.
It involves an alternation of haploid and diploid generations of cells.
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Sex is not absolutely necessary. Single-celled organisms can reproduce by simple mitotic division, and many plants propagate vegetatively, by forming multicellular offshoots that later detach from the parent. Likewise, in the animal kingdom, a solitary multicellular Hydra can produce offspring by budding (Figure 20-1
). Sea anemones and marine worms can split into two half-organisms, each of which then regenerates its missing half. There are even species of lizards that consist only of females and reproduce without mating. Although such asexual reproduction is simple and direct, it gives rise to offspring that are genetically identical to the parent organism. In sexual reproduction, on the other hand, the genomes from two individuals are mixed to produce offspring that differ genetically from one another and from both their parents. This mode of reproduction apparently has great advantages, as the vast majority of plants and animals have adopted it. Even many procaryotes and other organisms that normally reproduce asexually engage in occasional bouts of sexual reproduction, thereby creating offspring with new combinations of genes. This chapter describes the cellular machinery of sexual reproduction. Before discussing in detail how the machinery works, however, we shall pause briefly to consider why it exists and what benefits it brings.
It involves an alternation of haploid and diploid generations of cells.
). Genomes mix when two haploid cells fuse to form a diploid cell. Later, new haploid cells are generated when a descendant of this diploid cell divides by the process of meiosis. During meiosis, the chromosomes of the double chromosome set exchange DNA by genetic recombination before being shared out, in new combinations, into single chromosome sets. Because each single chromosome set will contain genes originating from one ancestral cell of the previous haploid generation mixed with genes from the other ancestral cell, each cell of the new haploid generation will receive a novel assortment of genes. Thus, through cycles of haploidy, cell fusion, diploidy, and meiosis, old combinations of genes are broken up and new combinations are created.
The haploid cells are shown in red and the diploid cells in blue. Cells in higher eucaryotic organisms usually proliferate in the diploid phase to form a multicellular organism; only the gametes are haploid, and they fuse at fertilization to form a diploid zygote, which develops into a new individual. In some lower eucaryotes, by contrast, the haploid cells proliferate, and the only diploid cell is the zygote, which exists transiently after mating.
).
Although many sperm are bound to the egg, only one will fertilize it, as we discuss later. (Courtesy of David Epel.)
). During the diploid phase that follows the 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.
This extravagant plumage serves solely to attract females for the purpose of sexual reproduction. (© Cyril Laubscher.)
). What benefits does it bring, and why did it evolve? Through genetic recombination, sexual individuals produce 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 to 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. Sexual reproduction also allows the many deleterious mutations that accumulate randomly to be eliminated, while permitting the rare advantageous mutations that arise in separate individuals to be combined in a single individual.
Whatever the benefits 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 now turn to the cellular mechanisms of sex, beginning with the events of meiosis, which segregates the chromosomes into new sets, as the diploid cells in the germ line divide to produce haploid gametes. We then focus our discussion on mammals. We consider the diploid cells of the germ line that give rise to the gametes and how the sex of a mammal is determined. Finally, we discuss the nature of the gametes themselves, as well as the process of fertilization, in which two gametes fuse to form a zygote, which develops into a new diploid organism.
Sexual reproduction has been favored by evolution probably because the random recombination of genetic information improves the chances of producing at least some offspring that will survive in an unpredictably variable environment. The sexual reproductive cycle involves an 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 that inherit novel assortments of genes. Most of the life cycle of higher plants and animals is spent in the diploid phase; only a small proportion of the diploid cells (those in the germ line) undergo meiosis to produce haploid cells (the gametes), and the haploid phase is very brief.
).
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. (Mitosis, which refers to the nuclear division that occurs during an ordinary mitotic cell division (discussed in Chapter 18), is from the Greek word mitos, meaning “a thread.” The term refers to the threadlike appearance of the chromosomes as they condense during nuclear division—a process that occurs in both meiotic and mitotic 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 events of meiosis were finally established. More recent genetic and molecular studies have begun to identify the meiosis-specific proteins that cause the chromosomes to behave in a special way and mediate the genetic recombination events that occur in meiosis.
The set of chromosomes of a typical sexually-reproducing organism consists of autosomes, which are common to all members of the species, and sex chromosomes, which are differently allocated according to the sex of the individual. A diploid nucleus contains two closely similar versions of each chromosome. For each of the autosomal chromosome pairs, one member was initially inherited from the male parent (a paternal chromosome) and the other was initially inherited from the female parent (a maternal chromosome). The two versions, which are very similar but not identical in DNA sequence, are called homologs, and in most cells they maintain a completely separate existence as independent chromosomes.
After a chromosome is duplicated by DNA replication, the twin copies of the fully replicated chromosome at first remain tightly linked along their length and are called sister chromatids. In a mitotic cell division, the sister chromatids line up at the equator of the spindle with their kinetochores (protein complexes associated with the centromeres, discussed in Chapter 18) and attached microtubules pointing toward opposite poles. The sister chromatids then separate completely from each other at anaphase to become individual chromosomes. In this manner each daughter cell formed by a mitotic cell division inherits one copy of each paternal chromosome and one copy of each maternal chromosome and is therefore unchanged in its genetic composition from the parent cell.
