Langman's Medical Embryology PDF

Contents part one General Embryology........................................... 1 chapter 1 Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes.................

Contents part one General Embryology........................................... 1 chapter 1 Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes.......................................................... 3 chapter 2 First Week of Development: Ovulation to Implantation................... 31 chapter 3 Second Week of Development: Bilaminar Germ Disc...................... 51 chapter 4 Third Week of Development: Trilaminar Germ Disc....................... 65 chapter 5 Third to Eighth Week: The Embryonic Period............................... 87 chapter 6 Third Month to Birth: The Fetus and Placenta............................... 117 chapter 7 Birth Defects and Prenatal Diagnosis........................................ 149 part two Special Embryology............................................ 169 chapter 8 Skeletal System.................................................................. 171 ix x Contents chapter 9 Muscular System................................................................ 199 chapter 10 Body Cavities.................................................................... 211 chapter 11 Cardiovascular System......................................................... 223 chapter 12 Respiratory System............................................................. 275 chapter 13 Digestive System................................................................ 285 chapter 14 Urogenital System.............................................................. 321 chapter 15 Head and Neck.................................................................. 363 chapter 16 Ear................................................................................. 403 chapter 17 Eye................................................................................ 415 chapter 18 Integumentary System.......................................................... 427 chapter 19 Central Nervous System........................................................ 433 part three Appendix......................................................... 483 Answers to Problems........................................................... 485 Figure Credits................................................................... 499 Index.............................................................................. 507  Preface The ninth edition of Langman’s Medical Embryology adheres to the tradition established by the original publication—it provides a concise but thorough de- scription of embryology and its clinical significance, an awareness of which is essential in the diagnosis and prevention of birth defects. Recent advances in ge- netics, developmental biology, maternal-fetal medicine, and public health have significantly increased our knowledge of embryology and its relevance. Because birth defects are the leading cause of infant mortality and a major contributor to disabilities, and because new prevention strategies have been developed, under- standing the principles of embryology is important for health care professionals. To accomplish its goal, Langman’s Medical Embryology retains its unique ap- proach of combining an economy of text with excellent diagrams and scanning electron micrographs. It reinforces basic embryologic concepts by providing numerous clinical examples that result from abnormalities in developmental processes. The following pedagogic features and updates in the ninth edition help facilitate student learning: Organization of Material: Langman’s Medical Embryology is organized into two parts. The first provides an overview of early development from gametogenesis through the embryonic period; also included in this section are chapters on placental and fetal development and prenatal diagnosis and birth defects. The second part of the text provides a description of the fundamental processes of embryogenesis for each organ system. Molecular Biology: New information is provided about the molecular basis of normal and abnormal development. Extensive Art Program: This edition features almost 400 illustrations, includ- ing new 4-color line drawings, scanning electron micrographs, and ultrasound images. Clinical Correlates: In addition to describing normal events, each chapter con- tains clinical correlates that appear in highlighted boxes. This material is de- signed to provide information about birth defects and other clinical entities that are directly related to embryologic concepts. vii viii Preface Summary: At the end of each chapter is a summary that serves as a concise review of the key points described in detail throughout the chapter. Problems to Solve: These problems test a student’s ability to apply the infor- mation covered in a particular chapter. Detailed answers are provided in an appendix in the back of the book. Simbryo: New to this edition, Simbryo, located in the back of the book, is an interactive CD-ROM that demonstrates normal embryologic events and the origins of some birth defects. This unique educational tool offers six original vector art animation modules to illustrate the complex, three-dimensional as- pects of embryology. Modules include normal early development as well as head and neck, cardiovascular, gastrointestinal, genitourinary, and pulmonary system development. Connection Web Site: This student and instructor site (http://connection. LWW.com/go/sadler) provides updates on new advances in the field and a syl- labus designed for use with the book. The syllabus contains objectives and definitions of key terms organized by chapters and the “bottom line,” which provides a synopsis of the most basic information that students should have mastered from their studies. I hope you find this edition of Langman’s Medical Embryology to be an excellent resource. Together, the textbook, CD, and connection site provide a user-friendly and innovative approach to learning embryology and its clinical relevance. T. W. Sadler Twin Bridges, Montana p a r t o n e General Embryology 1  c h a p t e r 1 Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes Primordial Germ Cells Development begins with fertilization, the pro- cess by which the male gamete, the sperm, and the female gamete, the oocyte, unite to give rise to a zygote. Gametes are derived from primordial germ cells (PGCs) that are formed in the epiblast during the second week and that move to the wall of the yolk sac (Fig. 1.1). During the fourth week these cells begin to migrate from the yolk sac toward the developing gonads, where they arrive by the end of the fifth week. Mitotic divisions increase their number during their migration and also when they arrive in the gonad. In preparation for fertilization, germ cells undergo gametogenesis, which includes meiosis, to reduce the number of chromosomes and cytodifferentiation to complete their maturation. CLINICAL CORRELATE Primordial Germ Cells (PGCs) and Teratomas Teratomas are tumors of disputed origin that often contain a variety of tissues, such as bone, hair, muscle, gut epithelia, and others. It is thought that these tumors arise from a pluripotent stem cell that can differentiate into any of the three germ layers or their derivatives. 3 4 Part One: General Embryology Figure 1.1 An embryo at the end of the third week, showing the position of primordial germ cells in the wall of the yolk sac, close to the attachment of the future umbilical cord. From this location, these cells migrate to the developing gonad. Some evidence suggests that PGCs that have strayed from their normal mi- gratory paths could be responsible for some of these tumors. Another source is epiblast cells migrating through the primitive streak during gastrulation (see page 80). The Chromosome Theory of Inheritance Traits of a new individual are determined by specific genes on chromosomes inherited from the father and the mother. Humans have approximately 35,000 genes on 46 chromosomes. Genes on the same chromosome tend to be inher- ited together and so are known as linked genes. In somatic cells, chromosomes appear as 23 homologous pairs to form the diploid number of 46. There are 22 pairs of matching chromosomes, the autosomes, and one pair of sex chro- mosomes. If the sex pair is XX, the individual is genetically female; if the pair is XY, the individual is genetically male. One chromosome of each pair is derived from the maternal gamete, the oocyte, and one from the paternal gamete, the Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 5 sperm. Thus each gamete contains a haploid number of 23 chromosomes, and the union of the gametes at fertilization restores the diploid number of 46. MITOSIS Mitosis is the process whereby one cell divides, giving rise to two daughter cells that are genetically identical to the parent cell (Fig. 1.2). Each daughter cell receives the complete complement of 46 chromosomes. Before a cell enters mitosis, each chromosome replicates its deoxyribonucleic acid (DNA). During this replication phase the chromosomes are extremely long, they are spread diffusely through the nucleus, and they cannot be recognized with the light mi- croscope. With the onset of mitosis the chromosomes begin to coil, contract, and condense; these events mark the beginning of prophase. Each chromo- some now consists of two parallel subunits, chromatids, that are joined at a narrow region common to both called the centromere. Throughout prophase the chromosomes continue to condense, shorten, and thicken (Fig. 1.2A), but only at prometaphase do the chromatids become distinguishable (Fig. 1.2B). During metaphase the chromosomes line up in the equatorial plane, Figure 1.2 Various stages of mitosis. In prophase, chromosomes are visible as slen- der threads. Doubled chromatids become clearly visible as individual units during metaphase. At no time during division do members of a chromosome pair unite. Blue, paternal chromosomes; red, maternal chromosomes. 