The prenatal period and placental physiology
Overview of pregnancy
The duration of pregnancy averages 266 days (38 weeks) after ovulation, or 280 days (40 weeks) after the first day of the last menstrual period (Figure 3-1). This equals 10 lunar months, or just over 9 calendar months. During these months, the almost solid uterus, with a cavity of 10 mL or less, develops into a large, thin-walled organ. The total volume of the contents of the uterus is 5 L or more at term, 500 to 1000 times the original capacity.33
Most of the changes encountered during pregnancy are progressive and can be attributed to either hormonal responses or physical alterations secondary to fetal size. The preimplantation endocrine system controls the reproductive cycle. In the woman, this involves the cyclic release of pituitary gonadotropins and secretion of estrogen and progesterone by the ovary (see Chapter 2).
First trimester
Physical signs associated with pregnancy include Goodell’s sign (softening of the cervix and vagina with increased leukorrheal discharge), Hegar’s sign (softening and increased compressibility of the lower uterine segment), and Chadwick’s sign (bluish purple discoloration of the vaginal mucosa, cervix, and vulva) by 8 weeks. Although a presumptive sign of pregnancy, Chadwick’s sign is only useful in primiparous women. By 8 to 10 weeks, fetal heart tones can be auscultated by Doppler ultrasonography. Real-time ultrasound can pick up fetal heart movements earlier. Maternal cardiovascular changes are also occurring, with stroke volume and cardiac output increasing and systemic vascular resistance decreasing. These changes contribute to increased renal plasma flow and glomerular filtration.33 Weight gain during the first trimester is usually small.
Second trimester
The increasing vascularity of the vagina and pelvic viscera may result in increased sensitivity and heightened arousal and sexual interest. Mucorrhea is not uncommon as a result of the hyperactivity of the vaginal glandular tissues. This change may increase the pleasure experienced during sexual intercourse. Spontaneous orgasm and multiple orgasms may occur as a result of the increased congestion. Leukorrhea often occurs, with thick, white, acidotic (pH of 3.5 to 6.0) discharge that may contribute to inhibition of pathogenic colonization of the vagina.33 Perineal structures also enlarge as a result of the vasocongestion, increased vascularity, hypertrophy of the perineal body, and fat deposition that began during the first trimester.
Third trimester
The heart is displaced slightly to the left as a result of the increased pressure from the enlarged uterus. Blood pressure rises slightly, and cardiac output remains unchanged. Blood volume peaks at 28 to 34 weeks’ gestation. Dependent edema frequently occurs as blood return from the lower extremities is reduced. Increasing pelvic congestion, relaxation of the smooth muscle in the veins, and the increased pressure of the growing fetus may result in varicosities of the perineum and rectum. Constipation and obesity may lead to development of engorged blood vessels.33
The increased elasticity of connective and collagen tissue leads to relaxation and hypermobility of the pelvic joints. Separation of the symphysis pubis results in instability of the sacroiliac joint. The center of gravity shifts lower with development of a progressive lordosis to compensate for the anterior shift of the uterus. Balance is maintained by an enhanced cervicodorsal curvature, leading to difficulty in walking and the characteristic waddling gait. Stress on the ligaments and muscles of the middle and lower back and spine may lead to discomfort and back pain.33
Conception
For conception to occur, a precise set of sequential events must take place. The probability of a viable conception per menstrual cycle is less than 40% to 50%.25,103 The process of conception and fetal survival is selective, as evidenced by implantation failures and the approximately 50% anomaly rate encountered in spontaneously aborted fetuses.103,130 Gametogenesis is described in Chapter 1. The ovarian and endometrial cycles necessary for conception and early support of the fertilized ovum, as well as follicle maturation, are described in Chapter 2. This section will examine ovulation, sperm transport, fertilization, cleavage, and zygote transport.
Ovulation
The ovary is responsible for two important functions: gametogenesis and steroid hormone synthesis. Integration of ovarian steroid synthesis, follicle maturation, ovulation, and corpus luteum function is essential for fertilization and implantation. Estrogen and progesterone have significant effects on tubal and uterine motility, endometrial proliferation, and the properties of the cervical mucus.33 In order for fertilization to take place, the oocyte must become “fertilization competent” (see Chapter 1). The close proximity of the oocytes and follicular cells in the ovary allows the follicle and oocyte to communicate bidirectionally via gap junctions (intracellular membrane channels) in order to work together to control meiotic arrest and resumption, follicle maturation; ovulation; and corpus luteum formation, function, and regression.26 The hypothalamus and anterior pituitary regulate these latter morphologic changes through secretion of gonadotropin-releasing hormone (GnRH) and gonadotropins. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) act synergistically (see Chapter 2). Endocrine interactions in follicle maturation are illustrated in Figure 3-2.
