INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 38: 979-986, 2016

The molecular basis of fertilization (Review)

1
Institute of Biology, Medical Chemistry and Biotechnology, National Hellenic Research Foundation,
Athens 116 35; 2Laboratory of Clinical Virology, School of Medicine, University of Crete, Heraklion 71003, Greece

Received April 13, 2016; Accepted August 2, 2016

DOI: 10.3892/ijmm.2016.2723

Abstract. Fertilization is the fusion of the male and female zygote (a diploid cell) from which the new organism will result.
gamete. The process involves the fusion of an oocyte with a During sexual intercourse, millions of sperm are deposited into
sperm, creating a single diploid cell, the zygote, from which the vagina. A number of these will die in the acidic environment.
a new individual organism will develop. The elucidation of However, many will survive due to the protective elements
the molecular mechanisms of fertilization has fascinated provided in the fluids surrounding them. Soon afterwards, the
researchers for many years. In this review, we focus on this sperm have to swim through the cervical mucus, towards to the
intriguing process at the molecular level. Several molecules uterus and then on to the fallopian tubes. As they swim towards
have been identified to play a key role in each step of this these, they decrease in number, in an attempt to make it through
intriguing process (the sperm attraction from the oocyte, the the mucus. Inside the uterus, the contractions of the uterus
sperm maturation, the sperm and oocyte fusion and the two assist the journey of the sperm towards the egg. Fertilization
gamete pronuclei fusion leading to the zygote). Understanding takes place in the ampulla of the oviduct. If the oocyte is not
the molecular mechanisms of the cell‑cell interactions will fertilized here, it slowly passes to the uterus, where it becomes
provide a better understanding of the causes of fertility issues degenerated and is absorbed. Achieving fertilization requires
due to fertilization defects. the activation of sperm and oocyte maturation. However, the
oocyte/sperm interaction depends on a number of changes
occurring in the egg and the sperm.
Contents The egg complex following ovulation enters the oviduct
consisting of three components: i) the oocyte (egg) arrested
1. Introduction at metaphase of meiosis II; ii) the extracellular matrix of the
2. Molecules synthesized and secreted from the oocyte, which egg or zona pellucida consisting of three glycoproteins (ZP1,
orient and stimulate sperm ZP2 and ZP3), synthesized and secreted by the oocyte; iii) the
3. The three stages of fertilization cumulus oophorus, consisting of granulosa cells enriched with
4. Reorganization and partition of the zygote hyaluronic acid (1,2). Cumulus cells support fertilization, and
5. Fertilization failure in vitro fertilization can be achieved more efficiently with them
than without them (3-5).
The journey of the sperm is facilitated by ovarian hormones
1. Introduction that affect the structure, composition and activity of the secre-
tory epithelia of the cervix, the uterus and the fallopian tubes
Fertilization is a sequence of coordinated molecular events and the contractility of these elements. The estrogen hormones,
involving the merging of the sperm with the egg, the fusion favor these factors, while progesterone does not. Oxytocin,
of the pronuclei and the intermingling of the maternal and which is secreted during intercourse by stimulation of the
paternal chromosomes. The first form of human life is the posterior pituitary, causes the contraction of the uterus and
the fallopian tubes, as well as by the prostaglandins that affect
the contractility of the uterus and fallopian tubes. During the
ovulation period, the uterus becomes more sensitive to prosta-
glandins.
Correspondence to: Dr Vasilis Zoumpourlis, Institute of Biology, Oocytes acquire the ability to fuse with sperm when they
Medical Chemistry and Biotechnology, National Hellenic Research
reach 20 µm in diameter and they are arrested at the prophase
Foundation, 48 Vassileos Constantinou Avenue, Athens 116 35,
Greece of meiosis II (6). Spermatozoa undergo a series of events during
E‑mail: [email protected] maturation in order to acquire motility and fertility, as they move
from the proximal towards the distal end of the epididymis.
Key words: fertilization, zygote, gamete fusion, sperm capacitation, Only spermatozoa that have passed through the epididymis are
cortical reaction mature enough to be capable of motility. Moreover, via deposi-
tion of new proteins in the nucleus, the DNA becomes more
condensed. The sperm head decreases in size and becomes
more compact. The above is crucial for the subsequent correct
980 GEORGADAKI et al: MOLECULAR BASIS OF FERTILIZATION

decondensation of the paternal DNA in the maternal oocyte. when resact is added to a solution with spermatozoa, the latter
The ability for motility is achieved, but at the same time is undergoes helical movements directed towards the point of
inhibited by the milieu. Finally, the structure of the plasma resact (http://worms.zoology.wisc.edu/dd2/echino/fert/chemo-
membrane is altered by the addition of glycoproteins and other taxis/chemotaxis.html).
proteins. This affects the motility, the capacitation ability and The sperm receptor for resact is a transmembrane protein.
the ability for the acrosome reaction (http://www.embryology. Once it binds resact on the extracellular side, it causes a confor-
ch/anglais/dbefruchtung/bereitstell02.html#genitaltrakt). mational change on the cytoplasmic side, and it activates the
Sperm is the male gamete and is derived from the Greek receptor's enzymatic activity. This triggers the mitochondrial
word ‘sperma’ (meaning ‘seed’). It consists of three parts, a ATP‑generating apparatus, as well as the dynein ATPase that
head (containing DNA and enzymes in order to penetrate the stimulates flagellar movement in the sperm (9,10).
oocyte), a midpiece (containing cellular elements, centrioles, Speract (sperm activating peptide) consists of ten amino
microtubules and a number of mitochondria for energy produc- acids and appears to promote the increase in pH and promotes
tion required to promote sperm) and a tail (enabling sperm sperm movement. The speract receptor is localized to the
movement) (http://www.newworldencyclopedia.org/entry/ sperm tail (11), which contains the axoneme, the organelle of
Sperm). motility. The signaling in the sperm tail is central to motility
The head of normal sperm has an oval shape, a length of regulation. There is evidence that speract can modulate sperm
3‑5 µm, a width of 2‑3 µm and is 1.5 µm thick. It consists of motility (12).
