Table Of ContentZOOLOGICAL SCIENCE 10: 179-182 (1993) 1993Zoological SocietyofJapan
[RAPID COMMUNICATION]
Sperm Behavior in the Micropyle of the Medaka Egg
Sumio Ishijima, Yukihisa Hamaguchi and Takashi Iwamatsu1
Biological Laboratory, Faculty of Science, Tokyo Institute of Technology,
O-okayama, Meguro-ku, Tokyo 152 and department of Biology,
Aichi University of Education, Kariya 448, Japan
—
ABSTRACT Sperm behavior in the micropyle of the exact behavior of spermatozoa in the micropyle.
medakaeggwasexaminedusingvideomicroscopy. Sper- Recent videomicroscopy and the transparency of
matozoa swimming near the egg, whose progressive the medaka eggenabled usto recordon videotape
speed was similar to that of spermatozoa freely swim-
the movement ofthe sperm head in the micropyle
ming in a medium, entered the micropylar canal after
being trapped in the funnel-shaped micropylarvestibule of the medaka eggs.
or entered directly. The spermatozoa stalled in the The purpose of the present study is to record
micropylarvestibuleenteredthe micropylarcanalwith a and analyze the behavior of spermatozoa in the
time lag ofless than 0.5sec. Proceeding at a fairly high micropyle ofthe medakaegg. On the basis ofthis
speedforashorttime,thespermatozoagraduallyslowed
down when the flagellar movement of the spermatozoa data, the mechanism for blocking polyspermy in
became obstructed by the surface of the micropylar the medaka egg is discussed.
canal, finally reaching the plasma membrane. The
spermatozoawhichdirectlyenteredthemicropylarcanal
MATERIALS AND METHODS
maintained progressive speed until their flagellar move-
mentbecameobstructedbythesurfaceofthemicropylar
Medaka oocytes and spermatozoa were pre-
canal. They reached the plasma membrane through the
micropylarcanalinthesamemannerasthespermatozoa paredbythemethoddescribedearlier[4] andwere
experiencingcollision with the surface ofthe micropylar kept in Ringer's solution (6.5 g NaCl, 0.4g KC1,
vestibule. 0.113g CaCl2 and0.15g MgS04-7H2 perliterof
deionizedwater, pH=7.3adjustedwithNaHC03)
before use. In each experiment, the gameteswere
INTRODUCTION
freshly prepared and used within 30min. The
In teleost fish, spermatozoa can reach the egg's observation chamber was constructed by gluing
plasma membrane only by means of a narrow two strips of silicon rubber (1 mm thick) onto a
channel, the micropyle of the vitelline envelope. glass slide parallel to each other. An egg was put
Since the base of the micropyle is as wide as the in the chamber with a small amount of Ringer's
diameter of the sperm heads, spermatozoa must solution, and the chamber was covered with a
pass in single file through the micropyle. This has coverslip after removal of the Ringer's solution
been thought to be the mechanism for blocking aroundtheegg. Toobserve anoblique viewofthe
polyspermy [2] because medaka eggs do not show egg micropyle, the egg was rotated by sliding the
a rapid electrical block to polyspermy [6]. coverslip. Sperm movement was observed using a
Since observation of sperm entry into the Nikon Optiphot microscope equipped with a plan
micropyle is difficult, little is known about the 40X objective, 10X eyepieces and a National
video camera (WV-1300A, Matsushita Com-
Accepted November 20, 1992 munication Industrial Co., Ltd, Yokohama,
Received October 19, 1992 Japan). While observing the egg displayed on a
180 S. Ishijima, Y. Hamaguchi and T. Iwamatsu
National TN-96 monitor (Matsushita Communica- spermatozoa may be critical for sperm progress in
tion Industrial Co., Ltd), approximately 0.1 ml of the small diameter of the micropyle (see below).
sperm suspensionwasintroduced intothe observa-
tion chamber from an open end. The image was Progressive speed of medaka spermatozoa in the
recorded on video tape with a Panasonic video micropyle
tape recorder (NV-FS900, Matsushita Com- Mostspermatozoa attachedtothe surface ofthe
munication Industrial Co., Ltd). micropylar vestibule, stayed there for approx-
Fordetailedfield-by-field analysis, imagesofthe imately 424 msec (S.D.=243 msec, n=20) and
head were traced from the video monitor onto then entered the micropylar canal, although some
transparent plastic sheets using a fine-point mar- spermatozoa entered directly. Both the progres-
ker. The progressive speed of free-swimming sivespeedofthe medakaspermatozoaandthe size
spermatozoa was determined by measuring the of the micropyle of the medaka egg varied; the
distance between the positions of the sperm head micropyle had a mean diameter (dv) of 5.30//m
at the beginning and the end of a time interval. (S.D.=1.25fixn, n=21) at the end of its vestibule
and a mean diameter (dc) of 3.19//m (S.D.=0.84
/um, n=21) at the base of the micropylar canal
RESULTS AND DISCUSSION
(Fig. 1). Thus, the profile oftheprogressive speed
of medaka spermatozoa in the micropyle varied.
Movement characteristics ofmedaka spermatozoa In general, three phases in the time course of the
Medaka spermatozoa swam at a maximum trans- progressive speed were recognized (Fig. 2). The
verse displacement of the sperm flagella of5.14+ first phase was a decrease in the progressive speed
1.73//m (mean+S.D., n=16) while rotating ab- ofthe spermatozoa from the free-swimming speed
out their longitudinal axis at a rate of 3.77per
secondaspreviouslyreportedbyIshijimaetal. [3].
