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Initial stages of sperm-egg fusion in the freshwater teleost Rhodeus ocellatus ocellatus.

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THE ANATOMICAL RECORD 229:195-202 (1991)
Initial Stages of Sperm-Egg Fusion in the
Freshwater Teleost, Rhodeus ocellatus ocellatus
Department of Biology, Aichi University of Education, Kariya City, Aichi 448, Japan
The morphology of spermatozoa and the initial stages of spermegg fusion a t fertilization were investigated ultrastructurally in the rose bitterling, Rhodeus ocellatus ocellatus. Each spermatozoon is composed of a spherical
head without a n acrosome, two centrioles, a large mitochondrion, and a flagellum.
Freeze-fracture of spermatozoa illustrates that specialized arrays of intramembranous particles (IMPS) are present on the protoplasmic facing (PF) surface of the
head plasma membrane a t the portion slightly in front of the centrioles. The
specialized arrays, whose functions are uncertain, are parallelogram-like in shape.
The distribution of the particles is random and less compact in other areas of the
head plasma membrane. The number of particles on the PF surface is larger than
that on the extracellular facing (EF) surface. The complementary structures of the
specialized arrays are also found on a similar portion of the EF surface. An ultrastructural study clearly shows fusion of gamete plasma membranes a t the initial
stages of sperm entry into the egg. Membrane fusion is first observed in eggs fixed
10 seconds after insemination in fresh water. The fusion site is the microvillus
membrane of a sperm entry site on the egg and the head membrane of the spermatozoon. The plasma membrane fusion of gametes is discussed relative to the
distribution of the IMPs and the fusion site.
Fertilization is a phenomenon of cell fusion. The primary morphological indication of sperm entry into the
egg is the fusion of the involved plasma membranes.
Consequently, the fertilizing spermatozoon carries a
male nucleus with some cytoplasmic components into
the egg. In marine invertebrates and mammals, initial
stages of sperm-egg fusion have been investigated by
electron microscopy. The results showed that the membrane fusion site of spermatozoa is restricted within
specially fixed portions (Colwin and Colwin, 1967;
Yanagimachi and Noda, 1970; Bedford and Cooper,
1978). Recently, a distribution of intramembranous
particles (IMPs) of the sperm plasma membrane in various animals has been investigated by freeze-fracture
(the millipede, Reger and Fitzgerald, 1979; the zebrafish, Kessel et al., 1983; the ascidian, Rosati, 1985;
the horseshoe crab, Tilney, 1985; the mammal, see
Toyama and Nagano, 1988). From the relationship between the fusion site of the plasma membrane and the
distribution of the particles, i t was shown that the
membrane fusion site is particle-free (cf. Yanagimachi,
A teleostean fish egg is enveloped by a thick egg
membrane (chorion) with a micropyle. As the fertilizing spermatozoon attaches to the egg plasma membrane (of the sperm entry site) directly beneath the
micropyle, the fusion site of the egg plasma membrane
is destined to become the sperm entry site. Though
plasma membrane fusion also occurs in fish gametes,
the initial stages of the fusion event have not been
researched so far.
The first objective of this study was to obtain decisive
ultrastructural evidence of the initial stages of gamete
c 1991 WILEY-LISS,
membrane fusion in teleostean fish. The second was to
investigate the distribution of the IMPs of fish spermatozoa.
Adult male and female rose bitterlings, R. ocellatus
ocellatus, were purchased from a fish farmer in Toyohashi City. They were kept in glass containers with
freshwater bivalves under conditions of light 14 hours
and dark 10 hours a t 20-23°C. Mature eggs were obtained by manually pressing the abdomens of females
having a lengthened ovipositor. Mature spermatozoa
were obtained from males by a similar method.
Unfertilized eggs kept in physiological saline were
washed with water (pH adjusted to 7.2 by Nil0 NCl)
which was boiled,once and treated with a sperm suspension (5 x 10- concentration). At various time intervals (1,3, 5, 10 seconds) after the start of insemination, the eggs were fixed with modified Karnovsky’s
fixative containing 3%sucrose for 4 hours a t 4°C. After
being washed with 5% sucrose-0.1 M phosphate buffer
(pH 7.2), they were postfixed with 1% OsO4-3g sucrose-0.1 M phosphate buffer for 1.5 hours a t 4°C. For
scanning electron microscopy (SEM), some of the eggs
were dehydrated in a n alcohol series followed by
isoamylacetate and then dried with liquid CO, in a
critical point dryer (Hitachi, HCP-2). The dried eggs
were coated with gold by ion sputter (JEOL, JFC-1100)
Received March 16, 1990; accepted J u n e 26, 1990
and examined with a JEOL, JEM-100B electron microscope with attached scanning devices. For superficial
observations of spermatozoa, those in physiological saline were put on a thin glass coated with 0.1% polylysine for 15 minutes. They were fixed with modified
Karnovsky's fixative containing 3% sucrose then
treated similar to the eggs except for the use of goldpalladium instead of gold. For transmission electron
microscopy, some of the eggs were dehydrated in a n
alcohol-acetone series and were embedded in Quetol
812 (Nisshin EMco., Tokyo). Ultra-thin sections were
stained with uranyl acetate and lead citrate, then examined with JEOL, JEM-100B, and JEM-2000FX electron microscopes.
