Molecular Biology of the Cell - Volume 3 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

From Cells to Multicellular Organisms
Germ Cells and Fertilization
Gametes

In embryos of all vertebrates at an early stage of development, certain Cells become segregated as precursors of future gametes. Such primordial Germ Cells migrate into the developing Gonads (Ovaries in females, Testes in males), where, following a period of mitotic proliferation, they undergo Meiosis and differentiate into mature gametes. Subsequently, the fusion of an egg and a sperm following mating initiates the developmental process of the embryo, which in turn forms its own primordial germ cells, thus initiating a new cycle.

It remains unclear precisely what causes certain cells in a mammalian embryo to become germ cells, but it is known that in at least one Organism, the determining factor is a specific component (or components) of the egg Cytoplasm: in Drosophila, a specialized region of the cytoplasm—the polar plasm located at the posterior pole of the egg—contains small RNA-rich granules (polar granules). Cells that form in this region of the egg and incorporate the polar granules become primordial germ cells and ultimately migrate to the gonads, where they develop into gametes. If polar plasm is transplanted into the anterior pole of an egg, cells that would otherwise have become somatic cells are induced to develop into germ cells.

15.3.1. In higher animals, the egg is the only Cell from which a new individual can develop

In at least one respect, eggs are the most remarkable of all animal cells: upon activation, they can give rise to an entire new organism, sometimes within just a few days or weeks. In higher animals, this is the exclusive privilege of the egg. Activation is normally the result of the fusion of the egg with a sperm during Fertilization, although, as we shall see later, in many cases eggs can be activated by alternative and often strikingly simple means (see Section 15.4.3). Egg activation triggers a developmental program whose progressive unfolding leads to The formation of a new individual.

In most non-mammalian animals, the early stage of egg development consists primarily of a series of rapid cell divisions, or Cleavage, during which the overall mass of the embryo generally remains unchanged. As cleavage proceeds, the resulting cells become progressively smaller until they reach the typical size of a mature somatic cell. Although massive amounts of DNA and Proteins are synthesized during early cleavage, there is little or no need for RNA Synthesis (Gene METABOLISM/31.html">Transcription) at this stage. This is because, prior to fertilization, The Cell accumulates vast reserves of messenger RNAs, Ribosomes, Transfer RNAs, and all the precursors required for macromolecular synthesis, as well as specialized mRNAs encoding proteins essential for the early Selection/3.html">Stages of development.

Particularly large stores of nutrients are required by eggs that undergo a prolonged period of embryonic development outside the parental organism; consequently, the eggs of amphibians and birds are considerably larger than, for example, mammalian eggs. Freshwater and marine invertebrates that develop from small eggs outside the parent (such as sea urchins) typically transform rapidly into free-feeding larvae.

Within the adult organism, an egg has the potential to give rise to a cell of virtually any type. Nevertheless, it is by no means an undifferentiated cell; rather, it is highly specialized to carry out a single function—building a new individual. Before discussing how the egg develops up to the point where it is ready for fertilization, let us briefly examine some of its unique features.

15.3.2. Eggs are highly specialized cells capable of independent development, containing abundant nutrient reserves and a protective coat [10]

The most obvious distinguishing feature of an egg is its large size. A typical egg is spherical or oval, with a diameter of about 100 µm in humans and sea urchins, 1 to 2 mm in frogs and fish, and measuring in centimeters in birds and reptiles (recall that the diameter of a typical somatic cell is only about 20 µm) (Figs. 15-21 and 15-22). The Nucleus can be equally impressive in size; for instance, in a 1500 µm frog oocyte, the nuclear diameter is about 400 µm, serving as a reservoir for proteins in anticipation of the rapid divisions that immediately follow fertilization.

The Nutritional Requirements of the cell are met primarily by yolk—a protoplasmic material rich in Lipids and proteins. Yolk is typically packaged into discrete structures known as yolk platelets or granules. In eggs that develop outside the mother and give rise to large animals, yolk may account for more than 95% of the total volume, whereas in mammals, whose embryos derive most of their Nutrition from the mother, yolk occupies less than 5% of the egg's volume.

Another major specialized Structure OF THE egg is its outer coat—an extracellular covering composed largely of glycoprotein molecules, some of which are secreted by the egg itself while others are produced by surrounding cells. In many species, this coat features an inner layer immediately adjacent to the egg Plasma Membrane, termed the zona pellucida in mammals (Fig. 15-23) and the vitelline layer in other vertebrates and invertebrates (such as sea urchins). This layer protects the egg from mechanical damage; in some species, it also acts as a species-specific sperm barrier, allowing penetration only by sperm of the same or very closely related species (see Section 15.4.2). In non-mammalian animals, neighboring cells frequently deposit additional layers over the vitelline layer. For example, as frog eggs pass from the Ovary through the oviduct (the tube through which eggs are expelled), their coats acquire several extra layers of a gelatinous substance secreted by epithelial Cells of the oviduct. Similarly, a chicken egg acquires its 'albumen' and hard shell as it traverses the oviduct (following fertilization), whereas insect eggs are encased in a thin, tough protective shell called the chorion, secreted by the follicular cells surrounding each egg.