Usually only one follicle matures and is ovulated, although the exact mechanism for this is unknown. At the beginning of the menstrual cycle, up to 15 to 20 primary (pre-antral) follicles are stimulated by FSH, but only 6 to 12 enlarge. Of these growing follicles, several develop into antral follicles. Eventually one follicle becomes dominant and begins to function independent of FSH. This follicle secretes inhibin, which inhibits pituitary FSH. Because the other maturing follicles are still dependent on FSH, which is now decreased, they begin to regress and degenerate.25 The process of follicular development and maturation is described in the section on the Ovarian Cycle in Chapter 2. The ovary during reproduction is illustrated in Figure 2-21.
In the fully developed follicle (Graafian or tertiary follicle), multiple layers of granulosa cells line the antral side of the basement membrane (membrane granulosa), and a cumulus of granulosa cells surrounds the oocyte. Proliferation of these cells is stimulated by growth differentiation factor 9 (GDF9), which is a member of the transforming growth factor β family.130 GDF9 is also important for oocyte maturation and differentiation. Oxygen and nutrients diffuse across the granulosa cells to the oocyte. Antral fluid contains proteins, enzymes, proteoglycans, and hormones such as FSH and steroids.25 The oocyte is surrounded by the zona pellucida, which contains sperm receptors. The external theca layers around the follicle consist of the outer theca externa (capsular like covering) and vascularized, glandular inner theca interna. Under the stimulation of FSH, the theca and granulosa cells produce large amounts of estrogen (primarily estradiol), which peak about 24 hours before ovulation. Production of estrogen stimulates proliferation of the endometrium, thinning of cervical mucus, and LH secretion.130
LH levels rise, which increases production of progesterone and inhibin A by the dominant follicle through interaction of LH with LH receptors on granulosa cells. The rise in progesterone occurs 12 to 24 hours before ovulation and elicits a rapid and marked surge in LH secretion, paralleling the mid-cycle FSH peak (see Chapter 2). The LH peak is essential for ovulation, which occurs 28 to 36 hours later.25,33 The mid-cycle surge of LH initiates ovulation by stimulating prostaglandin (PGE and PGF) synthesis, leading to formation of collagenase and other proteolytic enzymes with disruption of the gap junctions between the oocyte and follicular cells.25 The LH surge also increases concentration of maturation promoting factors, which disrupts meiotic inhibition and initiates completion of the first meiotic division.130 The oocyte completes its first meiotic division 10 to 12 hours before ovulation, forming the secondary oocyte (23 chromosomes plus most of the cell cytoplasm) and first polar body (23 chromosomes and minimal cytoplasm). The small polar body is nonfunctional and degenerates (see Chapter 1). The LH surge also causes a decrease in estradiol production.
Ovulation begins with a protrusion or bulge on the ovarian wall. A small avascular spot (stigma) develops, forms a vesicle, and ruptures, extruding the secondary oocyte, follicular fluid, and surrounding cells. Rupture is thought to be caused by enzymatic digestion of the follicular wall via the action of proteases (e.g., collagenase, plasmin, and hyaluronic acid), which dissolve connective tissues.58,103 The oocyte is surrounded by the zona pellucida and corona radiata (radially arranged granulosa cells). The second meiotic division begins with ovulation, then arrests in metaphase.130 The second meiotic division is not completed until fertilization. The oocyte is swept by the fimbriae into the fallopian tube. Muscular contraction of the tube and, primarily, beating of the cilia move the ovum along the tube to the ampulla (the usual site of fertilization). If unfertilized, the ovum usually dies within 24 hours.103
Corpus luteum
After ovulation, the follicular walls and theca collapse inward and become vascularized (Figure 3-3). The granulosa cells undergo a luteinizing process to form the corpus luteum. The corpus luteum secretes progesterone, beginning within 30 to 40 hours of the LH surge. A small amount of estrogen is secreted by the theca cells.25 If fertilization has taken place, implantation occurs during the latter part of this week. Around the time of implantation, the trophoblast tissue secretes human chorionic gonadotropin (hCG), a luteotropin that stimulates the corpus luteum to continue to function. hCG may alter the metabolism of the uterus to prevent the release of substances that result in luteal regression. The corpus luteum can only produce progesterone for about 10 days without hCG stimulation.25 If implantation does not occur, hCG is not produced, the corpus luteum begins to regress, undergoing apoptosis (mediated by uterine leutolytic factors such as prostaglandins), and involution begins.25 The decline in steroid hormones results in menstruation.
