Book - Comparative Embryology of the Vertebrates 2-5

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Nelsen OE. Comparative embryology of the vertebrates (1953) Mcgraw-Hill Book Company, New York.

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Part II - The Period of Fertilization

Part II - The Period of Fertilization: 4. Transportation of the Gametes (Sperm and Egg) from the Germ Glands to the Site where Fertilization Normally Occurs | 5. Fertilization


A. Definition of Fertilization

The union or fusion (syngamy) of the oocyte or egg (female gamete) with the sperm (male gamete) to form a zygote is known as fertilization. From this zvgotic fusion the new individual arises^ Strictly speaking, the word fertilization denotes the process of making the egg fruitful (i.e., develop) by means of the sperm's contact with the egg, and as such may not always imply a fusion of the sperm with the egg. In certain types of hybrid crosses, such as in the toad egg (Bufo) inseminated with urodele sperm (Triton), egg activation may occur without fusion of the sperm nucleus with the egg nucleus. Ordinarily, however, the word fertilization denotes a fusion of the two gametes (see Wilson, '25, pp. 460-461).

The word zygote is derived from a basic Greek word which means to join or yoke together) The word is particularly appropriate in reference to the behavior of the nuclei of the two gametes during fertilization. (For, during gametic union, the haploid group of chromosomes from one gamete is added to the haploid group from the other, restoring the diploid or normal number of chromosomes^^ln most instances, each chromosome from one gamete has a mate or homologue composed of similar genes in the other gamete. Therefore, the union of the two haploid groups of chromosomes forms an integrated association in which pairs of similar genes are yoked together to perform their functions in the development of the new individual.

In most animal species aside from the union of the chromosome groups, there is a coalescence of most of the cytoplasm of the male gamete with that of the female gamete as the entire sperm generally enters the egg (figs. 115, 118), However, in some species the tail of the sperm may be left out, e.g., rabbit, starfish, and sea urchin, while in the marine annelid. Nereis, the head of the sperm alone enters, the middle piece and tail being left behind.

The morphological fusion of the two sets of nucleoplasms and cytoplasms of the gametes during fertilization is made possible by certain physiological changes which accompany the fusion process. These changes begin the instant that a sperm makes intimate contact with the surface of the oocyte (or egg). As a result, important ooplasmic activities are aroused within the egg which not only draw the sperm into the ooplasm but also set in motion the physicochemical machinery which starts normal development. The initiation of normal development results from the complete activation of the egg. Partial activation of the egg is possible, and in these instances, various degrees of development occur which are more or less abnormal. Partial activation of the egg happens in most instances when the various methods of artificial activation (see p. 217) are employed

While the main processes of activation leading to development are concerned with the organization and substances of the egg, one should not overlook the fact thatTr/ie sperm also is activated (and in a sense, is fertilized) during the fusion process. Sperm activation is composed of two distinct phases:

( 1 ) Before the sperm makes contact with the oocyte or egg, it is aroused by environmental factors to swim and move in a directed manner and is attracted to the oocyte or egg by certain chemical substances secreted by the latter; and

( 2 ) after its entrance into the egg's substance, the sperm nucleus begins to enlarge and its chromosomes undergo changes which make it possible for them to associate with the egg chromosomes in the first cleavage spindle. Also, the first cleavage amphiaster in the majority of animal species appears to arise within the substance of the middle piece of the sperm as a result of ooplasmic stimulation^

In the process of normal fertilization it is clear, therefore, that two main conditions are satisfied:

(1) There is a union of two haploid chromosome groups, one male and the other female, bringing about the restoration of a proper diploid genic balance; and

(2) an activation of the substances of the fused gametes, both cytoplasmic and nuclear, is effected, resulting in the initiation of normal development.

The biochemical and physiological factors which accomplish the union of the haploid chromosome groups and the activation of the gametes are the objectives of one of the main facets of embryological investigation today.

B. Historical Considerations Concerning Gametic Fusion and Its Significance

The use of the word "fertilization" in the sense of initiating development and the idea of making fertile or fruitful, which the word arouses in one's mind, reaches back to the dawn of recorded history. (The concept of this fruitfulness as being dependent upon the union of one sex cell with another sex cell and of the fusion of the two to initiate the development of a new individual originated in the nineteenth century. However, Leeuwenhoek, in 1683, appears to have been the first to advance the thesis that the egg must be impregnated by a seminal animalcule (i.e., the sperm) in order to become fruitfi^ but the real significance of this statement certainly was not appreciated by him.

Moreover, to Leeuwenhoek, the idea behind the penetration of the egg by the seminal animalcula was to supply nourishment for the latter, which he believed was the essential element in that it contained the preformed embryo in an intangible way. That is, the sperm animalcule of the ram contains a lamb, which does not assume the external appearance of one until it has been nourished and grown in the uterus of the female (Cole, '30, pp. 57, 165). It should be added parenthetically that actual presence of the little animalcules as living entities had previously been called to Leeuwenhoek's attention in 1677 by a Mr. Ham (Cole, '30, p. 10).

In the years that followed Leeuwenhoek the exact interpretation to be applied to the seminal animalcules (sperm) was a matter of much debate. Many maintained that they were parasites in the seminal fluid, the latter being regarded as the essential fertilizing substance in the male semen.An 1827, von Baer, who regarded the sperm as parasites, named them spermatozoa, that is, parasitic animals in the spermatic flui^ (Cole, '30, p. 28). Finally, in the years from 1835-1841, Peltier, Wagner, Lallemand, and Kolliker, established the non-parasitic nature of the sperm.(^Kolliker in 1841 traced their origin from testicular tissue^) and thus settled the argument once and for all as to the true nature of the seminal animals or sperm.

Various individuals have laid claim to the honor of being the first to describe the sperm's entry into the egg at fertilization, but the studies of Newport and Bischoff (1853, 1854) resulted in the first exact descriptions of the process. (See Cole, '30, pp. 191-195.) Thus the general proposition set forth by Leeuwenhoek 170 years earlier became an accepted fact, although the illumination of the details of sperm and egg behavior during fertilization really began with the studies of O. Hertwig in 1875. The more important studies which have shed light upon the problems involved in gametic fusion are presented below:

(1) O. Hertwig, 1875, 1877, in the former paper, described the fusion of the egg and sperm pronuclei in the Mediterranean sea urchin, Toxopneustes lividus. One aspect of the work published in 1877 was concerned with the formation of the polar bodies in Haemopis and Nephelis. In a part of the latter publication O. Hertwig presented descriptions of sperm migration from the periphery of the egg and the ultimate association of the sperm and egg pronuclei during the fertilization in the frog, Rana temporaria (fig. 1191, J).

(2) Fol, 1879, contributed detailed information relative to the actual entrance of the sperm into the sea-urchin egg and showed that in the eggs of various animal species only one sperm normally enters. He also described the formation of the fertilization membrane in the egg of the sea urchin, Toxopneustes lividus.

(3) Mark, 1881, made important contributions relative to the formation of the polar bodies in the slug, Deroceras laeve (Umax campestris). He also presented information which showed that the egg and sperm pronuclei, although associated near the center of the egg during fertilization, do not actually form a fusion nucleus in this species as described for the sea urchin by O. Hertwig. This is an important contribution to the fertilization problem, as fusion nuclei are not formed in all animal species.

(4) Van Beneden, 1883, in his studies on maturation of the egg and fertilization in A scans megalocephala, demonstrated that half of the chromatin material of the egg nucleus was discharged in the maturation divisions. (He erroneously thought, however, that the female ejected the male chromosomes at this time, and in the male, the reverse process occurred.) (See fig. 133C, D.) He demonstrated also that the two pronuclei in Ascaris do not join to form a fusion nucleus at fertilization. His work revealed further that the male and female pronuclei each contributes the haploid or half the normal number of chromosomes at fertilization and that each haploid group of chromosomes enters the equatorial plate of the first cleavage spindle as an independent unit (fig. 133F-1). Upon the equatorial plate each chromosome divides and contributes one chromosome to each of the two daughter nuclei resulting from the first cleavage division. This contribution of the haploid number (half the typical, somatic number) of chromosomes from each parent Van Beneden assumed to be a fundamental principle of the fertilization process. This principle was definitely established by later workers and it has become known as Van Beneden's Law.

(5) Boveri, 1887 and the following years, further established the fact of Van Beneden's Law and also demonstrated that half of the chromosomes of the cells derived from the zygote are maternal and half are paternal in origin. (Fig. 133 is derived from Boveri's study of Ascaris.) In '00 and '05 he emphasized the importance of the centrosome and centrioles and presented the theory that the centrosome contributed by the sperm to the egg at the time of fertilization constituted the dynamic center of division which the egg lacked; hence, it was a causal factor in development. This latter concept added new thinking to the fertilization problem, for O. Hertwig, 1875, had suggested that the activation of the egg was due to the fusion of the egg and sperm nuclei. The centrosome theory of Boveri eventually became one of the foremost theories of egg activation (see end of chapter).

(6) During the last five years of the nineteenth century, intensive studies on artificial activation of the egg (artificial parthenogenesis) were initiated. This matter is discussed on page 217 in the section dealing with artificial activation.

(7) Another attack on the problem of fertilization and its meaning had its origin in the “idioplasm theory” of Nageli. This theory (1884) postulated an “idioplasm” carried by the germ cells which formed the essential physical basis of heredity. A little later O. Hertwig, Kolliker, and especially Weismann, identified the idioplasm of Nageli with the chromatin of the nucleus. In the meantime, Roux emphasized the importance of the chromatin threads of the nucleus and stated that the division of these threads by longitudinal splitting (separation) during mitosis implied that different longitudinal areas of these threads embodied different qualities, (See Wilson, '25, p. 500.) In harmony with the foregoing ideology and as a result of his own intensive work on maturation and fertilization in Ascaris and also upon other forms, Boveri came to the conclusion in 1902 and 1907 (Wilson, '25, p. 916) that development was dependent upon the chromosomes and further that the individual chromosomes possessed different qualities.

(8) As a result, the field of biological ideas was at this time well plowed and ready for another important suggestion. This came in '01 and '02 when McClung offered the view that the accessory chromosome described by Henking (1891) as the x-chromatin-element or nucleolus was, in the germ cell of the male grasshopper, a sex chromosome which carried factors involved in the determination of sex. McClung first made this suggestion and stated a definite hypothesis, immediately stimulating work by others; in a few years McClung's original suggestion was well rounded out and the complete cycle of sex chromosomes in the life history was formulated. E. B. Wilson led this work, and the theory that he formulated became the assured basis of cytological and genetical sex studies constituting one of the greatest present day advances in zoology. “McClung's anticipation of this theory is a striking example of scientific imagination applied to painstaking observation” (Lillie, F. R., '40).

Not only were the sex chromosomes studied, but other chromosomes as well, and an intense series of genetical investigations were initiated by Morgan and his students and others which succeeded in tying a large number of hereditary traits to individual chromosomes and also to definite areas or parts of the chromosomes. Thus the assumptions of Roux and Boveri were amply demonstrated. Moreover, these observations established experimental proof for the concept that in the gametic fusion which occurs during fertilization, the chromosomes pass from one generation to the next as individual entities, carrying the hereditary substances from the parents to the offspring. The heredity of the individual was in this way demonstrated to be intimately associated with the reunion of the haploid groups of chromosomes in the fertilization process.

C. Types of Egg Activation

1. Natural Activation of the Egg

(^Natural parthenogenesis, i.e., the development of the egg spontaneously without fertilization was suggested by Goedart, in 1667, for the moth, Orgyia gnastigma, and by Bonnet, in 1745, in his study of reproduction in the aphid.' (See Morgan, '27, p. 538.) Since this discovery by Goedart and Bonnet, many observations and cytological studies have shown thatflhere are two kinds of eggs which are capable of natural parthenogenesis:

  1. That which occurs in the so-called, non-sexual egg, i.e., the egg which has not undergone the maturation divisions and, hence, has the diploid number of chromosomes; and
  2. that which results in the sexual egg, i.e., the egg which has experienced meiosis (Chap. 3 ) and thus has the reduced or haploid number of chromosome (Sharp, '34, pp. 409, 410).

Parthenogenesis from a non-sexual egg is found in daphnids, aphids, flatworms, and certain orthopterans. In the case of the sexual egg, parthenogenesis normally occurs in bees, wasps, ants, some true bugs, grasshoppers, and arachnids. Jn this type of egg, development may result with or without fertilization i^For example, in the honeybee. Apis mellifica, haploid males arise from eggs which are not fertilized, workers and queens from fertilized eggs.

Extensive studies of the animal kingdom as a whole have demonstrated, however(^that the majority of oocytes or eggs depend upon the fertilization process for activation. Consequently, eggs may be regarded in the following light :fSome eggs are self-activating and develop spontaneously, while others require sperm activation before development is initiat^ However, the differences between these two types of eggs may not be as real as it appears, for it is probable that subtle changes in the environment of the so-called selfactivating or parthenogenetic eggs are sufficient to activate them, whereas in those eggs which require fertilization a strong, abrupt, stimulus is requisite to extricate them from their blocked condition and to start development. This idea is enhanced by the information obtained from the methods employed in the studies on artificial activation of the egg of different animal species.

2. Artificial Activation of the Egg

a. Object of Studies in Artificial Parthenogenesis

“The ultimate goal in the study of artificial parthenogenesis is the discovery of the chemical and physical forces which are assumed to cause the initiation of development” (Heilbrunn, T3). A brief resume of some of the results obtained in the studies on artificial activation of the egg is considered in the following paragraphs.

b. Some of the Procedures Used in Artificial Activation of the Egg

Ichomiroff, 1885 (Morgan, '27, p. 538), stated that eggs from virgin silkworm moths could be activated by rubbing or by treatment with sulfuric acid. Somewhat later Mead, 1896-1897, published results of studies on artificial parthenogenesis in the annelid worm, Chaetopterus. The egg of this worm has the germinal vesicle intact when it is deposited in sea water. Almost immediately after entrance into sea water, the germinal vesicle breaks down, and the chromatin proceeds to form the spindle for the first maturation division. At this point, however, it stops and awaits the entrance of the sperm for further activation. Thus, the immersion of the Chaetopterus egg in sea water under normal spawning conditions partially activates the egg' (Mead, 1897). Mead attempted by artificial means to complete this activation initiated by the sea water. In doing so, he took eggs from normal sea water, which thus had the first polar spindle, and placed them in sea water to which Va to Vi per cent of potassium chloride had been added. Eggs thus treated proceeded to form normal polar bodies and the beginnings of the first cleavage occurred. Development ceased, however, at this point. These eggs were further activated, but not completely activated. Two steps in partial activation are here demonstrated:

  1. When the egg is spawned into sea water, the nuclear membrane breaks down and the first polar spindle is formed; and
  2. by the immersion in hypertonic sea water the first and second maturation divisions occur, and the first cleavage begins.