In contrast, a haploid gamete produced from a diploid cell through meiosis must contain half the original number of chromosomes. It must contain only one chromosome in place of each homologous pair of chromosomes, so it 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 homologs recognize each other and become physically connected side-by-side along their entire length before they line up on the spindle. How the maternal and the paternal copy of each chromosome recognize each other is still uncertain. In many organisms, the initial association (a process called pairing) seems to be mediated by complementary DNA base-pair interactions at numerous and widely dispersed sites along the chromosomes.
For clarity, only one pair of homologous chromosomes is shown. Each chromosome has been duplicated and exists as attached sister chromatids before pairing with its homologous chromosome (homolog), thereby forming a structure containing four chromatids known as a bivalent. As shown by the formation of chromosomes that are part red and part black, chromosome pairing in meiosis leads to genetic recombination between homologous chromosomes, as explained later.
). To produce haploid gametes, however, another cell division is required.
).
As in the previous figure, only one pair of homologous chromosomes is shown. In meiosis, after DNA replication, two nuclear (and cell) divisions are required to produce the haploid gametes. Each diploid cell that enters meiosis therefore produces four genetically different haploid cells, whereas each diploid cell that divides by mitosis produces two genetically identical diploid cells.
.
Occasionally during meiosis, chromosomes fail to separate normally into the four haploid cells, a phenomenon known as nondisjunction. In such abnormal meiotic divisions some of the haploid cells that are produced lack a chromosome, while others have more than one copy. The resulting gametes form abnormal embryos, most of which die. Some survive, however: Down syndrome in humans, for example, is caused by an extra copy of chromosome 21, resulting from nondisjunction during meiotic division I or II. The vast majority of such segregation errors occur during meiosis in females, and the error rate increases with advancing maternal age. The frequency of missegregation in human oocytes is remarkably high (about 10% of meioses), and this is thought to be one reason for the high rate of miscarriages (spontaneous abortions) in early pregnancy.
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 at fertilization, two kinds of randomizing genetic reassortment have occurred during meiosis.
(A) The independent assortment of the maternal and paternal homologs during the first meiotic division produces 2n different haploid gametes for an organism with n chromosomes. Here n = 3, and there are eight different possible gametes. (B) Crossing-over during meiotic prophase I exchanges segments of homologous chromosomes and thereby reassorts genes on individual chromosomes. Because of the many small differences in DNA sequence that always exist between any two homologs, both mechanisms increase the genetic variability of organisms that reproduce sexually.
). In humans, for example, each individual can produce at least 223 = 8.4 × 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 (prophase 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 20-8B.
A single crossover event has occurred earlier in prophase to create one chiasma. Note that the four chromatids are arranged as two distinct pairs of sister chromatids. As in mitosis, the sister chromatids in each pair are tightly connected along their entire lengths, as well as at their centromeres, by proteins called cohesins. The entire unit of four chromatids is referred to as a bivalent. The combination of the chiasma and the tight attachment of the sister chromatids holds the two duplicated homologs together.
(A) In this drawing, chromatid 1 has undergone an exchange with chromatid 3, and chromatid 2 has undergone exchanges with chromatid 3 and 4. Note that the sister chromatids of the same chromosome do not exchange with each other. (B) Light micrograph of a grasshopper bivalent with three chiasmata. (B, courtesy of Bernard John.)
). Each of the two chromatids of a duplicated chromosome can cross over with either of the two chromatids of the other chromosome in the bivalent, as illustrated in Figure 20-10.
At this stage of meiosis, each pair of duplicated homologs is held together by at least one chiasma. Many bivalents contain more than one chiasma, indicating that multiple crossovers can occur between homologs.
). Before anaphase I, the two poles of the spindle pull on the duplicated homologs in opposite directions, and the chiasmata resist this pulling. 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 many of the resulting gametes contain too many or too few chromosomes.
). In Drosophila, for example, if a meiosis-specific cohesin is defective, sister chromatids separate prior to metaphase I and, as a consequence, the homologs segregate abnormally.
The ungluing of the sister chromatid arms allows the duplicated homologs to separate at anaphase I, while an ungluing of the chromosomes at their centromeres allows the sister chromatids to separate at anaphase II. In contrast, at anaphase in mitosis, both the arms and the centromeres come apart at the same time (discussed in Chapter 18).
, the arms of sister chromatids suddenly become unglued at the start of anaphase I, when the cohesins holding the arms together are degraded, allowing the duplicated homologs to separate and be pulled to opposite poles of the spindle. The sister chromatids of each duplicated homolog remain attached at the centromere by meiosis-specific cohesins, which are degraded at anaphase of meiotic division II (anaphase II); only then can the sister chromatids separate.
). In budding yeasts, a meiosis-specific protein located at the kinetochores of meiosis I chromosomes has been shown to be required for this special behavior.We have seen that duplicated homologous chromosomes must pair and form at least one chiasma during the first meiotic division if they are to segregate accurately between the daughter cells. But what happens to the sex chromosomes? Female mammals have two X chromosomes, which can pair and segregate like other homologs. But males have one X and one Y chromosome, and these chromosomes are not homologous. Yet, they must pair and then cross over 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 crossovers are possible because of a small region of homology between the X and the Y at one end of these chromosomes. The two chromosomes pair and cross over in this region during prophase I. The chiasmata resulting from this genetic recombination keep the X and Y chromosomes connected 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.