Many eggs (including those of mammals) contain specialized secretory vesicles located beneath The Plasma Membrane in the outer, or cortical, layer of the cytoplasm (Fig. 15-24). Upon egg activation by a sperm, these cortical granules release their contents via exocytosis, which modifies The properties of the egg coats so that additional sperm can no longer penetrate the egg (see Section 15.4.5).

While cortical granules are usually distributed fairly uniformly throughout the egg cortex, other cytoplasmic components may be arranged in a highly asymmetrical manner. For example, in the frog egg, the bulk of the yolk is concentrated at one pole (the vegetal pole), whereas the nucleus lies closer to the opposite pole (the animal pole). The polarity of the egg, which is largely dependent on environmental cues, often determines the polarity of the developing embryo (see Section 16.1.1).

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Fig. 15-21. Three different eggs shown at actual size. The diameter of a human egg is 0.1 mm.

Fig. 15-22. Relative sizes of various eggs compared with the size of a typical somatic cell.

15.3.3. The egg passes through several stages in its development [10, 11]

A developing egg is called an oocyte; its differentiation into a mature egg (or ovum), which involves a series of highly specialized stages, deviates from standard cell cycles. As we have already learned, instead of continuing to divide by conventional mitosis, germ cells undergo their final two divisions via the more complex process of meiosis. Furthermore, oocytes have evolved specialized mechanisms that allow them to arrest meiotic progression: oocytes are arrested in prophase I for extended periods during which they grow substantially in size, and later, in anticipation of fertilization, many species temporarily halt development at metaphase II.

Although the details of egg development (oogenesis) vary among species, the fundamental stages are similar (Fig. 15-25). Primordial germ cells migrate into the developing gonad and differentiate into oogonia; following a period of mitotic multiplication, oogonia differentiate into primary oocytes, which enter the first meiotic division. DNA Replication takes place, and each chromosome subsequently consists of two chromatids; homologous Chromosomes pair along their entire length, and Crossing-over occurs between the chromatids of the paired chromosomes. At this stage, prophase is arrested for a variable period, ranging from days to many years depending on the organism. During this prolonged prophase (or sometimes upon the attainment of sexual maturity), primary oocytes acquire their external coats and cortical granules, and accumulate ribosomes, yolk, Glycogen, lipids, and messenger RNAs that will later direct the synthesis of proteins required for early embryonic growth and the initiation of the developmental program. In many oocytes, this transcriptional activity is reflected in the visible structure of the still-paired chromosomes: they uncoil to form lateral loops, acquiring the characteristic 'lampbrush' appearance typical of chromosomes actively engaged in RNA synthesis (see Section 9.2.4).

The next phase of development, termed egg maturation, does not begin until sexual maturity is reached. Driven by Hormonal Influences (see below), the first meiotic division ensues: chromosomes recondense, the nuclear envelope breaks down (an event conventionally taken as the onset of maturation), and at anaphase I, replicated homologous chromosomes segregate into daughter nuclei, each now containing half the original chromosome number. However, cytoplasmic division is extremely asymmetric, so that by the end of the First Division two cells of vastly different sizes are produced: a small polar body and a large secondary oocyte, which retains all the developmental potential. At this stage, each chromosome still consists of two sister chromatids; they remain in this state until the second meiotic division, when the chromatids separate from one another through a process resembling mitotic anaphase (see Section 15.2.7). Following the final chromosomal segregation in anaphase II, the cytoplasm of the large secondary oocyte divides asymmetrically once again, giving rise to a mature egg (or ovum) and another small polar body, leaving both cells with a haploid Complement of single chromosomes (see Fig. 15-25). Thanks to these two asymmetric cytoplasmic divisions, oocytes retain their large size despite undergoing two meiotic divisions. All polar bodies are very small and gradually degenerate.

Fig. 15-23. A. Scanning Cytology/cytology/93.html">ELECTRON MICROGRAPHS OF a hamster egg. The zona pellucida is clearly visible; in micrograph B, this coat (to which numerous sperm are attached) has been partially reflected to reveal the underlying egg plasma membrane with its numerous microvilli. (M. Phillips, J. Ultrastruct. Res., 72, 1-12, 1980.)

Fig. 15-24. Schematic section through a sea urchin egg, showing the arrangement of granules. Note that the vitelline layer is covered by a jelly coat up to 30 µm thick.

Fig. 15-25. Various stages of oogenesis. Oogonia develop from primordial germ cells that migrate to the ovary during early Embryogenesis. Following a series of mitotic divisions, oogonia enter the first meiotic division, at which stage they are referred to as primary oocytes. In mammals, primary oocytes form very early and remain arrested in prophase I (between the 3rd and 8th months of Human embryonic development) until the female reaches sexual maturity. Thereafter, under hormonal influence, a small number of oocytes periodically mature, completing the first meiotic division to become secondary oocytes; the latter undergo the second meiotic division to become mature eggs. The stage at which the egg is released from the ovary and fertilized varies among animals. In most vertebrates, oocyte maturation arrests at metaphase II, and the secondary oocyte completes meiosis only after fertilization. All polar bodies eventually degenerate. However, in most animals, including mammals, polar bodies remain within the egg coat, and in some species, the first polar body undergoes one division before degenerating.

