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Fertilization in ascidians: apical processes and gamete fusion in Ciona
intestinalis spermatozoa
MAKOTO FUKUMOTO
Biological Institute, College of General Education, Nagoya City University, Nagoya 467, Japan
Summary
This paper describes the fine structure of the
spermatozoan apex and its morphological
changes during the process of fertilization in
Ciona intestinalis.
At the apex of the head an acrosome is present
in the form of a flattened vesicle containing
moderately electron-dense material with an electron-dense plate in its centre. Contiguous to the
acrosome, an apical substance is located in the
anterior-most tip of the sperm head.
The spermatozoa that bind to and penetrate the
chorion of both normal and caffeine-treated eggs
have an intact acrosome and do not show an
observable alteration of the plasmalemma enclosing the apex of the head. On the other hand,
spermatozoa in the perivitelline space of both
normal and caffeine-treated eggs lack an acrosome. Instead of an acrosome, apical processes
are observed at the apex of the spermatozoa in the
perivitelline space of both normal and caffeinetreated eggs. Gamete fusion seems to occur between the egg membrane and some of these apical
processes at the tip of the sperm head. The apical
process reported here is thought to be analogous
to the acrosomal process in other marine invertebrates. The apical processes that are induced in
the perivitelline space have not been previously
described in ascidians.
Introduction
through the chorion in Halocynthia mretzi (Ishikawa
et al. 1978; Fuke-Tsukamoto, 1983). Test cells are
located in the perivitelline space between the egg
surface and the chorion. The role of the test cells in
fertilization still remains enigmatic. Such an elaborate
set of egg investments leads to the supposition that
spermatozoa of ascidians may be highly modified to
effect penetration through the envelope and achieve
successful fusion with the female gamete. Recent
studies have shown that ascidian spermatozoa have a
small acrosome (Cloney & Abbott, 1980; Fukumoto,
1985, 1986). Furthermore, there is evidence that spermatozoa of ascidians contain proteases that act as lysins
(Woollacott, 1977; Hoshi et al. 1981; Sawada et al.
1982, 1984a,b). It is not clear how the acrosome
participates in the process of fertilization and where the
lysins are located in the spermatozoa. In order to
resolve these issues it is necessary to investigate the
morphological changes at the apex of spermatozoa
during the process of fertilization.
In spite of a fairly large number of descriptive and
experimental studies on ascidian fertilization, our
knowledge of the events associated with fertilization is
still poor and fragmentary in both biochemical and
morphological aspects.
The ascidian egg is enclosed by a relatively thick and
tough chorion (vitelline coat) to which spermatozoa
bind as a prerequisite for fertilization (Rosati & DeSantis, 1978; DeSantisei al. 1980; Honegger, 1982; FukeTsukamoto, 1983; Hoshi et al. 1985) and which might
participate in the block to polyspermy in Ascidia nigra
(Lambert, 1986). The chorion, which is externally
decorated by a single layer of highly vacuolated follicle
cells, presents a barrier to successful penetration by the
spermatozoa. The follicle cells are thought to be
involved in sperm attraction in Ciona intestinalis
(Miller, 1975), in egg flotation in Corella willmeriana
(Lambert & Lambert, 1978) and in sperm penetration
Journal of Cell Science 89, 189-196 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Key words: fertilization, ascidians, Ciona intestinalis,
spermatozoa.
189
With the electron microscope it is difficult to detect
gamete fusion and morphological changes in spermatozoa during the process of fertilization, especially in the
perivitelline space, because of the limited number of
spermatozoa that can pass through the chorion, even if
inseminated at high concentrations of sperm. Recently
the author has found that many spermatozoa penetrate
the chorion and reach the perivitelline space when
caffeine- or theophylline-treated eggs are inseminated
by fairly high concentrations of spermatozoa, resulting
in polyspermic fertilization. This has provided us with
useful material for observing how gamete fusion occurs
and any structural changes taking place in C. intestiualis spermatozoa prior to gamete fusion in the perivitelline space.
This paper describes the apical processes of C.
intestinalis spermatozoa in the perivitelline space in
both normal and caffeine-treated eggs and gamete
fusion in caffeine-treated polyspermic eggs.
Materials and methods
Ciona intestinalis was collected on the coast of Chita Peninsula, Aichi Prefecture, Japan.
