Dec 25, 2016

The Cytological Studies of Oogenesis and Fertilization of Ferns

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.

 Formation of the Separation Cavity
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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