Fertilization And The Establishment Of Pregnancy.
The egg
At ovulation, the egg is arrested
in metaphase of the second meiotic division (Chapter 4). It is surrounded by a
proteinaceous sphere called the zona pellucida. Those granulosa cells
that adhered to the surface of the zona pellucida and were expelled with the
egg from the ovary remain attached as the cumulus. Sperm that fertilize the egg
must first negotiate these surrounding layers before they can penetrate the egg
cell membrane. The oocyte will remain viable for at least 6–24 h once ovulated.
The sperm
With coitus, millions of sperm
are deposited in the upper vagina. Most will never arrive at the site of
fertilization. Abnormal sperm can rarely make this long trip successfully and even
most of the healthy spermatozoa die along the way. The vast majority leak from
the vagina upon liquification of the semen. Only a small proportion enters the
cervix, where sperm will be found within minutes of coitus. Here they can
survive within the epithelial crypts for hours. Sperm cannot traverse the cervix into the uterine cavity unless the
cervical mucous is receptive. This typically occurs at midcycle when estrogen
levels are high and progesterone is low. Estrogen softens the cervical stroma
and makes cervical secretions thin and watery. Progesterone has opposite
effects, a combination hostile to spermatozoa.
In the best of conditions, it
takes 2–7 h for sperm to move through the uterus to the site of fertilization
within the oviduct. Sperm transport results from self-propulsion, aided by the
ciliary beating of cells within the uterine lining. Typically, only several
hundred sperm reach the oviducts, where they will linger in a quiet state until
ovulation occurs. After ovulation, these spermatozoa are reactivated and begin
moving toward the egg. The signal that attracts the sperm to the egg is
unknown. Human spermatozoa can survive for approximately of 24–48 h in the
female reproductive tract.
Freshly ejaculated spermatozoa
are not capable of fertilizing an egg. They acquire the ability to penetrate
the cell layers surrounding the oocyte through a process known as capacitation.
Although capacitation
can be induced in vitro under the proper culture conditions, it occurs in
vivo within the female reproductive tract. During capacitation, the
glycoprotein coat that adheres to the spermatozoa cell membranes is initially
removed, initiating changes in the surface charge of the sperm membrane and
reorganization of that membrane. Capacitated sperm change their tail movements
from regular undulating waves to whip-like, thrashing movements that propel the
sperm forward. At the biochemical level, capacitated sperm acquire increased
calcium sensitivity and elevated internal cAMP levels. Capacitation takes
several hours both in vivo and in vitro.
Sperm capacitation allows for the
acrosome reaction. In the absence of an acrosome reaction, a sperm is
incapable of penetrating the zona pellucida. Contact of an intact, capacitated
sperm with the zona pellucida of an egg allows interaction of a specific sperm
cell surface glycoprotein, ZP3, with specific zona protein. These interactions
are likely mediated by the sugars on sperm–egg binding proteins. ZP3-binding
induces further calcium influx into the spermatozoa and intracellular cAMP
levels rise. The acrosome swells, its outer membrane fuses with the sperm
plasma membrane, and the enzymatic contents of the acro-some are released into
the extracellular space surrounding the head of the sperm. This exposes the
inner acrosomal membrane and another zona-binding protein, ZP2, to the oocyte
zona. ZP2 binding holds sperm near the egg. Proteolytic enzymes released from
the acrosome then facilitate penetration of the zona pellucida by the
whiplashing sperm. Complete penetration of the zona takes about 15 minutes.
Fertilization
Penetration of the zona pellucida
allows contact between spermatozoa and the oocyte membrane (Fig. 16.1). The
germ cell membranes fuse almost immediately and the sperm cell stops moving.
The sperm nucleus enters the egg cytoplasm.
Three important events are
triggered within the oocyte by the rise in intracellular calcium that occurs in
the oocyte upon fusion of sperm and egg cell membranes. The egg cell membrane
depolarizes, preventing membrane fusion with additional spermatozoa. This is
the primary block to polyspermy. It assures that only one male
pronucleus is available for fusion with the female pronucleus and protects the
diploid status of the zygote. The second event is known as the cortical
reaction. Cortical granules lie just beneath the egg cell membrane, and
with the cortical reaction they fuse with the membrane and release their
contents into the zona pellucida. This hardens the zona and impairs the ability
of sperm to bind to it – a secondary block to polyspermy. The third
event involves resumption of the second meiotic division of the egg. The second
polar body is formed and extruded from the egg, thereby assuring that the
female pronucleus is haploid. Again, the diploid zygote is protected. Failure
to preserve the diploid state of the conceptus is a frequent cause of early
pregnancy failure (Chapter 36).
