Fertilization, union of a spermatozoal nucleus, of paternal origin, with an egg nucleus, of maternal origin, to form the primary nucleus of an embryo. In all organisms the essence of fertilization is, in fact, the fusion of the hereditary material of two different sex cells, or gametes, each of which carries half the number of chromosomes typical of the species. The most primitive form of fertilization, found in micro-organisms and protozoans, consists of an exchange of genetic material between two cells.
The first significant event in fertilization is the fusion of the membranes of the two gametes resulting in the formation of a channel that allows the passage of material from one cell to the other. Fertilization in advanced plants is preceded by pollination, during which pollen is transferred to, and establishes contact with, the female gamete or macrospore. Fusion in advanced animals is usually followed by penetration of the egg by a single spermatozoon. The result of fertilization is a cell (zygote) capable of undergoing cell division to form a new individual.
The fusion of two gametes initiates several reactions in the egg. One of these causes a change in the egg membrane(s), so that the attachment of and penetration by more than one spermatozoon cannot occur. In species in which more than one spermatozoon normally enters an egg (polyspermy), only one spermatozoal nucleus actually merges with the egg nucleus. The most important result of fertilization is egg activation, which allows the egg to undergo cell division. Activation, however, does not necessarily require the intervention of a spermatozoon; during parthenogenesis, in which fertilization does not occur, activation of an egg may be accomplished through the intervention of physical and chemical agents. Invertebrates such as aphids, bees, and rotifers normally reproduce by parthenogenesis.
In plants certain chemicals produced by the egg may attract spermatozoa. In animals, with the possible exception of some coelenterates, it appears likely that contact between eggs and spermatozoa depends on random collisions. On the other hand, the gelatinous coats that surround the eggs of many animals exert a trapping action on spermatozoa, thus increasing the chances for successful sperm-egg interaction.
The eggs of marine invertebrates, especially echinoderms, are classical objects for the study of fertilization. These transparent eggs are valuable for studies observing living cells and for biochemical and molecular investigations because the time of fertilization can be accurately fixed, the development of many eggs occurs at about the same rate under suitable conditions, and large quantities of the eggs are obtainable. The eggs of some teleosts and amphibians also have been used with favourable results, and techniques for fertilization of mammalian eggs in the laboratory may allow their use even though only small numbers are available.
Maturation of the egg
Maturation is the final step in the production of functional eggs (oogenesis) that can associate with a spermatozoon and develop a reaction that prevents the entry of more than one spermatozoon; in addition, the cytoplasm of a mature egg can support the changes that lead to fusion of spermatozoal and egg nuclei and initiate embryonic development.
Certain components of an egg’s surface, especially the cortical granules, are associated with a mature condition. Cortical granules of sea urchin eggs, aligned beneath the plasma membrane (thin, soft, pliable layer) of mature eggs, have a diameter of 0.8–1.0 micron (0.0008–0.001 millimetre) and are surrounded by a membrane similar in structure to the plasma membrane surrounding the egg. Cortical granules are formed in a cell component known as a Golgi complex, from which they migrate to the surface of the maturing egg.
The surface of a sea urchin egg has the ability to affect the passage of light unequally in different directions; this property, called birefringence, is an indication that the molecules comprising the surface layers are arranged in a definite way. Since birefringence appears as an egg matures, it is likely that the properties of a mature egg membrane are associated with specific molecular arrangements. A mature egg is able to support the formation of a zygote nucleus; i.e., the result of fusion of spermatozoal and egg nuclei. In most eggs the process of reduction of chromosomal number (meiosis) is not completed prior to fertilization. In such cases the fertilizing spermatozoon remains beneath the egg surface until meiosis in the egg has been completed, after which changes and movements that lead to fusion and the formation of a zygote occur.