The corpus luteum is essential for continuation of the pregnancy until the placenta has developed the capacity to secrete estrogens and progesterone. Removal of the corpus luteum prior to this time usually leads to a miscarriage.130 From 6 to 10 weeks, there is a transition period in which both the placenta and corpus luteum are producing hormones; by 7 weeks the placenta is capable of producing sufficient progesterone to maintain pregnancy if needed. At 6 to 8 weeks, there is a dip in progesterone levels, indicating a decline in corpus luteum functioning. This is followed by a secondary rise in progesterone (presumably as a result of placental takeover) without a rise in the metabolite 17α-hydroxyprogesterone (secreted by the corpus luteum). Around 32 weeks there is a more gradual rise in this metabolite, indicating increased placental utilization of fetal precursors.
Sperm transport
Spermatozoa have not completely differentiated when they are released into the lumen of the seminiferous tubules (see Chapter 1). They are nonmotile and incapable of fertilization. Mature sperm have a condensed and genetically inactive nucleus. Reactivation of the nucleus occurs once the sperm enters the cytoplasm of the ovum.161 Sperm are moved down the seminiferous tubules and through the epididymis and vas deferens by (1) the pressure of additional sperm forming behind them, (2) seminal fluid, and (3) peristaltic action. Biochemical and morphologic maturation of the sperm occurs during their 14 to 21 day passage through the epididymis. Further modifications occur after ejaculation so that the sperm can bind to the zona pellucida of the ovum. Sperm are stored in the vas deferens and epididymis before ejaculation. Ejaculation occurs through the urethra with contraction of the ampulla and the ejaculatory duct upon orgasm.
The volume of ejaculate ranges from 2 to 6 (mean 3.5) mL and usually contains 100 million sperm per mL.103 Men with less than 10 million sperm per mL are likely to be sterile.103 Some spermatozoa are immature, senescent, or abnormal, and generally only the normal and strongest sperm are able to complete the journey within the female reproductive tract to the upper end of the fallopian tube. As sperm move along the epididymis, they begin to gain motility. Sperm become fully motile in the semen after entering the female reproductive tract.161 Semen provides fructose for energy and an alkaline pH for protection against the acid environment of the vagina; it also dilutes the sperm to improve motility. Sperm move at 2 to 3 mm per minute. Motility is slower in the acidic vaginal environment and faster in the alkaline uterine environment.103 Failure of sperm to achieve motility is a cause of male infertility; for potential fertility, at least 40% should be motile by 2 hours after ejaculation.103
The neck and midpiece of the spermatozoa contain a pair of centrioles, the base of the tail apparatus, and the mitochondrial sheath. The mitochondria are arranged in a tight helical spiral around the anterior portion of the flagellum (tail). Mitochondria supply the adenosine triphosphate (ATP) required for independent motility. Sperm must reach the ovum within an allotted time or they exhaust their energy supply and die. Sperm survival in the uterus is relatively short because phagocytosis by leukocytes begins within a few hours. Sperm retain their ability to fertilize the ovum for 1 to 3 days.132 However, most sperm do not survive for more than 48 hours.25,103
Once deposited at the external cervical os, some ejaculated sperm cross the cervical mucus facilitated by a decrease in mucus viscosity at mid-cycle (9 to 16 days), allowing for more rapid migration. Within minutes, these sperm enter the uterine cavity, although some get caught in cervical crypts and endometrial glands. The cervical crypts provide a short-term reservoir or storage site from which sperm are gradually released; this may increase the chance of fertilization.103 Uterine motility, stimulated by prostaglandins in seminal fluid that cause smooth muscle contraction, facilitates initial sperm transport.68 Other sperm move more slowly (2 to 3 mm/hr) or are stored in cervical crypts and slowly released.25
Sperm chemotaxis (organized movement of the sperm toward the ovum) is stimulated by chemoattractants in follicular fluid, and possibly the cumulus oorphus and ovum. Other components of follicular fluid that may also act as chemoattractants include heparin, progesterone, atrial natriuretic peptide, epinephrine, oxytocin, calcitonin, and acetylcholine.44 Capacitated sperm appear to be responsive to a sperm chemotrophic factor and other chemicals released by the follicle ovum and use these substances to “find” the ovum.132
Fertilization
The process of fertilization has been defined in three different ways: (1) the instant of sperm and ovum fusion, (2) time from sperm-ovum fusion to development of the male and female pronuclei, and (3) time from sperm-ovum fusion to the first mitotic division (about 24 hours). Fertilization begins with contact between the sperm and secondary oocyte, arrested in the metaphase of the second meiotic division (see Chapter 1). Fertilization usually occurs in the upper third of the fallopian tube, usually in the ampulla. Before fertilization, the sperm must undergo two final maturational changes: capacitation and the acrosome reaction.
