Fertilization

FERTILIZATION, the union of an egg with a spermatozoon, characterizes sexual reproduction, which represents one of the most common methods of reproduction in nature. The phenomena of conjugation (see) in protozoa and the exchange of nuclei occurring during this process should already be considered as a prototype of those phenomena observed during fertilization in higher animals and plants. This especially applies to the phenomenon of gamete copulation, in which both gametes can be identical in size (isogametes) or different (heterogametes). A new organism develops from such a cell formed by the fusion of two gametes, an example of which can be seen especially in algae. In the case of heterogamy, we have a picture very reminiscent of the phenomena of fertilization, wherein one of the gametes (microgamete) is, as it were, a prototype of the spermatozoon, while the other (macrogamete), overloaded with nutrients, largely resembles an egg. There exists a huge variety of forms of sexual reproduction ensuring the meeting of the egg and the spermatozoon. In cases where the union of these cells is carried out inside the female's body, one speaks of internal fertilization; otherwise, of external fertilization. Thus, in many amphibians (axolotl, etc.), spermatophores serve the purposes of fertilization; these are deposited by the male and then taken up by the female through the cloaca into the body, where fertilization occurs. Many marine and freshwater animals lay eggs and sperm into the surrounding environment, where the meeting and union of the germ cells take place. No less diverse, especially in plants, are the adaptations preventing the meeting of the egg with the spermatozoon in cases where the latter originate from the same individual. However, autosterility in animals and plants is a far from universal phenomenon, and self-fertilization is frequently observed in various hermaphroditic species (worms, snails, higher plants). In the practice of animal husbandry and plant cultivation, methods of artificial fertilization (see Insemination) are frequently resorted to, which allows for the selection of appropriate sires and provides a number of other practical advantages. Methods of artificial fertilization are diverse and hinge on the problem of sperm conservation, protection against self-fertilization (in plants), etc. The meeting of the egg with the spermatozoon and their subsequent union is usually carried out with great speed. The surface of the egg is soon strewn with a multitude of spermatozoa, which in this process largely lose their characteristic motility. The penetration of the spermatozoon into the egg in many species is carried out through a tiny opening in the egg—the micropyle. However, cases are known where the spermatozoon penetrates the egg bypassing the micropyle. In other animals (Nereis, etc.), a small conical section of the egg—the receptive cone—stretches out toward the spermatozoon, which, as it were, swallows the spermatozoon and then retracts back into the egg. However, the most common occurrence is apparently the passage of the spermatozoon through the undifferentiated membrane of the egg, and thus the site of the spermatozoon's entry into the egg is, for the most part, impossible to predict in advance. Often the entire spermatozoon penetrates the egg; sometimes only its head (nucleus) together with the cytoplasmic part containing the centrosome (and sometimes mitochondria); however, cases are also known where only the head of the spermatozoon penetrates the egg. Thus, the most important role in the processes unfolding in the egg after the introduction of the spermatozoon can be attributed to the nucleus of the latter and the centrosome. These processes are quite thoroughly described for many animal species. The head of the spermatozoon, having penetrated inside the egg, turns 180° and moves further into the egg with the centrosome in front. The path of the spermatozoon through the egg protoplasm until it meets the egg nucleus has the appearance of a curve, which can be decomposed into two component straight lines, often diverging from each other at a large angle. One of them bears the name of the path of penetration and is marked, for example, in the frog by a strip of pigment granules moving from the periphery of the egg. The path of penetration is for the most part directed more or less radially, i.e., toward the center of the egg. The other component part of the spermatozoon's trajectory bears the name of the path of copulation. The egg nucleus usually remains at rest until the direction of the path of copulation is indicated, and only after this does the migration of the egg nucleus to a specific place in the protoplasm begin, where the union or copulation of both nuclei occurs. However, the amphikaryon (see), or cleavage nucleus, formed from the fusion of the male and female pronuclei, also often performs a certain path in the egg, bearing the name of the path of cleavage, since the localization of the amphikaryon determines the direction of the first plane of cleavage of the egg. The causes of the described nuclear migrations are unknown. It is possible that influences from the egg cytoplasm play a role here in part, and in part the mutual attraction of the male and female pronuclei. The nature of the changes undergone by the spermatozoon and the egg also depends on the degree of maturity of the egg. In cases where the maturation processes (see Ovulation) have not yet finished in the egg by the moment of the spermatozoon's introduction, the spermatozoon provides the impetus for the formation of both polar or directive bodies (or the second one, if the first has already been expelled before fertilization, e.g., in Amphioxus), and only after this do the processes described above occur. In connection with this, the spermatozoon also manages to undergo more drastic changes before fusion with the female pronucleus—its head swells significantly, usually reaching the size of the egg nucleus. The fate of the centrosome introduced by the spermatozoon has been clarified for many cases. It divides in two and gives rise to the asters of the first cleavage spindle. However, in some cases, the latter are formed anew, possibly around the original centers. In any case, it can be considered