7. Artificial Activation and Conduction of Fertilization-wave*
* Partly extracted from T. Yamamoto, 1961: Physiology of Fertilization in Fish Eggs. Intern. Rev. Cytol. 12 361-405, edited by G.H. Boure and J.F. Danielli. Academic Press Inc., New York.
Unfertilized eggs of most fishes manifest activation on immersion not only in their spawning environment but also in isotonic salt solutions. Such eggs are inadequate for physiological analysis of artificial activation. The Oryzias ova provide an excellent material for this purpose because the majority of ripe ova remain normal in isotonic Ringer's solution and only a few eggs show autoactivation. If eggs are not activated within 5 minutes after removal from the ovary, they remain normal for a long period and can be used in experiments as well as for artificial activation. The ova are allowed to stand at least 30 minutes before the experiments, and all the experiments are performed in the Ringer's solution. It was found that the cortical response, essentially similar to that occurring in fertilization, can be induced by a number of stimulating agents.
Pricking activation
The breakdown of the cortical alveoli and subsequent separation of the chorion are induced by pricking with a fine glass needle. A needle, the tip of which is 15- 20 micron in diameter, is preferable. When the animal pole is pricked, the breakdown of cortical alveoli begins at the point of pricking and the change proceeds toward the vegetal pole and is completed at the vegetal pole just as in the case of fertilization. A puncture at the vegetal pole causes the direction of the breakdown process to take a reverse course, i.e., the process starts at the vegetal pole and ends near the animal pole. Pricking at the equator also causes the breakdown which begins at the site of pricking. In a preliminary report (Yamamoto, 1939a), it was stated that the process of the breakdown ends at the antipode. Later observation (Yamamoto, 1944a) disclosed that it does not end at the antipode in a strict sense. The process of the alveolar breakdown proceeds more quickly near the animal pole than near the vegetal pole, with the result that it ends at the point halfway between the antipode and the vegetal pole (Fig. 7-1). Exactly the same results were obtained by Thomopoulos (1953b) in the egg of the stickleback, Gasterosteus aculeatus.
Fig. 7-1. Sequence of cortical changes in the egg of the medaka, induced by prick-ing with a needle. Cortical alveoli are represented by dots and oil droplets as white circles. Yamamoto, 1944a.
A parallel phenomenon was found to occur in the brook lamprey Lampetra planeri (now L. reissneri) (Yamamoto, 1944c, 1947b). In this case the sequence of release of alveolar colloid and subsequent separation of the chorion was observed because cortical alveoli themselves were invisible. After the completion of the experiment, a short article by Pirlot (1925) came to my attention. He performed a pricking experiment on the egg of the river lamprey, Petromyzon fluviatilis, and observed the same phenomenon. I regret that this paper escaped my attention until the publication of my papers.
Chemical activation
The ripe unfertilized Oryzias ova can be activated through the action of such naturally occurring surface-active substances as sodium taurocholate (1%), sodium glycocholate (1%), sodium oleate (1%), saponin (0.1%), and digitalin (0.01%). Detergents such as Aerosol OT (0.01%), Monogen (0.5%), Nekal BX (0.05%), and Labolan (0.05%) are also effective activators (Yamamoto, 1944a, 1945, 1947a, b). The breakdown of the cortical alveoli starts near the animal pole and ends at the vegetal pole in Oryzias egg. It is postulated that these surface-active substances induced stimulation by emulsifying the protoplasmic surface.
Yamamoto (1951a) demonstrated that the vapors of such lipid solvents as chloroform, ether, benzene, toluene, and isoamyl alcohol bring about activation of Oryzias ova although they are not excellent activators. In most cases only incomplete breakdown of the cortical alveoli results. It is interesting that while a short exposure to vapors of these agents brings about complete breakdown of the cortical alveoli in a few eggs, longer exposure always induces incomplete breakdown in most eggs. This may show that in short exposure the ovum is stimulated but the subsequent excitation process is not yet inhibited. In longer exposure the excitation process is more or less inhibited. The most important fact revealed is that acetone, a neutral fat solvent, is ineffective in inducing activation either when an ovum is exposed to its vapor or in its dilute solution. Montalenti (1936) showed that chloroform vapor induces membrane separation in lamprey eggs (Lampetra planeri).
Inorganic acids such as HCl, H2SO4, and HNO3 in the concentration of 10-3 N in Ringer's are found to be excellent activators in Oryzias eggs. Lower fatty acids such as formic, acetic, and propionic acids (N/50) are also effective, in this decreasing order. In contrast to their effect on echinoderm ova, butyric and valeric acids are not activators. They exert only an inhibitory effect on the cortical change. As stated in the preceding chapter, the activation involves a chain of reactions, i.e., reception of stimulation and evocation of excitation followed by breakdown of cortical alveoli. All lower fatty acids have an inhibitory effect on the latter or secondary phase. Of the homologous series of fatty acids, formic, acetic, and propionic acids have a stimulating effect in the primary phase in this decreasing order. These facts may be interpreted as follows: with the homologous series under consideration, the stimulatory effect in the early phase of activation seems to be in the decreasing order of formic, acetic, propionic, butyric, and valeric, but the inhibitory effect in the later phase is considered to be in the reverse order. With butyric and valeric acids no stimulation may be brought about because their stimulating effect is so small that the egg does not reach the level of threshold of stimulation. Since their inhibitory effect on the latter process is very strong, they fail to elicit activation (Yamamoto, 1953).
