Popular Science Monthly/Volume 86/June 1915/Fertilization and Artificial Parthenogenesis of the Egg

by Jesse Francis McClendon

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FERTILIZATION AND ARTIFICIAL PARTHENOGENESIS OF THE EGG

By Dr. J. F. McCLENDON

UNIVERSITY OF MINNESOTA MEDICAL SCHOOL

TICHOMIROFF, in 1886, was the first to use the term artificial parthenogenesis, referring to acceleration in the development of the naturally parthenogenetic eggs of the silkworm by methods found effective in hastening development in fertilized eggs of the same species. To-day the term is applied to development of eggs not usually parthenogenetic, although a few such might develop in nature under accidentally abnormal conditions.

The exact extent of development that is to be dignified by this term is a matter of dispute, some claiming it should be possible to produce an adult reproductive organism by artificial parthenogenesis. Though Delage obtained two sea urchins in this manner and more than one observer has so produced frogs, none of these reproduced a second generation, a fact not hard to understand on remembering that normally fertilized eggs of many animals have never been reared to maturity and reproductive activity under observation. Loeb considers a swimming larva to be the goal of the investigator. But it is interesting to note that the "swimming larvæ" of the marine worm Chætopterus, which he produced from unfertilized eggs, were shown by F. Lillie to be abnormal, unsegmented or poorly segmented eggs that had developed cilia.

We may consider for a moment what signifies development in the egg. The egg of any animal is in the beginning a single cell and undergoes a certain development before normal fertilization. Some animals reproduce parthenogenetically for several generations (i. e., plant lice) and the silkworm eggs, noted before, undergo more or less development if fertilization fails to occur. The eggs of most animals, however, do not segment (produce an embryo) before fertilization. Though in many of these same species (for example, sea urchin) the eggs (during maturation) undergo before fertilization two very unequal cell divisions, resulting in the formation of "polar bodies," the unfertilized egg is generally considered a single cell, since the "polar bodies" seem to have no further part in the formation of the embryo. The positive sign of development in the mature egg seems to be segmentation. We may therefore consider artificial parthenogenesis to be demonstrated by the segmentation of unfertilized eggs which do not normally segment until fertilized.

R. Hertwig was the first to observe the segmentation of unfertilized sea-urchin eggs, which normally die if not fertilized. Morgan, in 1899, produced segmentation in unfertilized sea-urchin (Arbacia) eggs, by immersing them in sea water to which a dry salt had been added, such a solution being called hypertonic sea water, and having a greater osmotic pressure than ordinary sea water. Loeb, immediately afterwards, produced swimming larvæ in sea water made hypertonic by the addition of magnesium chloride. Since then many investigators have studied the subject. Eggs of various animals have been made parthenogenetic by putting them in solutions containing salts, acids or alkalis, sugar, fat solvents, blood sera, alkaloids, or by means of asphyxiation, or by mechanical, thermal or electric changes. The concentration of the solution in which the eggs are treated may be the same as that of the fluid in which they normally live, or it may be of a greater or less concentration.

It has been fairly well demonstrated that the artificial agents—used in producing parthenogenesis—act primarily on the surface of the egg, and R. Lillie supposes they tend to increase its permeability. Loeb recognizes a "superficial cytolysis," the exact nature of which is, however, unknown. When cells containing soluble coloring matters undergo cytolysis, the colored substances come out of the cells. Cytolysis has, therefore, been considered to consist of, or be accompanied by, an increase in permeability of the protoplasm.

The electric conductivity method of Kohlrausch suggested itself to the writer as the best way of settling this question of the permeability of the egg. The principle of the method lies in the fact that an electric current is carried through wet substances by the movement of electrically charged atoms or "ions." If the permeability increases, the ions move faster and the current is greater. The use of this method showed that the permeability increased immediately after fertilization, on the application of agents producing parthenogenesis.^[1] These results were confirmed by Gray,^[2] who observed further that the permeability decreased again about fifteen minutes after fertilization. Lyon and Shackell^[3] observed that the permeability of the egg to certain dyes increased on fertilization, and also that more of the red substances came out of the fertilized than out of the unfertilized eggs. E. N. Harvey observed independently that the permeability of the eggs to certain dyes and caustic soda increased on fertilization. Finally, Glaser has recently concluded from his experiments that fertilization increases the egg's permeability.

