Sadler's Langman's Medical Embryology - Introduction to Molecular Regulation and Signaling PDF

Sadler_Chap01.indd 1 8/25/2011 3:23:38 PM Sadler_Chap01.indd 2 8/25/2011 3:23:44 PM  Chapter 1 Introduction to Molecular Regulation and...

Sadler_Chap01.indd 1 8/25/2011 3:23:38 PM Sadler_Chap01.indd 2 8/25/2011 3:23:44 PM  Chapter 1 Introduction to Molecular Regulation and Signaling M olecular biology has opened the doors to string of DNA and is referred to as heterochro- new ways to study embryology and to matin. For transcription to occur, this DNA enhance our understanding of normal and must be uncoiled from the beads. In this uncoiled abnormal development. Sequencing the human state, chromatin is referred to as euchromatin. genome, together with creating techniques to Genes reside within the DNA strand and investigate gene regulation at many levels of com- contain regions called exons, which can be plexity, has taken embryology to the next level. translated into proteins, and introns, which are Thus, from the anatomical to the biochemical to interspersed between exons and which are not the molecular level, the story of embryology has transcribed into proteins (Fig. 1.2). In addition progressed, and each chapter has enhanced our to exons and introns, a typical gene includes the knowledge. following: a promoter region that binds RNA There are approximately 23,000 genes in polymerase for the initiation of transcrip- the human genome, which represents only one tion; a transcription initiation site; a transla- fifth of the number predicted prior to comple- tion initiation site to designate the first amino tion of the Human Genome Project. Because acid in the protein; a translation termination of various levels of regulation, however, the codon; and a 3′ untranslated region that includes number of proteins derived from these genes is a sequence (the poly A addition site) that assists closer to the original predicted number of genes. with stabilizing the mRNA, allows it to exit the What has been disproved is the one-gene–one- nucleus, and permits it to be translated into pro- protein hypothesis. Thus, through a variety of tein (Fig. 1.2). By convention, the 5′ and the 3′ mechanisms, a single gene may give rise to many regions of a gene are specified in relation to the proteins. RNA transcribed from the gene. Thus, DNA is Gene expression can be regulated at several transcribed from the 5′ to the 3′ end, and the levels: (1) different genes may be transcribed, (2) promoter region is upstream from the tran- nuclear deoxyribonucleic acid (DNA) transcribed scription initiation site (Fig. 1.2). The promoter from a gene may be selectively processed to regu- region, where the RNA polymerase binds, usu- late which RNAs reach the cytoplasm to become ally contains the sequence TATA, and this site messenger RNAs (mRNAs), (3) mRNAs may be selectively translated, and (4) proteins made from the mRNAs may be differentially modified. Histone complex DNA GENE TRANSCRIPTION Genes are contained in a complex of DNA and proteins (mostly histones) called chromatin, and Nucleosome its basic unit of structure is the nucleosome (Fig. 1.1). Each nucleosome is composed of an H1 octamer of histone proteins and approximately histones Linker 140 base pairs of DNA. Nucleosomes themselves DNA are joined into clusters by binding of DNA exist- ing between nucleosomes (linker DNA) with Figure 1.1 Drawing showing nucleosomes that form the other histone proteins (H1 histones; Fig. 1.1). basic unit of chromatin. Each nucleosome consists of an Nucleosomes keep the DNA tightly coiled, such octamer of histone proteins and approximately 140 base that it cannot be transcribed. In this inactive state, pairs of DNA. Nucleosomes are joined into clusters by chromatin appears as beads of nucleosomes on a linker DNA and other histone proteins. 3 Sadler_Chap01.indd 3 8/25/2011 3:23:44 PM  4 Part 1 General Embryology Promoter region Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 Intron 3 Exon 4 3' untranslated region TATA Translation Enhancer Translation Transcription box initiation sequence termination termination codon Poly A site addition site Figure 1.2 Drawing of a “typical” gene showing the promoter region containing the TATA box; exons that contain DNA sequences that are translated into proteins; introns; the transcription initiation site; the translation initiation site that designates the code for the first amino acid in a protein; and the 3′ untranslated region that includes the poly A addition site that participates in stabilizing the mRNA, allows it to exit the nucleus, and permits its translation into a protein. is called the TATA box (Fig. 1.2). In order to appropriate tissue. Enhancers act by altering bind to this site, however, the polymerase requires chromatin to expose the promoter or by facilitat- additional proteins called transcription factors ing binding of the RNA polymerase. Sometimes, (Fig. 1.3).Transcription factors also have a specific enhancers can inhibit transcription and are called DNA-binding domain plus a transactivat- silencers. This phenomenon allows a transcrip- ing domain that activates or inhibits transcrip- tion factor to activate one gene while silencing tion of the gene whose promoter or enhancer it another by binding to different enhancers. Thus, has bound. In combination with other proteins, transcription factors themselves have a DNA- transcription factors activate gene expression binding domain specific to a region of DNA plus by causing the DNA nucleosome complex to a transactivating domain that binds to a promoter unwind, by releasing the polymerase so that it or an enhancer and activates or inhibits the gene can transcribe the DNA template, and by pre- regulated by these elements. venting new nucleosomes from forming. Enhancers are regulatory elements of DNA DNA Methylation Represses that activate utilization of promoters to control Transcription their efficiency and the rate of transcription from Methylation of cytosine bases in the promoter the promoter. Enhancers can reside anywhere regions of genes represses transcription of those along the DNA strand and do not have to reside genes. Thus, some genes are silenced by this close to a promoter. Like promoters, enhancers mechanism. For example, one of the X chro- bind transcription factors (through the transcrip- mosomes in each cell of a female is inactivated tion factor’s transactivating domain) and are used (X chromosome inactivation) by this meth- to regulate the timing of a gene’s expression and ylation mechanism. Similarly, genes in different its cell-specific location. For example, separate types of cells are repressed by methylation, such enhancers in a gene can be used to direct the that muscle cells make muscle proteins (their same gene to be expressed in different tissues. promoter DNA is mostly unmethylated), but not The PAX6 transcription factor, which partici- blood proteins (their DNA is highly methylated). pates in pancreas, eye, and neural tube develop- In this manner, each cell can maintain its char- ment, contains three separate enhancers, each acteristic differentiated state. DNA methylation of which regulates the gene’s expression in the is also