Having considered the general way in which chromosomes behave and segregate during meiosis, we now return to the process of genetic recombination that occurs during the long prophase of meiotic division I and has such an important role in reassorting genes during gamete formation.
A series of complex events occurs during the long prophase of meiotic division I: duplicated homologous chromosomes pair, genetic recombination is initiated between nonsister chromatids, and each pair of duplicated homologs assembles into an elaborate structure called the synaptonemal complex. In some organisms, genetic recombination begins before the synaptonemal complex assembles and is required for the complex to form; in others, the complex can form in the absence of recombination. In all organisms, however, the recombination process is completed while the DNA is held in the synaptonemal complex, which serves to space out the crossover events along each chromosome.
(A) A single bivalent is shown. The pachytene stage is defined as the period during which a fully formed synaptonemal complex exists. At leptotene, the two sister chromatids condense, and their chromatin loops each extend from a common protein axis (red). As meiosis progresses, the two homologs become tightly connected by proteins that form the central region of the synaptonemal complex, composed of a central element (blue), transverse filaments (thin black lines), and the lateral elements (red) that anchor the chromatin loops. In the gametes of many female animals, but not those of mammals, the subsequent diplotene stage is an enormously prolonged period of cell growth, during which the chromosomes are decondensed and very active in transcription. Diplotene ends with diakinesis—the stage of transition to metaphase—in which the chromosomes recondense and transcription halts. In male gametes, diplotene and diakinesis are briefer and less distinct. (B) An electron micrograph of a synaptonemal complex from a meiotic cell at pachytene in a lily flower. (B, courtesy of Brian Wells.)
).
Only a short section of the long ladderlike complex is shown. A similar synaptonemal complex is present in organisms as diverse as yeasts and humans.
). The sister chromatids in each homolog are kept tightly packed together, with their DNA extending from their own side of the protein ladder in a series of loops. In the central region, a central element is connected by transverse filaments to lateral elements that run along each pair of sister chromatids, forming the sides of the ladder.
Several protein components of the synaptonemal complex have been identified and localized to specific structures of the complex. Yeast mutants that lack specific components have provided insights into the functions of the complex and some of its proteins. One yeast protein, for example, seems to nucleate the assembly of the lateral elements: if this protein is defective, these elements fail to form. Another yeast protein helps to form the transverse filaments: if this protein is absent, homolog pairing occurs without intimate synapsis, while an abnormally long mutant form of the protein creates a larger than normal separation between the two lateral elements of the synaptonemal complex.
The crossover events that take place during the prophase of meiotic division I can occur nearly anywhere along a chromosome. They are not distributed uniformly, however: there are recombination “hot spots,” where double-stranded DNA breaks seem to be preferentially induced by the meiotic endonuclease called Spo11. Moreover, both genetic and cytological experiments indicate that the occurrence of one crossover event decreases the probability of a second occurring at a nearby chromosomal site. This “interference” seems to ensure that the limited number of crossovers are spread out so that even small chromosomes get at least one, as required for the homologs to segregate normally. Although the molecular basis of the interference is unknown, the synaptonemal complex is thought to mediate the process.
). These nodules contain Rad51, which is the eucaryotic version of the RecA protein, which mediates general recombination in E. coli (discussed in Chapter 5). They seem to mark the site of a multienzyme “recombination machine” that interacts with local regions of DNA on the maternal and paternal chromatids across the 100-nm-wide synaptonemal complex.
There are two main types of recombination nodule. Early nodules are present before pachytene and are thought to mark the sites of the initial DNA-strand-exchange events of the recombination process. Late nodules are less numerous, are present during pachytene, and are thought to mark the sites where the initial strand-exchange events are being resolved as stable crossovers. Proteins known to be involved in general recombination have been identified in recombination nodules, and there is a strong correspondence between the number and distribution of late nodules and the number and distribution of crossovers. Moreover, meiosis-specific versions of proteins involved in mismatch DNA repair (discussed in Chapter 5) are also located in late nodules, where they help to resolve recombination intermediates as stable crossovers.
The occurrence of crossovers has enabled geneticists to map the relative positions of genes on chromosomes, as we now explain. Such maps have been crucial in the cloning of human disease genes.
On average, a human chromosome participates in two or three crossover events during meiosis, and every chromosome participates in at least one. Thus, whereas two genes very close to each other on a chromosome almost always end up together in the same gamete after meiosis, two genes located at the opposite ends of a chromosome are no more likely to end up together than are genes located on different chromosomes. One can therefore determine whether two genes—a gene with a mutant form causing congenital deafness, for example, and a second gene with a mutant form causing muscular dystrophy—are located close together on the same chromosome. This is done by measuring the frequency with which a child inherits the mutant forms of both genes from a parent that carries one mutant and one nonmutant version of each of them. If the two mutant genes are on different chromosomes, one will be inherited without the other 50% of the time, as chromosomes are independently segregated at meiosis. The same result is expected, however, if the two mutant genes are far apart on the same chromosome, as one or more crossover events will separate them at meiosis. To determine whether genes are on the same chromosome and, if so, how close they are to one another, human geneticists measure the frequency of coinheritance of many genes in large numbers of families. In this way, they can discover not only the neighbors of a particular gene but also the neighbors of the neighbors and thereby work their way down an entire chromosome. By this means, they have defined 24 linkage groups, one corresponding to each human chromosome (22 autosome pairs plus 2 sex chromosomes).