This experiment by Mead was one of a number of pioneering studies made during this period in an endeavor to activate artificially the egg. Another such experiment was reported by R. Hertwig (1896), using eggs of the sea urchin. In a strychnine solution the nucleus underwent changes preparatory to the first division. Also, Morgan (1896) found it possible to produce cleavage, normal and abnormal, if unfertilized eggs of the sea urchin, Arbacia, were placed in sea water to which certain amounts of sodium chloride had been added and then were returned to normal sea water. Morgan ('00) later found that magnesium chloride added to sea water induced cleavage in eggs treated for various intervals. Loeb, in 1899 (see Loeb, '06), initiated a series of experiments on activation of the sea-urchin egg. As a result of many similar experiments, Loeb reached the conclusion that membrane formation was the essential part of the activation process in that it stimulates the formation of the membrane by initiating cytolysis of the egg (see Loeb's theory at end of chapter). Consequently, he sought substances which would elicit membrane formation and found that monobasic fatty acids, such as butyric, acetic, formic, or valerianic, would produce membrane formation, and, also, that ether, bile salts, saponin, etc., would do the same. However, although these substances aroused certain initial activities of the egg, Loeb found it necessary to apply a so-called corrective treatment of hypertonic sea water to arrest the cytolytic effect of the first treatment, which, according to his belief, restored respiration to its normal level. As a result, Loeb perfected a treatment as follows which produced development in a considerable number of sea-urchin eggs so exposed (Loeb, '06):

  1. Unfertilized eggs were placed for Vi to IVi min. in 50 cc. of sea water to which 3 cc. of N/10 solution of butyric or other monobasic fatty acid had been added. This treatment effected membrane formation when the eggs were returned to normal sea water, provided the eggs had been exposed long enough to the acid.
  2. The eggs were allowed to remain in normal sea water for 5 to 10 min. and then were subjected to the corrective treatment in hypertonic sea water, made by adding 15 cc. of 2 Vi N. NaCl solution to 100 cc. normal sea water, and allowed to remain for 20 to 50 min. Lesser times of exposures also were used successfully.
  3. Following this treatment, the eggs were returned to normal sea water.

An example of an entirely different method from the solution technics employed above on the sea-urchin egg is that of Guyer ('07) and especially Bataillon ('10, 'll, '13) on the egg of the frog. The method employed by the latter with success was as follows: Eggs were punctured with a fine glass or platinum needle and then covered for a time with frog blood. Puncturing alone is not sufficient; a second factor present in the blood is necessary for successful parthenogenetic development. The number of actual developments procured by this method is small, however. In many cases an early cleavage or larval stage is reached, but the advanced tadpole state or that of the fully developed frog is quite rare.

The method introduced by Bataillon is still used extensively in studies on artificial parthenogenesis in the frog. Recently, Shaver ('49) finds that the “second factor” is present on certain cytoplasmic granules obtained by centrifugal fractionation. Heat at 60° C. and the enzyme, ribonuclease, destroy the second-factor activity. Successful second-factor granules were obtained from blood, early frog embryos, and “extracts of testis, brain, lung, muscle and liver.” This author also reports that heparin suppresses parthenogenetic cleavage.

In some of these parthenogenetically stimulated eggs of the frog, the diploid chromosome relationships appear to be restored during early cleavage; in others both diploid and triploid cells may be present. Some of these tadpoles may be completely triploid (Parmenter, '33, '40). However, a large percentage remain in the haploid condition (Parmenter, '33).

A third method of approach in stimulating parthenogenetic development was used by Pincus ('39) and Pincus and Shapiro ('40) on the rabbit. In the former work, Pincus reports the successful birth of young from tubal eggs activated by exposure to a temperature of 47° C. for three minutes. The treated eggs were transplanted into the oviducts of pseudopregnant females. In the latter work, eggs were exposed to a cooling temperature in vivo, that is, the eggs were allowed to remain in the Fallopian tube during exposure to cold. The birth of one living female was reported from such parthenogenetic stimulation.

The foregoing experiments illustrate three different procedures used on three widely separated animal species, namely, changing the external chemical environment of the egg, a tearing or injuring of the egg's surface followed by the application of substances obtained from living tissues, and, finally, changing the physical environment of the egg. To these three general approaches may be added that of mechanical shaking. For example, Mathews ('01 ) states that mechanical shaking of the eggs of the starfish, Asterias jorbesi, results in the development of a small percentage of eggs to the free-swimming blastula stage.

Some of the recent work on the initiation of development and in stimulating cells to divide has emphasized the importance of cellular injury as a factor. Little is known concerning the mode of action of the injuring substances. Harding ('51) concludes that an acid substance is released as the result of “injury” and that this acid substance causes “an increase in protoplasmic viscosity and initiates cell division” in the sea-urchin egg. (Cf. theory of R. S. Lillie at end of chapter.)

That no single method has been found which activates eggs in general is not surprising. The eggs of different species are not only in different states of maturation (i.e., development) when normally fertilized (fig. 137), but they behave differently during the normal fertilization process. In some eggs, such as the egg of Chaetopterus, there is only a slight change within the egg cortex during fertilization, whereas in the egg of the teleost fish, the egg of the frog, or in the egg of the urochordate, Styela, marked cortical changes involving mass movements of protoplasmic materials can be demonstrated.

c. Results Obtained by the Work on Artificial Parthenogenesis

The question naturally arises: What has the work on artificial activation of the egg contributed to the solution of the problems involved in egg activation? It has not, of course, solved the problem, but it has contributed much toward a better understanding of the processes concerned with egg activation and of the general problem of growth stimulation including cell division. We may summarize the contributions of this work as follows:

  1. It has demonstrated that the egg in its normal development reaches a condition when a factor (or factors) inhibits further development. That is, it becomes blocked in a developmental sense.
  2. It has shown that this inhibited state may be overcome and development initiated by appropriate types of treatment.
  3. It has revealed that activation of the egg is possible only at the time when normal fertilization occurs in the particular species. In other words, activation is possible only when favorable conditions are developed in the egg — conditions which enable it to respond to the activating stimulus.
  4. It has demonstrated that one of the primary conditions necessary for the initiation of division or cleavage of the egg is an initial increase in the viscosity of the egg's cytoplasm.
  5. Certain experiments suggest that chemical compounds, such as heparin or heparin-like substances, may suppress cleavage and cell division, presumably due to their ability to decrease the viscosity of the egg.
  6. It therefore follows that substances and conditions which tend to increase the egg's viscosity tend to overcome the inhibited state referred to in ( 1 ) above and thus initiate development.
  7. Recent evidence suggests that substances which produce cell injury tend to initiate cell division in the egg. As states of injury have been shown to produce growths of various kinds during embryonic development and also during the post-embryonic period, it is probable that the principles involved in egg activation are similar to those which cause differentiation and growth in general.
  8. A common factor, therefore, involved in egg stimulation and other types of growths, including tumor-like growths, is the liberation of some substance in the egg or in a cell which overcomes an inhibiting factor (or factors) and thus frees certain morphogenetic or developmental conditions within the egg or within a cell. Once the inhibiting or blocking condition is overcome, differentiation and growth begin.
  9. Finally, the work on artificial parthenogenesis has demonstrated that the egg is an organized system which, when properly stimulated, is able to produce a new individual without the aid of the sperm cell. This does not mean that the sperm is not an important factor in normal fertilization, but rather, that the egg has the power to regulate its internal conditions in such a way as to compensate for the absence of the sperm.

D. Behavior of the Gametes During the Fertilization Process

The activities of the gametes during the fertilization process may be divided for convenience into two major steps:

  1. activities of the gametes which bring about their physical contact with each other, and
  2. activities which result in the actual fusion of the gametes after this contact is made.

Before considering these two major steps, we shall first observe certain of the characteristics of the two gametes when they are about to take part in the fertilization process.

1. General Condition of the Gametes when Deposited within the Area where Fertilization is to Occur

a. Characteristics of the Female Gamete

1) Oocyte Stage of Development. In the case of most animal species, the female gamete is in the oocyte stage when it enters into the fertilization process. (See Chap. 3; also fig. 137.)) In the dog and fox the female gamete is in the primary oocyte stage, and both maturation processes happen after sperm entrance (fig. 115). In the protochordate, Styela, the first maturation spindle already is formed when the sperm enters (fig. 116A-D), and(^n Amphioxus the first polar body has been given off, and the second maturation spindle is developed when the sperm enters (fig. il7A-D). The last condition probably holds true for most vertebrate species (figs. 1 18B; 1 19D). However, in the invertebrates, the sea-urchin egg experiences both maturation divisions normally before sperm entry.

2) Inhibited or Blocked Condifion(^hen the female gamete thus reaches a state of development determined for the species, its further development is blocked or inhibited, and its future development depends on the circumvention of this state of inhibition^ If not fertilized or artificially aroused when this inhibited state is reached, the oocyte or egg begins cytolysis. Eggs fertilized, when these degenerative (cytolytic) conditions are initiated, fail to develop normally^ If allowed to continue, cytolysis soon produces a condition in which development is impossible, and dissolution of the egg results.

3) (Ebw Level of Respiration. While the egg is in this inhibited or arrested state awaiting the event of fertilization, respiration is carried on at a steady but low levels This respiratory level varies in different animal species (fig. 120). That this respiration rate may not be the direct cause of egg inhibition, is shown by the fact that the rate of respiration does not always increase immediately following fertilization in all species (fig. 120). (Consult Brachet, J., '50, p. 105.) Among the vertebrates, the low rate of oxygen consumption of the unfertilized egg has been shown to continue for some time after fertilization in the toad and frog egg and also in the egg of the teleost fish, Fundulus heteroclitus. However, in the case of the egg of the lamprey the respiration rate rises after fertilization (Brachet, J., '50, p. 108).

Fig. 115. (A) Early fertilized egg in upper Fallopian tube of the bitch (dog). Observe the female nucleus before the first maturation division together with the sperm head and tail. Note that the sperm, as in other mammals, enters the nuclear pole of the egg. Observe further that the zona pellucida and the ooplasm are contiguous. (B) Section of the egg of the dog, taken from the upper part of the Fallopian tube. Observe the following features: (1) The sperm pronucleus is forming; (2) the egg nucleus has now entered the meta^hase of the first maturation division; (3) the ooplasm of the egg has shrunk away from the zona pellucida and a space is present between the egg and the zona. This space is the perivitelline space, containing an ooplasmic exudate. (C) Section of the egg in the Fallopian tube of the bitch, showing the formation of the first polar body.

Loss of Permeability. A final characteristic of the female gamete immediately before fertilization is the loss of permeability of the egg surface to various substanceD Correlated with this fact is the presence of definite ooplasmic or other egg membranes associated with the egg surface. The relationship between the ooplasmic surface of the egg and these membranes is altered greatly after fertilization when the egg and the membranes tend to separate. To what extent the loss of permeability of the egg surface is caused by the intimate association of these membranes with the egg surface is problematical. The evidence to date suggests that under normal circumstances they are integrated with the conditions which restrict permeability and egg activation.

b. Characteristics of the Male Gamete

LJn contrast to the inertia and metabolic quietude experienced by the ^male gamete, the gamete of the male experiences quite opposite conditions. ^When the sperm, for instance, is brought into an environment which favors motility, such as the posterior region of the vas deferens of the mammal, it becomes highly motile and continues this motility in the female genital trac^ Similarly, the normal sperm of other vertebrate species is a very active cell when placed in the normal fertilization area (fig. 121, primary phase of fertilization). To quote from J. Brachet ('50), page 91: “This very active cell has an intense metabolism and the maintenance of this latter (condition) is indispensable to the continuance of motility.” As mentioned previously (Chap. 1 ),^his high respiratory metabolism at least in some species is supported partially by the utilization of a simple sugar in the seminal fluid as the sperm ‘(fs rich in oxidative enzymes and in hydrogen transporters'^ (J. Brachet, '50).

2. Specific Activities of the Gametes in Effecting Physical Contact of the Egg with the Sperm

(Consult fig. 121, primary phase of fertilization.)

While the gametes are in the condition mentioned, they are physiologically adapted to fulfill certain definite activities which enhance their contact with each other and bring about actual union in the fertilization process.

a. Activities of the Female Gamete in Aiding Sperm and Egg Contact

l)CFormation of Egg Secretions Which Influence the Sperm. The activities of the female gamete at this time are concerned mainly with the effusion of certain egg secretions. These secretions are known as gynogamic substances, or gynogamones. They are elaborated by the egg when the latter becomes physiologically mature, i.e., when it becomes fertilizablev(fig. 137).

A study of the natural secretions of the egg in relation to the fertilization process has occupied the attention of numerous investigators. These studies began during the early part of the twentieth century. (in reference to the egg, two main groups of substances have been recognized:

  1. the fertilizin complex, and
  2. substances which induce the spawning reactions in the male.

a) Fertilizin Comple?^ Some of the earliest studies upon fertilizin substances were made by von Dungern in '02, Schiicking in '03, and De Meyer in Tl. In these experiments an egg-water solution was obtained by allowing ripe eggs of the sea urchin to stand in sea water for a period of time or by disintegrating the eggs. All of these observers found tha^some substance from

Fig. 116. Fertilization and maturation of the egg in the urochordate, Styela (Cynthia) partita, (After Conklin, '05.) (A) Egg shortly after spawning but before sperm entrance. polar bodies (P.B.); fusion of chromosomal vesicles to form egg pronucleus (E.N.). (M) Movement of sperm nucleus, aster, and of surrounding yellow-pigmented and clear protoplasm to the posterior pole of the egg. The copulation path of egg pronucleus (E.N.) to meet the sperm nucleus is in progress. (N) Sperm aster has divided; egg pronucleus progresses along its copulation path toward posterior pole of egg to meet the male pronucleus. (O) Egg and sperm pronuclei are making contact with each other.

