Abstract
The present paper reports some new
advances on the sexual reproduction of ferns, including, mainly, the detailed
processes of the formation of the archegonia, the egg development and
fertilization. The archegonia of the ferns are derived from the archegonial
initial cell under the growing point. The initial cell has dense cytoplasm and
a large centrally placed nucleus. The initial cell gives rise to a tier of
three cells, by two divisions, middle of which is the primary cell. The primary
cell undergoes two unequal divisions, and forms a neck canal cell, a ventral
cell and an egg cell. During maturation, the egg cell becomes progressively
isolated from the adjacent cells by forming a separation cavity and an egg
envelope. It is proved that the advanced ferns form a fertilization pore in the
upper egg envelope. It is discovered that the ventral canal cell takes part in
formation of the fertilization pore. The fertilization experiment indicated
that the spermatozoid penetrate the egg through the fertilization pore. Immediate
shrinkage of the egg at the moment of the sperm penetration and formation of a
large sac blocking the fertilization pore are proposed to be used to prevent
polyspermy.
Introduction
Pteridophytes are spore-bearing vascular
plants. They are considered to be transitional taxa between bryophytes and seed
plants. Pteridophytes are usually divided into ferns (macrophyll pteridophytes)
and fern allies (microphyll pteridophytes) according to the traditional
viewpoint. Recent phylogenetic investigations revealed a basal dichotomy within
vascular plants, separating the lycophytes from other vascular plants (the
euphyllophytes) (Smith et al. 2006). Thus, the euphyllophytes comprise two
clades: the spermatophytes (seed plants) and ferns (the monilophytes), which
have about 9000 species, including horsetails, whisk ferns, and all
eusporangiate and leptosporangiate ferns (Pryer et al. 2004). The ferns are
divided into two categories according to the spore types: the homosporous
ferns, which have homosporous spores, including all horsetails, whisk ferns,
eusporangiate, and almost all the leptosporangiate ferns; and heterosporous
ferns, which have large and small spores, and only include the Marsileaceae,
Salviniaceae and Azollaceae. The ferns have independently-living gametophytes,
also known as the prothallus. The gametophyte propagates by sexual
reproduction, which includes spermatogenesis, oogenesis, fertilization and
embryo development. The spermatogenesis has been studied in many aspects and
with numerous species of ferns (Bell 1974, 1975; Bell et al. 1971; Bell and
Duckett 1976; Doonan et al. 1986; Duckett 1973; Hoffman 1995; Myles and Hepler
1977; Kotenko 1990; Renzaglia and Garbary 2001). Spermatogenesis in ferns can
usually be divided into two stages. The first stage is from the initial spermatogenous
cell to the spermatocytes (spermatid mother cell); and the second stage is the
differentiation of the spermatocytes. The differentiation of the
spermatocytesis a complex process, which includes a multilayered structure
(MLS) and microtubular ribbon (MTR) that are formed de novo; mitochondrial
fusion and nuclear shaping. And finally a spiral spermatozoid is formed. However,
the oogenesis and fertilization are less reported. The present paper reports
our recent investigations on the oogenesis and fertilization of ferns.
Oogenesis ‘Formation of the archegonia and the egg cells’
Archegonia of the ferns are usually produced
on the lower surface just behind the apical notch of the gametophyte (Fig. 1A).
So far, formation of the archegonia of many species, including Ceratopteris
thalictroides, Phymatosorus hainanensis, Adiantum flabellulatum
and Pteridium aquilinum var. latiusculum have been
investigated (Yang et al. 2009; Cao et al. 2010a; Dai et al. 2010; Huang
et al. 2011). These studies showed that the archegonia are derived from
the initial cell under the lower surface just behind the apical notch of the
gametophyte (Fig. 1B). The main features of the initial cell are dense cytoplasm
and central placed nucleus in contrast to the somatic cells. The vacuoles
in the initial cell are asymmetrically distributed. Large vacuoles are located
in the lower part, but small vacuoles lie in the upper part of the cell (Cao
et al. 2011). The chloroplasts in the initial cell, lacking well-developed lamellae
and containing little starch, are usually smaller than those in the somatic
cells. The initial cell forms three cells by two periclinal divisions (Fig.
1C). The upper cell becomes the neck jacket initial; the middle is the primary
cell and the lower cell becomes the jacket cells in the future. The primary
cell is a square with almost equivalent height and width. The nucleus is
larger and the chromatin becomes more dispersed than those in the
adjacent cells. Soon, the primary cell enlarges and its upper surface protrudes
upwards (Fig. 1D). Before division of the primary cell, the neck initial
cell divides into a rosette of four cells by two anticlinal divisions (Fig.