responsible for genomic imprinting in RNA Polymerase II RNA Polymerase II DNA TATA Transcription Transcription RNA transcript factor protein initiation site complex Figure 1.3 Drawing showing binding of RNA polymerase II to the TATA box site of the promoter region of a gene. This binding requires a complex of proteins plus an additional protein called a transcription factor. Transcription factors have their own specific DNA-binding domain and function to regulate gene expression. Sadler_Chap01.indd 4 8/25/2011 3:23:53 PM  Chapter 1 Introduction to Molecular Regulation and Signaling 5 which only a gene inherited from the father or have to be cleaved to become active, or they the mother is expressed, while the other gene might have to be phosphorylated. Others need is silenced. Approximately 40 to 60 human to combine with other proteins or be released genes are imprinted and their methylation pat- from sequestered sites or be targeted to specific terns are established during spermatogenesis and cell regions. Thus, there are many regulatory lev- oogenesis. Methylation silences DNA by inhib- els for synthesizing and activating proteins, such iting binding of transcription factors or by alter- that although only 23,000 genes exist, the poten- ing histone binding resulting in stabilization of tial number of proteins that can be synthesized is nucleosomes and tightly coiled DNA that cannot probably closer to five times the number of genes. be transcribed. INDUCTION AND ORGAN OTHER REGULATORS OF GENE FORMATION EXPRESSION Organs are formed by interactions between cells The initial transcript of a gene is called nuclear and tissues. Most often, one group of cells or tissues RNA (nRNA) or sometimes premessenger RNA. causes another set of cells or tissues to change nRNA is longer than mRNA because it con- their fate, a process called induction. In each such tains introns that are removed (spliced out) as interaction, one cell type or tissue is the inducer the nRNA moves from the nucleus to the cyto- that produces a signal, and one is the responder plasm. In fact, this splicing process provides a to that signal. The capacity to respond to such means for cells to produce different proteins from a signal is called competence, and competence a single gene. For example, by removing different requires activation of the responding tissue by a introns, exons are “spliced” in different patterns, competence factor. Many inductive interac- a process called alternative splicing (Fig. 1.4). tions occur between epithelial and mesenchymal The process is carried out by spliceosomes, cells and are called epithelial–mesenchymal which are complexes of small nuclear RNAs interactions (Fig. 1.5). Epithelial cells are joined (snRNAs) and proteins that recognize specific together in tubes or sheets, whereas mesenchymal splice sites at the 5′ or the 3′ ends of the nRNA. cells are fibroblastic in appearance and dispersed Proteins derived from the same gene are called in extracellular matrices (Fig. 1.5). Examples of splicing isoforms (also called splice vari- epithelial–mesenchymal interactions include the ants or alternative splice forms), and these following: gut endoderm and surrounding mes- afford the opportunity for different cells to use enchyme to produce gut-derived organs, includ- the same gene to make proteins specific for that ing the liver and pancreas; limb mesenchyme cell type. For example, isoforms of the WT1 gene with overlying ectoderm (epithelium) to pro- have different functions in gonadal versus kidney duce limb outgrowth and differentiation; and development. endoderm of the ureteric bud and mesenchyme Even after a protein is made (translated), there from the metanephric blastema to produce may be post-translational modifications that nephrons in the kidney. Inductive interactions affect its function. For example, some proteins can also occur between two epithelial tissues, 5' untranslated Tissue specific 3' untranslated region Exons Exon (bone) Introns region Hypothetical gene Protein I Protein II (bone) Protein III Figure 1.4 Drawing of a hypothetical gene illustrating the process of alternative splicing to form different proteins from the same gene. Spliceosomes recognize specific sites on the initial transcript of nRNA from a gene. Based on these sites, different introns are “spliced out” to create more than one protein from a single gene. Proteins derived from the same gene are called splicing isoforms. Sadler_Chap01.indd 5 8/25/2011 3:23:53 PM  6 Part 1 General Embryology to interact with other cells, or by juxtacrine interactions, which do not involve diffusable Mesenchyme proteins. The diffusable proteins responsible for paracrine signaling are called paracrine fac- tors or growth and differentiation factors Epithelium (GDFs). Signal Transduction Pathways Paracrine Signaling Figure 1.5 Drawing illustrating an epithelial–mesenchymal Paracrine factors act by signal transduction interaction. Following an initial signal from one tissue, a pathways either by activating a pathway directly second tissue is induced to differentiate into a specific or by blocking the activity of an inhibitor of a structure. The first tissue constitutes the inducer, and the pathway (inhibiting an inhibitor, as is the case second is the responder. Once the induction process is with hedgehog signaling). Signal transduction initiated, signals (arrows) are transmitted in both directions pathways include a signaling molecule (the to complete the differentiation process. ligand) and a receptor (Fig. 1.6). The receptor spans the cell membrane and has an extracel- such as induction of the lens by epithelium of lular domain (the ligand-binding region), a the optic cup. Although an initial signal by the transmembrane domain, and a cytoplasmic inducer to the responder initiates the inductive domain. When a ligand binds its receptor, it event, crosstalk between the two tissues or cell induces a conformational change in the recep- types is essential for differentiation to continue tor that activates its cytoplasmic domain. Usually, (Fig. 1.5, arrows). the result of this activation is to confer enzy- matic activity to the receptor, and most often CELL SIGNALING this activity is a kinase that can phosphorylate other proteins using ATP as a substrate. In turn, Cell-to-cell signaling is essential for induction, phosphorylation activates these proteins to phos- for conference of competency to respond, and for phorylate additional proteins, and thus a cascade crosstalk between inducing and responding cells. of protein interactions is established that ulti- These lines of communication are established by mately activates a transcription factor. This paracrine interactions, whereby proteins syn- transcription factor then activates or inhibits thesized by one cell diffuse over short distances gene expression. The pathways are numerous and Ligand Receptor complex Cell membrane P P Activated P P (kinase) region P Activated protein Nuclear Cytoplasm pores P Activated protein complex Activated protein P complex acts as a transcription factor Nucleus Figure 1.6 Drawing of a typical signal transduction pathway involving a ligand and its receptor. Activation of the receptor