The spindle fibers of the first maturation division are forming, and the nucleoplasm is located toward the animal pole. (B) Egg showing the spindle for first maturation division. Observe the sperm nucleus just inside the ooplasmic membrane near the midvegetal pole of the egg. The nucleoplasm (karyoplasm) of the female nucleus has spread into a thin cap at the animal pole. (C) Metaphase of first division spindle (1, D.S.) nearly parallel to the surface of the egg; no centrosomes are present. (D) Higher powered representation of sperm a little later than that shown in (B). The aster for the first cleavage spindle is forming in the middle piece of the sperm. (E) Slightly more advanced stage than that shown in (B). Collection of yellow-pigmented, peripheral protoplasm (PL.) is shown at bottom of the egg. (F) Anaphase of second polar spindle. Sperm aster enlarging. (See (G) and (H).) (G) Separation of first polar body, (H) Metaphase of second polar spindle, paratangential in position. (1) First polar body (1 P.B.) formed; chromatin of second spindle (CHR.j. Sperm has revolved 180®; sperm aster enlarging. (J) Telophase of second polar spindle. Sperm aster enlarges, and sperm nucleus assumes vesicular condition. (K) Separation of second polar body. (L) Two the egg, when present in dilute solution, caused the sperm of the sea urchin to loose their motility and to become clumped together or agglutinate^A little later, F. R. Lillie, '13, '14, '15, studied the activity of the egg water of the sea urchin, Arbacia, extensively. Lillie associated the egg secretion found in the egg water with a definite theory concerning the mechanism of fertilization. He called ^e substance given off when the sea-urchin egg is allowed to stand in sea water, “fertilizin; for, according to his results, washed eggs deprived of this egg secretion fail to fertilize. Only ripe eggs give off fertilizin according to his observations. Lillie found further that the activities of the sperm, introduced by means of a pipette into the egg-water solution are changed greatly. At first they are activated, to be followed by an agglutination. Moreover, a drop of egg water introduced into a sperm suspension activates the sperm and appears to influence them chemically, causing them to be attracted to the drop. (Lillie therefore concluded that fertilizin has a threefold action upon the sperm:

  1. that it activates the sperm (that is, stimulates their movement),
  2. attracts the sperm by a positive chemotaxis, and
  3. agglutinates the sperm, that is, causes the sperm to associate in clumps.)

The agglutination effect F. R. Lillie found is reversible in most sea-urchin sperm, providing the egg water containing fertilizin is not allowed to act too long. On the other hand, in the annelid, Nereis, agglutination of the sperm is "essentially permanent" (Lillie, F. R., '13). Lillie placed most emphasis upon the “agglutinin” factor in the egg water. He further postulated that fertilizin not only affected the sperm, but dso activates the egg to cause its development (see theory at end of chapter ).(j^illie also obtained another substance from crushed or laked eggs which combines “with the agglutinating group of fertilizin, but which is separate from it as long as the egg is inactive.” This substance present within the egg he called "anti- fertilizing".

(P) Pronuclei associate and begin to form early prophase conditions of the first cleavage.

(Q) Metaphase of first cleavage. (R) Anaphase of first cleavage. (S) Late anaphase of first cleavage.

Fig. 117, (See facing page for legend.)

Since the time of F. R. Lillie's original contribution, the subject of fertilizin and antifertilizin has been actively investigated by various students of the problem. Some investigators criticized the conclusipns drawn by Lillie, but more recent work substantiates them. For examplei^M. Hartmann, et al. ('40), working on the sea urchin, Arbacia pustulosa, and Tyler and Fox ('40) and Tyler ('41), using eggs from Strongylocentrotus purpuratus, find that fertilizing is present and that it is associated with the jelly layer around the egg. Tyler ('41) concludes:

  1. When fertilizin is present in the form of a gelatinous coat around the egg, it enhances fertilization;
  2. when present only in solution around the egg after the gelatinous coat is removed, it hinders fertilization by agglutinating the sperm; and
  3. that fertilizin is not entirely essential since eggs can be fertilized when the jelly coat is removed, but a greater number of sperm are needed under these circumstances^

Tyler also has detected antifertilizin below the surface of the egg and by crushing the eggs was able to show thal(^ntifertilizin from the interior of the egg is able to neutralize the fertilizin of the jelly coat surrounding the eg^ (Tyler, '40, '42). In Germany, Hartmann and his associates (see Hartmann, M., et al., '39, a and b, '40) have demonstrated that by exposing fertilizin to heat or light one may separate the “agglutinating factor” from the “activating factor.” Heat at 95° C. destroys the “agglutinating factor,” while exposure to bright light causes the “chemotactic” and “activating” factors to disappear. The factual presence of the egg products, fertilizin and antifertilizin, postulated by Lillie thus is well established.

Fertilizin appears to be widely distributed as an egg secretion among animals, invertebrate and vertebrate. Among the latter it has been identified in cyclostomes, certain teleost fishes, and in the frog, Rana pipiens (Tyler, '48). Moreover, it is becoming increasingly clear that the term, fertilizin, as employed originally by F. R. Lillie, includes more than one secretion. How many separate enzymes or other substances may be included under the general terms of fertilizin and antifertilizin remains for the future to determine. Moreover, the exact presence of particular gynogamic substances in the egg secretions of different animal species may vary considerably. For example, the spermactivating principle may not be present in all animal species. In fact, there is good evidence to show that it is not present, for example, in all species of sea urchins.

Fig. 117. Fertilization and maturation of the egg in Amphioxus. (A, B, FI after Cerfontaine, '06; C-I after Sobotta, 1897.) (A) Metaphase of first maturation division before sperm entrance. (B) Anaphase of first maturation division before sperm entrance. (C) First polar body and metaphase of second maturation division before sperm entrance. Observe the first or primary fertilization membrane. (D) Sperm has entered near vegetal pole of egg. (E) Outer egg membrane has enlarged and is now much thinner; the second egg membrane is separating from the egg, and the second polar body is forming. (F) Outer and inner egg membranes have fused and expanded; pronuclei of sperm and egg are evident; the sperm aster is to be observed in connection with the sperm nucleus. (G) Meeting of the two pronuclei between the developing amphiaster. (H) Fusion nucleus complete. (I) Diploid chromosomes now evident preparatory to the first cleavage of the egg.

(The general term “gamones” (Hartmann, M., '40) has been applied to the substances produced by the gametes at the time of fertilization. The Hartmann school further has identified the factor responsible for chemotaxis and activation as “echinochrome A,” tl^t is, the bluish-red pigment of the egg, and have called it “Gynogamone IJ^This factor will attract sperm and stimulate their movements “at the enormous dilution^of 1 part in 2,500,000,000 parts of water” (Brachet, J., '50, p. 96). However, Tyler has not been able to detect echinochrome in the egg of the Pacific coast sea urchin, Strongylocentrotus. But the egg water of this species does activate the sperm of this species, which suggests that the activating factor may be something else than echinochrome. (Xo the agglutinating factor, M. Hartmann and his associates have given the name “Gynogamone Il.j^

The exact identity of these gamones with particular chemical substances present in the egg water at the present time is impossible. To quote from J. Brachet, '50, p. 99:

It is clear that research in this field is complicated by the fact that a number of agents activate the movements of sperm (alkalinity, glutathione, echinochrome (?), etc.) . . . There is strong evidence in favor of the protein nature of agglutinin, while the sperm-activating principle is probably a substance with a small molecule, its identity with echinochrome being doubtful at the present time.

bl Spawning-inducing Substances. In addition to the fertilizin substances which act in effecting the actual contact of the sperm with the egg, a spawning-inducing agent is present in the egg water of certain species. In the annelid, Nereis, for example, there is something present in the egg water which induces spawning in the male^^^ownsend ('39) suggested that this substance may be glutathione, but Tyler ('48) does not readily concur with this conclusion. A spawning-inducing agent is found also in the egg water of oysters (Galtsoff, '40). Among the vertebrates, the spawning behavior of the female appears to be the important factor in inducing the male reactiofQ

b. Activities of the Male Gamete in Aiding the Actual Contact of the Two Gametes

^^The activities of the male gamete, including those of seminal fluid, are much more complicated and devious than those of the female gamete. These activities entail:

( 1 ) production of certain sperm secretions,

(2) activities of large sperm numbers,

(3) presence of a healthy seminal plasma or protective substance for the sperm, and

(4) physical movements and functioning of specific parts of the sperm cell itself.

(1) Sperm Secretions. The sperm secretions are known as androgamic substances or androgamone£) These substances have been the object of much study since the initial endeavors of Fieri in 1899. (in recent years, three general types of substances have come to be recogniz^ in relation to the sperm of different species. These three groups of substances are:

(1) secretions which cause lysis,

(2) a substance or substances related specifically to the fertilization reaction (i.e., egg and sperm contact), and

(3) substances which bring about the spawning reaction in the female.

a) Secretions Producing Lysis. To cite the importance of lytic substances produced by the sperm, reference is made first to the situation in the amphibian, Discoglossus pictus. In this primitive anuran, the sperm, although about 2 mm. long, are almost incapable of motility. However, they do accumulate in the region of a thickened portion of the egg capsule which overlies a depressed area of the egg. They are capable of passing through this thickened area of jelly by the aid of a digestive enzyme probably associated with the acrosome (Hibbard, '28j^Hibbard also suggests that “nuclear fluids” accumulate in the bottom of the egg depression and these fluids attract the sperm to the thickened area of the capsule. If so, here is an example of two chemical substances, one elaborated by the egg and the other by the sperm, both working together to bring about fertilization. In substantiation of Hibbard's views of the presence of a lytic enzyme associated with the sperm of this species, Wintrebert ('29) found that extracts from the sperm contained an enzyme which is capable of digesting the inner jelly coat of the egg.

More recently, Tyler ('39) has found that sea-water extracts of frozen and thawed sperm of two mollusks (Megathura crenulata and Haliotis cracherodii) were able to dissolve the egg membranes of the respective species. Cross-species reactions were not obtained, however. Strong extracts of concentrated sperm suspensions bring about egg-membrane disappearance in less than one-half minute, but with the jelly coat present around the egg it takes about three minutes. Also, Runnstrom and his collaborators ('44, '45, a and b, '46) made methanol extracts of sea-urchin sperm which were able to liquefy the superficial cortical area of the egg.

A most interesting enzyme, known as hyaluronidase, has been extracted from mammalian testes and from mammalian sperm. This substance is capable of dispersing the follicle cells of the corona radiata present around most mammalian eggs when discharged from the ovary^^ ( Sheep and opossum eggs as well as those of the monotremes do not possess a layer of follicle cells around the newly ovulated egg.) (This dispersing effect aids fertilization, for it enables sperm to reach the egg surface before degeneration processes occur in the egg^ Rowlands ('44) effected artificial insemination in the rabbit with dilute sperm solutions by adding the enzyme hyaluronidase from other sperm to the dilute suspensions. Without the addition of hyaluronidase, fertilization did not result. In certain cases in women where artificial insemination was tried but failed when semen alone was used, the addition of hyaluronidase from bull testis to the semen produced successful fertilization (Leonard and Kurzrok, '46).

b) Secretions Related Specifically to the Fertilization Reactions. The substances mentioned in the preceding paragraphs are related to the general fertilization process, but they may not be related specifically to the reactions which bring the sperm in direct contact with the egg(ln the egg we have observed the presence of fertilizin which stimulates a series of sperm activities directed to this end. Similarly, in the male gamete, sperm of various species seem capable of producing “androgamic substances which neutralize, in part, the action of the gynogamic substances and thus assure the precise mechanism necessary for precise fusion of the gamete$i^ (J. Brachet, '50).

An introductory study by Frank ('39) suggests the presence of a sperm substance which reacts directly with the fertilizin complex of the egg. It was shown by this investigator that an extract from the sperm of the sea urchin, Arbacia, is:

( 1 ) (able to destroy the sperm agglutinating factor when added to a solution of fertilizin derived from the sea-urchin egg, and

(2) possesses the power to agglutinate eggs of the same species

Other students of the problem have found a similar substance associated with the sperm. (See Hartmann, Schartau, and Wallenfels, '40; Southwick, '39; Tyler, '40.) (The general term “sperm antifertilizin” has been given to this substanc^(or substances) by Tyler and O'Mel veney ('41) (Sperm antifertilizin unites with fertilizin produced by the egg, with the resulf that the sperm is entrapped at the egg's surface. Tyler and O'Melveney ('41) regard the reaction between antifertilizin of the sperm and fertilizin of the egg to be the “initial (perhaps essential) step in the union of the gametes whereby the spermatozoon is entrapped by the . . . fertilizin, on the egg./

C) Secretions Which Induce The Spawning Reaction In The Female. Galtsoff ('38) has shown that the presence of sperm of the oyster, Ostrea virginica, “easily induces spawning in oysters. He also found that the spawning reaction is specific in that sperm of different species cannot provoke it. The active principle of the sperm suspension is thermolabile and insoluble in water. However, it may be readily extracted in 95 per cent ethyl alcohol and benzene.

To what extent spawning-inducing substances may be present in other animal species is questionable, but it may not be an uncommon phenomenon, especially in sedentary species, such as the oyster and other mollusks. In the vertebrate group, surface contact of the male and female bodies is an important factor in many cases.

2) Relation and Function of Sperm Number in Effecting the Contact of the Sperm with the Egg. In the preceding chapter, sperm transport is considered. This transportation journey is an efficient one with regard to the end achieved, namely, contact of a single sperm with an egg, but from the viewpoint of sperm survival it may appear as waste and caprice. This fact is especially true in those forms utilizing fertilization where only one or a very few eggs are fertilized. It has been shown by Walton ('27) in experiments dealing with artificial insemination in the rabbit, when dilution of the sperm is such that the number falls below 3,000 to 4,000 per cc., fertilization does not take place. Recent observations by Farris ('49) on the human suggest that numbers of sperm below 80,000,000 per cc. are precarious when conception is the end to be achieved. (For the total number of sperm ejaculated by certain males during a single copulation, see Chap. l.)(j\lthough exceedingly large numbers of sperm are deposited in the posterior area of the female reproductive tract, the number becomes less and less as the ovarian end of the duct is reached. The ability of effective sperm transport within the female tract probably varies considerably in different speeies and with different females in the same species^j^he rat and the dog appear to be more efficient in this respect than the rabbit.

The relation of sperm numbers to the efficiency of the fertilization process is not to be considered merely as a mechanical hit and miss device, whereby the presence of a greater number of sperm may assure an accurate “hit” or sperm-egg collision (Rothschild, Lord, and Swann, '51). Hammond ('34) has shown in the rabbit that fertilization is not effected by the few sperm which reach the region of the egg first, but by the later aggregations of numbers of sperm. The work on hyaluronidase mentioned on page 229 suggests strongly that one object of the excess sperm is to transport hyaluronidase to the vicinity of the egg. The presence of this enzyme close to the egg possibly facilitates the passage through the cells of the corona radiata and also through the zona pellucida of the single sperm which makes contact with the egg in the process of fertilization (Tyler, '48).(The general result should be regarded as the working of a cooperative enterprise, where many sperm aid in the dissolution of the interference in order that one sperm may reach the egg's surface.

3) Influences of the Seminal Plasma in Effecting Sperm Contact with the Egg. The importance of the seminal plasma (i.e., the fluid part of the semen; see Chap. 1) cannot be overestimated (Mann, '49). It is, to a great extent, ^he natural environment and at the same time the nutritive medium for the sj^rm during the transport from the male ducts through the external medium or within the lower region of the female genital tract. Its functions may be stated as follows:

(1) It increases the motility of the sperm;

(2) it has a high buffering capacity, which protects the sperm from injurious acids or other injurious substances; and

(3) it is a vehicle for nutritive substances, such as fructose, vitamin C, and the B complex which provide nourishment for the sperm

The B group of vitamins may be directly related to sperm motility. Other substances, such as iron, copper, etc., are present. One should consider the seminal plasma, therefore, as a most important association of substances which aids in producing a protective environment for the sperm while the latter is in migration to the egg.