The progressive speed of medaka spermatozoa
swimming near the micropyle was 74.0+14.6jum/
sec (mean+S.D., n=19), whereas thatofsperma-
tozoa freely swimming in the medium was 74.9+
31.3/mi/sec (mean+S.D., n=25). No difference
was found between them, suggesting that sperm
movement, especially progressive speed, is not
stimulated by the presence of the micropyle.
The progressive speed of medaka spermatozoa
was lowascomparedto that (191.4//m/sec) ofsea
urchin spermatozoa [1], whose flagellum is similar
in size to that of medaka spermatozoa. The
progressive speed is roughly proportional to the
square ofthemaximumtransversedisplacementof
the flagellum. The ratio of the progressive speed
to the square ofthe maximum transverse displace-
ment of the flagellum does not differ between
medaka(2.8; maximumtransversedisplacementof
5.14jum) and sea urchin spermatozoa (3.0; max-
imum transverse displacementof8.0^m) [1]. This Fig. 1. A phase-contrast videomicrograph of the mic-
suggests that the low progressive speed ofmedaka ropyle of the medaka egg. dv, diameter of the
spermatozoa is mainly due to the narrow ampli- mmiiccrrooppyyllaarr vceasntailbualteitasteintsd.basTe;wodcs,pdeirammaettoezrooafltihnee
tude oftheir flagellar waves. The small transverse up along the micropylar canal (arrowheads). Bar,
displacement of the beating flagella of medaka 20,£/m.
Spermatozoa in Micropyle of Medaka Egg 181
IOOr
o
0)
\in
E
-a
a>
50
s-
o
0.5 1.0 1.5 2.0 2.5
Time (sec)
Fig. 2. A typical profile ofthe time course ofthe progressive speed ofmedaka spermatozoa in the micropyle.
ioo r
o
a> 80
\in
E
60 -
Q>
a>
a.
in
0) 40 -
_>
in
in
0)
l.
oo
o 20
10
Inner diameter of the micropyle ( (jm )
Fig. 3. The relationship between the progressive speed and the diameter of the micropyle. Two examples are
shown.
182 S. Ishijima, Y. Hamaguchi and T. Iwamatsu
to nearlyzero; the spermatozoawhich swam freely mean diameter ofthe micropyle at its end (3.19+
were trapped in the micropylar vestibule and 0.84//m) is almost the same as that ofthe head of
wanderedthroughitorceasedanyprogressforless spermatozoa (3.71+0.60jum, n=17), so that
than 0.5 sec as mentioned above. The second sperm mustpassinsingle file through the micropy-
phase was the recovery of the progressive speed; le. The decrease in progressive speed (Figs. 2 and
the spermatozoa entered the micropylar canal and 3), especially slow progression of further sperm,
proceeded without obstruction from the micropy- may contribute to the effective polyspermy block.
lar canal. The final phase was the decrease of the There was no difference in sperm behavior in the
progressive speed until it was reduced to zero; the micropyle between the spermatozoa entering im-
spermatozoa made theirwaythroughthe micropy- mediately or later. Many spermatozoa could pass
lebecause theflagellarmovementofthespermato- through the micropyle which had been isolated
zoa was mechanically inhibited by the surface of from the medaka egg (data not shown). These
the micropylar canal, and finally reached the egg's observations suggest that substances inhibit sperm
plasma membrane. The inhibitory effect of the behaviorarenotsecretedbytheplasmamembrane
micropyle on the progressive speed is shown in and the micropyle.
Fig. 3. Progressive speed rapidly decreased with a Most medaka spermatozoa swimming freely in
decrease in the diameter ofthe micropyle. It took themedium rolled in aclockwisedirectionon their
689+428msec (n=16) for the spermatozoa to longitudinal axis when an observerviewed the cell
pass through the micropylarcanal (the second and fromtheanteriorendsalthoughsome spermatozoa
third phases). rolled in the other direction [3]. In contrast, the
No difference was found in the time course of sense of the micropyle is counterclockwise [5].
the spermatozoasecondarilyenteringthe micropy- Therefore, it is unlikely that the chirality of the
le compared to that of the first (data not shown). micropyle is involved in sperm movement when
Most spermatozoa were trappedin the micropy- passing through the micropyle.
lar vestibule before entering the micropylar canal,
suggesting that the micropylar vestibule is very REFERENCES
useful in capturing the spermatozoa.
In sperm-egg interaction, there are various 1 Gray J, Hancock GJ (1955) J Exp Biol 32: 802-814
mechanisms blocking polyspermy [2]. In medaka, 2 Hart NH (1990) Int Rev Cytol 121: 1-66
the micropyle plays an important role in blocking 3 HIsahmiajgimuachS,iHYam(a1g99u2c)hiJ MESx,p BNiaorlus1e63M:,15I-s3h1ijima SA,
polyspermy because only one spermatozoon 4 Iwamatsu T (1975) Bull Aichi Univ Educ (Nat Sci)
reached the plasma membrane in our study. The 24: 113-144 (in Japanese)
blocking of the polyspermy with the micropyle is 5 Iwamatsu T, Onitake K, Yoshimoto Y, Hiramoto Y
probablyduetothemechanicalinhibitionofsperm (1991) Develop Growth Differ 33: 479-490
progression of the surface of the micropyle. The 6 Nuccitelli R (1980) Develop Biol 76: 499-504