For fine-structural observations on differentiated spermatozoa, the sperm duct, which is filled compactly with
differentiated spermatozoa, was fixed in 3 % glutaraldehyde
in 0-1 M-cacodylate buffer (pH 7-4) for 1 h at room temperature. For observations on the spermatozoa suspended in both
normal sea water and caffeine-containing sea water (S mMcaffeine), sperm that were carefully obtained from the sperm
duct by pipetting were suspended for lOmin in normal sea
water or caffeine-containing sea water. They were centrifuged and fixed in 3 % glutaraldehyde in 0-1 M-cacodylate
buffer (pi I 7-4) for 30min at room temperature. For observations on both normally fertilized and polyspermic eggs, the
eggs obtained from the oviduct by careful puncture with a
razor were divided into two groups. One group was suspended in normal sea water for 5 min and then cross-fertilized
with a fairly high concentration of spermatozoa (approx. 108
sperm ml"'). The other group was suspended in caffeinecontaining sea water (5 mM-caffeine) for 5 nun (caffeine
pretreatment) and cross-fertilized, after removal of surplus
caffeine-containing sea water, with the same concentrations
of spermatozoa. These eggs were fixed at 3-5 min after
insemination in 3 % glutaraldehyde in 0-1 M-cacodylate buffer
(pH 7-4) for 30 min at room temperature.
These specimens were prepared for electron microscopy
according to a method described by Fukumoto (1981).
Electron micrographs were taken on a Hitachi H-300 electron
microscope operated at 75 kV.
Results
Fine structure of the apex of spermatozoa
The spermatozoon of C. intestinalis has architectural
features that are characteristic of the ascidian spermatozoa described by Cloney & Abbott (1980) and
190
M. Fukumoto
Fukumoto (1986). It has a moderately elongated head,
approximately 5 jitm long, with a wedge-shaped tip, and
a single mitochondrion, which is closely applied laterally to the nucleus (Fig. 1).
In appropriate sagittal (Fig. 2) and transverse
(Fig. 3) sections through the apex of the head an
acrosome is seen. The acrosome is a flattened vesicle,
about 150 nm X 160 nm X 60 nm, filled with moderately electron-dense material with an electron-dense
plate in its central region. An accumulation of granular
material, approximately 5-7 nm in diameter, is observed at the anterior tip of the head contiguous to the
acrosome (Figs 2, 4), and is referred to here as apical
substance. Such an accumulation of electron-dense
substance was first reported at the apex of the differentiated spermatozoa in Ascidia callosa, where it was
assumed to correspond to the periacrosomal substance
found in some animal species (Cloney & Abbot, 1980).
In C. intestinalis, Rosati et al. (1985) found similar
electron-dense material at the apex of the sperm head.
The inner and the outer nuclear membranes under the
acrosome make close contact with each other to form a
specialized structure that appears to form a pedestal for
the acrosome (Figs 3, 4, arrows). A fuzzy material
decorates the external surface of the plasmalemma
(Figs 2, 3, 4).
Apical processes and gamete fusion
The binding of the spermatozoa to the chorion seems to
be established by the fuzzy material on the plasmalemma of the apex adhering to the outer fibrous
structures of the chorion (Figs 7, 8). There is evidence
that this binding is mediated by an enzyme-substrate
reaction between a-L-fucosidase on spermatozoa and
fucosylated glycoproteins located on the fibrous structures of the chorion (Hoshi et al. 1985). An intact
acrosome has been recognized at the apex of spermatozoa that are suspended in both normal and caffeinecontaining sea water (Figs 5, 6), and it binds to and
passes through the chorion in both normal and caffeine-treated eggs (Figs 7, 8, 9, 10).
Several processes that have been designated apical
processes in the present paper have been found protruding from the apical region of the spermatozoa in
the perivitelline space of both normally fertilized (Figs
11, 12) and caffeine-treated eggs (Figs 13, 14). These
processes typically are about 100 nm in length and
40 nm in diameter (Figs 12, 14).
It is also interesting that gamete fusion probably
occurs between the egg plasmalemma and one or more
of the apical processes of the spermatozoa (Figs 15, 16,
17, arrows).
Fig. 1. Sagittal section through a differentiated spermatozoon. »i, mitochondrion; n, nucleus. X9500.
Fig. 2. Sagittal section through the anterior region of a differentiated spermatozoon, a, acrosome; as, apical substance;
fu, fuzzy material. X 114000.