In most vertebrates, oocyte maturation proceeds to metaphase II and arrests at this stage. Upon ovulation, the secondary oocyte is released from the ovary and, if fertilization occurs, completes meiosis.

15.3.4. The Egg Achieves Large Size Through Special Mechanisms [10, 11]

A small somatic cell with a diameter of 10–20 μm typically takes about a day to double its mass in preparation for division. At the same rates of macromolecular synthesis, that same cell would take a very long time to reach the thousandfold greater mass characteristic of a mammalian egg (100 μm in diameter) or the millionfold greater mass of an insect egg (1000 μm in diameter). Meanwhile, some insects live for only a few days yet manage to produce eggs exceeding 1000 μm in diameter. Clearly, their eggs must possess special mechanisms to attain such a large size.

One factor contributing to this growth is the presence of extra gene copies in the cell. In the oocytes of many animals, the completion of meiosis is delayed until almost the very end of maturation, so that these oocytes retain a duplicated diploid set of chromosomes throughout most of their growth period. Thus, they contain twice as much DNA for RNA synthesis as an average somatic cell in the Cell Cycle phase. In some eggs, the accumulation of extra DNA goes even further, leading to the formation of numerous additional copies of specific genes. As we saw in Chapter 9, to produce a sufficient number of ribosomes for Protein Synthesis, somatic cells in most organisms require 10 to 500 copies of the ribosomal RNA genes. Because eggs require an even greater number of ribosomes for protein synthesis during early embryogenesis, rRNA genes are amplified in the eggs of many animals; for instance, fish and amphibian eggs contain 1 to 2 million copies of such genes (Fig. 15-26).

The growth of many eggs depends to some extent on the biosynthetic activity of other cells. For example, yolk—a major component of large eggs—is typically synthesized outside the ovary and subsequently transferred to the oocyte. In birds, amphibians, and insects, yolk proteins are produced in Liver cells (or their functional equivalents), which secrete these substances into the bloodstream. Oocytes residing in the ovaries extract these future yolk protein components from the extracellular fluid via receptor-mediated endocytosis (see Fig. 6-72). Oocyte nutrition may also be supported by accessory cells located within the ovary. Depending on the organism, two Different types of ovarian accessory cells perform this function during oogenesis. Some invertebrates possess nurse cells: they typically surround the oocyte and are connected to it by cytoplasmic bridges through which macromolecules can pass directly into its cytoplasm. Nurse cells synthesize components for insect eggs (such as ribosomes, mRNA, proteins, etc.) that would otherwise be produced by the vertebrate egg itself.

Fig. 15-26. Micrograph of an isolated nucleus from a frog egg. The nucleus is stained with cresyl violet to reveal numerous nucleoli—the result of extensive ribosomal RNA gene Amplification. (D. D. Brown, I. Dawid, Science, 160, 273-275, 1968.)

Fig. 15-27. This diagram illustrates how a single Drosophila oogonium gives rise to 15 nurse cells and one oocyte, all interconnected by cytoplasmic bridges. With each mitosis, all cells divide once: in the first mitosis, cell 1 produces cells 1 and 2; In the second mitosis, cell 1 produces cells 1 and 3, cell 2 produces cells 2 and 4, and so on. Because cytoplasmic bridges form wherever spindle remnants connect two daughter cells during telophase, these bridges link only those cells produced by a shared mitosis. Only cell 1 or 2 develops into the egg; this may be because only these cells are connected by intercellular bridges to four other cells. An unusual feature of these divisions is that the cell size does not double before mitosis, so with each division the cells become progressively smaller. Later, during egg maturation, the nurse cells become exceptionally large; they synthesize massive amounts of macromolecules and Organelles such as ribosomes and Mitochondria, pumping them into the oocyte through the cytoplasmic bridges.

In some species, nurse cells derive from the same oogonium as the connected oocyte. For instance, in Drosophila, an oogonium undergoes four mitotic divisions to yield 16 cells. One of these cells becomes the egg, while the others differentiate into nurse cells, remaining interconnected with each other and with the egg via cytoplasmic bridges (Fig. 15-27). The nurse cells undergo multiple rounds of DNA replication without Cell Division, causing each cell to progressively grow very large, with a DNA content a thousand times normal (such DNA is organized into polytene chromosomes; see Section 9.2.5). All 15 nurse cells, containing hundreds to thousands of genome equivalents, synthesize Materials required for a single egg.

Another class of cells that helps nourish developing oocytes is follicular cells, found in both vertebrates and invertebrates. They form an epithelial layer surrounding the oocyte (Fig. 15-28) and are coupled to it via Gap Junctions, which permit the passage of small molecules but not macromolecules (Section 14.1.5). Although such cells cannot supply the oocyte with pre-made macromolecules through these junctions, they likely provide small precursor molecules from which macromolecules are built. In addition, follicular cells frequently secrete macromolecules that either become part of the egg envelope or enter the growing oocyte via endocytosis.