the nucleus and the acrosome, which are covered by the nuclear
membrane and the metacrosomic sheath. The nucleus contains 3. The three stages of fertilization
the male genetic material. The DNA of the sperm has a slightly
different structure from the DNA of a diploid (somatic) cell, Sperm preparation: Capacitation and acrosome reaction. The
meaning that the DNA is concentrated in the smallest possible phase of sperm maturation is known as activation (sperm capac-
volume, so as to save space. The acrosome contains proteolytic itation). It occurs in the genital tract of the female, and acts as a
enzymes, such as acrosin, trypsin, hyaluronidase and proteases, preparatory step for the acrosome reaction. Activation does not
which are released during the acrosome reaction and hydrolysis include morphological changes and is accompanied by hyper-
in granular cells and the zona pellucida of the egg in order to activation of the sperm which is a strong, non‑linear motion (2).
assist the penetration of the sperm and the merger with the An important role is played by proteolytic enzymes (13).
egg (7). In mammals, the ejaculated sperm is motile. However, their
The neck or mid‑section of the sperm connects the head to ability to fertilize an oocyte is reduced. The latter may occur
the tail (length, 7.5 µm; and width, 1 mm). It contains the axial after removing inhibitory factors, such as surface‑attached
filament surrounded by fibrils with circular layout and one to glycoproteins, seminal plasma proteins and the depletion
two centrosomes. The neck contains mitochondria, enzymes of of membrane cholesterol. This final state of activated sperm
glycolysis and oxidation systems and provides the necessary is known as hyperactivation, and is a high energy phase of
energy for survival and mobility. vigorous flagellar movement and swimming capacity (14).
The tail (length, 40‑50 mm) consists of a total of ten pairs of Capacitation involves a number of processes, such as
fibrils (a central and nine peripheral) and is responsible for the functional coupling of the signal transduction pathways that
typical sperm motility. regulate the initiation of acrosome reactions by ZP3; altera-
The sperm can survive for 3-5 days inside the vagina, tions in flagellar motility that may be required to penetrate the
particularly in the vagina or mucous membranes in the upper zona pellucida; and the development of the capacity to fuse
part of the female genital tract. Fertilization in this case is with eggs (15). The above-mentioned events are followed by
possible if the sperm remain alive. A single sperm is sufficient alterations in metabolism, membrane biophysical characteris-
to fertilize the egg. Sperm survive outside the female body for tics, changes in the protein phosphorylation state, elevations
up to several hours. When the sperm meets the oocyte, they in intracellular pH and calcium levels, and hyperpolarization
swim around it. Subsequently, one of them adheres to the head of membrane potential. In vivo, many factors are likely to
of the oocyte and begins rotational movements about the longi- mediate the activation of sperm. Sterol binding proteins, such
tudinal axis. The whole sperm then penetrates the oocyte, with as high density lipoprotein, have been identified in the fallo-
the acrosome penetrating the egg cover [http://www.yourarticle pian tubes and can accelerate the efflux of cholesterol from
library.com/biology/human-reproduction/structure-of-human- the sperm (16). Furthermore, progesterone can regulate some
sex-gametes-spermatozoan-and-ovum-biology/26913/; http:// aspects of sperm activation. It is present in the environment of
www.onmed.gr/ygeia/story/329099/poso-zei-to-sperma-mesa- the fallopian tubes, derived both from follicular fluid and cells
ston-kolpo#ixzz44lwgUQgb (in Greek)]. of the cumulus oophorus (17). Glucose is known to be essential
for successful capacitation. It functions not only as an energy
2. Molecules synthesized and secreted from the oocyte, molecule allowing spermatozoa to swim, but it also enables the
which orient and stimulate sperm spermatozoa to fertilize eggs (18).
The agents of the oocyte mucous that cause the acrosome
Resact (respiratory activating peptide) is a peptide of 14 amino reaction are specific for each species. In mammals, sperm
acids, isolated from the egg jelly of the sea urchin, Arbacia recognizes and binds to ZP3 glycoprotein of the zona pellu-
punctulata (8). It is probably recognized by the guanylate cida. The O‑polysaccharide connections to the core protein of
cyclase (guanylate cyclase), a protein of the cell membrane of the ZP3 seem to be necessary in this step. It is believed that the
sperm, which causes an increase in Ca2+ levels. Experimentally, terminal galactose of O‑linked oligosaccharides reacts with
 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 38: 979-986, 2016 981

galactosyltransferase (Mr 56 kDa), a cell membrane protein in Evans and Florman, in Nature Cell Biology and Nature
the anterior end of the sperm head. The acrosome reaction of Medicine (15), propose a model according to which in the
the sperm is caused by the core protein of ZP3. Once the core sperm head, the receptor TPC2‑ZP3 is triggered by its asso-
protein of ZP3 is damaged, this reaction is inhibited, but not the ciation with the zona pellucida. This results in calcium entry
binding of the sperm to the zona pellucida. The sperm reaching through T‑type channels, leading to a transient increase in
the transparent zone is connected via SED1 protein to ZP3. As calcium levels in the cytoplasm and the activation of phospholi-
a result of irreversible binding of the sperm to the egg, the zona pase C (PLC) via protein G. PLC is divided into IP3 and DAG.
pellucida triggers the acrosome reaction. This causes Ca+ entrance and leads to the acrosome reaction.
The outer plasma membrane of the acrosome fuses at In the capacitation process, phosphatidyl‑inositol‑3‑
multiple sites with the plasma membrane and the contents of kinase (PI3K) is phosphorylated/activated via a protein
the acrosome are released. After the acrosome reaction, it is kinase A (PKA)‑dependent cascade, and is downregulated by
believed that the protein pre‑acrosin of the acrosomal vesicle PKCa. At the beginning of capacitation, PKCa is active and
binds to ZP2, and becomes activated, forming the enzyme results in PI3K inactivation. PKCa and PP1c2 are degraded by
acrosin which digests the transparent area at this point. a PKA‑dependent mechanism, and this allows the activation
Digestion of the zona pellucida is followed by the fusion of the of PI3K. The activation of PKA, which depends on cAMP
membrane of the sperm to the egg membrane, which seems to produced by the bicarbonate‑dependent soluble adenylyl
be caused by the PH‑30 sperm protein (19). cyclase, leads to an increase in actin polymerization, an
PH‑30 is a transmembrane glycoprotein consisting of an essential step so as to reach hyperactivated motility, which is
α and β subunit. The α subunit (289 amino acids) contains a necessary for successful fertilization (25).
sequence between 90 and 111 which has many similarities The sperm‑specific CatSper ion channels have been
to the ‘fusion peptide’ E2 glycoprotein of the rubella virus. suggested to control the intracellular Ca 2+ concentration
The β subunit (353 amino acids) contains the tripeptide RGD and, thereby, the swimming behavior of spermatozoa (26).