Fig. 3. Transverse section through the apex of a differentiated spermatozoon at the level of an acrosome. Arrow indicates a
pedestal for the acrosome. a, acrosome;/«, fuzzy material; n, nucleus. XI14000.
Fig. 4. Transverse section through the apex of a differentiated spermatozoon at the level of the apical substance. Arrow
indicates a pedestal for the acrosome. as, apical substance;fu, fuzzy material; n, nucleus. X 114000.
Fettilization in ascidians
191
Discussion
Apical processes have been recognized at the apex of
the spermatozoa in the perivitelline space of both
normal and caffeine-treated eggs. Although the number of spermatozoa in the perivitelline space is limited
in normally inseminated eggs, apical processes have
been observed in almost all spermatozoa in appropriate
sections. Furthermore, we have observed a fairly large
number of spermatozoa in the perivitelline space of
caffeine-treated eggs. In these cases, all spermatozoa in
favourably oriented sections have apical processes. On
---2. r - :
as
192
M. Fukumoto
the other hand, these processes have never been
observed at the apex of spermatozoa that have been
suspended for 10 min in normal sea water (Fig. 5) or in
caffeine-containing sea water (Fig. 6). Moreover,
100% of caffeine-treated eggs can be fertilized and
develop normally if they are inseminated by 106
sperm ml" 1 , a concentration of spermatozoa that can
fertilize 100 % of normal eggs. These facts suggest that
the apical processes reported here have not been
induced by caffeine and that caffeine does not have a
harmful effect, at this concentration (5mM), on the
process of fertilization and development in C. intestinalis. Because these processes are observed in many
spermatozoa in both normal and caffeine-treated eggs,
these structural changes most probably occur in C.
intestinalis spermatozoa as one of the prerequisite steps
during the process of normal fertilization.
As the processes are believed to function in fusion
with the egg plasmalemma, they might be analogous to
the acrosomal processes of other marine invertebrates.
A vesicle is found contiguous to the region where
gamete fusion might occur (Figs 16, 17). At present,
the significance of this vesicle is unknown.
In spite of intensive observations of many spermatozoa in the perivitelline space in both normal and
caffeine-treated eggs, an acrosome has not been found,
except for a possible residue of an acrosome in a few
cases (Fig. 13, arrow). Furthermore, the apical substance sometimes has a fibrillar appearance (Figs 10,
14), which suggests that a molecular reorganization,
such as, for example, polymerization of actin, might
have occurred. These facts suggest that the formation
of the apical processes occurs as changes are taking
place in the acrosome and apical substance. In this
Fig. 5. Sagittal section through the apex of a
spermatozoon suspended in normal sea water, a, acrosome;
as, apical substance;/^, fuzzy material. X120000.
Fig. 6. Sagittal section through the apex of a
spermatozoon suspended in caffeine-containing sea water.
a, acrosome; as, apical substance;fu, fuzzy material,
x 120 000.
Fig. 7. Sagittal section through the apex of a
spermatozoon that is binding to the chorion of a normal
egg (non-caffeine-treated egg), a, acrosome; as, apical
substance; ch, chorion. X120000.
Fig. 8. Sagittal section through the apex of a
spermatozoon that is binding to the chorion of a caffeinetreated egg. a, acrosome; as, apical substance; ch, chorion.
X120000.
Fig. 9. Sagittal section through the apex of a
spermatozoon that is penetrating the chorion of a normal
egg (non-caffeine-treated egg), a, acrosome; as, apical
substance; ch, chorion. X 120000.
Fig. 10. Sagittal section through the apex of a
spermatozoon that is penetrating the chorion of a caffeinetreated egg. a, acrosome; as, apical substance; ch, chorion.
X120000.
context it is of particular interest to note the finding
that actin is probably present at the apex of the sperm
head in Boltenia and Cnemidocarpa (Lambert & Lambert, 1984), although negative results have been obtained in Ascidia ceratodes (Lambert & Lambert,
1984) and in Phallusia mammillata (Honegger,
19866). Although we have no supporting evidence, it
seems safe to assume that the factor(s) that elicit(s) the
structural changes at the apex of the spermatozoa
exist(s) in the perivitelline space of C. intestinalis.