Fig. 15-28. Transmission electron micrograph of a primordial rabbit follicle, consisting of a central oocyte surrounded by a single layer of flattened follicular cells. The primordial follicle is enclosed in a basement membrane (barely visible in the micrograph), which is in turn surrounded by ovarian Connective Tissue. Note that at this developmental stage, the primary oocyte lacks a zona pellucida and cortical granules. (J. Van Blerkom, P. Motta, Cellular Basis of Mammalian Reproduction, Baltimore: Urban and Schwarzenberg, 1979.)

The surrounding accessory cells enable the resumption of oocyte development; in both vertebrates and amphibians/invertebrates, it is the accessory cells that respond to polypeptide Hormones (gonadotropins), thereby overcoming prophase I arrest and priming the oocytes for further maturation.

15.3.5. Oocyte Maturation in Vertebrates Is Triggered by a Decrease in Intracellular Cyclic AMP Concentration [12]

In many animals, the completion of meiotic division I does not occur until sexual maturity, when gonadotropins appear in the Blood. These polypeptide hormones stimulate ovarian accessory cells to release a secondary messenger, which in turn acts on the oocytes to induce maturation. For most vertebrates, egg maturation encompasses the developmental period of the primary oocyte from the first meiotic division up to the subsequent arrest at metaphase II; the resulting secondary oocyte remains at this stage until fertilization, which triggers the completion of meiosis (Fig. 15-25).

Oocyte maturation has been studied most extensively in amphibians. In these animals, pituitary-controlled gonadotropins act on follicular cells surrounding the oocytes, initiating the release of the steroid hormone progesterone. Like other Steroid Hormones, progesterone diffuses across the Plasma Membranes of most target cells and binds to intracellular receptor proteins that regulate the transcription of specific genes (see Section 12.2.1). However, in oocyte maturation, progesterone appears to act differently: it is thought to bind to plasma membrane receptor proteins. This leads to the inactivation of adenylate cyclase in the oocyte plasma membrane, resulting in a drop in cytosolic cyclic AMP (cAMP) concentration and a corresponding decrease in cAMP-dependent protein kinase (A-kinase) activity (see Section 12.4.1).

There is compelling evidence that a decrease in A-kinase activity is both necessary and sufficient to trigger the oocyte maturation program. Microinjection of a catalytically active kinase fragment that Functions independently of cyclic AMP (see Section 12.4.2) inhibits progesterone-induced maturation, whereas microinjection of a specific kinase inhibitor initiates maturation even in the absence of progesterone. Experiments on mouse oocytes, also performed using microinjection techniques, have demonstrated that a similar Intracellular Signaling pathway operates in mammals. Although A-kinase substrates have not yet been identified, they are hypothesized to be Phosphoproteins involved in the meiotic arrest system responsible for holding the oocyte in prophase I. According to this hypothesis, The activity of these phosphoproteins is governed by A-kinase-mediated phosphorylation; a drop in cyclic AMP levels lifts the meiotic block and prompts its resumption.

15.3.6. Oocyte Maturation Is Associated with the Activation of Maturation-Promoting Factor [13]

The events following the drop in cyclic AMP levels are not yet fully elucidated, but they ultimately result in the Activation of a protein complex known as Maturation-Promoting Factor (MPF), which is believed to be essential for exit from meiotic prophase I. As discussed in Chapter 13, MPF has been remarkably conserved throughout eukaryotic evolution. Its pivotal role in the standard cell division cycle stems from its ability to drive cells from the G2 phase into the M phase (see Section 13.1.10). As previously mentioned, meiotic prophase I, despite its traditional name, closely resembles the G2 phase of a standard cell cycle: DNA replication has already occurred and is transcriptionally active, the nuclear envelope remains intact, and the mitotic spindle has not yet formed. Furthermore, much like the G2-to-M transition in dividing somatic cells, the transition from prophase I to the M phase of meiosis is triggered by MPF. In fact, MPF was originally discovered in frog oocytes as a maturation-inducing factor. Mature frog oocytes arrest at metaphase II, a stage characterized by high MPF levels (see below). When a small amount of cytoplasm from such a mature oocyte is injected into an immature oocyte, the MPF contained in the injection disrupts the nuclear membrane and induces chromosome Condensation—classic M-phase effects indicative of oocyte maturity.

A remarkable property of MPF is that it can be serially transferred from one oocyte to another indefinitely without loss of activity, despite the fact that such serial transfers result in enormous dilutions of the MPF solution. This indicates that MPF injections stimulate the recipient oocyte to synthesize even more MPF. This amplification occurs even in enucleated oocytes and in the presence of Protein Synthesis Inhibitors, suggesting that MPF likely activates preexisting MPF precursors rather than inducing de novo synthesis.

What mechanism underlies this autocatalytic self-amplification loop? Although MPF is not yet fully understood in molecular detail, evidence indicates that it is phosphorylated, that phosphorylation plays a crucial role in turning on MPF activity, and that at least one MPF component is a protein kinase. Thus, MPF can phosphorylate—and thereby activate—its own components. In frog oocytes, progesterone-induced inactivation of A-kinase initiates a complex cascade of events (coupled with protein synthesis) that ultimately activates small amounts of MPF, which in turn activate further MPF. Extensive phosphorylation of nuclear envelope and chromosomal structural proteins is characteristic of M-phase events (see Section 13.5.11), suggesting that MPF may also promote the synthesis of an unidentified protein kinase responsible for driving these downstream modifications (Fig. 15-29).