The importance of the environment of the sperm and also that of the egg cannot be overemphasized. If normal fertilization is to be effected, optimum conditions for both sperm and egg must be present. An example of this fact is shown by the observations of Reighard on fertilization of the walleyed pike. (See Morgan, *27, p. 18.) The best results with the eggs of this teleost fish were obtained when the eggs were fertilized as soon as they entered the water from the female genital tract. After two minutes only 40 per cent of the eggs segment, and after ten minutes no eggs segment. For many fish, “dry fertilization” gives the best results. Dry fertilization consists in stripping the female to force out the eggs into a dry container and then stripping the milt (seminal fluid) from the male directly over the eggs. The eggs are then placed in water after a few minutes. This work suggests strongly that a deleterious environment for either the egg or the sperm is disturbing to the fertilization process.

4) Roles Played by Specific Structural Parts of the Sperm in Effecting Contact with the Egg: a) Role of the Flagellum. As indicated in the foregoing paragraphs, when the sperm cells have reached the normal fertilization site, the activities which bring about actual contact of the sperm with the egg largely is a sperm problem. Aside from enzymes elaborated by the sperm, sperm motility is extremely important in achieving this end. Although sperm may appear to swim rather aimlessly, vigorous, healthy sperm do lash forward more or less in a straight line for some distance; ill-developed or otherwise impaired sperm may simply swim round and round or move forward feeblyi^n the case of flagellate sperm, the structure which makes the forward swimming movement possible is the flagellum or tail (figs. 74, 77, 78, 79). A two-tailed sperm or one in which the flagellate mechanism is not well developed would be at a disadvantage in this race to reach the confines of the egg. ^rachet ('50) considers the rate of metabolism necessary to support the acTivities of the tail or flagellum in sperm movement as directly comparable to that of muscle.

An interesting peculiarity of a different type of sperm mechanism useful iit' achieving contact with the egg's surface is that of the so-called “rocket sperm” of certain decapod Crustacea described by Koltzoff (fig. 75). After attachment of the sperm to the egg by its tripod-like tips, the caudal compartment, containing a centriole and the acfosome, explodes. “Koltzoff considers that the force of the explosion drives the sperm upon, or even into, the egg” (Wilson, '25, p. 299).

b) Role of the Acrosome in the Egg-sperm Contact. The acrosome of the sperm (fig. 78) has long been regarded as a structure which has a function in the reactions involved in fertilization. The older conception of Waldeyer that the acrosome was a perforating device which enabled the sperm to pass through the egg membranes and thus to enter the egg is untenable in the light of later observation. Many years ago Bowen ('24) though admitting a minor mechanical role for the acrosome, emphasized thati^e acrosome essentially is a secretory product whose principal function is to initiate the physicochemical reactions of fertilization^ It should be recalled in this connection that Hibbard ('28) and also ParatX'33, a and b) have attributed to the acrosome of the anuran, Discoglossus, ^e ability of carrying or producing an enzyme which enables it to reach the egg's surface through the jelly surrounding the egg) Parat further suggested that the acrosome in this species contains a “proteolytic enzyme” which, when introduced into the egg, results in development.

The concept of a proteolytic enzyme associated with the acrosome of Discoglossus is interesting in the light of the suggestion by Leuchtenberger and Schrader ('50) that the mucolytic enzyme, hyaluronidase, in the bull sperm may be associated with the acrosome. Both of the above suggestions need more work before it can be stated with certainty that the acrosome is connected with either of these enzymes in the above species. However, these suggestions do serve to emphasize the possibility that^lie acrosome may be an enzyme-producing or enzyme-carrying device which enables the sperm to make its way through the egg's surroundings to the egg surface, and also, that it may play a part in egg activation^

5) Summary of the Activities of the Egg and Sperm in Bringing About the Primary or Initial Stage of the Fertilization Process, Namely, that of Egg and Sperm Contact.

a) The secretion of fertilizin by the egg:

(1) activates the sperm to increased motility, and

(2) through chemotaxis, entices the sperm to move in the direction of the egg.

b) In moving toward the egg the lytic substances elaborated by the sperm enable it to “plow” through the gelatinous envelopes and cellular barriers to the surface of the egg. This movement undoubtedly is aided by movement of the flagellum in some species, but not in all (see Discoglossus) . The acrosome of the sperm may function at this time either as an instrument carrying lytic substances or as one which actually manufactures these substances. The presence of large numbers of sperm near the egg may aid sperm penetration to the egg's surface by contributing lytic substances to the environment around the egg which aid in the removal of membranes and other barriers surrounding the egg.

c) The antifertilizin of the sperm may then unite with the fertilizin of the egg (probably with the agglutinin factor) ; this reaction presumably agglutinates the sperm to the egg's surface.

d) An egg-surface, liquefying factor, androgamone III, has been isolated by Runnstrom, et al. ('44), from sea-urchin sperm (Runnstrom, '49, p. 270). A similar “sperm lysin” has been isolated also from mackerel testes. This work suggests that a specific sperm lysin may be involved in the activation processes within the egg cortex. (See theory of fertilization according to J. Loeb at end of chapter.)

e) Lastly, in certain animal species, substances may be present in the seminal fluid which induce the spawning reaction in the female, while in the egg secretion of certain species, a factor may be present which induces spawning in the male.

3. Fusion of the Gametes or the Second Stage of the Process of Fertilization

(Xhe actual fusion or union phase of fertilization begins once the sperm has made contact with the egg^fig. 121 B). From this instant the rest of the fertilization story becomes essentially an egg problem. The egg up to the time of sperm contact literally has been waiting, discharging fertilizin substances into the surrounding medium. However, when a sperm has made successful contact with the surface of the egg, ^e waiting period of the egg is over, its work begins, the fusion of the two gametes ensues, and the drama of a new life is initiated!

The following events of the fusion process may be listed — events which occur quite synchronously, once the mechanisms involved in egg activation and gametic fusion are set in motion:

(a) The separation of an egg membrane (fertilization membrane, vitelline membrane, chorion, zona pellucida, etc.) from the egg's surface and the exudation of fluid-like substances 'from the egg's surface.

(b) A fertilization cone may be elaborated in some species.

(c) Changes in the physicochemical activities of the egg.

(d) The maturation division (or divisions) is completed in most eggs.

(e) Profound cytoplasmic movements occur ^iC'fliany eggs which bring about various degrees of localization of cytoplasmic substances; these substances orient themselves into a pattern definite for the species. In some species a cytoplasmic pattern composed of future, organforming substances is rigidly established and definitely correlated with the first cleavage of the egg (Styela); in others it is less rigid (frog); and in still others it appears gradually during cleavage of the egg (teleost fishes).

(f) The sperm nucleus enlarges, and the middle-piece area in most animal species develops a cleavage aster.

(g) The copulation movements of the egg and sperm pronuclei take place. These movements bring about the association of the two pronuclei near the center of the protoplasm of the egg which is actively concerned with the cleavage phenomena.

(h) The pronuclei may fuse to form a fusion nucleus or they may associate less intimately. Regardless of the exact procedure of nuclear behavior, the female and male haploid chromosome groups eventually become associated in the first cleavage spindle to form one harmonious diploid complex of chromosomes, composed (in most cases) of paired chromosomal mates or homologues.

(i) The first cleavage plane is established.

4. Detailed Description of the Processes Involved in Gametic Union as Outlined Above

a. Separation and Importance of a Protective Egg Membrane, Exudates, etc.

The term “fertilization membrane” is applied to the egg (vitelline) membrane which, in many species, becomes apparent only at the time of fertilization. In many other eggs a definite and obvious vitelline membrane is present before the egg is fertilized and in many respects functions similarly to the more dramatically formed fertilization membrane. Both types of membrane fulfill definite functions during fertilization and early development. The fertilization men^brane which forms only as a distinct membrane during fertilization was observed first by Fol, in the autumn of 1876, in the starfish egg (Fol, '79). In the cephalochordate, Amphioxus, two definite membranes separate from the egg's surface. One membrane forms just before the sperm enters the egg, while the second membrane separates from the egg after the sperm enters. Both membranes soon fuse and expand to a considerable size, leaving a perivitelline space between them and the egg; the latter space is filled with fluid, the perivitelline flui^(fig. 117B-F, I). In the urochordate, Styela, no such membrane arises from the egg's surface, but the chorion previously formed by the follicle cells serves to fulfill the general functions of a fertilization membrane (figs. 91B,i^[J6) Jin teleost fishes, the egg emits a considerable quantity of perivitelline fluid at the time of fertilization, effecting a slight shrinkage in egg size with the production of a space filled with this fluid between the egg's surface and the zona radiata (fig. 122A-C). The zona radiata thus functions as a fertilization membrane. In the gobiid fish, Bathygobius soporator, the chorion and/or vitelline membrane expands greatly after the egg is discharged into sea water, and an enlarged capsule is soon formed which assumes the size and shape of the future embryo at the time of hatching (fig. 123). (See Tavolga, '50.) In the brook lamprey, according to Okkelberg ('14), shrinkage of the egg at fertilization is considerable, amounting to about 14 per cent of its original volume. A slight egg shrinkage with

Fig. 118. Fertilization in the guinea pig. (After Lams, Arch. Biol., Paris, 28, figures slightly modified.) (A) Spindle of first maturation division. (B) Second maturation division completed; head of sperm in cytoplasm beginning to swell. (C) Sperm pronucleus, with tail still attached, greatly enlarged; female pronucleus small. (D) Pronuclei ready to fuse; chromatin material (chromosomes) evident within. (E) First cleavage spindle. (F) First cleavage completed. Observe deutoplasmic and cytoplasmic globules which have been exuded into the space between the blastomeres and the zona pellucida. (G) Four-cell cleavage stage. Observe that the zona pellucida encloses the four blastomeres and the cytoplasmic globules which have been exuded. The zona functions to keep the entire mass intact.

the emission of fluid is present in the amphibia and the egg thus is enabled to revolve within a relatively thick vitelline membrane. The latter membrane expands gradually during development, and is associated intimately with the surrounding jelly membranes secreted by the oviduct. In the reptiles and birds, the separation of the egg from the vitelline membrane or zona radiata and the formation of the perivitelline space is less precipitous. In the egg of the bird (e.g., pigeon or hen) (fig. 126), a vitelline space filled with fluid appears during the latter phase of oocyte growth in the ovary which separates the surface ooplasm of the egg from the vitelline membrane. The egg is free to revolve in this vitelline space. In the prototherian mammals, the zona pellucida evidently functions in a manner similar to that of the bird or reptile (figs. 46, 127) ^However, in the metatherian and eutherian mammalia, the zona pellucida becomes separated from the ooplasm of the egg's surface with the subsequent development of a perivitelline space at fertilization or during early cleavage (figs. 115, 118, 124, 125).^

It is to be observed, therefore, that there are two general groups of egg or vitelline membranes in the phylum Chordata which assume an important role at fertilization and during the earlier part of embryonic development:

(1 ) those membranes which become separated from the egg surface in a somewhat dramatic manner at fertilization, and

(2) membranes which separate gradually during the late phases of ovarian development and during early embryonic development.

In the former group are to be found the egg membranes of the eggs of Amphioxus, teleost and many other fishes, and the amphibia; in the latter group are the membranes of eggs of Styela, elasmobranch fishes, reptiles, birds, and prototherian mammals. The higher mammalian eggs appear to occupy an intermediate position. >

The separation of the so-called fertilization membrane has been most intensively studied in certain invertebrate forms. As a matter of interest, some of the processes involved in membrane elevation in various invertebrate eggs are herewith described briefly.

In the nematode, Ascaris, the egg exudes a jelly-like substance after the sperm has entered. This substance hardens to form a thin, tough membrane which later thickens and expands. The egg also appears to shrink, leaving an enlarged perivitelline space between the egg surface and the outer hardened membrane (figs. 128, 133C-E).

The formation of the fertilization membrane in Echinarachnius, a genus of sea urchins, was the subject of intensive study by Just (T9). In this species the egg is larger than that of the sea urchin, Arbacia. According to Just's account, the fertilization membrane starts as a “blister” at the point of sperm contact; from this area it spreads and rapidly becomes lifted off from the general surface of the egg. Heilbrunn ('13) studied the fertilization membrane of the sea urchin's egg before fertilization and describes it as a vitelline membrane, “probably a gel or semi-gel” which is present at the surface of the egg. It becomes visible as a distinct membrane when lifted off from the egg's surface after fertilization. As this elevation occurs, according to Runnstrom, cortical granules are exuded from the surface of the egg, accompanied by a general contraction of the egg surface. These cortical granules later become merged with the vitelline membrane to form a relatively thick structure (fig. 129). (See Runnstrom, '49.) Fluid collects between the egg surface and the fertilization membrane.

On the other hand, in the annelid worm, Nereis, there is a complicated reaction at the egg's surface at the time of fertilization (Lillie, F. R., '12). In this egg a definite membrane is present around the newly laid egg. When a sperm has made an intimate contact with the egg's surface, the cortical layer of the egg exudes a substance which passes through the membrane to the outside; this substance turns into jelly on coming in contact with sea water (fig. 130B). The jelly layer carries away the excess sperm from the egg's surface. A striated area then appears between the vitelline membrane and the surface of the egg. This area, shown in fig. 130B as the cortical layer, represents the collapsed walls of small spaces of the superficial layer of the cortex of the egg which exude their contents through the vitelline membrane to form the surrounding jelly. The egg then forms a new ooplasmic surface beneath the collapsed walls of the small spaces of the original cortex (fig. 13(^, ooplasmic membrane).

All of these changes and reactions, namely, the formation of the fertilization membrane, the exudation of cortical granules, and the emission of a fluid or jelly together with the shrinkage of the egg result from changes which occur in the outer layer of the egg's protoplasm or cortex, and consequently may be classified as cortical changesj)The activation of the egg at the time of fertilization or during artificial stimulation thus appears to be closely integrated with cortical phenomena. It is debatable whether these changes are the result of activation or are a part of the “cause” of activation.