(arginine‑glycine‑aspartic acid) in the last 90 amino acids The CatSper channel seems to be activated by progesterone
of the N‑terminus. The tripeptide RGD is the active site in human spermatozoa (29), which is interesting from the
which can be recognized by integrins of the cell membrane sperm‑egg interaction viewpoint, as cumulus cells are known
of the egg. This recognition is followed by the fusion of the to produce progesterone (5).
membranes, which is caused by the α  subunit of PH‑30.
Both subunits derive from precursors that undergo proteo- Sperm‑egg binding and fusion. The plasma membrane of the
lytic modification during the differentiation of sperm in oocyte consists of two sites, a microvilli-free region and a
the testes and their maturation in the epididymis. PH‑30 is microvilli-rich region. The fusion of sperm and oocyte takes
located in the cell membrane in the anterior portion of the place in the microvilli-rich region (28,29). The interaction
side part of the mature sperm head (19,20). The acrosome between sperm and oocyte is preceded by acrosomal exocy-
reaction involves the fusion of the acrosome membrane with tosis, triggered by sperm and ZP binding. It involves sperm
the overlying plasma membrane of the sperm and vesicle attachment to the oocyte and cell‑cell adhesion, leading to
formation. The result is the release of peptidases from the membrane fusion of the two gametes (30). The inner acrosomal
acrosomal vesicle that digest the mucous coat. The fusion of membrane of the sperm, being exposed following the acrosome
the membranes appears to be caused by the entry of Ca+2 ions reaction, comes into contact with the oocyte membrane (31).
and the exit of K+ ions. The sperm plasma membrane that The equatorial segment adheres to the posterior head of the
does not participate in the acrosome reaction and overlying sperm and fuses with the oocyte membrane (32,33).
the acrosome is known as the equatorial region. This seems Several molecules have been identified in sperm and
to be the site of sperm and egg fusion (2). An acrosomal fila- oocytes, with a crucial role in gamete binding. Fertilin α,
ment, is formed by the polymerization of G (Globular) actin fertilin  β and cyritestin are also known as ADAM1, ADAM2
to F (Fibrilar) actin. Desmin (30.5 kDa) is exposed to the and ADAM3, respectively. Members of this family consist of
acrosomal filament. The polymerization of actin is induced a signal sequence domain, a metalloprotease domain, a disin-
by the increase in pH (~7.4), which results from the input of tegrin‑like domain, a cysteine-rich domain and an epidermal
Na+ and H+ output. The activation of dynein ATPase and the growth factor-like repeat. A number of studies have revealed
use of ATP results in an increased movement of the sperm. a role of ADAM1, ADAM2 and ADAM3 in sperm oocyte
cAMP levels increase (approximately 400-fold) in the cyto- binding (30). Fertilin β has been shown not to be essential for
plasm of sperm. This increase marks the beginning of the plasma membrane binding and fusion. Studies on fertilin β and
process of chromatin decondensation of the sperm, before cyritestin with knockout mice have shown a reduced adhesion
the formation of the zygote nucleus. to the oocyte plasma membrane; however, some sperm adhere
P4 (progesterone) is a well-studied molecule involved in the and they fuse with the oocyte (15,34). Moreover, they show
modulation of sperm function. Its role in sperm capacitation poor adhesion pattern to the ZP (34‑36).
has been revealed; however, the exact mechanisms involved are As regards oocytes, integrins found on the egg surface are
not yet fully understood. Progesterone is involved not only in thought to be receptors for sperm ADAMs. Studies have revealed
capacitation, but also in other events, such as hyperactivated that α6β1 integrin is an egg receptor for fertilin β (37‑39).
motility, flagellar bending, in chemotaxis, in acrosome reaction Other studies have indicated that α9β1 integrin is a receptor
and in sperm‑ZP binding and sperm‑oocyte fusion. Its effects for fertilin β (40,41) CD46 has been found to be expressed in
on sperm function have been studied in humans, as well as in rodents on the acrosomal membrane of sperm (42). CD46 inter-
other mammals (mouse, boar) (21-24). acts directly with β1 integrin and indirectly with tetraspanins
982 GEORGADAKI et al: MOLECULAR BASIS OF FERTILIZATION

in human cells (43). However, the key role of CD46 appears to proteasome system enables sperm to penetrate the chorion
stabilize the acrosomal membrane (44). or participate in the process as sperm‑related ‘moveable’
As an integrin‑associated protein, in the oocyte, CD9 binding proteins (52). Sperm hyaluronidases are believed
is essential for sperm‑egg interactions. It is a member of to play an intriguing role in fertilization in mammals, and
a tetraspanin protein family expressed on the mouse egg sperm‑specific SPAM1 and HYAL5 hyaluronidase have been
surface (45). The role of CD9 in sperm oocyte fusion has been suggested to be involved in sperm‑ZP binding in mice. Recent
demonstrated in a number of studies with CD9-null oocytes studies have shown that hyaluronidases are not required for
which showed reduced ability for strong sperm adhesion (46). fertilization (61,62).
