REVIEW
A comparison of early molecular fertilization mechanisms
in animals and flowering plants
Mihaela L. Ma´rton Æ Thomas Dresselhaus
Received: 4 October 2007 / Accepted: 4 December 2007 / Published online: 9 January 2008
Ó Springer-Verlag 2007
Abstract Since the first description of the double fertilization
process in flowering plants (angiosperms) 110 years
ago (Nawaschin in Bull Acad Imp Sci St Petersburg 9:377–
382, 1898), little progress has been made during the following
100 years to understand the underlying molecular
mechanisms. This seems to be in strong contrast to our
steadily increasing knowledge of single fertilization in
animals, where a large number of key players and the
corresponding molecular mechanisms have been unclosed
since the mid-1970s. Are the identified fertilization
mechanisms ubiquitously dispersed, occurring also in
higher plants? The past few years have seen a number of
discoveries indicating that general principles of fertilization
mechanisms in animals and flowering plants are more
conserved than previously thought. Here, we compare the
development and morphology of animal and flowering
plant gametes, discuss cell–cell communication events
between gametes and gametophytes as well as their physical
interaction and fusion during single and double
fertilization, respectively.
Keywords Fertilization Á Cell-cell communication Á
Gamete interaction Á Chemotaxis Á Species-specificity
Introduction
In comparison to the diplontic life cycle of most animals,
where only the gametes are in the haploid state, flowering
plants undergo a haplodiplontic life cycle, in which
the gametes are not the direct result of a meiotic division.
The haplodiplontic life cycle of plants alternates between a
multicellular diploid organism, the sporophyte, and a
multicellular haploid organism, the gametophyte (Greek
phyton, ‘‘plant’’). After meiosis, the sporophytes of higher
plants give rise to sexually differentiated types of spores,
microspores and megaspores. These spores divide mitotically
and develop into haploid gametophytes, whose main
function in angiosperms is to produce two male and female
gametes, respectively, and to manage the double fertilization
process (Drews et al. 1998; Raven et al. 1999; Lord
and Russell 2002; Boavida et al. 2005). The male gametophyte
(pollen or pollen tube) delivers two sperm cells to
fertilize egg and central cell, respectively (double fertilization),
which thereafter develop into a diploid embryo and
typically triploid endosperm. While plant gametes are
derived after meiosis from gametophytic vegetative cells
during flower development in adult plants, animal gametes
are generated from a founder population of primordial
germ cells (PGCs) that are determined early in embryogenesis
and set aside for gamete production during
adulthood (Aflatoonian and Moore 2006; Gilbert 2006).
Thus, flowering plants differ from animals, not only by an
alternation of two multicellular bodies (gametophyte and
sporophyte), but also by a different sexual life history.
Despite these major differences, mechanisms of cell–
cell communication, cell fusion and prevention of polyfusion
evolved long before the plant and animal lineage
separated more than 1,500 million years ago (MYA), when
the first single-celled green algal antecedent to plants
Communicated by Scott Russell.
M. L. Ma´rton
Developmental Biology and Biotechnology,
Biocenter Klein Flottbek, University of Hamburg,
Ohnhorststrasse 18, 22609 Hamburg, Germany
T. Dresselhaus (&)
Cell Biology and Plant Physiology, University of Regensburg,
Universita¨tsstrasse 31, 93053 Regensburg, Germany
e-mail: thomas.dresselhaus@biologie.uni-regensburg.de
123
Sex Plant Reprod (2008) 21:37–52
DOI 10.1007/s00497-007-0062-8
originated (a primitive eukaryote containing simple green
plastids; Yoon et al. 2004), and long before the first
multicellular organisms appeared, some 1,000 MYA (Kirk
2005). Life existed exclusively in water and it is assumed
that haploid cells of the same type fused randomly to form
a diploid cell (zygote). After genetic recombination the
zygote released haploid offspring that occasionally displayed
a higher fitness. Thus, the evolution of sex was
disadvantageous for the single cell, but advantageous for
the cell population. Taking into consideration that different
cell types and species co-existed in the same environment,
there also appeared the necessity to evolve species-specific
communication and fusion mechanisms as well as the
formation of mobility. It is therefore not a surprise that, for
example, simulation experiments indicate that evolution of
chemotactic cell behavior occurs quickly in evolutionary
times, requiring only few proteins (Soyer et al. 2006). As
another consequence, lower plants such as algae, mosses
and ferns, similarly to animals, use mobile sperm cells for
reproduction. The first land plants probably occurred 490–
425 MYA, from a single common ancestor (Sanderson
2003) that had to adapt ways to keep from drying out and to
reproduce. Under humid environmental conditions, swimming
sperm cells still use flagella to arrive at the egg cells.
However, the most recent and most successful plant lineage,
the angiosperms, which originated some 180–
140 MYA (Magallo´n and Sanderson 2005, Bell et al. 2005)
and have captured almost every ecological environment,
were forced to evolve new reproduction mechanisms such
as precociously forming enclosed male germ cells portable
over large distances, highly protected female gametes, as
well as a sexual mode to nourish the developing embryo,
and a protected, provisioned seedling. The latter need for
nutrition is achieved by a novel, second fertilization
product, the endosperm.
Considering that the sexual life cycle of flowering plants
is fundamentally different and arguably more complex than
in animals, can we expect that basic fertilization mechanisms
and the molecular nature of key regulators are still
conserved? If so, can plant reproduction biologists learn
from their colleagues working with animal model systems
to understand fertilization in plants?
A complete comparison of all fertilization mechanisms
between plants and animals including also algae, mosses,
ferns and gymnosperms as well as the numerous evolutionary
aspects would go far beyond the scope of this
review. We therefore restrict our comparative review to
early molecular fertilization mechanisms in flowering
plants and animals, reporting mainly about gamete morphology
and function, as well as gamete cross-talk and
fusion. Late fertilization events that include prevention of
polyspermy, nuclear migration and karyogamy as well as
egg activation are compared in the corresponding reviews
elsewhere in this issue (Spielman and Scott 2008; Kranz
and Scholten 2008; Curtis and Grossniklaus 2008,
respectively).
Gamete development and morphology
Male and female gametes are unique from all other cells
and are also different from each other. Animal male
gametes, known formally as sperm or spermatozoa, comprise
a head, containing the acrosome and the haploid
nucleus, and the flagellum, consisting of the neck, the
midpiece, the tail and the end piece (Clermont and Leblond
1955; Gilbert 2006; Fig. 1a, b). The acrosome plays a
critical role in facilitating sperm fusion during fertilization
and is derived from the Golgi apparatus. It contains
enzymes needed to digest extracellular coats surrounding
the egg or oocyte and thus enables the sperm nucleus to
enter the egg cytoplasm. In sea urchins and several other
species, recognition between sperm and egg involves
molecules of the acrosomal process. Animal sperm are able
to swim actively towards the female gamete via chemotropism
(see below). The ways sperm cells are propelled
vary according to how the species has adapted to its own
environmental conditions. In most species, each sperm is
able to travel long distances relative to its size by whipping
its flagellum. Microtubules represent the structural basis of
the flagellum, driven with energy for flagellar motion
derived from mitochondrial ATP and powered by a dynein
motor ATPase (Shinyoji et al. 1998).