almost indisputable that the centrosome of the spermatozoon plays the primary role in the formation of the cleavage spindle asters, and this fact (see below) served as the basis for one of the theories of fertilization. The other component part of the spermatozoon's neck—the mitochondria—can likewise penetrate inside the egg and, during its subsequent cleavages, pass into individual blastomeres. However, it is apparently impossible to attribute any special role to the mitochondria either in the phenomenon of fertilization or in the transmission of hereditary properties. The introduction of the spermatozoon into the egg is accompanied by a number of other changes in the egg besides those described, of which the formation of the fertilization membrane is of the greatest interest. According to some authors (see below), the fertilization membrane prevents the introduction of other spermatozoa into the egg and thus ensures monospermy (see below). The formation of the membrane occurs very soon after the introduction of the spermatozoon, and in the sea urchin, for example, the entire surface of the egg is covered with small droplets, which, mixing with each other, form larger and larger droplets and finally a continuous, rather voluminous membrane. Usually, the formation of the membrane begins at the site of the spermatozoon's introduction and from there spreads further over the entire surface of the egg. There are indications that in connection with the secretion of the membrane, a transparent layer forms on the surface of the egg beneath it, which sometimes allows the egg (e.g., a frog's) to rotate freely around its axis. The formation of the fertilization membrane is a quite reliable sign of fertilization having occurred. Another sign can be the aforementioned rotation of the egg (frog) with the animal (containing black pigment) pole upward. The nature of the forces ensuring the meeting and subsequent union of the egg with the spermatozoon has not been sufficiently clarified. A certain role is attributed to the chemotaxis (see) of spermatozoa, which are attractively acted upon by certain chemical substances (malic acid, etc.). This phenomenon especially characterizes the spermatozoa of plants (mosses, ferns). However, the theory of the 'physiological trap' should be recognized as more correct. According to this concept, spermatozoa accidentally approach the surface of the egg during their active movement, after which they to a significant extent lose their activity under the influence of either substances secreted by the egg or due to their characteristic positive thigmotaxis, i.e., the tendency to attach to a solid substrate. Some are inclined to explain the further penetration of the spermatozoon into the egg, given its reduced motility, by the activity of the egg itself, which is especially clearly visible in the example of the participation of the receptive cone in the process of fertilization (see above). Frank Lillie attributes the primary role in the process of fertilization to fertilizin—a hypothetical substance contained in infusions of mature unfertilized eggs in seawater. Fertilizin enhances the motility of spermatozoa and subsequently causes their agglutination. Lillie believes that fertilizin acts in an attractive manner on spermatozoa. Not limiting himself to this highly controversial role of fertilizin, Lillie constructed an entire theory of fertilization based on the existence of fertilizin (see below). Proceeding from the theory of fertilizin, Lillie denies the role of the fertilization membrane in ensuring monospermy. He asserts that the fertilized egg loses the ability to secrete fertilizin outward, and by this explains the impossibility of secondary fertilization of the egg.

Along with normal monospermy, in some cases di- and polyspermy are observed, which sometimes represent a normal phenomenon (selachians, etc.), but are mostly pathological. Of several spermatozoa that have penetrated the egg, usually only one fuses with the female pronucleus, however, the others also form cleavage figures, splitting the egg simultaneously into several blastomeres. In connection with this, the uniform distribution of the constituent parts in the egg and the normal course of its development are disturbed. Di- and polyspermic eggs usually die quickly. Phenomena of partial fertilization in those cases where the spermatozoon does not penetrate inside the egg (heterogeneous crossing), but only touches its surface, deserve attention. In these cases, the formation of a fertilization membrane occurs, and inside the egg—some preparatory processes of cleavage. However, as a rule, the development of the egg stops at this point. A similar phenomenon is observed when an unfertilized egg is exposed to certain artificially parthenogenetic agents (see Parthenogenesis). Finally, the phenomena of merogony are no less important in theoretical terms. Here, the spermatozoon penetrates an egg cell that has been artificially deprived of its nucleus, and yet the development of the egg is practically not disturbed. Experiments of this kind serve to clarify the comparative role of the male and female nucleus in the transmission of hereditary traits to offspring. They also play a certain role in theories of fertilization (see below). Chronologically, the first scientific theory of fertilization can be considered the theory of the Hertwig brothers (O. and R. Hertwig). This is the theory of amphimixis (see), or nuclear fusion, according to which the essence of fertilization consists in the fusion of the male and female pronuclei, which ensures the transmission of the traits of both parents to the offspring. While not denying the enormous significance of this phenomenon for the phenomena of heredity, one cannot, however, ascribe the primary significance in the mechanism of the fertilization process to the fusion of nuclei. The very fact of merogony (see above) shows that the activation of the egg can occur even in the presence of only one of the nuclei (the female one). Experiments in artificial parthenogenesis show, on the other hand, that the presence of the male pronucleus is likewise not mandatory for the activation of the egg. Another theory, also morphological, belongs to Boveri. He proceeds from the fact that the egg is normally devoid of