There is another proof that the egg of one species (the frog) becomes more permeable to salt on beginning development. The writer observed that the frog's egg may be made parthenogenetic by means of a momentary electric shock. Unfertilized eggs of a frog were divided into two equal lots, placed in distilled water, and one lot shocked electrically. It was found that three times as much salt diffused out of the shocked eggs as out of the control. Since the salt must have come from the interior of the eggs, the experiment seems to prove that the eggs must have been permeable to it. The shocked eggs began to segment and behaved in other ways as if normally fertilized.

There seems to be no doubt that the permeability of the egg is increased by agents producing parthenogenesis, but just how this influences the egg's development is not absolutely settled, because of the many processes in development which are very far from being solved. Some of these processes have been the subject of numerous investigations. The outward change in form during the segmentation of the egg is caused by changes in surface tension. Granted that fertilization alters the permeability of the egg, it may be that the changes in permeability influence the surface tension.^[4] The unfertilized eggs of sea urchins, frogs and many other animals are surrounded by jelly-like coats, the inner layer of which lies close to the egg. On fertilization, the jelly is pushed out by the perivitelline fluid exuding from the egg, the space occupied by the fluid being called the perivitelline space. The inner layer of the jelly looks like a distinct membrane and is called the "fertilization membrane." Loeb considers its formation of great importance. Biataszewicz has shown that the frog's egg shrinks as this fluid is "secreted." Glaser has observed the same phenomenon in the sea urchin's egg, though the shrinkage is so slight that other observers deny its taking place. Granting that the perivitelline fluid comes from the egg, the increase in permeability would facilitate its migration.

If the jelly be removed from the sea urchin's egg prior to fertilization, no "fertilization membrane" appears. Presumably the fluid is secreted but lost in the surrounding water.^[5] Though the membrane helps to protect the embryo, its existence is not absolutely essential, since eggs lacking it (due to the removal of the jelly) have been known to develop. Many observations and experiments have demonstrated to the writer that the tough "fertilization membrane" of the sea-urchin's egg does not exist (at least in its final condition) before fertilization. The increase in permeability allows the escape of the perivitelline fluid which, according to the hypothesis advanced in 1911, interacts with the jelly and forms the "fertilization membrane."^[6] Elder, in 1913, came to hold the same view. E. N. Harvey, though believing that the membrane is not present before fertilization, considers the jelly unnecessary for its formation, holding that the membrane substance hardens on contact with sea water. He admits that unfertilized eggs from which the jelly is removed soon lose their power of forming membranes on fertilization, but says they do not lose it immediately. Perhaps he left a thin film of jelly adhering to the eggs or had not removed the water containing the dissolved jelly. This dissolved jelly may be in time decomposed by bacteria and thus prevent membrane formation. If eggs with jelly remain in sea water fifty-two hours, they do not form membranes on fertilization.

When a sea urchin's egg is fertilized, an increase in the rate of respiration occurs, as shown by O. Warburg. This may be due to some physical change, and is to be expected, since the egg passes from a state of inactivity to one of activity. When the starfish egg is liberated from the body of the female into the sea, it becomes active to the extent of extruding the polar bodies. Loeb and Wasteneys found that respiration was high at the time of formation of the polar bodies in the starfish egg, and continued about the same level, whether fertilized or not. The egg may pass through an inactive stage while in the ovary, with corresponding low respiration. Coming in contact with sea water may stimulate it toward development, with resulting maturation and increased respiration, though the stimulus is not sufficient to cause segmentation. This is in harmony with the fact that much weaker stimuli cause segmentation in starfish than are required by sea urchin eggs. The frog's egg resembles the former and Batallion has shown that the slight prick of a needle is sufficient to cause the frog's egg to segment, while needles have been thrust by the writer all the way through sea urchin's eggs without causing either segmentation or death.

O. Warburg has shown that the respiration of all developing eggs is high, regardless of the methods used to cause segmentation. Respiration is therefore essential to development. Cleavage once started may be slowed or stopped entirely without materially decreasing respiration, indicating that respiration is not a result of cleavage. In order to discuss the relation of respiration to development, it is necessary to go more into detail on the general question of respiration.