In contrast to animal sperm, male gametes of flowering
plants are non-motile and are transported towards the
female gametes via the pollen tube (Fig. 1c). Here, we
describe male gamete development in flowering plants only
briefly, as excellent reviews can be found elsewhere that
describe also the evolution of aflagellate sperm cells (for an
overview see Renzaglia and Garbary 2001; Morrow 2004;
Boavida et al. 2005). Angiosperms contain two male
gametes (sperm cells) that play a central role in double
fertilization. The mature male gametophyte, also referred
to as the pollen grain or microgametophyte, develops
within the anther locules and is composed of two or three
haploid cells: a vegetative cell that encloses either one
generative cell (bicellular pollen grain) or two sperm cells
(tricellular pollen grain). In the case of the tricellular pollen
grain, sperm cells are formed after the second mitotic
division within the anther (for example in grasses and
crucifers), whereas in the bicellular pollen grain the second
mitotic division occurs during pollen tube growth, as in
many Solanaceae species (McCormick 2004). The vegetative
cell coordinates pollen tube growth and thus the
delivery of the two male gametes to the female gametophyte
(Weterings and Russell 2004).
38 Sex Plant Reprod (2008) 21:37–52
123
The animal female gamete, called the ovum (a mature
egg with a haploid nucleus, as in sea urchins) or the oocyte
(an egg at an earlier stage of development arrested at
meiosis II, as in mammals), is non-motile and huge in
comparison to the male gamete. While the sperm has
eliminated most of its cytoplasm during maturation, the
egg or oocyte contains a large mass of cytoplasm storing
nutritive and regulatory proteins, ribosomes, tRNAs and
mRNAs needed to accomplish protein synthesis during
embryo development. In many species, morphogenetic
factors are also present in different regions in the egg
cytoplasm. Furthermore, many egg cells contain protective
agents (e.g. ultraviolet filters, DNA repair enzymes) that
are needed for their survival and, later on, for embryo
survival in particular environments. Each egg contains,
within this huge mass of cytoplasm, a large nucleus, which
in some species (like sea urchins) is already haploid at the
time of fertilization, whereas in other species (like many
worms and most mammals) is still in meiosis. The egg cell
membrane plays the most important role during the fertilization
process by regulating the secretion of sperm
attractants, the flow of certain ions and fusing with the
sperm cell membrane. Outside of the cell membrane is an
extracellular layer that is often involved in sperm–egg
recognition (Correia and Carroll 1997). In most animals,
this structure is usually called the vitelline envelope,
whereas the surrounding layer in mammals is a separate
and thick extracellular matrix called the zona pellucida
(Fig. 1a, b). Many types of egg cells also possess outside
the vitelline envelope a layer of egg jelly, which contains
glycoproteins and which is most commonly used either to
attract or to activate sperm. Mammalian egg cells are
additionally surrounded by a layer of cells called the
cumulus, with this innermost layer adjacent to the zona
pellucida called the corona radiata. Directly beneath the
egg plasma membrane lies the cortex, a thin shell of gellike
cytoplasm containing high concentrations of globular
actin molecules. These actin molecules polymerize during
Fig. 1 Models comparing sperm attraction in marine invertebrates,
mammals and flowering plants. a Eggs (ovums) of marine invertebrates
such as sea-urchins secrete species-specific peptides (dots) to
attract sperm cells. Short-range sperm guidance occurs along a
concentration gradient. Sperm of the same species swim towards the
egg via chemotaxis (left, one sperm penetrates the jelly egg coat),
while sperm of other marine animals are not attracted (right).
b Chemotaxis and probably also thermotaxis takes place inside the
mammalian oviduct once sperm have passed the uterus. Dots indicate
a concentration gradient of signaling ligand molecules secreted by
the ovulated egg and/or surrounding cumulus cells. c Pollen tube
guidance precedes double fertilization in flowering plants. A pollen
tube harboring two sperm cells and a large vegetative nucleus has left
the placenta to grow along the placental surface and the funiculus into
the micropyle following gradients generated by the maternal tissues
of the ovule as well as by the female gametophyte. A Polygonum-type
embryo sac containing the egg apparatus (egg cell and two synergids),
a central cell with two polar nuclei and three antipodal cells is
surrounded by nucellus cells (light green) and the two layers of the
inner and outer integuments. The nucellus is disintegrated in some
plant species generating a naked egg apparatus at the micropylar
region. Abbreviations: AN antipodal cells, CC central cell, CU
cumulus cells, EC egg cell or ovum, FU funiculus, OD oviduct, OV
ovary, PL placenta, PT pollen tube, SC sperm cell, SY synergid, VN
vegetative nucleus, YE egg jelly, ZP zona pellucida
b
Sex Plant Reprod (2008) 21:37–52 39
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fertilization and form microfilaments that are necessary for
cell division and are used as well to extend the egg surface
into small projections called microvilli, which may aid in
sperm entry into the cell. The final structural feature of the
egg cell that serves as a critical function during fertilization
is a set of membrane-bound structures, called cortical
granules that accumulate within the cortex. These are
homologous to the acrosomal vesicle of the sperm and are
Golgi-derived organelles that contain proteolytic enzymes
and mucopolysaccharides, needed to prevent polyspermy
(Yanagimachi 1994; Gilbert 2006).
The female gametophyte of flowering plants, also
referred to as the embryo sac or megagametophyte, constitutes
the structural setting for double fertilization
(Nawaschin 1898). More than 15 different patterns of
female gametophyte development have been described.
The most frequently exhibited pattern, occurring in about
70% of the angiosperms, is known as the Polygonum
type (Fig. 1c), which was first described in Polygonum
divaricatum (Strasburger 1879). The development of the
Polygonum-type female gametophyte occurs over two
phases referred to as megasporogenesis (megaspore formation
during meiosis when three of the four megaspores
undergo programmed cell death) and megagametogenesis
(embryo sac development) (Huang and Russell 1992;
Yadegari and Drews 2004). After megasporogenesis, the
functional megaspore undergoes three cycles of mitosis,
producing an eight-nucleate syncytium. Phragmoplasts and
cell plates form between sister and non-sister nuclei after
the third mitosis, and the female gametophyte cells become
partly surrounded by cell walls (Webb and Gunning 1994).
The three cells at the micropylar pole differentiate into the
egg apparatus, which consists of an egg cell that is flanked
by two synergids (Punwani and Drews 2008, this issue).