is conferred by binding to the ligand. Typically, the activation is enzymatic involving a tyrosine kinase, although other enzymes may be employed. Ultimately, kinase activity results in a phosphorylation cascade of several proteins that activates a transcription factor for regulating gene expression. Sadler_Chap01.indd 6 8/25/2011 3:23:53 PM  Chapter 1 Introduction to Molecular Regulation and Signaling 7 complex and in some cases are characterized by a channel, and these channels are “connected” one protein inhibiting another that in turn acti- between adjacent cells. vates another protein (much like the situation It is important to note that there is a great with hedgehog signaling). amount of redundancy built into the process of signal transduction. For example, paracrine sig- Juxtacrine Signaling naling molecules often have many family mem- Juxtacrine signaling is mediated through sig- bers such that other genes in the family may nal transduction pathways as well but does not compensate for the loss of one of their coun- involve diffusable factors. Instead, there are three terparts. Thus, the loss of function of a signaling ways juxtacrine signaling occurs: (1) A protein protein through a gene mutation does not neces- on one cell surface interacts with a receptor on sarily result in abnormal development or death. an adjacent cell in a process analogous to para- In addition, there is crosstalk between pathways, crine signaling (Fig. 1.6). The Notch pathway such that they are intimately interconnected. represents an example of this type of signal- These connections provide numerous additional ing. The Notch receptor protein extends across sites to regulate signaling. the cell membrane and binds to cells that have Delta, Serrate, or Jagged proteins in their cell membranes. Binding of one of these pro- Paracrine Signaling Factors teins to Notch causes a conformational change There are a large number of paracrine signaling in the Notch protein such that part of it on the factors acting as ligands, which are also called cytoplasmic side of the membrane is cleaved.The GDFs. Most are grouped into four families, and cleaved portion then binds to a transcription fac- members of these same families are used repeat- tor to activate gene expression. Notch signaling edly to regulate development and differentiation is especially important in neuronal differentia- of organ systems. Furthermore, the same GDFs tion, blood vessel specification, and somite seg- regulate organ development throughout the ani- mentation. (2) Ligands in the extracellular matrix mal kingdom from Drosophila to humans. The secreted by one cell interact with their receptors four groups of GDFs include the fibroblast on neighboring cells. The extracellular matrix growth factor (FGF), WNT, hedgehog, and is the milieu in which cells reside. This milieu transforming growth factor-b (TGF-b) consists of large molecules secreted by cells families. Each family of GDFs interacts with its including collagen, proteoglycans (chondroi- own family of receptors, and these receptors are tin sulfates, hyaluronic acid, etc.), and gly- as important as the signal molecules themselves coproteins, such as fibronectin and laminin. in determining the outcome of a signal. These molecules provide a substrate for cells on Fibroblast Growth Factors which they can anchor or migrate. For example, Originally named because they stimulate the laminin and type IV collagen are components growth of fibroblasts in culture, there are now of the basal lamina for epithelial cell attach- approximately two dozen FGF genes that have ment, and fibronectin molecules form scaffolds been identified, and they can produce hundreds for cell migration. Receptors that link extracellu- of protein isoforms by altering their RNA splic- lar molecules such as fibronectin and laminin to ing or their initiation codons. FGF proteins cells are called integrins. These receptors “inte- produced by these genes activate a collection grate” matrix molecules with a cell’s cytoskel- of tyrosine receptor kinases called fibro- etal machinery (e.g., actin microfilaments) blast growth factor receptors (FGFRs). In thereby creating the ability to migrate along turn, these receptors activate various signaling matrix scaffolding by using contractile proteins, pathways. FGFs are particularly important for such as actin. Also, integrins can induce gene angiogenesis, axon growth, and mesoderm dif- expression and regulate differentiation as in the ferentiation. Although there is redundancy in case of chondrocytes that must be linked to the the family, such that FGFs can sometimes sub- matrix to form cartilage. (3) There is direct trans- stitute for one another, individual FGFs may be mission of signals from one cell to another by responsible for specific developmental events. For gap junctions. These junctions occur as chan- example, FGF8 is important for development of nels between cells through which small mol- the limbs and parts of the brain. ecules and ions can pass. Such communication is important in tightly connected cells like epithelia Hedgehog Proteins of the gut and neural tube because they allow The hedgehog gene was named because it coded these cells to act in concert. The junctions them- for a pattern of bristles on the leg of Drosophila selves are made of connexin proteins that form that resembled the shape of a hedgehog. In Sadler_Chap01.indd 7 8/25/2011 3:23:54 PM  8 Part 1 General Embryology mammals, there are three hedgehog genes, apoptosis (programmed cell death) in the Desert, Indian, and sonic hedgehog. Sonic hedgehog interdigital spaces and in other cell types. is involved in a number of developmental events including limb patterning, neural tube induc- tion and patterning, somite differentiation, gut Summary regionalization, and others. The receptor for the hedgehog family is Patched, which binds to a During the past century, embryology has pro- protein called Smoothened. The Smoothened gressed from an observational science to one protein transduces the hedgehog signal, but it involving sophisticated technological and molec- is inhibited by Patched until the hedgehog pro- ular advances. Together, observations and mod- tein binds to this receptor. Thus, the role of the ern techniques provide a clearer understanding paracrine factor hedgehog in this example is to of the origins of normal and abnormal develop- bind to its receptor to remove the inhibition of a ment and, in turn, suggest ways to prevent and transducer that would normally be active, not to treat birth defects. In this regard, knowledge of activate the transducer directly. gene function has created entire new approaches to the subject. WNT Proteins There are approximately 23,000 genes in There are at least 15 different WNT genes that the human genome, but these genes code for are related to the segment polarity gene, wingless approximately 100,000 proteins. Genes are in Drosophilia.Their receptors are members of the contained in a complex of DNA and proteins frizzled family of proteins. WNT proteins are called chromatin, and its basic unit of structure involved in regulating limb patterning, midbrain is the nucleosome. Chromatin appears tightly