Capacitation involves removal of the glycoprotein coat and seminal plasma proteins from the plasma membrane over the acrosome (head of the sperm), which allows the acrosome reaction to occur. Capacitation takes about 7 hours and usually occurs in the fallopian tubes while the sperm are attached to the tubal epithelial lining, but may begin while the sperm is still in the uterus.25 This process is stimulated by substances in the female genital tract and follicular fluid.58,103 For example, albumin in genital tract secretions stimulates loss of fatty acids and cholesterol from the sperm plasma membrane. This increases permeability of the sperm plasma membrane and initiates capacitation and the acrosome reaction.
Capacitated sperm are chemotaxically active.44 Approximately 2% to 14% of sperm are capacitated at any time, with continued replacement of sperm that lose their capacitation with newly capacitated sperm. Each sperm can only become capacitated once in its lifespan.44 This constant replacement of capacitated sperm extends the time when fertilization is possible by continuous production of “ripe” sperm.44 Thus after ejaculation precapacitated, capacitated, and postcapacitated sperm, as well as sperm that have undergone the acrosomal reaction, can be found within the female genital tract.
Of the millions of sperm in the ejaculate, only up to 300 to 500 sperm are found in the fallopian tubes at any given time.25,130 The ampulla of the ovulatory tube has more sperm than the ampulla of the nonovulatory tube. Although it takes only one sperm to penetrate the ovum, it appears that several hundred are necessary to effect passage of the spermatozoa through the corona radiata to the ovum. The number of spermatozoa that are ejaculated does not appear to influence the number of sperm that enter the fallopian tubes unless very low counts occur.
The acrosome reaction with release of enzymes through small holes in the acrosomal membrane must occur for successful penetration of the corona radiata and zona pellucida by the sperm. The acrosome is a saclike structure on the head of the sperm containing many enzymes, including acid glycohydrases, proteases, phosphatases, esterases, and hyaluronidase.103 Acrosin, a serine protease, may be most important.25 The capacitated sperm binds to the zona pellucid of the ovum, initiating the acrosome reaction (sperm activation).155 The sperm penetrates the zona pellucida and binds to the outer membrane of the oocyte (Figure 3-4).
The zona pellucida is an ovum-specific extracellular membrane composed of three glycoproteins (ZP1, ZP2, ZP3) that act as ligands (molecules that bind to receptors) for sperm receptors. ZP3 mediates sperm binding and the acrosomal reaction.130 Roles of the zona pellucida include sperm activation (acrosome reaction), preventing fertilization by more than one sperm, protecting the ovum before fertilization and protecting the fertilized ovum until shortly before implantation.155 Binding of the sperm to ZP3 is mediated by a sperm surface protein (SED1).132 Once a sperm has bound to ZP3, a zonal reaction occurs with release of lysosomal enzymes. This reaction causes physicochemical alterations in the zona pellucida that make it impenetrable to other sperm.
