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 coatthe 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 56 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).

Once a capacitated sperm has penetrated the layer of follicle cells, it binds to the zona pellucida (see Figure 20-21). 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).

The zona pellucida of mammalian eggs is composed mainly of three glycoproteins, all of which are produced exclusively by the growing oocyte. Two of them, ZP2 and ZP3, assemble into long filaments, while the other, ZP1, cross-links the filaments into a three-dimensional network. The protein ZP3 is crucial: female mice with an inactivated ZP3 gene produce eggs lacking a zona and are infertile. ZP3 is responsible for the species-specific binding of sperm to the zona, at least in mice. Several proteins on the sperm surface that bind to specific O-linked oligosaccharides on ZP3 have been implicated as ZP3 receptors, but the contribution of each is uncertain. On binding to the zona, the sperm is induced to undergo the acrosome reaction, in which the contents of the acrosome are released by exocytosis (Figure 20-31). 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 spermegg 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 spermegg 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 fusesa condition called polyspermymultipolar 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 Ca²⁺ wave or oscillations activate the egg to begin development, and they initiate the cortical reaction, in which the cortical granules release their contents by exocytosis. If the cytosolic concentration of Ca²⁺ is increased artificiallyeither 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 contents of the cortical granules include various enzymes that are released by the cortical reaction and change the structure of the zona pellucida. The altered zona becomes "hardened," so that sperm no longer bind to it, and it therefore provides a 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 20-32).

The Mechanism of SpermEgg Fusion Is Still Unknown

After a sperm has penetrated the extracellular coat of the egg, it interacts with the egg plasma membrane overlying the tips of microvilli on the egg surface (see Figure 20-30). 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.

Fertilin is composed of two glycosylated transmembrane subunits called a and b, which are held together by noncovalent bonds (Figure 20-33). 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 proteinsso-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 a 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 a chain can induce membrane fusion in a test-tube, consistent with the possibility that fertilin helps to mediate spermegg 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.

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 has broken down in preparation for the zygote's first mitotic division (Figure 20-34).

In most animals, including humans, the sperm contributes more than DNA to the zygote. It also donates a centriolean organelle that is lacking in unfertilized human eggs. The sperm centriole enters the egg along with the sperm nucleus and tail and a centrosome forms around it. In humans, it replicates and helps organize the assembly of the first mitotic spindle in the zygote (Figure 20-35). 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 biologythe 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.