Further Development 7.1.3 On the Nature of the Process of Fertilization

and the Artificial Production of Normal Larvae (Plutei) from the Unfertilized Eggs of the Sea Urchin

Jacques Loeb, 1899

American Journal of Physiology 3: 135-138

1. FORMER researches had led me to suspect that changes in the state of matter (liquefactions and solidifications) might play an important rdle in the mechanics of life-phenomena. While studying tbe absorption of liquids by muscle I found that, to all appearances, a n/8 solution of CaCl2 favors the formation of solid compounds in the muscle, while an equimolecular solution of KCl favors the formation of more liquid compounds. I propose to substitute in the future the n/8 solution of NaCl for the 0.7 per cent. solution. It is time that we were rid of percentage solutions in physiology. Na ions rank between the K and Ca ions. In these phenomena, however, much depends upon the concentration of the salts. We know that the enzymes of coagulation and liquefaction are greatly influenced in their action by the Ca, Na, K, and Mg ions. Ca favors coagulation, and Mg does the reverse. Between these come the two other ions. In this case also much depends upon the concentration. I have made a series of studies on the mechanics of life phenomena, which will be published shortly in this Journal. I wish now to deal only with one part of these studies, namely, that referring to the nature of the process of fertilization.

I found that in 5/8 n solutions (approximately the concentration of sea water) of CaCl2, NaCl, KCl, and MgCl2 the segmentation of fertilized eggs of sea-urchins (Arbacia) proceeded best in MgCl2, next best in KCl, while CaCl2 proved to be the most injurious in the series.

Seven years ago I, and later Norman, found that if the concentration of sea-water be raised sufficiently by the addition of certain salts, a segmentation of the nucleus takes place without any segmentation of the protoplasm. Such eggs, however, when brought back into normal sea-water, divide into as many cells as there are preformed nuclei. This year I tried the effects of equimolecular solutions of MgCl2, KCl, NaCl, and CaCl2 upon this process of nuclear division (in which the nuclear membrane is apparently liquefied), and found that the influence of the four salts (or rather kations) followed the order mentioned above. We know that enzymes as a rule require a slight degree of acidity or alkalinity for their action. I showed last year that the addition of a small amount of H ions to sea-water retards or prevents segmentation, while a small amount of HO ions favors and accelerates the development of the Arbacia egg.

2. It has been known for some time that the unfertilized eggs of echinoderms, worms, and arthropods begin to segment when left for a comparatively long time in sea-water. This has generally been considered a pathological phenomenon. Mead succeded in causing a segmentation of the unfertilized egg of a marine worm, Chaetopterus, by the addition of a very small amount of KCl to sea-water. Morgan tried the effect of more concentrated sea-water on the unfertilized egg of sea-urchins, with result similar to those obtained by me previously with the same methods in fertilized eggs. If the unfertilized eggs are brought back from the more concentrated sea-water into normal sea-water, they break up into as many cells as there are nuclear masses preformed in the more concentrated solution. But in none of these cases did the cell-divisions of the unfertilized eggs lead to the formation of a blastula. A heap of cells, at the best about sixty, were formed, and then everything stopped. We cannot utilize these observations for the theory of fertilization, for the simple reason that the essential element of the process of fertilization, namely, the formation of an embryo, was lacking. In the case of tumors or galls we have cell-division and even growth, and yet these cell-divisions do not result in the formation of an embryo.

3. Some recent observations suggested to me that something in the constitution of the sea-water prevented the unfertilized eggs of marine animals from developing parthenogenetically. Last year I found that the striped muscles of a frog beat rhythmically (like the heart) if put into a n/8 NaCl or NaBr solution. It is only the presence of K and Ca ions in the blood that prevents striated muscles from contracting rhythmically in the body. Romanes had observed that if the margin (with the nerve ring) in Hydromedusae be cut off, the center no longer contracts rhythmically. I found this summer that this is due solely to the presence of K and Ca ions in the sea-water. In a 5/8 n solation of NaCl or still better of NaBr, the center continues to beat spontaneously. In applying this and my more recent observations on the relative influence of the various ions upon segmentation to the problem of artificial parthenogenesis it seemed to me that by making two changes in the constitution of sea-water the eggs of the sea-urchin might be able to produce perfect embryos without being fertilized. These changes were either a reduction of the Na and Ca ions or an increase in the Mg (or K) ions or both. I think that a great number of variations in this sense might bring about the desired effect, but the end of the season allowed me to try only a limited number of variations. Without going into details (which may be reserved for the full report) I will state briefly that the mixture of about 5O per cent. 20/8 n MgCl2 with about 50 percent of sea-water was able to bring about the same effect as the entrance of a spermatozoon. Loeb writes in 1903: "The subsequent experiments proved that the increase in the concentration of the sea-water caused the development ot the eggs. I was aware of this possibility and was looking for it, but was misled through an error in the preparation of the solutions (which I had intrusted to others). This error was afterward discovered and corrected." The unfertilized eggs were left in such a solution for about two hours. When brought back into normal sea-water they began to segment and form blastulae, gastrulae, and plutei, which were normal in every respect. The only difference was that fewer eggs developed, and that their development was slower than in the case of the normal development of fertilized eggs. With each experiment a series of control experiments was made to guard against the possible presence of spermatozoa in the sea-water. Unfertilized eggs of the same female were brought into normal sea-water, and in solutions with too little MgCl2. Neither in the normal sea-water nor in any of these solutions with too little MgCl2 did one single egg develop into a blastula or show anything more than the beginning of a segmentation after a long time.

4. From these experiments it follows that the unfertilized egg of the sea-urchin contains all the essential elements for the production of a perfect pluteus. The only reason that prevents the sea-urchin from developing parthenogenetically under normal conditions is the constitution of the sea-water. The latter either lacks the presence of a sufficient amount of the ions that are necessary for the mechanics of cellzdivision (Mg, K, H0, or others), or it contains too large a quantity of ions that are unfavorable to this procees (Ca, Na or others), or both. All the spermatozoon needs to carry into the egg for the process of fertilization are ions to supplement the lack of the one or counteract the effects of the other class of ions in the sea water, or both. The spermatozoon may, however, carry in addition a number of enzymes or other material. The ions and not the nucleins in the spermatozoon are essential to the process of fertilization (which may interest those who believe with me that physiologists ought to pay a little more attention to inorganic chemistry). I have no doubt that the same principles hold good for the process of fertilization of other, if not all the marine animals, although the ions involved will probably differ in various species.

Finally we may ask the question, whether we may expect to produce artificial parthenogenesis in mammalians. JanUsik has found segmentation in the unfertilized eggs of mammalians. This is similar to the fact mentioned above, that the unfertilized eggs of sea-urchins may show a segmentation if they stay long enough in the sea-water. I consider it possible that only the ions of the blood prevent the parthenogenetic origin of embryos in mammalians, and I think it further not impossible that a transitory change in the ions of the blood may also allow complete parthenogenesis in mammalians.