The sperm head traverses the perivillous space between the plasma membranes and zona pellucida and attaches to the surface of the oocyte, and their plasma membranes fuse. This process is mediated by integrins (adhesion molecules) on the ovum surface along with FERTILINβ (also known as ADAM2), IZUMO and other substances produced by the sperm.132 The head and tail of the sperm enter the oocyte, leaving the outer plasma membrane of the sperm attached to the outer membrane of the oocyte. The ovum has a layer of cortical secretory granules along the inside of its plasma membrane. After sperm entry, the sperm-ovum interaction releases a wave of calcium along the zona pellucida resulting in fusion of the cortical granules with the plasma membrane of the ovum and release of hydrolytic enzymes, proteases, and polysaccharides into the perivillous space.25 This modifies the zona pellucida glycoproteins, preventing activation and entry of other sperm.154
After entering the cytoplasm of the oocyte, the sperm undergoes rapid morphologic changes. The tail of the sperm degenerates and the head enlarges to form the male pronucleus. Each pronucleus has 23 chromosomes (22 autosomes and 1 sex chromosome). Sex of the offspring is determined by the male and depends on whether the sperm that enters the ovum contains an X or Y chromosome. The sperm nucleus becomes reactivated so that it can again synthesize DNA and RNA.161 This processing involves removal of the nuclear membrane with exposure of the sperm chromatin to the cytoplasm of the ovum. The nuclear protein is remodeled and the nucleus decondenses, becoming larger and more spherical. A new nuclear envelope develops, forming the male pronucleus and activating DNA transcription and replication. This is thought to be mediated by factors in the cytoplasm of the ovum. This process takes about 3 to 4 hours, during which the developing male pronucleus gradually approaches the female pronucleus.161
The ovum must be metabolically activated. Entry of the sperm into the ovum triggers two events: (1) the cortical and zonal reactions described earlier, which blocks entry of other sperm; and (2) a transient increase in intracellular calcium accompanied by an increase in oxidative metabolism.25 The increased calcium stimulates the oocyte to complete its second meiotic division with extrusion of the second polar body into the perivitelline space. The nucleus enlarges and is called the female pronucleus. The oocyte is now mature and metabolically active.154 Failure of calcium signaling can lead to complete failure (triploidy) or partial failure (abnormalities of chromosomal number of the second meiotic division, cleavage arrest, and alterations in development of the inner cell mass and trophectoderm [trophoblast]). These alterations can result in implantation failure and miscarriage.143
The female and male pronuclei approach each other, their membranes disintegrate, and the nuclei fuse (see Figure 3-4). Chromatin strands intermingle, and the diploid number (46) of chromosomes is restored. The zygote (from the Greek, meaning “yoked together”) is formed, and mitotic division (cleavage) begins. The zygote measures 0.2 mm in diameter and carries the genetic material necessary to create a unique human being. Fertilization results in species variation, with half of the chromosomes coming from the mother and half from the father, mixing the genes each parent originally received from their parents.103
Cleavage and zygote transport
Cleavage involves a series of rapid mitotic cell divisions that begins with the first mitotic division of the zygote and ends with formation of the blastocyst. Cleavage is under the control of mitosis-promoting or maturation-promoting factor (MPF).25 The zygote divides into two daughter cells (blastomeres) about 30 hours after fertilization; each of these cells divides into two smaller cells, which also divide, and so forth (see Figure 3-3). The dividing cells are contained by the zona pellucida and become progressively smaller with each subsequent division, with no change in the total mass of the zygote. The trophoblast secretes an immunosuppressant protein called early pregnancy factor (EPF) by 24 to 48 hours after fertilization. Pregnancy tests within the first 10 days after fertilization use EPF in maternal serum.103
Cell division occurs every 12 to 24 hours. By 3 to 4 days, the zygote has divided into 8 to 16 blastomeres. Around the eight- to nine-cell stage, the blastomeres realign and form a tight ball of cells mediated by cell surface adhesion glycoproteins. This process, called compaction, allows increased interaction between cells needed for formation of the inner cell mass. This occurs via gap and tight junctions.25
The zygote remains in the ampulla for the first 24 hours, then is propelled down the fallopian tube by ciliary action over the next few days. At the 12- to 16-cell stage (about 3 days after fertilization), the zygote becomes a solid cluster of cells called the morula (from the Latin word for “mulberry,” which it resembles).25 The zygote reaches the uterine cavity 3 to 4 days after fertilization (about 90 hours or 5 days after follicle rupture). Development is now under control of the embryonic genome. Fluid (which provides nutrients) from the uterine cavity enters the morula as the blastocyst is formed.