Using such measurements, geneticists have constructed detailed genetic maps of the entire human genome, in which the distance between each pair of neighboring genes is displayed as the percentage recombination between them. The standard unit of genetic distance is the centimorgan (cM), which corresponds to a 1% probability that two genes will be separated by a crossover event during meiosis. A typical human chromosome is more than 100 centimorgans long, indicating that more than one crossover is likely to occur on a typical human chromosome.
Another way to construct a genetic map is to measure the coinheritance of short DNA sequences (called DNA markers) that differ between individuals in the population—that is, that are polymorphic (see p. 464). Genetic maps constructed in this way have two advantages over genetic maps constructed by tracing the phenotypes of individuals that inherit mutant genes. First, they can be more detailed, as there are large numbers of DNA markers that can be measured. Second, they can reveal the real distance in nucleotide pairs between the markers, so that genetic distances in centimorgans can be compared directly with true physical distances along a chromosome.
The DNA markers shown are various genes. A indicates a region where the genetic map is contracted owing to decreased frequency of crossing-over. B indicates a region where the genetic map is expanded owing to increased frequency of crossing-over.
. As the entire DNA sequence of this organism's genome is known, the physical map indicates the true distances between the DNA markers. The regions of the genetic map that are expanded in comparison with the physical map indicate recombination “hotspots,” where crossovers during meiosis occur with an unusually high frequency. Regions that are contracted indicate recombination “coldspots,” where crossovers occur with unusually low frequency. Human genetic maps show similar expansions and contractions. A likely explanation for the hotspots is that they contain an abundance of sites where the DNA helix is cut by the meiotic endonuclease (Spo11) that creates the double-strand DNA breaks that begin the recombination process (see Figure 5-56).
).
(A) Approximate times for a male mammal (mouse). (B) Approximate times for the male tissue of a plant (lily). Times differ for male and female gametes (sperm and eggs, respectively) of the same species, as well as for the same gametes of different species. Meiosis in a human male, for example, lasts for 24 days, compared with 12 days in the mouse. In all species, however, meiotic prophase I is always much longer than all the other meiotic stages combined.
).
).
). In mitosis, however, the sister chromatids are glued together along their length, as well as at the centromere, and both types of contact are released at the start of anaphase. In meiosis, by contrast, the sister chromatids come apart in two steps—their arms have separated at anaphase I, while their centromeres remain attached, separating only at anaphase II (see Figures 20-7 and 20-11).
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 mammals. As we shall see, by the end of meiosis a mammalian egg is fully mature, whereas a sperm that has completed meiosis has only just begun its differentiation. Before discussing these gametes, however, we consider how certain cells in the mammalian embryo become committed to developing into germ cells and how these cells then become committed to developing into either sperm or eggs, depending on the sex of the individual.
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. As it enters this prophase, each chromosome consists of two tightly joined sister chromatids. The two replicated homologs present in each diploid nucleus then pair to form a bivalent, consisting of four chromatids. Chromosomal crossover events occur during this time. Each results in the formation of a chiasma, which helps hold each pair of homologs together during metaphase I. Crossing-over has an important role in reassorting genes during gamete formation, and it allows geneticists to map the relative positions of genes on chromosomes. The pairing of homologs culminates in the formation of a synaptonemal complex, which somehow serves to spread out the crossover events along the chromosomes. At anaphase of the first meiotic cell division, the arms of the sister chromatids suddenly become unglued, causing one member of each chromosome pair, still composed of a pair of sister chromatids linked at their centromeres, to be distributed to each daughter nucleus. A second cell division cycle, without DNA replication, then rapidly ensues; in anaphase II, each sister chromatid separates from its sister and is segregated into a separate haploid nucleus.
Sexual reproductive strategies can vary enormously between different organisms. In the rest of this chapter, we focus mainly on the strategies used by mammals.
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, which will form the ovaries in females and the testes in males. After a period of mitotic proliferation, the primordial germ cells undergo meiosis and differentiate into mature gametes—either eggs or sperm. Later, the fusion of egg and sperm after mating initiates embryogenesis. The subsequent production in this embryo of new primordial germ cells begins the cycle again.
In this section, we consider how mammalian primordial germ cells arise, how the sex of a mammal is determined, and how sex determination dictates whether the primordial germ cells develop into sperm or eggs.
(A) Drawing showing the final stages of migration through the hindgut into the two genital ridges, each of which will develop into a gonad—either an ovary or a testis. (B) Micrograph showing migrating primordial germ cells in an early mouse embryo. The primordial germ cells are stained with a monoclonal antibody (in green) that specifically labels these cells at this stage of embryogenesis. The remaining cells in the embryo are stained with a lectin that binds to sialic acid, which is found on the surface of all cells. (C) Drawing corresponding to the micrograph shown in (B). (B, courtesy of Robert Anderson and Chris Wylie.)
). As the primordial germ cells migrate through the embryo, they are signaled to survive, proliferate, and migrate by various extracellular proteins produced by adjacent somatic cells.
After the primordial germ cells enter the developing mouse gonad, which at this stage is called the genital ridge, they continue to proliferate for 2 or 3 more days. At this point, they commit to a developmental pathway that will lead them to become either eggs or sperm, depending not on their own sex chromosome constitution but on whether the genital ridge has begun to develop into an ovary or a testis, respectively. The sex chromosomes in the somatic cells of the genital ridge determine which type of gonad the ridge becomes. A single gene on the Y chromosome has an especially important role in this decision.
