Chapter 20:  Germ Cells and Fertilization

Introduction

The sexual reproductive cycle involves an alternation of haploid generations of cells, each carrying a single set of chromosomes, with diploid generations of cells, each carrying a double set of chromosomes. The mixing of genomes is achieved by fusion of two haploid cells to form a diploid cell. Later, new haploid cells are generated when a descendant of this diploid cell divides by the process of meiosis (Figure 20-2). During meiosis the chromosomes of the double chromosome set exchange DNA by genetic recombination before being shared out, in fresh combinations, into single chromosome sets. In this way each cell of the new haploid generation receives a novel assortment of genes, with some genes on each chromosome originating from one ancestral cell of the previous haploid generation and some from the other. Thus, through cycles of haploidy, cell fusion, diploidy, and meiosis, old combinations of genes are broken up and new combinations are created.

In Multicellular Animals the Diploid Phase Is Complex and Long, the Haploid Simple and Fleeting

Cells proliferate by mitotic division. In most organisms that reproduce sexually, this proliferation occurs during the diploid phase. Some primitive organisms, such as fission yeasts, are exceptional in that the haploid cells proliferate mitotically and the diploid cells, once formed, proceed directly to meiosis. A less extreme exception occurs in plants, where mitotic cell divisions occur in both the haploid and the diploid phases. In all but the most primitive plants, however, the haploid phase is very brief and simple, while the diploid phase is extended into a long period of development and proliferation. For almost all multicellular animals, including vertebrates, practically the whole of the life cycle is spent in the diploid state: the haploid cells exist only briefly, do not divide at all, and are highly specialized for sexual fusion (Figure 20-3).

Haploid cells that are specialized for sexual fusion are called gametes. Typically, two types of gametes are formed: one is large and nonmotile and is referred to as the egg (or ovum); the other is small and motile and is referred to as the sperm (or spermatozoon) (Figure 20-4). During the diploid phase that follows fusion of gametes, the cells proliferate and diversify to form a complex multicellular organism. In most animals a useful distinction can be drawn between the cells of the germ line, from which the next generation of gametes will be derived, and the somatic cells, which form the rest of the body and ultimately leave no progeny. In a sense, the somatic cells exist only to help the cells of the germ line (the germ cells) survive and propagate.

Sexual Reproduction Gives a Competitive Advantage to Organisms in an Unpredictably Variable Environment ¹

The machinery of sexual reproduction is elaborate, and the resources spent on it are large. What benefits does it bring, and why did it evolve? Through genetic recombination sexual individuals beget unpredictably dissimilar offspring, whose haphazard genotypes are at least as likely to represent a change for the worse as a change for the better. Why, then, should sexual individuals have a competitive advantage over individuals that breed true, by an asexual process? This problem continues to perplex population geneticists, but the general conclusion seems to be that the reshuffling of genes in sexual reproduction helps a species survive in an unpredictably variable environment. If a parent produces many offspring with a wide variety of gene combinations, there is a better chance that at least one of the offspring will have the assortment of features necessary for survival.

Many other ideas have been proposed to explain the competitive advantages of sexual reproduction. One of these suggests how one of the first steps in the evolution of sex might have occurred. Evolution depends to a large extent on competition among individuals carrying alternative alleles, or variants, created by mutation of particular genes. Suppose that two individuals in a population each undergo a beneficial mutation affecting a different genetic locus and therefore a different function. In a strictly asexual species each of these individuals will give rise to a clone of mutant progeny, and the two clones will compete until one or the other triumphs: one of the two beneficial mutations will spread through the population, while the other will eventually be lost. But suppose that one of the original mutants has evolved a genetically determined mechanism that enables it occasionally to incorporate genes from other cells. During the period of competition acquisition of genes from a cell of the competing clone is likely to create a cell that carries both beneficial mutations. Such a cell will be the most successful of all, and its success will ensure the propagation of the trait that enabled it to incorporate genes from other cells. This rudimentary sexual capability will thus be favored by natural selection.

Whatever the origins of sex may be, it is striking that practically all complex present-day organisms have evolved largely through generations of sexual, rather than asexual, reproduction. Asexual organisms, although plentiful, seem mostly to have remained simple and primitive.

We shall now examine the detailed cellular mechanisms of sex, beginning with the events of meiosis, in which genetic recombination occurs and diploid cells of the germ line divide to produce haploid gametes. Then we shall consider the gametes themselves and, finally, the process of fertilization, in which the gametes fuse to form a new diploid organism.

Summary

Sexual reproduction involves a cyclic alternation of diploid and haploid states: diploid cells divide by meiosis to form haploid cells, and the haploid cells from two individuals fuse in pairs at fertilization to form new diploid cells. In the process, genomes are mixed and recombined to produce individuals with novel assortments of genes. Most of the life cycle of higher plants and animals is spent in the diploid phase; the haploid phase is very brief. Sexual reproduction has probably been favored by evolution because the random recombination of genetic information improves the chances of producing at least some offspring that will survive in an unpredictably variable environment.

Introduction

The realization that germ cells are haploid, and must therefore be produced by a special type of cell division, came from an observation that was also among the first to suggest that chromosomes carry genetic information. In 1883 it was discovered that, whereas the fertilized egg of a particular worm contains four chromosomes, the nucleus of the egg and that of the sperm each contain only two chromosomes. The chromosome theory of heredity therefore explained the long-standing paradox that maternal and paternal contributions to the character of the progeny seem often to be equal, despite the enormous difference in size between the egg and sperm (see Figure 20-4).

The finding also implied that germ cells must be formed by a special kind of nuclear division in which the chromosome complement is precisely halved. This type of division is called meiosis, from the Greek, meaning diminution. (There is no connection with the term mitosis, which is from the Greek mitos, meaning a thread, and refers to the threadlike appearance of the chromosomes as they condense during nuclear division - a process that occurs in both ordinary and meiotic divisions.) The behavior of the chromosomes during meiosis turned out to be considerably more complex than expected. Consequently, it was not until the early 1930s, as a result of painstaking cytological and genetic studies, that the essential features of meiosis were established.