development, and some aspects of somite and coiled as beads of nucleosomes on a string and urogenital differentiation among other actions. is called heterochromatin. For transcription to occur, DNA must be uncoiled from the beads The TGF-b Superfamily as euchromatin. Genes reside within strands The TGF-b superfamily has more than 30 mem- of DNA and contain regions that can be trans- bers and includes the TGF-bs, the bone mor- lated into proteins, called exons, and untranslat- phogenetic proteins, the activin family, the able regions, called introns. A typical gene also Müllerian inhibiting factor (MIF, anti-Mül- contains a promoter region that binds RNA lerian hormone), and others. The first member polymerase for the initiation of transcription; a of the family, TGF-b1, was isolated from virally transcription initiation site, to designate the transformed cells.TGF-b members are important first amino acid in the protein; a translation ter- for extracellular matrix formation and epithelial mination codon; and a 3′ untranslated region branching that occurs in lung, kidney, and salivary that includes a sequence (the poly A addition gland development. The BMP family induces site) that assists with stabilization of the mRNA. bone formation and is involved in regulating cell The RNA polymerase binds to the promoter division, cell death (apoptosis), and cell migration region that usually contains the sequence TATA, among other functions. the TATA box. Binding requires additional pro- teins called transcription factors. Methylation Other Paracrine Signaling Molecules of cytosine bases in the promoter region silences Another group of paracrine signaling molecules genes and prevents transcription. This process is important during development are neurotrans- responsible for X chromosome inactivation mitters, including serotonin and norepinephrine, whereby the expression of genes on one of the X that act as ligands and bind to receptors just as pro- chromosomes in females is silenced and also for teins do.These molecules are not just transmitters genomic imprinting in which either a paternal for neurons, but also provide important signals for or a maternal gene’s expression is repressed. embryological development. For example, sero- Different proteins can be produced from a tonin (5HT) acts as a ligand for a large number of single gene by the process of alternative splicing receptors, most of which are G protein–coupled that removes different introns using spliceo- receptors. Acting through these receptors, 5HT somes. Proteins derived in this manner are called regulates a variety of cellular functions, including splicing isoforms or splice variants. Also, cell proliferation and migration, and is impor- proteins may be altered by post-translational tant for establishing laterality, gastrulation, heart modifications, such as phosphorylation or development, and other processes during early cleavage. stages of differentiation. Norepinephrine also acts Induction is the process whereby one group through receptors and appears to play a role in of cells or tissues (the inducer) causes another Sadler_Chap01.indd 8 8/25/2011 3:23:54 PM  Chapter 1 Introduction to Molecular Regulation and Signaling 9 group (the responder) to change their fate. The neurotransmitters, such as serotonin (5HT) capacity to respond is called competence and and norepinephrine, also act through para- must be conferred by a competence factor. crine signaling, serving as ligands and binding to Many inductive phenomena involve epithelial– receptors to produce specific cellular responses. mesenchymal interactions. Juxtacrine factors may include products of the Signal transduction pathways include a extracellular matrix, ligands bound to a cell’s signaling molecule (the ligand) and a recep- surface, and direct cell-to-cell communications. tor. The receptor usually spans the cell mem- brane and is activated by binding with its specific ligand. Activation usually involves the capacity Problems to Solve to phosphorylate other proteins, most often as a kinase. This activation establishes a cascade of 1. What is meant by “competence to respond” enzyme activity among proteins that ultimately as part of the process of induction? What activates a transcription factor for initiation of tissues are most often involved in induction? gene expression. Give two examples. Cell-to-cell signaling may be paracrine, 2. Under normal conditions, FGFs and their involving diffusable factors, or juxtacrine, receptors (FGFRs) are responsible for growth involving a variety of nondiffusable factors. of the skull and development of the cranial Proteins responsible for paracrine signaling are sutures. How might these signaling pathways called paracrine factors or growth and dif- be disrupted? Do these pathways involve ferentiation factors (GDFs). There are four paracrine or juxtacrine signaling? Can you major families of GDFs: FGFs, WNTs, hedge- think of a way that loss of expression of one hogs, and TGF-bs. In addition to proteins, FGF might be circumvented? Sadler_Chap01.indd 9 8/25/2011 3:23:54 PM  Amniotic cavity Head end of embryo Tail end Future umbilical cord Heart Allantois Primordial germ cells in wall of yolk sac Yolk sac Sadler_Chap02.indd 10 8/25/2011 8:21:41 PM  Chapter 2 Gametogenesis: Conversion of Germ Cells into Male and Female Gametes 11 THE CHROMOSOME THEORY OF chromosomes are extremely long, they are spread INHERITANCE diffusely through the nucleus, and they cannot be recognized with the light microscope. With Traits of a new individual are determined by spe- the onset of mitosis, the chromosomes begin to cific genes on chromosomes inherited from the coil, contract, and condense; these events mark father and the mother. Humans have approxi- the beginning of prophase. Each chromosome mately 23,000 genes on 46 chromosomes. Genes now consists of two parallel subunits, chroma- on the same chromosome tend to be inherited tids, that are joined at a narrow region common together and so are known as linked genes. to both called the centromere. Throughout In somatic cells, chromosomes appear as 23 prophase, the chromosomes continue to con- homologous pairs to form the diploid num- dense, shorten, and thicken (Fig. 2.3A), but only ber of 46. There are 22 pairs of matching chro- at prometaphase do the chromatids become dis- mosomes, the autosomes, and one pair of sex tinguishable (Fig. 2.3B). During metaphase, the chromosomes. If the sex pair is XX, the indi- chromosomes line up in the equatorial plane, vidual is genetically female; if the pair is XY, the and their doubled structure is clearly visible individual is genetically male. One chromosome (Fig. 2.3C). Each is attached by microtubules of each pair is derived from the maternal gamete, extending from the centromere to the centriole, the oocyte, and one from the paternal gamete, forming the mitotic spindle. Soon, the centro- the sperm. Thus, each gamete contains a hap- mere of each chromosome divides, marking the loid number of 23 chromosomes, and the union beginning of anaphase, followed by migration of the gametes at fertilization restores the dip- of chromatids to opposite poles of the spindle. loid number of 46. Finally, during