Gametogenesis, Fertilization and Early Embryogenesis in Mammals with Special Reference to Goat: A Review
Embryo in vitro Production (IVP) and associated
Assisted Reproductive Technologies (ARTs) in goat, e.g., estrus synchronization
and superovulation, Laparoscopic Ovum Pick-up (LOPU), in vitro
Maturation (IVM), in vitro Fertilization (IVF), Intracytoplasmic
Sperm Injection (ICSI) and in vitro Culture (IVC) attained significant
attention in the recent years. However, for the success in any IVP protocols
in goat sound knowledge of physiology of gametogenesis, fertilization
and early embryogenesis in vivo is crucial because in vivo
information form the basis and guide for any in vitro experiment.
Unlike human, laboratory animals, cattle and sheep, fewer studies have
been conducted in gametogenesis, fertilization and early embryo development
in goat. Data for sheep and cattle are mostly used as a basis for goat
IVP studies. Therefore, the current study is intended to review gametogenesis,
sperm oocyte interaction, fertilization and early embryogenetic process
in mammals with special reference to goat.
In vitro Embryo Production (IVEP) or in vitro Production
(IVP) technique is currently in the central focus among all the Assisted
Reproductive Technologies (ARTs) in human as well as in animals. Therefore,
the physiology of normal fertilization should be considered as a must
for the development of any IVEP or IVP protocols especially for in
vitro Fertilization (IVF) or Intracytoplasmic Sperm Injection (ICSI)
techniques. Gametogenesis, fertilization and early embryogenesis or pre-implantation
embryo development are crucial periods for normal development of an embryo
afterwards. Until now researches in mammalian gametogenesis, fertilization
and early embryogenesis mainly based on the laboratory animals and human
(Tulsiani et al., 1997; Kupker et al., 1998; Elder and Dale,
2000). Compared with laboratory and farm animals like cattle, sheep and
pig, studies in goat are scarce (Hafez and Hafez, 2000a; Miyano and Hirao,
2003). Therefore, the current paper will briefly review some aspects of
gametogenesis, fertilization and early embryogenesis in mammals with special
reference to goat.
Genetically and functionally competent gametes are a prerequisite for
normal fertilization and early embryo development. The first phase in
the sexual reproduction of an organism is gametogenesis, a process of
formation of gametes from the germ cells in the testes and ovaries. This
process is termed as spermatogenesis in the male and oogenesis in the
female. It is the fundamental biological process in both the sexes and
the key event of gametogenesis is the halving of the number of chromosomes
to produce haploid germ cells (sperm and oocyte) through meiosis. Thus,
in goat, where the chromosome number of somatic cells is 60, each sperm
and each oocyte has only 30 chromosomes. However, until this point spermatogenesis
and oogenesis resume their similarity. After this, in the male, each primary
spermatocyte divides meiotically and produces four spermatids, each destined
to become a functional sperm. In the female, on the other hand, of the
four cells produced from each primary oocyte only one finally becomes
a functional oocyte. A schematic diagram of gametogenesis in mammals has
been illustrated in Fig. 1.
|| Schematic diagram of gametogenesis in mammals. Modified
from Hickman et al. (1986)
In mammals, the male and female gametes originate from the embryonic
yolk sac. The gametes initially colonize in the primitive gonadal ridge
at the early stage of pregnancy by migration through the developing mesentery
of the embryo, where germ cells associate with somatic cells; it continues
with their multiplication, growth and maturation. Finally, migrate to
the pelvic and inguinal regions to form the ovary or testis, respectively
and ends at fertilization. Mammalian spermatogenesis and oogenesis are
briefly described below:
Spermatogenesis: Competent sperm are required for the successful
contribution of the paternal genotype to embryo development. The process
of spermatogenesis results in the formation of the haploid male gamete
required for fertilization of an oocyte. Spermatogenesis is a continual
and complex process that involves three major steps: (a) proliferation-
multiplication of spermatogonia by the process of mitosis (spermatocytogenesis);
(b) meiosis (spermiogenesis)- meiotic divisions whereby the chromosome
number is reduced from diploid to haploid and (c) differentiation- transformation
of the round spermatid into the complex structure of the spermatozoon
(reviewed in Barth and Oko, 1989; De Kretser et al., 1998; Elder
and Dale, 2000). Spermatogenesis in buck and other male mammals begins
at puberty through the proliferation of interphase germ cells, continues
throughout adult life and takes place inside the seminiferous tubules
of the testes. The age of puberty for male goat or buck ranged between
4 and 6 months (Jainudeen et al., 2000). The walls of the seminiferous
tubules contain the differentiating sex cells arranged in a stratified
layer, 5 to 8 cells deep. The outermost layers contain spermatogonia,
which have increased in number by spemtocytogenesis. The spemtocytogenesis
begins with mitosis of the diploid A spematogonia in the basal compartment
of the Sertoli cells. The A spematogonia differentiate into B spermatogonia
which enter their final mitotic division prior to their entrance into
the pre-leptotene phase of first meiosis (meiosis 1). Meiosis occurs in
the adluminal compartment of the Sertoli cell and results in the formation
of primary spermatocytes. Condensation of chromosomes occurs during the
leptotene phase followed by the zygotene phase. The long pachytene phase
involves the crossing over of chromosomal material and is most susceptible
to damage. Meiosis progresses through the diplotene phase, diakinesis,
Metaphase I (MI) and anaphase, finally resulting in secondary spermatocytes
(2c, 1n). Each secondary spermatocyte then enters the second meiotic division
(meiosis 2), without the intervention of a resting period, resulting in
spermatids with a haploid number of chromosomes and DNA content (1c, 1n),
required for fertilization. The round-shaped spermatids proceed through
spermiogenesis and metamorphically changed into highly specialized motile
cells. Spermiogenesis consists of the Golgi phase, the acrosomal cap phase,
the acrosomal phase and the complex maturation stage involves development
of the sperm tail (reviewed by Barth and Oko, 1989). Deviations from the
process of normal spermatogenesis result in abnormal morphology or dysfunction
of the sperm cell. The progression from spermatogonia to mature spermatozoa
in mammals require approximately 60 to 70 days, with at least three mitotic
and two meiotic divisions during spermatogenesis (Kupker et al.,
1998). Finally, the newly formed sperm are released into the lumen of
the seminiferous tubules. These sperm are immotile and still immature
in terms of fertilizing capability. Therefore, to acquire motility as
well as maturation they need to pass down the epididymis to the ejaculatory
duct, which is known as epididymal maturation (Elder and Dale, 2000).