The blastocyst consists of four distinct components: (1) zona pellucida, a thick glycoprotein membrane that is beginning to stretch and thin; (2) trophectoderm (trophoblast), a one-cell-thick outer layer of flattened cells that will form the placenta and chorion; (3) inner cell mass (embryoblast), a one- or two-cell-thick, crescent-shaped cluster of cells that will form the embryo; and (4) fluid-filled blastocyst cavity.103 The zona pellucida protects the zygote from adhering to the mucosa of the fallopian tube and from rejection by the maternal immune system (see Chapter 13). Position of individual cells and gene transcription factors influence which cells become trophoblast and which become inner cell mass. For example, Oct4 and Nanog are transcription factors found in all blastomeres in the morula. In the cells that become the inner cell mass, these transcription factors continue to be expressed, but are turned off in the cells of the future trophoblast.132 If these gene transcription factors are deficient, all or most of the cells in the blastocyst become part of the trophectoderm, resulting in a molar pregnancy (see Gestational Trophoblast Disease). The blastocyst floats free in the uterine cavity from 90 to 150 hours after ovulation, then begins to implant 6 to 7 days after fertilization (Figure 3-5).
Genetic control of development
Embryonic development combines growth, differentiation, and organization of cellular components at all levels. As development progresses, differential synthesis is established, resulting in cellular differentiation. Growth is the process of creating more of a substance that is already present through increase in cell size and number. In contrast, differentiation is the creation of new types of substances, cells, tissues, and organs that were not previously present. Organization is the process by which these elements are coordinated into functional integrated units. Morphogenesis is the production of a special form, shape, or structure of a cell or group of cells and occurs by the precise organization of cell populations into distinct organs.71
The mechanisms controlling morphogenesis are complex and incompletely understood.25 Development is controlled by developmental gene families within the embryo.21,25,31,52,85 Much of the knowledge of developmental genes to date comes from animal models. Often the names of these genes or their products reflect characteristics of the animals or situations in which they were first identified. Developmental genes control the definition of body axes (ventral/dorsal, anterior/posterior, left/right, medial/dorsal)—and the arrangement of different cells to form tissues and organogenesis.151 These processes involve the coordination of signaling molecules and other proteins, DNA transcription factors, extracellular matrix components, enzymes, and transport systems.40,73 Development genes are involved in cell differentiation and proliferation into adulthood and if later altered can lead to malignancies.56 Imprinted genes (see box on p. 9) are also important in prenatal and placental development as well as in development and function of metabolic processes.121
Although the first cell divisions after fertilization are under maternal genetic control, by the two- to four-cell stage, the embryonic genome is activated and is producing many intercellular signaling proteins and transcription factors.40,61 Transcription factors are proteins that turn other genes on and off, thus controlling expression of these genes. Positive feedback induces further production of regulatory proteins and gene transcription factors that influence that cell or other target cells. Negative feedback results in the production of inhibitors. This process is controlled by interactions of developmental genes with environmental factors that turn the gene on and off at precise intervals. Each gene can produce multiple isotypes, each isotype producing a different product. The different isotypes are produced by the splicing and reorganization of exons within a given gene (see Figure 1-3).85 As a result, a single gene can guide the production of many different forms of mRNA and formation of proteins with unique biologic functions. Thus individual developmental genes may have different functions at different stages of development and with development of specific organs.21,25,31,52
Developmental genes produce signaling molecules and transcription factors. Transcription factors remain within the cell and bind to DNA at the promoter or enhancer regions of specific genes or regulate mRNA production.25,56 Often a cascade is set up, wherein the transcription factor turns on various genes which in turn can regulate other genes. Initially these regulatory genes send out signals that induce expression of other genes, which in turn induce expression of still other genes and so forth until genes that encode development of specific structures or functions of cells or tissues within the embryo are expressed.132 The proteins produced regulate cell activities, such as causing a cell to differentiate in a specific way, and are modulated by positive and negative feedback loops.52 Examples of developmental gene families include the homeobox (HOX) and PAX gene families. For example, the HOX genes are involved with craniocaudal organization; the PAX gene family is involved with development of the urogenital system, central nervous system (CNS), thyroid gland, and eye, among other sites.25