Cyclic fluctuations in MPF activity are widely considered the decisive regulatory mechanism governing the standard mitotic cell cycle, determining when a cell enters and exits mitosis (see Section 13.1.10). MPF appears to undergo analogous cyclic changes during oocyte maturation. An initial rise in MPF levels drives the oocyte through prophase and into metaphase I of meiosis. Subsequent inactivation of MPF allows the oocyte to complete nuclear division, whereas a renewed surge in MPF triggers the transition into M phase of the second meiotic division. A meiotic II arrest system (sometimes identified as cytostatic factor, or CSF, whose properties remain poorly understood) is thought to prevent MPF inactivation at this stage, thereby arresting mature eggs at metaphase II. Fertilization—likely by raising cytosolic Ca²⁺ concentrations (see Section 15.4.5)—allows the oocyte to complete the second meiotic division and, alongside recurring cycles of MPF activation and inactivation that drive repeated mitotic divisions, initiates embryonic development.

Fig. 15-29. A model illustrating how progesterone-induced maturation-promoting factor (MPF) may trigger the transition of a frog oocyte from prophase I to metaphase. According to this hypothesis, progesterone indirectly stimulates the synthesis of a specific enzyme that activates small amounts of MPF. This leads not only to the activation of much larger quantities of MPF (a strongly amplified response), but also to the activation of protein Kinases that phosphorylate nuclear membranes and chromosomal proteins. As a result, the nuclear envelope breaks down, chromosomes condense, and the cell enters metaphase. Subsequent inactivation of MPF leads to the re-Formation of the nuclear envelope, chromosome decondensation, and allows the cell to enter the second meiotic division (not shown in the figure). A similar mechanism likely underlies the maturation of mammalian oocytes. One of the components of MPF has been identified as a protein kinase homologous to the Yeast protein kinase encoded by the cdc2/28 gene, which plays a pivotal role in regulating the yeast cell cycle.

15.3.7. The majority of human oocytes degenerate in the ovary without ever maturing [14]

Although the molecular mechanisms underlying the key events of oocyte development and maturation appear remarkably similar across all vertebrates, the timing of these processes and The Nature of the extracellular signals that trigger them can vary considerably. For instance, in humans, these signals are significantly more complex and far less understood than in amphibians. Furthermore, the number of mature eggs produced by these two species differs strikingly. In frogs, oocytes develop from oogonia during each breeding season throughout the animal's reproductive life; hundreds of these oocytes divide and undergo ovulation in response to hormonal stimulation. In contrast, in human females, several million oocytes are formed from oogonia only During the first few months of embryonic development, and all but a few hundred of them eventually degenerate in the ovary without maturing.

Fig. 15-30. Micrograph of a maturing follicle in a rabbit ovary. The primary oocyte is larger than the younger oocytes shown in Fig. 15-28 and already contains cortical granules (hardly visible in this micrograph) as well as a narrow zona pellucida; the oocyte is surrounded by layers of follicular cells. The inner follicular cells extend processes that traverse the zona pellucida and form gap junctions with the oocyte (although these structures are not clearly visible in the photograph). The entire follicle is enclosed by a basal lamina. (J. Van Blerkom, P. Motta. Cellular Basis of Mammalian Reproduction, Baltimore, Urban and Schwarzenberg, 1979.)

Primary oocytes in a newborn girl arrest in prophase I of meiosis (much like amphibian primary oocytes), and the vast majority of them are surrounded by a single layer of follicular cells; such an oocyte, together with these follicular cells, constitutes a primordial follicle (Fig. 15-28). Sometime before birth, a small fraction of the primordial follicles periodically begins to grow, transforming into developing follicles: their cells enlarge and proliferate, forming a multilayered sheath around the primary oocyte. While remaining in prophase I, the oocyte itself grows and develops a zona pellucida and cortical granules (Fig. 15-30). In some of the developing follicles, a fluid-filled cavity, or antrum, forms, turning them into antral follicles (Fig. 15-31). What triggers the initial growth of individual primordial follicles and their transition into developing follicles remains unclear. However, continued development likely depends on pituitary gonadotropins [primarily follicle-stimulating hormone (FSH)] and estrogens secreted by the follicular cells themselves.

All developing follicles continue to grow and eventually either degenerate or release the oocytes enclosed within them during ovulation. However, oocyte maturation (completion of prophase I) and ovulation only occur after the onset of Puberty. From this time onward, once a month (roughly in the middle of the Menstrual cycle), a sharp surge in the level of another pituitary gonadotropin—luteinizing hormone (LH)—stimulates the growth of 15,000 to 20,000 developing follicles in the ovary. Of all these follicles, only one completes its development: the primary oocyte within it matures, reaches metaphase II of meiotic division, and arrests at this stage. Meanwhile, the single stimulated follicle rapidly enlarges and ruptures at The surface of the ovary, releasing the secondary oocyte, which at this point is still enclosed within a layer of follicular cells embedded in a jelly-like matrix (Fig. 15-32). Completion of meiosis in the released oocyte is only initiated if it is fertilized by a sperm within roughly 24 hours (or slightly longer).