During epididymal maturation, the sperm shed the cytoplasmic droplet,
undergo modifications in the protein, carbohydrate and glycoprotein composition
of the plasma membrane and acquire a net negative charge (Yanagimachi,
1994; Harrison, 1996). Sperm motility and capacitation are suppressed
during storage in the cauda epididymis, which is characterized by low
pH, low Ca2+, low Na+ and increased K+
(Dalvit et al., 1995; Jones and Murdoch, 1996). Disruptions in
the epididymal environment may result in abnormalities of sperm function.
Immediately after ejaculation, the sperm are incapable of fertilizing
the oocyte and, therefore, to acquire fertilization potential, they need
to undergo functional changes (or capacitation), which occur inside the
female reproductive tract.
Although in comparison with the oocytes, the spermatozoa are very small,
however, they are essentially very long and compact cells with a few highly
specialized cytoplasmic structures, the flagellum for motility and the
acrosome, which is instrumental in sperm-oocyte binding and fusion. Morphologically
and functionally, a goat spermatozoon is composed of four regions: (a)
the head containing nucleus and the acrosome, (b) the neck containing
centrioles, (c) the middle piece containing mitochondria and d) the tail
piece or flagellum. A goat spermatozoon is approximately 60 μm long
of which head and tail piece are 7.69 and 52 μm long, respectively
(Gravance et al., 1995). Frozen-thawed goat spermatozoa from proven
quality Jermasia buck (Jermasia is a synthetic breed developed by the
University of Malaya) have been shown in Fig. 2a, b.
Oogenesis: The maternal contribution to the development of the
embryo is determined during formation and maturation of the female gamete,
the oocyte. The ability of the oocyte to achieve sperm-oocyte fusion is
acquired early in oogenesis or the process of oocyte formation. The oogenetic
products synthesized during oocyte growth must also be sufficient to support
embryonic development from fertilization until the activation of the embryonic
genome (Olszanska and Borgul, 1993). Ultimately, the nuclear and ooplasmic
maturity of the oocyte influences the success of fertilization and embryo
development. In mammals, oogenesis commences during early fetal development,
stops at birth and continues during puberty throughout the reproductive
life of the female. After continuation of meiosis, the oogenesis process
until completion is very fast. Oogenesis in mammals includes seven steps:
(a) generation of primordial germ cells (PGCs), (b) migration of PGCs
to the prospective gonads, (c) colonization of the gonads by PGCs, (d)
differentiation of PGCs to oogonia, (e) proliferation of oogonia, (f)
initiation of meiosis and (g) arrest at the diplotene stage of first meiotic
prophase or prophase 1 (reviewed in Van den Hurk and Zhao, 2005).
Oogonia are the early germ cells in the ovary, which increase in number
by mitosis. Oogonial multiplication begins during early fetal development
and ends months to years later in the sexually mature adult (Picton et
al., 1998). Once mitosis ceases, the oogonia then grow in size and
enter the prophase of the first meiotic division at approximately day
55 of gestation in the ewe (McNatty et al., 1995) and are then
referred to as primary oocytes (Wassarman and Albertini, 1994). Each oogonium
or primary oocyte contains the diploid number of chromosome. The primary
oocyte which is transformed from each oogonium is a cell which becomes
enclosed in a follicle, known as primordial follicle. In goat, sheep and
cow, large population (approximately 100,000) of primordial or pre-antral
follicles with meiotically incompetent oocytes are present in the ovaries
(Miyano, 2003; Miyano and Hirao, 2003; Zhou and Zhang, 2006). Most of
them are lost at various stages of development owing to atresia and only
a very minority of oocytes becomes available for ovulation. In contrast,
Ariyaratna and Gunawardana (1997) found in their histological study that
one pair of ovaries of Batu goat (a Sri Lankan goat breed) aged between
18 to 36 months contained 35,092 primordial follicles (which are 90% of
total ovarian follicle population), 10.67 normal and 20.42 atretic vesicular
(antral) follicles (1-6 mm diameter). At birth, all oocytes from growing
and dominant follicles are arrested at the diplotene stage of prophase
1 (Van den Hurk and Zhao, 2005). This dictyate stage is characterized
by the enclosure of the chromosomes within the large nucleus, also known
as the Germinal Vesicle (GV) (Elder and Dale, 2000). The oocytes remain
in the arrested state until a few hours before ovulation. Surprisingly,
the oocytes may stay at this arrested stage for a longer period of time
depending on the species, waiting for the signal to resume growth and
subsequent development occurs at puberty. The age of puberty for female
goat or doe is ranged between 5 and 7 months (Jainudeen et al.,
2000). The reason for storing the oocytes in this remarkable frozen meiotic
state is unknown (Johnson and Everitt, 1980).