telophase, chromosomes uncoil and lengthen, the nuclear envelope reforms, Mitosis and the cytoplasm divides (Fig. 2.3D–F ). Each Mitosis is the process whereby one cell divides, daughter cell receives half of all doubled chromo- giving rise to two daughter cells that are geneti- some material and thus maintains the same num- cally identical to the parent cell (Fig. 2.3). Each ber of chromosomes as the mother cell. daughter cell receives the complete complement of 46 chromosomes. Before a cell enters mitosis, Meiosis each chromosome replicates its deoxyribonu- Meiosis is the cell division that takes place in the cleic acid (DNA). During this replication phase, germ cells to generate male and female gametes, Chromosome Double-structured Centriole chromosome A B C Prophase Prometaphase Metaphase D E F Anaphase Telophase Daughter cells Figure 2.3 Various stages of mitosis. In prophase, chromosomes are visible as slender 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. Sadler_Chap02.indd 11 8/25/2011 8:21:45 PM  12 Part 1 General Embryology sperm and egg cells, respectively. Meiosis requires paired homologous chromosomes (Fig. 2.4C). two cell divisions, meiosis I and meiosis II, Segments of chromatids break and are exchanged to reduce the number of chromosomes to the as homologous chromosomes separate. As separa- haploid number of 23 (Fig. 2.4). As in mitosis, tion occurs, points of interchange are temporarily male and female germ cells (spermatocytes united and form an X-like structure, a chiasma and primary oocytes) at the beginning of (Fig. 2.4C). The approximately 30 to 40 cross- meiosis I replicate their DNA so that each of overs (one or two per chromosome) with each the 46 chromosomes is duplicated into sister meiotic I division are most frequent between chromatids. In contrast to mitosis, however, genes that are far apart on a chromosome. homologous chromosomes then align them- As a result of meiotic divisions: selves in pairs, a process called synapsis. The Genetic variability is enhanced through pairing is exact and point for point except for crossover, which redistributes genetic the XY combination. Homologous pairs then material separate into two daughter cells, thereby reduc- random distribution of homologous chro- ing the chromosome number from diploid to mosomes to the daughter cells haploid. Shortly thereafter, meiosis II separates Each germ cell contains a haploid number sister chromatids. Each gamete then contains of chromosomes, so that at fertilization the 23 chromosomes. diploid number of 46 is restored. Crossover Polar Bodies Crossovers, critical events in meiosis I, are the Also during meiosis, one primary oocyte gives interchange of chromatid segments between rise to four daughter cells, each with 22 plus A B C D Pairing begins Pairing of Chiasma Pulling apart of chromosomes formation double-structured chromosomes Anaphase of 1st meiotic division E Cells contain 23 Cells resulting double-structured from 1st chromosomes meiotic division F Cells contain 23 Cells resulting single chromosomes from 2nd meiotic division G Figure 2.4 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,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. Sadler_Chap02.indd 12 8/25/2011 8:21:45 PM  Primary oocyte after DNA These cells contain replication 46 double-structured Primary spermatocyte chromosomes after DNA replication First Maturation Division Secondary 23 double-structured oocyte chromosomes Secondary spermatocyte Second Maturation Division Mature oocyte 23 single (22 + X) chromosomes (22 + X) (22 + Y) A Polar bodies B Spermatids (22 + X) Sadler_Chap02.indd 13 8/25/2011 8:21:45 PM  Primary oocyte or spermatocyte after DNA duplication 46 double-structured chromosomes Normal meiotic Nondisjunction Nondisjunction division 1st meiotic division 2nd meiotic division 2nd meiotic 2nd meiotic 2nd meiotic division division division 24 22 22 24 22 24 23 single chromosomes chromosomes chromosomes A B C Sadler_Chap02.indd 14 8/25/2011 8:21:48 PM  A 14 21 t(14;21) B Sadler_Chap02.indd 15 8/25/2011 8:21:49 PM  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y A B Sadler_Chap02.indd 16 8/25/2011 8:21:50 PM Sadler_Chap02.indd 17 8/25/2011 8:21:53 PM  A B C D Sadler_Chap02.indd 18 8/25/2011 8:21:55 PM Sadler_Chap02.indd 19 8/25/2011 8:22:01 PM Sadler_Chap02.indd 20 8/25/2011 8:22:03 PM  A B Mitotic Mitotic division division A B C Primordial Oogonium Primary oocyte germ cell in prophase Sadler_Chap02.indd 21 8/25/2011 8:22:03 PM  22 Part 1 General Embryology Surface epithelium of ovary Resting primary oocyte Primary oocyte (diplotene stage) in prophase Follicular cell Flat epithelial cell Oogonia Primary oocytes in prophase of 1st meiotic division A B C 4th month 7th month Newborn Figure 2.17 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. follicular epithelial cells (Fig. 2.17B). A primary by follicular cells. The total number of primary oocyte, together with its surrounding flat epithelial oocytes at birth is estimated to vary from 600,000 to cells, is known as a primordial follicle (Fig. 2.18A). 800,000. During childhood, most oocytes become atretic; only approximately 40,000 are present by the Maturation of Oocytes Continues at Puberty beginning of puberty, and fewer than 500 will be Near the time of birth, all primary oocytes have ovulated. Some oocytes that reach maturity late in started prophase of meiosis I, but instead of proceed- life have been dormant in the diplotene stage of the ing into metaphase, they enter the diplotene stage, first meiotic division for 40 years or more before a resting stage during prophase that is character- ovulation. Whether the diplotene stage is the most ized by a lacy network of chromatin (Fig. 2.17C). suitable phase to protect the oocyte against environ- Primary oocytes remain arrested in prophase and do not mental influences is unknown.The fact that the risk finish their first meiotic division before puberty is reached. of having children with chromosomal abnormalities This arrested state is produced by oocyte matu- increases with maternal age indicates that primary ration inhibitor (OMI), a small peptide secreted oocytes are vulnerable to damage as they age. A B C Figure 2.18 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. Sadler_Chap02.indd 22 8/25/2011 8:22:06 PM  Chapter 2 Gametogenesis: Conversion of Germ Cells into Male and Female Gametes 23 At puberty, a pool of growing follicles is As development continues, fluid-filled spaces established and continuously maintained from appear between granulosa cells. Coalescence of the supply of primordial follicles. Each month, these spaces forms the antrum, and the follicle is 15 to 20 follicles selected from this pool begin to termed a vesicular or an antral follicle. Initially, mature. Some of these die, while others begin to the antrum is crescent-shaped, but with time, it accumulate fluid in a space called the antrum, enlarges (Fig. 2.19). Granulosa cells surrounding thereby entering the antral or vesicular stage the oocyte remain intact and form the cumulus (Fig. 2.19A). Fluid continues to accumulate such oophorus. At maturity, the mature vesicular that, immediately prior to ovulation, follicles