1D). The primary cell divides asymmetrically to form two cells. The cell
towards the neck of the archegonium is a mononucleate neck canal cell (mnc)
(Fig. 1E). The lower cell, obtaining more cytoplasm from its mother cell, is
named as the central cell (cc) (Fig. 1E). The central cell also divides asymmetrically
to form a small ventral canal cell (VCC) and a large egg cell, which possesses
most of the cytoplasm (Fig. 1F). Soon after the egg is formed, the nucleus of
the mononucleate neck canal cell divides into two, without cell wall formation
between the two nuclei, which resulting in a binucleate neck canal cell (NCC)
(Fig. 1G). Thus, an archegonium contains an axial row of three cells, i.e., the
egg cell, the ventral canal cell, and the neck canal cell (Fig. 1G).
Development of the Egg Cell
Investigations of the egg development of
the ferns are few in contrast to the spermatogenesis because of the simplicity
of oogenesis. Oogenesis that has been investigated includes Pteridium
aquilinum (Bell and Mühlethler 1962; Bell and Duckett 1976), Histiopteris
incisa (Bell 1980), two species of Osmunda (Bao et al. 2003; Cao et
al. 2012a), and Dryopteris crassirhizoma (Bao et al. 2005). These
investigations revealed that the egg of advanced ferns is surrounded by a
conspicuous extra egg membrane. This same structure has been regarded as a
venter coat, covering the top of the egg, in the ferns Athyrium filix-femina
(Fasciati et al. 1994) and Ceratopteris richardii (Lopez- Smith and
Renzaglia 2008). The nuclear evaginations are formed during maturing of the egg
development. Recently, we discovered a fertilization pore in the mature egg of
the ferns C. thalictroides, A. flabellulatum, Plagiogyria euphlebia,
and P. aquilinum var. latiusculum (Cao et al. 2009, 2010b, 2010c,
2011, 2012b). And the detailed development of oogenesis has been investigated with
the mode species of Ceratopteris thalictroides and Pteridium
aquilinum (Cao et al. 2010b, 2012b).
Young Egg Stage
The inner three cells are closely
appressed to the wall of the archegonial jacket cells. There are well developed
plasmodesmata between the egg and the VCC (Fig. 2A), but these are absent between
the inner cells and the jacket cells. The nucleus of the new egg is spherical
and contains two or more irregular nucleoli. Abundant vesicles, with a diameter
of 0.5–1 μm, are distributed principally in the lateral side of the egg
cytoplasm. Plastids, containing fewer starch grains and lamellae, lie closely
to the nucleus. Mitochondria are distributed randomly throughout the cytoplasm
of the egg. At this stage the organelles in the canal cells are similar to the
egg.
The first detectable change in oogenesis
is the formation of a separation cavity, which forms initially around the periphery
in the upper surface of the egg in C. thalictroides, A. flabellulatum,
P. euphlebia, and P. aquilinum (Cao et al. 2010b, 2010c, 2011,
2012b). The plasma lemma of the egg is dissociated from the wall in the
periphery. Simultaneously, the plasmodesmata disappear from the wall of the
separation region. However, plasmodesmata still connect the egg and the VCC in
the central region. Subsequently, the separation cavity expands centripetally
and the connection region becomes correspondingly decreased. However, a pore
region with a diameter about 2–3 μm persistently connects the egg and the VCC.
There are well developed plasmodesmata in the pore region (Fig. 2B). For the P.
euphlebia and P. aquilinum, a temporary wall between the egg and the
VCC becomes thickened except in the pore region during formation of the
separation cavity. This wall always lies closely to the VCC (Cao et al. 2011,
2012b). The biological significance of the temporary cell wall may lie in the
isolation of the egg cell and ensures the independent development of the sex
cells (Cao et al. 2011). At this stage, the Golgi bodies in the egg cytoplasm
increase in number, especially, in the upper side of the egg, which may take
part in formation of the separation cavity.
Formation of the Egg Envelope and the Fertilization Pore
It is shown that an egg envelope is formed
outside the mature egg in the advanced ferns, such as P. aquilinum, H.
incise, Dryopteris crassirhizoma, C. thalictroides, A.
flabellulatum, P. euphlebia, and P. aquilinum (Bell and
Duckett 1976; Bell 1980; Cao et al. 2008, 2010c, 2011, 2012b). And the egg
envelope on the upper surface of the egg is especially thick in contrast to the
side and lower part of the egg. However, the primary fern Osmunda japonica has
no typical egg envelope outside the mature egg (Cao et al. 2012a). In the C.
thalictroides and A. flabellulatum, the egg envelope may be formed by
the endoplasmic reticula, which are attached to the inner surface of the plasma
lemma. And some lipid materials may take part in formation of the egg envelope
(Cao et al. 2008, 2010c). But in P. euphlebia and P. aquilinum, formation
of the egg envelope is accompanied by the decreasing of the amorphous materials
in the separation cavity. So it is considered that the egg envelope is formed
by amorphous materials depositing on the outer surface of the plasma lemma (Cao
et al. 2011, 2012b). The thickness of the upper egg envelope increases from the
periphery to the center and its maximum thickness reaches to about 0.5 μm.