During cellularization, two nuclei, one from each pole
(referred to as polar nuclei), migrate towards the egg
apparatus and fuse before or after fertilization to generate
the diploid secondary nucleus of the central cell (Diboll
1968; Webb and Gunning 1994; Christensen et al. 1997;
Kranz et al. 1998). The synergids form the filliform
apparatus, a complex consisting mainly of cell wall
invaginations, at their micropylar end. The three cells
opposite to the egg apparatus form antipodal cells. In some
species, like Arabidopsis, antipodal cells undergo programmed
cell death before fertilization (Murgia et al. 1993;
Christensen et al. 1997), whereas in other species, such as
maize, they proliferate, generating a cell cluster of up to 60
cells (Diboll and Larson 1966; Huang and Sheridan 1994;
Kiesselbach 1999). Thus, in most angiosperms, the haploid
mature Polygonum-type female gametophyte is embedded
in several maternal diploid cell layers of the ovule and
consists of four distinct types of cells, including the two
female gametes, the egg cell and the central cell. During
double fertilization, the egg cell gets fertilized by one of
the male gametes and develops into the diploid embryo,
while the central cell is fertilized by the second sperm
giving rise to a triploid endosperm.
Despite these obvious differences between plant and
animal gametes, there also exist a number of common
features. Heteromorphism, for example, the formation of at
least two different types of sperm or pollen grains, occurs
in both animal and flowering plant species. In animals,
sperm heteromorphism is typically related to the generation
of one fertile morph and one (or more) sterile morph(s),
whereas in plants two or more pollen morphs (one of which
can be either sterile or fertile) are produced in all flowers
but sometimes in different anthers (Till-Bottraud et al.
2005). This example of convergence suggests a general
evolutionary response to sexual selection, either to increase
the success of one male’s sperm or pollen in competition
with others, or directly mediate interactions between male
and female gametes. Female gametes of both kingdoms are
generally very large and immobile polar cells containing
not only a store of RNAs and protein for subsequent
development, but also morphogen gradients, generating
daughter cells with a different fate after asymmetric cell
divisions. In animals, this is generally achieved by polar
anchoring of transcripts encoding transcriptional regulators,
components of signal transduction chains or RNAbinding
proteins and encoded proteins, respectively, in
cytoplasmic regions of the egg cytoplasm (for review, see
Gilbert 2006). The first examples indicating that similar
mechanisms exist in plants are, for example, transcripts for
the Homeobox containing transcriptional regulators WOX2
and WOX8, which are expressed in the egg cell, but are
differentially distributed to apical and basal daughter cells
after the asymmetric division of the zygote (Haecker et al.
2004). Direct communication pathways between male and
female gametes of marine invertebrate species have been
known already for more than 40 years (Miller 1985);
however, higher animals, including mammals, as well as
plants, seem to employ helper cells (cumulus cells in
mammals and synergids in flowering plants) to attract and
guide the male gametes and gametophytes, respectively
(for review, see Eisenbach and Giojalas 2006; Punwani and
Drews 2008, this issue). The next chapter therefore discusses
cell–cell communication and chemotaxis between
gametes in animals and flowering plants.
Cross-talk between gametes
Cell–cell communication plays an elementary role in various
developmental processes either between neighboring
cells or between cells that are separated at some distance.
At fertilization, cell–cell communication between male and
40 Sex Plant Reprod (2008) 21:37–52
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female gametes is critical both in animal and plant systems.
Although there are several marine invertebrates that
accomplish internal cross-fertilization via copulation or
pseudocopulation or spermcast mating (Bishop and
Pemberton 2006), external fertilization is a more widely
recognized mode of animal mating in invertebrates species,
and this review will only refer to the marine broadcast
spawners with external fertilization. In these animals, the
encounter between their male and female gametes takes
place in a tumultuous, aqueous environment shared with
other species that may shed their sex cells at the same time.
Hence, such organisms have to solve two problems: in such
a dilute concentration, sperm must find a way to meet the
eggs from their own species and not to fertilize eggs of
another species. In mammals, where fertilization is internal,
semen is placed inside the female genital tract,
requiring less specificity. However, despite the ingenious
design, in fact, only few of the ejaculated sperm (in
humans, only *1 of every million sperm enter the Fallopian
tubes) ever arrive near enough to the egg to be
contenders for fertilization (Williams et al. 1993; Eisenbach
and Tur-Kaspa 1999). The number of sperms capable
of fertilizing the egg is even smaller. Many do not possess
the capacity to bind and fertilize the egg cell and, hence,
unlike spermatozoa of marine species, mammalian spermatozoa
must first undergo a process of ripening, known as
capacitation, which takes place in the female reproductive
tract. The percentage of capacitated sperm is low (*10%
in humans) and, therefore, the chances that such low
numbers of sperm will successfully reach the egg by
chance, without a guidance mechanism, are very small
(Cohen-Dayag et al. 1995; Giojalas et al. 2004; Eisenbach
and Giojalas 2006). A ripening process has also been
observed in flowering plants. Pollen tubes of Torenia
fournieri and Arabidopsis thaliana grown in vitro are
unable to reach the female gametophyte, whereas pollen
tubes emerging from a cut style precisely find their way to
the female gametophyte, indicating the existence of competence
mediation by the stigma and/or style (Higashiyama
et al. 1998; Palanivelu and Preuss 2006; Higashiyama and
Hamamura 2008, this issue). However, in contrast to animals,
fertilization in plants is more efficient and complex,
involving more interaction partners (Dresselhaus 2006).
Male gametophytes (pollen tubes) in compatible pollination
systems are guided to the female gametophyte
(embryo sac) by a multistep process that includes promoting
of tube growth and guidance by the sporophytic
tissues to verify that almost every ovule is reached by at
least one pollen tube. It is therefore not surprising that
transcriptome analysis of Arabidopsis pollen grains reveal
a very high percentage of transcripts encoding proteins
involved in signaling processes (Pina et al. 2005), indicating
that communication is a major task of the growing
pollen tube. Moreover, communication at different levels
also exists between the male and female gametophytes and
has been distinguished as funicular and micropylar guidance
(Higashiyama et al. 2003). Additionally, it can also be
expected that direct communication between male and
female gametes takes place during sperm delivery in the
receptive synergid to verify that both female gametes are
fertilized. Preferential fertilization in Plumbago supports
this hypothesis (Russell 1985), and first transcriptome
analyses of isolated female gametophytes and egg cells
suggest the presence of a high number of proteins required
for signaling processes (Ma´rton et al. 2005; Sprunck et al.
2005; Yang et al. 2006).
Animals evolved diverse mechanisms of sperm guidance.
In many species, sperm are guided toward eggs by
chemotaxis (Table 1), which is the movement of cells up
a concentration gradient toward a chemical attractant
Table 1 Signaling ligands and receptors involved in animal sperm chemotaxis
Ligand Source Receptor Species Reference
Resact Egg Resact receptor/mGC Sea urchin (A. punctulata) Kaupp et al. (2003, 2006);
Bo¨hmer et al. (2005)
Asterosap Egg Asterosap receptor/mGC Starfish (A. amurensis) Nishigaki et al. (1996);
Bo¨hmer et al. (2005)
Speract Egg Speract receptor Sea urchin (S. purpuratus) Dangott et al. (1989);
Kaupp et al. (2006)
RANTES Follicular fluid RANTES receptor/mGC Human Isobe et al. (2002)
ANP Follicular fluid ANP receptor/mGC Human Silvestroni et al. (1992);
Zamir et al. (1993)
NO Ovary NO receptor/mGC Human Miraglia et al. (2007)
Bourgeonal, Lyral NK G-protein-ass. odorant
receptors
Human and mouse Spehr et al. (2003);
Fukuda et al. (2004)
Progesterone Follicular fluid,
Cumulus
NK Human and rabbit Villanueva-Diaz et al. (1995);
Teves et al. (2006)
NK not known, mGC membrane guanylyl cyclase, ANP atrial natriuretic peptide, NO nitric oxide
Sex Plant Reprod (2008) 21:37–52 41
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(chemoattractant). Several studies have shown that chemoattractant
signals which initiate sperm chemotaxis are
synthesized and secreted by the egg, or in some cases by
somatic cells associated with the egg, and are believed
to exert influence over short distance to enhance the
efficiency of sperm–egg contact (Miller 1985; KirkmanBrown
et al. 2003; Kaupp et al. 2008).