The particular activity of egg behavior at the time of fertilization which

Fig. 119. Fertilization phenomena in the egg of Rana pipiens. (Drawings B, D-G made from prepared slides by the courtesy of Dr. C. L. Parmenter.) (A) Semidiagrammatic representation of the egg shortly before ovulation. The germinal vesicle has broken down, and the chromosomes in diakinesis have migrated toward the apex of the animal pole preparatory to the first maturation spindle formation shown in (B). (B)

First polar spindle. Tetrad condition of chromosomes in process of separation into the respective dyads. (C) Polar view of egg after first maturation division. Compare with (D), which represents a section of a comparable condition. (D) Lateral view of spindle of second maturation division. First polar body present in a slight depression at animal pole. The egg is spawned in this condition. (E) Second polar body shown in a depression of the animal pole. Within the superficial ooplasm of the egg, the reorganized female pronucleus is shown. (F) Meeting of the two pronuclei is shown in this section of the egg at the bottom of the female copulation path or “egg streak,” E.S. (G) Two pronuclei in contact (shown in F) under higher magnification. (H) Entrance and copulation paths of sperm nucleus. (Modified from Rugh: The Frog, Philadelphia, The Blakiston Co., 1951.) (1) Sperm-entrance path, copulation path, and meeting of pronuclei. (From

O. Hertwig, 1877.) (J ) First cleavage path, showing daughter nuclei. (From O. Hertwig, 1877.) (K) External, lateral view of the egg just before first cleavage. Arrows show direction of pigment migration with resulting formation of gray crescent.

appears to be common to the eggs of many species (sea urchin, cyclostomatous and teleost fishes, frog, and mammal) is the contraction of the egg's surface, together with the exudation of various substances from the egg. (See, in this connection, the fertilization theory of Bataillon at the end of this chapter.) It is this behavior of the egg's surface which makes the fertilization membranes and other egg membranes more apparent; it represents one of the essential and immediate activities associated with egg activation. Separation of the various egg membranes at the time of fertilization appears to be secondary to this primary activity.

Aside from the immediate functions at the time of fertilization, the activities of the various types of vitelline membranes are concerned mainly with nutritional, environmental, and protective conditions of the early embryo. The presence of a fluid in the perivitelline space between the membrane and the developing egg affords a favorable environment for early developmental processes. Moreover, it permits the egg to rotate when its position is disturbed, a proper developmental orientation being maintained. A further accommodation is evident in that it permits the developing egg to exude substances, including yolk, into the surrounding area, which may be retained in the immediate environment of the egg and later utilized in a nutritional way. If the surrounding vitelline membrane were not present, this material, solid or fluid, would be dissipated. For example, in the early cleavage stage of the opossum or guinea-pig egg, yolk material is discharged into the area surrounding the early blastomeres (figs. 118, 125). The exuded yolk and dissolved substances later come to lie in the cavity within the blastomeres and, thereby, may be used for nutritional purposes. Also, in some forms, such as the opossum, the early blastomeres utilize the zona pellucida as a framework upon which they arrange themselves along its inner aspect during the development of the early blastula. This apparent independence of the early cleavage blastomeres in the opossum and their lack of cohesiveness is evident in other mammals, also. The tendency of the blastomeres in mammals in general to separate from each other emphasizes the importance of the zona as a capsule which functions to hold the blastomeres together.

Fig. 120. Effects of fertilization on oxygen consumption in various marine eggs. (After J. Brachet, '50; data supplied by Whitaker.)

The surrounding egg membrane, in many cases, may act osmotically to permit a nice balance between the developing egg and the substances outside of the membrane. For example, in birds, the egg and its contained embryo together with its immediate environment are largely maintained as a physicochemical system due to the osmotic properties of the zona radiata or vitelline membrane. This membrane separates the watery albumen from the nutritive yolk material. These two substances have different osmotic conditions. Consequently, the vitelline membrane must maintain the proper conditions between these two general areas, and it performs this function in an admirable fashion. It should be emphasized further that the vitelline membrane in the chick's egg is a living membrane, and consequently its osmotic properties are different from that of a non-living membrane, such as a collodion membrane. If the egg and albumen of the hen's egg are separated by a thin collodion membrane, for example, they will reach an osmotic equilibrium more rapidly than when separated by the thin vitelline membrane. If, however, the vitelline membrane is isolated from its normal relationships in the egg, it behaves similarly to a collodion membrane. It is best to regard the vitelline membrane, the yolk, and the albumen of the bird's egg as forming an harmonious system, in which all parts are responsible for the maintenance of the necessary conditions for development. (Consult Romanoff and Romanoff, '49, pp. 388-391.)

Undoubtedly in other eggs, such as that of the frog, the delicate relationship existing between the egg, the perivitelline fluid, the vitelline membrane, and the surrounding external medium forms a complete unit for the proper maintenance of developmental conditions. In most eggs the vitelline or similar membranes maintain the protective funetion until a relatively late period in development.

b. Fertilization Cone or Attraction Cone

VThe fertilization cone results from specialized activity of the surface of the egg (egg cortex) at the point of sperm contact (fig. 130)')This structure has been described in various invertebrate eggs, such as those of th€(sea urchins, annelid worms, mollusks, and in some of the ascidians among the protochordatay In the annelid worm. Nereis virens, as the sperm makes its way through the egg membrane, a cone of cortical ooplasm flows out to meet the sperm, making an intimate contact with the perforatorium (acrosome) of the sperm (fig. 130B, C). When this contact is made, the extended cone withdraws again gradually, and appears to pull the sperm head into the egg's substance (fig. 130D-G).An the egg of the sea urchin, Toxopneustes variegatus, a protoplasmic prominence appears only after a sperm begins to pass into the egg. It persists until about the time that the pronuclei unite?( Wilson and Mathews, 1895). (See fig. 131B-F.) A prominent fertilization cone is found also in the starfish, Asterias forbesi (Wilson and Mathews, 1895). In the vertebrate group, fertilization cones are not generally observed, but the protoplasmic bridge from the egg membrane to the ooplasmic surface in Petromyzon evidently fulfills the functions of a cone (fig. 134C).

Fig. 121. {See facing page for legend.)

(^he formation of the fertilization cone and its withdrawal again, suggests that ooplasmic movements are concerned mainly with the sperm's entry into the interior of the egg. These movements appear to be aroused by some stimulus emanating from the sperm as it contacts the egg's surface. That is to say, although the sperm becomes immobile once it has touched the egg's surface, various stimuli, chemical and/or physical, issue from the sperm into the egg substance. Here these stimuli inaugurate movements in the ooplasm which draw the sperm into the egg. This modern view thus emphasizes motility of the cortical area of the egg as the factor which conveys the sperm into the interior of the egg. It suggests further that the older view of sperm entry which was presumed to result from sperm motility alone does not agree with the actual facts demonstrated by observation.

c. Some Changes in the Physiological Activities of the Egg at Fertilization

The separation of the egg membrane from the egg surface, the emission of fluid substances from the egg's surface into the perivitelline space, and contraction of the egg's surface have been noted above. Associated with these immediate results of sperm contact with the egg, a pronounced movement of cytoplasmic substances within the egg can be demonstrated in many species. Examples of cytoplasmic movements within the ooplasm of the egg are given below in the descriptions of the fertilization processes which occur in various chordate species.

Accompanying the above-mentioned activities, pronounced changes of a metabolic nature occur. In the egg of the frog and toad, for example, there is little change in the oxygen consumption during fertilization, although there

Fig. 121. Two stages of fertilization in animals. (A) In the primary phase of fertilization (“external fertilization” of F. R. Lillie), the sperm is activated to greater motility by the environmental factors encountered at the fertilization site, including the gynogamic substances secreted by the egg. It is also drawn to the egg by a positive chemotaxis. The lytic substances (androgamic substances) enable the stimulated sperm to make its way more easily through the jelly membranes and ooplasmic membranes surrounding the egg to the egg's surface. At the egg's surface the interaction of gynogamic and androgamic substances brings about the agglutination of the sperm to the egg's surface. This initiates stage B, on the secondary phase of the fertilization process (“internal fertilization” of F. R. Lillie). (B) Secondary phase of fertilization or fusion of the gametes. (See text for further description.) This stage begins when the sperm has made contact with the egg and terminates when the first cleavage spindle has formed.

Fig. 122. Changes during fertilization in the egg of Fundulus heteroclitus. (A) Egg before fertilization. (B, C) Changes in the egg shortly after sperm entrance into the egg. In (B) is shown the contraction of the egg from the vitelline membrane, the disappearance of the yolk plates, and the formation of the peri vitelline space. In (C) is shown the migration of the peripheral cytoplasm toward the point where the sperm has entered the egg, forming a cytoplasmic or polar cap.

is a pronounced drop in the respiratory quotient, presumably indicating a change in the character of oxygen consumptioi^ (Brachet, J., '50, p. 106). Fertilization does not change the rate of oxygen consumption in the teleost fish, Fundulus heteroclitus, but, in the lamprey, oxygen consumption is increased (Brachet, J., '50, p. 108).^lso, in the egg of the sea urchin, following artificial activation or normal fertilization, there is a considerable increase in oxygen cons umptioji3( fig. 120). In the unfertilized and fertilized egg of the starfish (Asterias) apparently there is no change in the rate of oxygen metabolism. (In the eggs of certain sea urchins it has been shown by Runnstrom and co-worlOTs (Runnstrom, '49, p. 306) that acid formation occurs following fertilization. It is of brief duration} (Constllt also Brachet, J., '50, p. 120, for references.) Other changes have been described, such as an increase in viscosity of the egg (Heilbrunn, '15), and an increase in permeability of the egg membran#( Heilbrunn, '15). Fertilization may produce a higher dispersity of the egg colloidal material, at least in some specie^/ Changes of a metabolic nature, therefore, are a part of the fertilization picture. (The reader should consult Brachet, J., '50, Chap. 4, for a thorough discussion of physiological changes at fertilization.)

d. Completion of Maturation Divisions, Ooplasmic Movements, and Copulatory Paths of the Male and Female Pronuclei in Eggs of Various Chordate Species

A description of the maturation processes, ooplasmic movements, and the behavior of the male and female pronuclei in the fertilization processes of various chordate species is given below. It should be observed that all of these events occur rather synchronously in the urochordate, Styela, and in the egg of the frog, while in others, such as the prototherian mammal, Echidna, they may come to pass in sequence.

1) Fertilization in Styela (Cynthia) partita: a) Characteristics of the Egg Before Fertilization. The living, fully formed, primary oocyte of the urochordate, Styela (Cynthia) partita, is about 150 /x in diameter. It possesses at this time three areas which can be distinguished with clearness, namely, a peripheral transparent layer which contains a sparsely distributed yellow pigment, a central mass of gray-appearing yolk, and the area of the germinal vesicle, located near the future animal pole of the egg (fig. 132A).

^^he first steps leading to the maturation divisions of the chromatin material take place before sperm entrance, at the time the egg is spawned or shortly before. At this time the wall of the germinal vesicle (i.e., the nuclear membrane) breaks down, and the contained clear cytoplasm moves up to the animal pole of the egg where it spreads out to form a disc. The chromosomes then line up on the metaphase plate of the first maturation spindle; they remain thus in the metaphase of the first maturation until the sperm enters (fig. 116A, B).

b) Entrance of the Sperm. The sperm enters the egg (i.e., the primary oocyte) at the future vegetal (vegetative) pole,)either exactly at the pole or a little to one side (fig. 116B). Sperm entrance at this pole probably is due to a fundamental structural and physiological condition which in turn reflects a definite polarity of the egg. (Qnly one sperm normally enters the egg, but several sperm may penetrate through the chorion into the perivitelline space,

c) Cytoplasmic Segregation. A striking series of changes appear within the cytoplasm of the egg immediately following sperm entrance. The yellowpigmented, peripheral layer of protoplasm flows toward the point of sperm entrance (i.e., the vegetal pole) and collects into a “deep, orange-yellow spot” which surrounds the sperm (fig. 132B, C, peripheral protoplasm). It later spreads again and then covers most of the lower or vegetal pole of the egg. Accompanying the flow of yellow peripheral protoplasm toward the vegetal egg pole, most of the clear protoplasm of the germinal vesicle (i.e., the nuclear plasm mentioned above) flows with the yellow protoplasm tovi#rd the vegetal pole. The clear protoplasm, to some extent, tends to mingle with the yellowpigmented, peripheral protoplasm. In figure 132C, the clear protoplasm may be observed as a clear area above the yellow-pigmented protoplasm.

The sperm pronucleus next moves upward away from the vegetal pole and toward one side of the egg to a point which marks the posterior pole of the egg and future embryo (fig. 116M). The clear protoplasm and the yellowpigmented protoplasm move upward with the sperm (fig. 132D). The yellowpigmented protoplasm at this time forms a yellow crescent just below the egg's equator, and the middle point of this crescent marks the posterior end of the future embryo (fig. 132D, E). A distinct crescent of clear protoplasm appears just above the yellow crescent at this time (fig. 132D-F). The crescent substance is therefore plainly differentiated at once into clear and yellow protoplasm, which remain distinct throughout the entire development (Conklin, '05, p. 21).

The yolk material, which at first is centrally located in the egg, moves toward the animal pole when the clear and yellow-pigmented protoplasms migrate to the vegetal pole. As the yellow and clear protoplasmic crescents are formed, the yolk material moves to occupy its ultimate position at the vegetal pole of the egg (fig. 132D). Later when the first cleavage division occurs, another crescentic area, the gray crescent, appears on the side of the egg opposite the yellow crescent.

As a result of the segregation of ooplasmic materials, four definite areas are localized:

( 1 ) a vegetal, yolk-laden area,

(2) a gray crescent,

(3) the yellow and clear protoplasmic crescents opposite the latter, and finally

(4) the more or less homogeneous cytoplasm at the animal pole of the egg.

The movements of cytoplasmic materials in the cephalochordate, Amphioxiis, are similar to those in Styela (Conklin, '32).

d) CopuLATORY Paths and Fusion of the Gametic Pronuclei. The entrance of the sperm into the egg substance, its migratory movements in the ooplasm, its meeting with the egg pronucleus, and final fusion or association of the pronuclei afford an interesting problem. The factors governing the movements of the female and male pronuclei are unknown, although the movements in many eggs are spectacular. The movements of the pronuclei in Styela partita offer an excellent illustration of the copulatory migrations of the pronuclei within the cytoplasm of the egg.

The sperm enters the egg of Styela partita, as stated previously, at the vegetal pole near the midpolar area or a little to one side (fig. 116B). The sperm moves inward through the yellow-pigmented protoplasm and eventually becomes surrounded with the yellow and clear protoplasms (figs. 132C; 1 1 6B-F ) . This initial pathway through the superficial protoplasm of the egg constitutes the penetration path of the sperm (Wilhelm Roux). The sperm head in the meantime begins to swell and becomes vesicular (figs. 116F, J; 133B-G, Ascaris). The nucleus and the middle piece of the sperm with its forming aster now rotate 180 degrees, so that the aster lies anterior to the nucleus as it migrates within the egg (fig. 116F, I, J). The sperm aster thus precedes the pronucleus as the latter moves through the cytoplasm (fig. 1 16M) .

