23 FERTILIZATION, PLACENTATION, AND LACTATION

FERTILIZATION

Two events must occur before fertilization: (1) sperm maturation in the epididymis and (2) sperm capacitation in the female reproductive tract.

Sperm released from the testis and entering the epididymal duct have circular motion. After a 2-week maturation process, following epididymal transit and storage in the tail or cauda of the epididymis, sperm acquire forward motility necessary for fertilization. After ejaculation, sperm undergo a capacitation process in the uterus and fertilization of the ovum or egg takes place in the oviduct.

Essentially, a fertilizing sperm must complete both maturation and capacitation before sperm-egg fusion. Capacitation is a biochemical event that can be induced in vitro, a procedure that permits in vitro fertilization. During capacitation, non-covalently bound epididymal and seminal glycoproteins are removed from the sperm plasma membrane. Membrane permeability to Ca²⁺ increases. An influx of Ca²⁺ maximizes intracellular cyclic adenosine monophosphate (cAMP) levels, resulting in an increase in sperm motility. This process is known as hyperactivation.

We have seen that the sperm head consists of three components: (1) the condensed elongated nucleus, (2) the acrosomal sac bound to the acroplaxome, a cytoskeletal plate anchoring the acrosome to the nuclear envelope, and (3) the plasma membrane. The head-tail coupling apparatus, containing a pair of centrioles and associated proteins, links the sperm head to its tail.

The condensed nucleus consists of DNA surrounded by very basic protamines. Nucleosomes are not present because somatic histones have been replaced by protamines that protect and stabilize the DNA during fertilization.

The acrosomal sac is formed by three constituents (Figure 23-1): (1) the outer acrosomal membrane, (2) the inner acrosomal membrane, and (3) hydrolytic enzymes (mainly hyaluronidase and acrosin, the latter derived from the precursor proacrosin). The thin portion of the acrosomal sac, extending toward the tail, is the equatorial segment.

Figure 23-1 Acrosome reaction

The three main events during fertilization are, sequentially the acrosome reaction, sperm binding to ZP3, a glycoprotein of the zona pellucida (ZP), and sperm-egg fusion (Figure 23-2).

Figure 23-2 Fertilization

In the proximity of the ovum, and in the presence of Ca²⁺, the sperm plasma membrane fuses with the outer acrosomal membrane. This event is known as the acrosome reaction. Small openings created by membrane fusion facilitate the release of hydrolytic enzymes (see Figures 23-1 and 23-2). The equatorial region of the acrosome does not participate in the plasma membrane-outer acrosome membrane fusion process. Male infertility may occur when the acrosome reaction fails to occur or takes place before the sperm reaches the egg.

Two membrane proteins have been shown to be essential for sperm-egg fusion, Izumo in the sperm and CD9 in the egg. Izumo is a sperm-specific membrane protein of the immunoglobulin superfamily that localizes to the sperm plasma membrane after the acrosomal reaction. CD9 is a member of the tetraspanin super family of transmembrane proteins (see Box 23-A). Izumo and CD9 may be involved in the organization or stabilization of plasma membrane protein complexes essential for the sperm-egg fusion reaction. Other proteins, such as ADAMs (a disintegrin and metalloprotease), may participate in this reaction. We discussed in Chapter 1, Epithelium, how the disintegrin domain of ADAMs participates in the shedding of the ectoplasmic portion of transmembrane proteins.

Box 23-A Tetraspanins

• Tetraspanins, first discovered on the human leukocyte surface, have four transmembrane domains, two extracellular loops (small and large), and short intracytoplasmic N- and C-terminal tails.

• The transmembrane domains enable the association of additional tetraspanins to assemble the tetraspanin web in which integrins are included. Tetraspanins function as surface organizers by grouping and interconnecting specific cell surface proteins.

• The large extracellular loop is involved in protein-protein interaction with laterally positioned proteins.

• The intracellular short tails are linked to intracellular cytoskeletal and signaling molecules.