6 Part One: General Embryology and their doubled structure is clearly visible (Fig. 1.2C ). Each is attached by microtubules extending from the centromere to the centriole, forming the mi- totic spindle. Soon the centromere of each chromosome divides, marking the beginning of anaphase, followed by migration of chromatids to opposite poles of the spindle. Finally, during telophase, chromosomes uncoil and lengthen, the nuclear envelope reforms, and the cytoplasm divides (Fig. 1.2, D and E ). Each daughter cell receives half of all doubled chromosome material and thus maintains the same number of chromosomes as the mother cell. MEIOSIS Meiosis is the cell division that takes place in the germ cells to generate male and female gametes, sperm and egg cells, respectively. Meiosis requires two cell divisions, meiosis I and meiosis II, to reduce the number of chromosomes to the haploid number of 23 (Fig. 1.3). As in mitosis, male and female germ cells (spermatocytes and primary oocytes) at the beginning of meiosis I replicate their DNA so that each of the 46 chromosomes is duplicated into sister chro- matids. In contrast to mitosis, however, homologous chromosomes then align themselves in pairs, a process called synapsis. The pairing is exact and point for point except for the XY combination. Homologous pairs then separate into two daughter cells. Shortly thereafter meiosis II separates sister chromatids. Each gamete then contains 23 chromosomes. Crossover Crossovers, critical events in meiosis I, are the interchange of chromatid seg- ments between paired homologous chromosomes (Fig. 1.3C ). Segments of chromatids break and are exchanged as homologous chromosomes separate. As separation occurs, points of interchange are temporarily united and form an X-like structure, a chiasma (Fig. 1.3C ). The approximately 30 to 40 crossovers (one or two per chromosome) with each meiotic I division are most frequent between genes that are far apart on a chromosome. As a result of meiotic divisions, (a) genetic variability is enhanced through crossover, which redistributes genetic material, and through random distribu- tion of homologous chromosomes to the daughter cells; and (b) each germ cell contains a haploid number of chromosomes, so that at fertilization the diploid number of 46 is restored. Polar Bodies Also during meiosis one primary oocyte gives rise to four daughter cells, each with 22 plus 1 X chromosomes (Fig. 1.4A). However, only one of these develops into a mature gamete, the oocyte; the other three, the polar bodies, receive little cytoplasm and degenerate during subsequent development. Similarly, one primary spermatocyte gives rise to four daughter cells, two with 22 plus 1 Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 7 Figure 1.3 First and second meiotic divisions. A. Homologous chromosomes approach each other. B. Homologous chromosomes pair, and each member of the pair consists of two chromatids. C. Intimately paired homologous chromosomes interchange chromatid fragments (crossover). Note the chiasma. D. Double-structured chromosomes pull apart. E. Anaphase of the first meiotic division. F and G. During the second meiotic division, the double-structured chromosomes split at the centromere. At completion of division, chromosomes in each of the four daughter cells are different from each other. X chromosomes and two with 22 plus 1 Y chromosomes (Fig. 1.4B ). However, in contrast to oocyte formation, all four develop into mature gametes. CLINICAL CORRELATES Birth Defects and Spontaneous Abortions: Chromosomal and Genetic Factors Chromosomal abnormalities, which may be numerical or structural, are important causes of birth defects and spontaneous abortions. It is estimated that 50% of conceptions end in spontaneous abortion and that 50% of these 8 Part One: General Embryology Figure 1.4 Events occurring during the first and second maturation divisions. A. The primitive female germ cell (primary oocyte) produces only one mature gamete, the ma- ture oocyte. B. The primitive male germ cell (primary spermatocyte) produces four sper- matids, all of which develop into spermatozoa. abortuses have major chromosomal abnormalities. Thus approximately 25% of conceptuses have a major chromosomal defect. The most common chro- mosomal abnormalities in abortuses are 45,X (Turner syndrome), triploidy, and trisomy 16. Chromosomal abnormalities account for 7% of major birth defects, and gene mutations account for an additional 8%. Numerical Abnormalities The normal human somatic cell contains 46 chromosomes; the normal ga- mete contains 23. Normal somatic cells are diploid, or 2n; normal gametes are haploid, or n. Euploid refers to any exact multiple of n, e.g., diploid or triploid. Aneuploid refers to any chromosome number that is not euploid; it is usually applied when an extra chromosome is present (trisomy) or when one is missing (monosomy). Abnormalities in chromosome number may origi- nate during meiotic or mitotic divisions. In meiosis, two members of a pair of homologous chromosomes normally separate during the first meiotic divi- sion so that each daughter cell receives one member of each pair (Fig. 1.5A). Sometimes, however, separation does not occur (nondisjunction), and both members of a pair move into one cell (Fig. 1.5, B and C ). As a result of nondisjunction of the chromosomes, one cell receives 24 chromosomes, and the other receives 22 instead of the normal 23. When, at fertiliza- tion, a gamete having 23 chromosomes fuses with a gamete having 24 or Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 9 Figure 1.5 A. Normal maturation divisions. B. Nondisjunction in the first meiotic divi- sion. C. Nondisjunction in the second meiotic division. 22 chromosomes, the result is an individual with either 47 chromosomes (trisomy) or 45 chromosomes (monosomy). Nondisjunction, which occurs during either the first or the second meiotic division of the germ cells, may involve the autosomes or sex chromosomes. In women, the incidence of chromosomal abnormalities, including nondisjunction, increases with age, especially at 35 years and older. Occasionally nondisjunction occurs during mitosis (mitotic nondisjunc- tion) in an embryonic cell during the earliest cell divisions. Such conditions produce mosaicism, with some cells having an abnormal chromosome num- ber and others being normal. Affected individuals may exhibit few or many of the characteristics of a particular syndrome, depending on the number of cells involved and their distribution. Sometimes chromosomes break, and pieces of one chromosome attach to another. Such translocations may be balanced, in which case breakage and reunion occur between two chromosomes but no critical genetic material is lost and individuals are normal; or they may be unbalanced, in which case part of one chromosome is lost and an altered phenotype is produced. For example, unbalanced translocations between the long arms of chromosomes 14 and 21 during meiosis I or II produce gametes with an extra copy of chro- mosome 21, one of the causes of Down syndrome (Fig. 1.6). Translocations 10 Part One: General Embryology A 14 21 t(14;21) Figure 1.6 A. Translocation of the long arms of chromosomes 14 and 21 at the cen- tromere. Loss of the short arms is not clinically significant, and these individuals are clinically normal, although they are at risk for producing offspring with unbalanced translocations. B. Karyotype of translocation of chromosome 21 onto 14, resulting in Down syndrome. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 11 Figure 1.7 Karyotype of trisomy 21 (arrow), Down syndrome. are particularly common between chromosomes 13, 14, 15, 21, and 22 be- cause they cluster during meiosis. TRISOMY 21 (DOWN SYNDROME) Down syndrome is usually caused by an extra copy of chromosome 21 (tri- somy 21, Fig. 1.7). Features of children with Down syndrome include growth retardation; varying degrees of mental retardation; craniofacial abnormalities, including upward slanting eyes, epicanthal folds (extra skin folds at the medial corners of the eyes), flat facies, and small ears; cardiac defects; and hypotonia (Fig. 1.8). These individuals also have relatively high incidences of leukemia, infections, thyroid dysfunction, and premature aging. Furthermore, nearly all develop signs of Alzheimer’s disease after age 35. In 95% of cases, the syndrome is caused by trisomy 21 resulting from meiotic nondisjunction, and in 75% of these instances, nondisjunction occurs during oocyte formation. The incidence of Down syndrome is approximately 1 in 2000 conceptuses for women under age 25. This risk increases with maternal age to 1 in 300 at age 35 and 1 in 100 at age 40. In approximately 4% of cases of Down syndrome, there is an unbal- anced translocation between chromosome 21 and chromosome 13, 14, or 15 (Fig. 1.6). The final 1% are caused by mosaicism resulting from mitotic 12 Part One: General Embryology Figure 1.8 A and B. Children with Down syndrome, which is characterized by a flat, broad face, oblique palpebral fissures, epicanthus, and furrowed lower lip. C. Another characteristic of Down syndrome is a broad hand with single transverse or simian crease. Many children with Down syndrome are mentally retarded and have congenital heart abnormalities. nondisjunction. These individuals have some cells with a normal chromo- some number and some that are aneuploid. They may exhibit few or many of the characteristics of Down syndrome. TRISOMY 18 Patients with trisomy 18 show the following features: mental retardation, con- genital heart defects, low-set ears, and flexion of fingers and hands (Fig. 1.9). In addition, patients frequently show micrognathia, renal anomalies, syndactyly, and malformations of the skeletal system. The incidence of this condition is approximately 1 in 5000 newborns. Eighty-five percent are lost between 10 weeks of gestation and term, whereas those born alive usually die by age 2 months. TRISOMY 13 The main abnormalities of trisomy 13 are mental retardation, holo- prosencephaly, congenital heart defects, deafness, cleft lip and palate, and eye defects, such as microphthalmia, anophthalmia, and coloboma (Fig. 1.10). The incidence of this abnormality is approximately 1 in 20,000 live births, and over 90% of the infants die in the first month after birth. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 13 Figure 1.9 Photograph of child with trisomy 18. Note the prominent occiput, cleft lip, micrognathia, low-set ears, and one or more flexed fingers. Figure 1.10 A. Child with trisomy 13. Note the cleft lip and palate, the sloping forehead, and microphthalmia. B. The syndrome is commonly accompanied by polydactyly. KLINEFELTER SYNDROME The clinical features of Klinefelter syndrome, found only in males and usually detected at puberty, are sterility, testicular atrophy, hyalinization of the semi- niferous tubules, and usually gynecomastia. The cells have 47 chromosomes with a sex chromosomal complement of the XXY type, and a sex chromatin body (Barr body: formed by condensation of an inactivated sex chromo- some; a Barr body is also present in normal females) is found in 80% of cases (Fig. 1.11). The incidence is approximately 1 in 500 males. Nondisjunction of the XX homologues is the most common causative event. Occasionally, pa- tients with Klinefelter syndrome have 48 chromosomes: 44 autosomes and four sex chromosomes (XXXY). Although mental retardation is not generally 14 Part One: General Embryology Figure 1.11 Patient with Klinefelter syndrome showing normal phallus development but gynecomastia (enlarged breasts). part of the syndrome, the more X chromosomes there are, the more likely there will be some degree of mental impairment. TURNER SYNDROME Turner syndrome, with a 45,X karyotype, is the only monosomy compat- ible with life. Even then, 98% of all fetuses with the syndrome are sponta- neously aborted. The few that survive are unmistakably female in appearance (Fig. 1.12) and are characterized by the absence of ovaries (gonadal dysgen- esis) and short stature. Other common associated abnormalities are webbed neck, lymphedema of the extremities, skeletal deformities, and a broad chest with widely spaced nipples. Approximately 55% of affected women are mono- somic for the X and chromatin body negative because of nondisjunction. In 80% of these women, nondisjunction in the male gamete is the cause. In the remainder of women, structural abnormalities of the X chromosome or mitotic nondisjunction resulting in mosaicism are the cause. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 15 Figure 1.12 Patient with Turner syndrome. The main characteristics are webbed neck, short stature, broad chest, and absence of sexual maturation. TRIPLE X SYNDROME Patients with triple X syndrome are infantite, with scanty menses and some degree of mental retardation. They have two sex chromatin bodies in their cells. Structural Abnormalities Structural chromosome abnormalities, which involve one or more chro- mosomes, usually result from chromosome breakage. Breaks are caused by environmental factors, such as viruses, radiation, and drugs. The result of breakage depends on what happens to the broken pieces. In some cases, the broken piece of a chromosome is lost, and the infant with partial deletion of a chromosome is abnormal. A well-known syndrome, caused by partial dele- tion of the short arm of chromosome 5, is the cri-du-chat syndrome. Such children have a catlike cry, microcephaly, mental retardation, and congenital heart disease. Many other relatively rare syndromes are known to result from a partial chromosome loss. Microdeletions, spanning only a few contiguous genes, may result in microdeletion syndrome or contiguous gene syndrome. Sites where these deletions occur, called contiguous gene complexes, can be identified by high-resolution chromosome banding. An example of a microdeletion 16 Part One: General Embryology Figure 1.13 Patient with Angelman syndrome resulting from a microdeletion on mater- nal chromosome 15. If the defect is inherited on the paternal chromosome, Prader-Willi syndrome occurs (Fig. 1.14). occurs on the long arm of chromosome 15 (15q11–15q13). Inheriting the deletion on the maternal chromosome results in Angelman syndrome, and the children are mentally retarded, cannot speak, exhibit poor motor devel- opment, and are prone to unprovoked and prolonged periods of laughter (Fig. 1.13). If the defect is inherited on the paternal chromosome, Prader-Willi syndrome is produced; affected individuals are characterized by hypotonia, obesity, mental retardation, hypogonadism, and cryptorchidism (Fig. 1.14). Characteristics that are differentially expressed depending upon whether the genetic material is inherited from the mother or the father are examples of genomic imprinting. Other contiguous gene syndromes may be inherited from either parent, including Miller-Dieker syndrome (lissencephaly, devel- opmental delay, seizures, and cardiac and facial abnormalities resulting from a deletion at 17p13) and most cases of velocardiofacial (Shprintzen) syndrome (palatal defects, conotruncal heart defects, speech delay, learning disorders, and schizophrenia-like disorder resulting from a deletion in 22q11). Fragile sites are regions of chromosomes that demonstrate a propensity to separate or break under certain cell manipulations. For example, fragile sites can be revealed by culturing lymphocytes in folate-deficient medium. Although numerous fragile sites have been defined and consist of CGG re- peats, only the site on the long arm of the X chromosome (Xq27) has been Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 17 Figure 1.14 Patient with Prader-Willi syndrome resulting from a microdeletion on pater- nal chromosome 15. If the defect is inherited on the maternal chromosome, Angelman syndrome occurs (Fig. 1.13). correlated with an altered phenotype and is called the fragile X syndrome. Fragile X syndrome is characterized by mental retardation, large ears, promi- nent jaw, and pale blue irides. Males are affected more often than females (1/1000 versus 1/2000), which may account for the preponderance of males among the mentally retarded. Fragile X syndrome is second only to Down syndrome as a cause of mental retardation because of chromosomal abnor- malities. Gene Mutations Many congenital formations in humans are inherited, and some show a clear mendelian pattern of inheritance. Many birth defects are directly attributable to a change in the structure or function of a single gene, hence the name single gene mutation. This type of defect is estimated to account for approximately 8% of all human malformations. 18 Part One: General Embryology With the exception of the X and Y chromosomes in the male, genes exist as pairs, or alleles, so that there are two doses for each genetic determinant, one from the mother and one from the father. If a mutant gene produces an abnormality in a single dose, despite the presence of a normal allele, it is a dominant mutation. If both alleles must be abnormal (double dose) or if the mutation is X-linked in the male, it is a recessive mutation. Gradations in the effects of mutant genes may be a result of modifying factors. The application of molecular biological techniques has increased our knowledge of genes responsible for normal development. In turn, genetic analysis of human syndromes has shown that mutations in many of these same genes are responsible for some congenital abnormalities and childhood diseases. Thus, the link between key genes in development and their role in clinical syndromes is becoming clearer. In addition to causing congenital malformations, mutations can result in inborn errors of metabolism. These diseases, among which phenylketonuria, homocystinuria, and galactosemia are the best known, are frequently accom- panied by or cause various degrees of mental retardation. Diagnostic Techniques for Identifying Genetic Abnormalities Cytogenetic analysis is used to assess chromosome number and integrity. The technique requires dividing cells, which usually means establishing cell cultures that are arrested in metaphase by chemical treatment. Chromosomes are stained with Giemsa stain to reveal light and dark banding patterns (G-bands; Fig. 1.6) unique for each chromosome. Each band represents 5 to 10 × 106 base pairs of DNA, which may include a few to several hundred genes. Recently, high resolution metaphase banding techniques have been devel- oped that demonstrate greater numbers of bands representing even smaller pieces of DNA, thereby facilitating diagnosis of small deletions. New molecular techniques, such as fluorescence in situ hybridization (FISH), use specific DNA probes to identify ploidy for a few selected chro- mosomes. Fluorescent probes are hybridized to chromosomes or genetic loci using cells on a slide, and the results are visualized with a fluorescence microscope (Fig.1.15). Spectral karyotype analysis is a technique in which every chromosome is hybridized to a unique fluorescent probe of a different color. Results are then analyzed by a computer. Morphological Changes During Maturation of the Gametes OOGENESIS Maturation of Oocytes Begins Before Birth Once primordial germ cells have arrived in the gonad of a genetic female, they differentiate into oogonia (Fig. 1.16, A and B). These cells undergo a number Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 19 Figure 1.15 Fluorescence in situ hybridization (FISH) using a probe for chromosome 21. Two interphase cells and a metaphase spread of chromosomes are shown; each has three domains, indicated by the probe, characteristic of trisomy 21 (Down syndrome). Figure 1.16 Differentiation of primordial germ cells into oogonia begins shortly after their arrival in the ovary. By the third month of development, some oogonia give rise to primary oocytes that enter prophase of the first meiotic division. This prophase may last 40 or more years and finishes only when the cell begins its final maturation. During this period it carries 46 double-structured chromosomes. 20 Part One: General Embryology Resting primary oocyte Surface epithelium of ovary Primary oocyte in (diplotene stage) prophase Flat Follicular cell epithelial cell Oogonia Primary oocytes in prophase of 1st meiotic division A B C 4th month 7th month Newborn Figure 1.17 Segment of the ovary at different stages of development. A. Oogonia are grouped in clusters in the cortical part of the ovary. Some show mitosis; others have differentiated into primary oocytes and entered prophase of the first meiotic division. B. Almost all oogonia are transformed into primary oocytes in prophase of the first meiotic division. C. There are no oogonia. Each primary oocyte is surrounded by a single layer of follicular cells, forming the primordial follicle. Oocytes have entered the diplotene stage of prophase, in which they remain until just before ovulation. Only then do they enter metaphase of the first meiotic division. of mitotic divisions and, by the end of the third month, are arranged in clusters surrounded by a layer of flat epithelial cells (Fig. 1.17 and 1.18). Whereas all of the oogonia in one cluster are probably derived from a single cell, the flat epithelial cells, known as follicular cells, originate from surface epithelium covering the ovary. The majority of oogonia continue to divide by mitosis, but some of them arrest their cell division in prophase of meiosis I and form primary oocytes (Figs. 1.16C and 1.17A). During the next few months, oogonia increase rapidly in number, and by the fifth month of prenatal development, the total number of germ cells in the ovary reaches its maximum, estimated at 7 million. At this time, cell death begins, and many oogonia as well as primary oocytes become atretic. By the seventh month, the majority of oogonia have degenerated except for a few near the surface. All surviving primary oocytes have entered prophase of meiosis I, and most of them are individually surrounded by a layer of flat epithelial cells (Fig. 1.17B). A primary oocyte, together with its surrounding flat epithelial cells, is known as a primordial follicle (Fig. 1.19A). Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 21 Figure 1.18 A. Primordial follicle consisting of a primary oocyte surrounded by a layer of flattened epithelial cells. B. Early primary or preantral stage follicle recruited from the pool of primordial follicles. As the follicle grows, follicular cells become cuboidal and begin to secrete the zona pellucida, which is visible in irregular patches on the surface of the oocyte. C. Mature primary (preantral) follicle with follicular cells forming a stratified layer of granulosa cells around the oocyte and the presence of a well-defined zona pellucida. Maturation of Oocytes Continues at Puberty Near the time of birth, all primary oocytes have started prophase of meiosis I, but instead of proceeding into metaphase, they enter the diplotene stage, a resting stage during prophase that is characterized by a lacy network of chro- matin (Fig. 1.17C ). Primary oocytes remain in prophase and do not finish their first meiotic division before puberty is reached, apparently because of oocyte maturation inhibitor (OMI), a substance secreted by follicular cells. The total number of primary oocytes at birth is estimated to vary from 700,000 to 2 million. During childhood most oocytes become atretic; only approximately 400,000 are present by the beginning of puberty, and fewer than 500 will be ovulated. Some oocytes that reach maturity late in life have been dormant in the diplotene stage of the first meiotic division for 40 years or more before ovulation. Whether the diplotene stage is the most suitable phase to protect the oocyte against environmental influences is unknown. The fact that the risk of having children with chromosomal abnormalities increases with maternal age indicates that primary oocytes are vulnerable to damage as they age. At puberty, a pool of growing follicles is established and continuously main- tained from the supply of primordial follicles. Each month, 15 to 20 follicles selected from this pool begin to mature, passing through three stages: 1) pri- mary or preantral; 2) secondary or antral (also called vesicular or Graafian); and 3) preovulatory. The antral stage is the longest, whereas the preovulatory stage encompasses approximately 37 hours before ovulation. As the primary oocyte begins to grow, surrounding follicular cells change from flat to cuboidal and proliferate to produce a stratified epithelium of granulosa cells, and the unit  AF Figure 1.19 A. Secondary (antral) stage follicle. The oocyte, surrounded by the zona pellucida, is off-center; the antrum has developed by fluid accumulation between in- tercellular spaces. Note the arrangement of cells of the theca interna and the theca externa. B. Mature secondary (graafian) follicle. The antrum has enlarged considerably, is filled with follicular fluid, and is surrounded by a stratified layer of granulosa cells. The oocyte is embedded in a mound of granulosa cells, the cumulus oophorus. C. Pho- tomicrograph of a mature secondary follicle with an enlarged fluid-filled antrum (cavity, Cav) and a diameter of 20 mm (×65). CO, cumulus oophorus; MG, granulosa cells; AF, atretic follicle. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 23 is called a primary follicle (Fig. 1.18, B and C ). Granulosa cells rest on a base- ment membrane separating them from surrounding stromal cells that form the theca folliculi. Also, granulosa cells and the oocyte secrete a layer of glycopro- teins on the surface of the oocyte, forming the zona pellucida (Fig. 1.18C ). As follicles continue to grow, cells of the theca folliculi organize into an inner layer of secretory cells, the theca interna, and an outer fibrous capsule, the theca externa. Also, small, finger-like processes of the follicular cells extend across the zona pellucida and interdigitate with microvilli of the plasma membrane of the oocyte. These processes are important for transport of materials from follicular cells to the oocyte. As development continues, fluid-filled spaces appear between granulosa cells. Coalescence of these spaces forms the antrum, and the follicle is termed a secondary (vesicular, Graafian) follicle. Initially, the antrum is crescent shaped, but with time, it enlarges (Fig. 1.19). Granulosa cells surrounding the oocyte remain intact and form the cumulus oophorus. At maturity, the sec- ondary follicle may be 25 mm or more in diameter. It is surrounded by the theca interna, which is composed of cells having characteristics of steroid se- cretion, rich in blood vessels, and the theca externa, which gradually merges with the ovarian stroma (Fig. 1.19). With each ovarian cycle, a number of follicles begin to develop, but usu- ally only one reaches full maturity. The others degenerate and become atretic (Fig. 1.19C ). When the secondary follicle is mature, a surge in luteinizing hormone (LH) induces the preovulatory growth phase. Meiosis I is completed, resulting in formation of two daughter cells of unequal size, each with 23 double- structured chromosomes (Fig. 1.20, A and B). One cell, the secondary oocyte, receives most of the cytoplasm; the other, the first polar body, receives prac- tically none. The first polar body lies between the zona pellucida and the cell Secondary oocyte Zona pellucida Granulosa cells in division A B C Primary oocyte in division Secondary oocyte and Polar body in division polar body 1 Figure 1.20 Maturation of the oocyte. A. Primary oocyte showing the spindle of the first meiotic division. B. Secondary oocyte and first polar body. The nuclear membrane is absent. C. Secondary oocyte showing the spindle of the second meiotic division. The first polar body is also dividing. 24 Part One: General Embryology membrane of the secondary oocyte in the perivitelline space (Fig. 1.20B ). The cell then enters meiosis II but arrests in metaphase approximately 3 hours before ovulation. Meiosis II is completed only if the oocyte is fertilized; oth- erwise, the cell degenerates approximately 24 hours after ovulation. The first polar body also undergoes a second division (Fig. 1.20C). SPERMATOGENESIS Maturation of Sperm Begins at Puberty Spermatogenesis, which begins at puberty, includes all of the events by which spermatogonia are transformed into spermatozoa. At birth, germ cells in the male can be recognized in the sex cords of the testis as large, pale cells sur- rounded by supporting cells (Fig. 1.21A). Supporting cells, which are derived from the surface epithelium of the gland in the same manner as follicular cells, become sustentacular cells, or Sertoli cells (Fig. 1.21C ). Shortly before puberty, the sex cords acquire a lumen and become the seminiferous tubules. At about the same time, primordial germ cells give rise to spermatogonial stem cells. At regular intervals, cells emerge from this stem cell population to form type A spermatogonia, and their production marks the initiation of spermatogenesis. Type A cells undergo a limited num- ber of mitotic divisions to form a clone of cells. The last cell division pro- duces type B spermatogonia, which then divide to form primary sperma- tocytes (Figs. 1.21 and 1.22). Primary spermatocytes then enter a prolonged Figure 1.21 A. Cross section through primitive sex cords of a newborn boy showing primordial germ cells and supporting cells. B and C. Two segments of a seminiferous tubule in transverse section. Note the different stages of spermatogenesis. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 25 Type A dark spermatogonia Type A pale spermatogonia Type A pale spermatogonia Type A pale spermatogonia Type A pale spermatogonia Type B spermatogonia Primary spermatocytes Secondary spermatocytes Spermatids Residual bodies Spermatozoa Figure 1.22 Type A spermatogonia, derived from the spermatogonial stem cell popu- lation, represent the first cells in the process of spermatogenesis. Clones of cells are established and cytoplasmic bridges join cells in each succeeding division until individ- ual sperm are separated from residual bodies. In fact, the number of individual inter- connected cells is considerably greater than depicted in this figure. 26 Part One: General Embryology Resting primary Secondary Type B spermatocyte spermatocyte spermatogonium Spermatid division A B C D Mitotic 1st meiotic 2nd meiotic division division Figure 1.23 The products of meiosis during spermatogenesis in humans. prophase (22 days) followed by rapid completion of meiosis I and formation of secondary spermatocytes. During the second meiotic division, these cells immediately begin to form haploid spermatids (Figs. 1.21–1.23). Throughout this series of events, from the time type A cells leave the stem cell popula- tion to formation of spermatids, cytokinesis is incomplete, so that successive cell generations are joined by cytoplasmic bridges. Thus, the progeny of a sin- gle type A spermatogonium form a clone of germ cells that maintain contact throughout differentiation (Fig. 1.22). Furthermore, spermatogonia and sper- matids remain embedded in deep recesses of Sertoli cells throughout their development (Fig. 1.24). In this manner, Sertoli cells support and protect the germ cells, participate in their nutrition, and assist in the release of mature spermatozoa. Spermatogenesis is regulated by luteinizing hormone (LH) production by the pituitary. LH binds to receptors on Leydig cells and stimulates testosterone production, which in turn binds to Sertoli cells to promote spermatogenesis. Follicle stimulating hormone (FSH) is also essential because its binding to Sertoli cells stimulates testicular fluid production and synthesis of intracellular androgen receptor proteins. Spermiogenesis The series of changes resulting in the transformation of spermatids into sperma- tozoa is spermiogenesis. These changes include (a) formation of the acrosome, which covers half of the nuclear surface and contains enzymes to assist in pen- etration of the egg and its surrounding layers during fertilization (Fig. 1.25); (b) condensation of the nucleus; (c) formation of neck, middle piece, and tail; and (d) shedding of most of the cytoplasm. In humans, the time required for a spermatogonium to develop into a mature spermatozoon is approximately 64 days. When fully formed, spermatozoa enter the lumen of seminiferous tubules. From there, they are pushed toward the epididymis by contractile elements in the wall of the seminiferous tubules. Although initially only slightly motile, spermatozoa obtain full motility in the epididymis. Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 27 Late spermatids Early spermatids Primary spermatocyte Sertoli cell Junctional complex Type A pale spermatogonia Type A dark spermatogonia Type B spermatogonia Basal lamina Peritubular cells Figure 1.24 Sertoli cells and maturing spermatocytes. Spermatogonia, spermatocytes, and early spermatids occupy depressions in basal aspects of the cell; late spermatids are in deep recesses near the apex. CLINICAL CORRELATES Abnormal Gametes In humans and in most mammals, one ovarian follicle occasionally contains two or three clearly distinguishable primary oocytes (Fig. 1.26A). Although these oocytes may give rise to twins or triplets, they usually degenerate before reaching maturity. In rare cases, one primary oocyte contains two or even three nuclei (Fig. 1.26B). Such binucleated or trinucleated oocytes die before reaching maturity. In contrast to atypical oocytes, abnormal spermatozoa are seen fre- quently, and up to 10% of all spermatozoa have observable defects. The head or the tail may be abnormal; spermatozoa may be giants or dwarfs; and sometimes they are joined (Fig. 1.26C ). Sperm with morphologic abnor- malities lack normal motility and probably do not fertilize oocytes. 