||Microphotographs of goat sperm. (a) Frozen-thawed goat
sperm from Jermasia buck of proven quality, (b) a single motile normal
goat sperm before being immobilized for ICSI procedure. Scale bar
represents 10 μm
Oocyte growth and development: The growth and development of an
oocyte occurs inside an ovarian follicle and oocyte undergoes a progressive
series of morphological modifications as it grows and proceeds through
the different stages of development (Eppig et al., 1994). Although
data are lacking for doe, Ariyaratna and Gunawardana (1997) indicated
from their study that follicular morphology and activity are similar in
does and ewes. In the ewe, primordial, primary and secondary follicles,
respectively, appear in the fetal ovary at days 75, 100 and 120 (McNatty
et al., 1995). Once a primordial follicle oocyte is activated to
grow, it embarks on a complex journey that involves numerous molecular
and morphological changes to both the oocyte and the follicle. The modifications
are carefully orchestrated and require sensitive communication between
the oocyte and surrounding Granulosa Cells or GCs (Fair, 2003). These
structural rearrangements facilitate the increasing energy and nucleic
acid synthesis requirements of the developing oocyte and are a prerequisite
to the oocyte achieving meiotic competence and embryo developmental potential.
The first sign of morphological change when the oocyte begins to grow
is turning of the flat GCs to cuboidal which is known as primary follicle.
After completion of the morphological change, the GCs proliferate actively,
which cause the follicles to develop and increase in size. Through a series
of mitotic division of GCs, unilaminar primary follicles are converted
to multilaminar secondary follicles, followed by the antral or tertiary
follicles (Miyano and Hirao, 2003). The antral follicle is a highly complex
unit consisting of several layers of GC surrounding a fluid-filled cavity
or antrum` in which the oocyte surrounded by somatic cells is bathed.
In the doe, antrum formation began when the GCs are about six cell layers
in thickness and the Zona Pellucida (ZP) is visible at this stage (Ariyaratna
and Gunawardana, 1997). The fluid found in the antrum` is known
as Follicular Fluid (FF). During this growth phase there is a major increase
in ooplasmic organelles. The follicle provides a microenvironment for
oocyte growth, development and is responsible for the production of hormones.
The walls of mature preovulatory follicles consist of membrana granulosa
(mural granulosa), theca interna and theca externa. The GCs are cells
of epithelial origin essential for the growth and survival of the oocyte.
The GCs consist of the corona radiata; Cumulus Cells (CCs), membrana granulosa
and antral granulosa cells. The CCs surround the oocyte, which nourish
the oocyte, are involved in oocyte growth, maturation (Buccione et
al., 1990) and participate in the formation of the ZP (made of a translucent
acellular layer of glycoprotein). In addition, these cells have also been
implicated in the modulation or generation of oocyte maturation inhibitors
(Tsafriri et al., 1982; Eppig and Downs, 1984). The CCs in close
contact with the oocyte are known as corona radiata. They are in close
contact with the oocyte through ooplasmic extensions or processes across
the ZP (De Loos et al., 1991), which are known as gap junctions.
The heterologous gap junctions provide the basis for extensive network
of intracellular communication among GCs. Normally CCs or corona cells
surrounding the goat oocyte shed ≥30 h after ovulation (Harper, 1982).
Oocyte maturation: As mentioned earlier that oocytes are arrested
at the diplotene stage of the prophase 1 at birth, they resume meiosis
after a long quiescent phase at puberty which involve sequential sub-cellular
and molecular transformations by various components of the follicle. During
postnatal life, starting from puberty, ovarian follicles continue to grow,
mature and either ovulate or regress. Follicles are recruited continuously
until the original store is exhausted. Reinitiation of meiosis in the
fully-grown oocyte is the first sign of oocyte maturation, which involves
condensation of interphase chromatin, breakdown of nuclear membrane (germinal
vesicle breakdown: GVBD), spindle formation and chromosome segregation.
In vivo, resumption of meiosis is initiated by a preovulatory LH
surge and only occurs in fully grown, meiotically competent oocytes from
dominant follicles. Small oocytes in primordial and primary follicles
have no ability to resume meiosis. Oocytes acquire the competence to resume
meiosis when their size exceeds 80% of their final diameter; they then
become gradually competent to progress to metaphase 2 (MII) as the diameter
increases to over 90% of the maximum (Miyano and Hirao, 2003). The diameter
of mature goat oocytes excluding ZP (ooplasm) ranged from 119-146 μm
(De Smedt et al., 1994; Crozet et al., 2000). Diameter of
mature oocyte in different animals and human is presented in Table
1. During this follicle and oocyte growth phase, oocytes not only
acquire competency to resume meiosis, but also acquire ooplasmic maturity,
also known as oocyte capacitation, both of which are required to ensure
normal fertilization and embryo development (Gosden et al., 1997;
Hyttel et al., 1997). From in vitro studies, it is found
that goat oocytes acquired the ability to initiate meiotic resumption
in early antral follicles of 0.5 to 0.8 mm in diameter and to reach MI
in follicles of 1.0 to 1.8 mm in diameter (De Smedt et al., 1994).