• Tetraspanins behave as metastasis suppressor molecules. Decreased expression of tetraspanins correlates with increased invasive and metastasis potential.

Sperm-egg fusion causes a local mild depolarization of the egg plasma membrane that generates a calcium wave across the egg’s cytoplasm within 5 to 20 seconds. An increase in calcium concentration amplifies the depolarization signal and triggers the oocyte to resume cell division, complete meiosis II by releasing the second polar body into the perivitelline space, and start the early embryogenesis developmental program.

Zona pellucida during fertilization

The plasma membrane of mammalian eggs is surrounded by a 6- to 7-μm-thick zona pellucida (plural zonae pellucidae), a glycoprotein coat produced mainly by the primary oocyte during folliculogenesis, as early as during the primary follicle stage. The zona pellucida has important roles in fertilization and implantation of the embryo in the endometrium. In vitro fertilization overcomes most forms of infertility (see Box 23-B)

Box 23-B In vitro fertilization

• Fertilization of human sperm and eggs in vitro consists in the following steps:

• Preovulatory oocytes (about 10 or more) are collected by laparoscopy or transvaginally guided by ultrasound imaging, following stimulation of the ovaries by gonadotropin-releasing hormone and follicle-stimulating hormone administration. Oocytes are retrieved 34 to 38 hours after injection of human chorionic gonadotropin to mimic the luteinizing hormone surge.

• The oocytes are incubated overnight with motile sperm in a defined culture medium to achieve fertilization in vitro. Embryos can then be transferred to the patient.

• Alternatively, in cases of a severe male infertility factor, a sperm can be injected into the oocyte by the process of intracytoplasmatic sperm injection (ICSI).

• In cases of azoospermia (absence of sperm in the ejaculate), sperm can be obtained surgically from the epididymis or the testes and used for ICSI.

• Embryos can be tested in vitro for the presence of a genetic or chromosomal abnormality by a procedure known as preimplantation genetics diagnosis. A sample can be a blastomere of the embryo, a piece of the trophoectoderm or even the oocyte polar body. Unaffected embryos can then be transferred to the patient.

• An excess of embryos can be cryopreserved in liquid nitrogen for later use. Propanediol or dimethylsulphoxide can be used as cryoprotectant for pre-blastocyst embryos, and glycerol is used for blastocysts.

The zona pellucida is composed of three glycoproteins (see Figure 23-2): ZP1, a dimer of 200 kd; ZP2, 120 kd; and ZP3, 83 kd. ZP2 and ZP3 interact to form a long filament complex interconnected by ZP1 dimers at regular intervals.

There are four functional aspects related to ZP3 that you should remember: (1) ZP3 is responsible for sperm binding, mediated by O-oligosaccharides linked to ZP3 with binding affinity to sperm receptors. (2) Only acrosome-reacted sperm can interact with ZP3. (3) ZP3 is essential for species specific sperm binding, preventing sperm from a different species from fertilizing the egg. (4) After the first sperm fertilizes the egg, proteases released from the cortical granules present in the egg remove oligosaccharides from ZP3 and partially cleave ZP2. This process, called cortical reaction, prevents polyspermy, an egg to be fertilized by more than one sperm. Polyspermy results in nonviable zygotes.

PLACENTATION

The placenta and embryonic-fetal membranes (amnion, chorion, allantois, and yolk sac) protect the embryo-fetus and provide for nutrition, respiration, excretion, and hormone production during development. The membranes are formed by the embryo. Both the embryo and the maternal endometrium begin to form the placenta as soon as the blastocyst implants in the endometrium.

You have learned in your Embryology course the pre-implantation developmental steps of embryo following fertilization in the ampulla of the oviduct. As you may recall, the first rounds of cell divisions of the zygote (the fertilized egg) are designated cleavage. The daughter cells are named blastomeres. The embryo consists of a compact or ball structure, called morula, once it has attained an 8-cell number.