28 Part One: General Embryology Figure 1.25 Important stages in transformation of the human spermatid into the sper- matozoon. Figure 1.26 Abnormal germ cells. A. Primordial follicle with two oocytes. B. Trinucle- ated oocyte. C. Various types of abnormal spermatozoa. Summary Primordial germ cells appear in the wall of the yolk sac in the fourth week and migrate to the indifferent gonad (Fig. 1.1), where they ar- rive at the end of the fifth week. In preparation for fertilization, both male and female germ cells undergo gametogenesis, which includes meio- sis and cytodifferentiation. During meiosis I, homologous chromosomes pair and exchange genetic material; during meiosis II, cells fail to replicate DNA, and each cell is thus provided with a haploid number of chromosomes and half the amount of DNA of a normal somatic cell (Fig. 1.3). Hence, ma- ture male and female gametes have, respectively, 22 plus X or 22 plus Y chromosomes. Birth defects may arise through abnormalities in chromosome number or structure and from single gene mutations. Approximately 7% of major Chapter 1: Gametogenesis: Conversion of Germ Cells Into Male and Female Gametes 29 birth defects are a result of chromosome abnormalities, and 8%, are a re- sult of gene mutations. Trisomies (an extra chromosome) and monosomies (loss of a chromosome) arise during mitosis or meiosis. During meiosis, ho- mologous chromosomes normally pair and then separate. However, if sepa- ration fails (nondisjunction), one cell receives too many chromosomes and one receives too few (Fig. 1.5). The incidence of abnormalities of chromo- some number increases with age of the mother, particularly with mothers aged 35 years and older. Structural abnormalities of chromosomes include large deletions (cri-du-chat syndrome) and microdeletions. Microdeletions involve contiguous genes that may result in defects such as Angelman syn- drome (maternal deletion, chromosome 15q11–15q13) or Prader-Willi syn- drome (paternal deletion, 15q11–15q13). Because these syndromes depend on whether the affected genetic material is inherited from the mother or the father, they also are an example of imprinting. Gene mutations may be dom- inant (only one gene of an allelic pair has to be affected to produce an al- teration) or recessive (both allelic gene pairs must be mutated). Mutations re- sponsible for many birth defects affect genes involved in normal embryological development. In the female, maturation from primitive germ cell to mature gamete, which is called oogenesis, begins before birth; in the male, it is called spermatoge- nesis, and it begins at puberty. In the female, primordial germ cells form oogonia. After repeated mitotic divisions, some of these arrest in prophase of meiosis I to form primary oocytes. By the seventh month, nearly all oogo- nia have become atretic, and only primary oocytes remain surrounded by a layer of follicular cells derived from the surface epithelium of the ovary (Fig. 1.17). Together, they form the primordial follicle. At puberty, a pool of growing follicles is recruited and maintained from the finite supply of primor- dial follicles. Thus, everyday 15 to 20 follicles begin to grow, and as they ma- ture, they pass through three stages: 1) primary or preantral; 2) secondary or antral (vesicular, Graafian); and 3) preovulatory. The primary oocyte re- mains in prophase of the first meiotic division until the secondary follicle is mature. At this point, a surge in luteinizing hormone (LH) stimulates pre- ovulatory growth: meiosis I is completed and a secondary oocyte and polar body are formed. Then, the secondary oocyte is arrested in metaphase of meiosis II approximately 3 hours before ovulation and will not complete this cell division until fertilization. In the male, primordial cells remain dormant until puberty, and only then do they differentiate into spermatogonia. These stem cells give rise to primary spermatocytes, which through two successive meiotic divisions produce four spermatids (Fig. 1.4). Spermatids go through a series of changes (spermiogenesis) (Fig. 1.25) including (a) formation of the acrosome, (b) condensation of the nucleus, (c) formation of neck, middle piece, and tail, and (d) shedding of most of the cytoplasm. The time required for a spermatogonium to become a mature spermatozoon is approximately 64 days. 30 Part One: General Embryology Problems to Solve 1. What is the most common cause of abnormal chromosome number? Give an example of a clinical syndrome involving abnormal numbers of chromosomes. 2. In addition to numerical abnormalities, what types of chromosomal alterations occur? 3. What is mosaicism, and how does it occur? SUGGESTED READING Chandley AC: Meiosis in man. Trends Genet 4:79, 1988. Clermont Y: Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and sper- matogonial renewal. Physiol Rev 52:198, 1972. Eddy EM, Clark JM, Gong D, Fenderson BA: Origin and migration of primordial germ cells in mammals. Gamete Res 4:333, 1981. Gelchrter TD, Collins FS: Principles of Medical Genetics. Baltimore, Williams & Wilkins, 1990. Gorlin RJ, Cohen MM, Levin LS (eds): Syndromes of the Head and Neck. 3rd ed. New York, Oxford University, 1990. Heller CG, Clermont Y: Kinetics of the germinal epithelium in man. Recent Prog Horm Res 20:545, 1964. Johnson MH, Everett BJ: Essential Reproduction. 5th ed. London, Blackwell Science Limited, 2000. Jones KL (ed): Smith’s Recognizable Patterns of Human Malformation. 4th ed. Philadelphia, WB Saunders, 1988. Larsen WJ, Wert SE: Roles of cell junctions in gametogenesis and early embryonic development. Tissue Cell 20:809, 1988. Lenke RR, Levy HL: Maternal phenylketonuria and hyperphenylalaninemia: an international survey of untreated and treated pregnancies. N Engl J Med 303:1202, 1980. Pelletier RA, We K, Balakier H: Development of membrane differentiations in the guinea pig sper- matid during spermiogenesis. Am J Anat 167:119, 1983. Russell LD: Sertoligerm cell interactions: a review. Gamete Res 3:179, 1980. Stevenson RE, Hall JG, Goodman RM (eds): Human Malformations and Related Anomalies. Vol I, II. New York, Oxford University Press, 1993. Thorogood P (ed): Embryos, Genes, and Birth Defects. New York, Wiley, 1997. Witschj E: Migration of the germ cells of the human embryos from the yolk sac to the primitive gonadal folds. Contrib Embryol 36:67, 1948.  c h a p t e r 2 First Week of Development: Ovulation to Implantation Ovarian Cycle At puberty, the female begins to undergo regular monthly cycles. These sexual cycles are controlled by the hypothalamus. Gonadotropin-releasing hor- mone (GnRH) produced by the hypothalamus acts on cells of the anterior pituitary gland, which in turn secrete gonadotropins. These hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), stimulate and control cyclic changes in the ovary. At the beginning of each ovarian cycle, 15 to 20 primary (preantral) stage follicles are stimulated to grow under the influence of FSH. (The hormone is not necessary to promote development of primordial follicles to the primary follicle stage, but without it, these primary follicles die and become atretic.) Thus, FSH rescues 15 to 20 of these cells from a pool of continuously forming primary follicles (Fig. 2.1). Under normal conditions, only one of these follicles reaches full maturity, and only one oocyte is discharged; the others degenerate and become atretic. In the next cycle, another group of primary follicles is recruited, and again, only one follicle reaches maturity. Consequently, most follicles degenerate without ever reaching full maturity. When a follicle becomes atretic, the oocyte and surrounding follicular cells degenerate and are replaced by connective tissue, forming a corpus atreticum. FSH also stimulates maturation of follicular (granulosa) cells surrounding the oocyte. In turn, proliferation of these cells is mediated by growth differentiation 31 32 Part One: General Embryology Zona pellucida Antrum Granulosa Primary oocyte Theca cells externa Theca interna Primordial follicle Primary follicle Secondary follicle Figure 2.1 From the pool of primordial follicles, every day some begin to grow and de- velop into secondary (preantral) follicles, and this growth is independent of FSH. Then, as the cycle progresses, FSH secretion recruits primary follicles to begin development into secondary (antral, Graafian) follicles. During the last few days of maturation of sec- ondary follicles, estrogens, produced by follicular and thecal cells, stimulate increased production of LH by the pituitary (Fig. 2.13), and this hormone causes the follicle to enter the preovulatory stage, to complete meiosis I, and to enter meiosis II where it arrests in metaphase approximately 3 hours before ovulation. factor-9 (GDF-9), a member of the transforming growth factor-β (TGF-β) family. In cooperation, granulosa and thecal cells produce estrogens that (a) cause the uterine endometrium to enter the follicular or proliferative phase; (b) cause thinning of the cervical mucus to allow passage of sperm; and (c) stimulate the pituitary gland to secrete LH. At mid-cycle, there is an LH surge that (a) ele- vates concentrations of maturation-promoting factor, causing oocytes to com- plete meiosis I and initiate meiosis II; (b) stimulates production of progesterone by follicular stromal cells (luteinization); and (c) causes follicular rupture and ovulation. OVULATION In the days immediately preceding ovulation, under the influence of FSH and LH, the secondary follicle grows rapidly to a diameter of 25 mm. Coincident with final development of the secondary follicle, there is an abrupt increase in LH that causes the primary oocyte to complete meiosis I and the follicle to enter the preovulatory stage. Meiosis II is also initiated, but the oocyte is arrested in metaphase approximately 3 hours before ovulation. In the meantime, the sur- face of the ovary begins to bulge locally, and at the apex, an avascular spot, the stigma, appears. The high concentration of LH increases collagenase activity, resulting in digestion of collagen fibers surrounding the follicle. Prostaglandin levels also increase in response to the LH surge and cause local muscular con- tractions in the ovarian wall. Those contractions extrude the oocyte, which together with its surrounding granulosa cells from the region of the cumulus  Chapter 2: First Week of Development: Ovulation to Implantation 33 Antrum Granulosa cells Luteal cells Ovarian stroma Theca interna Theca externa 1st Blood polar vessels body Oocyte in 2nd meiotic Cumulus oophorus Fibrin division cells A Preovulatory follicle B Ovulation C Corpus luteum Figure 2.2 A. Preovulatory follicle bulging at the ovarian surface. B. Ovulation. The oocyte, in metaphase of meiosis II, is discharged from the ovary together with a large number of cumulus oophorus cells. Follicular cells remaining inside the collapsed folli- cle differentiate into lutean cells. C. Corpus luteum. Note the large size of the corpus luteum, caused by hypertrophy and accumulation of lipid in granulosa and theca interna cells. The remaining cavity of the follicle is filled with fibrin. oophorus, breaks free (ovulation) and floats out of the ovary (Figs. 2.2 and 2.3). Some of the cumulus oophorus cells then rearrange themselves around the zona pellucida to form the corona radiata (Figs. 2.4–2.6). CLINICAL CORRELATES Ovulation During ovulation, some women feel a slight pain, known as middle pain because it normally occurs near the middle of the menstrual cycle. Ovulation is also generally accompanied by a rise in basal temperature, which can be monitored to aid in determining when release of the oocyte occurs. Some women fail to ovulate because of a low concentration of gonadotropins. In these cases, administration of an agent to stimulate gonadotropin release and hence ovulation can be employed. Although such drugs are effective, they often produce multiple ovulations, so that the risk of multiple pregnancies is 10 times higher in these women than in the general population. CORPUS LUTEUM After ovulation, granulosa cells remaining in the wall of the ruptured follicle, together with cells from the theca interna, are vascularized by surrounding ves- sels. Under the influence of LH, these cells develop a yellowish pigment and change into lutean cells, which form the corpus luteum and secrete the hor- mone progesterone (Fig. 2.2C ). Progesterone, together with estrogenic hor- mones, causes the uterine mucosa to enter the progestational or secretory stage in preparation for implantation of the embryo. 34 Part One: General Embryology Figure 2.3 A. Scanning electron micrograph of ovulation in the mouse. The surface of the oocyte is covered by the zona pellucida. The cumulus oophorus is composed of granulosa cells. B. Scanning electron micrograph of a rabbit oocyte 1.5 hours after ovulation. The oocyte, which is surrounded by granulosa cells, lies on the surface of the ovary. Note the site of ovulation.  