Although 86% of goat oocytes from follicles larger than 2 mm progress
to MII (De Smedt et al., 1992), only a small proportion of them
can support embryonic development (Crozet et al., 1993). In cattle,
oocytes originating from follicles larger than 6 mm in diameter yield
a significantly higher percentage of blastocyst than the smaller follicles
(Lonergan et al., 1994). In an in vitro study, a significantly
higher oocyte maturation, morula and blastocyst development rates were
achieved with goat oocytes originating from larger than 5 mm follicles
compared with medium (>3-5) and smaller (2-3) follicles (Crozet et
al., 1995). To reach the maturity, goat oocytes (ooplasm) grow from
29.6 μm in primordial follicles to 119 to 146 μm (De Smedt et
al., 1994; Crozet et al., 2000) in antral follicles (>2
mm in diameter). The ZP of a goat oocyte from antral follicle bigger than
2 mm is about 3.2 μm thick (Ariyaratna and Gunawardana, 1997). The
growth of goat oocyte in relation to follicular growth in the ovary as
described by Ariyaratna and Gunawardana (1997) has been presented in Table
Before and at the time of LH surge, the oocyte is surrounded by a compact
CCs investment. The oocyte undergoes a series of changes in its nucleus,
ooplasm and organization of the plasma membrane (oolemma) during the period
between the LH surge and ovulation, which is known as oocyte maturation.
Completion of the meiosis 1 takes place when oocytes have undergone extensive
growth in cellular interaction with GCs and theca cells. The oocyte undergoes
asymmetric cytokinesis and extrudes the first polar body (PB-1) containing
a haploid chromosome complement (Kupker et al., 1998). Immediately
after the meiosis 1 is completed, the meiosis 2 is initiated and the oocytes
are again arrested at MII stage until fertilization, when an activation
stimulus provided by sperm penetration triggers the completion of the
meiotic cycle and initiates embryonic development. Ooplasmic maturation
is required to acquire to the conditions to block polyspermy in case of
fertilization, to decondense penetrated spermatozoon and to form pronucleus
(PN). It includes redistribution of cell organelles, migration of mitochondria
to perinuclear position and accumulation of granules along the oolemma
(Van den Hurk and Zhao, 2005). The endpoint of this, in vivo, is
the ovulation and release from the follicle of a MII oocyte with potential
to support normal embryonic development (Elder and Dale, 2000). However,
the doe ovulates two to three mature oocytes in each estrous cycle (Jainudeen
et al., 2000). Compared with human or mouse, goat oocyte is dark
in opacity (Betteridge, 2003) due to very dense ooplasm consisted of concentrated
lipid materials (Keskintepe et al., 1997). The ooplasmic opacity
in different animals and human has been presented in Table
1. A developmentally competent immature goat Cumulus-Oocyte Complex
(COC) and mature goat oocyte have been illustrated, respectively, in Fig.
|| Mature oocyte size and opacity in different animals
|Oocyte diameter without ZP (ooplasmic diameter) was
calculated by deducting average ZP thickness from oocyte diameter
with ZP. aBelongs to Batu breed (a Srilankan goat breed)
|| Growth of goat (Batu breed) oocyte (Mean ± SD)
in relation to follicular growth in the ovary
|In each column, values with different superscript letters
are different (p<0.05)
||Developmentally competent (a) immature and (b) mature
goat oocytes recovered through Laparoscopic Ovum Pick-Up (LOPU) technique
from estrus synchronized and superstimulated doe. Scale bar represents
ACTIVATION OF SPERM AND SPERM-OOCYTE INTERACTION
Fertilization encompasses a series of different steps which have to be
performed in a well orchestrated way to create a new individual. These
include sperm capacitation, sperm binding and penetration of the ZP, traversing
the perivitelline space (PVS), binding and fusion with oolemma, activation
of the oocytes and decondensation of the sperm head to form the Male PN
(MPN). In mammals, fertilization process is internal and the male gametes
(spermatozoa) must be introduced into the female reproductive tract at
coitus or artificially. As already mentioned, when the ejaculated spermatozoa
are deposited into the female reproductive tract at that time they have
no fertilizing capability. Therefore, the spermatozoa need to gain this
ability, or in other word, need to be activated or made in ready state
first before they can interact with oocyte to ensure fertilization success.
The known physiological events occurring during sperm activation and sperm-oocyte
interaction have been reviewed mainly based on mice models (Bedford, 1982;
Yanagimachi, 1994; Tulsiani et al., 1997; Bedford, 1998).
Activation of spermatozoa: In mammalian species, activation of
spermatozoa follows several physiological and structural changes upon
exposure to environmental signals in the female reproductive tract before
they interact with the oocytes towards a successful fertilization. These
include various alterations in the plasma membrane and intracellular components
and changes in motility pattern and metabolism of the spermatozoa. Two
main processes, namely capacitation and acrosome reaction, must occur
to activate the spermatozoa before the sperm interact with the oocyte.