Sperm chemotaxis differs among animal species and
was first discovered in the mid-1960s in marine invertebrate
animals. Most of our current understanding of sperm
guidance originates from those studies, based mainly on
sea urchin and starfish as model organisms. In these species,
the aim of sperm chemotaxis is to recruit as many
spermatozoa as possible to the eggs and to prevent crossspecies
fertilization, meaning that a chemoattractant for
one marine species is usually not recognized by the spermatozoa
of another marine species (Miller 1985). Up to
now, around 80 peptides have been identified, mainly from
cnidaria and echinodermata, that affect sperm motility in a
species-specific fashion and are called sperm-activating
peptides (SAPs) (Miller 1985; Suzuki 1995). From these,
the chemotactic function has been so far clearly proved
only for two SAPs. One is resact, a 14 amino-acid peptide
that was isolated from the egg jelly of the sea urchin
Arbacia punctulata and whose gradient extends at least
1 mm around the egg (Ward et al. 1985; Vacquier 1998;
Kaupp et al. 2003; Bo¨hmer et al. 2005). The other one is
asterosap, a 34 amino-acid peptide from the egg jelly coat
of starfish Asterias amurensis (Nishigaki et al. 1996;
Bo¨hmer et al. 2005). Binding of these peptides to specific
sperm receptors belonging to the membrane guanylate
cyclase family (localized along the length of the sperm
flagellum) stimulates an increase of intracellular cyclic
guanosine monophosphate (cGMP) and mediates ion fluxes
across the sperm membrane. In turn, this affects flagellar
motion and finally determines the direction of movement
(Ramarao and Garbers 1985; Bentley et al. 1986; Nishigaki
et al. 2000; Matsumoto et al. 2003; Neill and Vacquier
2004; Kaupp et al. 2008). Thus, cGMP is considered to
play a pivotal role in the motility response of sea urchin
sperm to chemoattractants.
One of the most intensely studied SAPs and the first to
be purified and characterized is speract, a decapeptide that
is released by eggs of another sea urchin, Strongylocentrotus
purpuratus (Suzuki 1995). Like resact, speract
activates a cGMP-signaling pathway (Cook and Babcock
1993; Matsumoto et al. 2003; Solzin et al. 2004). However,
the speract receptor, a 77 kDa plasma membrane receptor
localized exclusively in the sperm flagellum, has been
reported to be unrelated to guanylyl cyclase (Dangott et al.
1989; Cardullo et al. 1994). The speract-induced motility
changes in S. purpuratus sperm closely resemble those of
other marine species whose sperm undergo chemotaxis, but
attempts have failed to demonstrate its chemotactic activity
(Solzin et al. 2004; Wood et al. 2007). It was suggested that
resact, speract and other SAPs function through a common
signal transduction pathway to stimulate and orient sperm
motility, and that this signal involves changes in intracellular
pH, in the concentrations of cGMP, cAMP as well as
Na+
and Ca2+
ions, in membrane potential and in the
phosphorylation pattern of several proteins (Ward and
Kopf 1993; Cook et al. 1994; Darszon et al. 2005, 2006;
Wood et al. 2007). Hence, although the function of most
SAPs has not been firmly established, it is tacitly assumed
that they are involved in chemotaxis (Table 1). The
sequence of events and the specific function of several
cellular reactions, however, are still controversial (Kirkman-Brown
et al. 2003; Eisenbach 2004; Kaupp et al.
2006, 2008).
In mammals, in which fertilization takes place inside
the oviducts of the female, two active mechanisms of
sperm guidance have been shown: chemotaxis and
thermotaxis (the directed movement of cells along a temperature
gradient). Mammalian sperm chemotaxis is still
surrounded by a great deal of controversy and has been
demonstrated in vitro primarily in humans (Ralt et al. 1991;
1994), frogs (Al-Anzi and Chandler 1998), mice (Oliveira
et al. 1999) and rabbits (Fabro et al. 2002) (for reviews, see
Eisenbach 1999, 2004; Eisenbach and Giojalas 2006). In
all of these species, only capacitated spermatozoa, which
represents a small fraction of sperm population, are
chemotactically responsive (Cohen-Dayag et al. 1995;
Eisenbach 1999; Fabro et al. 2002; Giojalas et al. 2004).
Up to now, only a few putative chemoattractants are known
to satisfy the main criterion for sperm chemotaxis, namely,
the accumulation of sperm at the optimal chemoattractant
concentration (Eisenbach 1999, 2004). Recent findings
indicate that sperm chemoattractants are secreted both prior
to ovulation within the follicle and after egg maturation
outside the follicle, and that there are two chemoattractant
sources: the mature egg and the surrounding cumulus cells
(Table 1; Ralt et al. 1991, 1994; Oliveira et al. 1999; Fabro
et al. 2002; Sun et al. 2005). The 8-kDa chemokine Regulated
on Activation, Normal T Expressed and Secreted
Chemokine (RANTES), which is produced in human
follicular fluid, prior to ovulation, was shown to be a
chemoattractant for human spermatozoa (Isobe et al. 2002).
Whether RANTES is also secreted in the female genital
tract after ovulation is still not known. In contrast to
echinoderms, the molecular mechanisms of sperm chemotaxis
in mammals are largely unknown, and the role of
cGMP in human sperm functions still needs to be clarified.
Atrial natriuretic peptide (ANP), a polypeptide hormone
produced in the human follicular fluid and a ligand for
mGC, has been observed to attract human spermatozoa in
vitro and has been proposed as a chemotactic signal in
42 Sex Plant Reprod (2008) 21:37–52
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mammals (Silvestroni et al. 1992; Zamir et al. 1993).
However, this hypothesis is not yet confirmed. Recently,
Miraglia et al. (2007) suggested that nitric oxide (NO),
known to be synthesized in the ovarian cells of different
mammalian species, may behave as a chemoattractant
for human spermatozoa and that the signal transduction
involved is a cGMP-signaling pathway. In addition,
because human sperm are also able to synthesize and
release NO, it was hypothesized that during chemotaxis a
complex interplay may occur, not only between spermatozoa,
oocytes and other cells of the reproductive tract, but
also among spermatozoa themselves (Miraglia et al. 2007).
