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4. Rhythmical Contractile Movements

As early as 1840, renowned Italian embryologist Rusconi first observed 'rotations' of the egg of the pike (Esox lucius), which begin a half hour after fertilization. If one observes a fixed point continuously, it moves regularly describing circles or ellipses. He misleadingly regarded them as the phenomena caused by ciliary movements. The phenomena since have been observed by Aubert (1854) and Lereboullet (1854) in the same form. Reichert (1856) pointed out that rotations are not due to ciliary movements and he (1857) actually observed contractile changes on the uncovered yolk sphere. It is evident that 'rotations' are caused by rhythmic contractile movements of egg protoplasm surrounding the yolk.

Table 4-1. Fishes which manifest rhythmical contractile movements in their developmental stages. Yamamoto, 1941.

A review on these movements was given by the writer (Yamamoto, 1941). Table 4-1 shows species which manifest rhythmical contractile movements in their developmental stages. It will be seen that stages as well as parts of eggs which manifest the movements vary from species to species.

In the egg of the goldfish, Carassius auratus, the movements are seen only on the uncovered yolk sphere in stages from cleavage to the closure of blastopore. No movements can be seen on the expanding blastoderm and the extra- embryonic surface of the covered yolk sac. (Yamamoto, 1934a). Obviously, it is the superficial protoplasmic layer of the uncovered yolk sphere that contracts.

In the egg of the medaka, Oryzias latipes, by contraries, the uncovered yolk sphere shows no movements and the expanding blastoderm and the extra- embryonic surface covered by epiboly manifest movements. Loci of contractions eggs of this type seem to be the periblast beneath the blastoderm and its continued protoplasmic layer covered by epiboly. The active contractions of underlying protoplasmic layer cause passive changes in form of the blastoderm and the covered yolk sac. In the pike (Esox lucius) and the sticklebacks (Gasterosteus and Pygosteus), movements of both Carassius- and Oryzias types appear.

Fig. 4-1. Rhythmical contractile movements of Oryzias egg at the stage of 1/2 epiboly. es, embryonic shield. Yamamoto, 1931a.

Most researches on these movements were casual observations. The outstanding work has been performed by Ransom (1854, 1866, 1867), an English author, using eggs of the pike and sticklebacks as materials. It is remarkable, indeed that besides detailed observations he performed such experiments as the effects of CO2 and chloroform, temperature, oxygen deficiency and electricity on these movements as early as in the nineteenth century.

Fig. 4-2. An alternate contractions in one day Oryzias embryo (reared at 25 oC). a-c, dextral wave; d-f, sinistral wave. Yamamoto, 1931a.

In Oryzias egg, Amemiya (1928) first reported his casual observation on the rhythmical contractile movements. In the same year, the writer independently observed the same phenomena. In the blastoderm of Oryzias, the rhythmical contractile movements first appear when it covers one third of the yolk sphere (Stage 14-15). At this stage the contraction waves arise at the edge of the blastoderm opposite the place where the embryonic shield is situated, and pass along the median surface of the blastoderm, finally disappearing on the opposit edge. After the blastoderm encloses half of the yolk sphere, the direction of contraction waves so changes that they travel along the edge of the blastoderm in a circular fashion (Fig. 4-1). In later stages the contractions originate from the extra-embryonic region near the embryo, encircle the egg and disappear on the opposite side. The waves proceed in either dextral or sinistral direction in reference to the embryo. The number of the waves of the same direction which take place in succession varies greatly. Generally the older the embryo the smaller the number. Thus, after the closure of the blastoderm, one dextral and one sinistral wave appear alternately (Fig. 4-2). Sometimes two waves of opposite directions may start simultaneously. The movements continue until the embryo reached stage 28.

The writer has performed a series of experimental studies on the phenomena (Yamamoto, 1931a, b, 1933a, b, 1934a, b, 1936a, b, 1938a, b, 1939a, b, 1940). Of these studies relationship between temperature and frequency of the rhythmical movements seem to be worthy of special attention.