Fig. 15-31. Micrograph of an antral follicle in a rabbit ovary. Multiple layers of follicular cells are now visible. The oocyte nuclei did not lie within the plane of the section and are therefore not visible in the photograph. For comparison, note the much smaller primordial follicle in the upper right corner. (J. Van Blerkom, P. Motta. Cellular Basis of Mammalian Reproduction, Baltimore, Urban and Schwarzenberg, 1979.)

Fig. 15-32. Schematic representation of human oocyte development stages. 1. Before birth, a small fraction of primordial follicles sequentially begins to grow, now referred to as developing follicles. 2. Some of the developing follicles accumulate fluid, transforming into antral follicles. 3. Following puberty, once a month, a surge of luteinizing hormone (LH) prompts about 20 antral follicles to undergo accelerated growth; however, only one of these follicles completes maturation and undergoes ovulation. The primary oocyte within this follicle completes the first meiotic division, producing a polar body and becoming a secondary oocyte (Fig. 15-25). 4. The secondary oocyte, arrested at metaphase of meiosis II, along with the polar body and some surrounding follicular cells, is released when the follicle ruptures at the ovarian surface. The secondary oocyte only completes the second meiotic division if fertilization occurs. Following ovulation, the emptied follicle transforms into an endocrine structure (the corpus luteum), which secretes progesterone, thereby preparing the Uterus to receive the fertilized oocyte. If fertilization does not take place, the corpus luteum regresses, and the inner lining of the uterus is shed and expelled from the body during menstruation.

How does the mid-cycle LH surge initiate oocyte maturation? It appears that the follicular cells surrounding the oocyte normally exert an inhibitory effect on its maturation, since oocytes spontaneously resume meiosis when removed from antral follicles and deprived of stimulating hormones in the culture medium. The LH surge likely stimulates egg maturation by lifting this inhibitory influence exerted by the follicular cells.

One of the puzzling Features of human egg maturation is that only a tiny fraction of the numerous antral follicles present in the ovaries at the time of the monthly LH surge begin to grow, and of these growing follicles, only a single one completes maturation and releases an oocyte; the rest are destined to degenerate. Presumably, immediately after the maturation of the "chosen" follicle reaches a certain critical point, a feedback mechanism comes into play, preventing any other follicle from maturing and ovulating during the same cycle. This means that over the course of a roughly 40-year reproductive lifespan, a woman releases only 400 to 500 eggs. The remaining 3 million primary oocytes present at birth simply degenerate. It remains a mystery why such an enormous number of eggs are formed only to perish in the ovaries.

Secondary oocytes released from the ovary (at ovulation) toward the end of the reproductive lifespan originate from primary oocytes that have spent 40 to 50 years arrested in prophase I. Defects accumulating in the egg over this prolonged period may account for the high incidence of genetic abnormalities among children born to older mothers. For instance, 1% of children born to women over 40 suffer from Down syndrome, caused by trisomy 21 (resulting from non-disjunction of the homologous chromosomes during the first meiotic division of the maturing oocyte) (Fig. 15-33).

15.3.8. Sperm are exquisitely adapted for delivering their DNA into the egg [15]

In most species, There are two distinct types of gametes that differ strikingly from one another. While the egg is one of the largest cells in the organism, the sperm (or spermatozoon) is typically the smallest. Both egg and sperm are optimally, yet differently, adapted to propagate their respective genes. The egg ensures the survival of maternal genes by storing abundant nutrients required for GROWTH AND DEVELOPMENT, and by providing a robust protective envelope. In contrast, the sperm is typically optimized for the task of fertilization and the transmission of paternal genes by being exceptionally motile and streamlined for rapid movement. Sperm operate under conditions of fierce competition, and the vast majority perish in the line of duty: out of the billions of sperm produced by a man during his reproductive life, only a handful ever reach their destination and fertilize an egg.

Fig. 15-33. The incidence of trisomy 21 (Down syndrome) in newborns as a function of maternal age. (After: L. Mastroianni, Jr. and J. D. Digger, eds. Fertilization and Embryonic Development in Vitro, p. 260, Plenum, 1981.)

Fig. 15-34. Schematic Longitudinal section of a human spermatozoon.

A typical sperm (Fig. 15-34) is a cell equipped with a powerful flagellum that propels it through fluid media; in the process, the sperm is "stripped of all excess," lacking organelles such as ribosomes, The Endoplasmic reticulum, or the Golgi apparatus, which are unnecessary for delivering DNA to the egg. On the other hand, sperm contain numerous mitochondria positioned where they can most efficiently power the flagellum. A sperm typically consists of two morphologically and functionally distinct parts enclosed within a single plasma membrane: a HEAD, containing an exceptionally condensed haploid nucleus, and a tail, which propels the entire cell toward the egg and facilitates the passage of the head through the egg investments. The DNA within the nucleus is inactive and packed extremely tightly, reducing its volume to a minimum. The chromosomes of many sperm lack even the Histones characteristic of somatic cells, relying instead on simple, highly positively charged proteins.