Meiosis Involves Two Nuclear Divisions Rather Than One

With the exception of the chromosomes that determine sex (the sex chromosomes), a diploid nucleus contains two closely similar versions of each of the other chromosomes (the autosomes), one from the male parent (paternal chromosome) and one from the female parent (maternal chromosome). The two versions are called homologues, and in most cells they maintain a completely separate existence as independent chromosomes. When each chromosome is duplicated by DNA replication, the twin copies of the fully replicated chromosome at first remain closely associated and are called sister chromatids. In an ordinary cell division (described in Chapter 18) the sister chromatids line up on the spindle during mitosis with their kinetochore fibers pointing toward opposite poles. The sister chromatids then separate from each other at anaphase to become individual chromosomes. In this manner each daughter cell formed by ordinary cell division inherits one copy of each paternal chromosome and one copy of each maternal chromosome.

In contrast, a haploid gamete produced by the divisions of a diploid cell during meiosis must contain half the original number of chromosomes - only one chromosome in place of each homologous pair of chromosomes - so that the gamete is endowed with either the maternal or the paternal copy of each gene but not both. This requirement makes an extra demand on the machinery for cell division. The mechanism that has evolved to accomplish the additional sorting requires that homologues recognize each other and become physically paired before they line up on the spindle. This pairing of the maternal and the paternal copy of each chromosome is unique to meiosis. How the correct chromosomes recognize each other is still unclear, as will be discussed later.

Given a mechanism for pairing the maternal and paternal homologues and for their subsequent separation on the spindle, cells could, in principle, carry out meiosis by a simple modification of a single mitotic cell cycle in which chromosome duplication (S phase) was omitted: if the unduplicated homologues paired before M phase, the ensuing cell division would then produce two haploid cells directly. For unknown reasons, the actual meiotic process is more complex. Before the homologues pair, each one replicates to produce two sister chromatids as in an ordinary cell division. It is only after DNA replication has been completed that the special features of meiosis become evident. Rather than separating, the sister chromatids behave as a unit, as if chromosome duplication had not occurred: each duplicated homologue pairs with its partner, forming a structure called a bivalent, which contains four chromatids. The pairing, as we shall see, allows genetic recombination to occur, whereby a fragment of a maternal chromatid may be exchanged for a corresponding fragment of a homologous paternal chromatid. The bivalents line up on the spindle, and at anaphase the two duplicated homologues (each consisting of two sister chromatids) separate and move to opposite poles. Because the joined sister chromatids behave as a unit, each daughter cell inherits two copies of one of the two homologues when the meiotic cell divides; these two copies are identical except where genetic recombination has occurred (Figure 20-5). The two progeny of this division (division I of meiosis) therefore contain a diploid amount of DNA but differ from normal diploid cells in two ways: (1) both of the two DNA copies of each chromosome derive from only one of the two homologous chromosomes in the original cell (except where there has been genetic recombination), and (2) these two copies are inherited as closely associated sister chromatids, as if they were a single chromosome (see Figure 20-5).

Formation of the actual gamete nuclei can now proceed simply through a second cell division, division II of meiosis, without further DNA replication. The chromosomes align on a second spindle and the sister chromatids separate, as in normal mitosis, to produce cells with a haploid DNA content. Meiosis thus consists of two cell divisions following a single phase of DNA replication, so that four haploid cells are produced from each cell that enters meiosis. Meiosis and mitosis are compared in Figure 20-6.

Occasionally, the meiotic process occurs abnormally and homologues fail to separate - a phenomenon known as nondisjunction. In this case some of the haploid cells that are produced lack a chromosome, while others have more than one copy. Such gametes form abnormal embryos, most of which die. Some survive, however: Down's syndromein humans, for example, is caused by an extra copy of chromosome 21 resulting from nondisjunction during meiotic division I or II.

Genetic Reassortment Is Enhanced by Crossing-over Between Homologous Nonsister Chromatids ³

Unless they are identical twins, which develop from a single zygote, no two offspring of the same parents are genetically the same. This is because, long before the two gametes fuse, two kinds of randomizing genetic reassortment have occurred during meiosis.

One kind of reassortment is a consequence of the random distribution of the maternal and paternal homologues between the daughter cells at meiotic division I, as a result of which each gamete acquires a different mixture of maternal and paternal chromosomes. From this process alone, one individual could, in principle, produce 2 ⁿ genetically different gametes, where n is the haploid number of chromosomes (Figure 20-7A). In humans, for example, each individual can produce at least 2²³ = 8.4 x 10⁶ genetically different gametes. But the actual number of variants is very much greater than this because a second type of reassortment, called chromosomal crossing-over, occurs during meiosis. It takes place during the long prophase of meiotic division I, in which parts of homologous chromosomes are exchanged. On average, between two and three crossover events occur on each pair of human chromosomes during meiotic division I. This process scrambles the genetic constitution of each of the chromosomes in gametes, as illustrated in Figure 20-7B.

Chromosomal crossing-over involves breaking the DNA double helix in a maternal chromatid and in a homologous paternal chromatid so as to exchange fragments between the two nonsister chromatids in a reciprocal fashion by a process known as general genetic recombination. The molecular details of this process are discussed in Chapter 6. The consequences of each crossover event can be observed cytologically at the latest stages of prophase of meiotic division I, when the chromosomes are highly condensed. At this stage the sister chromatids are tightly apposed along their entire length. The two duplicated homologues (maternal and paternal) are seen to be physically connected at specific points. Each connection, called a chiasma (plural chiasmata), corresponds to a crossover between two nonsister chromatids (Figure 20-8).

At this stage of meiosis, each pair of duplicated homologues, or bivalent, is held together by at least one chiasma. Many bivalents contain more than one chiasma, indicating that multiple crossovers can occur between homologues.