Testes fixed for 4 hours with modified Karnovsky's
fixative were washed with 0.1 M phosphate buffer (pH
7.2) and kept in 30% glycerin-0.1 M phosphate buffer
for about 2 hours. The testes were frozen in degassed
liquid nitrogen and fractured at about - 125"C, using a
freeze-fracture apparatus (JEOL, JFD-7000). Platinumicarbon followed by carbon was coated onto the
fractured surfaces of the testes. Replica membranes
were separated from the testies in bleach, rinsed in
distilled water, mounted on 150 mesh grids and observed with the electron microscopes.
Sperm Morphology
Each spermatozoon of the rose bitterling is composed
of a head, a middle piece, and a tail (Fig. 1).The spermatozoa have no acrosome structure in the head (Fig.
1A). Each has a large mitochondrion and centrioles in
the middle piece (Fig. 1).The proximal portion of the
flagellum is surrounded by plasma membrane (sleeve,
Fig. l A , S).
SEM observations showed that the superficial structures of the round shaped heads were smooth without
remarkable characters (Fig. lB,C).
The distribution of IMPs in the sperm plasma membrane was examined by freeze-fracture. Each portion,
the head, middle piece and flagellum, of each fractured
spermatozoa was easily differentiated (Figs. 2,3). Specialized arrays, like the parallelogram shapes of IMPs
on the protoplasmic facing (PF) surfaces, were seen in
a portion of sperm heads on the flagellar side shown in
Fig. 1A,B (Fig. 2A-D, arrows). The specialized arrays
of IMPs were positioned slightly in front of the centrioles. The extent of their distribution varied depending
on individual differences in spermatozoa (Fig. 2A-C).
The average size of the IMPs was about 11.7 i 3.5 nm
(means of 54 particles 2 standard deviation). The number of the IMPs per 0.25 km2 was about 43 2 8 (means
of 130 areas i standard deviation) on the P F surface of
Fig. 1. Spermatozoa of R. ocellatus ocellatus. A: An electron micrograph of a spermatozoon. C, centriole (basal body); CF, centriolar
fossa; F, flagellum; M, mitochondrion; N, nucleus; S , sleeve. x 27,000.
B: A S E M photo of a spermatozoon viewed from the flagellar side
(mof A ) . F, flagellum; H, head. x 30,000. C: A S E M photo of a spermatozoon viewed from the mitochondrial side ( m o f A). H, head; MP,
middle piece. x 30,000.
Fig. 2. Freeze-fracture replicas of the PF surface plasma membranes
of spermatozoa. A,B:These photos show the distribution of the IMPS
on the sperm membrane viewed from the flagellar side. Specialized
arrays (arrows) of the IMPs were found on the sperm head slightly in
front of centrioles. A ) x 34,000. B) x 42,500. C: The distribution of
IMPs on the apical-side membrane of the sperm head. x 24,000. D
Higher magnification ofthe specialized arrays of the IMPs. x 120,000.
E: The distribution of the IMPs on the head and middle piece membranes viewed from the mitochondrial side. Note that they are scattered. x 34,000.
Fig 2
Flg. 3. Freeze-fracture replicas of t h e E F surface plasma membranes
of spermatozoa. A Parallelogram-like structures (arrows) were found
on the complementary portion of t h e PF surface. x 40,000. B: Higher
magnification of t h e parallelogram-like structures. x 102.000. C: The
distribution of the IMPS on the head and middle piece membranes
viewed from the mitochondria1 side. There a r e fewer IMPS on the E F
surface than on the PF surface. x 50,000.
TABLE 1. Number of eggs with a spermatozoon at
various time intervals after insemination
time (seconds)
No. of
the head and about 41
8 on the middle piece. No
significant difference in number was recognized among
the different areas of the head and middle piece. There
were no specialized arrays of the IMPs on the mitochondrial side shown in Fig. 1A,C (Fig. 2E). Complementary structures (Fig. 3B) of the specialized arrays
of the IMPs were observed on the extracellular facing
(EF) surface in similar positions to the specialized arrays on the PF surface (Fig. 3A,C). Fewer IMPs were
observed on the E F surface than the PF (Figs. 2 , 3).