Chapter 2: First Week of Development: Ovulation to Implantation 35 Figure 2.4 Relation of fimbriae and ovary. Fimbriae collect the oocyte and sweep it into the uterine tube. OOCYTE TRANSPORT Shortly before ovulation, fimbriae of the oviduct begin to sweep over the surface of the ovary, and the tube itself begins to contract rhythmically. It is thought that the oocyte surrounded by some granulosa cells (Figs. 2.3 and 2.4) is carried into the tube by these sweeping movements of the fimbriae and by motion of cilia on the epithelial lining. Once in the tube, cumulus cells withdraw their cytoplasmic processes from the zona pellucida and lose contact with the oocyte. Once the oocyte is in the uterine tube, it is propelled by cilia with the rate of transport regulated by the endocrine status during and after ovulation. In humans, the fertilized oocyte reaches the uterine lumen in approximately 3 to 4 days. CORPUS ALBICANS If fertilization does not occur, the corpus luteum reaches maximum develop- ment approximately 9 days after ovulation. It can easily be recognized as a yel- lowish projection on the surface of the ovary. Subsequently, the corpus luteum shrinks because of degeneration of lutean cells and forms a mass of fibrotic 36 Part One: General Embryology A B Figure 2.5 A. Scanning electron micrograph of sperm binding to the zona pellucida. B. The three phases of oocyte penetration. In phase 1, spermatozoa pass through the corona radiata barrier; in phase 2, one or more spermatozoa penetrate the zona pellu- cida; in phase 3, one spermatozoon penetrates the oocyte membrane while losing its own plasma membrane. Inset. Normal spermatocyte with acrosomal head cap.  Chapter 2: First Week of Development: Ovulation to Implantation 37 Figure 2.6 A. Oocyte immediately after ovulation, showing the spindle of the second meiotic division. B. A spermatozoon has penetrated the oocyte, which has finished its second meiotic division. Chromosomes of the oocyte are arranged in a vesicular nucleus, the female pronucleus. Heads of several sperm are stuck in the zona pellucida. C. Male and female pronuclei. D and E. Chromosomes become arranged on the spindle, split longitudinally, and move to opposite poles. F. Two-cell stage. scar tissue, the corpus albicans. Simultaneously, progesterone production de- creases, precipitating menstrual bleeding. If the oocyte is fertilized, degener- ation of the corpus luteum is prevented by human chorionic gonadotropin (hCG), a hormone secreted by the syncytiotrophoblast of the developing em- bryo. The corpus luteum continues to grow and forms the corpus luteum of pregnancy (corpus luteum graviditatis). By the end of the third month, this structure may be one-third to one-half of the total size of the ovary. Yellowish luteal cells continue to secrete progesterone until the end of the fourth month; thereafter, they regress slowly as secretion of progesterone by the trophoblastic component of the placenta becomes adequate for maintenance of pregnancy. Removal of the corpus luteum of pregnancy before the fourth month usually leads to abortion. Fertilization Fertilization, the process by which male and female gametes fuse, occurs in the ampullary region of the uterine tube. This is the widest part of the tube and 38 Part One: General Embryology is close to the ovary (Fig. 2.4). Spermatozoa may remain viable in the female reproductive tract for several days. Only 1% of sperm deposited in the vagina enter the cervix, where they may survive for many hours. Movement of sperm from the cervix to the oviduct is accomplished primarily by their own propulsion, although they may be as- sisted by movements of fluids created by uterine cilia. The trip from cervix to oviduct requires a minimum of 2 to 7 hours, and after reaching the isth- mus, sperm become less motile and cease their migration. At ovulation, sperm again become motile, perhaps because of chemoattractants produced by cu- mulus cells surrounding the egg, and swim to the ampulla where fertilization usually occurs. Spermatozoa are not able to fertilize the oocyte immediately upon arrival in the female genital tract but must undergo (a) capacitation and (b) the acrosome reaction to acquire this capability. Capacitation is a period of conditioning in the female reproductive tract that in the human lasts approximately 7 hours. Much of this conditioning, which occurs in the uterine tube, entails epithelial interactions between the sperm and mucosal surface of the tube. During this time a glycoprotein coat and seminal plasma proteins are removed from the plasma membrane that overlies the acrosomal region of the spermatozoa. Only capacitated sperm can pass through the corona cells and undergo the acrosome reaction. The acrosome reaction, which occurs after binding to the zona pellucida, is induced by zona proteins. This reaction culminates in the release of enzymes needed to penetrate the zona pellucida, including acrosin and trypsin-like sub- stances (Fig. 2.5). The phases of fertilization include phase 1, penetration of the corona ra- diata; phase 2, penetration of the zona pellucida; and phase 3, fusion of the oocyte and sperm cell membranes. PHASE 1: PENETRATION OF THE CORONA RADIATA Of the 200 to 300 million spermatozoa deposited in the female genital tract, only 300 to 500 reach the site of fertilization. Only one of these fertilizes the egg. It is thought that the others aid the fertilizing sperm in penetrating the barriers protecting the female gamete. Capacitated sperm pass freely through corona cells (Fig. 2.5). PHASE 2: PENETRATION OF THE ZONA PELLUCIDA The zona is a glycoprotein shell surrounding the egg that facilitates and main- tains sperm binding and induces the acrosome reaction. Both binding and the acrosome reaction are mediated by the ligand ZP3, a zona protein. Release of acrosomal enzymes (acrosin) allows sperm to penetrate the zona, thereby coming in contact with the plasma membrane of the oocyte (Fig. 2.5). Per- meability of the zona pellucida changes when the head of the sperm comes in contact with the oocyte surface. This contact results in release of lysosomal  Chapter 2: First Week of Development: Ovulation to Implantation 39 enzymes from cortical granules lining the plasma membrane of the oocyte. In turn, these enzymes alter properties of the zona pellucida (zona reaction) to prevent sperm penetration and inactivate species-specific receptor sites for spermatozoa on the zona surface. Other spermatozoa have been found embed- ded in the zona pellucida, but only one seems to be able to penetrate the oocyte (Fig. 2.6). PHASE 3: FUSION OF THE OOCYTE AND SPERM CELL MEMBRANES The initial adhesion of sperm to the oocyte is mediated in part by the interac- tion of integrins on the oocyte and their ligands, disintegrins, on sperm. After adhesion, the plasma membranes of the sperm and egg fuse (Fig. 2.5). Because the plasma membrane covering the acrosomal head cap disappears during the acrosome reaction, actual fusion is accomplished between the oocyte mem- brane and the membrane that covers the posterior region of the sperm head (Fig. 2.5). In the human, both the head and tail of the spermatozoon enter the cytoplasm of the oocyte, but the plasma membrane is left behind on the oocyte surface. As soon as the spermatozoon has entered the oocyte, the egg responds in three ways: 1. Cortical and zona reactions. As a result of the release of cortical oocyte granules, which contain lysosomal enzymes, (a) the oocyte membrane becomes impenetrable to other spermatozoa, and (b) the zona pellu- cida alters its structure and composition to prevent sperm binding and penetration. These reactions prevent polyspermy (penetration of more than one spermatozoon into the oocyte). 2. Resumption of the second meiotic division. The oocyte finishes its sec- ond meiotic division immediately after entry of the spermatozoon. One of the daughter cells, which receives hardly any cytoplasm, is known as the second polar body; the other daughter cell is the definitive oocyte. Its chromosomes (22 + X) arrange themselves in a vesicular nucleus known as the female pronucleus (Figs. 2.6 and 2.7). 3. Metabolic activation of the egg. The activating factor is probably car- ried by the spermatozoon. Postfusion activation may be considered to encompass the initial cellular and molecular events associated with early embryogenesis. The spermatozoon, meanwhile, moves forward until it lies close to the female pronucleus. Its nucleus becomes swollen and forms the male pronu- cleus (Fig. 2.6); the tail detaches and degenerates. Morphologically, the male and female pronuclei are indistinguishable, and eventually, they come into close contact and lose their nuclear envelopes (Fig. 2.7A). During growth of male and female pronuclei (both haploid), each pronucleus must replicate its DNA. If it does not, each cell of the two-cell zygote has only half of the normal amount of DNA. Immediately after DNA synthesis, chromosomes organize on 40 Part One: General Embryology Figure 2.7 A. Phase contrast view of the pronuclear stage of a fertilized human oocyte with male and female pronuclei. B. Two-cell stage of human zygote. the spindle in preparation for a normal mitotic division. The 23 maternal and 23 paternal (double) chromosomes split longitudinally at the centromere, and sister chromatids move to opposite poles, providing each cell of the zygote with the normal diploid number of chromosomes and DNA (Fig. 2.6, D and E ). As sister chromatids move to opposite poles, a deep furrow appears on the surface of the cell, gradually dividing the cytoplasm into two parts (Figs. 2.6F and 2.7B ). The main results of fertilization are as follows: r Restoration of the diploid number of chromosomes, half from the fa- ther and half from the mother. Hence, the zygote contains a new combi- nation of chromosomes different from both parents. r Determination of the sex of the new individual. An X-carrying sperm produces a female (XX) embryo, and a Y-carrying sperm produces a male (XY) embryo. Hence, the chromosomal sex of the embryo is determined at fertilization. r Initiation of cleavage. Without fertilization, the oocyte usually degener- ates 24 hours after ovulation. CLINICAL CORRELATES Contraceptive Methods Barrier techniques of contraception include the male condom, made of latex and often containing chemical spermicides, which fits over the penis; and the female condom, made of polyurethane, which lines the vagina. Other barriers placed in the vagina include the diaphragm, the cervical cap, and the contraceptive sponge. The contraceptive pill is a combination of estrogen and the progesterone analogue progestin, which together inhibit ovulation but permit menstruation.  