Intercellular signaling molecules (first messengers), many of which are growth factors, influence other cells by binding to receptor molecules (Figure 3-6). Signaling molecules act in a paracrine fashion, that is, they are secreted into the spaces surrounding the cell where the molecules are produced and diffuse between cells in that area.73,130 These molecules act to bind (as ligands) to receptor molecules on cell membranes. After a series of protein interactions, a transcription factor is activated and a signal is sent to the cell nucleus and a gene is expressed resulting in production of specific proteins needed to guide development.25,130 Receptor molecules can be intracellular or on the cell surface. Extracellular receptors are binding sites for ligands (hormones, growth factor, or cytokine). Binding to the receptor alters the receptor and stimulates an intercellular response (signal transduction) either directly via a protein kinase or indirectly via a second messenger such as cyclic adenosine monophosphate (cAMP). Signaling molecules may also act by inhibiting other signaling molecules.25 Justacine signaling also occurs via three mechanisms: (1) interaction of a protein on one cell membrane with a receptor on the surface of another cell; (2) via gap junctions (see Chapter 4 for a discussion of gap junctions), or (3) interaction of extracellular matrix ligands (collagen, proteoglycans, fibronectin, and laminin) with receptors on neighboring cells.130Major signaling pathways are Wnt, Hedgehog, transforming growth factor β (TGF-β) family, tyrosine kinase, Notch, Integrin, and retinoic acid signaling.73,130,132 The Wnt family is involved in the dorsal/ventral axis and formation of the midbrain, muscles, gonads, and kidneys. Alterations are associated with tumors and possibly congenital anomalies.73 The hedgehog family, such as the sonic hedgehog (Shh) gene, is involved in patterning of many tissues and organs, including axis formation, motor neuron induction, somite differentiation, neural tube induction and patterning, and limb patterning.25,56,130,132 Mutations are associated with central nervous system (e.g., holoprosencephaly), axial skeletal, and limb abnormalities as well as with some basal cell carcinomas.25,73 The TGF-β family includes TGF-β, which is important in mesoderm induction and myoblast proliferation, activin (granulose cell proliferation, mesoderm induction), inhibin (inhibition of gonadotropin secretion by the pituitary), müllerian inhibitory substance (regression of the paramesonephric duct (see Chapter 1), bone morphogenetic proteins, and decapentaplegic (limb development).21,25,31,52,130,132 The TGF-β family is also involved in angiogenesis, axon growth, mesoderm differentiation and epithelial branching in the lung and kidneys.130 Defects in TGFβ signaling can lead to vascular and skeletal disorders as well as pulmonary hypertension and cancer.132
Tyrosine kinase signaling involves growth factors (GFs) such as fibroblast growth factor (FGF), epidermal GF (EGF), insulin-like GFs, platelet derived GFs, and vascular endothelial GF.130,132 FGFs are found in bone, so mutations in FGF receptor genes can lead to skeletal dysplasia and disorders such as achondroplasia, Crouzon syndrome, Apert syndrome, and some forms of craniosyntosis.73 Defects in Notch signaling can lead to skeletal disorders such as Alagille syndrome and spondylocostal dysostosis as well as cancers such as T-cell acute lymphoblastic leukemia.132 Integrins are receptors that are involved in linking the extracellular matrix and cell cytoskeleton and in signal transduction that can lead to changes in cell size, shape, and position.132 Defective integrin signaling can lead to alterations in skin and connective tissue such as epidermolysis bullosa and cancers of the breast, intestine, and reproductive organs.132 Retinoic acid (derived from vitamin A) acts as a morphogen, which is “a diffusible substance that determines cell fate during development in a concentration-dependent manner.”132, p. 161
Alterations in developmental genes and their products can result in congenital anomalies through various mechanisms.41 Mechanical failures involve defects in structural genes such as collagen resulting in qualitative or quantitative differences. For example, collagen mutations are seen in osteogenesis imperfecta, Apert syndrome, and epidermolysis bullosa; a fibrillin defect is seen in Marfan syndrome. Alterations in cell numbers due to regulatory failures can lead to overgrowth, such as occurs in Beckwith-Wiedemann syndrome, or undergrowth, such as occurs with some forms of microcephaly. Failure of cell migration during development leads to anomalies such as lissencephaly (failure of neuronal migration) or Hirschsprung’s disease (failure of neural crest cells to migrate and form ganglia). Failure of the developmental switch, turning genes on and off, can upset the development timetable and also lead to defects.41
Mechanisms of morphogenesis
Cell differentiation
Initially all cells are similar and unspecialized, but each must eventually become 1 of 350 different cell types found in the human body.139 Cells pass through two phases in order to become specialized. In the first phase (determination), the cell becomes restricted in its developmental capabilities and loses the ability to develop in alternative ways. Cell determination occurs for the first time in the blastocyst, with formation of the inner cell mass (which forms the embryo) and trophoblast (which becomes the placenta). In the second phase (differentiation), cells develop distinctive morphologic and functional characteristics. Initially, cell position determines the fate of the cell. Specific differentiation is regulated by interactions between cell populations and is controlled by HOX and other gene families that are switched on to produce specific signaling molecules in a sequential manner.139 Cell differentiation often involves induction (see below) in which one tissue signals (induces) a second tissue (responder) to differentiate into a specific structure. Signals are sent between cells in both directions (cross-talk) to complete the differentiation.130