CD9-deficient mice show reduced fertility (47‑49). Another It is important to mention that the oviductal environment
tetraspanin member, CD81, is expressed on the oocyte surface and its secretions also play an essential role in the transport
and interacts with CD9 (50). Deletion of the CD81 gene results and interaction of male and female gametes. The expression of
in a reduction in fertility. However, its role in sperm oocyte lactoferrin, a human oviductal protein, is able to inhibit gamete
interaction has not yet been fully elucidated (51,52). interaction in vitro and it seems to be involved in the regula-
CRISP1, a sperm protein expressed by the cumulus cells tion of the reproductive process through a role in polyspermy
surrounding the oocyte, stimulates sperm orientation through prevention (63). It has also been shown that lactoferrin causes a
the modulation of sperm hyperactivation and it seems to decrease in sperm α‑D‑mannose binding sites and an increase
regulate CatSper (53). Recent studies have revealed an in tyrosine phosphorylation of sperm proteins, thus implying
oocyte‑derived chemotactic activity associated with a hydro- that this protein is able to modulate the parameters of sperm
phobic non‑peptide molecule in human sperm (54) and an function (64).
association between follicle rupture and uterine contractions
with the success of human in vivo insemination, suggesting the Cortical reaction: Meiosis resumption of oocyte and activa‑
existence of possible chemoattractive substances in the female tion of the zygote. Once the sperm fuses with the oocyte, the
tract (55). It is not clear if there is a binding partner on the beating of the tail stops immediately. The fusion of sperm and
oocyte membrane for sperm-associated CRISP protein, along the oocyte membrane appears to cause the polymerization of
with the exact mechanisms through which gamete binding is actin and microvilli extension. The sperm instead is drawn
achieved. into the oocyte by elongation and fusion of the microvilli of
The sperm‑specific protein, Izumo, is essential for the egg. As a result, the sperm nucleus and other organelles are
sperm‑egg plasma membrane binding and fusion (56). IZUMO incorporated into the oocyte cytoplasm. The actin filaments are
interacts directly with some molecules on the oolema. On the essential for the attraction of the sperm into the oocyte. The
oocyte side, Juno is a member of the folate receptor family cytoplasm swells and forms colliculus (~7 microns in length
and recognizes the sperm IZUMO, facilitating fertilization. and 2 microns wide) resembling a so-called fertilization cone.
It has been revealed that mice lacking Juno on the surface of In some invertebrates and amphibians, there are certain areas of
their egg cells are infertile as their egg cells do not fuse with the oocyte membrane for the binding and fusion of the sperm.
normal sperm. This finding demonstrates the essential role of Seconds after fertilization, the membrane potential of the
Juno in the fertility of female mice (57). Other studies have oocyte undergoes a large depolarization via a massive influx
shown that a helical dimer of fragments of the N‑terminus of Na+ ions. The depolarization of the egg takes about a minute
domain of IZUMO is required for sperm‑oocyte fusion (58). to repolarize via K+ leakage. This is the fast block to poly­
IZUMO forms complexes with other proteins on the sperm spermy: sperm cannot fuse to a membrane that is not ‑70 mV
surface via its N‑terminal domain which forms dimers (56). (https://structureandfunction.wordpress.com/2013/02/28/the-
Therefore, IZUMO plays a key role in organizing and stabi- fast-and-slow-blocks-to-polyspermy-and-egg-activation/).
lizing a protein-like complex crucial for membrane fusion. CG distribution and the cortical reaction following calcium
Inoue  et al (56) found a protein, angiotensin-converting oscillation are crucial steps in the prevention of polyspermy.
enzyme 3 (ACE3) on the sperm acrosomal cap capable of Calcium released from the endoplasmic reticulum is depen-
interacting with IZUMO (59). dent on the quality and quantity of the mitochondria, which
Studies have revealed the rapid loss of Juno from the oocyte are markers of oocyte quality, since a low mitochondrial DNA
membrane soon after fertilization. This implies that Juno is copy number results in poor oocyte developmental compe-
essential for the process of fertilization, thus, being the basis tence (65,66).
for polyspermy block in mammals. One possible explana- PLCζ from spermatozoa is considered to be the responsible
tion for this process is that Juno is shed in vesicles following activator during this process (67); however, other factors may
fertilization, generating a zone of ‘decoy oocyte’ confined also be involved (5,68).
within the perivitelline space that could bind acrosome‑reacted The cortical reaction is a process through which cortical
sperm and therefore avoid supernumerary sperm entry (60). granules from the oocyte are released preventing polyspermy.
Although it has been validated that Juno protein as the first The fast block of polyspermy immediately prevents additional
cell surface receptor conserved in mammals, the interaction sperm getting attached to the oocyte. On the other hand, the
between IZUMO1 and Juno seems to be a necessary and an cortical reaction establishes a permanent barrier to sperm entry
essential adhesion step; however, its role in the gamete fusion and functions as the main part of the slow block of polyspermy
mechanism is not yet clear (57). in many animals. The cortical reaction is propagated over the
Two molecules, trypsin‑like acrosin and spermosin surface of the egg by a wave of Ca++.
proteases have been proposed to be involved in the first In this process, secretory vesicles located in the region
physical contact of the two gametes, suggesting that a below the plasma membrane of the oocyte, are fused with
 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 38: 979-986, 2016 983

Figure 1. The events taking place in fertilization. (A) Sperm preparation‑capacitation: Molecules (resact, speract) secreted from the oocyte, orient and stimulate
sperm (guanylate cyclase). (B) Acrosome reaction: release of hydrolytic enzymes. The sperm via SED1 protein is connected to ZP3. (C) Fusion of sperm with
plasma membrane of the oocyte: sperm pre‑acrosin binds to ZP2. Proteins of sperm IZUMO, ADAMs 1, ADAMs 2, ADAMs 3 and CRISP1 bind to receptors
on the oocyte (Juno, integrins, CD9, CD81). Other molecules identified playing role in gamete fusion are: Trypsin‑like acrosin, spermosin, SPAM1, HYAL5,
ACE3. (D) Cortical Reaction: Ca+2 release/wave of Ca+2 and formation of fertilization cone. Enzymes released by cortical granules, digest sperm receptors ZP2
and ZP3 (block of polyspermy). (E) Sperm chromatin decondensation to form male pronucleus: The oocyte nucleus completes the 2nd meiosis and eliminates
the 2nd polar body.