The surfaces of most animal eggs are surrounded by envelopes, which may be soft, gelatinous coats (as in echinoderms and some amphibians) or thick membranes (as in fishes, insects, and mammals). In order to reach the egg surface, therefore, spermatozoa must penetrate these envelopes; indeed, spermatozoa contain enzymes (organic catalysts) that break them down. In some cases (e.g., fishes and insects) there is a channel, or micropyle, in the envelope, through which a spermatozoon can reach the egg.
The jelly coats of echinoderm and amphibian eggs consist of complex carbohydrates called sulfated mucopoly-saccharides; it is not yet known if they have a species-specific composition. The envelope of a mammalian egg is more complex. The egg is surrounded by a thick coat composed of a carbohydrate protein complex called zona pellucida. The zona is surrounded by an outer envelope, the corona radiata, which is many cell layers thick and formed by follicle cells adhering to the oocyte before it leaves the ovarian follicle.
Although it once was postulated that the jelly coat of an echinoderm egg contains a substance (fertilizin) thought to have an important role not only in the establishment of sperm-egg interaction but also in egg activation, fertilizin now has been shown identical with jelly-coat material, rather than a substance continuously secreted from it. Yet there is evidence that the egg envelopes do play a role in fertilization; i.e., contact with the egg coat elicits the acrosome reaction (described below) in spermatozoa.
Events of fertilization
The acrosome reaction of spermatozoa is a prerequisite for the association between a spermatozoon and an egg, which occurs through fusion of their plasma membranes. After a spermatozoon comes in contact with an egg, the acrosome, which is a prominence at the anterior tip of the spermatozoa, undergoes a series of well-defined structural changes. A structure within the acrosome, called the acrosomal vesicle, bursts, and the plasma membrane surrounding the spermatozoon fuses at the acrosomal tip with the membrane surrounding the acrosomal vesicle to form an opening. As the opening is formed, the acrosomal granule, which is enclosed within the acrosomal vesicle, disappears. It is thought that dissolution of the granule releases a substance called a lysin, which breaks down the egg envelopes, allowing passage of the spermatozoon to the egg. The acrosomal membrane region opposite the opening adheres to the nuclear envelope of the spermatozoon and forms a shallow outpocketing, which rapidly elongates into a thin tube, the acrosomal tubule that extends to the egg surface and fuses with the egg plasma membrane. The tubule thus formed establishes continuity between the egg and the spermatozoon and provides a way for the spermatozoal nucleus to reach the interior of the egg. Other spermatozoal structures that may be carried within the egg include the midpiece and part of the tail; the spermatozoal plasma membrane and the acrosomal membrane, however, do not reach the interior of the egg. In fact, whole spermatozoa injected into unfertilized eggs cannot elicit the activation reaction or merge with the egg nucleus. As the spermatozoal nucleus is drawn within the egg, the spermatozoal plasma membrane breaks down; at the end of the process, the continuity of the egg plasma membrane is re-established. This description of the process of sperm-egg association, first documented for the acorn worm Saccoglossus (phylum Enteropneusta), generally applies to most eggs studied thus far.
During their passage through the female genital tract of mammals, spermatozoa undergo physiological change, called capacitation, which is a prerequisite for their participation in fertilization; they are able to undergo the acrosome reaction, traverse the egg envelopes, and reach the interior of the egg. Dispersal of cells in the outer egg envelope (corona radiata) is caused by the action of an enzyme (hyaluronidase) that breaks down a substance (hyaluronic acid) binding corona radiata cells together. The enzyme may be contained in the acrosome and released as a result of the acrosome reaction, during passage of the spermatozoon through the corona radiata. The reaction is well advanced by the time a spermatozoon contacts the thick coat surrounding the egg itself (zona pellucida). The pathway of a spermatozoon through the zona pellucida appears to be an oblique slit.
Association of a mammalian spermatozoon with the egg surface occurs along the lateral surface of the spermatozoon, rather than at the tip as in other animals, so that the spermatozoon lies flat on the egg surface; several points of fusion occur between the plasma membranes of the two gametes (i.e., the breakdown of membranes occurs by formation of numerous small vesicles).