Induction
Induction is the process by which cells in one part of the embryo influence cells around them to develop in a specific way. Induction requires inductors, or cells that stimulate reactions in surrounding cells via signal transduction and induced tissue, which is made up of cells that have the capacity or competence to respond to these protein signals via cell membrane receptor molecules (see Figure 3-6). At some point, inductors and inducers lose their ability to perform these actions.31
Secondary induction is a cascade of developmental events and is a common way many parts of the embryo are formed. For example, in the nervous system, the notochord is a primary inductor or organizer for brain development. The forebrain reacts to secondary inductors in the mesoderm to form the optic cup, which then induces adjacent ectoderm to form the eye lens. The eye lens then induces epidermis around it to form the corneal epithelium. Other examples of induction are in the formation of the gastrointestinal system, where the gut endoderm induces the surrounding mesenchyme to differentiate into organs such as the liver or pancreas (see Chapter 12) or in the renal system where the utereic bud causes the surrounding mesenchyme to become nephrons (see Chapter 11).130
If any of these steps is interfered with, the next stage in development may not occur, or it may occur abnormally. If these alterations occur early in the developmental sequence, complete organ agenesis may result.103 An example of chemical pathway activity during secondary induction is the interaction of activin and TGF-β, which influences branching of epithelial tubes in the kidney, pancreas, and salivary glands.85,130
Programmed cell death
Programmed cell death or apoptosis is a precisely timed event—under genetic control and cell feedback mechanisms—that occurs in many of the embryonic tissues as part of normal development. The process involves the release of lysosomal hydrolytic enzymes that dissolve cells, thereby altering the tissues. This mechanism is responsible for lumen formation in solid tubes (trachea and parts of the gut) and the disappearance of the webbing between the fingers and toes. If enzyme release is inhibited, syndactyly, some forms of bowel atresia, or imperforate anus may result. If enzyme activity is increased, micromelia (shortened limbs) may result.139
Cell migration
During development some cells move around in a fashion similar to that of an amoeba. This process is dependent on microtubular and microfilament elongation and contraction. Migration involves the elongation of the leading edge of the cell, followed by adhesion of the cell to a new contact point. Contraction of the cell toward the new adhesion site results in movement of the cell. Alterations or interference may limit cell migration and result in a defect. Hirschsprung’s disease (absence of intestinal ganglion cells) results from failure of neural crest cells to migrate. From 3 to 6 months’ gestation, millions of neurons and glial cells within the central nervous system migrate from their point of origin in the periventricular area to their eventual loci in the cerebrum and cerebellum. Alterations in this migration can result in alterations in CNS organization and function (see Chapter 15).103,139
Overview of embryonic development
Preembryonic development occurs from the time of conception and zygote formation until 2 weeks’ gestation. By the time of implantation, the inner cell mass consists of 12 to 15 cells. At about 7 days, the first of three germ cell layers that give rise to the embryo—the hypoblast, or primitive endoderm—appears.25 During the second week the bilaminar embryo develops as the inner cell mass differentiates to form the epiblast along the inner part of the amniotic cavity.
The developing organism appears as a flat disk with a connecting stalk that will become part of the umbilical cord. Cytotrophoblast cells around the inner wall of the blastocyst cavity form the primitive yolk sac (see Figure 3-5, C) and extraembryonic coelom, which serves as transfer interface and nutrient reservoir.69 Connective tissue (extraembryonic mesoderm) fills in the space between the cytotrophoblast cells and the extraembryonic coelomic membrane. Near the end of the second week, cavities appear in the extraembryonic mesoderm and fuse to form the extraembryonic coelom. A secondary yolk sac (see Figure 3-5, D) develops from the primary sac (which gradually disintegrates) and provides for early nutrition of the embryo. Part of it is eventually incorporated into the primitive gut. The fluid in this cavity is an ultrafiltrate of maternal serum with placental and secondary yolk sac products.69 An endodermal cell thickening (prochordal plate) appears at one end of the disk and is the future site of the mouth and cranial region.
The embryonic period lasts from 2 weeks after fertilization until the end of the eighth week. This period is the time of organogenesis. Figure 3-7 summarizes the major stages in development of specific organ systems. Development of specific organ systems is described in detail in Chapters 8 to 20. This section provides an overview of major events during embryogenesis.