The claim that the acrosome reaction occurs at the
surface of the chorion after the specific binding of
spermatozoa has been made repeatedly for C. intestinalis (Rosati & DeSantis, 1978; DeSantis et al. 1980,
1983; Monroy & Rosati, 1983; Rosati et al. 1985). In
spite of intensive observations on a large number of
spermatozoa that are bound to the chorion, the acrosome reaction has not been observed, as has been
reported (Fukumoto, 1984a). In Phallusia mammillata, Honegger (1986a,b) found several vesicles (up to
eight) at the apex of the sperm head. He reported that
some of these vesicles fuse with the sperm membrane
and release their contents prior to the passage of the
sperm through the chorion and that their remains can
still be recognized in the spermatozoa in the perivitelline space. He has speculated that these vesicles contain
chorion lysin(s) and /or additional enzymes that participate in the process of sperm-egg fusion. Honegger
(1986a,b) has suggested that sperm-egg fusion in P.
mammillata occurs between the plasma membrane of
the post-acrosomal region of the sperm head and the
egg membrane, as observed in mammalian fertilization. In contrast to the findings in P. mammillata,
gamete fusion between the sperm and the egg plasma
membranes in C. intestinalis most probably occurs at
the anterior tip of the sperm head by means of the
apical process(es) that protrude(s) from the apical
region of the spermatozoa.
In C. intestinalis, I have reported acrosome fragmentation, which has been referred to as an acrosome
reaction. The acrosome transforms into smaller vesicles immediately after spawning or suspension in sea
water (Fukumoto, 1984a). In the present study, however, such a change has not been recognized in acrosomes of spermatozoa that are suspended in normal or
caffeine-containing sea water, or binding to and passing
through the chorion. The reason for this discrepancy is
not clear, but it is probable that I mistook the apical
substance, which sometimes appears as small vesicles
(Fig. 5) and figs 4, 6, 8, 9, 14 (Fukumoto, 1984a), for
acrosome fragmentation because I overlooked the apical substance at that time. Further studies to clarify the
precise reason for this discrepancy are in progress. In
any case, the spermatozoa can pass through the chorion
without releasing the contents of the acrosome and
without any observable changes in the plasmalemma
Fertilization in ascidians
193
Fig. 11. Transverse section, probably at the level of the apical substance, at the apex of a spermatozoon that is in the
perivitelline space of a normal egg. as, apical substance", es, egg surface; n, nucleus; pr, processes. X 120000.
Fig. 12. Longitudinal section through the apex of a spermatozoon that is in the perivitelline space of a normal egg.
pr, processes. X 120000.
Fig. 13. Sagittal section through the apex of a spermatozoon that is in the perivitelline space of a caffeine-treated egg.
Arrow indicates probable remnant of the acrosome. ep, egg plasmalemma; pr, processes. X120000.
Fig. 14. Sagittal section through the apex of a spermatozoon that is in the perivitelline space of a caffeine-treated egg.
Electron-dense material is present at the apex, pr, processes. X 120 000.
enclosing the apex of the sperm head (Fukumoto,
1984fl, and the present study).
The findings in the present study are in good
agreement with the hypotheses that ascidian spermatozoa contain a poorly developed acrosome that reacts at
an appropriate step in fertilization and participates
194
M. Fukumoto
mainly in the fusion of the gamete plasma membranes,
and that the chorion lysin(s) are intercalated into the
plasmalemma enclosing the sperm head (Fukumoto,
1983, 19846).
The author is most grateful to Professor Gary Freeman and
Dr Judy Lundelius, of the University of Texas at Austin, for
V?
17
Fig. IS. Sagittal section through the head of a spermatozoon that is in contact with the egg surface in a caffeine-treated
egg. es, egg surface. X 10 000.
Fig. 16. Enlargement of the apical region of the sperm head shown in Fig. IS. Arrow indicates the region where the egg
and the sperm plasma membranes are probably confluent, ep, egg plasmalemma; v, vesicle. X 120000.
Fig. 17. Longitudinal section through the apex of a spermatozoon whose plasmalemma is probably confluent with the egg
membrane at two places (arrows) in a caffeine-treated egg. ep, egg plasmalemma; v, vesicle. X120000.
Fertilization in ascidians
L95
their valuable suggestions and for reading the manuscript. He
also thanks Miss Mieko Matsushima and Mr Toshihiro
Nagao, medical students of Nagoya City University, for their
assistance in preparing the manuscript.
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