Specificity of sperm–egg interaction
Although fertilization is strictly species-specific, very little is known about the molecular basis of such specificity. The egg coats may have a role. Among the echinoderms solutions of the jelly coat clump, or agglutinate, only spermatozoa of their own species. In both echinoderms and amphibians, however, slight damage to an egg surface makes fertilization possible with spermatozoa of different species (heterologous fertilization); this procedure has been used to obtain certain hybrid larvae.
The eggs of ascidians, or sea squirts, members of the chordate subphylum Tunicata, are covered with a thick membrane called a chorion; the space between the chorion and the egg is filled with cells called test cells. The gametes of ascidians, which have both male and female reproductive organs in one animal, mature at the same time; yet self-fertilization does not occur. If the chorion and the test cells are removed, however, not only is fertilization with spermatozoa of different species possible, but self-fertilization also can occur.
Prevention of polyspermy
Most animal eggs are monospermic; i.e., only one spermatozoon is admitted into an egg. In some eggs, protection against the penetration of the egg by more than one spermatozoon (polyspermy) is due to some property of the egg surface; in others, however, the egg envelopes are responsible. The ability of some eggs to develop a polyspermy-preventing reaction depends on a molecular rearrangement of the egg surface that occurs during egg maturation (oogenesis). Although immature sea urchin eggs have the ability to associate with spermatozoa, they also allow multiple penetration; i.e., they are unable to develop a polyspermy-preventing reaction. Since the mature eggs of most animals are fertilized before completion of meiosis and are able to develop a polyspermy-preventing reaction, specific properties of the egg surface must have differentiated by the time meiosis stops, which is when the egg is ready to be fertilized.
In some mammalian eggs defense against polyspermy depends on properties of the zona pellucida; i.e., when a spermatozoon has started to move through the zona, it does not allow the penetration of additional spermatozoa (zona reaction). In other mammals, however, the zona reaction either does not take place or is weak, as indicated by the presence of numerous spermatozoa in the space between the zona and egg surface. In such cases the polyspermy-preventing reaction resides in the egg surface. Although the eggs of some kinds of animals (e.g., some amphibians, birds, reptiles, and sharks) are naturally polyspermic, only one spermatozoal nucleus fuses with an egg nucleus to form a zygote nucleus; all of the other spermatozoa degenerate.
Formation of the fertilization membrane
The most spectacular changes that follow fertilization occur at the egg surface. The best known example, that of the sea urchin egg, is described below. An immediate response to fertilization is the raising of a membrane, called a vitelline membrane, from the egg surface. In the beginning the membrane is very thin; soon, however, it thickens, develops a well-organized molecular structure, and is called the fertilization membrane. At the same time an extensive rearrangement of the molecular structure of the egg surface occurs. The events leading to formation of the fertilization membrane require about one minute.
At the point on the outer surface of the sea urchin egg at which a spermatozoan attaches, the thin vitelline membrane becomes detached. As a result the membranes of the cortical granules come into contact with the inner aspect of the egg’s plasma membrane and fuse with it, the granules open, and their contents are extruded into the perivitelline space; i.e., the space between the egg surface and the raised vitelline membrane. Part of the contents of the granules merge with the vitelline membrane to form the fertilization membrane; if fusion of the contents of the cortical granules with the vitelline membrane is prevented, the membrane remains thin and soft. Another material that also derives from the cortical granules covers the surface of the egg to form a transparent layer, called the hyaline layer, which plays an important role in holding together the cells (blastomeres) formed during division, or cleavage, of the egg. The plasma membrane surrounding a fertilized egg, therefore, is a mosaic structure containing patches of the original plasma membrane of the unfertilized egg and areas derived from membranes of the cortical granules. The events leading to the formation of the fertilization membrane are accompanied by a change of the electric charge across the plasma membrane, referred to as the fertilization potential, and a concurrent outflow of potassium ions (charged particles); both of these phenomena are similar to those that occur in a stimulated nerve fibre. Another effect of fertilization on the plasma membrane of the egg is a several-fold increase in its permeability to various molecules; this change may be the result of the activation of some surface-located membrane transport mechanism.
