The physiology of fertilization in the medaka (Oryzias latipes)

Esperimentnl

387

Cell Research, 10, 387-393 (1956)

THE PHYSIOLOGY

OF FERTILIZATION (ORYZIAS LATIPES)l

IN THE MEDAKA

T.YAMAMOTO Biological

Institute,

Faculty

of Science,

Nagoya

University,

Nagoya, Japan

ALTIIOVGII extensive

studies have been done on the physiology of fertilization in invertcbratc ova, only a few works have appeared on the physiological change occurring in fish eggs at the time of fertilization. This is due to lack of appropriate materials as well as to inadequate methods. It has long been kno\vn that ripe unfertilized ova of teleosts lose their fertilizability within a short period after they are immersed into water in which fish are living. For instance, ripe unfertilized eggs of the medaka (Oryzias latipes), a fresh-water cyprinodont, become incapable of fertilization within a few minutes after they have been immersed into fresh water. For this reason physiological analysis of the fertilization reaction has rarely been performed. Although the “dry method” is excellent for practical use, it is not adequate for the physiological analysis of the fertilization reaction. Runnstrijm [8] seems to have been the first to observe that ova of a salmonid fish long retain fertilizability in an isotonic salt solution. The fertilization reaction in ova of sahnonid fish, however, does not occur in the isotonic solution. The egg of the medaka was found to be isotonic to M 7.5 NaCl [16]. The writer [ 13, 171 found that unfertilized ova of the medaka retain their fertilization capacity for a number of hours after they have been kept in an isotonic Ringer’s solution (M/7.5 NaCl 100 parts + M/7.5 KC1 2.0 parts + M/l1 CaCl, 2.1 parts, pH 7.3 by NaHCO,). Furthermore ova are capable of fertilization and normal development in Ringer’s solution. The female medaka exhibits a maturation cycle of 24 hours and lays a batch of eggs at dawn of every day during the breeding season. In order to obtain ripe unfertilized eggs for research purposes, previously egg-laying females must be isolated from males a day before the experiment is to be performed. It is recommended that females be sacrificed to obtain ripe ova because ordinary stripping method frequently injures ova and causes them to manifest mechanical activation. When ripe ova removed from isolated ovary are immersed into Ringer’s solution, only a few ripe ova (5-6 per cent) exhibit auto-activation while the majority remain unchanged. Non-activated 1 This paper is a contribution the Physiology of Fertilization, 24-6~3702

to the reports presented Palermo, April 1955.

in connection

with the Symposium

Experimental

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10

T. Yamamoto ova can be used in fertilization and artificial activation for a number of hours. This property is different from that of ova of Cyprinidae and others (except Salrnonidae) in which 100 per cent of ripe ova manifest auto-activation even in Ringer’s solution. The ova of the medaka, therefore, afford us unique material for physiological analysis of the fertilization reaction among teleostean eggs.

Cortical Changes at the Time of Fertilization

and Artijcial

Stimulation

Kagan [3] found in Fundulus heteroclitus that the “platelets” embedded in the cortex of unfertilized egg disappear after fertilization. The “platelets” seem to be identical to the cortical alveoli. Tchou and Chen [lo] may have been the first to observe the wave-like breakdown of the cortical alveoli at the time of fertilization in the egg of the goldfish (Carassius auratus). Independently, the writer [ 13, 171 observed the same phenomenon in the egg of the medaka. In ripe unfertilized eggs, the cortical alveoli are evenly embedded in the protoplasmic layer except for a small area around the animal pole. The first visible change at fertilization is a wave-like breakdown of the cortical alveoli beginning near the animal pole and ending at the vegetal pole. The change is subsequently followed by the separation of the egg membrane. The formation of the perivitelline space is a consequence of the breakdown of the cortical alveoli. The release of a colloid from the cortical alveoli into the interstice between the plasma membrane and the egg membrane is observable by means of a special method [23]. This is in harmony with previous observations that the egg membrane, which is a dialyzing membrane, is separated by a colloid osmotic pressure [14] and that a slight but significant decrease in the volume of the egg proper takes place after fertilization [15]. Hence it has become clear that the membrane separation is due to a colloid osmotic pressure of the perivitelline fluid which is derived from the content of the cortical alveoli. Essentially similar cortical response can be induced by a number of stimulating agents. When the cortex of either the animal or vegetal pole is pricked with a glass needle, the tip of which is 15-20,~ in diameter, the breakdown of the cortical alveoli begins at the site of stimulation and ends at the antipode. Pricking at the equator also causes the alveolar breakdown at the site of stimulation. The process of the breakdown in this case, however, does not end at the antipode, in a strict sense. The alveolar breakdown near the animal pole proceeds quicker than that near the vegetal pole, with the result that it ends at the point halfway between the antipode and the vegetal pole [ 171. The ripe unfertilized ova can be activated thermally by temporary treatment Experimental