Aristotle believed that the temperature of the male during sexual intercourse determined the sex of offspring: the higher the temperature, the greater the chance of producing a male. We now know that the sex of a mammal is determined by its sex chromosomes, rather than by the environment (although for some animals, such as crocodiles and many fish, the opposite is true). Female mammals have two X chromosomes in all of their somatic cells, whereas males have one X and one Y. The Y chromosome is the determining factor. Individuals with a Y chromosome develop as males no matter how many X chromosomes they have, whereas individuals without a Y chromosome develop as females, even if they have only one X chromosome. The sperm that fertilizes the egg determines the sex of the resulting zygote: eggs have a single X chromosome, whereas the sperm can have either an X or a Y.
The Sry gene, injected into the nucleus of an XX female zygote, caused the transgenic embryo produced to develop into a male. The external genitalia of the transgenic mouse are indistinguishable from those of a normal XY male mouse. (From P. Koopman et al., Nature 351:117–121, 1991. © Macmillan Magazines Ltd.)
). Such mice, however, cannot produce sperm, in part, at least, because the presence of two X chromosomes suppresses sperm development.
Sry is expressed only in a subset of the somatic cells of the developing gonad, and it causes these cells to differentiate into Sertoli cells, which are the main type of supporting cells found in the testis. The Sertoli cells direct sexual development along a male pathway by affecting other cells in the genital ridge in at least four ways:
They stimulate the newly arriving primordial germ cells to develop along a pathway that produces sperm.
They secrete anti-Müllerian hormone, which suppresses the development of the female reproductive tract by causing the Müllerian duct to regress (this duct otherwise gives rise to the oviduct, uterus, and upper part of the vagina).
They stimulate particular somatic cells that lie adjacent to the developing gonad to migrate into the gonad and form critical connective tissue structures that are required for normal sperm production.
They help to induce other somatic cells in the developing gonad to become Leydig cells, which secrete the male sex hormone testosterone; this hormone is responsible for inducing all male secondary sexual characteristics. These include the structures of the male reproductive tract, such as the prostate and seminal vesicles, which develop from another duct, called the Wolffian duct system. This duct system degenerates in the developing female because it requires testosterone to survive and develop. The testosterone also masculinizes the early developing brain and thereby plays a major part in determining male sexual identity and orientation, and thereby behavior: female rats that are treated with testosterone around birth, for example, later display malelike sexual behavior.
). In the absence of Sry, the primordial germ cells commit to egg development, and the somatic cells develop into either follicle cells, which support egg development, or theca cells, which secrete estrogen. Whereas Leydig cells begin secreting testosterone in the fetus, theca cells do not begin secreting estrogen until puberty.
). Spermatogenesis also depends on testosterone secreted by Leydig cells, located between the seminiferous tubules. (B) Some of these cells are self-renewing stem-cell spermatogonia, whereas others are maturing spermatogonia; after a number of mitotic divisions, the maturing spermatogonia stop dividing by mitosis and enter meiosis to become primary spermatocytes. Eventually, sperm are released into the lumen. In man, it takes about 24 days for a spermatocyte to complete meiosis to become a spermatid and another 5 weeks for a spermatid to develop into a sperm. Sperm undergo further maturation and become motile in the epididymis; only then are they fully mature sperm.
, and see Figure 20-28), and the animal develops as a female.
If the genital ridges are removed before they have started to develop into testes or ovaries, a mammal develops into a female, regardless of the sex chromosomes it carries. It seems that female development is the “default” pathway of sexual development in mammals.
A small number of cells in the gastrulating mammalian embryo are signaled by their neighbors to become primordial germ cells. These cells migrate into the genital ridges, which develop into the gonads. Here, the primordial germ cells start to develop into either eggs, if the gonad is becoming an ovary, or sperm, if the gonad is becoming a testis. A developing gonad will develop into an ovary unless its somatic cells contain a Y chromosome, in which case it develops into a testis. The Sry gene on the Y chromosome is responsible for this testis-determining function: it is expressed in a subset of somatic cells in the developing gonad, and it induces these cells to differentiate into Sertoli cells. The Sertoli cells in turn produce the signal molecules that promote the development of male characteristics, suppress the development of female characteristics, and induce the primordial germ cells to commit to sperm development.
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. In some organisms, however, the sperm itself is not strictly required, and an egg can be activated artificially by a variety of nonspecific chemical or physical treatments. Indeed, some organisms, including 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, it is itself a highly specialized cell, uniquely equipped for the single function of generating a new individual. The cytoplasm of an egg can even reprogram a somatic cell nucleus so that the nucleus can direct the development of a new individual. That is how the famous sheep Dolly was produced. The nucleus of an unfertilized sheep egg was destroyed and replaced with the nucleus of an adult somatic cell. An electric shock was used to activate the egg, and the resulting embryo was implanted in the uterus of a surrogate mother. The resulting normal adult sheep had the genome of the donor somatic cell and was therefore a clone of the donor sheep.
In this section, we briefly consider some of the specialized features of an egg before discussing how it develops to the point of being ready for fertilization.
The human egg is 0.1 mm in diameter.