Fig. 4-3. Relationship between temperature and frequency of rhythmical contractile movements in eggs of Oryzias latipes. Based on the data of Yamamoto, 1931b. Original.

Time required for one cycle and frequency of the rhythmical movements as influenced by temperature are shown in Figure 4-3. Yamamoto (1931b) showed that the frequency of the movements increases exponentially with temperature according the Arrhenius (1915)-Crozier (1924) equation V=ke-µ/RT where V is the rate (frequency), k a constant, R(=2) is gas constant, T is absolute temperature and u (myu) is the temperature characteristic which represents apparent activation energy in calories per mol of the reaction. The equation can be expressed also as

Since physiological processes involve series of chemical reactions, u (myu)is interpreted as the energy of activation of the slowest or the 'master' reaction in the catenary reactions (Crozier, 1924; Crozier and Hoagland, 1934). The idea of the 'master' reaction in biological processes was criticized by Burton (1936). Since the significance of u (myu) is not yet satisfactorily interpreted, Buchanan and Fulmer (1930) recommended to use the equation in the formV=ke-A/T employing the constant A instead of u (myu)/2.

Nevertheless, aside from its theoretical significance, the Arrhenius-Crozier equation in quite adequate in classifying or characterizing vital activities. There is a host of evidence to indicate that vital activities underlying mechanisms of which are closely allied each other have similar temperature characteristics.

Fig. 4-4. Arrhenius-Crozier plots of some biological phenomena in the medaka showing that u (myu)s of the rhythmical contractile movements are practically coincide with those of the developmental rates and quite different from those of heart rates of embryos and adult as well as opercular breathing rhythm. R.M.1,2,3 = Rhythmical contractile movements of 1-day, 2-day and 3-day embryos; D.R.3,8,15,21 = Developmental rates (reciprocals of time) based on the time required to reach 3, 8, 15, and 21 somite stage from fertilization (Shirai's data). O.B.R. = Opercular breathing rhythm; H.R.a,4,8 = heart rates of adults, 4-day and 8-day embryos, respectively. Yamamoto, 1938b, 1939b.

Arrhenius-Crozier plots of four physiological activities, viz. opercular breathing rhythm (O.B.R.), heart beats (H.B.), the rhythmical movements (R.M.) and rates of developments (D.R.) in the medaka are given in Fig. 4-4. (Yamamoto, 1938b, 1939b). Data for developmental rates are those by Shirai (1937). It will be seen that inclinations of lines representing u (myu)s for the rhythmical movements are distinctly different from those for the opercular breathing rhythm and heart beats of embryos and adults and remarkably agree with those for the rate of development. The two activities are common in having two critical temperatures or 'breaks'.

The u (myu)s for the rhythmical movements are 10.3 ~ 10exp3 for upper range, 21.6 ~ 10exp3 for intermediate range, and 28 ~10exp3 for lower range of temperatures whereas those for the rate of development are 9.8 ~10exp3, 20.3 ~ 10exp3 and 29~10exp3 for respective temperature ranges.

It may be remarked that these values are practically coincide with the 'peaks' of the frequency polygons of temperature characteristics for the rates of development of several animals as marshaled by Needham (1931). The u (myu)s for the respiratory movement and heart beats of the medaka, on the other hand, are well coincide with 'peaks' of Crozier's frequency polygons for all vital activities, especially for heart rates and other activities associated with biological oxidation (Crozier, 1926).

Fig. 4-5. Time course of the action of M/1 KCl solution on the rhythmical contractile movements (circle) and heart beats (solid circle) in two-day embryos reared at 28 oC. ~ denotes stoppage of movements. Yamamoto, 1939a.

In this connection, it is interesting to note that the reaction of these movements to KC1 is quite different from that of heart beats (Yamamoto, 1939a). Figure 4-5 shows that action of KC1 solution on the rhythmical movements and the heart beats of the same eggs. While the rate of the rhythmical movements gradually increases before their arrest, that of the heart beats gradually decreases before the stoppage. It may be noticed that the rhythmical movements continue a half hour after the stoppage of the heart beats. All the facts seem to indicate that the rhythmical movements are phenomena closely associated with the developmental processes and quite different from activities associated with oxidative processes.