With the movement of the clear and pigmented protoplasmic substances upward toward the equator and to the point marking the future posterior end of the embryo, the sperm pronucleus and aster move upward. This latter movement of the sperm constitutes the copulation path, and it is formed at a sharp angle to the penetration path (figs. 116M, 139B)^he egg chromatin in the meantime undergoes its first and second maturation divisions (fig. 116F-L). After the second polar body has been formed, the haploid number of chromosomes reform the egg nucleus, now called the female pronucleus (fig. 116L, M). The latter then moves downward through the yolk along its copulation path to meet the sperm pronucleus near the posterior pole of the egg (figs. 116M-P; 139B). The actual meeting place in the clear cytoplasm is about halfway between the posterior pole and the center of the egg (fig. 139B) .

Shortly before the pronuclei meet, the sperm aster divides, each aster moving to opposite poles of the sperm pronucleus (fig. 116N). The two pronuclei now meet between the amphiaster of the first cleavage (fig. 1160, P) and thus become enclosed by the amphiaster spindle (fig. 116P). Following this association, the entire complex migrates toward the center of the egg together with a mass of clear cytoplasm. Some of the yellow protoplasm also migrates slightly centerward. The latter movement of the pronuclei toward the center of the egg is called the cleavage path. In the new position, slightly posterior to the egg's center, the pronuclei form an intimate association (figs. 116P, 139). The chromosomes then make their appearance, the nuclear membranes disappear, and the chromosomes line up in the metaphase plate of the first cleavage spindle preparatory to the first cleavage (fig. 116Q). The first cleavage plane always bisects the midplane of the future embryo and hence bisects the yellow and clear protoplasmic crescents (figs. 116R, S; 132F, G).

2) Fertilization of Amphioxus. The fertilization stages of Amphioxus are shown in figures 117A-I; 139C. The general process of fertilization in this species appears much the same as in Styela. However, in Amphioxus the fertilization phenomena cannot be studied as readily for a pigmented material is not formed in the peripheral cytoplasm. According to Conklin ('32), the general movements of the cytoplasmic substances resemble those of Styela. It is to be observed, however, that the copulation paths of the sperm and egg pronuclei, and also the cleavage path of the two pronuclei, are different slightly in Amphioxus from those present in Styela (fig. 139B, C).

3) Fertilization of the Frog's Egg. The egg of Rana pipiens is spherical and approximately 1.75 mm. in diameter as it lies in the uterine portion of the oviduct just before spawning. The size, however, may vary considerably. It has a darkly pigmented animal pole and a lightly colored vegetal pole. The first maturation division occurs when the egg is ovulated or shortly after ovulation during its passage through the peritoneal cavity en route to the oviduct (fig. 119B, C). The secondary oocyte then enters the oviduct, and during its passage posteriad in the latter, the maturation spindle of the second maturation division is formed (fig. 119D). The egg is in this condition when it is spawned. Immediately upon its entrance into the water, it is fertilized by the sperm from the amplectant male.

The sperm enters the egg at a point about 20 to 30 degrees down from the midregion of the animal pole. As it penetrates through the cortex of the egg, a trail of dark pigment from the egg's periphery flows in after the sperm (fig. 119H, I). This initial entrance path of the sperm constitutes the penetration path. After making its initial entrance, the sperm begins to travel toward its meeting place with the female pronucleus. This secondary path is the copulation path of the sperm (fig. 1 1 91). If the sperm should continue more or less in a straight line toward the egg pronucleus, the penetration path and copulation path would be continuous. However, if the sperm should veer away at an angle from the original penetration path in its journey to meet the female pronucleus, the copulation path would be at an angle to the penetration path.

The second maturation division of the oocyte occurs in about 20 to 30 minutes after sperm entrance with a surrounding temperature approximating 22° C. After the female pronucleus is organized, it migrates along its copulation path toward the meeting place with the sperm pronucleus, located near the center of the animal pole cytoplasm of the egg (fig. 1 19F, G).

Shortly after the sperm penetrates the egg, it revolves 180 degrees, and the middle-piece area travels foremost. This revolving movement, whereby the middle-piece area assumes a foremost position, is similar to that which occurs in the protochordates. Stye la and Amphioxus. This revolving movement appears to be characteristic of all sperm after entering the egg. (See figs. 116, 117, 131.) The sperm pronucleus gradually enlarges as it continues along the copulation path, and the first cleavage amphiaster arises in relation to the middle-piece region.

Fusion of the two pronuclei occurs at about one and one-half to two hours after fertilization at a normal room temperature of about 22° C. (fig. 119G). At about two and three-quarter hours after fertilization the first cleavage furrow begins (figs. 119J; 142A).

As stated above, the peripheral egg cytoplasm with its pigment tends to flow into the interior of the egg, following the trail of the sperm and thus forms a pigmented trail. The migration of the superficial cytoplasm with its pigmented granules is general over the upper pole of the egg and its direction of flow is toward the point of sperm penetration (see arrows, fig. 119K). Consequently, jaX a point on the egg's surface opposite the point of sperm entrance, the peripheral area of the egg becomes lighter in color and assumes a gray appearance. This area is crescentic in shape and is known as the gray crescent (fig. IlOK).

The formation of the gray crescent occurs in the cytoplasmic area just above the margin where the yellow-white vegetal pole material merges with the darkly pigmented animal pole. The gray crescent is continuous with the lighter vegetal pole material and is seen most clearly during the first cleavage of the egg. The plane which bisects the gray crescent into two equal halves represents the future median plane of the embryo.

In the frog, Rana jusca, Ancel and Vintemberger ('33) have shown that extensive movements of egg-surface materials accompanies the formation of the gray crescent. Sperm contact with the egg's surface thus appears to set in motion ooplasmic substances which fix the final symmetry of the egg and the future embryo.

4) Fertilization of the Teleost Fish Egg. When the egg of the teleost fish is spawned, the yolk lies near the center of the egg, and its yolk-free cytoplasm forms a peripheral layer. Around the egg the yolk-free cytoplasm is somewhat more abundant in the region where the egg nuclear material is situated. This concentration of the peripheral cytoplasm at the nuclear pole is more evident in the eggs of some species than in others. The area of nuclear residence is situated near the micropyle in many teleost eggs, but not in all. For example, the concentration of cytoplasm with the contained nuclear material is located in Bathygobius soporator at the opposite end to the micropyle (Tavolga, '50). (See fig. 123A.)

The sperm enters the egg through the micropyle (figs. 122, 123, 134A), and the actual processes of fertilization are initiated when the sperm makes contact with the peripheral ooplasm near the point where the egg nuclear material is located. This normally occurs in about a minute or less after the egg reaches the water. Within a few minutes the second polar body is given off. Meanwhile, the peripheral cytoplasm flows toward the area where the sperm has made contact, and a protoplasmic cap forms at this pole (figs. 122C; 123B-D). The remainder of the egg, with the exception of a thin layer of surface protoplasm, contains the deutoplasmic or yolk material. The egg is converted in this manner from a more or less centrolecithal egg into a strongly telolecithal egg. (Compare with Styela and Amphioxus.)

While these events are progressing, the egg as a whole contracts slightly, and a fluid is given off into the forming perivitelline space between the egg's surface and the vitelline membrane (fig. 122B, C). (However, a space between the egg membrane and the egg is evident to some extent in certain teleost eggs before the sperm enters the egg (fig. 123A).) The egg is now free to rotate within the perivitelline space, being cushioned and bathed by the perivitelline fluid.

The expansion of the vitelline membrane of the egg in certain teleosts is both dramatic and prophetic of the future shape of the embryo (fig. 123B-H).

Fig. 123. Development of the gobiid fish, Bathygobius soporator. (After Tavolga, '50, slightly modified.) (A) Freshly stripped egg. Adherent filaments at proximal end of chorion; micropyle at distal end. Peripheral cytoplasm partly concentrated at the pole of the egg containing the female nucleus. (B) Fifteen minutes after fertilization; cytoplasm concentrating at nuclear pole of the egg; chorion expanding into shape of future embryo. (C) Twenty minutes after fertilization; second polar body given off. (D) Twenty-five minutes after fertilization. (E) Ninety minutes after fertilization. (F) Seventeen hours after fertilization. (G) Twenty-four hours after fertilization. (H) Thirty hours after fertilization. (I) Thirty-six hours after fertilization. (J) Ninetysix hours after fertilization. (K) Ninety-six hours after fertilization. Hatching. (L) Three days after hatching, temperature 27 to 29°,

In demersal eggs, that is, eggs which sink to the bottom, the protoplasmic cap tends to assume an uppermost position. In pelagic eggs, i.e., eggs which float in the water, the protoplasmic disc turns downward since it is the heaviest part of the egg.

After the polar bodies are given off, the egg-chromatin material reforms the female pronucleus. The latter and the sperm pronucleus migrate to a position near the center of the protoplasmic disc. The first cleavage plane is established within thirty minutes to an hour following sperm entrance.

5) Fertilization in the Egg of the Hen and the Pigeon. Fertilization in the hen's egg occurs without any demonstrable movement of cytoplasmic materials, as manifested in the eggs of Styela, Amphioxus, frog, and teleost fish. The egg is strongly telolecithal, and the true protoplasm or blastodisc, which takes part in active development, is a flattened mass about 3 mm. in width. The germinal vesicle in the mature egg is approximately 350 /x in diameter and about 90 /x in thickness (fig. 126A). Approximately 24 hours before ovulation occurs, the wall of the germinal vesicle begins to break down, and the contained nuclear sap spreads in the form of a thin sheet below the ooplasmic membrane overlying the blastodisc (fig. 126B). (See Olsen, '42.)

Changes in the chromatin material of the germinal vesicle are synchronized with the breakdown of the membranous wall of the vesicle. The chromatin material, extremely diffuse during the period when the yolk material was formed and the egg as a whole was growing rapidly, contracts and assumes the character of thickened chromosomes in the tetrad condition. The diffuse diplotene state thus passes into the diakinesis stage (figs. 126C; 135A, show the breakdown of the nuclear wall and appearance of chromosomes in the pigeon).

Fig. 124. Fertilization stages in the rabbit egg. (A, B after Pincus, '39.) (A) Second polar body exuded; male and female pronuclei. (B) Twenty-two hours after copulation, showing two pronuclei close together. (C) Coagulated plug in infundibular portion of Fallopian tube, containing eggs. This plug is dissolved by sperm during fertilization process.

Fig. 125. Fertilization in the opossum. (A after McCrady, '38, from Duesberg; B-F after Hartman, '16.) (A) Conjugate sperm of opossum. (B) Ovarian egg showing discus proligerus around the egg; first polar body extruded; chromosomes of egg nucleus evident. (C) Tubal ovum. (D) Uterine ovum with pronuclei near center of the egg. (E) First cleavage spindle of uterine egg. (F) Two-cell stage, showing zona pellucida and exuded yolk material lying in perivitelline space.

Fig. 126. Maturation and fertilization in the hen's egg. (Drawings from photomicrographs by Olsen, '42.) (A) Cross section of germinal vesicle of almost mature egg, showing the general position and condition of the intact germinal vesicle. (B) Egg just prior to ovulation. Germinal vesicle spreading laterally as a thin layer below the ooplasmic membrane. (C) Chromatin material near center of disintegrating germinal vesicle (G.V.) of an egg estimated to be one hour prior to ovulation. (D) First polar body (1 P.B.) of recently ovulated egg. (E) Cross section of blastodisc of recently ovulated egg showing male pronucleus (^), female pronucleus (9), and second polar body (2 P.B.).

The first maturation division occurs and the first polar body is extruded shortly before ovulation (fig. 126D). The second maturation spindle is then formed. In this state the egg is ovulated. From four to six sperm penetrate into the egg shortly after it enters the infundibulum of the oviduct. The latter events are consummated within fifteen minutes after ovulation. The second maturation division then occurs, followed by the discharge of the second polocyte, which becomes manifest about the time of, or shortly before, the fusion of a single male pronucleus with the female pronucleus (fig. 126E). Thus, although polyspermy is the rule, only one sperm pronucleus takes part in the syngamic process.

After the two pronuclei become closely associated, the chromosomes become evident, the nuclear membranes disintegrate, and the first cleavage spindle is formed in about five and one-quarter hours after the sperm enters the egg (Olsen, '42).

In the egg of the pigeon, according to Harper ('04), the germinal vesicle breaks down, and the first polar spindle forms in the egg just before ovulation (fig. 135A, B). Fertilization then occurs just as the egg (in reality the primary oocyte) enters the oviduct. Normally from 15 to 20 sperm enter the blastodisc of the pigeon's egg. However, only one sperm pronucleus associates with the female pronucleus. Consequently, unlike the condition in the hen's egg, both maturation divisions occur and the first and second polar bodies are given off after sperm entrance (fig. 135C, D). Following the maturation divisions, the two pronuclei proceed to associate (fig. 135E, F). The first cleavage nucleus is shown in fig. 135G with two accessory sperm nuclei shown to the extreme left of the figure.

6) Fertilization in the Rabbit. In the rabbit, ovulation occurs around 10 to 1 1 hours after copulation. It takes about four hours for the sperm to travel to the upper parts of the Fallopian tube. (See Chap. 4.) The sperm thus lie waiting for about six to seven hours before the eggs are ovulated. When the eggs are discharged from the ovary, each egg is surrounded by its cumulus cells. The latter form the corona radiata, surrounding the zona pellucida (fig. 124A). As the eggs are discharged from their follicles, an albuminous substance from the follicles forms a clot, and several eggs are included within this clot (fig. 124C). A sperm, therefore, must make its way through the substance of the clot, as well as between the cells of the corona radiata, and then through the zona pellucida to reach the egg. This feat is accomplished partly by its own swimming efforts and partly also by means of an enzyme (or enzymes) which dissolves a pathway for the sperm. (See hyaluronidase, etc., mentioned on pp. 229.) The ferment hyaluronidase, associated with the sperm, frees the eggs from the albuminous clot and aids in the dissolution of the corona radiata cells, so that each egg lies free in the Fallopian tube, surrounded by the zona pellucida. It may be that some other lytic substance associated with the sperm also is active in aiding the sperm to reach the egg's surface.

Fig. 127. Maturation and fertilization in Echidna. (Courtesy, Flynn and Hill, '39.) (A) Oocyte, diameter 3.9 by 3.6 mm. Section of upper pole of egg showing saucer-shaped germinal vesicle lying in the germinal disc. (B) First polar spindle of egg just previous to ovulation. (C) First polar body and chromatin of female nucleus just previous to formation of second polar spindle shown in (D). (D) Second polar spindle of newly ovulated egg. Sperm presumably enters germinal disc at this time but possibly may wait until condition shown in (E) in some instances. (E) Second polar body and female pronucleus. (F) Male and female pronuclei. (G, H) Fusion stages of pronuclei.