 

Oxidation or Cell Respiration

As is well known, the heat of a flame is unnecessary for the burning (oxidation) of many substances. For example, coal oxidizes slowly in the air, decreasing in weight, a fact which has led to efforts to preserve its fuel value by keeping it under water. Naturally, even slow combustion generates heat, and if the heat be confined, results in spontaneous combustion, i. e., the raising of the temperature to the flame point. Many substances burned in the body—sugar, for example—may undergo slow oxidation in alkaline solution in the presence of atmospheric oxygen ${\ce {(O2)}}$. In the body, however, it is burned at a faster rate, leading to the conclusion that some other substance or substances are necessary. The search for such substances has led to the discovery of so called oxidizing enzymes, which oxidize many organic substances. It is characteristic of an enzyme, however, that it accelerates but one reaction. For the complete oxidation of grape sugar, for instance, it is supposed that a series of enzymes is necessary. This must remain for some time a supposition, as no pure substance or mixture of soluble substances has been extracted from the body that will completely oxidize grape sugar.

It might be concluded that "life" is essential to such oxidations, but such is not the case. In some instances ground up tissue, free from entire cells, absorbs oxygen and gives out ${\ce {CO2}}$ at a rapid rate. It is evident that some substances are completely oxidized in the process. The question has been raised as to whether the cell structure which has not been completely destroyed in grinding the tissue be necessary for the oxidation. In certain experiments Harden and McLean failed to observe respiration in juice pressed out of muscles and other tissues. Warburg and Meyerhof ground nucleated red-blood corpuscles with sand, finding that the mass did not absorb oxygen or give out ${\ce {CO2}}$, whereas the original cells did. Warburg tried to destroy the structure completely by grinding corpuscles in a steel box; with steel spheres rotating at such high speed it was found necessary to cool the box with ice in order to prevent injury to the corpuscles by heat (Barnard & Hewlett apparatus). All microscopic structure was destroyed and respiration ceased.

In other experiments, Warburg ground up liver cells, passing the juice through a Berkefeld filter. The respiration of the juice was but five per cent. of that of the corresponding amount of liver cells. But when a coarser filter was used which allowed the passage of cell granules, the oxidation was found to increase to twenty per cent. of that of intact cells.

If blood corpuscles be placed in water, or in certain solutions, the hemoglobin passes out of them, they become pale and are called "ghosts." This liberation of the hemoglobin, known as "laking," is a kind of cytolysis. Warburg laked nucleated red-blood corpuscles of a goose, finding that respiration continued in the "ghosts," but did not occur in the fluid procured by laking.

Such experiments seem to show that the presence of solid structures, granules, etc., accelerates the respiration, since no substances were eliminated in the process of grinding. It is possible that the solid structures act in the same way, as does finely divided platinum (called platinum black), which accelerates certain chemical reactions by the condensation of the reacting substances on the surface of the platinum, and their consequent increase in concentration. This process of condensation on surfaces is called adsorption. Warburg supposes that the oxidizing enzymes, oxidizable substances and oxygen are condensed on "surfaces," thus causing the oxidation rate to increase, but what surfaces he means it is difficult to determine, in some places apparently referring to surfaces of granules or colloidal particles, in others to cell or nuclear surfaces.

The adsorption of easily adsorbed substances may retard or prevent entirely the adsorption of others less readily adsorbed. Warburg found that anesthetics reduced the respiration of a mass of cell granules, presumably by driving the enzymes or oxidizable substances from their surfaces. He further observed that animal charcoal in water oxidized oxalic acid to ${\ce {CO2}}$, whereas if anesthetics were added the oxidation was reduced.

Warburg and Meyerhof found that the respiration of sea-urchins' eggs was not entirely destroyed by grinding with sand, presumably because the cell granules were left intact. They explain it, however, as an auto-oxidation or spontaneous oxidation of lecithin in the presence of iron salts, the oxidation taking place in the test tube. Warburg found iron and lecithin in the sea-urchin eggs and observed that if the total lecithin that could be extracted from a mass of eggs were mixed with a dilute solution of iron chloride, the oxidation was as great as that of the mass of ground cells. From his data we conclude that the mass of ground unfertilized eggs undergoes the same oxidation as does the same mass of cells if it were fertilized before grinding. Warburg interprets this as indicating that the oxidation of unfertilized eggs is due to auto-oxidation of lecithin, and that the increase in oxidation on fertilization is due to increase in structure (surfaces). Since mechanical agitations, however, may cause the eggs to develop, it is possible that the grinding first stimulated each egg to as great respiration as that of a fertilized egg, but the crushing and subsequent mixing of substances reduced the oxidation. It is interesting to note that, whereas unfertilized as well as fertilized eggs absorb oxygen and give off ${\ce {CO2}}$, ground eggs or lecithin and iron mixtures do not give off ${\ce {CO2}}$, indicating oxidation is not complete.