the oocyte plasma membrane. This results in the release of 4,5‑biphosphate (PIP2) in diacyglycerol (DAG) and inositol
contents of the cortical granules, modifying the extracellular 1,4,5‑triphosphate (IP3). PIP2 and DAG are second messen-
matrix so as to be impenetrable to other sperm. The cortical gers. IP3 binds to its receptors in the endoplasmic reticulum,
granules contain hydrolytic enzymes, such as proteases that at the entry point of the sperm. This results in the release of
clip perivitelline proteins, peroxidases that harden the vitelline Ca+2 from the endoplasmic reticulum at this point. Ca+2 ions
envelope and glycosaminoglycans that attract water into the bind to sensitive Ca+2 receptors in the endoplasmic reticulum
perivitelline space, causing it to expand and form the hyaline around the cortical granules. Ca+2 binding to these receptors
layer. Calcium ions from the cortical smooth endoplasmic results in the release of more Ca+2, which spreads like a wave in
reticulum are responsible for cortical granule rupture. It is the region (cortex) of the egg and causes the rupture of cortical
believed that this is provoked by the activation of a G-protein granules. DAG activates PKC which phosphorylates protein
in the plasma membrane of the oocyte. The sperm binds to exchange ions Na+ to H+. The protein that exchanges ions, is
ZP3 which is consistent with the G‑protein and becomes activated by Ca+2 and the result of this activation is the entrance
active. Bindin binds the sperm to the oocyte cell membrane, to Na+ and H+ output and an increase in pH from 6.8 to 7.3 in the
but another sperm factor activates a receptor, which in turn egg. This ‘wakes up’ the oocyte from the metabolic inertia (69).
activates the G‑protein. G‑proteins of the spermatozoon itself The last phase of oocyte activation is the resumption and
(activated when sperm was in contact with the mucous of the completion of meiosis. This leads to polar body extrusion,
egg shell with the initiation of the acrosome reaction) activate cleavage of the zygote and embryonic cell divisions. There
enzymes, which results in increased Ca+2 levels in the egg. are widespread changes in molecules, such as proteins and
In mammals, the cortical reaction modifies the zona pellu- RNAs that are not necessarily involved in meiosis resumption.
cida, leading to the block of polyspermy. Several enzymes are Changes in proteomes and their composition have been revealed,
released by the cortical granules, leading to the digestion of showing significant degradation of maternal proteins. This is
the sperm receptor glycoproteins ZP2 and ZP3, so that they due to protein degradation, phosphorylation, post‑translational
can no longer bind spermatozoon (https://en.wikipedia.org/ modifications and new translation of maternal RNAs (70).
wiki/Cortical_reaction). Cytoplasmic polyadenylation involves the elongation of the
It is interesting to know hows sperm factor causes the poly(A) tail after the export of mRNAs to the cytoplasm. It has
release of Ca+2. Activated G‑protein activates the enzyme, been observed and described in the oocytes and early embryos
PLC. The latter cleaves the lipid phosphotidylinositol of many animal species, from invertebrates to mammals, and
984 GEORGADAKI et al: MOLECULAR BASIS OF FERTILIZATION

is universally considered to be a regulatory mechanism for of sperm is the level of the lateral symmetry normally marked
protein expression from specific mRNAs. The mediators of with the first cleavage.
this process (cytoplasmic polyadenylation elements and their The gray crescent is a region of zygote in which early
binding proteins) have been described in detail and some new morphogenetic movements will begin to form the blastopore,
findings have been reviewed recently (71,72). which marks the beginning of the process of gastrulation in
The sperm nucleus undergoes a series of changes, including the early embryo. Following fertilization, a sequence of cell
chromatin decondensation and the formation of a new nuclear divisions results in the formation of the early embryo. The
envelope, to form the male pronucleus. The latter uses micro- zygote begins to divide mitotically (cleavage), and the resulting
tubules to migrate to the center of the cell, where it fuses with cells are termed blastomeres. As long as cleavage progresses,
the female pronucleus to form a diploid nucleus. Other sperm the cells proliferate and become progressively smaller, and the
organelles (e.g., mitochondria) persist during the early cleavage fetal size and general shape remains the same (73).
stages of the embryo and they may play a role in develop- The first cleavage begins as ‘a ring’ (cleavage furrow) around
ment (19). the zygote and is parallel to the polar axis (animal‑vegetal pole)
The beginning, condensation of chromatin seems to of the zygote and normal to the longitudinal axis (axis joining
happen with two histone phosphorylations of the sperm and the poles of the spindle) of the mitotic spindle. The ring consists
their exchange with histones of the oocyte. Chromatin is of actin‑myosin found in the cortical separating the zygote
condensed following interaction with histones H3 and H4, and inwardly. The myosin plays the role of ATPase and controls the
X and Y sperm-specific proteins. In the cytoplasm of the ovum actin. The level of the second cleavage is parallel to the polar
is the protein nycleoplasmin connected with histones H2A axis. At this stage, the embryo consists of four blastomeres,
and H2B. As nucleoplasmin has greater affinity for the extending from the animal to the vegetal pole. Cleavage is a
X and Y sperm proteins, the exchange of histones H2A/H2B with fractionating process, resulting in the formation of an eight
X and Y proteins occurs. This exchange allows the de‑concen- -cell stage and in a 16‑32 cell so-called morula (69).
tration and sharpness of the chromatin nucleus of the sperm.
The process of decompression begins from the periphery to the 5. Fertilization failure
center of the nucleus. When this process is finished, the vesicles
of the nuclear membrane of the sperm bind to vesicles from Some fertility issues may arise from failure in fertilization events,
the endoplasmic reticulum of the egg. The membrane of the in the molecular base, described in this review. Defects in the
pronucleus of the sperm is produced. While the pro nucleus of expression of receptors in the oocyte cell membrane (e.g., CD9),
the sperm is formed, the nucleus of the oocyte completes the destruction of the gamete fusion mechanism, insufficient or
second division of meiosis and eliminates the second polar body. abnormal variation of Ca2+ and failure of exocytosis of cortical
After entering the oocyte, the nucleus and the sperm centro- granules, inadequate completion of second meiotic division of
some rotate 180˚, so that the centrosome is positioned between the oocyte, defective sperm chromatin decondensation, defective
the nucleus of the sperm and ovum. The centrosome organizes pronucleus due to abnormal remodeling of chromatin (multiple
microtubules that attach and pull the pronucleus of the sperm pronuclei) or abnormal formation/function of the mitotic
and the egg. Both pronuclei migrate towards each other and they spindle, may account for infertility.
are directed towards the center of the cytoplasm of the ovum.