Upon entry into the egg, sperm
cytoplasm mixes with that of the egg and the sperm nuclear membrane breaks
down. A new membrane forms around the sperm chromatin, forming the male
pronucleus. A new oocyte nuclear membrane also forms around the female
pronucleus. DNA synthesis begins during this period as the haploid pronuclei
prepare for the first mitotic division of the zygote. The pronuclear membranes
break down, the parental chromosomes mix and the metaphase mitotic spindle
forms. At about 24 h after fertilization, the chromosomes separate and the
first cell division occurs.
During the first few embryonic
cell divisions, no new mRNA is synthesized from the nuclear DNA of the
conceptus. The embryo stays the same total size and the size of each individual
cell decreases accordingly. Thus, the
early embryo uses only maternal cell components to develop and important
signals must be transmitted to the embryo through the oocyte cytoplasm. These
signals likely reside in mitochondrial DNA, which is replicated during
early embryonic cell division. In fact, mitochondrial DNA is quite stable and
can be traced through generations to determine maternal lineage.
Establishment of pregnancy
After fertilization, a successful
pregnancy must implant within the wall of the uterus and inform the mother that
pregnancy adaptations must occur. Without these two important events, the
zygote will simply wash out of the uterus with the next menses.
The cleaving zygote floats in the
oviduct for approximately 1 week, progressing from the 16-cell stage through
the solid morula (mulberry) stage to the 32–64 cell blastocyst stage.
The latter stage requires formation of the fluid-containing blastocyst cavity.
The blastocyst contains two distinct differentiated embryonic cell types: the
outer trophectoderm cells and the inner cell mass. The trophectoderm
cells will eventually form the placenta. The inner cell mass will form the
fetus and fetal membranes. It is at the blastocyst stage that the conceptus
enters the uterus.
During the time that it spends in
the oviduct, the conceptus remains surrounded by the zona pellucida. After
about 2 days in the uterus, the blastocyst will lose or “hatch” from the zona
pellucida. On hatching, the trophectodermal cells of the blastocyst
differentiate into trophoblast cells. These simultaneous processes allow
trophoblast cells to make direct contact with the uterine luminal epithelial
cells. The blastocyst attaches to and invades the uterine lining. Within hours,
the surface epithelium immediately underlying the conceptus becomes eroded and
nearby cells lyse, releasing primary metabolic substrates used by the
blastocyst. The endometrium undergoes dramatic biochemical and morphologic
changes called decidualization, a process beginning at the point of
attachment and spreading in a concentric wave from the point of implantation.
Thedecidualized endometrium will heal over the conceptus so that the entire
implantation becomes buried within the endometrium.
As the embryo invades maternal
tissues the trophoblast cells further differentiate into two layers: inner cytotrophoblast
cells and an outer syncytiotrophoblast (Chapter 17). The
syncytiotrophoblast is a continuous, multinucleated layer that covers the
interstitial space and arises from fusion of the underlying cytotrophoblast
progenitor cells. Syncytiotrophoblast is active in placental hormone secretion
and in nutrient transport from mother to fetus. A separate subset of cytotrophoblast
cells acquires invasive properties and traverses endometrial stroma to reach
maternal blood vessels, including the spiral arteries of the endometrium.
Appropriate invasion and subsequent remodeling of the spiral arteries by these
extravillous cytotrophoblast cells is key to a normal pregnancy outcome
(Chapter 38).
A number of growth factors are
integral to successful implantation: (i)
leukemia inhibitory factor, a cytokine; (ii) the integrins, which mediate
cell–cell interactions; and (iii) transforming growth factor beta (TGF-β),
which stimulates syncytium formation and inhibits trophoblast invasion.
Epidermal growth factor and interleukin 1β are also important mediators of
invasion.
Implantation occurs about 7–10
days after ovulation. If the conceptus is to survive more than 14 days after
ovulation, the ovarian corpus luteum must continue to secrete progesterone. Human
chorionic gonadotropin (hCG) produced by the developing trophoblast and
secreted into the maternal bloodstream acts like luteinizing hormone,
supporting the corpus luteum by inhibiting luteal regression (Chapters 14 and
18).