Yamada (1954) reported that activation of Oryzias eggs can be induced by such oxidizing agents as KMnO4 and H2O2.
Thermal activation
Ripe unfertilized Oryzias eggs in Ringer's solution can be activated by temporary exposure to high temperature (45 oC., 1/2-4 minutes). It should be noted that the breakdown of cortical alveoli with subsequent separation of the chorion always begins near the animal pole and ends at the vegetal pole, notwithstanding the fact that the thermal effect must be the same at all points of the cortical layer. This means that the cortical layer at the animal pole is most sensitive to thermal treatment.
Fig. 7-2. Sequence of cortical changes in Oryzias egg activated with electric current. Cortical alveoli are represented by dots and oil droplets by circles. Yamamoto, 1949a.
Electric activation
Ripe unfertilized eggs of Oryzias in Ringer's solution can be activated by an electric current. When the potential gradient in the electric field is higher than 2 volts per centimeter, the breakdown of the cortical alveoli with subsequent separation of the chorion begins at the anodal and cathodal sides. When the axis of the egg connecting the animal and vegetal poles is transverse (90 o) to the lines of electric current, the anodal effect occurs earlier than the cathodal effect (Fig. 7-2). By changing the axis of the egg to the direction of the electric current, it is found that the reaction time for the anodal effect is shortest when the animal pole is directed to the anode and longest when the vegetal pole is faced to the anode. The same is true of the cathodal effect. The cortical change proceeds more rapidly near the animal pole than the vegetal pole (Yamamoto, 1944a, 1949a). Nearly the same results were obtained in the ova of the brook lamprey (Yamamoto, 1944c, 1947b).
Photodynamic activation
When unfertilized eggs of Oryzias are immersed in such fluorescent dyes as rhodamine B and eosin in Ringer's, and exposed to ultraviolet rays, a number of eggs are activated (Yamamoto, 1950).
Supersonic activation
Takashima et al (1952) reported activation of Oryzias eggs with supersonics. The activated eggs revealed cortical changes.
Thus, artificial activation is elicited by various kinds of agents. We cannot find any common factor among these agents. Obviously, effective stimulants trigger off the reactive system in the ovum.
Gradient of irritability and excitation-conduction
Pricking and electric stimulation revealed the existence of the regional difference in physiological properties in the eggs of the medaka (Oryzias) and the brook lamprey (Lampetra). The response time of Oryzias eggs for pricking with the same needle (20 micron tip) is shortest at the animal pole (10 seconds at 28-30 oC.), longest (22 seconds at 28-30 oC.) at the vegetal pole, and intermediate at the equator. In Lampetra eggs, it is 38 seconds at the animal pole and 89 seconds at the vegetal pole at 17-18 oC. Therefore, the response time at the vegetal pole is about twice that at the animal pole.
The results of electric stimulation revealed the same fact. When the axis connecting the animal and vegetal poles is transverse to the lines of the electric current, the cortical change starts earlier at the anodal side than at the cathodal side. As stated before the response time at the anodal side is shortest when the animal pole is directed to the anode and longest when the vegetal pole is faced to the anode. The same is true with the response time at the cathodal side. These experiments indicate that there exists on the surface of the unfertilized egg a steep gradient or polarity of sensitivity which is highest at the animal pole and lowest at the vegetal pole.
This fact interprets satisfactorily the phenomenon observed in thermal activation in which the cortical response starts at the animal pole in spite of the fact that the heat acts equally on the entire surface of the egg. The revelation of highest sensitivity at the animal pole seems to have significance in fertilization when we take into consideration that the animal pole is the very region at which the sperm attacks the ovoplasm. Figures 7-1 and 7-2 indicate that the cortical change proceeds earlier near the animal pole than near the vegetal pole. This means that the ripe unfertilized egg is an irritable system with a gradient of irritability and excitation- conduction, which is highest at the animal pole and lowest at the vegetal pole.
Decrement conduction of fertilization-wave or impulse
The following experiments performed on Oryzias eggs indicate that the fertilization-wave or impulse propagates itself with diminishing intensity and velocity so that the all-or-none principle cannot be applicable (Yamamoto, 1944b).