It is astonishing that Ransom as early as 1867, performed experiments concerning the effect of oxygen lack on the movements using eggs of the stickleback and the pike as materials. He observed movements in the egg of stickleback in water from which oxygen was previously removed by boiling. However, when the eggs of the pike were placed in the oxygen free water on which olive oil was placed to prevent oxygen diffusion from outside, the movements were arrested. He concluded that oxygen is sine qua non for the contractile movements. Such a study will give us some hints as to the energy source of these movements. Accordingly, the writer (Yamamoto, 1936b) studied the effect of anaerobiosis on these movements. Oxygen was removed by bubbling of nitrogen gas purified according to Michaelis and Flexner (1928) by passing over copper heated in an electric furnace to remove the trace of oxygen. The final trace of oxygen is usually removed about 30 minutes after the bubbling of nitrogen gas.

Fig. 4-6. Time course of anaerobic movements of the rhythmical contractile movements of eggs stained with methylene and recovery process. Yamamoto, 1936b.

Under the anaerobic condition, the movements persist about one hour and half at 28 oC but their rate and amplitude gradually fall down until they are completed arrested. When air is admitted the recovery soon takes place. It was found that recovery occurs even after 20 hour's anaerobiosis. Fig. 4-6 shows the anaerobic movements and the recovery process of the eggs which have previously been stained vitally with methylene blue (rH2 14.4). Methylene blue within the egg is reduced to its leucobase in about 40 minutes after N2 bubbling.

Noteworthy fact is that the movements can last even after methylene blue is reduced to its leucobase within the egg. This shows that they can persist in practically complete absence of oxygen in the medium as well as within the egg.

The same experiment was performed on eggs vitally stained with other oxidation-reduction dyes. It was found that oxidation-reduction indicators having rH2 values higher than 8, viz. toluylene blue, thionine, brilliant cresyl blue, toluidine blue, nile blue sulphate, and cresyl violet, are reduced before the movements are arrested. The indicators having rH2 values lower than 6, viz. phenosafranine, safranine T and neutral red, are not reduced for several hours after the anaerobic stoppage of the movements

Oxidation-reduction indicators having rH2 values higher than 22, viz. dimethyl-p-phenylendiamine and Bindschedler's green, are reduced in the egg even in aerobic condition. Apparent aerobic oxidation-reduction potential of the interior of Oryzias egg lies between rH2 18-22. Under anaerobiosis rH2 values gradually decreases. Apparent anaerobic oxidation-reduction potential are between rH2 6-9 at the pHs of the yolk (6.2-6.6) and the cytoplasm (6.6-7.0). The rhythmical movements last until the rH2 of the interior of the egg become at the level of 6-9 under anaerobiosis.

It has been concluded that oxygen is not directly required for the mobile mechanism, but it is necessary for the continuation of movements. Gradual decrease in the rate of the movements under anaerobic condition may be due to an accumulation of some waste products by anaerobiosis, which may be eliminated by aeration. The energy for the movements seems to be supplied in the initial phase by some hydrolytic process which require no oxygen, while oxygen is necessary for the later phases. Kuhl (1938) who observed the rhythmical movements in the egg of the white fish, Coregonus, regarded them as a kind of respiratory movements, ficilitating excretion of carbonic acid from the perivitelline space and absorption of oxygen into it.

However, physiological studies on the movements of Oryzias egg indicated that they cannot be regarded as respiratory movements. It is generally known that respiratory movements in animals usually increase in rate under low oxygen tension. As stated before, the rhythmical movements decrease in rate before anaerobic stoppage. The temperature characteristics for the rhythmical movements of Oryzias egg are coincide with those for developmental rates and are essentially different from respiratory movements.

The writer has made no approach to the contractile mechanism of the movements. No contractile proteins were known at that time.

