benner DB. 1991. Movement of spermatozoa of Megaselia
scalaris (Diptera:Brachycera:Cyclorrhapha:Phoridae) in artificial and natural fluids. J Morphol 210:85–99.
Dallai R, Afzelius BA. 1984. Paired spermatozoa in Thermobia
(Insecta, Thysanura). J Ultrastruct Res 86:67–74.
Dallai R, Afzelius BA. 1985. Membrane specializations in the paired
spermatozoa of dytiscid water beetles. Tissue Cell 17:561–572.
Dallai R, Afzelius BA. 1987. Sperm ultrastructure in the water beetles
(Insecta, Coleoptera). Boll Zool 4:301–306.
Friedländer M, Gitay H. 1972. The fate of the normal-anucleated
spermatozoa in inseminated females of the silk worm Bombyx
mori. J Morphol 138:121–130.
Gray J. 1928. Ciliary movement. London: Cambridge University Press.
95
Gray J. 1962. Flagellar propulsion. In Bishop DW, editor. Spermatozoan motility. Washington, DC: American Association for the
Advancement of Science. p 1–12.
Holwill MEJ, McGregor JL. 1976. Effects of calcium on flagellar
movement in the trypanosome Crithidia oncopelti. J Exp Biol
65:229–242.
Ishida S, Yamashita Y, Teshirogi W. 1991. Analytical studies of the
ultrastructure and movement of the spermatozoa of freshwater
triclads. Hydrobiologia 227:95–104.
Ishijima S. 1992. Methods for analyzing sperm motility. In Mohri H,
Morisawa M, Hoshi M, editors. Spermatology. Tokyo: University of Tokyo Press. p 212–224 (in Japanese).
Ishijima S, Hamaguchi Y. 1992. Relationship between direction of
rolling and yawing of golden hamster and sea urchin spermatozoa. Cell Struct Funct 17:319–323.
Ishijima S, Hamaguchi Y. 1993. Calcium ion regulation of chirality of
beating flagellum of reactivated sea urchin spermatozoa. Biophys J 65:1445–1448.
Ishijima S, Oshio S, Mohri H. 1986. Flagellar movement of human
spermatozoa. Gamete Res 13:185–197.
Ishijima S, Hamaguchi MS, Naruse M, Ishijima SA, Hamaguchi Y.
1992. Rotational movement of a spermatozoon around its long
axis. J Exp Biol 163:15–31.
Ishijima S, Ishijima SA, Afzelius B. 1994. Movement of Myzostomum
spermatozoa: calcium ion regulation of swimming direction.
Cell Motil. Cytoskeleton 28:135–142.
Kaneda Y. 1965. Movement of sperm tail of frog. J Fac Sci Univ Tokyo,
Sec.IV, 10:427–440.
Kennedy JJ, Keegan BF. 1992. The encapsular developmental sequence of the mesogastropod Turritella communis (Gastropoda:
Triitellidae). J Mar Biol Ass UK 72:783–805.
Koike K, Nishiwaki S. 1980. The ultrastructure of dimorphic spermatozoa in two species of the Strombidae (Gastropoda: Prosobrachia). Venus 38:259–275.
Machin KE. 1958. Wave propagation along flagella. J Exp Biol
35:796–806.
Mann T, Lutwak-Mann C. 1981. Male reproductive function and
semen. Berlin: Springer-Verlag.
Moore HDM, Taggart DA. 1995. Sperm pairing in the opossum
increases the efficiency of sperm movement in a viscous
environment. Biol Reprod 52:947–953.
Okuno M, Hiramoto Y. 1976. Mechanical stimulation of starfish sperm
flagella. J Exp Biol 65:401–413.
Phillips DM. 1972. Comparative analysis of mammalian sperm motility. J Cell Biol 53:561–573.
Reger JF, Fitzgerald ME. 1979. The fine structure of membrane
complexes in spermatozoa of the millipede, Spirobolus sp., as
seen by thin-section and freeze-fracture techniques. J Ultrastruct Res 67:95–108.
Rothschild Lord 1949. Measurement of sperm activity before artificial
insemination. Nature 163:358.
Taggart DA, Johnson JL, O’Brien HP, Moore HDM. 1993. Why do
spermatozoa of American marsupials form pairs? A clue from the
analysis of sperm-pairing in the epididymis of the grey short-tailed
opossum, Monodelphis domestica. Anat Rec 236:465–478.
Taylor G. 1952. The action of waving cylindrical tails in propelling
microscopic organisms. Proc R Soc London Ser A 211:225–239.
Temple-Smith PD, Bedford JM. 1980. Sperm maturation and the
formation of sperm pairs in the epididymis of the opossum,
Didelphis virginiana. J Exptl Zool 214:161–171.
Wilson EB. 1925. The cell in development and heredity. New York:
MacMillan Inc.
Woolley DM, Bozkurt HH. 1995. The distal sperm flagellum: its
potential for motility after separation from the basal structures. J
Exp Biol 198:1469–1481.