Meiotic Chromosome Pairing Culminates in the Formation of the Synaptonemal Complex ⁴

Elaborate morphological changes occur in the chromosomes as they pair (synapse) and then begin to unpair (desynapse) during the first meiotic prophase. This prophase is traditionally divided into five sequential stages - leptotene, zygotene, pachytene, diplotene, and diakinesis- defined by these morphological changes. The most striking event is the initiation of intimate chromosome synapsis at zygotene, when a complex structure called the synaptonemal complex begins to develop between the two sets of sister chromatids in each bivalent. Pachytene is said to begin as soon as synapsis is complete, and it generally persists for days, until desynapsis begins the diplotene stage, in which the chiasmata are first seen (Figure 20-9).

Genetic recombination requires a close apposition between the recombining chromosomes. The synaptonemal complex, which forms just before pachytene and dissolves just afterward, keeps the homologous chromosomes in a bivalent together and closely aligned, and it has been suggested that it may play a part in the recombination process. It consists of a long ladderlike protein core, on opposite sides of which the two homologues are aligned to form a long linear chromosome pair. The sister chromatids in each homologue are kept tightly packed together, and their DNA extends from the same side of the protein ladder in a series of loops (Figure 20-10).

It is not known how homologous chromosomes become aligned. It is unlikely that continuous connections all along the interacting chromosomes are involved, since the chromatin of one homologue is well separated from the chromatin of its partner in the synaptonemal complex. It has been proposed that the initial interaction between homologous chromosomes is mediated by complementary DNA base-pair interactions at discrete sites along the chromosomes. This recognition may occur at zygotene or even earlier, when the chromosomes are not very condensed; following chromosome condensation, the formation of the synaptonemal complex would then pack the remaining portions of the chromosomes together.

Recombination Nodules Are Thought to Mediate Chromatid Exchanges ⁵

Although the synaptonemal complex may provide the structural framework for recombination events, it probably is not the engine that brings them about. The active recombination process is thought to be mediated instead by recombination nodules, which are very large protein-containing assemblies with a diameter of about 90 nm. (For comparison, a large globular protein molecule of molecular weight 400,000 has a diameter of about 10 nm.) Recombination nodules sit at intervals on the synaptonemal complex, placed like basketballs on a ladder between the two homologous chromosomes (see Figure 20-10). They are thought to mark the site of a large multienzyme "recombination machine," which brings local regions of DNA on the maternal and paternal chromatids together across the 100-nm-wide synaptonemal complex.

The evidence that the recombination nodule serves this function is indirect: (1) The total number of nodules is about equal to the total number of chiasmata seen later in prophase. (2) The nodules are distributed along the synaptonemal complex in the same way that crossover events are distributed. Like the crossover events themselves, for example, the nodules are absent from those regions of the synaptonemal complex that hold heterochromatin together. Moreover, both genetic and cytological measurements indicate that the occurrence of one crossover event prevents a second crossover event occurring at any nearby chromosomal site; similarly, the nodules tend not to occur very near one another. (3) Some Drosophila mutations cause an abnormal distribution of crossover events along the chromosomes, as well as a greatly diminished recombination frequency. In these mutants correspondingly fewer recombination nodules are found, with a changed distribution that parallels the changed crossover distribution. This correlation strongly suggests that a recombination nodule determines the site of each crossover event. (4) Genetic recombination is thought to involve a limited amount of DNA synthesis at the site of each crossover event (discussed in Chapter 6). Electron microscopic autoradiography shows that radioactive DNA precursors are preferentially incorporated into pachytene DNA at or near recombination nodules.

Because there are about as many recombination nodules as crossover events, it seems that recombination nodules are extremely efficient in causing the chromatids on opposite homologues to recombine. Little is known, however, about their structure or mechanism of action.

Chiasmata Play an Important Part in Chromosome Segregation in Meiosis

In addition to reassorting genes, chromosomal crossing-over is crucial in most organisms for the correct segregation of the two homologues to separate daughter nuclei. This is because the chiasma created by each crossover event plays a role analogous to that of the centromere in an ordinary mitotic division, holding the maternal and paternal homologues together on the spindle until anaphase I. In mutant organisms that have a reduced frequency of meiotic chromosome crossing-over, some of the chromosome pairs lack chiasmata. These pairs fail to segregate normally, and a high proportion of the resulting gametes contain too many or too few chromosomes - an example of nondisjunction.

There are at least two major differences in the way chromosomes separate in meiotic division I and in normal mitosis (see Figure 20-6). (1) During normal mitosis (and in meiotic division II, which resembles a normal mitosis) the sister chromatids are held together only at the centromere; the kinetochores (protein complexes associated with the centromeres, discussed in Chapter 18) on each sister chromatid have attached kinetochore fibers pointing in opposite directions, so that the chromatids are drawn into different daughter cells at anaphase. At metaphase I of meiosis, by contrast, the kinetochores on both sister chromatids appear to have fused so that their attached kinetochore fibers all point in the same direction and the arms of the sister chromatids are closely apposed; moreover, the homologous maternal and paternal chromosomes are held together at the chiasmata. (2) During normal mitosis (and meiotic division II) the movement of chromatids to the poles is triggered by a mechanism that detaches the two sister kinetochores from each other (thus beginning anaphase), allowing the sister chromatids to segregate into different daughter cells. In anaphase I of meiosis, however, movement to the poles is initiated by the disruption of the poorly understood forces keeping the arms of sister chromatids together and by the simultaneous dissolution of the chiasmata linking the homologous maternal and paternal chromosomes; consequently, the sister chromatids remain paired, but the maternal and paternal homologues segregate into different daughter cells. The difference between the way chromosomes separate in meiotic divisions I and II are illustrated in Figure 20-11.