Comparative studies on several species of fishes, however, gave some hints as to the significance of these movements. First, no movements were observed by myself in the eggs of the pond smelt (Hypomesus olidus), the paradise fish (Macropodus chinensis) and the goby (Tridentiger trigonocephalus). These eggs have a large amount of protoplasm and a small amount of yolk. The ratio of protoplasm to yolk is compared at one-celled stage when the blastodisc is fully formed. Generally speaking, eggs having lower ratios of plasma/yolk manifest the movements in longer period of development. These facts suggest that the movements may be necessary for absorption of yolk in embryogenesis. They are unnecessary for eggs having a small amount of yolk.

Secondly, the eggs of the trouts (Oncorhynchus and Salvelinus) show movements only in limited stages of development despite they have a low plasma/yolk ratio. This may be accounted by the fact that they have vitelline veins with an elaborated branchings. Vitelline veins may serve to facilitate absorption of yolk. The same argument may be held on the egg of Fundulus heteroclitus, in which no rhythmical movements have been reported despite its low plasma/yolk ratio (see Fig. 48 in Kellicott, 1913). The egg has vitelline veins with elaborate branchings (see Oppenheimer, 1937).

In short, the rhythmical movements occur more vigorously in eggs with large amount of yolk relative to protoplasm and with simpler vitelline vessels. These facts suggest that the movements may act as stirrer in order to facilitate its absorbtion by the absorbing system. In heavily yolk-laden eggs, it is suspected that yolk underneath the egg surface would be liable to become dilute. On account of the rhythmical movements yolk of normal concentration will be always supplied to the absorbing system.

References Amemiya, I., 1928 Egg of the medaka, Oryzias latipes, as an educational material (in Japanese). Toyo Gakugei Zassi, 8:571-579.

Arrhenius, S., 1915 Quantitative laws in biological Chemistry. London.

Aubert, H., 1854 Beitrage zur Entwickelungsgeschickte der Fische. Zeit. Wiss. Zool., 5: 94.

Buchanan, R.E., and E.I. Fulmer, 1930 Physiology and biochemistry of bacteria. Vol. 2: 33. Baltimore.

Burton, A.C., 1936 The basis of the principle of the master reaction in biology. J. Cell. Comp. Physiol. 9: 1-14.

Crozier, W.J., 1924 On biological oxidation as functions of temperature. J. Gen. Physiol., 7: 189-216.

Crozier, W.J., 1926 The distribution of temperature characteristics for biological processes; critical increments for heart rates. J. Gen. Physiol., 9: 531.

Crozier, W.J. and H. Hoagland, 1934 The study of living organisms. In A handbook of general experimental psychology edited by C.H. Murchison. Worcester.

His, W., 1938 Untersuchungen ueber das Ei und die Entwickelung der Knochenfische. Leipzig.

Ihring, V., 1937 Bewegung des Ei-inhaltes zweier Brasilianischer Suesswasserfische. Zool. Anz., 120: 45.

Kellicott, W.E., 1913 A textbook of general embryology. Henry Holt and Co. New York.

Kuhl, W., 1939 Zeitrafferfilm-untersuchungen ueber die rhythmischen Bewegungen des Blaufelchen-und Gangfischeies (Coregonus Wartmanni Bloch und C. macrophthalmus Nuesslin) wahrend der Entwicklung. Zeit. Wiss. Zool. 152:305.

Lereboullet, A., 1854 Embryogenies des vertebres et des animaux articules.- Ouelques propositions sur l'embryologie des poissons, particulierement du brochet et de la perche, et sur l'embryogenie de l'ecrevisse. C. R. Acad. Sci., 38: 978.

Michaelis, L., and L.B. Flexner, 1928 Oxidation-reduction systems of biological significance. I. The reduction potential of cysteine, its measurements and significance. J. Biol. Chem. 79: 689.

Needham, J., 1931 Chemical embryology. Vol. 1, p. 524. Oppenheimer, J.M., 1937 The normal stages of Fundulus heteroclitus. Anat. Rec. 68: 1-15.

Ransom, W.H., 1854 On the impregnation of the ovum in the sticklebach. Proc. Roy. Soc., London, 7: 168.