Capacitation: The spermatozoa must reside a minimum period in
the female reproductive tract before gaining the ability to fertilize
oocytes (Austin, 1951; Chang, 1951). It is speculated that during this
time, glycoproteins from the sperm surface are removed, thus exposing
receptor sites that can respond to oocyte signals and lead to acrosome
reaction. This process allows the spermatozoa to bind to the ZP, which
subsequently leads to the Acrosome Reaction (AR), penetration of the ZP
and fertilization of the oocyte. In another word, the spermatozoa gains
the capacity to fertilize oocyte and, therefore, is termed as capacitation
(reviewed in Markert, 1983; Elder and Dale, 2000). This phenomenon is
first noted by Chang (1951) and Austin (1951) and the term capacitation`
was referred by Austin (1951). The time required for sperm capacitation
varies among different species of animals and ranges from less than one
hour in male mouse to 6 h in man (Elder and Dale, 2000). The changes that
occur during capacitation in the sperm plasma membrane include modification
of ion channels, increased adenylate cyclase and cyclic adenosine monophosphate
(cAMP), changes in surface glycoprotein moieties that lead to changes
in lectin binding patterns and also enzymatic modification of surface
proteins such as sugar transferases. Metabolic changes include increased
glycolytic activity and oxygen consumption, hyperactivation associated
with activation of adenylate cyclase system and loss of Zn+2
ions that lead to increased nuclear stability (Yanagimachi, 1994; Tulsiani
et al., 1997). A number of enzymes and factors that present in
the female reproductive tract have been implicated to affect sperm capacitation,
such as arylsulphatase, fucosidase and taurine. However, till to date,
the exact mechanism on how these enzymes and factors capacitate sperm
remains unknown (reviewed in Elder and Dale, 2000).
During capacitation, sperm motility is apparently regulated by changes
in the intracellular concentration of calcium ions through a calcium channel
(named CatSper) that is only expressed in the tail of mature sperm (Ren
et al., 2001). It is thought that the capacitation phenomenon is
necessary for spermatozoon to fuse with the oolemma, but not for subsequent
steps of fertilization such as DNA decondensation and PN formation. This
is supported by in vitro experiments in Xenopus laevis,
an amphibian species (Brun, 1974), mouse (Kimura and Yanagimachi, 1995)
and human (Palermo et al., 1992) in which injection of intact sperm
into the ooplasm resulted in development of normal offspring. However,
discrepancies also exists, for example, the capacitation step appears
to be critical in cattle (Goto et al., 1990; Sutovsky et al.,
1997), goat (Keskintepe et al., 1997; Wang et al., 2003)
and in other farm animals where ICSI is problematic (Catt and Rhodes,
1995). The failure of ICSI in some farm animals may be due to the failure
of removal of the perinuclear theca, a cytoskeletal capsule present between
the sperm membrane and the nuclear envelope (Sutovsky et al., 1997).
This structure could block the access of cytoplasmic factors involved
in decondensation of the sperm nucleus. This is supported by results where
artificially capacitated bull spermatozoa resulted in PN formation and
production of a normal calf after ICSI (Goto et al., 1990). Capacitation
may be attained in vivo or in vitro. Different in vitro
conditions are used to capacitate spermatozoa from various species
(Yanagimachi, 1994). Capacitation is temperature-dependant and only occurs
at 37° to 39°C.
Acrosome reaction: The first interaction between the spermatozoon
and the oocyte is at the ZP, an extracellular coat that surrounds all
mammalian oocytes (Wassarman et al., 2001). The sperm nucleus is
covered by membrane bound secretory vesicle known as the acrosome which
is a large, Golgi-derived lysosome-like organelle that overlies the nucleus
in the apical region of the sperm head (Kaji and Kudo, 2004). The acrosome
or its surrounding membranes contains a large array of hydrolytic enzymes
including hyaluronidase, acrosin, serine protease, proacroson, phosphatase,
arylsulphatase, colagenase, phospholipase C and β-galactosidase (reviewed
in Elder and Dale, 2000). The spermatozoon interacts with the ZP by the
plasma membrane overlying the acrosome. The specific binding of the spermatozoon
to the ZP induces in the acrosome a calcium-mediated signal transduction
process that leads to AR. The AR brings exposure of the inner acrosomal
membrane to the outside by breaking the organelle and involves fusion
of the outer acrosomal membrane with the overlying plasma membrane allowing
the acrosomal content to be released (Wassarman et al., 2001).
This process is necessary for the spermatozoon to acquire fusibility with
the oolemma (Kaji and Kudo, 2004). The AR is accompanied by modifications
in the sperm plasma membrane, which exposes receptors for ZP binding and
possibly factors exposed on the equatorial segment in preparation for
sperm-oocyte fusion (Kupker et al., 1998). The acrosomal enzymes
react with the extracellular matrix (or ZP) of oocyte and digest the ZP
at the point of spermatozoon contact. After losing its acrosomal content
the spermatozoon retains only the inner acrosomal membrane that makes
direct contact with the ZP during penetration. Following fusion, the sperm
plasma membrane remains in the ooplasm that indicate the point of fusion.