Cell diversity is achieved in the blastocyst, when the trophectoderm and inner cell mass are recognized. In the late blastocyst, the trophectoderm is referred to trophoblast and is distributed in two regions: in direct contact with the inner cell mass, the polar trophoblast, and surrounding the blastocyst cavity, the mural trophoblast. The blastocyst hatches from its zona pellucida at 6 to 7 days and the the differentiation of the inner cell mas proceeds.

Implantation of the blastocyst

The implantation of the blastocyst into a nurturing endometrium involves (1) the initial unstable adhesion of the blastocyst to the endometrial surface, called apposition, followed by a stable adhesion phase and (2) the decidualization of the endometrial stroma (Figure 23-3).

Figure 23-3 Implantation of the blastocyst

The timing of preimplantation and implantation is extremely precise (see Box 23-C). So is the preparation of the implantation site.

Box 23-C Timetable of implantation

• Fertilization occurs in the oviduct or fallopian tube within 24 to 48 hours after ovulation.

• The development of the fertilized egg, called zygote, to the morula stage, occurs as the embryo—surrounded by the zona pellucida—travels through the fallopian tube.

• The morula appears in the uterine cavity about 2 to 3 days after fertilization.

• The embryo, now a blastocyst, hatches from the zona pellucida 72 hours after entering the uterine cavity.

• Implantation occurs 6 or 7 days after fertilization. Implantation involves two stages: (1) apposition of the blastocyst to the endometrial surface and (2) implantation of the blastocyst mediated by penetrating trophoblastic cells.

• The blastocyst is entirely embedded in the receptive endometrium by day 10 after fertilization. Uterine receptivity–corresponding to days 20 to 24 of a regular 28-day menstrual cycle—is defined by the optimal state of endometrial maturation for the implantation of the blastocyst. Uterine receptivity consists in a vascular and edematous endometrial stroma, secretory endometrial glands, and apical microprocesses, the pinopodes, on the apical domain of the luminal endometrial lining cells.

• Differentiated syncytiotrophoblastic cells invade part of the myometrium (interstitial invasion), as well as local uterine blood vessels (endovascular invasion).

• The uteroplacental circulation is established when trophoblastic cells are in direct contact with maternal blood (see Box 23-D).

On day 4 of pregnancy, the embryo—at the blastocyst stage—is within the uterine cavity. The coordinated effect of ovarian estrogens and progesterone has already conditioned the endometrium for implantation, including an increase in endometrial vascular permeability at the implantation site.

The blastocyst hatches from the zona pellucida and exposes its trophoblast epithelial lining to the uterine luminal epithelium. If zona pellucida hatching fails to occur, the embryo will not implant. Failure of the uterine stroma to undergo decidualization can lead to spontaneous abortion.

Trophoblast-mediated attachment and subsequent implantation depend on two conditions: (1) the apical surface of the endometrial epithelial cell must display membrane bound and soluble forms of heparin-bound epidermal growth factorlike factor (HB-EGF), a member of the transforming growth factor-α family; and (2) the surface of the trophoectoderm cells must autophosphorylate epidermal growth factor receptor (EGF-R) and have heparan sulfate proteoglycan (also called perlecan) to strongly bind to HB-EGF.

At implantation (see Figure 23-3), cytoplasmic processes of trophoblastic cells interact with small processes on the apical surface of the endometrial epithelial cells, called pinopodes, and penetrate the intercellular spaces of the endometrial cells. Penetration is facilitated by a decrease in the number of desmosomes linking the endometrial epithelial cells that undergo apoptosis.

As you recall, the endometrial lamina propria has undergone a decidual transformation during the secretory phase of the menstrual cycle. This primary decidual zone is remodeled by the action of metalloproteinases (see Figure 23-5), and a secondary decidual zone houses the implanting embryo.

Differentiation of the trophoblast

The trophoblast differentiates into (1) an inner layer of mitotically active mononucleated cytotrophoblastic cells and (2) an outer layer of multinucleated syncytiotrophoblastic cells at the embryonic pole, facing the endometrium. The syncytiotrophoblast mass invades the endometrium (formed by glands, stroma, and blood vessels) and rapidly surrounds the entire embryo.