Chapter 2: First Week of Development: Ovulation to Implantation 41 Both hormones act at the level of FSH and LH, preventing their release from the pituitary. The pills are taken for 21 days and then stopped to allow men- struation, after which the cycle is repeated. Depo-Provera is a progestin compound that can be implanted subder- mally or injected intramuscularly to prevent ovulation for up to 5 years or 23 months, respectively. A male “pill” has been developed and tested in clinical trials. It contains a synthetic androgen that prevents both LH and FSH secretion and either stops sperm production (70–90% of men) or reduces it to a level of infertility. The intrauterine device (IUD) is placed in the uterine cavity. Its mech- anism for preventing pregnancy is not clear but may entail direct effects on sperm and oocytes or inhibition of preimplantation stages of development. The drug RU-486 (mifepristone) causes abortion if it is administered within 8 weeks of the previous menses. It initiates menstruation, possibly through its action as an antiprogesterone agent. Vasectomy and tubal ligation are effective means of contraception, and both procedures are reversible, although not in every case. Infertility Infertility is a problem for 15% to 30% of couples. Male infertility may be a result of insufficient numbers of sperm and/or poor motility. Normally, the ejaculate has a volume of 3 to 4 ml, with approximately 100 million sperm per ml. Males with 20 million sperm per ml or 50 million sperm per total ejaculate are usually fertile. Infertility in a woman may be due to a number of causes, including occluded oviducts (most commonly caused by pelvic inflam- matory disease), hostile cervical mucus, immunity to spermatozoa, absence of ovulation, and others. In vitro fertilization (IVF) of human ova and embryo transfer is a frequent practice conducted by laboratories throughout the world. Follicle growth in the ovary is stimulated by administration of gonadotropins. Oocytes are recovered by laparoscopy from ovarian follicles with an aspirator just before ovulation when the oocyte is in the late stages of the first meiotic division. The egg is placed in a simple culture medium and sperm are added immediately. Fertil- ized eggs are monitored to the eight-cell stage and then placed in the uterus to develop to term. Fortunately, because preimplantation-stage embryos are resistant to teratogenic insult, the risk of producing malformed offspring by in vitro procedures is low. A disadvantage of IVF is its low success rate; only 20% of fertilized ova implant and develop to term. Therefore, to increase chances of a successful pregnancy, four or five ova are collected, fertilized, and placed in the uterus. This approach sometimes leads to multiple births. Another technique, gamete intrafallopian transfer (GIFT), introduces oocytes and sperm into the ampulla of the fallopian (uterine) tube, where 42 Part One: General Embryology fertilization takes place. Development then proceeds in a normal fashion. In a similar approach, zygote intrafallopian transfer (ZIFT), fertilized oocytes are placed in the ampullary region. Both of these methods require patent uterine tubes. Severe male infertility, in which the ejaculate contains very few live sperm (oligozoospermia) or even no live sperm (azoospermia), can be overcome using intracytoplasmic sperm injection (ICSI). With this technique, a single sperm, which may be obtained from any point in the male reproductive tract, is injected into the cytoplasm of the egg to cause fertilization. This approach offers couples an alternative to using donor sperm for IVF. The technique carries an increased risk for fetuses to have Y chromosome deletions but no other chromosomal abnormalities. Cleavage Once the zygote has reached the two-cell stage, it undergoes a series of mitotic divisions, increasing the numbers of cells. These cells, which become smaller with each cleavage division, are known as blastomeres (Fig. 2.8). Until the eight-cell stage, they form a loosely arranged clump (Fig. 2.9A). However, after the third cleavage, blastomeres maximize their contact with each other, form- ing a compact ball of cells held together by tight junctions (Fig. 2.9B). This process, compaction, segregates inner cells, which communicate extensively by gap junctions, from outer cells. Approximately 3 days after fertilization, cells of the compacted embryo divide again to form a 16-cell morula (mulberry). Inner cells of the morula constitute the inner cell mass, and surrounding cells compose the outer cell mass. The inner cell mass gives rise to tissues of the Figure 2.8 Development of the zygote from the two-cell stage to the late morula stage. The two-cell stage is reached approximately 30 hours after fertilization; the four-cell stage, at approximately 40 hours; the 12- to 16-cell stage, at approximately 3 days; and the late morula stage, at approximately 4 days. During this period, blas- tomeres are surrounded by the zona pellucida, which disappears at the end of the fourth day.  Chapter 2: First Week of Development: Ovulation to Implantation 43 A B Figure 2.9 Scanning electron micrographs of uncompacted (A) and compacted (B) eight-cell mouse embryos. In the uncompacted state, outlines of each blastomere are distinct, whereas after compaction cell-cell contacts are maximized and cellular outlines are indistinct. embryo proper, and the outer cell mass forms the trophoblast, which later contributes to the placenta. Blastocyst Formation About the time the morula enters the uterine cavity, fluid begins to penetrate through the zona pellucida into the intercellular spaces of the inner cell mass. Gradually the intercellular spaces become confluent, and finally a single cavity, the blastocele, forms (Fig. 2.10, A and B ). At this time, the embryo is a blastocyst. Cells of the inner cell mass, now called the embryoblast, are at one pole, and those of the outer cell mass, or trophoblast, flatten and form the epithelial wall of the blastocyst (Fig. 2.10, A and B). The zona pellucida has disappeared, allowing implantation to begin. In the human, trophoblastic cells over the embryoblast pole begin to pen- etrate between the epithelial cells of the uterine mucosa about the sixth day (Fig. 2.10C ). Attachment and invasion of the trophoblast involve integrins, ex- pressed by the trophoblast, and the extracellular matrix molecules laminin and fibronectin. Integrin receptors for laminin promote attachment, while those for fibronectin stimulate migration. These molecules also interact along signal transduction pathways to regulate trophoblast differentiation so that implanta- tion is the result of mutual trophoblastic and endometrial action. Hence, by the end of the first week of development, the human zygote has passed through the morula and blastocyst stages and has begun implantation in the uterine mucosa. 44 Part One: General Embryology A Figure 2.10 A. Section of a 107-cell human blastocyst showing inner cell mass and trophoblast cells. B. Schematic representation of a human blastocyst recovered from the uterine cavity at approximately 4.5 days. Blue, inner cell mass or embryoblast; green, trophoblast. C. Schematic representation of a blastocyst at the ninth day of development showing trophoblast cells at the embryonic pole of the blastocyst penetrating the uterine mucosa. The human blastocyst begins to penetrate the uterine mucosa by the sixth day of development. CLINICAL CORRELATES Abnormal Zygotes The exact number of abnormal zygotes formed is unknown because they are usually lost within 2 to 3 weeks of fertilization, before the woman re- alizes she is pregnant, and therefore are not detected. Estimates are that as many as 50% of pregnancies end in spontaneous abortion and that  Chapter 2: First Week of Development: Ovulation to Implantation 45 half of these losses are a result of chromosomal abnormalities. These abortions are a natural means of screening embryos for defects, reducing the incidence of congenital malformations. Without this phenomenon, approximately 12% instead of 2% to 3% of infants would have birth defects. With the use of a combination of IVF and polymerase chain reaction (PCR), molecular screening of embryos for genetic defects is being conducted. Single blastomeres from early-stage embryos can be removed and their DNA amplified for analysis. As the Human Genome Project provides more sequenc- ing information and as specific genes are linked to various syndromes, such procedures will become more commonplace. Uterus at Time of Implantation The wall of the uterus consists of three layers: (a) endometrium or mu- cosa lining the inside wall; (b) myometrium, a thick layer of smooth mus- cle; and (c) perimetrium, the peritoneal covering lining the outside wall (Fig. 2.11). From puberty (11–13 years) until menopause (45–50 years), the endometrium undergoes changes in a cycle of approximately 28 days under hormonal control by the ovary. During this menstrual cycle, the uterine en- dometrium passes through three stages, the follicular or proliferative phase, Preovulatory Figure 2.11 Events during the first week of human development. 1, Oocyte immedi- ately after ovulation. 2, Fertilization, approximately 12 to 24 hours after ovulation. 3, Stage of the male and female pronuclei. 4, Spindle of the first mitotic division. 5, Two- cell stage (approximately 30 hours of age). 6, Morula containing 12 to 16 blastomeres (approximately 3 days of age). 7, Advanced morula stage reaching the uterine lumen (approximately 4 days of age). 8, Early blastocyst stage (approximately 4.5 days of age). The zona pellucida has disappeared. 9, Early phase of implantation (blastocyst approx- imately 6 days of age). The ovary shows stages of transformation between a primary follicle and a preovulatory follicle as well as a corpus luteum. The uterine endometrium is shown in the progestational stage. 46 Part One: General Embryology Maturation of follicle Ovulation Corpus luteum Corpus luteum of pregnancy Implanted embryo Implantation begins Gland Compact layer Spongy layer Basal layer 0 4 14 28 Menstrual phase Follicular or Progestational or Gravid phase proliferative phase secretory phase Figure 2.12 Changes in the uterine mucosa correlated with those in the ovary. Implan- tation of the blastocyst has caused development of a large corpus luteum of pregnancy. Secretory activity of the endometrium increases gradually as a result of large amounts of progesterone produced by the corpus luteum of pregnancy. the secretory or progestational phase, and the menstrual phase (Figs. 2.11– 2.13). The proliferative phase begins at the end of the menstrual phase, is under the influence of estrogen, and parallels growth of the ovarian follicles. The se- cretory phase begins approximately 2 to 3 days after ovulation in response to progesterone produced by the corpus luteum. If fertilization does not occur, shedding of the endometrium (compact and spongy layers) marks the begin- ning of the menstrual phase. If fertilization does occur, the endometrium assists in implantation and contributes to formation of the placenta. At the time of implantation, the mucosa of the uterus is in the secretory phase (Figs. 2.11 and 2.12), during which time uterine glands and arteries become coiled and the tissue becomes succulent. As a result, three distinct layers can be recognized in the endometrium: a superficial compact layer, an intermediate spongy layer, and a thin basal layer (Fig. 2.12). Normally, the human blastocyst implants in the endometrium along the anterior or posterior wall of the body of the uterus, where it becomes embedded between the openings of the glands (Fig. 2.12). If the oocyte is not fertilized, venules and sinusoidal spaces gradually be- come packed with blood cells, and an extensive diapedesis of blood into the tissue is seen. When the menstrual phase begins, blood escapes from super- ficial arteries, and small pieces of stroma and glands break