During the formation of the egg envelope,
the pore region still connects the egg cell and the VCC. It is striking that
sheets of ER are not deposited on the inner surface of the pore region. Eventually,
a fertilization pore is formed at the pore region (Fig. 2C, D). The only
membrane covering the fertilization pore is plasma lemma. So far, the
fertilization pore have been discovered in C. thalictroides (Cao et al.
2009), A. flabellulatum (Cao et al. 2010c), P. euphlebia (Cao et
al. 2011), P. aquilinum (Cao et al. 2012b), Coniogramme emeiensis (Wang
et al. 2012a), Cibotium barometz (Wang et al. 2012b). However, no
fertilization pore was discovered in the primary fern O. japonica (Cao
et al. 2012a). It possibly indicated that the fertilization pore is a derived
structure in the advanced ferns, which may be in favor of preventing
polyspermy.
Nuclear Behavior and Evagination
The nuclear behavior is noticeable. The rounded
nucleus of the advanced ferns in the young egg becomes gradually cup-shaped and
has an irregular surface (Cao et al. 2010b, 2010c, 2011, 2012b). The primitive
fern Osmunda possesses a rounded nucleus (Cao et al. 2012a). Moreover,
most advanced ferns produces obvious nuclear evaginations during the maturing
of the egg, like that of the ferns P. aquilinum (Bell and Mülethaler
1962; Bell and Duckett 1976), Dryopteris fi lix-mas (Cave and Bell
1975), Histiopteris incisa (Bell 1980), Dryopteris crassirhizoma (Bao
et al. 2005) and Adiantum (Cao et al. 2010c). In Ceratopteris
thalictroides, the nucleus also becomes highly irregular, but it does not
produce evaginations during oogenesis. It is suggested that the nuclear
behavior and evaginations may have some significance in assessing the affiliation
of the ferns. The advanced ferns are inclined to produce more complicated
evaginations (Cao et al. 2010b, 2011, 2012b).
The Function of the Ventral Canal Cell in Oogenesis
The function of the VCC in oogenesis is
less mentioned in previous reports. Our recent investigations of C.
thalictroides, A. flabellulatum, and P. euphlebia showed that
there is persistent connection of the egg and the VCC through the pore region,
which suggests that the VCC plays an important role in oogenesis. The
well-developed plasmodesmata in the pore region undoubtedly indicate
informational and material communication between the egg cell and VCC, which
consequentially influence the development of the egg envelope and the formation
of the fertilization pore. It is suggested that the VCC can absorb materials
from the egg through the plasmodesmata in C. thalictroides (Cao et al.
2010b). Or the VCC disturbs the activities of the endoplasmic reticula beneath
the pore region, which leads to no formation of the egg envelop in P. aquilinum
(Cao et al. 2012b). Finally a fertilization pore is developed from the
connection region.
Fertilization and Zygote Development of the Fern
The fertilization and zygote development,
including the approach of sperm to the egg, the fusion of gametes, and the
prevention of polyspermy are poorly understood in ferns. The cytological
processes, including the egg penetration, male nuclear decondensation, nuclear
fusion, digestion of male organelles, rebuilding of plasma lemma and cell wall
of zygote, are described in detail.
Fertilization and Preventing Polyspermy
The fertilization process occurs inside
the archegonium of gametophytes. So we cannot see how the sperm enter the egg directly.
Through investigations of the fertilization of C. thalictroides, the
detailed processes of fertilization and zygote development are described (Cao
et al. 2010d). It is indicated that the fertilization pore is an entrance, through
which the spermatozoid can penetrate the egg (Fig. 3A). After the spermatozoid
enters the egg, the egg envelope is still intact, which indicated that the
spermatozoid penetration is restricted exclusively to the fertilization pore.
So far, such a fertilization experiment has not been reported in other ferns so
far investigated.
How the egg prevent polyspermy is
interesting. In our investigations on the fertilization of C. thalictroides,
although several spermatozoids were observed in the cavity above the egg, only
one of these is able to penetrate the egg. Penetration of the egg by more than
one spermatozoid was not encountered in any of the specimens examined. The
mechanism of preventing polyspermy may be attributable to the sac that
containing numerous small vesicles, which are seen to block the fertilization
pore persistently (Cao et al. 2009, 2010d). Perhaps, the shrinkage of the
fertilized egg may also result in the pyknosis of the protoplasm, which may
also contribute to prevent polyspermy.