In vitro studies in flowering plants have shown that NO
has a negative chemotropic effect on pollen tube guidance
(Prado et al. 2004) indicating that plant male gametophytes
are also sensitive to NO. Other general candidate attractants
include Ca2+
ions, of which high concentrations have
been measured in synergids and pollen tube tips and which
were shown to be able to reorient pollen tube growth. The
role of Ca2+
ions during fertilization has been discussed
excellently in reviews elsewhere (Malho´ et al. 2000;
Dumas and Gaude 2006). Gamma-amino butyric acid
(GABA), known as the most important inhibitory neurotransmitter
in the nervous system of animals (Boehm et al.
2006), has been shown recently to be involved in longrange
pollen tube guidance (Palanivelu et al. 2003). Until
now, nothing is known in plants about the involvement of
such general molecules in gamete cross-talk.
A general chemoattractant secreted by the cumulus cells
in mammals is progesterone, whose release does not
involve a cGMP-signaling pathway. Interestingly, progesterone
has been identified as being a chemoattractant for
both human and rabbit spermatozoa (Villanueva-Diaz et al.
1995; Jaiswal et al. 1999; Teves et al. 2006). This finding
corroborates well with the lack of chemotaxis-related
species-specificity previously reported for follicular fluids
and conditioned media of several mammals and suggests
that at least some of the mammalian sperm chemoattractants,
which originate in the female genital tract, are
common or very similar (Sun et al. 2003, 2005). Hence, in
mammals, in contrast to marine species, sperm competition,
if it exists, is limited to semen from different
individuals of the same species (Gomendio et al. 1998;
Eisenbach and Giojalas 2006). Bourgeonal and lyral are
two ligands of G-protein-associated odorant receptors
recently found to be chemoattractants for human and
mouse spermatozoa and that like progesterone do not
involve a cGMP-signaling pathway. However, it seems that
they are not the physiological sperm chemoattractants,
because they are probably not secreted in the female
genital tract (Spehr et al. 2003; Fukuda et al. 2004). The
chemoattractant secreted from the egg is not yet known but
it is presumed to be different and more potent or more
concentrated than those secreted from the cumulus cells.
Thus, mammalian sperm chemotaxis in vivo seems to be a
two-step process: first chemoattraction to the cumulus
followed by chemoattraction to the oocyte (Sun et al.
2005). However, it is not yet known whether the cumulus
cells secrete other chemoattractants besides progesterone
or whether the chemoattractant secreted from the egg is
species-specific or common. Sperm guidance seems to be
much more elaborate than originally thought and chemotaxis
seems to extend over a much longer distance. One of
the reasons why there are so many chemoattractants is that
spermatozoa might undergo a multistep chemotaxis as they
travel through the female genital tract, and each step might
sequentially guide them to the next chemoattractant source.
Another reason is that different spermatozoa might respond
to different chemoattractants, resulting in sperm selection.
Thus, chemoattractant-specific sperm selection, if it exists
in mammals, might be involved in sperm competition or
might enable the female to choose sperm (Eisenbach and
Giojalas 2006).
A similar multistep process seems to exist also in
flowering plants: general molecules (see earlier) might
be involved in long-range pollen tube chemotaxis that
involves the secretion of specific promoting factors from
the style (for review, see Higashiyama and Hamamura
2008, this issue), while short-range chemotaxis is mediated
by the synergids, which are the most active secretory cells
of the female gametophyte, and probably also by the other
cells of the female gametophyte. The last step is likely to
be controlled by the two female gametes themselves.
However, there is no evidence that egg or central cell
secreted molecules exist as would be required to attract
either one or the other of the two sperm cells or both.
Nevertheless, the synergids contain a large number of
secretory vesicles and masses of rER (Diboll and Larson
1966; Mansfield et al. 1991) and have been shown to be
involved in short-range and probably ovular attraction
of pollen tubes (Higashiyama and Hamamura 2008, this
issue). Thus, plant synergids might be functionally equivalent
to mammalian cumulus cells. Several studies have
been performed to demonstrate the existence of speciesspecific
guidance signal(s) in flowering plants. Crosses of
A. thaliana, for example, with pollen of other Brassicaceae
species showed germination and growth of pollen tubes
through the transmitting tissue, but not towards the female
gametophyte, indicating that it secretes species-specific
signaling molecule(s) (Shimizu and Okada 2000). Interspecific
cross-pollination of T. fournieri with related
species showed similar results, supporting the hypothesis
that species-specific signaling of the female gametophyte
exist (Higashiyama et al. 2006). Until now, only one small
secreted protein (EA1) has been identified in maize that
fulfils all of the criteria of a species-specific signaling
Sex Plant Reprod (2008) 21:37–52 43
123
molecule, as it is secreted by the egg apparatus into the
micropylar region of the ovule, is degraded after fertilization
and RNA silencing experiments resulted in the loss
of short-range micropylar pollen tube guidance (Ma´rton
et al. 2005; Table 3). Interestingly, Arabidopsis and other
dicotyledonous plant species do not possess EA1-homologs.
Homologs occurring in grass species display amino
acid variation in the conserved C-terminal region of the
peptide that is predicted to contain the mature peptide
ligand (M. Ma´rton and T. Dresselhaus, unpublished results)
supporting the hypothesis that short-range pollen tube
guidance is species-specific in plants. Biotests using the
mature EA1 peptide of maize and other grasses should help
in answering this question.
In mammals, a second active mechanism of sperm
guidance exists called thermotaxis (Fig. 1b), which appears
to be an essential mechanism in guiding spermatozoa
released from the cooler reservoir site toward the warmer
fertilization site. Bahat et al. (2003) showed that rabbit and
human spermatozoa can sense small temperature differences
(0.5°C and, perhaps, even lower) and respond to it by
thermotaxis. The temperature difference between the site
of the sperm reservoir and the fertilization site is generated
at ovulation by a temperature drop at the former. Similar to
mammalian chemotaxis, only capacitated spermatozoa are
thermotactically responsive (Bahat et al. 2003). Several
in vitro studies indicate that capacitated spermatozoa are
guided from the storage site to the egg primarily by a
combination of chemotaxis and thermotaxis, assisted perhaps
by oviductal contractions (Cohen-Dayag et al. 1995;
Bahat et al. 2003). Thus, thermotaxis appears to be a longrange
guidance mechanism, in addition to chemotaxis,
which seems to be short-range and likely occurs at close
proximity to the oocyte and within the cumulus mass. Both
mechanisms probably have a similar function—to guide
capacitated, ready-to-fertilize spermatozoa towards the
oocyte, and they may complement each other, meaning that
each mechanism is functional in a region where the other
mechanism is ineffective. The molecular mechanism of
sperm thermotaxis is not yet known, but if it shares some
of the chemotaxis signaling pathway, it may involve,
for example, cGMP-mediated transient elevation of intracellular
Ca2+
(Bahat and Eisenbach 2006).
It is unlikely however that thermotaxis is involved in
long-range guidance in flowering plants. Mo´l et al. (2000)
reported that egg cell maturation is essentially accelerated
after pollination in maize, but removal of silk directly after
pollination did not repress accelerated egg maturation,
suggesting an electric nature of the pollen signal. Even
wounding using sea sand is able to promote egg maturation
(R. Mo´l, personal communication), indicating the potential
existence of long-range signals unrelated to peptides/
proteins.