away. During the following 3 or 4 days, the compact and spongy layers are expelled from the  Chapter 2: First Week of Development: Ovulation to Implantation 47 Figure 2.13 Changes in the uterine mucosa (endometrium) and corresponding changes in the ovary during a regular menstrual cycle without fertilization. uterus, and the basal layer is the only part of the endometrium that is retained (Fig. 2.13). This layer, which is supplied by its own arteries, the basal arteries, functions as the regenerative layer in the rebuilding of glands and arteries in the proliferative phase (Fig. 2.13). Summary With each ovarian cycle, a number of primary follicles begin to grow, but usually only one reaches full maturity, and only one oocyte is discharged at ovulation. At ovulation, the oocyte is in metaphase of the second meiotic division and is surrounded by the zona pellucida and some granulosa cells (Fig. 2.4). Sweeping action of tubal fimbriae carries the oocyte into the uterine tube. 48 Part One: General Embryology Before spermatozoa can fertilize the oocyte, they must undergo (a) capac- itation, during which time a glycoprotein coat and seminal plasma proteins are removed from the spermatozoon head, and (b) the acrosome reaction, during which acrosin and trypsin-like substances are released to penetrate the zona pellucida. During fertilization, the spermatozoon must penetrate (a) the corona radiata, (b) the zona pellucida, and (c) the oocyte cell membrane (Fig. 2.5). As soon as the spermatocyte has entered the oocyte, (a) the oocyte finishes its second meiotic division and forms the female pronucleus; (b) the zona pellucida becomes impenetrable to other spermatozoa; and (c) the head of the sperm separates from the tail, swells, and forms the male pronucleus (Figs. 2.6 and 2.7). After both pronuclei have replicated their DNA, paternal and maternal chromosomes intermingle, split longitudinally, and go through a mitotic division, giving rise to the two-cell stage. The results of fertilization are (a) restoration of the diploid number of chromosomes, (b) determination of chromosomal sex, and (c) initiation of cleavage. Cleavage is a series of mitotic divisions that results in an increase in cells, blastomeres, which become smaller with each division. After three divisions, blastomeres undergo compaction to become a tightly grouped ball of cells with inner and outer layers. Compacted blastomeres divide to form a 16-cell morula. As the morula enters the uterus on the third or fourth day after fertilization, a cavity begins to appear, and the blastocyst forms. The inner cell mass, which is formed at the time of compaction and will develop into the embryo proper, is at one pole of the blastocyst. The outer cell mass, which surrounds the inner cells and the blastocyst cavity, will form the trophoblast. The uterus at the time of implantation is in the secretory phase, and the blastocyst implants in the endometrium along the anterior or posterior wall. If fertilization does not occur, then the menstrual phase begins and the spongy and compact endometrial layers are shed. The basal layer remains to regenerate the other layers during the next cycle. Problems to Solve 1. What are the primary causes of infertility in men and women? 2. A woman has had several bouts of pelvic inflammatory disease and now wants to have children. However, she has been having difficulty becoming pregnant. What is likely to be the problem, and what would you suggest? SUGGESTED READING Allen CA, Green DPL: The mammalian acrosome reaction: gateway to sperm fusion with the oocyte? Bioessays 19:241, 1997. Archer DF, Zeleznik AJ, Rockette HE: Ovarian follicular maturation in women: 2. Reversal of estrogen inhibited ovarian folliculogenesis by human gonadotropin. Fertil Steril 50:555, 1988. Barratt CLR, Cooke ID: Sperm transport in the human female reproductive tract: a dynamic inter- action. Int J Androl 14:394, 1991.  Chapter 2: First Week of Development: Ovulation to Implantation 49 Boldt J, et al: Carbohydrate involvement in sperm-egg fusion in mice. Biol Reprod 40:887, 1989. Burrows TD, King A, Loke YW: Expression of integrins by human trophoblast and differential adhesion to laminin or fibronectin. Hum Reprod 8:475, 1993. Carr DH: Chromosome studies on selected spontaneous abortions: polyploidy in man. J Med Genet 8:164, 1971. Chen CM, Sathananthan AH: Early penetration of human sperm through the vestments of human egg in vitro. Arch Androl 16:183, 1986. Cowchock S: Autoantibodies and fetal wastage. Am J Reprod Immunol 26:38, 1991. Edwards RG: A decade of in vitro fertilization. Res Reprod 22:1, 1990. Edwards RG, Bavister BD, Steptoe PC: Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (Lond) 221:632, 1969. Egarter C: The complex nature of egg transport through the oviduct. Am J Obstet Gynecol 163:687, 1990. Enders AC, Hendrickx AG, Schlake S: Implantation in the rhesus monkey: initial penetration of the endometrium. Am J Anat 167:275, 1983. Gilbert SF: Developmental Biology. Sunderland, MA, Sinauer, 1991. Handyside AH, Kontogianni EH, Hardy K, Winston RML: Pregnancies from biopsied human preim- plantation embryos sexed by Y-specific DNA amplification. Nature 344:768, 1990. Hertig AT, Rock J, Adams EC: A description of 34 human ova within the first 17 days of development. Am J Anat 98:435, 1956. Johnson MH, Everitt BJ: Essential Reproduction. 5th ed. London, Blackwell Science Limited, 2000. Liu J, et al: Analysis of 76 total fertilization failure cycles out of 2732 intracytoplasmic sperm injection cycles. Hum Reprod 10:2630, 1995. Oura C, Toshimori K: Ultrasound studies on the fertilization of mammalian gametes. Rev Cytol 122:105, 1990. Pedersen RA, We K, Balakier H: Origin of the inner cell mass in mouse embryos: cell lineage analysis by microinjection. Dev Biol 117:581, 1986. Reproduction (entire issue). J NIH Res 9:1997. Scott RT, Hodgen GD: The ovarian follicle: life cycle of a pelvic clock. Clin Obstet Gynecol 33:551, 1990. Wasserman PM: Fertilization in mammals. Sci Am 259:78, 1988. Wolf DP, Quigley MM (eds): Human in Vitro Fertilization and Transfer. New York, Plenum, 1984. Yen SC, Jaffe RB (eds): Reproductive Endocrinology: Physiology, Pathophysiology, and Clinical Man- agement. 2nd ed. Philadelphia, WB Saunders, 1986.  c h a p t e r 3 Second Week of Development: Bilaminar Germ Disc This chapter gives a day-by-day account of the major events of the second week of development. However, embryos of the same fertilization age do not necessarily develop at the same rate. Indeed, con- siderable differences in rate of growth have been found even at these early stages of development. Day 8 At the eighth day of development, the blastocyst is partially embedded in the endometrial stroma. In the area over the em- bryoblast, the trophoblast has differentiated into two layers: (a) an inner layer of mononucleated cells, the cytotrophoblast, and (b) an outer multinucleated zone without distinct cell bound- aries, the syncytiotrophoblast (Figs. 3.1 and 3.2). Mitotic figures are found in the cytotrophoblast but not in the syncytiotrophoblast. Thus, cells in the cytotrophoblast divide and migrate into the syncytiotro- phoblast, where they fuse and lose their individual cell membranes. Cells of the inner cell mass or embryoblast also differentiate into two layers: (a) a layer of small cuboidal cells adjacent to the blastocyst cavity, known as the hypoblast layer, and (b) a layer of high columnar cells adjacent to the amniotic cavity, the epiblast layer (Figs. 3.1 and 3.2). Together, the layers form a flat disc. At the same time, a small cavity appears within the epiblast. This cavity enlarges to become the 51 52 Part One: General Embryology Figure 3.1 A 7.5-day human blastocyst, partially embedded in the endometrial stroma. The trophoblast consists of an inner layer with mononuclear cells, the cytotrophoblast, and an outer layer without distinct cell boundaries, the syncytiotrophoblast. The em- bryoblast is formed by the epiblast and hypoblast layers. The amniotic cavity appears as a small cleft. Figure 3.2 Section of a 7.5-day human blastocyst (×100). Note the multinucleated ap- pearance of the syncytiotrophoblast, large cells of the cytotrophoblast, and slit-like amniotic cavity. amniotic cavity. Epiblast cells adjacent to the cytotrophoblast are called am- nioblasts; together with the rest of the epiblast, they line the amniotic cavity (Figs. 3.1 and 3.3). The endometrial stroma adjacent to the implantation site is edematous and highly vascular. The large, tortuous glands secrete abundant glycogen and mucus.  Chapter 3: Second Week of Development: Bilaminar Germ Disc 53 Figure 3.3 A 9-day human blastocyst. The syncytiotrophoblast shows a large number of lacunae. Flat cells form the exocoelomic membrane. The bilaminar disc consists of a layer of columnar epiblast cells and a layer of cuboidal hypoblast cells. The original surface defect is closed by a fibrin coagulum. Day 9 The blastocyst is more deeply embedded in the endometrium, and the penetra- tion defect in the surface epithelium is closed by a fibrin coagulum (Fig. 3.3). The trophoblast shows considerable progress in development, particularly at the embryonic pole, where vacuoles appear in the syncytium. When these vac- uoles fuse, they form large lacunae, and this phase of trophoblast development is thus known as the lacunar stage (Fig. 3.3). At the abembryonic pole, meanwhile, flattened cells probably originating from the hypoblast form a thin membrane, the exocoelomic (Heuser’s) mem- brane, that lines the inner surface of the cytotrophoblast (Fig. 3.3). This mem- brane, together with the hypoblast, forms the lining of the exocoelomic cavity, or primitive yolk sac. Days 11 and 12 By the 11th to 12th day of development, the blastocyst is completely embedded in the endometrial stroma, and the surface epithelium almost entirely covers 54 Part One: General Embryology Figure 3.4 Human blastocyst of approximately 12 days. The trophoblastic lacunae at the embryonic pole are in open connection with maternal sinusoids in the endometrial stroma. Extraembryonic mesoderm proliferates and fills the space between the exo- coelomic membrane and the inner aspect of the trophoblast. the original defect in the uterine wall (Figs. 3.4 and 3.5). The blastocyst now produces a slight protrusion into the lumen of the uterus. The trophoblast is characterized by lacunar spaces in the syncytium that form an intercommuni- cating network. This network is particularly evident at the embryonic pole; at the abembryonic pole, the trophoblast still consists mainly of cytotrophoblastic cells (Figs. 3.4 and 3.5). Concurrently, cells of the syncytiotrophoblast penetrate deeper into the stroma and erode the endothelial lining of the maternal capillaries. These capil- laries, which are congested and dilated, are known as sinusoids. The syncytial lacunae become continuous with the sinusoids and maternal blood enters the lacunar system (Fig. 3.4). As the trophoblast continues to erode more and more sinusoids, maternal blood begins to flow through the trophoblastic system, es- tablishing the uteroplacental circulation. In the meantime, a new population of cells appears between the in- ner surface of the cytotrophoblast and the outer surface of the exocoelomic  Chapter 3: Second Week of Development: Bilaminar Germ Disc 55 Figure 3.5 Fully implanted