The first maturation division of the egg occurs as the egg is being ovulated. The egg remains in this condition until the sperm enters, which normally occurs within two hours after ovulation. Thus, sperm entrance into the rabbit's egg presumably is much slower than in the case of the hen's egg, possibly due to the albuminous and cellular barriers mentioned above. Several sperm may penetrate through the zona pellucida into the perivitelline space, but only one succeeds in becoming attached to the egg's surface (Pincus, '39). The sperm tail is left behind in the perivitelline fluid, and the sperm head and middle piece “appear to be drawn into the egg cytoplasm rather rapidly” (Pincus and Enzmann, '32). The second polar body is then extruded, a process which ordinarily is completed about the thirteenth hour following copulation (fig. 124A). About three or four hours later (that is, about 17 hours after copulation) the two pronuclei are formed and begin to approach one another, and at 20 to 23 hours after copulation the pronuclei have expanded to full size and come to lie side by side (fig. 124B). The migration of the pronuclei to the center of the egg thus consumes about four to six hours. The spindle for the first cleavage division generally is found from 21 to 24 hours after copulation (Pincus, '39). (Consult also Gregory, '30; Lewis and Gregory, '29.)

7) Fertilization in the Echidna^ a Prototherian Mammal. The egg of the Tasmanian anteater, Echidna, when it reaches the pouch is about 15 by 13 mm. in diameter. This measurement, of course, is only approximate, and it includes the egg proper plus its external envelopes of albumen and the leathery shell. (The egg of Ornithorhynchus is slightly larger, approximating 17 by 14 mm.) At the time of fertilization in the upper portion of the Fallopian tube, the fresh ovum of Echidna without its external envelopes measures about four to 4.5 mm. in diameter.

The fully developed eggs of the monotreme (prototherian) mammals are strongly telolecithal, with a small disc of true protoplasm situated at one pole as in the bird or reptile egg. In Echidna aculeata this disc measures about 0.7 mm. in diameter during the maturation stages. Just before the onset of the maturation divisions of the nucleus, the germinal vesicle is saucer-shaped and lies in the midportion of the upper part of the disc (fig. 127A). The first maturation division (fig. 127B, C) occurs before ovulation, while the second maturation division (fig. 127D, E) occurs after ovulation. There is some evidence that the second maturation division, in some cases, may precede the actual entrance of the sperm into the germinal disc (Flynn and Hill, '39). In figure 127F-H, the stages in the association of the male and female pronuclei are shown.

As fertilization is accomplished, a rearrangement and movement of the ooplasmic substances of the germinal disc occur. As a result, the blastodisc, circular during the maturation period, becomes transformed into an ovalshaped affair with the polar bodies situated on one end (fig. 136). The first plane of cleavage is indicated by numerals I-I, and the second plane of cleavage by numerals II-II. A distinct bilateral symmetry thus is established by the rearrangement of ooplasmic materials during the fertilization process. (Compare with Styela, Amphioxus, and frog.)

£. Significance of the Maturation Divisions of the Oocyte in Relation to Sperm Entrance and Egg Activation

As indicated in the foregoing pages, the maturation divisions of the oocyte vary greatly in different animal species. Figure 137 shows that the time of sperm entrance in the majority of eggs occurs either before or during the maturation divisions, that is, when the female gamete is in the primary or secondary oocyte condition. In some animals, however, the sperm enters normally after the two maturation divisions are completed.

The correlation between the maturation period of the egg and sperm entrance indicates that the breakdown of the germinal vesicle and the accompanying maturation divisions has a profound effect upon the egg. This conclusion is substantiated by experimental data. For example, A. Brachet ('22) and Runnstrbm and Monne ('45, a and b), working on the sea-urchin egg, found

Fig. 128. Formation of the vitelline membrane in the egg of A scans after fertilization. (After Collier, '36.) (A) Heavy cell wall (vitelline membrane) is beginning to thicken.

(B) Cell wall is reaching condition of maximum thickness. (C) Egg contracts away from vitelline membrane, leaving perivitelline space filled with fluid-like substance, forming the typical fertilized egg of Ascaris as ordinarily observed.

that several sperm enter the egg in the sea urchin if insemination is permitted before the maturation divisions occur. The immature egg, therefore, lacks the mechanism for the control of sperm entrance. Moreover, A. Brachet ('22) and Bataillon ('29) demonstrated that the sperm nuclei and asters behave abnormally under these conditions, and normal development is impossible. Runnstrom and Monne have further shown for the sea-urchin egg that the normal fertilization process, permitting the entrance of but one sperm, requires a mechanism which is built up gradually by degrees during the time when the maturation divisions of the egg occur, even extending to a necessary short period after the divisions are completed. Not only is the mechanism which permits but one sperm to enter the egg established at this time in the seaurchin egg, but Runnstrom and Monne further conclude, p. 25, “that the cytoplasmic maturation” which occurs at the period of the maturation divisions, “involves the accumulation at the egg surface of substances which participate in the activating reactions.” It appears, therefore, that the breakdown of the germinal vesicle together with the phenomena associated with the maturation divisions is an all-important period of oocyte development, controlling sperm entrance on the one hand and, on the other, presumably being concerned with formation of substances which permit egg activation.

F. Micropyles and Other Physiologically Determined Areas for Sperm Entrance

A micropyle is a specialized structural opening in the membrane or membranes surrounding many eggs which permits the sperm to enter the egg. For example, in the eggs of teleostean fishes or in the eggs of cyclostomatous fishes, a small opening through the vitelline membrane (or chorion) at one pole of the egg permits the sperm to enter (figs. 93 A; 134A-F). On the other hand, many chordate eggs do not possess a specialized micropyle through the egg membrane. The latter condition is found in the protochordates, Styela and Arnphioxus, and in vertebrates in general other than the fish group. In Styela and Arnphioxus the sperm enters the vegetal pole of the egg, i.e., the pole opposite the animal or nuclear pole. In most of the vertebrate species the sperm enters the animal or nuclear pole of the egg usually to one side of the area where the maturation divisions occur (figs. 115, 118, 1191). In urodele amphibians, the passage of several sperm into the egg at the time of fertilization complicates the picture. However, the sperm which finally conjugates with the egg pronucleus is the one nearest the area where the egg pronucleus is located. The several sperm entering other parts of the egg ultimately degenerate (fig. 138). Presumably this condition is present in reptiles and birds where many sperm normally enter the egg at fertilization.

In conclusion, therefore, it may be stated that the point of sperm entrance in chordate eggs in general appears to be definitely related to one area of the egg, either by the presence of a morphologically developed micropyle or off from the plasma surface of the egg, cortical granular material is exuded from the egg cortex and passes out across the perivitelline space toward the fertilization membrane. (2) Cortical granules begin to consolidate with the vitelline membrane. (3) Fully developed fertilization membrane is formed by a union of the vitelline membrane with the cortical granules derived from the egg cortex.

Fig. 129. Formation of the fertilization membrane in the mature egg of the sea urchin. (A) Surface of the egg and surrounding jelly coat before fertilization. (After Runnstrom, '49, p. 245.) (Bl, 2, 3, 4, 5) Point x marks the point of sperm contact. The fertilization membrane separates from the egg at the point of sperm contact and spreads rapidly around the egg from this point. (After Just, '39, p. 106.) (C) Membrane formation in greater detail. (After Runnstrom, '49, p. 276.) (I) As the vitelline membrane is lifted

by some physiological condition inherent in the organization of the egg. In the majority of chordate eggs, the place of sperm penetration is at that pole of the egg which contains the egg nuclear material, although in some, such as in the gobiid fish (fig. 123), the micropyle, permitting the sperm to get through the egg membrane, may be situated at a point opposite the nuclear pole of the egg.

G. Monospermic and Polyspermic Eggs

In the eggs of most animal species only one sperm normally enters the egg. Such eggs are known as monospermic eggs. Among the chordates, the eggs of Styela, Amphioxus, frog, toad, and mammals are monospermic. Abnormal cleavage and early death of the embryo is the general result of dispermy and polyspermy in frogs (Brachet, A., '12; Herlant, 'll). In those chordates whose eggs possess much yolk, the eggs are normally polyspermic, and several sperm enter the egg at fertilization, although only one male pronucleus enters into syngamic relationship with the egg pronucleus; the other sperm soon degenerate and die in most cases (fig. 138). (See Fankhauser, '48.) In some urodele amphibia, it appears that syngamic conjugation of more than one sperm pronucleus with the egg pronucleus may occur in certain instances and may give origin to heteroploidy, and development may be quite normal (Fankhauser, '45). Examples of normal polyspermic eggs are: birds, reptiles, tailed amphibia, elasmobranch fishes, and possibly some teleost fishes. Among the invertebrates, polyspermy is found in some insects and in the Bryozoa. In the sea urchin, polyspermy may occur, but abnormal embryos are the rule in such cases as indicated above. Similar conditions are found in certain other invertebrates (Morgan, '27, pp. 84-86).

Two explanations of normal polyspermy are suggested:

( 1 ) The presence of a superabundance of yolk hinders the operation of the mechanism whereby the egg inhibits the entrance of extra sperm; the egg, therefore, falls back upon a second line of defense within its own substance which excludes the sperm from taking part in or hindering the normal functioning of the syngamic nucleus in its relation to development; and

(2) a large amount of yolk makes it advantageous to the egg for extra sperm to enter, as they may contribute enzymes or other substances which enable the egg better to carry on the metabolism necessary in utilizing yolk material.

H. Importance of the Sperm Aster and the Origin of the First Cleavage Amphiaster

One of the older views of fertilization maintained that the egg possessed the cytoplasm but lacked a potent centrosome or “cell center” capable of giving origin to the first cleavage amphiaster, whereas the sperm possessed a dynamic centrosome with its included centriole but lacked sufficient cytoplasm for division or cleavage. Consequently, fertilization brought together a relationship necessary for cleavage and development. This idea was first set forth by Boveri (see fertilization theories at the end of this chapter).

Fig, 130. Fertilization in Nereis. (After F. R. Lillie, '12.) (A) Sperm of Nereis, entire. (B) Egg of Nereis, 15 minutes after insemination. The fertilization cone is evident below point of sperm contact. Observe that the intact germinal vesicle is present in the center of the egg. It will break down as the sperm enters the egg (G). The cortical substance from the empty cortical compartments in the cortical layer shown around the periphery of the egg has passed out through the vitelline membrane to form the jelly layer around the egg. (C-G) Entrance of the sperm head into the ooplasm of the egg as the fertilization cone substance is withdrawn inward from the vitelline membrane. (C) Fifteen minutes after insemination. (D) Thirty-seven minutes after insemination. (E) Forty-eight and one-half minutes after insemination. (F) Fifty-four minutes after insemination. (G) Sperm head has completed penetration. Observe that the middle piece of the sperm remains outside, attached to the vitelline membrane. Anaphase of first maturation division.

In the majority of animals, the central body (i.e., the centrosome) with its surrounding aster, which ultimately divides and gives origin to the first cleavage amphiaster, does not arise until after the sperm has entered the egg. In these cases the aster complex arises in the middle piece of the sperm in close proximity to the nucleus. These facts are well illustrated in figures 116, 117, and 131. Many studies of the fertilization process and early cleavage bolster this general conclusion. There are some exceptions, however, to this rule. For example, Wheeler ( 1 895) in his studies of fertilization in Myzostoma glabrum demonstrated that the centrioles of the egg near the germinal vesicle give origin to the amphiaster concerned with polar body formation. Following the maturation divisions, the female pronucleus with its centrioles and forming amphiaster, migrates along the copulation path to meet the sperm pronucleus. The amphiaster and centrioles are closely adherent to the egg pronucleus during the migration of the latter. In the honeybee, Nachtsheim (M3) found a similar situation, while in the mollusk, Crepidula plana, Conklin ('04) found evidence which suggests that one aster of the cleavage amphiaster arises from the egg, whereas the other aster arises from the sperm, “although there is not positive evidence that they are directly derived from egg and sperm centrosomes.”

Where the egg develops as a result of artificial stimulation the first cleavage spindle arises without the aid of the sperm middle piece. In these instances the amphiaster probably is derived from the central body of the last maturation division, or, it may be, from certain asters or cytasters artificially induced in the egg cytoplasm by the activation process. The production of numerous asters in the cytoplasm of the egg by artificial stimulation has long been known (Mead, 1897; Morgan, 1899, '00).

The general conclusion to be extracted from the evidence at hand, therefore, suggests that the central body from the last maturation spindle or other artificially induced asters in the egg cytoplasm may form the amphiaster of the first cleavage spindle in the case of an emergency. Such an emergency arises in normal parthenogenesis or in cases of artificial activation (artificial parthenogenesis) of the egg. However, under the conditions of normal fertilization the sperm aster fulfills the role of developing the first cleavage amphiaster.

Regardless of the fact that the first cleavage amphiaster appears to be derived from the middle piece of the sperm, the influence of the egg protoplasm is undoubtedly an important factor in its formation. In the normal polyspermy of the newt, Triton (fig. 138; Fankhauser, '48), the sperm aster nearest the egg pronucleus enlarges and develops the amphiaster, whereas the more distantly located sperm asters fade and disintegrate. This fact suggests that some influence from the egg pronucleus stimulates the further development of the amphiaster in the sperm nearest the egg pronucleus. In experiments on insemination of egg fragments in the urodele, Triton, Fankhauser ('34) found that the sperm aster in that fragment which did not contain the egg nucleus failed to reach the size of the aster in the fragment containing the egg nucleus. He concludes, p. 204, “The interactions between the sperm complex and the cytoplasm of the egg seem, therefore, to be stimulated in the presence of the egg nucleus.”

On the other hand, the experiments on androgenesis by Whiting ('49) in Habrobracon, and the insemination of the “red halves” of the sea-urchin egg by Harvey ('40) demonstrate that the sperm aster can, without the egg pronucleus, produce the first cleavage amphiaster. However, the presence of a nucleoplasmic substance in both of these cases cannot be ruled out. For example, A. Brachet ('22) and Bataillon ('29), the former working on the sea-urchin egg and the latter on the eggs of two amphibian species, demonstrated that large, normal sperm asters and large vesicular sperm nuclei do not form until after the germinal vesicle breaks down and the egg becomes mature. Premature fertilization results in polyspermy, small sperm nuclei, and small sperm asters. In normal fertilization, therefore, it is very probable that the development of the sperm aster into a normal cleavage amphiaster is dependent:

( 1 ) upon the egg cytoplasm, and

(2) upon some factor contributed to the egg cytoplasm by the nuclear sap or from the chromosomes of the female nucleus at the time of the breakdown of the germinal vesicle or during the maturation divisions.