In summary, cross-talk between animal gametes and
plant gametophytes is very complex and involves speciesspecific
short-range guidance molecules predominantly
consisting of secreted peptides or proteins (Tables 1, 3).
The nature of long-range guidance molecules is mostly
unclear. Direct mechanisms of cross-talk between plant
gametes are unknown to date.
Gamete interaction and fusion
The direct interactions between male and female gametes
are among the most fascinating in cell biology and generally
first include cell–cell adhesion and then membrane
fusion between the two gametes. In animals, the interaction
between sperm and egg follows, in general, five
distinct steps: (1) sperm chemoattraction to the egg (discussed
in the previous chapter), (2) the acrosome reaction,
which causes acrosomal exocytosis and the release of
proteolytic enzymes, (3) sperm binding to the egg extracellular
envelope, which is the vitelline layer, in case of
marine species, or the zona pellucida in case of mammals,
(4) penetration of the sperm through this extracellular
envelope and (5) sperm adhesion to the egg and fusion of
their plasma membranes. However, in mammalian fertilization,
steps (2) and (3) are reversed, and the acrosome
reaction in mammals occurs upon sperm binding to the
zona pellucida (Vacquier 1998; Gilbert 2006).
The egg envelope and extracellular matrix of animals
play important roles in sperm–egg binding, induction of the
sperm acrosome reaction, egg activation and the block to
polyspermy. Studies with both mammalian and invertebrate
species have shown that carbohydrate moieties of
specific egg envelope glycoproteins bind to sperm surface
proteins. In marine invertebrates, such as sea urchins,
activation of spermatozoa triggering the acrosome reaction
results from their direct contact with a highly sulfated
fucose sulfate polymer in the jelly coat of the fully mature
ovum (Vacquier and Moy 1997). This interaction causes
adherence of numerous spermatozoa to the jelly coat, and
the opening of incurrent Ca2+
channels in their cell membranes
that permits calcium to enter the sperm head
(Hirohashi and Vacquier 2002). Here, calcium-mediated
fusion of the acrosomal membrane with the adjacent sperm
cell membrane appears to cause acrosomal exocytosis.
Interestingly, the ligand that induces the acrosome reaction
in sea urchins is a pure polysaccharide, with no associated
protein (Vacquier and Moy 1997). Egg jelly sulfated
polysaccharides have been isolated and characterized from
a number of species, and they are all species-specific
inducers of the sperm acrosome reaction (Alves et al.
1997). Such species-specificity seems to be determined by
the glycosidic linkage of the polymer and the pattern of
44 Sex Plant Reprod (2008) 21:37–52
123
sulfation of the sugar residues (Hirohashi et al. 2002;
Vilela-Silva et al. 2002; Biermann et al. 2004). Thus, the
activation of the acrosome reaction in sea urchins constitutes
a barrier to interspecies fertilizations (Vilela-Silva
et al. 2002; Moura˜o 2007).
In mammals, the species-specificity of fertilization is
thought to be determined, in large part, at the level of
sperm binding to the zona pellucida (ZP) of the egg. The
mammalian ZP is synthesized and secreted by growing
oocytes, and forms an extracellular glycoprotein matrix
with a variety of functions. Results obtained by many
scientists suggest that species-specific gamete recognition
occurs between defined carbohydrate structures of the ZP
and their corresponding receptors on the sperm plasma
membrane (Tulsiani et al. 1997; Wassarman et al. 2001;
Shur et al. 2006). Mouse ZP is composed of three families
of glycoproteins, ZP1, ZP2 and ZP3, of which only ZP3
was shown to possess sperm-binding activity with the egg
coat (Bleil and Wassarman 1980a, b, 1988). Initial gamete
adhesion is mediated by the sperm surface receptor, b1,
4-galactosyltransferase-I (GalT) that binds to specific oligosaccharide
chains on the ZP3 (Wassarman et al. 2001;
Rodeheffer and Shur 2002). Binding of ZP3 oligosaccharide
chains induces aggregation of GalT, thus activating,
directly or indirectly, acrosomal exocytosis (Macek et al.
1991; Gong et al. 1995). Recent studies suggest that there
are at least two distinct sperm–egg binding events in
mouse: a ZP3- and GalT-independent interaction responsible
for gametes adhesion, followed by a ZP3- and GalTdependent
interaction that facilitates acrosomal exocytosis
(Shur et al. 2006). At least two GalT-ZP3-independent
receptors have been identified so far: zonadhesin, originally
identified in pig, and mouse isolated SED1 (Secreted protein
containing a cleavable signal sequence, N-terminal
Notch-like type II EGF repeats and C-terminal Discoidin/
F5/8 Complement domains). Zonadhesin is a sperm protein
that binds to the ZP in a species-specific manner. It is
localized in the acrosomal matrix and, thus, may mediate
the binding of sperm to the egg coat during early stages of
acrosomal exocytosis (Gao and Garbers 1998; Shur et al.
2006). Sperm SED1 seems to be required for the initial
sperm adhesion to the egg coat, and in doing so, brings ZP3
oligosaccharides into close enough proximity to bind and
aggregate its receptor on the sperm membrane (i.e. GalT
among other candidate receptors). ZP3 binding activates
specific heterotrimeric G-proteins in the sperm cell membrane
and membrane calcium channels that culminate in
calcium-mediated acrosomal exocytosis, zona penetration
and oocyte activation (Shi et al. 2001; Shur et al. 2006).
The acrosome reaction is a crucial event for successful
fertilization and was first discovered in sea urchins, starfish
and several other marine invertebrates in the early 1950s.
In most marine invertebrates, the acrosome reaction is
characterized by two major physiological events: the fusion
of the acrosomal vesicle membrane with the sperm plasma
membrane (an exocytosis that causes the release or secretion
of the contents of the acrosomal vesicle) and the
extension of the acrosomal process (Colwin and Colwin
1963). In sea urchins, acrosomal exocytosis releases proteolytic
enzymes from the acrosomal vesicle that begin to
digest constituents of the jelly coat, making a path through it
to the egg surface. Once the sea urchin sperm has penetrated
the egg jelly and the acrosomal process of the sperm contacts
the surface of the egg, the acrosomal protein bindin
mediates the species-specific adhesion of sperm to egg
(Vacquier and Moy 1977; Vacquier et al. 1995; Gilbert
2006). Sea urchin bindins are not related to any other proteins,
but they do contain a central domain of 60 amino
acids that has been conserved for more than 150 MY
(Biermann 1998). The acrosomal process is formed by the
pH-dependent polymerization of globular actin molecules
into actin filaments. The process then extends, 1 mm from
the tip of the sperm head, projects through the remains of
the jelly coat towards the vitelline membrane of the egg, and
is covered by the bindin-coated membrane that will finally
fuse with the egg plasma membrane (Barre et al. 2003). The
interaction between the plasma membranes of sperm and
egg is a receptor-mediated event, with the egg receptor for
bindin recognizing and binding species-specifically to
sperm bindin (Kamei and Glabe 2003; Neill and Vacquier
2004). Thus, in sea urchins, species-specific recognition of
gametes occurs at the levels of sperm attraction, sperm
activation and sperm adhesion to the egg surface.