The blastocyst has a cavity containing fluid and the eccentric inner cell mass, which gives rise to the embryo and some extraembryonic tissues. The mural trophoblastic cells proximal to the inner cell mass begin to develop the chorionic sac. The chorionic sac consists of two components: the trophoblast and the underlying extraembryonic mesoderm.

Invasion of the endometrium and the inner third of the myometrium, a process called interstitial invasion, is determined by the action of secretory proteolytic enzymes released by the syncytiotrophoblast. Proteases erode the branches of the spiral uterine arteries to form spaces or lacunae of maternal blood within the syncytiotrophoblast mass. This endometrial eroding event, called endovascular invasion, initiates the primitive uteroplacental circulation and represents the starting point of the future intervillous space. Decidualization allows an orderly access of trophoblastic cells to the maternal nutrients by modulating the invasion of uterine spiral arteries.

The syncytiotrophoblast begins the secretion of human chorionic gonadotropin (hCG) into the maternal lacunae. The secretion of estrogens and progesterone by the corpus luteum is now under the control of hCG.

Role of the decidual cells during implantation

On the maternal side, decidual cells, close to the mass of invading syncytiotrophoblastic cells, degenerate and release glycogen and lipids, thus providing, together with maternal blood in the lacunae, the initial nutrients for embryonic development.

The decidua provides an immune-protective environment for the development of the embryo. The decidual reaction involves (1) the production of immunosuppressive substances (mainly prostaglandins) by decidual cells to inhibit the activation of natural killer cells at the implantation site and (2) infiltrating leukocytes in the endometrial stroma that secrete interleukin-2 to prevent maternal tissue rejection of the implanting embryo. Syncytiotrophoblastic cells do not express major histocompatibility complex class II. Therefore, the syncytiotrophoblast cannot present antigens to maternal CD4⁺ T cells.

Formation of primary, secondary, and tertiary villi

At the end of the second week, cytotrophoblastic cells proliferate under the influence of the extraembryonic mesoderm, and extend into the syncytiotrophoblast mass, forming the primary villi (Figure 23-4).

Figure 23-4 Primary and secondary chorionic villi

Figure 23-5 Tertiary chorionic villi (3rd week, late)

Primary villi represent the first step in the development of the chorionic villi of the placenta. In cross section, a primary villus is formed by a core of cytotrophoblastic cells covered by syncytiotrophoblast.

Early in the third week, the extraembryonic mesoderm extends into the primary villi, forming the secondary villi (see Figure 23-4). Secondary villi cover the entire surface of the chorionic sac. In cross section, a secondary villus is formed by a core of extraembryonic mesoderm surrounded by a middle cytotrophoblast layer and an outer layer of syncytiotrophoblast.

Soon after, cells of the extraembryonic mesoderm differentiate into capillary and blood cells, forming the tertiary villi (Figure 23-5). The difference between the secondary and tertiary villi is the presence of capillaries in the latter. The capillaries in the tertiary villi interconnect to form arteriocapillary networks leading to the embryonic heart.

In cross section, a tertiary villus is formed by a core of extraembryonic mesoderm with capillaries, surrounded by a middle cytotrophoblast layer and an outer layer of syncytiotrophoblast.

The following events occur as the chorionic tertiary villi continue to develop:

1. Cytotrophoblastic cells extend beyond the syncytiotrophoblast to form the cytotrophoblastic shell, attaching the chorionic sac to the endometrium.

2. Some villi, the stem or anchoring villi, attach to the cytotrophoblastic shell.

3. Branch or terminal villi grow from the sides of the stem villi and are in direct contact with maternal blood in the intervillous space.

The chorionic villi cover the entire chorionic sac until the beginning of the eighth week. Then, villi associated with the decidua capsularis degenerate, forming a smooth chorion (chorion laeve).