In mammals, Austin and Bishop (Austin and Bishop, 1958) first described
the AR. Although, AR is now considered as a separate process (Bavister,
2002), which triggers after capacitation, however, discrepancies exists
as some authors believe that it was a part of the capacitation process
(Austin and Bishop, 1958) and others considered AR as the final phase
of capacitation (Yanagimachi, 1969). The AR is considered as the final
prerequisite step in the sperm activation process before it gets the ability
to fuse with the oocyte. It is obligatory that this reaction only occur
in the presence of a rise in the intracellular calcium (Ca2+)
level (Florman and Babcock, 1991; Yanagimachi, 1994; Ben-Yosef and Shalgi,
1998; Wassarman, 1999). This increase of intracellular calcium can be
achieved in vitro by exposing sperm to Ca2+ ionophores
or phosphodiesterase inhibitors (Kupker et al., 1998). An artificially
high pH of 9.0-9.5 could also induce AR (Elder and Dale, 2000). Although,
it is not clear whether the AR is initiated whilst the sperm is interacting
with the cumulus mass, however, it is speculated that the reaction may
be started in the CCs as a major component of the cumulus matrix is hyaluronic
acid and the acrosome contains hyaluronidase. The AR is relatively rapid
once the correct trigger signals have been received and may take 2 to
15 min in vitro (Elder and Dale, 2000).
Sperm-oocyte interaction: As mentioned earlier, sperm-oocyte interaction
is the earliest step towards the fertilization process, which involves
the contact between the acrosome of the sperm and the ZP of the oocyte.
The acrosome is membrane-bound and contains lytic agents such as proteases,
sulphatases and glycosidases. On the other hand, the ZP of the oocyte
is made up of protein and carbohydrates in the form of glycoprotein units
that are probably stabilized by disulphide bonds. The principal types
of carbohydrate found are fucose and glycoprotein units, which are synthesized
by the oocyte itself (reviewed in Elder and Dale, 2000).
After completion of AR, the sperm penetrate the ZP. Following penetration,
the acrosome-reacted sperm passes through PVS and interacts with the sperm
plasma membrane of the oocyte (oolemma) (Schultz and Kopf, 1995). This
initial interaction involves specific recognition, binding and then fusion
between the sperm plasma membrane in the region of the equatorial segment
and the oolemma (Yanagimachi, 1994). The fusion site on the equatorial
segment may be excluded from the acrosome reaction and the sperm plasma
membrane is still intact on the apical part of the postacrosomal sheath
and the equatorial segment. The sperm tail is still beating outside the
ZP after its head and mid-piece have entered the PVS and the equatorial
segment has made an initial contact with the oolemma. The sperm head fuses
with microvilli on the oolemma (Kupker et al., 1998). The attachment
of the sperm head to the ZP inevitably alters the permeability of the
sperm plasma membrane, causing a transient change in the concentration
of several intracellular ions (Tulsiani et al., 1997). Once fusion
has occurred, the microvilli retract and draw most or the entire spermatozoon
into the ooplasm. As it is incorporated into oocyte, the spermatozoon
becomes immotile. Subsequently the entire spermatozoon including the tail
is drawn into the ooplasm (Kupker et al., 1998). This initiates
syngamy and further development of the zygote (Tulsiani et al.,
1997). The exact mechanism of sperm penetration of the ZP and the complementary
molecules that initiate penetration of the ZP by the sperm are not yet
known. However, it is generally believed that the process involves receptor-ligand
interaction between sperm-surface proteins and ZP glycoproteins. In rodents,
these consist of sperm membrane galactosyltransferase and N-acetylglucosamine
residues of the ZP. The receptor-ligand interaction induces the AR leading
to exocytosis and release of acrosomal enzymes from the acrosomal cap.
The specific sperm protein(s) responsible for sperm-oocyte interaction
are yet to be fully characterized. However, four ZP proteins with variable
roles in sperm-oocyte interaction have been isolated in mammals. In the
mouse, ZP3 plays the primary role, while ZP1 is the primary sperm receptor
in pig, rabbit and non-human primates (Dunbar et al., 1998). It
is not known yet whether such a characterization is available for the
goat. It is thought that the penetration of the ZP by the sperm is mediated
by lytic acrosomal enzymes (Tulsiani et al., 1997; Bedford, 1998)
or by the mechanical action of the sperm itself (Tulsiani et al.,
1997; Bedford, 1998). The latter author argues that available evidence
is in favor of mechanical instead of lytic penetration.
FERTILIZATION AND EARLY EMBRYOGENESIS
Fertilization in mammals is one of the most carefully regulated cell-cell
interactions in the animal body, involving two morphologically disparate
cells that must recognize, bind and fuse with each other in a very specific
way. It can be defined as the process of union of two germ cells, namely
spermatozoon and oocyte, whereby the somatic chromosome number is restored
and the development of a new and unique individual exhibiting characteristic
of the same species is initiated (Wassarman, 1999). By transferring genetic
information from one generation to the next, it ensures the immortality
of an individual and by creating variation it allows evolutionary forces
to operate (Elder and Dale, 2000).
As already mentioned earlier, before penetrating into the oocyte, various
biochemical changes occur in the spermatozoon including capacitation,
AR and sperm oocyte interaction. Following ZP penetration, a series of
events take place leading to syngamy and the production of the zygote.
These steps have been summarized previously (Bedford, 1982; Yanagimachi,
1994). In the following three sub-sections oocyte activation, PN formation
and cleavage as well as early embryonic development or embryogenesis in
mammals with special reference to goat is briefly discussed.