are (Graafian) follicle may be 25 mm or more in quite swollen and are called mature vesicular diameter. It is surrounded by the theca interna, follicles or Graffian follicles (Fig. 2.19B). The which is composed of cells having characteristics antral stage is the longest,whereas the mature vesic- of steroid secretion, rich in blood vessels, and the ular stage encompasses approximately 37 hours theca externa, which gradually merges with the prior to ovulation. ovarian connective tissue (Fig. 2.19). As primordial follicles begin to grow, sur- With each ovarian cycle, a number of follicles rounding follicular cells change from flat to begin to develop, but usually only one reaches cuboidal and proliferate to produce a stratified full maturity. The others degenerate and become epithelium of granulosa cells, and the unit atretic. When the secondary follicle is mature, a is called a primary follicle (Fig. 2.18B,C). surge in luteinizing hormone (LH) induces Granulosa cells rest on a basement membrane the preovulatory growth phase. Meiosis I is com- separating them from surrounding ovarian con- pleted, resulting in formation of two daughter cells nective tissue (stromal cells) that form the theca of unequal size, each with 23 double-structured folliculi. Also, granulosa cells and the oocyte chromosomes (Fig. 2.20A,B). One cell, the sec- secrete a layer of glycoproteins on the surface of ondary oocyte, receives most of the cytoplasm; the oocyte, forming the zona pellucida (Fig. the other, the first polar body, receives practically 2.18C). As follicles continue to grow, cells of none. The first polar body lies between the zona the theca folliculi organize into an inner layer of pellucida and the cell membrane of the secondary secretory cells, the theca interna, and an outer oocyte in the perivitelline space (Fig. 2.20B). The fibrous capsule, the theca externa. Also, small, cell then enters meiosis II but arrests in metaphase finger-like processes of the follicular cells extend approximately 3 hours before ovulation. Meiosis across the zona pellucida and interdigitate with II is completed only if the oocyte is fertilized; microvilli of the plasma membrane of the oocyte. otherwise, the cell degenerates approximately 24 These processes are important for transport of hours after ovulation. The first polar body may materials from follicular cells to the oocyte. undergo a second division (Fig. 2.20C). Theca interna Theca externa Follicular antrum Antrum Primary oocyte A B Zona pellucida Cumulus oophorus Figure 2.19 A. Vesicular (antral) stage follicle.The oocyte, surrounded by the zona pellucida, is off center; the antrum has developed by fluid accumulation between intercellular spaces. Note the arrangement of cells of the theca interna and the theca externa. B. Mature vesicular (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. Sadler_Chap02.indd 23 8/25/2011 8:22:07 PM  24 Part 1 General Embryology 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 2.20 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. Spermatogenesis Shortly before puberty, the sex cords Maturation of Sperm Begins at Puberty acquire a lumen and become the seminif- Spermatogenesis, which begins at puberty, erous tubules. At about the same time, includes all of the events by which sper- PGCs give rise to spermatogonial stem cells. matogonia are transformed into spermato- At regular intervals, cells emerge from this zoa. At birth, germ cells in the male infant stem cell population to form type A sper- can be recognized in the sex cords of the tes- matogonia, and their production marks tis as large, pale cells surrounded by support- the initiation of spermatogenesis. Type ing cells (Fig. 2.21A). Supporting cells, which A cells undergo a limited number of mitotic are derived from the surface epithelium of the divisions to form clones of cells. The last cell testis in the same manner as follicular cells, division produces type B spermatogonia, become sustentacular cells, or Sertoli cells which then divide to form primary sper- (Fig. 2.21B). matocytes (Figs. 2.21B and 2.22). Primary Spermatozoon Maturing spermatids Spermatids Primary spermatocyte in prophase Primordal Sertoli germ cell cells Sertoli cell A B Spermatogonial division Basement membrane Spermatogonia Figure 2.21 A. Cross section through primitive sex cords of a newborn boy showing PGCs and supporting cells. B. Cross section through a seminiferous tubule at puberty. Note the different stages of spermatogenesis and that developing sperm cells are embedded in the cytoplasmic processes of a supporting Sertoli cell. Sadler_Chap02.indd 24 8/25/2011 8:22:07 PM  Chapter 2 Gametogenesis: Conversion of Germ Cells into Male and Female Gametes 25 Type A dark spermatogonia Type A pale spermatogonia Type A pale spermatogonia Type B spermatogonia Primary spermatocytes Secondary spermatocytes Early spermatids Late spermatids Residual bodies Spermatozoa Figure 2.22 Type A spermatogonia, derived from the spermatogonial stem cell population, represent the first cells in the process of spermatogenesis. Clones of cells are established, and cytoplasmic bridges join cells in each succeeding divi- sion until individual sperm are separated from residual bodies. In fact, the number of individual interconnected cells is considerably greater than depicted in this figure. Sadler_Chap02.indd 25 8/25/2011 8:22:08 PM  26 Part 1 General Embryology Resting primary Secondary Type B spermatocyte spermatocyte spermatogonium Spermatid A B C D Mitotic 1st meiotic 2nd meiotic division division division Figure 2.23 The products of meiosis during spermatogenesis in humans. spermatocytes then enter a prolonged pro- binding to Sertoli cells stimulates testicular fluid phase (22 days) followed by rapid comple- production and synthesis of intracellular andro- tion of meiosis I and formation of secondary gen receptor proteins. spermatocytes. During the second mei- otic division, these cells immediately begin Spermiogenesis to form haploid spermatids (Figs. 2.21B to The series of changes resulting in the trans- 2.23). Throughout this series of events, from formation of spermatids into spermatozoa the time type A cells leave the stem cell popu- is spermiogenesis. These changes include lation to formation of spermatids, cytokinesis (1) formation of the acrosome, which cov- is incomplete, so that successive cell genera- ers half of the nuclear surface and contains tions are joined by cytoplasmic bridges. Thus, enzymes to assist in penetration of the egg the progeny of a single type A spermatogo- and its surrounding layers during fertilization nium form a clone of germ cells that maintain (Fig. 2.24); (2) condensation of the nucleus; contact throughout differentiation (Fig. 2.22). (3) formation of neck, middle piece, and tail; Furthermore, spermatogonia and spermatids and (4) shedding of most of the cytoplasm remain embedded in deep recesses of Sertoli as residual bodies that are phagocytized by cells throughout their development (Fig. Sertoli cells. In humans, the time required for 2.21B). In this manner, Sertoli cells support a spermatogonium to develop into a mature and protect the germ cells, participate in their spermatozoon is