Sizes are compared with that of a typical somatic cell.
). A typical somatic cell, by contrast, has a diameter of only about 10 or 20 μm (Figure 20-20).
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-enclosed, 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. In mammals, whose embryos are largely nourished by their mothers, there is little, if any, yolk.
(A) Scanning electron micrograph of a hamster egg, showing the zona pellucida. (B) A scanning electron micrograph of a similar egg in which the zona (to which many sperm are attached) has been peeled back to reveal the underlying plasma membrane, which contains numerous microvilli. The zona is made entirely by the developing oocyte. (From D.M. Phillips, J. Ultrastruct. Res. 72:1–12, 1980.)
). 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.
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).
Cortical granules are usually distributed evenly throughout the egg cortex, but in some organisms other cytoplasmic components have a strikingly asymmetrical distribution. Some of these localized components later serve to help establish the polarity of the embryo, as discussed in Chapter 21.
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 a prolonged period while the oocyte grows in size, and in many cases they later arrest in metaphase II while awaiting fertilization (although they can arrest at various other points, depending on the species).
Oogonia develop from primordial germ cells that migrate into the developing gonad early in embryogenesis. After a number of mitotic divisions, oogonia begin meiotic division I, after which they are called primary oocytes. In mammals, primary oocytes are formed very early (between 3 and 8 months of gestation in the human embryo) and remain arrested in prophase of meiotic division I until the female becomes sexually mature. At this point, a small number periodically mature under the influence of hormones, completing meiotic division I to become secondary oocytes, which eventually undergo meiotic division II to become mature eggs (ova). The stage at which the egg or oocyte is released from the ovary and is fertilized varies from species to species. In most vertebrates, oocyte maturation is arrested at metaphase of meiosis II and the secondary oocyte completes meiosis II only after fertilization. All of the polar bodies eventually degenerate. In most animals, the developing oocyte is surrounded by specialized accessory cells that help to isolate and nourish it (not shown).
. Primordial germ cells migrate to the forming gonad to become oogonia, which proliferate by mitosis for a period before differentiating into primary oocytes. At this stage (usually before birth in mammals), the first meiotic division begins: the DNA replicates so that each chromosome consists of two sister chromatids, the duplicated homologous chromosomes pair along their long axes, and crossing-over occurs between nonsister 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 a mitotic 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. In the case of large nonmammalian oocytes, they also 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 (see Figures 4-36 and 4-37).
). 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 undergoes a rapid maturation step that transforms it into an egg that is prepared for fertilization. If fertilization occurs, the egg is stimulated to complete meiosis.
A somatic cell with a diameter of 10–20 μm 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 thousand-fold greater mass of a mammalian egg with a diameter of 100 μm. It would take even longer to reach the million-fold greater mass of an insect egg with a diameter of 1000 μm. Yet some insects live only a few days and manage to produce eggs with diameters even greater than 1000 μm. 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. The oocytes of some species go to even greater lengths to accumulate extra DNA: they produce many extra copies of certain genes. We discuss in Chapter 6 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.
The nurse cells and the oocyte arise from a common oogonium, which gives rise to one oocyte and 15 nurse cells (only 7 of which are seen in this plane of section). These cells remain joined by cytoplasmic bridges, which result from incomplete cell division. Eventually the nurse cells dump their cytoplasmic contents into the developing oocyte and then kill themselves. The follicle cells develop independently (from mesodermal cells).
). For the insect oocyte, the nurse cells manufacture many of the products—ribosomes, mRNA, protein, and so on—that vertebrate oocytes have to manufacture for themselves.
(A) An early stage of primary oocyte development. Neither a zona pellucida nor cortical granules have developed, and the oocyte is surrounded by a single layer of flattened follicle cells. (B) A more mature primary oocyte, which is shown at a sixfold lower magnification because it is much larger than the oocyte in (A). This oocyte has acquired a thick zona pellucida and is surrounded by several layers of follicle cells and a basal lamina, which isolate the oocyte from the other cells in the ovary. The primary oocyte together with its surrounding follicle cells is called a primary follicle. The follicle cells are connected to one another and to the oocyte by gap junctions. (From The Cellular Basis of Mammalian Reproduction [J. Van Blerkom and P. Motta eds.]. Baltimore—Munich: Urban & Schwarzenberg, 1979.)
, and see Figure 20-23), 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).
Eggs develop in stages from primordial germ cells that migrate into the developing gonad early in development to become oogonia. After mitotic proliferation, oogonia become primary oocytes, which begin meiotic division I and then arrest at prophase I for days to 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.
In most species, there are just two types of gamete, 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, together with 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.
It is shown in longitudinal section.
). The DNA in the nucleus is extremely tightly packed, so that its volume is minimized for transport, and transcription is shut down. 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.
). This vesicle contains hydrolytic enzymes that may 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 acrosome reaction; in some sperm, this reaction also exposes or releases specific proteins that help bind the sperm tightly to the egg coat.
) results from the fusion of individual mitochondria during spermatid differentiation.
). These dense fibers are stiff and noncontractile, and it is not known what role they have 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 20-25 and 20-26).
) in several ways. (1) New cells enter meiosis continually from the time of puberty. (2) Each cell that begins meiosis gives rise to four mature gametes rather than one. (3) Mature sperm form by an elaborate process of cell differentiation that begins after meiosis is complete. (4) About twice as many cell divisions occur in the production of a sperm as in the production of an egg; in a mouse, for example, it is estimated that on average about 56 divisions occur from zygote to mature sperm, and about 27 divisions occur from zygote to mature egg.