Formation of the zygote nucleus
After its entry into the egg cytoplasm, the spermatozoal nucleus, now called the male pronucleus, begins to swell, and its chromosomal material disperses and becomes similar in appearance to that of the female pronucleus. Although the membranous envelope surrounding the male pronucleus rapidly disintegrates in the egg, a new envelope promptly forms around it. The male pronucleus, which rotates 180° and moves towards the egg nucleus, initially is accompanied by two structures (centrioles) that function in cell division. After the male and female pronuclei have come into contact, the spermatozoal centrioles give rise to the first cleavage spindle, which precedes division of the fertilized egg. In some cases fusion of the two pronuclei may occur by a process of membrane fusion; in this process, two adjoining membranes fuse at the point of contact to give rise to the continuous nuclear envelope that surrounds the zygote nucleus.
Biochemical analysis of fertilization
Many of the early studies on biochemical changes occurring during fertilization were concerned with the respiratory metabolism of the egg. The results, however, were deceiving; the sea urchin egg, for example, showed an increased rate of oxygen consumption as an immediate response to either fertilization or parthenogenetic activation, in apparent support of the idea that the essence of fertilization is the removal of a respiratory or metabolic block in the unfertilized egg. Extensive comparative studies have shown that the increased rate of oxygen consumption in fertilized sea urchin eggs is not a general rule; indeed, the rate of oxygen consumption of most animal eggs does not change at the time of fertilization and may even temporarily decrease.
At the time of fertilization the egg contains the components required to carry out protein synthesis, and hence development, through an early embryonic stage called the blastula. Most immediate post-fertilization protein synthesis is directed by molecules of ribonucleic acid, known as messenger RNA, that were formed during oogenesis and stored in the egg. In addition, protein synthesis up to the blastula stage (up to a much earlier stage in the mammalian embryo) is directed by the cell components called ribosomes, which are present in the unfertilized egg; new ribosomes, as well as molecules of another type of RNA involved in protein synthesis, and called transfer RNA, are synthesized at a later stage in embryonic development (gastrulation). Eggs fertilized and allowed to develop in the presence of the antibiotic actinomycin, which suppresses RNA synthesis, not only reach the blastula stage but their rate of protein synthesis is the same as that in untreated embryos.
Unfertilized sea urchin eggs, as well as those of other marine animals studied thus far, have a very low rate of protein synthesis, suggesting that something in the unfertilized egg inhibits its protein synthesizing machinery. Since the rate of protein synthesis increases immediately following fertilization, it may depend on some change in, or removal of, an inhibitor. In the sea urchin egg, for example, the low efficiency of the protein synthesizing apparatus apparently depends on certain properties of the ribosomes. Most of the ribosomes found in an unfertilized sea urchin egg are single ribosomes (so-called monosomes); soon after fertilization, however, the single ribosomes interact with messenger RNA molecules thus giving rise to the polyribosomes, which are the active units in protein synthesis. This process also occurs in eggs of a few other marine animals that have been studied. The protein-synthesizing inefficiency of unfertilized sea-urchin-egg ribosomes is caused by an inhibitor that is associated with them and interferes with the binding of messenger RNA molecules to the ribosomes; the inhibitor is removed almost immediately following fertilization, perhaps by enzymatic breakdown.
It thus appears that at least in the sea urchin egg the overall rate of protein synthesis is controlled at the ribosome level and that the first step in the activation of protein synthesis following fertilization is the “turning on” of the ribosomes.
In vertebrates such as amphibians, activation of protein synthesis takes place at the onset of egg maturation, apparently initiated by the action of a hormone, progesterone. The effect of progesterone is not mediated by the nucleus but is a direct effect on the cytoplasm.