Initial Stages of Sperm-Egg Fusion
In order to identify the membrane fusion between
sperm and egg, inseminated eggs had to be sectioned
along the micropyle and then examined by a transmission electron microscope. It was necessary first to investigate the timing of insemination to increase the
probability of observing a fusion event.
Accordingly, the presence of sperm a t a sperm entry
site on the plasma membrane directly beneath the micropyle was examined in eggs fixed at l - 10 seconds
after the start of insemination. Naturally, the number
of eggs with spermatozoa a t the micropyle increased
with the increase of insemination time (Table 1).Spermatozoa were observed at the sperm entry site or the
micropyle in 50% of the eggs after 3 seconds and nearly
all eggs after 10 seconds.
As the chorion of rose bitterling eggs is somewhat
thin, it was easy to visualize the sperm entry site superficially. In each egg the micropyle was situated in
the chorion of the animal pole where the chorion became most hollow. Some microvilli were found a t the
sperm entry site (Fig. 4A).
In eggs fixed 10 seconds after the start of insemination, fertilizing spermatozoa had attached to the microvilli of the sperm entry sites by their lateral head
portions (Figs. 4B-D, 5A-C). Figures 4 and 5 clearly
show that each fertilizing spermatozoon entered into
the egg by mutual plasma membrane fusion. The membrane fusion site was formed by the sperm entry site
microvilli of the egg and the head portion of the spermatozoon (Figs. 4B-D, 5A-C).
Plasma Membrane Fusion Site Between Sperm and Eggs
We quickly discovered that plasma membrane fusion
occurred in the sperm entry site microvilli of the eggs
(Figs. 4, 5). Yet, we found it necessary to obtain more
information on the precise site of fusion on the spermatozoa. In eggs fixed 30 seconds after the start of
insemination, sperm were observed by the SEM to be
attached to the sperm entry sites. Fixed eggs were dechorionated to facilitate our observations. Spermatozoa
attached to the microvilli of eggs by various portions of
their heads. We obtained the following results: sperm
attached to the eggs by the flagellar side (see Fig.
1A,B) of their heads in 69 of 99 eggs, by the mitochondrial side (see Fig. 1A,C) in 8 of 99 eggs and by some
point in between in 25 of 99 eggs.
Spermatozoa possess a male nucleus and some cytoplasmic components which differ slightly from species
to species. Though the spermatozoa of most animals
have a n acrosomal structure in the anterior portion of
their heads, those of teleostean fish are known to lack
the acrosomal structure (cf. Afzelius, 1978; Yanagimachi, 1988). The spermatozoa of the rose bitterling, a
teleostean fish, have also been found to lack the acrosoma1 structure (Ohta and Iwamatsu, 1983).Other features of these spermatozoa include the following: 1)
they have a large mitochondrion in their middle piece
and a sleeve which encircles the root of their flagella,
and 2) their flagella project from one side, not the center, of the head when viewed from the tail.
Freeze-fracture electron microscopy has shown that
the apical region of acrosomal membrane is almost free
of IMPs in marine invertebrates (cf. Rosati, 1985; Tilney, 1985). During the acrosome reaction, the fusion of
the acrosomal and plasma membranes occurs at a particle-free site. Mammalian spermatozoa are also known
to undergo a similar type of fusion (Friend e t al., 1977).
Generally, acrosome-reacted spermatozoa can fuse
with the egg. The fusion site on the plasma membrane
of these spermatozoa has been shown to be particle-free
(cf. Longo, 1987; Yanagimachi, 1988). Similarly, in rat
peritoneal mast cells, the area of fusion between vesicles and plasma membranes is known to be devoid of all
particles (Lawson et al., 1977). These observations suggest that plasma membrane fusion occurs at particlefree sites. Our present observations on freeze-fractured
spermatozoa showed the existence of specialized arrays
of IMPs in sperm heads. In contrast to the area of the
specialized arrays, the IMP distribution in the other
areas of the sperm head was not as compact, and a
notable particle-free zone was not present. Therefore, it
is difficult to suggest a possible membrane fusion site
by the distribution of the IMPs in the rose bitterling; a
situation different from other animals.
The specialized arrays of the IMPs were observed in
the sperm head portion in front of the centrioles. Their
pattern was parallelogram-like in shape. Kessel et al.
(1983) reported a similar structure in zebrafish spermatozoa. They found that unusual arrays of IMPs appeared as simple hexagons or parallelograms and were
localized in an equatorial position in the sperm heads.