Pairing of the Sex Chromosomes Ensures That They Also Segregate ⁶

We have explained how homologous chromosomes pair during meiotic division I so that they segregate accurately between the daughter cells. But what about the sex chromosomes, which in male mammals are not homologous? Females have two X chromosomes, which pair and segregate like other homologues. But males have one X and one Y chromosome, which must pair during the first metaphase of meiosis if the sperm are to contain either one Y or one X chromosome and not both or neither. The necessary pairing is made possible by a small region of homology between the X and the Y at one end of these chromosomes. In this region the two chromosomes pair and cross over during the first meiotic prophase. The chiasma corresponding to this small amount of genetic recombination is sufficient to keep the X and Y chromosomes paired on the spindle so that only two types of sperm are normally produced: sperm containing one Y chromosome, which will give rise to male embryos, and sperm containing one X chromosome, which will give rise to female embryos.

Meiotic Division II Resembles a Normal Mitosis

After the long prophase I (which can occupy 90% or more of meiosis) has ended, two successive cell divisions, without an intervening period of DNA synthesis, bring meiosis to an end (see Figure 20-6). The entire first meiotic cell cycle, which ends with an initial meiotic cell division, is called meiotic division I, and it is far more complex and requires much more time than the second meiotic cell cycle, called meiotic division II (Figure 20-12). Even the preparatory DNA replication during the first cell cycle tends to take much longer than a normal S phase, and cells can then spend days, months, or even years in the first meiotic prophase, depending on the species and on the gamete being formed. (Although it is traditionally called prophase, this prolonged phase of meiotic division I resembles the G₂ phase of an ordinary cell division in that the nuclear envelope remains intact and disappears only when the spindle fibers begin to form as prophase I gives way to metaphase I.)

After the end of meiotic division I, nuclear membranes re-form around the two daughter nuclei and a brief interphase begins. During this period the chromosomes may decondense somewhat, but usually they soon recondense and prophase II begins. As there is no DNA synthesis during this interval, in some organisms the chromosomes seem to pass almost directly from one division phase into the next. In all organisms prophase II is brief: the nuclear envelope breaks down as the new spindle forms, after which metaphase II, anaphase II, and telophase II usually follow in quick succession. As in an ordinary mitosis, a separate set of kinetochore fibers forms on each sister chromatid, and these two sets of fibers extend in opposite directions. Moreover, the two sister chromatids are kept together on the metaphase plate until they are released by the sudden separation of their kinetochores at anaphase (see Figure 20-11). Thus division II, unlike division I, closely resembles a normal mitosis. The difference is that one copy of each chromosome is present instead of two homologues. After nuclear envelopes have formed around the four haploid nuclei produced at telophase II, meiosis is complete (see Figure 20-6). The principles of meiosis are the same in plants and animals and in males and females. But the production of gametes involves more than just meiosis, and the other processes required vary widely among organisms and are very different for eggs and sperm. We shall focus our discussion of gametogenesis mainly on vertebrates. As we shall see, by the end of meiosis a vertebrate egg is fully mature (and in some cases even fertilized), whereas a sperm that has completed meiosis has only just begun its differentiation.

Summary

The formation of both eggs and sperm begins in a similar way, with meiosis. In this process two successive cell divisions following one round of DNA replication give rise to four haploid cells from a single diploid cell. Meiosis is dominated by prophase of meiotic division I, which can occupy 90% or more of the total meiotic period. Each chromosome as it enters this prophase consists of two tightly joined sister chromatids. Chromosomal crossover events occur during this prolonged prophase I, when homologous chromosomes are aligned in register. Each crossover event is thought to be mediated by a recombination nodule, and it results in the formation of a chiasma, which persists until anaphase I. In the first meiotic cell division one member of each chromosome pair, still composed of linked sister chromatids, is distributed to each daughter cell. A second cell division, without DNA replication, then rapidly ensues in which each sister chromatid is segregated into a separate haploid cell.

Introduction

In all vertebrate embryos certain cells are singled out early in development as progenitors of the gametes. These primordial germ cells migrate to the developing gonads, called the genital ridges, which will form the ovaries in females and the testes in males. After a period of mitotic proliferation, these cells undergo meiosis and differentiate into mature gametes - either eggs or sperm. Later, the fusion of egg and sperm after mating initiates embryogenesis, with the subsequent production in the embryo of new primordial germ cells, which begins the cycle again.

An Egg Is the Only Cell in a Higher Animal That Is Able to Develop into a New Individual

In one respect at least, eggs are the most remarkable of animal cells: once activated, they can give rise to a complete new individual within a matter of days or weeks. No other cell in a higher animal has this capacity. Activation is usually the consequence of fertilization- fusion of a sperm with the egg. The sperm itself, however, is not strictly required. An egg can be activated artificially by a variety of nonspecific chemical or physical treatments; a frog egg, for example, can be activated by pricking it with a needle. Indeed, some organisms, including even a few vertebrates such as some lizards, normally reproduce from eggs that become activated in the absence of sperm - that is, parthenogenetically.

Although an egg can give rise to every cell type in the adult organism (that is, it is totipotent), it is itself a highly specialized cell, uniquely equipped for the single function of generating a new individual. We shall now briefly consider some of its specialized features before discussing how it develops to the point at which it is ready for fertilization.

An Egg Is Highly Specialized for Independent Development, with Large Nutrient Reserves and an Elaborate Coat ⁷

The eggs of most animals are giant cells, containing stockpiles of all the materials needed for initial development of the embryo to carry it through to the stage where the new individual can begin feeding. Before this stage, the single giant cell cleaves into many smaller cells, but no net growth occurs. Mammals are an exception in that the embryo can start to grow early by taking up nutrients from the mother; thus a mammalian egg, though still a large cell, does not have to be as large as the egg of a frog or a bird, for example. In general, eggs are typically spherical or ovoid, with a diameter of about 100 microns in humans and sea urchins (whose feeding larvae are tiny), 1 microns to 2 microns in frogs and fishes, and many centimeters in birds and reptiles (Figures 20-13). A typical somatic cell, by contrast, has a diameter of only about 10 or 20 microns (Figure 20-14).