An acrosomal reaction seems not to exist in flowering
plants where the synergids play the key role in double
fertilization: synergids are required for short-range pollen
tube attraction (see above), pollen tube growth arrest and
pollen tube discharge as well as transportation of the two
sperm cells towards their targets, the female gametes (see
also Punwani and Drews 2008, this issue). Recently, a first
key regulator of pollen tube arrest and bursting was identified
in Arabidopsis as a plasma membrane-localized
receptor-like serine-threonine kinase (Escobar-Restrepo
et al. 2007). FERONIA (FER) belongs to the CrRLK1L-1
group of receptor-like kinases and accumulates asymmetrically
in the synergid membrane at the filiform apparatus,
which is the entry point of the pollen tube. A FERdependent
signaling pathway is proposed involving an
unknown ligand from the pollen tube. Activated FER
would cause the synergid to send another signal back to the
pollen tube, inducing both growth arrest and bursting.
Candidate pollen/sperm ligands have been reported by
Engel et al. (2003), but none has been shown yet to be
involved in pollen tube-synergid signaling.
As in animals, the actin cytoskeleton seems to play also
a key role in flowering plants. In the degenerated synergid,
Sex Plant Reprod (2008) 21:37–52 45
123
the actin cytoskeleton becomes reorganized during synergid
cell death soon after pollen tube discharge and forms
two actin coronas. The coronas extend from the middle of
the degenerated synergid, one extending to the vicinity of
the egg nucleus and the second towards the vicinity of the
egg and central cell extending to the polar nuclei (Huang
and Sheridan 1998; Huang et al. 1999; Fu et al. 2000; Ye
et al. 2002). It is thought that these coronas are involved in
actomyosin-mediated sperm transport to approximate male
and female gametes. It is still a matter of debate whether
sperm cells become activated after delivery, are attracted
each by only one female gamete and fuse preferentially
either with the egg or central cell. In Plumbago, which
contains dimorphic sperm cells, it was shown that the
mitochondria-rich sperm preferentially fertilized the central
cell and the plastid-enriched sperm the egg cell
(Russell 1985). Two distinguishable types of sperm cells
have also been observed in tobacco (Yang et al. 2005);
however, it is unclear whether they are required for preferential
fertilization. Genetic studies in maize involving
supernumerary B-chromosomes could not show a difference
(Faure et al. 2003). Recently, a mutant of Arabidopsis
known to produce pollen containing a single sperm-like cell
was used to pollinate embryo sacs. All sperm-like cells
fused with egg cells only (Nowack et al. 2006) indicating
that either egg cell fertilization occurs first due to the shorter
transport distance after delivery or due to the presence of
egg-adhesion molecules at the sperm surface. However, a
similar Arabidopsis mutant described by Chen et al. (2008)
adds more complexity to this process as the single spermlike
cell could not discriminate either female gamete.
In mammals, sperm must be acrosome-reacted to penetrate
the ZP and fuse with the egg (Florman and Ducibella
2006). As sperm undergo the acrosome reaction, they must
remain transiently attached to the ZP prior to initiation of
zona penetration. It has been shown that the binding of
acrosome-reacted sperm to the ZP seems to depend on ZP2,
because acrosome-reacted sperm lose their affinity for ZP3
and gain affinity for ZP2 (Bleil et al. 1988; Mortillo and
Wassarman 1991). During the acrosome reaction in
mammals, sperm membrane alterations occur that lead to
the subsequent relocalization of some membrane proteins
(e.g. the protein Izumo) into other membrane regions and
are required for fusion (Inoue et al. 2005). Acrosomal
exocytosis releases a variety of proteases that lyse the ZP,
thus creating a hole through which the sperm can travel
toward the egg (Yamagata et al. 1999; Gilbert 2006).
Once a sperm cell has undergone the acrosome reaction
and has managed to travel to the egg, fusion of the sperm
cell membrane with the egg cell membrane can finally
proceed. In sea urchins, sperm–egg fusion causes actin
polymerization in the egg to form a fertilization cone
(Terasaki 1996). Since the acrosomal process is formed by
polymerization of actin as well, a connection is then
formed by the actin from both gametes. This connection
expands the cytoplasmic bridge between the egg and the
sperm, and the sperm nucleus and tail can pass through this
bridge (Gilbert 2006). The fusion process, being an active
process, is often mediated by specific ‘‘fusogenic’’ proteins.
Among these, the sea urchin sperm protein bindin was
suggested to play a second role as a fusogenic protein, in
addition to its main role to recognize the egg (Ulrich et al.
1999).
In mammals only a few cell surface proteins have been
identified to date in both gametes as being essential for
gamete fusion (example candidates are listed in Table 2).
Interestingly, no mammalian homologs of invertebrate
sperm–egg fusion proteins have been discovered to date
(Primakoff and Myles 2007). Tetraspanins localizing to the
microvillar-rich region of the egg membrane are thought
to play an important role in mammalian gamete fusion as
they function primarily as organizers of networks of
Table 2 Candidate molecules mediating gamete interaction and fusion in animals
Molecule Source Species Function Reference
Sulfated
polysaccharides
Egg jelly Sea urchins Induction of acrosome reaction Alves et al. (1997)
Bindin Sperm (acrosome) Sea urchins Species-specific egg adhesion, fusion Vacquier and Moy (1977); Ulrich et al. (1999)
GalT/ZP3 Sperm/ZP Mouse Initial gamete adhesion Wassarman et al. (2001)
SED1 Sperm surface Mouse Binding to the egg coata
Shur et al. (2006)
Izumo Sperm surface Mouse Gamete fusiona
Inoue et al. (2005)
DE / CRISP-1 Sperm surface Rat Binding to the egg surface Cohen et al. (2000b), Ellerman et al. (2006)
Zonadhesin Sperm surface Pig Binding to the egg coata
Gao and Garbers (1998)
Tetraspannins Egg membrane Mammals Gamete fusiona
Primakoff and Myles (2007)
ZP zona pellucida
a
Not yet confirmed
46 Sex Plant Reprod (2008) 21:37–52
123
transmembrane and cytoplasmic proteins (Hemler 2003).
However, until now, it is not known how exactly tetraspanins
facilitate membrane fusion (Primakoff and Myles
2007). GPI-anchored proteins localized on the egg surface
are additional candidates mediating sperm–egg fusion;
however, a clear role in membrane fusion remains to be
demonstrated (Alfieri et al. 2003). The membrane protein
Izumo, which localizes to the sperm cell surface, was found
to be required for sperm–egg fusion either in in-vitro
assays (Okabe et al. 1988) or in knockout lines (Inoue et al.