Ransom, W.H., 1866 Observations on the ovum of osseus fishes. Ann. Mag. Nat. Hist., 3 ser., 18: 249.

Ransom, W.H., 1867 On the conditions of the protoplasmic movements in the eggs of osseus fishes. Jour. Anat., 1: 237.

Reichert, C.B., 1856 Ueber die Mueller-Wolff'schen Korper bei Fischembryonen und ueber die sogenannten Rotationen des Dotters im befruchteter Fischeier (Hecht). Muellers Arch. Anat. Physiol., (1856) : 124

Reichert, C.B., 1857 Der Nahrungsdotter des Hechteies, -eine kontraktile Substanz. Muellers Arch. Physiol., (1857) : 46.

Rusconi, M., 1840 Sopra la fecondazione artificiale nei pesci, e sopra alcune nuove esperienze intorno alla fecondazione artificiale nella rane. Muellers Arch. Anat., 1840, S. 185.

Shirai, K., 1937 The influence of temperature on embryonic development of Oryzias latipes (in Japanese). Hakubutugaku Zasshi (Mag. Nat. Hist. Tokyo), 35: 202-210.

Wintrebert, P., et Young-Ko-Ching 1926 La contraction protoplasmique des ebauches embryonnaires chez l'Epinoche et l'Epinochette. C. R. Acad. Sci., 183: 455.

Yamamoto, T., 1931a Studies on the rhythmical movements of the early embryos of Oryzias latipes. I. General description. Jour. Fac. Sci. Tokyo Imp. Univ. Sec. IV (Zool.), 2: 147-152.

Yamamoto, T., 1931b Ditto. II. Relation between temperature and frequency of the rhythmical contractions. Ibid., 2: 153-162.

Yamamoto, T., 1933a Ditto. III. Temperature and the amplitude of the contraction waves. Ibid., 3: 105-110.

Yamamoto, T., 1933b Ditto. IV. Temperature constants for the velocity of the wave and for the pause. Ibid., 3: 111-117.

Yamamoto, T., 1934a On the rhythmic movements of the egg of goldfish. Ibid., 3: 275-285.

Yamamoto, T., 1934b Studies on the rhythmical movements of the early embryo of Oryzias latipes. V. The action of electrolytes and osmotic pressure. Ibid., 3: 287-299.

Yamamoto, T., 1936a Ditto. VI. The action of hydrogen ion concentration. Ibid., 4: 221-232.

Yamamoto, T.,1936b Ditto. VII. Anaerobic movements and oxidation-reduction potential of the egg limiting the rhythmical movements. Ibid., 4: 233- 247.

Yamamoto, T., 1938a Contractile movement of the egg of a bony fish, Salanx microdon. Proc. Imp. Acad. (Tokyo), 14: 149-151.

Yamamoto, T., 1938b On the distribution of temperature constants in Oryzias latipes. Proc. Imp. Acad. (Tokyo), 14: 393-395.

Yamamoto, T., 1939a Studies on the rhythmical movements of the early embryo of Oryzias latipes. IX. Potassium poisoning of 'rhythmical movements' and of heart beat in Oryzias embryo. Jour. Fac. Sci. Tokyo Imp. Univ., Sec. IV (Zool.), 5: 211-219.

Yamamoto, T., 1939b Ditto. X. The distribution of temperature constants in Oryzias. Ibid., 5: 221-228.

Yamamoto, T., 1940 Rhythmical contractile movement of eggs of trouts. Anno. Zool. Japan., 19: 68-79.

Yamamoto, T., 1941 Rhythmical contractile movements in fish eggs (in Japanese), I, II, III. Dobutsu-gaku Zassi (Zool. Mag. Tokyo), 53: I, 384-357; II, 411- 418; III, 452-457.

Ziegler, H.E., 1882 Die embryonale Entwicklung von Salmo salar. Diss. Freiburg.

Ziegler, H.E., 1902 Lehrbuch der vergleichenden Entwickelungsgeschichte der niederen Wilbeltiere. Gustav Fischer, Jena.