approximately 74 days, and nutrition, and assist in the release of mature approximately 300 million sperm cells are spermatozoa. produced daily. Spermatogenesis is regulated by LH produc- When fully formed, spermatozoa enter the tion by the pituitary gland. LH binds to receptors lumen of seminiferous tubules. From there, they on Leydig cells and stimulates testosterone pro- are pushed toward the epididymis by contractile duction, which in turn binds to Sertoli cells to elements in the wall of the seminiferous tubules. promote spermatogenesis. Follicle-stimulating Although initially only slightly motile, spermato- hormone (FSH) is also essential because its zoa obtain full motility in the epididymis. Centriole Mitochondria Tail piece Golgi Golgi material Golgi material A Acrosomic B Ring structure granule Middle piece Acrosome C D Nucleus covered by the acrosome Figure 2.24 Important stages in transformation of the human spermatid into the spermatozoon. Sadler_Chap02.indd 26 8/25/2011 8:22:11 PM  A B C Primordial follicle with Trinucleated oocyte two oocytes Sadler_Chap02.indd 27 8/25/2011 8:22:12 PM  28 Part 1 General Embryology remain surrounded by a layer of follicular cells cells give rise to primary spermatocytes, which derived from the surface epithelium of the ovary through two successive meiotic divisions pro- (Fig. 2.17). Together, they form the primordial duce four spermatids (Fig. 2.5). Spermatids follicle. At puberty, a pool of growing follicles go through a series of changes (spermiogen- is recruited and maintained from the finite sup- esis) (Fig. 2.24), including (1) formation of ply of primordial follicles. Thus, every month, the acrosome; (2) condensation of the nucleus; 15 to 20 follicles begin to grow, and as they (3) formation of neck, middle piece, and tail; mature, they pass through three stages: (1) pri- and (4) shedding of most of the cytoplasm. mary or preantral, (2) vesicular or antral, and The time required for a spermatogonium to (3) mature vesicular or Graafian follicle. The become a mature spermatozoon is approxi- primary oocyte remains in prophase of the first mately 74 days. meiotic division until the secondary follicle is mature. At this point, a surge in LH stimulates preovulatory growth: Meiosis I is completed, and Problems to Solve a secondary oocyte and polar body are formed. 1. What is the most common cause of abnor- Then, the secondary oocyte is arrested in meta- mal chromosome number? Give an example phase of meiosis II approximately 3 hours before of a clinical syndrome involving abnormal ovulation and will not complete this cell division numbers of chromosomes. until fertilization. In the male, primordial cells remain dor- 2. In addition to numerical abnormalities, what mant until puberty, and only then do they types of chromosomal alterations occur? differentiate into spermatogonia. These stem 3. What is mosaicism, and how does it occur? Sadler_Chap02.indd 28 8/25/2011 8:22:15 PM Sadler_Chap03.indd 29 8/25/2011 8:23:34 PM Sadler_Chap03.indd 30 Hypothalamic impulses Pituitary gland 30 Part 1 General Embryology Gonadotropins FSH LH Maturation of follicle Ovulation Corpus luteum Degenerating corpus luteum 1 2 3 4 Figure 3.1 Drawing showing the role of the hypothalamus and pituitary gland in regulating the ovarian cycle. Under the influence of GnRH from the hypothalamus, the pituitary releases the gonadotropins, FSH, and LH. Follicles are stimulated to grow by FSH and to mature by FSH and LH. Ovulation occurs when concentrations of LH surge to high levels. LH also promotes development of the corpus luteum. 1, primordial follicle; 2, growing follicle; 3, vesicular follicle; 4, mature vesicular (graafian) follicle. 8/25/2011 8:23:37 PM  Chapter 3 First Week of Development: Ovulation to Implantation 31 Primary oocyte Granulosa Zona pellucida Antrum cells Theca externa Theca interna A Primordial follicle B Growing follicle C Vesicular follicle Figure 3.2 A. Primordial follicle. B. Growing follicle. C. Vesicular follicle. Every day from the pool of primordial follicles A, some begin to develop into growing follicles B, and this growth is independent of FSH.Then, as the cycle progresses, FSH secretion recruits growing follicles to begin development into vesicular (antral) follicles. C. During the last few days of maturation of vesicular follicles, estrogens, produced by follicular and thecal cells, stimulate increased production of LH by the pituitary gland (Fig. 3.1), and this hormone causes the follicle to enter the mature vesicular (graafian) stage, to complete meiosis I, and to enter meiosis II, where it is arrested in metaphase approximately 3 hours before ovulation. follicle. Coincident with final development of breaks free (ovulation) and floats out of the the vesicular follicle, there is an abrupt increase ovary (Fig. 3.3). Some of the cumulus oopho- in LH that causes the primary oocyte to com- rus cells then rearrange themselves around the plete meiosis I and the follicle to enter the zona pellucida to form the corona radiata preovulatory mature vesicular stage. Meiosis II (Figs. 3.2B to 3.6). is also initiated, but the oocyte is arrested in metaphase approximately 3 hours before ovula- Corpus Luteum tion. In the meantime, the surface of the ovary After ovulation, granulosa cells remaining in begins to bulge locally, and at the apex, an avas- the wall of the ruptured follicle, together with cular spot, the stigma, appears. The high con- cells from the theca interna, are vascularized centration of LH increases collagenase activity, by surrounding vessels. Under the influence of resulting in digestion of collagen fibers sur- LH, these cells develop a yellowish pigment and rounding the follicle. Prostaglandin levels also change into lutein cells, which form the corpus increase in response to the LH surge and cause luteum and secrete estrogens and progesterone local muscular contractions in the ovarian wall. (Fig. 3.3C). Progesterone, together with some Those contractions extrude the oocyte, which estrogen, causes the uterine mucosa to enter the together with its surrounding granulosa cells progestational or secretory stage in prepara- from the region of the cumulus oophorus tion for implantation of the embryo. Antrum Granulosa cells Luteal cells Ovarian stroma Theca interna Theca externa Blood vessels 1st polar body Oocyte in Cumulus 2nd meiotic oophorus Fibrin division cells A Mature vesicular follicle B Ovulation C Corpus luteum Figure 3.3 A. Mature vesicular 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 follicle differentiate into lutean cells. C. Corpus luteum. Note the large size of the corpus luteum, caused by hyper- trophy and accumulation of lipid in granulosa and theca interna cells. The remaining cavity of the follicle is filled with fibrin. Sadler_Chap03.indd 31 8/25/2011 8:23:38 PM  32 Part 1 General Embryology Figure 3.4 Relation of fimbriae and ovary. Fimbriae collect the oocyte and sweep it into the uterine tube. Oocyte Transport syncytiotrophoblast of the developing embryo. Shortly before ovulation, fimbriae of the uter- The corpus luteum continues to grow and forms ine tube sweep over the surface of the ovary, and the corpus luteum of pregnancy (corpus