I. Some Related Conditions of Development Associated with the Fertilization Process

1. Gynogenesis

The word gynogenesis means “female genesis.” Therefore, gynogenesis is the development of the egg governed by the female pronucleus alone. The male gamete may enter the egg but plays no further role (Sharp, '34, p. 406; Wilson, '25, p. 460). In the nematode, Rhabdites aberrans, the egg produces but one polar body, and diploidy is retained. The egg is penetrated by the sperm which takes no part in later development, as it degenerates upon entering the egg.

In the above instance, it is doubtful whether or not sperm is necessary to activate the egg. However, in the nematode, Rhabdites pellio, the egg is penetrated by the sperm which plays no further role in development. Nevertheless, in the latter instance, sperm entrance appears to be necessary for egg activation. A somewhat similar phenomenon may also occur in other animal species taking part in hybrid crosses, where some or all of the paternal chromosomes may be eliminated; activation normally occurs in these instances, and development results. Gynogenesis is experimentally produced in amphibia by radiating the sperm before fertilization. Development is carried on by the female pronucleus in the latter instance, although it may produce larvae which ultimately die. Parthenogenesis, natural and artificial, in all its essential features in a sense may be regarded as gynogenesis.

Fig. 131. Fertilization in the sea urchin, Toxopneustes variegatus. (After Wilson and Mathews, 1895.) (A) Sperm head and middle piece. (B) Fertilization cone (attrac tion cone; “cone of exudation” of Fol). The fertilization cone forms after the sperm head and middle piece have entered the egg, and persists through (F) when the pronuclei begin to come together. (C-J) Different stages in the fusion of the pronuclei. Observe that the sperm rotates at about 180® and that the sperm aster appears near base of nucleus (D, E). The aster grows rapidly (F, G) as the sperm pronucleus advances toward the female pronucleus, and appears between the two pronuclei in (G). In (H) the aster has divided, and the daughter asters are found at either end of the two fusing pronuclei. In (I, J) the two asters are at either end of the fusion nucleus. (J) Fusion nucleus between the amphiaster of the first cleavage.

2. Androgenesis

This form of development is experimentally produced by removal of egg pronucieus with a small pipette before nuclear syngamy occurs (Porter, '39) or by treating the egg with x-rays before fertilization (Whiting, '49). The male pronucleus seems incapable of bringing about normal and full development in amphibia, but in wasps, where the egg pronucleus has been destroyed by radiation, it has been successful (Whiting, '49).

Fig. 132. Movement of ooplasmic substances in the egg of Styela (Cynthia) partita at the time of fertilization. (All figures after Conklin, '05.) (A) Unfertilized egg after

the disappearance of the nuclear membrane of the germinal vesicle. The gray yolk is shown in the center of the egg, surrounded by the yellow-pigmented cytoplasm. The test cells and chorion surround the egg. (B) Egg five minutes after fertilization, showing the streaming of the peripheral protoplasm indicated by arrows toward the vegetal pole, where the sperm has entered. The gray yolk is shown in the upper part of the egg below the nuclear material. The clear protoplasm, derived from the nuclear sap of the germinal vesicle, also flows down with the peripheral protoplasm. (C) Side view of an egg after peripheral protoplasm has migrated to the vegetal pole of the egg. The clear protoplasm is shown at the upper edge of the yellow cap. The polar bodies are forming in the midpolar area (MP.) at the animal pole. (D) Side view of the egg showing the yellow crescent and the area of clear protoplasm above the yellow crescent. The yolk material is shown at the vegetal pole below and to one side of the yellow crescent. (E) Yellow crescent and clear protoplasm viewed from the posterior pole of the egg. Animal pole is above the crescent; yolk material is below. (F) Same view as (E) a little later, showing the external beginnings of the first cleavage. The polar bodies are shown above the crescent material. (G) View similar to that of (E, F) a little later. The first cleavage is complete. Observe that the clear protoplasm and the yellow crescent have been bisected equally. The cleavage plane corresponds to the median axis of the embryo.

3. Merogony

Merogony is the development of part of the egg, that is, an egg fragment. Egg fragments are obtained by shaking the egg to pieces, by cutting with a sharp instrument, or by the use of centrifugal force. Andromerogony is the development of a non-nucleate egg fragment after it has been fertilized by

Fig. 133. Stages in the fertilization of Ascaris. (After Boveri, 1887.) (A) Ameboid

sperm on the periphery of the egg; germinal vesicle in center of primary oocyte. (B) Sperm entered egg substance; germinal vesicle broken down and tetrads becoming evident.

(C) Sperm in center of the egg; first maturation division, showing tetrads on spindle.

(D) Second maturation division; first polar body at egg's surface. (E) First and second polar bodies shown; sperm aster forming in relation to sperm. (F) Second polar body; male and female pronuclei. (G) Male and female haploid chromosomes evident; amphiaster forming. (H) Chromosomes distinct, showing haploid condition. Observe amphiaster. (I) Amphiaster complete; metaphase of first cleavage.

Fig. 134. Micropyle and egg membranes of certain fishes. (A) Micropyle, egg membranes, and germinal vesicle in Lepidosteus. (Modified from three figures drawn by E. L. Mark, Bull. Mus. Comp. Zool. at Harvard College, 19: No. 1.) (B-F) Micropyle

and egg membranes of the cyclostome, Petromyzon planeri. (Slightly modified from Calberla, Zeit. Wiss. Zool., 30.) (B) Mature, unfertilized egg. (C) Sperm passes

through the micropyle and enters the protoplasmic strand, P.S. (D) Higher power view of sperm in protoplasmic strand; also observe that the egg is shrinking away from the egg membrane, forming the peri vitelline space. (E, F) Egg contracts away from the egg membrane, leaving the egg free to revolve within the membrane.

sperm. Development is not normal and does not go beyond the larval condition. Parthenogenetic merogony is the development of non-nucleate parts of the egg which have been artificially activated. Artificial activation of nonnucleate parts of the egg of the sea urchin, Arbacia, is possible by immersion of these parts of the egg for 10 to 20 minutes in sea water, concentrated to about one half of the original volume, or by the addition of sodium chloride to sea water to bring it to a similar hypertonicity (Harvey, '36, '38, '51).

These parthenogenetic merogons develop to the blastula stage only. Gynomerogony is the parthenogenetic development of an egg fragment containing the egg pronucleus.

Theories of Fertilization and Egg Activation

Boveri, T., 1887, 1895. In somatic mitoses, the division center or centrosome is handed down from cell to cell. In the development of the female gamete, the division center degenerates or becomes physiologically incapable of continuing the division of the egg either before or after the maturation divisions. The mature egg thus contains all the essentials for development other than a potent division center. The sperm, on the other hand, lacks the cytoplasmic conditions necessary for development, but possesses an active division center which it introduces into the egg at fertilization. Fertilization, therefore, restores the diploid number of chromosomes to the egg and introduces an active division center.

Fig. 135. Fertilization phenomena in the pigeon. (After Harper, '04.) (A) Germinal

vesicle of late ovarian egg. The chromatin material is shown in the center of the vesicle; the nuclear wall is beginning to break down. (B) Spindle of first maturation division. Egg just ovulated and entering the oviduct. Sperm enters the egg at this time. (C) Second polar spindle and first polar body. (D) First and second polar bodies; egg pronucleus reorganizing. (E) Two pronuclei approaching, preparatory to fusion. Sperm nucleus to the left. (F) Two pronuclei fusing. (G) Accessory sperm nuclei to the left of this figure; fusion nucleus to the right.

Fig. 136. Organization of germinal disc of the Echidna egg following fertilization.

(After Flynn and Hill, '39.)

Loeb, J., '13. Loeb believed that two factors were involved in egg activation: (a) Superficial cytolysis of the egg cortex which leads to a sudden increase in the oxidation processes of the egg, and (b) a factor which corrects cytolysis and excess oxidation, thus restoring the egg to normal chemical conditions. He placed great emphasis on superficial cytolysis of the cortex with the resultant elevation of the fertilization membrane.

Loeb suggested that in normal fertilization the sperm brings in a lytic principle which brings about cortical cytolysis, and a second substance which regulates oxidation.

For discussion of this theory, consult J. Brachet, '50, p. 138.

Bataillon, E., '10, 'll, '13, '16. Like Loeb, Bataillon emphasized two steps in the activation process of the egg: (a) First treatment, whether it is the puncture of the frog's egg by a fine needle or the butyric acid treatment of the egg of the sea urchin, according to the method of Loeb, causes; ( 1 ) elevation of fertilization membrane and the excretion of toxic substances from the egg, and (2) the formation of a monaster, (b) Second treatment, whether it is blood, in the case of the frog, or hypertonic sea water, as used by Loeb in the sea-urchin egg, introduces a catalyzer which converts the monaster into an amphiaster, and in this way renders the egg capable of cleavage.

Bataillon placed great emphasis upon the exudation (excretion) of substances into the perivitelline space and the elevation of the fertilization membrane. He believed that the unfertilized egg was inhibited because of an accumulation of metabolic products and that activation or fertilization led to a release of these substances to the egg's exterior.

For discussion, consult Wilson, '25, p. 484; J. Brachet, '50, p. 144.

Lillie, F. R., '14, '19. This author postulated that a substance, fertilizin, carried in the cortex of the egg, exerts two kinds of actions in the activation process: (1) An activating, attracting, and agglutinating action on the sperm, and (2) an aetivating effect on the egg itself. In essence, the egg is selffertilizing, for the fertilizing substance is present in the egg. The procedure is somewhat as follows: At the period optimum for fertilization, inactive fertilizin (i.e., inactive from the viewpoint of possessing the ability to activate the egg) is produced by the egg. Released into the surrounding water, it activates, attracts, and agglutinates the sperm at the egg's surface. As the sperm touches the egg, it unites with a part of the fertilizin molecule. The fertilizin molecule plus the sperm then have the ability to unite with an egg receptor, and the union of the fertilizin-sperm complex with the egg receptor, releases the activating principle within the egg, which spreads â€with extreme rapidity" around the egg cortex. The activating principle activates the egg as a whole, setting it in motion toward development. It is thought to work especially upon the cortex of the egg, producing cortical changes, including the formation of a fertilization membrane. Further, it agglutinates or immobilizes all other sperm around the egg. Consequently, polyspermy may be hindered by this agglutination effect and by the fertilization membrane. In regard to polyspermy, Lillie also postulated another substance, antifertilizin, within the egg which unites with the remaining fertilizin molecules in the egg the instant that one sperm has made successful union with a molecule of fertilizin, thus preventing other sperm from entering the egg.

Fig. 137. Maturation divisions of the oocyte relative to time of sperm entrance. (A) Sperm enters the primary oocyte before maturation divisions. In some, e.g.. Nereis, Thalassema, Ascaris, Fluty nereis, Myzostonui, etc., the sperm enters before the germinal vesicle breaks down; in Styela, Chaetopterus, pigeon, etc., the first maturation spindle is formed or forming; in the dog, the condition is somewhat similar to Nereis, Ascaris, etc. (B) Sperm enters the egg after first maturation division, i.e., in secondary oocyte stage (Asterias (starfish), Amphioxus, hen, rabbit, man, frog, salamander, newt, most vertebrates). (C) Sperm enters the egg after maturation divisions are completed, i.e., in the mature egg {Arhacia and other sea urchins; possibly in monotreme. Echidna, on occasion).

Fig. 138. Polyspermy in the European newt, Triton. (After Fankhauser, '48.) (A) Ten minutes after insemination at 23® C. Metaphase of second maturation division; four sperm have entered the egg, one of which is at the vegetal pole of the egg, and another between the two poles of the egg. (B) One hour and 30 minutes; second polar body given off; small egg pronucleus moves toward nearest sperm nucleus. The latter will become the principal sperm nucleus. Observe that accessory sperm nuclei are enlarging and a sperm aster is developed relative to each. (C) Two hours and 30 minutes. Egg and principal sperm pronuclei in contact; maximum development of sperm asters. (D) Three hours. Fusion of egg pronucleus and principal sperm pronucleus. Accessory sperm nucleus nearest to fusion nucleus shows signs of degeneration. Accessory sperm asters remain undivided, while principal sperm aster has formed an amphiaster. (E) Three hours and 30 minutes. Metaphase of first cleavage; all accessory sperm nuclei degenerating. (F) Four hours. Early telophase of first cleavage; remnant of accessory nuclei being pushed out of animal pole region by amphiaster and spindle of first cleavage division.

For discussion, see J. Brachet, '50, p. 143; Dalcq, '28.

Lillie, R. S., '41. Like Loeb, R. S. Lillie conceived of cortical changes as being the main aspect of activation, particularly changes such as a decrease in viscosity which permits interaction of various substances which normally are kept separated in the unactivated egg. Lillie's hypothesis may be stated as follows: An activating substance, comparable to a growth hormone or auxin, is formed in the egg. This substance may be called (A). The formation of (A) results from the interaction of two substances, (S) and (B), present in low concentrations in the egg. One of these substances, (S), is synthesized in the egg by treating the egg in various ways, such as immersion in sea water in the presence of oxygen. The other substance, (B), is freed from pre-existing combination by a simple splitting (hydrolytic) process initiated or catalyzed by acid. This reaction is independent of oxygen. The union of the two substances, (S) and (B), forms the activating substance, (A). Lillie thus believes in a single factor as the initiator of development. Complete activation of the egg results when (A) is produced in adequate concentration; partial activation occurs when it is present in quantity below the optimum concentration.

, For discussion, see Brachet, J., '50, p. 141.

Hcilbrunn, L. V., '15, '28, '43. This author believes that an increase in viscosity with resultant coagulation or gelation of egg cytoplasm is involved directly with the initiation of development. Heilbrunn regards this gelation process to be similar to the clotting of blood. He also regards calcium as the main agent in bringing about this effect, and therefore believes calcium to be concerned directly with egg activation. According to this view, calcium is bound to the proteins localized in the egg cortex. At the time of activation, artificially or by sperm contact, part of this calcium is liberated which in turn produces a coagulation of the cytoplasm, initiating development. Dalcq and his associates also have emphasized the importance of calcium in the activation process.

For discussion, see Brachet, J., '50, p. 146; Dalcq, '28; Runnstrom, '49.

Runnstrom, J., '49. Runnstrom more recently has contended that an inhibitor of proteolytic enzymes may be present in the vitelline membrane and cortex of the egg. He assumes that the inhibitor, possibly fertilizin, may be identical with a heparin-like substance. He further assumes that the inhibitor is bound to a kinase and is released when protein substances associated with the sperm unite with the inhibitor. - A kinase acting on a proenzyme may then be released ; the latter, i.e., the kinase, acts upon the proenzyme in the cortex of the egg, giving origin to an enzyme or enzymes which initiate development. Runnstrdm's position in essence is a modern statement of the inhibition theory of F. R. Lillie (see J. Morphol., vol. 22).


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