The membranes of the two pronuclei rupture and mixing of References
nuclear material is observed, resulting in the formation of the
diploid nucleus of the zygote. The fusion of the pronuclei occurs  1. Wassarman PM and Florman HM: Cellular mechanisms
in <1 h e.g., in Echinus (the meiotic division is finished before during mammalian fertilization. In: Handbook of Physiology:
Section 14‑Cell Physiology. Oxford University Press, New York,
ovulation). Approximately 12 h in mammals including most NY, pp885‑938, 1997.
eggs (e.g., human) is needed to complete the second division of  2. Yanagimachi R: The Physiology of Reproduction. Raven Press,
meiosis with the excretion of the second polar body (73). Fig. 1 New York, NY, pp189‑317, 1994.
 3. Jin M, Fujiwara E, Kakiuchi Y, Okabe M, Satouh Y, Baba SA,
illustrates the events taking place in fertilization. Chiba K and Hirohashi N: Most fertilizing mouse spermatozoa
begin their acrosome reaction before contact with the zona
4. Reorganization and partition of the zygote pellucida during in vitro fertilization. Proc Natl Acad Sci
USA 108: 4892‑4896, 2011.
 4. Tokuhiro K, Ikawa M, Benham AM and Okabe M: Protein
Once the sperm enters the oocyte, the cytoplasmic material is disulfide isomerase homolog PDILT is required for quality
reorganized. The cytoplasm of the oocyte contains cytoplasmic control of sperm membrane protein ADAM3 and male fertility
[corrected]. Proc Natl Acad Sci USA 109: 3850‑3855, 2012.
determinants that become distributed on different cells during  5. Okabe M: The cell biology of mammalian fertilization.
cleavage. The cytoplasmic determinants (e.g., Vg1 and bicoid) Development 140: 4471‑4479, 2013.
directly or indirectly activate or inactivate genes and control  6. Zuccotti M, Piccinelli A, Giorgi Rossi P, Garagna S and
Redi CA: Chromatin organization during mouse oocyte growth.
events of differentiation in an early embryo. The point through Mol Reprod Dev 41: 479‑485, 1995.
which sperm has penetrated the oocyte marks the bilateral  7. Nelson L: Enzymes associated with sperm cell function. In:
symmetry of the future adult organism. Following fertilization, Biology of Fertilization. Metz CB and Monroy A (eds). London
Academic Press, Orlando, FL, pp215‑131, 1985.
cortical rotation occurs. Tubulin microtubules are organized  8. Ward GE, Brokaw CJ, Garbers DL and Vacquier VD: Chemotaxis
as train tracks between the cortical and the endoplasm in the of Arbacia punctulata spermatozoa to resact, a peptide from the
vegetal pole. The result is the appearance of a gray area, the egg jelly layer. J Cell Biol 101: 2324‑2329, 1985.
 9. Shimomura H, Dangott LJ and Garbers DL: Covalent coupling
gray crescent, which marks the future dorsal side of the embryo. of a resact analogue to guanylate cyclase. J Biol Chem 261:
Between the midpoint of the gray crescent and the entry point 15778‑15782, 1986.
 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 38: 979-986, 2016 985

10. Cook SP and Babcock DF: Selective modulation by cGMP of the 34. Cho C, Bunch DO, Faure JE, Goulding EH, Eddy EM,
K+ channel activated by speract. J Biol Chem 268: 22402‑22407, Primakoff P and Myles DG: Fertilization defects in sperm from
1993. mice lacking fertilin β. Science 281: 1857‑1859, 1998.
11. Cardullo RA, Herrick SB, Peterson MJ and Dangott LJ: Speract 35. Shamsadin R, Adham IM, Nayernia K, Heinlein UAO,
receptors are localized on sea urchin sperm flagella using a fluo- Oberwinkler H and Engel W: Male mice deficient for germ‑cell
rescent peptide analog. Dev Biol 162: 600‑607, 1994. cyritestin are infertile. Biol Reprod 61: 1445‑1451, 1999.
12. Cook SP, Brokaw CJ, Muller CH and Babcock DF: Sperm 36. Nishimura H, Cho C, Branciforte DR, Myles DG and Primakoff P:
chemotaxis: Egg peptides control cytosolic calcium to regulate Analysis of loss of adhesive function in sperm lacking cyritestin
flagellar responses. Dev Biol 165: 10‑19, 1994. or fertilin beta. Dev Biol 233: 204‑213, 2001.
13. Talbot P and Franklin LE: Surface modification of guinea pig sperm 37. Almeida EA, Huovila AP, Sutherland AE, Stephens LE,
during in vitro capacitation: An assessment using lectin‑induced Calarco PG, Shaw LM, Mercurio AM, Sonnenberg A,
agglutination of living sperm. J Exp Zool 203: 1‑14, 1978. Primakoff P, Myles DG and White JM: Mouse egg integrin α 6
14. Lishko PV, Kirichok Y, Ren D, Navarro B, Chung JJ and β 1 functions as a sperm receptor. Cell 81: 1095‑1104, 1995.
Clapham DE: The control of male fertility by spermatozoan ion 38. Chen MS, Tung KS, Coonrod SA, Takahashi Y, Bigler D,
channels. Annu Rev Physiol 74: 453‑475, 2012. Chang A, Yamashita Y, Kincade PW, Herr JC and White JM:
15. Evans JP and Florman HM: The state of the union: the cell Role of the integrin‑associated protein CD9 in binding between
biology of fertilization. Nat Cell Biol 4: s57‑s63, 2002. sperm ADAM 2 and the egg integrin α 6β1: Implications for
16. Visconti PE, Ning X, Fornés MW, Alvarez JG, Stein P, murine fertilization. Proc Natl Acad Sci USA 96: 11830‑11835,
Connors SA and Kopf GS: Cholesterol efflux‑mediated signal 1999.
transduction in mammalian sperm: Cholesterol release signals 39. Chen H and Sampson NS: Mediation of sperm‑egg fusion:
an increase in protein tyrosine phosphorylation during mouse Evidence that mouse egg α6β1 integrin is the receptor for sperm
sperm capacitation. Dev Biol 214: 429‑443, 1999. fertilinbeta. Chem Biol 6: 1‑10, 1999.