In the sperm head, directly anterior to the nuclear membrane, lies a specialized secretory vesicle called the acrosome (Fig. 15-34). This vesicle contains hydrolytic Enzymes that enable the sperm to penetrate the outer egg coats. When the sperm head contacts the egg, the acrosomal contents are released via exocytosis (the acrosome reaction). In invertebrates, this reaction exposes specific proteins that firmly anchor the sperm to the egg coat (see Section 15.4.2).

The motile tail of the sperm is a long flagellum whose axoneme originates from a basal body located immediately behind the nucleus. As previously noted (Section 11.3.5), the axoneme consists of two single central microtubules surrounded by nine evenly spaced microtubule doublets. In some animals (including mammals), the sperm flagellum differs from other flagella in having an additional ring of nine outer dense fibers of unknown composition surrounding the axoneme, resulting in a 9 + 9 + 2 arrangement rather than the standard 9 + 2 structure (Figs. 15-35 and 15-36). These dense fibers are rigid and incapable of contraction; it remains unclear whether they contribute to the vigorous bending Movements of the flagellum generated by the sliding of adjacent microtubule doublets relative to one another (Section 11.3.6). Energy for flagellar movement is provided by the Hydrolysis of ATP synthesized by highly specialized mitochondria located precisely where they are needed most—in the anterior region of the tail known as the midpiece (Figs. 15-34 and 15-35).

Fig. 15-35. Transmission electron micrograph of a cross-section through the midpiece of a mammalian spermatozoon. The flagellum consists of an axoneme surrounded by nine dense fibers. The axoneme is composed of two single microtubules surrounded by nine doublet microtubules. The arrangement of the mitochondrion (shown in color) is ideally suited to supply the ATP required for flagellar movement; its unusual structure results from the fusion of individual mitochondria during spermiogenesis.

15.3.9. In many mammals, sperm are produced continuously [16]

There are several fundamental differences between the formation of egg cells (oogenesis) and sperm cells (Spermatogenesis) in mammals. For example, as we noted earlier, in human females a limited number of oocytes are formed from proliferating oogonia during early embryogenesis, which then undergo ovulation one by one at regular intervals. Spermatogenesis in males, on the other hand, begins only at puberty and then proceeds continuously within the epithelial lining of very long, highly convoluted tubules called seminiferous tubules, located in the testes. Immature germ cells, known as spermatogonia, lie at the very periphery of the tubule near the basement membrane, where they continuously divide by mitosis. Some of the daughter cells cease dividing and differentiate into primary spermatocytes. These cells enter prophase I of meiosis, during which Crossing Over occurs between their paired homologous chromosomes, and subsequently complete the first meiotic division to yield two secondary spermatocytes; in humans, each of these contains 22 duplicated autosomes and a single duplicated X or Y chromosome. Each chromosome still consists of two sister chromatids, and when both secondary spermatocytes undergo the second meiotic division, four spermatids with a haploid number of single chromosomes are produced. These haploid spermatids then undergo morphological differentiation to become mature spermatozoa, which are released into the lumen of the seminiferous tubule (Figs. 15-37 and 15-38) and later pass into the Epididymis—a coiled tube hugging the Testis—where the sperm are stored and continue to mature.

Like oogenesis, spermatogenesis is under hormonal control. Following the onset of puberty, the male Pituitary Gland begins secreting a hormone already mentioned in our Discussion of mammalian oogenesis: luteinizing hormone (LH). Stimulated by LH, Leydig cells located in the interstitial spaces between the seminiferous tubules secrete large amounts of the male sex hormone testosterone. Testosterone, in turn, initiates spermatogenesis, likely by acting on Sertoli cells, which form a continuous layer surrounding the developing sperm to protect and nourish them (Fig. 15-38).

One of the most intriguing and unique features of sperm development is that mitotic and meiotic divisions are not accompanied by complete, final cytoplasmic cleavage (cytokinesis); consequently, all differentiating daughter cells derived from a single spermatogonium remain connected by intercellular cytoplasmic bridges (Fig. 15-39). These bridges persist until the very final STAGES OF SPERM differentiation, right up until individual spermatozoa are released into the tubular lumen. A group of cells linked in this manner is called a syncytium. The presence of this structure accounts for the synchronous appearance of mature spermatozoa in any given region of the seminiferous tubule.

Fig. 15-36. Electron micrograph of a cross-section through a guinea pig sperm flagellum. Mitochondria are absent here because the section was taken through the tail region, posterior to the midpiece. In this region of the flagellum, two of the nine dense fibers terminate and fuse with the outer fibrous sheath. (Courtesy of Daniel S. Friend.)