2005). Izumo is a novel member of the Immunoglobulin
super-family (IgSF) proteins, with a C-transmembrane
domain, and shows a testis and sperm-specific expression
pattern. Like many cell surface IgSF proteins, Izumo could
act in cell–cell adhesion, but to date, no specific function
for Izumo in the fusion process has been determined. One
of the most-studied gamete surface proteins is the spermassociated
glycoprotein DE, a candidate molecule to
mediate sperm–egg fusion. DE is known as well as CRISP-
1 protein, being the first member of the Cysteine-RIch
Secretory Protein (CRISP) family to be described in the rat
epididymis (Cameo and Blaquier 1976; Cohen et al.
2000b). Several CRISPs with a high amino acid sequence
similarity have been identified in animals, plants and fungi,
but their function is still largely unknown. CRISP-1 proteins
are candidates to mediate gamete fusion in the rat,
mouse and human through their binding to complementary
sites on the egg surface (Cohen et al. 2000a, 2001). Deletion
mutants of protein DE showed that its binding site
resides in an evolutionarily conserved region of the CRISP
family (Ellerman et al. 2006).
In summary, our current knowledge of the key players
and the molecular mechanisms mediating gamete fusion
in mammals is far advanced compared with the current
understanding of gamete fusion in flowering plants
(Table 3). However, it was shown recently in A. thaliana
that disruption of a sperm-specific gene, referred to as
Generative Cell Specific 1 (GCS1), results in severe male
sterility due to disturbed pollen tube guidance and gamete
interaction (von Besser et al. 2006; Mori et al. 2006). The
main role of GCS1 seems to be its involvement in sperm–
egg attachment or membrane fusion as suggested by the
authors (Mori et al. 2006), and its function might be similar
to that of mammalian C-terminal transmembrane proteins
localized on the sperm surface, like Izumo (discussed
above). The encoded GCS1 protein was first identified
using generative cells (precursors of the two sperm cells)
isolated from Lilium longiflorum pollen. Molecular biological
and immunological assays indicate that GCS1 is
specifically expressed in the male gamete, accumulating
during late gametogenesis and is localized on the plasma
membrane of generative cells and the sperm cells, probably
anchored by the putative transmembrane domain identified
at the C-terminus. GCS1 homologues are present in various
species, including non-angiosperms.
Another transmembrane protein, LGC1, has been
reported in Lilium generative cells (Xu et al. 1999). LGC1
was identified as a male gamete-specific protein, which due
to its male gamete surface expression may have an
important function in female gamete recognition. However,
no evidence for such a function has still been obtained.
Transcriptomics approaches have identified a number of
candidate plasma membrane localized proteins in sperm
and egg cells of maize and Arabidopsis, respectively, (for
review, see Dresselhaus 2006). Although we are still
awaiting functional studies, a number of plant sperm and
egg marker lines are now available (see also Singh et al.
2008, this issue) that will aid to identify and characterize
more key players of early and late fertilization events.
Conclusions and prospects
In conclusion, early fertilization events are complex multistep
processes in both animals and flowering plants
requiring extensive cross-talk between the gametes and the
gametophytes, respectively. Long-range attraction of male
gametes/gametophytes involves thermotaxis in mammals
and possibly electric signals in plants; however, the
majority of known communication events are mediated by
chemotaxis. Many chemoattractants have been identified
during the past three decades in animals, and first chemoattractant
candidates have now been discovered also in
plants. Small common molecules such as NO and GABA
seem to be involved in long-range attraction/guidance,
whereas species-specific proteins/peptides seem to
Table 3 Signaling ligands and receptors involved in double fertilization of flowering plants
Ligand/receptor Source Species Function Reference
GCS1 Sperm A. thaliana, lily Gamete interactiona
Mori et al. (2006)
LGC1 Sperm Lily NK Xu et al. (1999)
EA1 Egg apparatus Maize Short-range PT attraction Ma´rton et al. (2005)
FERONIA Synergid A. thaliana PT arrest Escobar-Restrepo et al. (2007)
PT pollen tube, NK not known
a
Not yet confirmed
Sex Plant Reprod (2008) 21:37–52 47
123
represent the major mediators of short-range chemotaxis.
Whereas marine animals use small secreted peptides
directly derived from the egg cell, higher organisms
including mammals and flowering plants involve helper
cells such as cumulus and synergids as an additional step,
probably to enhance attractant concentrations for guidance
of male gametes and gametophytes, respectively. In this
sense, plant synergids seem to be functionally equivalent to
mammalian cumulus cells. In another sense, plant synergids
are probably also involved in the prevention of polyspermy
not only by blocking secretion of further attraction signals
but also by gametophyte arrival induced vesicle exocytosis
similar to the cortical reaction in animals (Spielman and
Scott 2008, this issue). However, more detailed cellular and
molecular data are required to sustain these hypotheses.
A major difference between animals and flowering
plants is the absence of flagellar or independent movement
in higher plant sperm cells and also, for example, the lack
of an acrosome reaction. Nevertheless, male gametophytes
of higher plants steadily release vesicles by exocytosis
from the pollen tube tip during growth through the female
tissues of the ovary (Campanoni and Blatt 2007). Interestingly,
both mammalian and flowering plant male
gametes/gametophytes undergo a ripening process inside
the female sexual organs. Another similarity is the polymerization/reorganization
of actin filaments during the
fertilization process. In animals, the acrosomal reaction and
sperm–egg fusion, respectively, cause actin polymerization
in the egg to form a fertilization cone as a prerequisite of
gamete fusion. In the degenerated synergid of flowering
plants, the actin cytoskeleton becomes reorganized during
cell death soon after pollen tube discharge and two actin
coronas are formed that are likely to be involved in sperm
transport and gamete fusion.
Marine organisms require species-specific attractants
and fusogenic molecules in order to assure intra-specific
fertilization. This seems to be less important in mammals
and flowering plants where sperm of the same species are
often either deposited inside or at the surface of female
sexual organs of the same species. Additionally a number
of molecular mechanisms have been evolved to inactivate
incompatible male gametes and gametophytes, respectively,
in order to prevent inter-specific fertilization. In
invertebrate marine animals, the interaction between the
plasma membranes of sperm and egg is a species-specific
receptor-mediated event, and it is therefore not a surprise
that mammalian homologs of invertebrate sperm–egg
fusion proteins have not been discovered. Plant receptors
involved in chemotaxis and gamete fusion are not known to
date. However, in contrast to animals, flowering plants
contain hundreds of predicted membrane bound receptorlike
kinases (Shiu et al. 2004) suggesting that these processes
are more complex and potentially involve more
interacting molecules. Membrane guanylate cyclases that
play a major role in animal sperm chemotaxis do not exist
in plants (Schaap 2005) indicating once more that although
principles of fertilization mechanisms in animals and
flowering plants are more similar than previously thought,
the precise molecular nature of the key players are
completely different. The identification and functional
characterization of these players remain a major challenge
of plant reproduction biologists for the next decade. The
identification of first candidates, already available transcriptomes
of male and female gametes and gametophytes,
as well as an increasing number of marker lines, will now
help in identifying and studying a larger number of fertilization
molecules to understand the molecular regulation of
fertilization also in flowering plants.
Acknowledgments We are grateful to Scott Russell for critical
reading of the manuscript and the German Research Foundation
(DFG, grant MA 3976/1-1) for financial support to M.L.M.
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