Activation of oocyte: Oocyte activation is the restoration of
metabolic activity in the quiescent oocyte, in other word; it is the process
of releasing the oocyte from the second meiotic arrest when the spermatozoon
fertilizes it. It is a cell signaling event that results in events including
the cortical granule reaction or Cortical Reaction (CR), decondensation
of the sperm nucleus, maternal RNA recruitment, resumption of meiosis
as evidenced by the extrusion of second polar body (PB-2) and later events
such as PN formation and initiation of DNA synthesis and cleavage (Yanagimachi,
1994). The very early cellular event observed in all activated mammalian
oocytes is an intracellular rise in Ca2+ concentration. In
human, this increase occurs within 1 to 3 min of sperm-oocyte fusion and
takes the form of a wave originating at the point of spermatozoon entry
(Reviewed in Ben-Yosef and Shalgi, 2001). The site of Ca2+ release
and sequestration is thought to be the endoplasmic reticulum, where inositol
1, 4, 5-triphosphate (IP3) receptors are present (Kline and
Kline, 1992). The first Ca2+ transient is followed by a series
of shorter Ca2+ transients of high amplitude (Ca2+
oscillations). In IVF studies, Ca2+ oscillations were observed
in all mammalian species including goat (Jellerette et al., 2006),
although their frequency is species specific (Ben-Yosef and Shalgi, 2001).
As fertilization progresses, the amplitude and frequency of the Ca2+
transients decrease, while the duration increases until an absolute cessation
of Ca2+ oscillations during entry into interphase and PN formation,
several hours after sperm entry (Jones et al., 1995). Calcium oscillations
require a continuous Ca2+ influx to refill endoplasmic reticulum
stores (Miyazaki, 1995). It was suggested that although a single rise
is sufficient to produce activation, oscillations might be required for
additional developmental events (Ozil, 1990).
There are two postulated hypotheses of oocyte activation mechanism, namely
receptor hypothesis` and sperm factor hypothesis, which describe
the contribution of the spermatozoa to successful oocyte activation (Kimura
et al., 1998; Fissore et al., 1998 ). In the receptor hypothesis,
it is the spermatozoon interacting with the oolemma that results in oocyte
activation. In this model, an oocyte surface receptor is coupled to a
G-protein (Miyazaki et al., 1993) or a tyrosine-kinase-mediated
(Ben-Yosef and Shalgi, 1998) signaling pathway and when activated by a
sperm, activates phospholipase C that results in the formation of IP3
and hence intracellular Ca2+ is released. The second hypothesis,
which is called the sperm factor hypothesis, suggests that the release
of intracellular Ca2+ is triggered by diffusible messenger(s)
in sperm cytoplasm which enters the ooplasm following sperm-oocyte fusion.
This hypothesis gained support with the development of ICSI technique
where initial Ca2+ rise, Ca2+ oscillations and full
oocyte activation occur after injecting sperm or sperm extract into the
ooplasm (Fissore et al., 1998).
Formation of pronuclei: Soon after oocyte activation through sperm
entry the PB-2 extrudes (Payne et al., 1997). The PB-2 is generally
extrudes immediately adjacent to the PB-1. The mammalian sperm nucleus
is packed with distinct protamines. When sperm enters the ooplasm, the
sperm nuclear envelope breaks down, the protamines are lost, the sperm
head enlarges in the ooplasm and chromosome or nuclear decondensation
takes place a few hours by reduction of disulphide bonds between protamines
by the action of glutathione (Kupker et al., 1998). Protamines
are replaced by the histones generated by the oocyte. Formation of MPN
takes place simultaneously with disappearance of the nuclear membrane,
decondensation of the chromosomes and reformation of the pronuclear membrane
that is supported by action of growth factors. This coincides with decondensation
of maternal chromatin and formation of the female PN (FPN). Usually, but
not always, the MPN appears near the site of sperm entry whereas the FPN
forms close to the PB-2 (Payne et al., 1997). At this stage (pronuclear
development), DNA synthesis and RNA transcription begin. The MPN and FPN
then merge (syngamy) to form the zygote. This marks the end of the fertilization
process and the beginning of embryonic development.
Cleavage and early embryogenesis: After the zygote stage, embryo
enters into several mitotic divisions. The zygote or one-cell stage embryo
is comparatively large, having a low nuclear to ooplasmic ratio. DNA replication
and cell divisions occur without an increase in cell mass to attain a
ratio similar to somatic cells. This process is referred to as cleavage
(Hafez and Hafez, 2000b). Once the diploid constitution of the species
has been restored by fertilization then the cleavage commences. Cleavage
of the zygote occurs by vertical division through the main axis of the
oocyte from the animal to vegetal pole (animal pole refers to the site
of PB extrusion and vegetal pole refers to the site of yolk reserve).
The cleavage furrow often goes through the region where the PN resided
at the initiation of syngamy. The resulting daughter cells are known as
blastomeres. The plane of second division is also vertical and passes
through the main axis but at a right angle to the initial plane of cleavage,
resulting in 4 blastomeres. The third cleavage division occurs approximately
at a right angle to the second, resulting in 8 blastomeres. This doubling
sequence is followed on through the remainder of early cleavage (Hafez
and Hafez, 2000b). Cleavage divisions are always mitotic and each blastomere
receives full assortment of chromosomes. Although the blastomeres undergo
mitosis like adult somatic cells and their number increases, however,
unlike somatic cells they do not retain the size of the parent cell. The
blastomeres resulting from each division are always approximately half
the size of the parent blastomere. Thus, the size of a pre-implantation
embryo remains the same even though the number of blastomeres increases.