The functional significance of these characteristic arrays of IMPs is quite unclear a t present. In the rose
bitterling, it is known that a sperm-stimulating factor
is localized in the chorion near a micropyle and attracts
spermatozoa to the egg (Suzuki, 1961). It may be that
the arrays of IMPs play the role of receptors to the
factor, yet, we have no convincing experimental data to
support or refute this. Another possibility is that they
play an essential role when a spermatozoon attaches to
the egg plasma membrane directly beneath the micropyle. Our present observatons on sperm-egg interactions showed many cases in which the fertilizing spermatozoon fused with the egg a t the membrane side
rr. OHTA
Fig. 4. Sperm-egg fusion in eggs fixed 10 seconds after insemination.
A: A SEM photo of a n unfertilized egg. CH, chorion; SS, sperm entry
site. x 8,800. B: A SEM photo in t h e vicinity of t he micropyle of a
fertilized egg. A fertilizing spermatozoon seems to attach the mi-
crovilli of t h e sperm entry site. x 8,800. C, D A fertilizing spermatozoon clearly fused by its head with the membrane of t h e microvilli.
C ) x 12,300. D ) x 38,500.
Fig. 5 . Another sperm-egg fusion event in an egg fixed 10 seconds
after insemination. A A SEM photo of a fertilizing spermatozoon
which seems to have attached the microvilli by the sperm head membrane on the flagellar side. x 10,000 B, C: A fertilizing spermatozoon
20 1
fused with the membrane of the microvilli by the sperm head membrane near the centrioles (arrows). MV, microvilli; ch, chorion. B)
x 28,000. C) x 52,500.
with the arrays of the IMPS. Therefore, the arrays of
the IMPS may play a
in egg recognition. To “lve
these problems, research using immunocytochemical
techniques is in progress.
A fertilizing spermatozoonpenetratesinto an egg by
gamete plasma membrane fusion. The membrane fusion occurs a t the inner aCrOSOma1 membrane covering
the acrosomal process in marine invertebrates (Colwin
and Colwin, 1967; cf. Yanagimachi, 1988) and in birds
(Okamura and Nishiyamaj
It Occurs at the postacrosomal region (Yanagimachi and Noda, 1970) or
over the equatorial segment (Bedford and Cooper,
1978) in mammals.
fishes, membrane fusion was
reported in the medaka (Iwamatsu and Ohta, 19781,
the rose bitterling (Ohta and Iwamatsu, 1983; Ohta,
1985), Fundulus heteroclitus (Brummett et al., 19851,
the common carp (Kudo and
1985)7and the ze(Wolenski and
However, data describing the very early stages of gamete membrane fusion have not been presented so far. The present
electron microscopic observations showed the following:
A spermatozoon primarily contacted the microvilli of a sperm entry site on the egg. 2 ) The sperm
plasma membrane fused with the plasma membrane of
the microvilli at the sperm head portion. 3) The membrane fusion had Occurred
within lo seconds after insemination.
In spermatozoa having an acrosomal structure, the
structure is located at the tip Of the head’ Just opposite
the site Of the centriolar fossa. However, the ’permatozoa of the rose bitterling have a spherical head and
no acrosomal structure. In addition, the single mitochondrion does not completely encircle the flagellum,
Because Of these morphological
expression of “apical” or “lateral” is not SO easy. At present, it
is difficult to determine the real fusion site of the sperm
marine invertebrates, electron microscop~cobservations have shown clear
fusion. From the
results of these studies, the time from insemination to
membrane fusion is known to be less than 9 seconds in
annelida and within 7 seconds in hemichordata (colwin and Colwin, 1967). We found that this event in the
rose bitterling required a similar amount of time.
Though the membrane fusion event had occurred in
eggs fixed 10
after insemination, it seemed to
begin earlier. In fact, Longo et al. (1986) reported that
membrane fusion occurred as quickly as 5 seconds after
the onset of sperm-induced conductance increase in sea
urchin eggs. However, the exacttirne between primary
Of the spermatozoon and membrane fusion is
uncertain in rose bitterling eggs.
Afzelius, B.A. 1978 Fine structure of the garfish spermatozoon. J.
Ultrastruct. Res., 64:309-314.
Bedford, J.M. and G.W. Cooper 1978 Membrane fusion events in the
fertilization of vertebrate eggs. In: Membrane Surface Reviews.
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Brummett, A.R., J.N. Dumont, and C.S. Richter 1985 Later stages of
sperm penetration and second polar body and blastodisc formation in the egg of Fundulus heteroclitus. J . Exp. zool., 234: 423439.
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The author wishes to express his thanks to Dr. T’
Iwamatsu ofthe Department of Biology, Aichi University of Education for reading the manuscript.
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freshwater, stage, initial, egg, teleost, ocellatum, fusion, rhodeus, sperm
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