a centrosome, whereas in the absence of the latter, cleavage of the egg is impossible. Boveri relies on the carefully studied behavior of the seminal centrosome in the egg. Even Fol, proceeding from the incorrect assumption about the existence of the egg's own division centers, described the so-called "quadrille of centers"—the pairwise fusion of seminal and egg centrosomes, similar to the fusion of pronuclei. However, subsequent studies have established with certainty the absence of a centrosome in the egg, and relying on this fact, as well as his own careful observations, Boveri constructed his theory. In his opinion, the egg contains everything necessary for its development, and it lacks only a centrosome. Boveri relied on observations proving the origin of the cleavage centrosome from the spermatid (this, however, cannot always be proven), on the independent behavior of the centrosome in the egg, on experiments of merogony, on experiments of one of the types of partial fertilization, in which the seminal aster and centrosome can lead an existence independent of the nucleus and cause cleavage of the egg before the fusion of both nuclei, etc. However, in light of new studies that have shown the possibility of the emergence of centers in the egg de novo under the influence of physical-chemical agents, and especially in connection with experiments of artificial parthenogenesis, the significance of the centrosome theory has fallen sharply, and at the present time it finds almost no adherents. Somewhat similar to the centrosome theory in its basic concept is the theory of fertilizin. Both proceed from the premise that a mature egg is a system developing at the expense of its own resources, and that it lacks only an insignificant factor: in one case—the centrosome, in the other—those substances that enter into combination with the fertilizin of the egg. This factor is introduced into the egg by the spermatozoon. The egg and the spermatozoon, according to Lillie, contain: the former—an ovophilic group, the latter—a spermophilic group, which combine with a particle of fertilizin, which plays the role of an amboceptor. In addition, there is also an antifertilizin, which combines with the free particles of fertilizin remaining after fertilization. The "state of fertilization" is characterized, according to Lillie, by the combination of the indicated substances. On the basis of this scheme, Lillie explains a whole range of phenomena: monospermy, specificity in fertilization (the impossibility of heterogeneous crossing), activation and agglutination of spermatozoa, etc. However, Lillie's scheme cannot claim either universality or, even less, reliability, since it is based on few facts (agglutination and activation of sperm by egg water), which can receive a different explanation. We still do not have a complete theory of fertilization, and only recently has it been possible to discover some individual aspects of this process with the help of physical-chemical methods. In this direction, many extremely interesting observations have been made, which deserve separate consideration. Very original are the observations of Chambers, obtained by the application of a delicate micro-manipulation technique. By isolating the cortical layer of the egg from its endoplasm, Chambers showed that endoplasm deprived of the cortical layer is incapable of forming a fertilization membrane. The basic processes of fertilization, therefore, play out in the surface cortical layer of the egg. In connection with this, studies clarifying the mechanism of the formation of the fertilization membrane are very important. At the present time, the majority of researchers incline to the opinion that the fertilization membrane is not formed anew, but pre-exists in the unfertilized egg and only detaches and swells during fertilization. This process, according to Peterfi, is connected with the swelling and subsequent contraction of the egg, which releases water and the hyaline layer located under the membrane, which causes its detachment. The swelling and subsequent contraction of the egg during fertilization, according to Peterfi, is connected with the thixotropic properties of the egg's colloids. These reversible changes in the egg's colloids are caused either by the mechanical action of the motile spermatozoon or by substances secreted by it. The changes in the egg's colloids are indicated by the sharply defined changes in the viscosity of the egg that occur after fertilization. The very appearance of the radiate aster in the egg indicates the existence of more liquid areas of protoplasm (rays) and denser ones (the spaces between them), as shown by Chambers' observations made with the help of a microneedle. More precise data from Heilbrunn, obtained by the method of centrifuging the egg, showed that the viscosity of the egg after fertilization increases by 2–3 times, and sometimes even by 6–8 times. This increase in the viscosity of the egg precedes its cleavage. During the formation of the spindle, it is small and increases only just before cleavage, after which it falls again. In the period between fertilization and the first cleavage of the egg, there also occurs a noticeable increase in the permeability of the egg, which can be proven by the penetration of various substances into it or by the method of swelling in a hypotonic medium. In connection with this, it is important to note the intensification of oxidative processes in the egg, which occurs after fertilization (according to Warburg—several times over). Finally, recent observations show that this burst of oxidative processes in the fertilized egg is accompanied by a burst of mitogenetic radiation. Thus, in the period between fertilization and the first cleavage of the egg, a certain "critical" period of its life occurs, characterized by the physical-chemical and chemical changes described above. The theory of fertilization will become complete only when these processes are studied in more detail. Let us also note that the study of these phenomena simultaneously sheds light on the mechanism of cell division in general, and conversely—the investigation of the latter allows one to a certain extent to conclude about the mechanism of the process of fertilization. Fertilization, artificial—see Insemination.