the tube itself begins to contract rhythmically. It luteum graviditatis). By the end of the third is thought that the oocyte, surrounded by some month, this structure may be one third to one granulosa cells (Figs. 3.3B and 3.4), is carried into half of the total size of the ovary.Yellowish luteal the tube by these sweeping movements of the cells continue to secrete progesterone until the fimbriae and by motion of cilia on the epithelial end of the fourth month; thereafter, they regress lining. Once in the tube, cumulus cells withdraw slowly as secretion of progesterone by the tro- their cytoplasmic processes from the zona pellu- phoblastic component of the placenta becomes cida and lose contact with the oocyte. adequate for maintenance of pregnancy. Removal Once the oocyte is in the uterine tube, it is of the corpus luteum of pregnancy before the propelled by peristaltic muscular contractions of fourth month usually leads to abortion. the tube and by cilia in the tubal mucosa with the rate of transport regulated by the endocrine FERTILIZATION status during and after ovulation. In humans, the fertilized oocyte reaches the uterine lumen in Fertilization, the process by which male and approximately 3 to 4 days. female gametes fuse, occurs in the ampullary region of the uterine tube. This is the wid- Corpus Albicans est part of the tube and is close to the ovary If fertilization does not occur, the corpus luteum (Fig. 3.4). Spermatozoa may remain viable in the reaches maximum development approximately female reproductive tract for several days. 9 days after ovulation. It can easily be recognized as Only 1% of sperm deposited in the vagina a yellowish projection on the surface of the ovary. enter the cervix, where they may survive for Subsequently, the corpus luteum shrinks because many hours. Movement of sperm from the of degeneration of lutean cells (luteolysis) and cervix to the uterine tube occurs by muscular forms a mass of fibrotic scar tissue, the corpus contractions of the uterus and uterine tube and albicans. Simultaneously, progesterone produc- very little by their own propulsion. The trip tion decreases, precipitating menstrual bleeding. from cervix to oviduct can occur as rapidly as If the oocyte is fertilized, degeneration of the 30 minutes or as slow as 6 days. After reach- corpus luteum is prevented by human chori- ing the isthmus, sperm become less motile and onic gonadotropin, a hormone secreted by the cease their migration. At ovulation, sperm again Sadler_Chap03.indd 32 8/25/2011 8:23:39 PM  Chapter 3 First Week of Development: Ovulation to Implantation 33 become motile, perhaps because of chemoat- ampulla is not an advantage, since capacitation has tractants produced by cumulus cells surrounding not yet occurred and such sperm are not capable the egg, and swim to the ampulla, where fertil- of fertilizing the egg. Much of this conditioning ization usually occurs. Spermatozoa are not able during capacitation occurs in the uterine tube and to fertilize the oocyte immediately upon arrival involves epithelial interactions between the sperm in the female genital tract but must undergo and the mucosal surface of the tube. During this (1) capacitation and (2) the acrosome reac- time, a glycoprotein coat and seminal plasma pro- tion to acquire this capability. teins are removed from the plasma membrane that Capacitation is a period of conditioning in overlies the acrosomal region of the spermato- the female reproductive tract that in the human zoa. Only capacitated sperm can pass through the lasts approximately 7 hours. Thus, speeding to the corona cells and undergo the acrosome reaction. A Corona radiata cells Phase 1 Phase 2 Polor body in division Acrosome Inner acrosomal membrane dissolves Sperm nucleus Secondary oocyte in Plasma 2nd meiotic division membrane Fusion oocyte and sperm cell membranes B Phase 3 Figure 3.5 A. Scanning electron micrograph of sperm binding to the zona pellucida. B. The three phases of oocyte pen- etration. In phase 1, spermatozoa pass through the corona radiata barrier; in phase 2, one or more spermatozoa penetrate the zona pellucida; in phase 3, one spermatozoon penetrates the oocyte membrane while losing its own plasma membrane. Inset shows normal spermatocyte with acrosomal head cap. Sadler_Chap03.indd 33 8/25/2011 8:23:40 PM  34 Part 1 General Embryology Figure 3.6 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,E. Chromosomes become arranged on the spindle, split longitudinally, and move to opposite poles. F. Two-cell stage. The acrosome reaction, which occurs after enzymes (acrosin) allows sperm to penetrate the binding to the zona pellucida, is induced by zona zona, thereby coming in contact with the plasma proteins.This reaction culminates in the release of membrane of the oocyte (Fig. 3.5). Permeability of enzymes needed to penetrate the zona pellucida, the zona pellucida changes when the head of the including acrosin- and trypsin-like substances sperm comes in contact with the oocyte surface. (Fig. 3.5). This contact results in release of lysosomal enzymes The phases of fertilization include from cortical granules lining the plasma mem- Phase 1, penetration of the corona radiata brane of the oocyte. In turn, these enzymes alter Phase 2, penetration of the zona pellucida properties of the zona pellucida (zona reaction) Phase 3, fusion of the oocyte and sperm cell to prevent sperm penetration and inactivate spe- membranes cies-specific receptor sites for spermatozoa on the zona surface. Other spermatozoa have been found Phase 1: Penetration of the Corona embedded in the zona pellucida, but only one Radiata seems to be able to penetrate the oocyte (Fig. 3.6). Of the 200 to 300 million spermatozoa normally deposited in the female genital tract, only 300 Phase 3: Fusion of the Oocyte and to 500 reach the site of fertilization. Only one Sperm Cell Membranes of these fertilizes the egg. It is thought that the The initial adhesion of sperm to the oocyte is others aid the fertilizing sperm in penetrating the mediated in part by the interaction of integrins barriers protecting the female gamete. Capacitated on the oocyte and their ligands, disintegrins, on sperm pass freely through corona cells (Fig. 3.5). sperm. After adhesion, the plasma membranes of the sperm and egg fuse (Fig. 3.5). Because the Phase 2: Penetration of the Zona plasma membrane covering the acrosomal head Pellucida cap disappears during the acrosome reaction, The zona is a glycoprotein shell surrounding the actual fusion is accomplished between the oocyte egg that facilitates and maintains sperm binding membrane and the membrane that covers the and induces the acrosome reaction. Both binding posterior region of the sperm head (Fig. 3.5). In and the acrosome reaction are mediated by the the human, both the head and the tail of the sper- ligand ZP3, a zona protein. Release of acrosomal matozoon enter the cytoplasm of the oocyte, but Sadler_Chap03.indd 34 8/25/2011 8:23:42 PM  A B Sadler_Chap03.indd 35 8/25/2011 8:23:43 PM Sadler_Chap03.indd 36 8/25/2011 8:23:46 PM  Chapter 3 First Week of Development: Ovulation to Implantation 37 Figure 3.8 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 is reach