The egg cytoplasm contains nutritional reserves in the form of yolk, which is rich in lipids, proteins, and polysaccharides and is usually contained within discrete structures called yolk granules. In some species each yolk granule is membrane-bounded, whereas in others it is not. In eggs that develop into large animals outside the mother's body, yolk can account for more than 95% of the volume of the cell, whereas in mammals, whose embryos are largely nourished by their mothers, there is little if any.

The egg coat is another peculiarity of eggs. It is a specialized form of extracellular matrix consisting largely of glycoprotein molecules, some secreted by the egg and others by surrounding cells. In many species the major coat is a layer immediately surrounding the egg plasma membrane; in nonmammalian eggs, such as those of sea urchins or chickens, it is called the vitelline layer, whereas in mammalian eggs it is called the zona pellucida(Figure 20-15). This layer protects the egg from mechanical damage, and in many eggs it also acts as a species-specific barrier to sperm, admitting only those of the same or closely related species (discussed below). Nonmammalian eggs often have additional layers overlying the vitelline layer that are secreted by surrounding cells. As frog eggs, for example, pass from the ovary through the oviduct (the tube that conveys them to the outside), they acquire several layers of gelatinous coating secreted by epithelial cells lining the oviduct. Similarly, the "white" (albumin) and shell of chicken eggs are added (after fertilization) as the eggs pass along the oviduct. The vitelline layer of insect eggs is covered by a thick, tough layer called the chorion, which is secreted by the follicle cells that surround each egg in the ovary.

Many eggs (including those of mammals) contain specialized secretory vesicles just under the plasma membrane in the outer region, or cortex, of the egg cytoplasm. When the egg is activated by a sperm, these cortical granules release their contents by exocytosis; the contents of the granules act to alter the egg coat so as to prevent more than one sperm from fusing with the egg (discussed below).

Eggs Develop in Stages ⁸

A developing egg is called an oocyte. Its differentiation into a mature egg (or ovum) involves a series of changes whose timing is geared to the steps of meiosis in which the germ cells go through their two final, highly specialized divisions. Oocytes have evolved special mechanisms for arresting progress through meiosis: they remain suspended in prophase I for prolonged periods while the oocyte grows in size, and in many cases they later arrest in metaphase II while awaiting fertilization.

While the details of oocyte development (oogenesis) vary in different species, the general stages are similar, as outlined in Figure 20-16. Primordial germ cells migrate to the forming gonad to become oogonia,which proliferate by ordinary cell division cycles for a period before differentiating into primary oocytes. At this stage the first meiotic division begins: the DNA replicates so that each chromosome consists of two chromatids, the homologous chromosomes pair along their long axes, and crossing-over occurs between the chromatids of these paired chromosomes. After these events the cell remains arrested in prophase of division I of meiosis (in a state equivalent, as we previously pointed out, to a G₂ phase of an ordinary division cycle) for a period lasting from a few days to many years, depending on the species. During this long period (or, in some cases, at the onset of sexual maturity) the primary oocytes synthesize a coat and cortical granules and, in the case of large nonmammalian oocytes, they accumulate ribosomes, yolk, glycogen, lipid, and the mRNA that will later direct the synthesis of proteins required for early embryonic growth and the unfolding of the developmental program. In many oocytes the intensive biosynthetic activities are reflected in the structure of the chromosomes, which decondense and form lateral loops, taking on a characteristic "lampbrush" appearance, signifying that they are very busily engaged in RNA synthesis (discussed in Chapter 8).

The next phase of oocyte development is called oocyte maturation and usually does not occur until sexual maturity, when it is stimulated by hormones. Under these hormonal influences the cell resumes its progress through division I of meiosis: the chromosomes recondense, the nuclear envelope breaks down (this is generally taken to mark the beginning of maturation), and the replicated homologous chromosomes segregate at anaphase I into two daughter nuclei, each containing half the original number of chromosomes. To end division I, the cytoplasm divides asymmetrically to produce two cells that differ greatly in size: one is a small polar body, and the other is a large secondary oocyte, the precursor of the egg. At this stage each of the chromosomes is still composed of two sister chromatids. These chromatids do not separate until division II of meiosis, when they are partitioned into separate cells by a process that is identical to a normal mitosis, as previously described. After this final chromosome separation at anaphase II, the cytoplasm of the large secondary oocyte again divides asymmetrically to produce the mature egg (or ovum) and a second small polar body, each with a haploid number of single chromosomes (see Figure 20-16). Because of these two asymmetrical divisions of their cytoplasm, oocytes maintain their large size despite undergoing the two meiotic divisions. Both of the polar bodies are small, and they eventually degenerate.

In most vertebrates oocyte maturation proceeds to metaphase of meiosis II and then arrests until fertilization. At ovulation the arrested secondary oocyte is released from the ovary, and if fertilization occurs, the oocyte is stimulated to complete meiosis.

Oocytes Grow to Their Large Size Through Special Mechanisms ^{8, 9}

A somatic cell with a diameter of 10 to 20 microns typically takes about 24 hours to double its mass in preparation for cell division. At this rate of biosynthesis such a cell would take a very long time to reach the hundredfold greater mass of a mammalian egg with a diameter of 100 microns or the 10⁵-fold greater mass of an insect egg with a diameter of 1000 microns. Yet some insects live only a few days and manage to produce eggs with diameters even greater than 1000 microns. It is clear that eggs must have special mechanisms for achieving their large size.

One simple strategy for rapid growth is to have extra gene copies in the cell. Thus the oocyte delays completion of the first meiotic division so as to grow while it contains the diploid chromosome set in duplicate. In this way it has twice as much DNA available for RNA synthesis as does an average somatic cell in the G₁ phase of the cell cycle. Some oocytes go to even greater lengths to accumulate extra DNA: they produce many extra copies of certain genes. We discuss in Chapter 8 how the somatic cells of most organisms require 100 to 500 copies of the ribosomal RNA genes in order to produce enough ribosomes for protein synthesis. Eggs require even greater numbers of ribosomes to support protein synthesis during early embryogenesis, and in the oocytes of many animals the ribosomal RNA genes are specifically amplified; some amphibian eggs, for example, contain 1 or 2 million copies of these genes.