(a) In normal fertilization the breakdown of the cortical alveoli begins near the animal pole and ends at the vegetal pole. In most cases the breakdown is complete even at the vegetal pole. In some cases, however, it is incomplete; viz., while the breakdown in almost all of the surface is complete, some alveoli at the vegetal pole do not break down. Whether the breakdown is complete or incomplete depends on the individual female from which the eggs are obtained. If one egg breaks down incompletely, all other eggs removed from the same female show the same incomplete breakdown. A few eggs from a batch were subjected to a trial insemination in order to see whether or not they would show incomplete breakdown; other eggs from the same batch were kept unfertilized. When the tested eggs showed incomplete breakdown, the following experiments were performed on the unfertilized eggs, which were divided into two groups. The eggs of one group were inseminated and the eggs of the other group were pricked at the animal pole with a glass needle having 15 micron tip point. It was found that the fertilized eggs showed incomplete breakdown while the pricked eggs showed complete breakdown, i.e., the cortical alveoli at the vegetal pole also broke down. This experiment may imply that the impulse elicited by the sperm is weaker than that provoked by pricking with a 15 micron needle point. Since the impulse provoked by the sperm is weak and since it conducts itself with diminishing intensity, it cannot cause the alveoli at the vegetal pole to break down. The impulse provoked by pricking with a 15 micron needle point is strong enough to cause the breakdown of the alveoli at the vegetal pole even though it propagates itself with decrement during passage through the cortex of the egg.
(b) That the fertilization-wave propagates itself with decrement is also confirmed in the fertilization and artificial activation in incompletely anesthetized eggs (Yamamoto, 1944b, 1949b). Complete anesthesia with either M/200 phenylurethane or M/200 chloretone (chlorobutanol) in Ringer's solution brings about reversible inhibition of both fertilization and artificial activation. Shorter exposure to anesthetics results in incomplete anesthesia. When these eggs are inseminated the breakdown of the cortical alveoli begins near the animal pole but it ends on the way between the animal pole and the vegetal pole. [In the summary of the writer's paper (1949b) the last sentence was erroneously deleted by the printer.] Thus, with these incompletely anesthetized eggs we are able to obtain fertilized or activated eggs in which the cortical alveoli in the animal hemisphere are broken down whereas those in the vegetal hemisphere remain unchanged. The consequence is that the perivitelline space is formed only on the animal hemisphere, and the eggs remain in this state for a considerable period. Later the perivitelline space is formed on the vegetal hemisphere also as the perivitelline colloid on the animal hemisphere diffuses between the cortex and the chorion on the vegetal hemisphere. This experiment proves that when the unfertilized eggs are partially anesthetized, the intensity of the impulse elicited by the sperm becomes so weak that it can only break down the alveoli in the animal hemisphere but cannot do it in the vegetal hemisphere through the decrement of the intensity of the fertilization- wave. If our interpretation is correct we would expect to duplicate this condition even in the normal egg by pricking at the animal pole with a needle of sufficiently small diameter. In fact, Hori (1960) was able to obtain a few Oryzias eggs in which the cortical response in the animal hemisphere was completed but was absent in the vegetal hemisphere.
(c) As has been mentioned before, there exists a steep gradient in irritability and excitation-conduction. Since the fertilization-wave propagates itself with decrement in such a heterobolic system, its velocity in a certain region varies with the direction of the wave. When the animal pole of Oryzias eggs is pricked with 15-20 micron needle point, the velocity at the middle region (AE) between the animal pole (A) and the equator (E) is 26 micron/second, while that at the middle region (VE) between the vegetal pole (V) and the equator (E) is 15 micron/second (29-33 oC.) When, however, the vegetal pole is pricked with a needle of the same size, the velocity at the VE is 22 micron/second and at the AE 19 micron/second. At the equator (E) the conduction velocity is about 20 micron/second in both directions (Yamamoto, 1956). The decrement conduction of the fertilization-wave in the direction from the animal pole to the vegetal pole is evident. The fact that in the direction from the vegetal pole to the animal pole the velocity at the VE is almost the same as that at the AE requires some consideration. The velocity at the AE is slower than would be expected on the basis of non-decrement conduction, which would require a velocity parallel to the irritability of the region. If the fertilization-wave were propagating without decrement against the gradient of increasing irritability, the velocity at the AE would become more repid than that at the VE when the fertilization-wave is passing through in the region of high irritability, as the nerve impulse recovers its normal velocity when it passes from the anesthetized region to the normal region. This is not the case in this heterobolic system. Actually, the velocity of the fertilization-wave does not increase when it passes through the region of high irritability. In other words, it is conducted with diminishing velocity.
The foregoing experiments indicate that the fertilization-wave propagates itself with decrement in respect to both its intensity and velocity. In other words, the all- or-none principle cannot be applicable. Hence, the ovum may be regarded as a heterobolic system, in contrast to nerve, which is an isobolic system. Allen (1954), in sea urchin eggs, also demonstrated that the fertilization-impulse propagates itself with diminishing intensity.
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