Features of the Fertilized Egg at Early Stage
As soon as the egg is fertilized, it
shrinks markedly. The volume of the fertilized egg decreases to almost one-half
that of the unfertilized egg. The protoplasm of the fertilized egg becomes dense
and opaque. The organelles, particularly the mitochondria and endoplasmic
reticula of the egg and most of the motile apparatus of the fertilizing
spermatozoid, including the flagella, microtubular ribbon, multilayered
structure, and mitochondrion, are scarcely identifiable. Only the starch grain-containing
plastids remain prominent in the egg cytoplasm. The egg envelope is still intact
except in the region of the fertilization pore where the plasma lemma has been
broken up after the spermatozoid enters the egg. At approximately 10–20 min
after fertilization, the fertilized egg begins to increase in volume. The
original obscure organelles in the cytoplasm become clear gradually.
Male Nuclear Decondensation and Nuclear Fusion
At approximately 20 min after
fertilization, a conspicuous feature of the male nucleus is that the MTr is
detaching itself from the male chromatin. Sometimes, the mitochondrion is
observed lying between the MTr and the male chromatin. It seems that the
mitochondrion is stripping off the MTr from the male chromatin (Cao et al.
2010d). Simultaneously, the male chromatin decondenses continuously and
eventually transforms into a fibrous form. When the male nucleus becomes dissociated
from the MTr, no envelope is observed at the surface of the male chromatin.
Approximately 30 min after fertilization, an envelope reappears around the male
chromatin. The electron-clear space can be observed in the male nucleus.
At approximately 45 min after fertilization,
the gametic nuclei begin to fuse with each other. The manner of nuclear fusion
of the ferns is interesting, because it involves a helical male nucleus fusing
with a highly irregular egg nucleus. Bell (1975) suggested that the anterior
end of the male nucleus first fuses with the female nucleus. Fasciati et al.
(1994) considered that the anterior parts of the spermatozoid cytoskeleton
served as a guiding structure inside which the male nucleus twists into the egg
nucleus like a corkscrew. Lopez-Smith and Renzaglia (2008) indicated that the
spermatozoid defines a circular space within which the sperm nucleus progressively
fuses with and enters the egg nucleus. But in C. thalictroides, the
nuclear fusion is possibly in a different manner. The ultrastructural
observation suggests the female nucleus fuses actively with the male nucleus.
The anterior end of the female nuclear protrusions seems to approach and wrap
the male nucleus (Cao et al. 2010d). However, the nuclear behaviors during
nuclear fusion need a further investigation.
Digestion of Male Organelles
The male organelles of the spermatozoid
that entered the cytoplasm of the egg include microtubular ribbon, flagellum,
and male mitochondrion. It is generally considered that the male organelles are
digested finally. Our investigations about C. thalictroides provide a
detailed degeneration process of the male organelles (Cao et al. 2010d). The
different organelles are digested at different time of zygote development. The
membranes outside the flagella and the MTr are digested shortly after
fertilization. The microtubules are digested about 3–6 h after fertilization.
Our observations show that the male mitochondria are digested between 6 and 9 h
after fertilization. ERs and vacuoles possibly participate in the digestion of
the male organelles. It is often observed that the male organelles are
enveloped by ER in the early stages.
Rebuilding of a Functional Zygote
At approximately 9 h after fertilization
the nucleus becomes to be round or elliptical. The male organelles cannot be detected
in any forms. However, the rebuilding of the functional zygote needs still more
than 24 hours (Cao et al. 2010d). During this time, the zygote undergoes
complex cytological changes. Firstly, the organelles in the zygote undergoes
rearrangement and polarity of the zygote is rebuilt. The zygote at the early
stage of the zygote development showed a conspicuous vertical polarity.
Vacuoles lie in the upper part of the zygote at early stage. However, the
zygote changes its vertical polarity into a horizontal polarity by the
rearrangement of the organelles. Vacuoles migrate to one lateral side of the
zygote and the nonvacuole organelles move into the opposite side of the zygote.
This may be important for the first division, which is always vertical to the
gametophyte. The cell toward the notch becomes the
stem polarity and the cell toward the posterior part of the gametophyte becomes
the root polarity.
Secondly, functional zygote rebuilding
depends on the formation of the plasma lemma. The egg cell loses its plasma
lemma and cell wall when the egg envelope and separation cavity has been
formed. However, a new plasma lemma is rebuilt inside the extra egg membrane
before the zygotic division. It is possible that ERs participate in the
formation of the plasma lemma, since ERs are frequently observed in the
periphery of the zygote and paralleling to the surface of the egg. Terasaki and
Jaffe (1991) also suggested that ER may be comparable to the plasma lemma in
complexity of function. Soon after the formation of the plasma lemma, a layer
of cell wall deposits between the newly-formed plasma lemma and the extra egg membrane.
The rebuilding of plasma lemma and the cell wall undoubtedly represents the
formation of the first functional cell of the sporophyte.
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