Oocytes may also depend partly on the synthetic activities of other cells for their growth. Yolk, for example, is usually synthesized outside the ovary and imported into the oocyte. In birds, amphibians, and insects yolk proteins are made by liver cells (or their equivalents), which secrete these proteins into the blood. Within the ovaries oocytes take up the yolk proteins from the extracellular fluid by receptor-mediated endocytosis (see Figure 13-28). Nutritive help can also come from neighboring accessory cells in the ovary. These can be of two types. In some invertebrates some of the progeny of the oogonia become nurse cells instead of becoming oocytes. These cells usually are connected to the oocyte by cytoplasmic bridges through which macromolecules can pass directly into the oocyte cytoplasm (Figure 20-17). For the insect oocyte the nurse cells manufacture many of the products - ribosomes, mRNA, protein, and so on - that a vertebrate oocyte has to manufacture for itself.

The other accessory cells in the ovary that help nourish developing oocytes are ordinary somatic cells called follicle cells, which are found in both invertebrates and vertebrates. They are arranged as an epithelial layer around the oocyte (Figure 20-18, and see Figure 20-17), to which they are connected only by gap junctions, which permit the exchange of small molecules but not macromolecules. While these cells are unable to provide the oocyte with preformed macromolecules through these communicating junctions, they may help to supply the smaller precursor molecules from which macromolecules are made. In addition, follicle cells frequently secrete macromolecules that contribute to the egg coat, or are taken up by receptor-mediated endocytosis into the growing oocyte, or act on egg cell-surface receptors to control the spatial patterning and axial asymmetries of the egg (discussed in Chapter 21).

Summary

Eggs develop in stages from primordial germ cells that migrate into the developing gonad very early in development to become oogonia. After mitotic proliferation oogonia become primary oocytes that begin meiotic division I and then arrest at prophase for days or years, depending on the species. During this prophase-I arrest period, primary oocytes grow, synthesize a coat, and accumulate ribosomes, mRNAs, and proteins, often enlisting the help of other cells, including surrounding accessory cells. In the process of maturation primary oocytes complete meiotic division I to form a small polar body and a large secondary oocyte, which proceeds into metaphase of meiotic division II. There, in many species, the oocyte is arrested until stimulated by fertilization to complete meiosis and begin embryonic development.

Introduction

In most species there are just two types of gametes, and they are radically different. The egg is among the largest cells in an organism, while the sperm (spermatozoon, plural spermatozoa) is often the smallest. The egg and the sperm are optimized in opposite ways for the propagation of the genes they carry. The egg is nonmotile and aids the survival of the maternal genes by providing large stocks of raw materials for growth and development, as well as providing an effective protective wrapping. The sperm, by contrast, is optimized to propagate the paternal genes by exploiting this maternal investment: it is usually highly motile and streamlined for speed and efficiency in the task of fertilization. Competition between sperm is fierce, and the vast majority fail in their mission: of the billions of sperm released during the reproductive life of a human male, only a few ever manage to fertilize an egg.

Sperm Are Highly Adapted for Delivering Their DNA to an Egg ¹⁰

Typical sperm are "stripped-down" cells, equipped with a strong flagellum to propel them through an aqueous medium but unencumbered by cytoplasmic organelles such as ribosomes, endoplasmic reticulum, or Golgi apparatus, which are unnecessary for the task of delivering the DNA to the egg. On the other hand, sperm contain many mitochondria strategically placed where they can most efficiently power the flagellum. Sperm usually consist of two morphologically and functionally distinct regions enclosed by a single plasma membrane: the tail, which propels the sperm to the egg and helps it burrow through the egg coat, and the head, which contains a condensed haploid nucleus (Figure 20-19). The DNA in the nucleus is extremely tightly packed, so that its volume is minimized for transport. The chromosomes of many sperm have dispensed with the histones of somatic cells and are packed instead with simple, highly positively charged proteins called protamines.

In the head of most animal sperm, closely apposed to the anterior end of the nuclear envelope, is a specialized secretory vesicle called the acrosomal vesicle (see Figure 20-19). This contains hydrolytic enzymes that help the sperm to penetrate the egg's outer coat. When a sperm contacts an egg, the contents of the vesicle are released by exocytosis in the so-called acrosomal reaction; in some sperm this reaction also exposes or releases specific proteins that help bind the sperm tightly to the egg coat.

The motile tail of a sperm is a long flagellum whose central axoneme emanates from a basal body situated just posterior to the nucleus. As described in Chapter 16, the axoneme consists of two central singlet microtubules surrounded by nine evenly spaced microtubule doublets. The flagellum of some sperm (including those of mammals) differs from other flagella in that the usual 9 + 2 pattern of the axoneme is further surrounded by nine outer dense fibers composed mainly of keratin (Figure 20-20). These dense fibers are stiff and noncontractile, and it is not known what part they play in the active bending of the flagellum, which is caused by the sliding of adjacent microtubule doublets past one another. Flagellar movement is driven by dynein motor proteins, which use the energy of ATP hydrolysis to slide the microtubules, as discussed in Chapter 16; the ATP is generated by highly specialized mitochondria in the anterior part of the sperm tail (called the midpiece), where the ATP is needed (see Figures 20-19 and 20-20).