17. Contreras HR and Llanos MN: Detection of progesterone 40. Eto K, Huet C, Ta r u i T, Kupr iya nov S, Liu H Z,
receptors in human spermatozoa and their correlation with Puzon‑McLaughlin W, Zhang XP, Sheppard D, Engvall E and
morphological and functional properties. Int J Androl 24: Takada Y: Functional classification of ADAMs based on a
246‑252, 2001. conserved motif for binding to integrin α 9β 1: Implications for
18. Goodson SG, Qiu Y, Sutton KA, Xie G, Jia W and O'Brien DA: sperm‑egg binding and other cell interactions. J Biol Chem 277:
Metabolic substrates exhibit differential effects on functional 17804‑17810, 2002.
parameters of mouse sperm capacitation. Biol Reprod 87: 75, 41. Zhu X and Evans JP: Analysis of the roles of RGD‑binding
2012. integrins, α(4)/α(9) integrins, α(6) integrins, and CD9 in the
19. Blobel CP, Myles DG, Primakoff P and White JM: Proteolytic interaction of the fertilin β (ADAM2) disintegrin domain with
processing of a protein involved in sperm‑egg fusion correlates the mouse egg membrane. Biol Reprod 66: 1193‑1202, 2002.
with acquisition of fertilization competence. J Cell Biol 111: 42. Mizuno M, Harris CL, Johnson PM and Morgan BP: Rat
69‑78, 1990. membrane cofactor protein (MCP; CD46) is expressed only
20. Primakoff P, Hyatt H and Tredick‑Kline J: Identification and in the acrosome of developing and mature spermatozoa and
purification of a sperm surface protein with a potential role in mediates binding to immobilized activated C3. Biol Reprod 71:
sperm‑egg membrane fusion. J Cell Biol 104: 141‑149, 1987. 1374‑1383, 2004.
21. López‑Torres AS and Chirinos M: Modulation of human sperm 43. Lozahic S, Christiansen D, Manié S, Gerlier D, Billard M,
capacitation by progesterone, estradiol, and luteinizing hormone. Boucheix C and Rubinstein E: CD46 (membrane cofactor
Reprod Sci 12: 1‑9, 2016. protein) associates with multiple beta1 integrins and tetraspans.
22. Roldan ER, Murase T and Shi QX: Exocytosis in spermatozoa Eur J Immunol 30: 900‑907, 2000.
in response to progesterone and zona pellucida. Science 266: 44. Johnson PM, Clift LE, Andrlíková P, Jursová M, Flanagan BF,
1578‑1581, 1994. Cummerson JA, Stopka P and Dvorakova‑Hortova K:
23. Wu JT, Chiang KC and Cheng FP: Expression of progesterone Rapid sperm acrosome reaction in the absence of acrosomal
receptor(s) during capacitation and incidence of acrosome reaction CD46 expression in promiscuous field mice (Apodemus).
induced by progesterone and zona proteins in boar spermatozoa. Reproduction 134: 739‑747, 2007.
Anim Reprod Sci 93: 34-45, 2006.
24. de Lamirande E, Harakat A and Gagnon C: Human sperm 45. Stein KK, Primakoff P and Myles D: Sperm‑egg fusion: Events
capacitation induced by biological fluids and progesterone, but at the plasma membrane. J Cell Sci 117: 6269‑6274, 2004.
not by NADH or NADPH, is associated with the production of 46. Jégou A, Ziyyat A, Barraud‑Lange V, Perez E, Wolf JP, Pincet F
superoxide anion. J Androl 19: 215-225, 1998. and Gourier C: CD9 tetraspanin generates fusion competent sites
25. Ickowicz D, Finkelstein M and Breitbart H: Mechanism of sperm on the egg membrane for mammalian fertilization. Proc Natl
capacitation and the acrosome reaction: Role of protein kinases. Acad Sci USA 108: 10946‑10951, 2011.
Asian J Androl 14: 816‑821, 2012. 47. Kaji K, Oda S, Shikano T, Ohnuki T, Uematsu Y, Sakagami J,
26. Chang H and Suarez SS: Two distinct Ca(2+) signaling pathways Tada N, Miyazaki S and Kudo A: The gamete fusion process is
modulate sperm flagellar beating patterns in mice. Biol defective in eggs of Cd9‑deficient mice. Nat Genet 24: 279‑282,
Reprod 85: 296‑305, 2011. 2000.
27. Brenker C, Goodwin N, Weyand I, Kashikar ND, Naruse M, 48. Le Naour F, Rubinstein E, Jasmin C, Prenant M and Boucheix C:
Krähling M, Müller A, Kaupp UB and Strünker T: The Severely reduced female fertility in CD9‑deficient mice.
CatSper channel: A polymodal chemosensor in human sperm. Science 287: 319‑321, 2000.
EMBO J 31: 1654‑1665, 2012. 49. Miyado K, Yamada G, Yamada S, Hasuwa H, Nakamura Y, Ryu F,
28. Ebensperger C and Barros C: Changes at the hamster oocyte Suzuki K, Kosai K, Inoue K, Ogura A, et al: Requirement of
surface from the germinal vesicle stage to ovulation. Gamete CD9 on the egg plasma membrane for fertilization. Science 287:
Res 9: 387‑397, 1984. 321‑324, 2000.
29. Johnson MH, Eager D, Muggleton‑Harris A and Grave HM: 50. Horváth G, Serru V, Clay D, Billard M, Boucheix C and
Mosaicism in organisation concanavalin A receptors on surface Rubinstein E: CD19 is linked to the integrin‑associated tetraspans
membrane of mouse egg. Nature 257: 321‑322, 1975. CD9, CD81, and CD82. J Biol Chem 273: 30537‑30543, 1998.