 

Relation of Oxidation to Permeability

R. Lillie supposed the oxidation within the unfertilized eggs to be suppressed by an accumulation of some end product of oxidation that could not escape. It is possible that such a substance might act like an anesthetic and suppress oxidation by adsorption to the granules. Lillie supposed this substance to be carbonic acid, but this is hard to believe when we bear in mind that the egg may be caused to develop by short exposure to carbonic acid. On fertilization this hypothetical substance would be liberated and could be collected. Glaser fertilized quantities of eggs in a small amount of sea water. On using the same water in which to develop other fertilized eggs, he found it inhibited their development, indicating the presence of an inhibiting substance that came out of the first eggs on fertilization. (Was this ${\ce {CO2}}$?)

Loeb's "improved method of artificial parthenogenesis" claims two treatments of the eggs to be necessary. They are first to be stimulated to development by use of fatty acid, or some other method, and then exposed to a hypertonic solution. The latter he calls a "corrective agent" and supposes that it changes the character of the oxidation in the egg, since he observes no effect on the rate of oxidation in the developing eggs. It is hard to conceive of such a change in "character," since oxidation means union with oxygen and there is but one kind of oxygen atom in combinations. The oxygen might attack different substances, but in such cases different amounts of heat would be given off, the heat of combustion of fats and carbohydrates, for instance, differing in amount. Meyerhof showed that the ratio of oxygen used to heat produced was the same for eggs in the hypertonic solution as in sea water. When we consider that by the use of either fatty acid or hypertonic solution alone, sea-urchin (Arbacia) eggs may be made to develop, it seems unnecessary to devote more time to their combined effect.

 

Relation of Anesthesia to Development of the Egg

Anesthetics have a depressant action on various cell activities when used in certain concentration. They decrease the respiration and rate of cleavage of sea-urchin eggs (and asphyxiation will cause cleavage to cease). It may therefore be supposed that it is the suppression of oxidation by anesthetics that suppresses cleavage. Warburg, however, caused the almost complete cessation of cleavage in sea-urchin eggs with anesthetics without appreciably lowering the respiration. It may be that the anesthetic acts in one part of the cell (on the surface of the granules) in suppressing oxidation, and in another (on the cell surface) in suppressing cleavage.

In 1909, while measuring the electric conductivity of sea urchins' eggs, the writer observed the decrease in conductivity on the addition of a certain per cent. of chloroform. This experiment was not repeated, but we may imagine that the chloroform decreased the permeability of the eggs to ions. Osterhout, becoming interested in the methods used, modified them for use with plants, and observed a decrease in electric conductivity of certain plants (kelp) when using a certain concentration of anesthetic, indicating that the anesthetic decreased permeability. R. Lillie found that anesthetics might antagonize the action of the pure salt solutions used to cause eggs to develop, presumably preventing the increase in permeability usually caused by the salt solution.

The use of fish eggs in settling this question presented itself to the writer. It was found that the eggs of the pike will develop in distilled water and are practically impermeable to salts—that is to say, that the salts which they contain diffuse out of them only in such small quantities as to render detection almost impossible even with as sensitive an instrument as the nephelometer. It was found, further, that pure solutions of sodium nitrate increased the permeability of the eggs to chlorides (since the chlorides diffused rapidly from the eggs). The use of anesthetics prevented the effect of nitrates on the permeability of the eggs, so that the chlorides failed to diffuse.^[7]

It is thus evident that the problem of parthenogenesis is closely interwoven with fundamental problems of physiology—stimulation, oxidation and anesthesia; and that the final elucidation of parthenogenesis and fertilization must wait on the solution of these other problems. On the other hand, the systematic study of parthenogenesis has already shed much light on general physiology, and progress will be more certain if all of these problems be kept before the mind of the investigator.

1. McClendon, Am. Jour. Physiol., 1910, 27, 240.
2. J. Gray, Jour. Marine Bio. Assn. United Kingdom, 1913, X., 50.
3. Science, 1910, 33, 249.
4. McClendon, Roux's Archiv, 1913, 37, 233.
5. McClendon, "On the Nature and Formation of the Fertilization Membrane," Internat. Zeit. f. Physik.-Chem. Biologie, 1914, Vol. 163.
6. McClendon, Science, 1912, 33, 387.
7. Science, 1914, Vol. 40, p. 214.