Sperm Are Produced Continuously in Many Mammals ¹¹

In mammals there are major differences in the way eggs are produced (oogenesis) and the way sperm are produced (spermatogenesis). In human females, for example, oogonia proliferate only in the fetus, enter meiosis before birth, and become arrested as oocytes in the first meiotic prophase, in which state they may remain for up to 50 years. Individual oocytes mature from this strictly limited stock and are ovulated at intervals, generally one at a time, beginning at puberty. In human males, on the other hand, meiosis and spermatogenesis do not begin in the testes until puberty and then go on continuously in the epithelial lining of very long, tightly coiled tubes, called seminiferous tubules. Immature germ cells, called spermatogonia, are located around the outer edge of these tubes next to the basal lamina, where they proliferate continuously by ordinary cell division cycles. Some of the daughter cells stop proliferating and differentiate into primary spermatocytes. These cells enter the first meiotic prophase, in which their paired homologous chromosomes participate in crossing-over, and then proceed with division I of meiosis to produce two secondary spermatocytes, each containing 22 duplicated autosomal chromosomes and either a duplicated X or a duplicated Y chromosome. The two secondary spermatocytes proceed through meiotic division II to produce four spermatids, each with a haploid number of single chromosomes. These haploid spermatids then undergo morphological differentiation into sperm (Figure 20-21), which escape into the lumen of the seminiferous tubule (Figure 20-22). The sperm subsequently pass into the epididymis, a coiled tube overlying the testis, where they are stored and undergo further maturation.

An intriguing feature of spermatogenesis is that the developing male germ cells fail to complete cytoplasmic division (cytokinesis) during mitosis and meiosis. Consequently, large clones of differentiating daughter cells descended from one maturing spermatogonium remain connected by cytoplasmic bridges, forming a syncytium (Figure 20-23). The cytoplasmic bridges persist until the very end of sperm differentiation, when individual sperm are released into the tubule lumen. This accounts for the observation that mature sperm arise synchronously in any given area of a seminiferous tubule. But what is the function of the syncytial arrangement?

Unlike oocytes, sperm undergo most of their differentiation after their nuclei have completed meiosis to become haploid. The presence of cytoplasmic bridges between them, however, means that each developing haploid sperm shares a common cytoplasm with its neighbors, so that it can be supplied with all the products of a complete diploid genome. Thus the diploid genome directs sperm differentiation just as it directs egg differentiation.

Summary

A sperm is usually a small, compact cell, highly specialized for the task of fertilizing an egg. Whereas in human females the total pool of oocytes is produced before birth, in males new germ cells enter meiosis continually from the time of sexual maturation, each primary spermatocyte giving rise to four mature sperm. Sperm differentiation occurs after meiosis, when the nuclei are haploid. Because the maturing spermatogonia and spermatocytes fail to complete cytokinesis, however, the progeny of a single spermatogonium develop as a large syncytium. This allows sperm differentiation to be directed by the products of both parental chromosomes.

Introduction

Once released, egg and sperm alike are destined to die within minutes or hours unless they find each other and fuse in the process of fertilization. Through fertilization the egg and sperm are saved: the egg is activated to begin its developmental program, and the nuclei of the two gametes come together to form the genome of a new organism. The mechanism of fertilization has been most intensively studied in marine invertebrates, especially sea urchins. In these organisms fertilization occurs in sea water, into which huge numbers of both sperm and eggs are released. Such external fertilization is more accessible to study than the internal fertilization of mammals, which occurs in the female reproductive tract following mating.

In the late 1950s, however, it became possible to fertilize mammalian eggs (more accurately, secondary oocytes - see Figure 20-16) in vitro, opening the way to an analysis of the cellular and molecular events in mammalian fertilization. Progress in understanding mammalian fertilization has brought substantial medical benefit: mammalian eggs that have been fertilized in vitro can develop into normal individuals when transplanted into the uterus; in this way many previously infertile women have been able to produce normal children. We shall focus our brief discussion, then, on mammalian fertilization.

Binding to the Zona Pellucida Induces the Sperm to Undergo an Acrosomal Reaction ¹³

Of the 300 million human sperm ejaculated during coitus, only about 200 reach the site of fertilization in the oviduct. Once there, the sperm must first migrate through the shell of follicular cells that surrounds the ovulated egg and then bind to and traverse the egg coat - the zona pellucida. Finally, it must bind and fuse with the egg plasma membrane. To become competent to accomplish these tasks, ejaculated mammalian sperm must normally be modified by secretions in the female reproductive tract, a process called capacitation, which requires about 5-6 hours in humans. Capacitation seems to involve both an alteration in the lipid and glycoprotein composition of the sperm plasma membrane and an increase in sperm metabolism and motility; its mechanism is unclear.

Once a capacitated sperm has penetrated the layer of follicle cells, it binds to the zona pellucida (see Figure 20-15). The zona pellucida usually acts as a barrier to fertilization across species, and removing it often removes this barrier. Human sperm, for example, will fertilize hamster eggs from which the zona pellucida has been removed with specific enzymes; not surprisingly, such hybrid zygotes do not develop. Zona-free hamster eggs, however, are used in fertility clinics to assess the fertilizing capacity of human sperm in vitro (Figure 20-24) .

The zona pellucida of mammalian eggs is composed of only three glycoproteins. Two of them, ZP2 and ZP3, assemble into filaments, while the other, ZP1, cross-links the filaments into a three-dimensional network. ZP3 acts as a sperm receptor: the species-specific binding of sperm to the zona pellucida is mediated by a molecule (which may be the enzyme galactosyl transferase) on the surface of the sperm head that binds to O-linked oligosaccharides on ZP3 in the zona. On binding, the sperm is induced to undergo the acrosomal reaction, in which the contents of the acrosome are released by exocytosis (Figure 20-25). In the mouse, at least, the trigger for the acrosomal reaction is ZP3 in the zona, which activates a complex intracellular signaling mechanism that induces an influx of Ca²⁺ into the sperm cytosol, which is thought to initiate exocytosis.

The acrosomal reaction releases proteases and hyaluronidase, which are essential for the penetration of the sperm through the zona pellucida, and it exposes other proteins on the sperm surface that bind to ZP2 and thereby help the sperm maintain its tight binding to the zona while boring through it. In addition, the acrosomal reaction exposes a protein in the sperm plasma membrane that mediates the binding and fusion of this membrane with that of the egg, as we see below.