), which escape into the lumen of the seminiferous tubule (Figure 20-28). The sperm subsequently pass into the epididymis, a coiled tube overlying the testis, where they undergo further maturation and are stored.
The progeny of a single maturing spermatogonium remain connected to one another by cytoplasmic bridges throughout their differentiation into mature sperm. For the sake of simplicity, only two connected maturing spermatogonia are shown entering meiosis, eventually to form eight connected haploid spermatids. In fact, the number of connected cells that go through two meiotic divisions and differentiate synchronously is very much larger than shown here. Note that in the process of differentiating, most of the spermatid cytoplasm is discarded as residual bodies.
). 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. In this way, it can be supplied with all the products of a complete diploid genome. Developing sperm that carry a Y chromosome, for example, can be supplied with essential proteins encoded by genes on the X chromosome. Thus, the diploid genome directs sperm differentiation just as it directs egg differentiation.
Some of the genes that regulate spermatogenesis have been conserved in evolution from flies to humans. The DAZ gene, for example, which encodes an RNA-binding protein and is located on the Y chromosome, is deleted in many infertile men, many of whom cannot make sperm. Two Drosophila genes that are homologous to DAZ are essential for spermatogenesis in the fly. RNA-binding proteins are especially important in spermatogenesis, because many of the genes expressed in the sperm lineage are regulated at the level of RNA translation.
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, with each diploid primary spermatocyte giving rise to four haploid mature sperm. The process of sperm differentiation occurs after meiosis is complete, requiring five weeks in humans. Because the maturing spermatogonia and spermatocytes fail to complete cytokinesis, however, the progeny of a single spermatogonium develop as a large syncytium. Sperm differentiation is therefore directed by the products from both parental chromosomes, even though each nucleus is haploid.
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 haploid nuclei of the two gametes come together to form the genome of a new diploid 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 has been more accessible to study than the internal fertilization of mammals, which normally occurs in the female reproductive tract after mating.
In the late 1950s, however, it became possible to fertilize mammalian eggs 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. As mentioned earlier, it is possible to use in vitro fertilization to produce a clone of a sheep, a pig, or a mouse by transferring the nucleus of one of its somatic cells into an unfertilized egg that has had its own nucleus removed or destroyed. There is no reason to doubt that a human could be cloned in the same way, although there are serious ethical arguments about whether this should ever be done, especially as the likelihood of producing an abnormal child is very high. In this section, we focus our discussion on the fertilization of mammalian eggs.
Of the 300,000,000 human sperm ejaculated during coitus, only about 200 reach the site of fertilization in the oviduct. There is evidence that chemical signals released by the follicle cells that surround the ovulated egg attract the sperm to the egg, but the nature of the chemoattractant molecules is unknown. Once it finds an egg, the sperm must first migrate through the layer of follicle cells and then bind to and cross the egg coat—the zona pellucida. Finally, the sperm must bind to and fuse with the egg plasma membrane. To become competent to accomplish these tasks, ejaculated mammalian sperm must normally be modified by conditions in the female reproductive tract, a process called capacitation, which requires about 5–6 hours in humans. Capacitation is triggered by bicarbonate ions (HCO3–) in the vagina, which enter the sperm and directly activate a soluble adenylyl cyclase enzyme in the cytosol. The cyclase produces cyclic AMP (discussed in Chapter 15), which helps to initiate the changes associated with capacitation. Capacitation alters the lipid and glycoprotein composition of the sperm plasma membrane, increases sperm metabolism and motility, and markedly decreases the membrane potential (that is, the membrane potential moves to a more negative value so that the membrane becomes hyperpolarized).
The zona pellucida of the egg has been removed, exposing the plasma membrane, which contains numerous microvilli. The ability of an individual's sperm to penetrate hamster eggs is used as an assay of male fertility; penetration of more than 10–25% of the eggs is considered to be normal. (Courtesy of David M. Phillips.)
). The zona usually acts as a barrier to fertilization across species, and removing it often eliminates this barrier. Human sperm, for example, will fertilize hamster eggs from which the zona has been removed with specific enzymes; not surprisingly, such hybrid zygotes fail to develop. Zona-free hamster eggs, however, are sometimes used in infertility clinics to assess the fertilizing capacity of human sperm in vitro (Figure 20-30).
In mice, a single glycoprotein in the zona pellucida, ZP3, is thought to be responsible for both binding the sperm and inducing the acrosome reaction. Note that a mammalian sperm interacts tangentially with the egg plasma membrane so that fusion occurs at the equator, rather than at the tip, of the sperm head. In mice, the zona pellucida is about 6 μm thick, and sperm cross it at a rate of about 1 μm/min.
). In the mouse, at least, the trigger for the acrosome reaction is ZP3 in the zona, which induces an influx of Ca2+ into the sperm cytosol; this in turn initiates exocytosis. An increase in cytosolic Ca2+ seems to be necessary and sufficient to trigger the acrosome reaction in all animals.