30. Evans JP: The molecular basis of sperm‑oocyte membrane inter- 51. Takahashi Y, Bigler D, Ito Y and White JM: Sequence‑specific
actions during mammalian fertilization. Hum Reprod Update 8: interaction between the disintegrin domain of mouse ADAM 3
297‑311, 2002. and murine eggs: Role of beta1 integrin‑associated proteins CD9,
31. Huang TTF Jr and Yanagimachi R: Inner acrosomal membrane CD81, and CD98. Mol Biol Cell 12: 809‑820, 2001.
of mammalian spermatozoa: Its properties and possible functions 52. Rubinstein E, Ziyyat A, Prenant M, Wrobel E, Wolf JP, Levy S,
in fertilization. Am J Anat 174: 249‑268, 1985. Le Naour F and Boucheix C: Reduced fertility of female mice
32. Yanagimachi R and Noda YD: Physiological changes in the lacking CD81. Dev Biol 290: 351‑358, 2006.
postnuclear cap region of mammalian spermatozoa: A necessary 53. Ernesto JI, Weigel Muñoz M, Battistone MA, Vasen G,
preliminary to the membrane fusion between sperm and egg M a r t í n e z ‑ L ó p e z P, O r t a G, F i g u e i r a s ‑ F i e r r o D,
cells. J Ultrastruct Res 31: 486‑493, 1970. De la Vega‑Beltran JL, Moreno IA, Guidobaldi HA, et al: CRISP1
33. Bedford JM, Moore HDM and Franklin LE: Significance of as a novel CatSper regulator that modulates sperm motility and
the equatorial segment of the acrosome of the spermatozoon in orientation during fertilization. J Cell Biol 210: 1213‑1224,
eutherian mammals. Exp Cell Res 119: 119‑126, 1979. 2015.
986 GEORGADAKI et al: MOLECULAR BASIS OF FERTILIZATION

54. Armon L, Ben‑Ami I, Ron‑El R and Eisenbach M: Human 64. Zumoffen CM, Gil R, Caille AM, Morente C, Munuce MJ and
oocyte‑derived sperm chemoattractant is a hydrophobic Ghersevich SA: A protein isolated from human oviductal tissue
molecule associated with a carrier protein. Fertil Steril 102: in vitro secretion, identified as human lactoferrin, interacts with
885‑890, 2014. spermatozoa and oocytes and modulates gamete interaction.
55. Blasco V, Prados N, Carranza F, González‑Ravina C, Pellicer A Hum Reprod 28: 1297‑1308, 2013.
and Fernández‑Sánchez M: Influence of follicle rupture and 65. Zumoffen CM, Massa E, Caille AM, Munuce MJ and
uterine contractions on intrauterine insemination outcome: A Ghersevich SA: Effects of lactoferrin, a protein present in the
new predictive model. Fertil Steril 102: 1034‑1040, 2014. female reproductive tract, on parameters of human sperm capaci-
56. Inoue N, Ikawa M, Isotani A and Okabe M: The immunoglobulin tation and gamete interaction. Andrology 3: 1068‑1075, 2015.
superfamily protein Izumo is required for sperm to fuse with 66. Tosti E and Ménézo Y: Gamete activation: Basic knowledge and
eggs. Nature 434: 234‑238, 2005. clinical applications. Hum Reprod Update 22: 420‑439, 2016.
57. Bianchi E, Doe B, Goulding D and Wright GJ: Juno is the egg 67. Nomikos M, Swann K and Lai FA: Starting a new life: sperm
Izumo receptor and is essential for mammalian fertilization. PLC‑zeta mobilizes the Ca2+ signal that induces egg activation
Nature 508: 483‑487, 2014. and embryo development: an essential phospholipase C with
58. Inoue N, Hamada D, Kamikubo H, Hirata K, Kataoka M, implications for male infertility. BioEssays 34: 126‑134, 2012.
Yamamoto M, Ikawa M, Okabe M and Hagihara Y: Molecular 68. Harada Y, Kawazoe M, Eto Y, Ueno S and Iwao Y: The Ca2+
dissection of IZUMO1, a sperm protein essential for sperm‑egg increase by the sperm factor in physiologically polyspermic newt
fusion. Development 140: 3221‑3229, 2013. fertilization: Its signaling mechanism in egg cytoplasm and the
59. Inoue N, Kasahara T, Ikawa M and Okabe M: Identification and species‑specificity. Dev Biol 351: 266‑276, 2011.
disruption of sperm‑specific angiotensin converting enzyme‑3 69. Abbott AL and Ducibella T: Calcium and the control of mammalian
(ACE3) in mouse. PLoS One 5: e10301, 2010. cortical granule exocytosis. Front Biosci 6: D792‑D806, 2001.
60. Bianchi E and Wright GJ: Izumo meets Juno: Preventing poly- 70. Krauchunas AR and Wolfner MF: Molecular changes during egg
spermy in fertilization. Cell Cycle 13: 2019‑2020, 2014. activation. Curr Top Dev Biol 102: 267‑292, 2013.
61. Sawada H, Mino M and Akasaka M: Sperm proteases and 71. Charlesworth A, Meijer HA and de Moor CH: Specificity factors
extracellular ubiquitin‑proteasome system involved in fertil- in cytoplasmic polyadenylation. Wiley Interdiscip Rev RNA 4:
ization of ascidians and sea urchins. Adv Exp Med Biol 759: 437‑461, 2013.
1‑11, 2014. 72. Sousa Martins JP, Liu X, Oke A, Arora R, Franciosi F, Viville S,
62. Zhou C, Kang W and Baba T: Functional characterization of Laird DJ, Fung JC and Conti M: DAZL and CPEB1 regulate
double‑knockout mouse sperm lacking SPAM1 and ACR or mRNA translation synergistically during oocyte maturation. J
SPAM1 and PRSS21 in fertilization. J Reprod Dev 58: 330‑337, Cell Sci 129: 1271‑1282, 2016.
2012. 73. Gilbert SF (ed): Developmental Biology. 10th edition. Sinauer
63. Yoon S, Chang KT, Cho H, Moon J, Kim JS, Min SH, Koo DB, Associates. Sunderlan, 2013.
Lee SR, Kim SH, Park KE, et al: Characterization of pig sperm
hyaluronidase and improvement of the digestibility of cumulus
cell mass by recombinant pSPAM1 hyaluronidase in an in vitro
fertilization assay. Anim Reprod Sci 150: 107‑114, 2014.