The Egg Cortical Reaction Helps to Ensure That Only One Sperm Fertilizes the Egg ¹⁴

Although many sperm can bind to an egg, normally only one fuses with the egg plasma membrane and injects its nucleus and other organelles into the egg cytoplasm. If more than one sperm fuses - a condition called polyspermy - multipolar or extra mitotic spindles are formed, resulting in faulty segregation of chromosomes during cell division; nondiploid cells are produced, and development quickly stops. Two mechanisms operate to ensure that only one sperm fertilizes the egg. A rapid depolarization of the egg plasma membrane, which is caused by the fusion of the first sperm, is thought to prevent further sperm from fusing and thereby acts as a fast primary block to polyspermy. But the membrane potential returns to normal soon after fertilization, so that a second mechanism is required to ensure a longer-term, secondary block to polyspermy. This is provided by the egg cortical reaction.

When the sperm fuses with the egg plasma membrane, it activates the inositol phospholipid cell-signaling pathway (discussed in Chapter 15) in the egg. This, in turn, causes a local increase in cytosolic Ca²⁺, which spreads through the cell in a wave. The rise in Ca²⁺ in the cytosol is thought to activate the egg and initiate the cortical reaction, in which the cortical granules release their contents by exocytosis. If the cytosolic concentration of Ca²⁺ is increased artificially - either directly by an injection of Ca²⁺ or indirectly by the use of Ca²⁺-carrying ionophores (discussed in Chapter 11) - the eggs of all animals so far tested, including mammals, are activated. Conversely, preventing the increase in Ca²⁺ by injecting the Ca²⁺ chelator EGTA inhibits activation of the egg in response to fertilization. The enzymes released by the cortical reaction change the structure of the zona pellucida, which becomes "hardened," so that sperm no longer bind to it, thereby providing a slow, secondary block to polyspermy. Among the changes that occur in the zona is the proteolytic cleavage of ZP2 and the hydrolysis of sugar groups on ZP3 (Figure 20-26).

A Transmembrane Fusion Protein in the Sperm Plasma Membrane Catalyzes Sperm-Egg Fusion ¹⁵

After a sperm penetrates the extracellular coat of the egg, it interacts with the egg plasma membrane overlying the tips of microvilli on the egg surface (see Figure 20-24). Neighboring microvilli then rapidly elongate and cluster around the sperm to ensure that it is held firmly so that it can fuse with the egg. After fusion, the entire sperm is drawn head-first into the egg as the microvilli are resorbed. In hamsters a single transmembrane protein called PH-30, which becomes exposed on the sperm surface during the acrosomal reaction, is thought to mediate both the binding of the sperm to the egg plasma membrane and the fusion of the two plasma membranes.

The protein is composed of two glycosylated transmembrane subunits called α and β, which are held together by noncovalent bonds (Figure 20-27). The extracellular domain of the alpha subunit contains a hydrophobic region of about 20 amino acid residues that resembles the fusogenic regions of viral fusion proteins, which mediate the fusion of enveloped viruses with the cells that they infect (discussed in Chapter 13). It has long been suspected that the various membrane fusions that occur within and between eucaryotic cells are catalyzed by fusion proteins resembling those present in enveloped viruses; PH-30 is the first such cellular protein to be defined.

The extracellular amino-terminal domain of the β subunit of PH-30 resembles a domain found in some proteins that bind to integrins, the cell-surface receptors that help animal cells to adhere to the extracellular matrix (discussed in Chapter 19). This and other indirect evidence suggest that the PH-30 β subunit binds to an integrin in the egg plasma membrane and thereby helps the sperm adhere to the surface of the egg in preparation for fusion.

As the cell biology of mammalian fertilization becomes better understood and the molecules that mediate the various steps in the process are defined, new strategies for contraception become possible. One approach currently being investigated, for example, is to immunize males or females with molecules that are required for reproduction in the hope that the antibodies produced will inhibit the activities of these molecules. In addition to the various hormones and hormone receptors involved in reproduction, ZP3 and PH-30 might be appropriate target molecules. An alternative approach would be to administer oligosaccharides or peptides corresponding to ligands, such as the postulated integrin-binding domain of PH-30, that operate in fertilization. Small molecules of this type might block fertilization by competing with the normal ligand.

The Sperm Provides a Centriole for the Zygote ¹⁶

Once fertilized, the egg is called a zygote. Fertilization is not complete, however, until the two haploid nuclei (called pronuclei) have come together and combined their chromosomes into a single diploid nucleus. In fertilized mammalian eggs the two pronuclei do not fuse directly as they do in many other species: they approach each other but remain distinct until after the membrane of each pronucleus breaks down in preparation for the first mitotic division (Figure 20-28).

In most animals, including humans, the sperm contributes more than DNA to the zygote: it also donates a centriole - an organelle that is curiously lacking in the unfertilized eggs of these animals; the egg has a centrosome, but this does not contain a centriole. The sperm centriole enters the egg along with the sperm nucleus and tail, and in some species it replicates and helps organize the assembly of the first mitotic spindle in the zygote (see Figure 20-28). This explains why multipolar or extra mitotic spindles form in cases of polyspermy, where several sperm contribute centrioles to the egg.

Fertilization marks the beginning of one of the most remarkable phenomena in all of biology - the process of embryogenesis, in which the zygote develops into a new individual. This is the subject of the next chapter.

Summary

Mammalian fertilization begins when the head of a sperm binds in a species-specific manner to the zona pellucida surrounding the egg. This induces the acrosomal reaction in the sperm, which releases the contents of its acrosomal vesicle, including enzymes that help the sperm digest its way through the zona to the egg plasma membrane in order to fuse with it. Fusion is catalyzed by a transmembrane protein located in the sperm plasma membrane. This event activates the egg to undergo the cortical reaction, in which cortical granules release their contents, including enzymes that alter the zona pellucida and thereby prevent the fusion of additional sperm. Development of the zygote begins after sperm and egg haploid pronuclei have come together, pooling their chromosomes to form a single diploid nucleus.