The embryo undergoes polarization and differences arise between the blastomeres
as cleavage stage progresses. This may be due to the unequal distribution
of ooplasmic components already laid down in the oocyte during oogenesis,
or due to new embryonic gene transcription that results in changes to
blastomeres. This way, each blastomere sets off on its own particular
program of development to give rise to specific cell lines, for example,
epithelium, muscle, nerve or other connective tissue. During cleavage
stage, individual blastomeres can be clearly detected when viewed under
the microscope. It is important to note that the precise cleavage of zygotes
and blastomeres into two equally sized daughter cells relies upon the
position of the spindle and the functional activity of cytoskeletal elements.
Slight variations in blastomere sizes within the same embryo are probably
unimportant, but major differences may indicate defects in underlying
cellular processes (Hardarson et al., 2001). Although oocyte size
(diameter of ooplasm) was measured in most of the species, there was paucity
of information on blastomeres size. A search of available literature revealed
one paper where morphometric measurements of human ICSI-derived cleaved
embryos were performed up to 4-cell stage. Using computer-controlled morphometric
analysis of blastomere size, it was found that average diameter of blastomeres
of 2-, 3-and 4-cell stage embryos were 80.1, 68.7 and 64.9 μm, respectively
(Hnida et al., 2004). The diameter of ooplasm of a fully grown
human oocyte or 1-cell embryo is 135 to 160 μm (Oppenheimer and Lefevre,
1989) which is about the size of a mature goat oocyte (119-146 μm)
(Crozet et al., 2000) (Table 1). It was found
from their study that blastomere size significantly affected by degree
of fragmentation and multinuclearity and that computer-assisted, multilevel
analysis of blastomere size may function as a biomarker for embryo quality
(Hnida et al., 2004). The chronology of embryo development from
2-cell stage to blastocyst hatching in different species of farm animals
including goat, rabbit, mouse and human has been presented in Table
3. In goat, initial and principal activations of embryonic genome
(cell stage) are reported to be occurring at 2-cell and 8-cell stage,
respectively (Kelk et al., 1994). After the embryo reaches to the
morula stage it is very difficult to discern the boundaries between blastomeres
as the embryo undergoes compaction in which the blastomeres flatten against
each other to form intercellular junctions between them. Depending upon
the species, there is a great deal of variation of the beginning of embryo
compaction stage. In goat, compaction of the embryo begins at 8-cell stage
(Sakkas et al., 1989). The data for other farm animals, rabbit,
mouse and human are depicted in Table 3. As the morula
progresses to further embryonic development, some morphological change
occurs and the morula progresses to blastocyst stage. The blastocyst is
characterized by the formation of an outer layer of trophectoderm cells,
Inner Cell Mass (ICM) and a fluid-filled cavity, known as blastocoel.
In goat, the blastocoel appears at 120 h post-fertilization (Sakkas et
al., 1989). Data for other farm animals, rabbit, mouse and human have
been depicted in Table 3.
|| Chronology of pre-implantation embryo development in
goat and other farm animals, rabbit, mouse and human
|N.B: Data mainly derived from in vivo studies;
h = hours; n/a = not applicable; aKelk et al. (1994);
bCrosby et al. (1988); cYang (1991)
The embryo is confined within the ZP (analogous to eggshell in bird,
reptiles or monotremes) throughout the early stages of development. This
keeps the blastomeres together prior to compaction and prevents embryos
from fusing together. During early cleavage, there is little increase
in metabolic rate, however, a sharp rise occurs between the morula and
blastocyst stage. The goat embryo stays in the oviduct for 48 to 72 h
from the time of ovulation (Moore, 1977). The trophectoderm cells of the
embryo secrete proteolytic enzymes that digest a passage through the ZP
and allow the blastocyst to hatch. The hatching of goat blastocyst occurs
around 168 h (7 days) after fertilization (Sakkas et al., 1989).
The times required to blastocyst hatching in case of other domestic animals,
rabbit, mouse and human are depicted in Table 3. The
exposed cells of the goat blastocyst then make firm physical contact with
the uterine wall, implantation stage begins, embryo becomes elongated
and the final implantation in the uterine endometrium occurs at day 16
post-fertilization (Gurdon et al., 1995).
Currently, goat producing developed countries focusing their researches
on ARTs for rapid increment of goat numbers as well as genetic improvements.
Besides, goats are found to be more appropriate animal for the production
of valuable recombinant pharmaceutical or biomedical protein through using
latest ARTs like transgenesis, NT or ICSI. Although, using modern ARTs
genetic and overall improvement in goat have been achieved in some extent,
however, it is still a long way to go. To get success in any in vitro
studies in any species of animals it crucial to know the normal fertilization
process that occurs in vivo. Although, previously a number of in
vivo fertilization studies have been performed in goat, however, compare
to cattle, sheep and laboratory animals it is still not enough. Most of
the description of goat gametogenesis, sperm-oocyte interaction, fertilization
and early embryogenesis are mainly based on the data derived from sheep
and in some extent cattle. Although, sheep and cattle are also ruminants,
however, it is to be remembered that they are different species and their
physiology is different from that of goat. Therefore, there is still a
need for study some basic in vivo aspects of gametogenesis, fertilization
and pre-implantation embryogentic process in goat which will not only
fill up gaps of information but also help in the successful deigning of
suitable ARTs for rapid improvement of goat industry.
The authors wish to thank Islamic Development Bank (IDB) for providing
an IDB Merit Scholarship to the first author. This work was supported
by grants from MOSTI Special Project (Grant No. 01-02-03-0696) and IPPP
(Grant No. Vote F-0179/2004D, 0145/2005D and P0170/2006C).
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