Fig. 15-37. Various stages of spermatogenesis. Spermatogonia develop from primordial germ cells that migrate into the testes during early embryonic development. When the animal reaches sexual maturity, the spermatogonia begin to proliferate rapidly; some of their progeny retain the capacity for continuous, unlimited division (stem-cell spermatogonia), while others (maturing spermatogonia), after a limited number of sequential mitoses, enter meiosis and differentiate into primary spermatocytes. Upon completion of the second meiotic division, primary spermatocytes give rise to haploid spermatids, which differentiate into mature spermatozoa. Spermatogenesis differs from oogenesis (Fig. 15-25) in several key respects: (1) after puberty, new cells continually enter meiosis; (2) each cell entering meiosis yields four mature gametes rather than one; and (3) mature sperm are formed after the completion of meiosis through a complex process of cellular differentiation.

Fig. 15-38. Highly simplified diagram of a cross-section through a mammalian seminiferous tubule. A. At all stages of spermatogenesis shown here, developing gametes are intimately associated with Sertoli cells; the latter are large cells spanning the distance from the basement membrane to the lumen of the seminiferous tubule. The progression of spermatogenesis is influenced by testosterone secreted by Leydig cells situated in the interstitial spaces between the seminiferous tubules. B. Dividing spermatogonia reside near the basement membrane. Some of these cells cease mitotic division and enter meiosis, becoming primary spermatocytes. Eventually, mature spermatozoa are released into the tubular lumen. In humans, completion of meiosis by a primary spermatocyte and its transformation into a spermatid takes about 24 days, and an additional 5 weeks are required for the spermatid to mature into a spermatozoon. Sperm undergo further maturation and acquire motility in the epididymis before achieving full functional maturity.

15.3.10. Sperm nuclei are haploid, yet the differentiation of these cells is directed by the diploid genome [17]

Unlike egg cells, spermatozoa undergo the major part of their differentiation after they have completed meiosis and become haploid. Because of cytoplasmic bridges, each developing haploid sperm has access to the full complement of products encoded by the complete diploid genome. The fact that sperm differentiation, like oocyte differentiation, is governed by the diploid genome is significant for two reasons. First, the original diploid genome typically carries A number of defective alleles—recessive lethal Mutations (Section 15.1.4); a haploid cell inheriting one of these defective alleles would almost certainly perish unless supplied with products of the normal allele transcribed in other nuclei that contain it. Second, in some organisms (such as humans), some sperm receive an X chromosome during meiosis, whereas others receive a Y chromosome. Because the X chromosome carries many vital genes absent from the Y chromosome, it is likely that without cytoplasmic bridges between developing sperm, those receiving a Y chromosome would fail to survive, resulting in an all-female next generation.

Indeed, there is direct experimental evidence that sperm differentiation is directed by products of the diploid genome. Some of this evidence comes from studies of Drosophila chromosome-malsegregation mutants in which chromosomes are unequally distributed among daughter cells during meiosis; as a result, some spermatozoa contain too few chromosomes, others too many, and some none at all. Strikingly, the differentiation of all these cells, even those completely lacking chromosomes, proceeds normally. This finding can be explained by the hypothesis mentioned above: products of the missing chromosomes could be supplied via diffusion through the cytoplasmic bridges connecting neighboring cells. Alternatively, instructions for sperm differentiation might be synthesized in advance—before meiosis—in diploid spermatogonia or primary spermatocytes (presumably as long-lived mRNAs), rendering transcription by the haploid genome unnecessary during differentiation itself. Regardless of which explanation is correct, it is clear that sperm differentiation utilizes products from both sets of chromosomes, even though the cell's own nucleus is haploid.

Fig. 15-39. This diagram illustrates how the progeny of a single maturing spermatogonium remain connected by cytoplasmic bridges throughout their entire differentiation into mature spermatozoa. For simplicity, only two connected maturing spermatogonia are shown entering mitosis, eventually producing eight interconnected haploid spermatids. In reality, the number of linked cells undergoing meiosis and differentiating together is significantly greater than shown here.

Summary

Egg cells develop from primordial germ cells that migrate into the ovary early in development and differentiate into oogonia. Following a period of mitotic proliferation, oogonia become primary spermatocytes—or rather, primary oocytes—which enter the first meiotic division and arrest in prophase I for a period lasting from days to years, depending on the species. During this arrest, the oocyte grows and accumulates ribosomes, mRNAs, and proteins, frequently drawing on neighboring cells, including surrounding auxiliary cells. Subsequent development (oocyte maturation) is regulated by polypeptide hormones (gonadotropins) that act on the auxiliary cells enclosing each oocyte, prompting them to induce maturation in a small subset of oocytes. These oocytes complete the first meiotic division to yield a small polar body and a large secondary oocyte, which then arrests at metaphase of the second meiotic division; in many species, the oocyte remains at this stage until fertilization triggers the completion of meiosis and the onset of embryonic development.

A spermatozoon is typically a small, highly compact cell specialized for the sole function of delivering its DNA into an egg. Whereas in many organisms the entire pool of oocytes is established early in female development, in males new germ cells continuously enter meiosis after puberty, with each primary spermatocyte giving rise to four mature spermatozoa. Sperm differentiation occurs post-meiotically, while the nuclei are haploid. However, because cytokinesis is incomplete during the mitotic divisions of spermatogonia and spermatocytes, the progeny of a single spermatogonium develop as a syncytium. Consequently, sperm differentiation can be controlled by gene products from both parental sets of chromosomes.