The acrosome reaction is required for fertilization. It exposes various hydrolytic enzymes that help the sperm tunnel through the zona pellucida, and it exposes other proteins on the sperm surface that bind to the ZP2 protein and thereby help the sperm maintain its tight binding to the zona while burrowing through it. In addition, the acrosome reaction exposes proteins in the sperm plasma membrane that mediate the binding and fusion of this membrane with that of the egg, as we discuss below. Although fertilization normally occurs by sperm—egg fusion, it can also be achieved artificially, by injecting the sperm into the egg cytoplasm; this is sometimes done in infertility clinics when there is a problem with sperm—egg fusion.
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 usually stops. Two mechanisms can operate to ensure that only one sperm fertilizes the egg. In many cases, a rapid depolarization of the egg plasma membrane, which is caused by the fusion of the first sperm, prevents 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 causes a local increase in cytosolic Ca2+, which spreads through the cell in a wave. In some mammalian eggs, the initial increase in Ca2+ is followed by prolonged Ca2+ oscillations. There is evidence that the Ca2+ wave or oscillations are induced by a protein that is introduced into the egg by the sperm, but the nature of the protein is unknown.
The released contents of the cortical granules both remove carbohydrate from ZP3 so it no longer can bind to the sperm plasma membrane and partly cleave ZP2, hardening the zona pellucida. Together these changes provide a block to polyspermy.
).
). 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 mouse sperm, a transmembrane protein called fertilin, which becomes exposed on the sperm surface during the acrosome reaction, helps the sperm bind to the egg plasma membrane and may also have a role in the fusion of the two plasma membranes.
The α and β subunits, which are both glycosylated (not shown), are noncovalently associated. Both subunits belong to the ADAM family of proteins, which includes proteins thought to function in either cell adhesion or the proteolytic processing of other transmembrane proteins (such as Notch, which is discussed in Chapter 15). The proteolytic domain that is normally present at the amino terminus of these proteins is removed from the fertilin protein during sperm maturation.
). The extracellular N-terminal domain of the fertilin subunits is thought to bind to integrins in the egg plasma membrane and thereby help the sperm adhere to the egg membrane in preparation for fusion. The integrin in the egg plasma membrane is associated with a member of the tetraspan family of membrane proteins—so-called because they have four membrane-spanning segments. Female mice that are deficient in this protein are infertile, as their eggs cannot fuse with sperm. The extracellular domain of the α subunit of fertilin contains a hydrophobic region that resembles the fusogenic region of viral fusion proteins, which mediates the fusion of enveloped viruses with the cells that they infect (discussed in Chapter 13). Synthetic peptides corresponding to this region of the fertilin α chain can induce membrane fusion in a test-tube, consistent with the possibility that fertilin helps to mediate sperm—egg fusion.
Male mice that are fertilin-deficient are infertile, and their sperm are eightfold less efficient than normal sperm in binding to the egg plasma membrane but only 50% less efficient in fusing with it. Surprisingly, these defects do not seem to be the main cause of the infertility. The fertilin-deficient sperm are even more impaired in their ability to bind to the zona pellucida and to migrate out of the uterus into the oviduct, where the egg is normally fertilized. Clearly, fertilin's roles in fertilization are more complex than originally suspected and are still not completely understood. The finding that fertilin-deficient sperm can still fertilize eggs in a test tube, albeit inefficiently, suggests that other sperm proteins normally help to mediate sperm binding and fusion to the egg plasma membrane.
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 fertilin might be appropriate target molecules. An alternative approach would be to administer oligosaccharides or peptides corresponding to ligands that operate in fertilization, such as the postulated integrin-binding domain of fertilin. Small molecules of this type block fertilization in a test-tube by competing with the normal ligand for its receptor.
The pronuclei migrate toward the center of the egg. When they come together, their nuclear envelopes interdigitate. The centrosome replicates, the nuclear envelopes break down, and the chromosomes of both gametes are eventually integrated into a single mitotic spindle, which mediates the first cleavage division of the zygote. (Adapted from drawings and electron micrographs provided by Daniel Szöllösi.)
).
Spindle microtubules are stained in green with anti-tubulin antibodies, and DNA is labeled in blue with a DNA stain. (A) A meiotic spindle in a mature, unfertilized oocyte. (B) This fertilized egg is extruding its second polar body and is shown about 5 hours after fusion with a sperm. The sperm head (left) has nucleated an array of microtubules. (C) The two pronuclei have come together. (D) By 16 hours after fusion with a sperm, the centrosome that entered the egg with the sperm has duplicated, and the daughter centrosomes have organized a bipolar mitotic spindle. The chromosomes of both pronuclei are aligned at the metaphase plate of the spindle. As indicated by the arrows in (C) and (D), the sperm tail is associated with one of the centrosomes. (From C. Simerly et al., Nat. Med. 1:47–53, 1995. © Macmillan Magazines Ltd.)
). This explains why multipolar or extra mitotic spindles form in cases of polyspermy, where several sperm contribute their 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.
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 acrosome reaction in the sperm, which releases the contents of its acrosomal vesicle, exposing enzymes that help the sperm to digest its way through the zona to the egg plasma membrane in order to fuse with it. The fusion of the sperm with the egg induces a Ca2+ signal in the egg. The Ca2+ signal 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. The Ca2+ signal also triggers the development of the zygote, which begins after sperm and egg haploid pronuclei have come together, and their chromosomes have aligned on a single mitotic spindle, which mediates the first division of the zygote.
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