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Fertilization

in fish eggs

at 45°C [13, 171. A number of chemical substances also induce the cortical changes. Surface active substances such as sodium taurocholate, sodium glycocholate, sodium oleate, saponin, digitalin and detergents (Aerosol OT, hlonogen, Nekal BX and Labolan) are effective activating agents [17, 191. A short exposure to vapors of lipid solvent such as chloroform, ether, benzene, toluene and iso-amyl alcohol induces almost complete activation while long exposure only results in partial activation [22]. The ripe ova can be activated by electric current. The breakdown of the cortical alveoli and subsequent separation of the egg membrane begin on the anodal and the cathodal sides. The propagation of the change underlying the alveolar breakdown is more rapid near the animal pole than near the vegetal pole [20]. Local stimulation induced by pricking and electric current indicates that there exists an excitation-conduction gradient which is the highest at the animal pole and the lowest at the vegetal pole. The reaction time at the vegetal pole for pricking is about twice that at the animal pole. By centrifugal force, the cortical alveoli are forced to accumulate in the centrifugal side. There is no relation between the egg axis and the direction of the accumulation. Centrifuged eggs retain their capacity for fertilization and for artificial activation. Even those centrifuged eggs which are alveolifree in the animal hemisphere can be fertilized. Alveoli-free cortex of centrifuged eggs can be stimulated by pricking. An important fact in fertilization of centrifuged eggs is that only the cortical alveoli embedded in the protoplasm break down while the alveoli which are accumulated in the yolk remain unchanged. This indicates that some sort of propagating change which underlies the alveolar breakdown takes place in the protoplasm either in the presence or the absence of the cortical alveoli, and that the change cannot propagate through the yolk. It has been postulated as the cause of the breakdown of the cortical alveoli that an “impulse” is provoked in the cortical layer by the sperm or activating agents, propagating in a wave-like manner. This impulse was termed the “fertilization-wave” by the writer in 1944 [17, 181. As an alternative view, an intra-cellular or an intra-cortical diffusion of a sperm substance or of a locally produced cortical substance might be supposed. That such a diffusion concept cannot be applicable is shown by the following heretofore unpublished experiment. When the vegetal pole, which has the lowest sensitivity, is pricked with a 5p-needle, most eggs show no cortical response. In such cases, not a single alveolus at the vegetal pole breaks down. Such eggs, however, show complete breakdown of the cortical alveoli on stimulation with a 15-20,u-needle or on repeated stimulation with a 5,u-needle. Experimental

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T. Yamamofo If some cortical substance which causes the breakdown of the cortical alveoli were produced at the site of pricking and diffuse either intra-celhrlarly or intra-cortically, some alveoli at the site of pricking would break down in response to the pricking with the 5,u-needle even in cases of unsuccessful stimulation. No such condition is realized. Hence the diffusion concept can be excluded. It may be remarked that the volume of the sperm of the medaka is roughly 1/1,000,000 that of the ovum. It is difficult to suppose a stoichiometrical reaction between an active substance of the sperm and a total of reactive substance in entire cortex of the ovum which would account for cortical changes in fertilization. If a chemical reaction between sperm and egg substances ever takes place, the reaction might be restricted only at the point of sperm attachment. Then cortical changes in the entire cortex proceed autonomously as the consequence of the propagating fertilization-wave in virtue of reactions of substances contained in the ovum itself. The occurrence of the fertilization-wave at the time of fertilization and activation in sea urchin eggs was confirmed by Sugiyama in 1953 [9] and by Allen in 1954 [a].

The Decrement Conduction of the Fertilization-

Wuve

The fertilization-wave is defined as an “impulse” which propagates along the cortical protoplasm as the cause of the breakdown of the cortical alveoli. Apparent wave-like breakdown of the cortical alveoli is a consequence of the propagation of this physiological wave. In normal fertilization the breakdown of the cortical alveoli begins at the animal pole and ends at the vegetal pole. In most cases the breakdown is complete. In some cases it is incomplete, i.e., the alveoli at the vegetal pole do not break down. This difference is due to the individuality of the fish from which ova are removed. If the breakdown in an egg is incomplete, all other eggs derived from the same female show the same result. The same batch of eggs which shows an incomplete breakdown in fertilization, manifests complete breakdown when the animal pole is pricked with a needle of 15,~ in diameter. This indicates that the intensity of the fertilization-wave provoked by the sperm is weaker than that produced by the 15,u-needle and that it conducts with diminishing intensity. The impulse provoked by the 15,u-needle is so strong that it can become the cause of the breakdown of the alveoli at the vegetal pole although it conducts with decrement during the passage through the cortex. In other words, the all-or-none principle cannot be applicable [ 181. This fact is also confirmed in fertilization and artificial activation of incompletely anesthetized eggs. Anesthesia with Experimental

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Fertilization

in fish eggs

391

either M/200 phenyl urethane or M/200 chloretone in Ringer’s solution brings about reversible inhibition in the fertilization reaction. When the eggs are completely anesthetized, no cortical response is provoked by the sperm or by activating agents such as chemical, mechanical and thermal stimuli. In incompletely anesthetized eggs such agents induce partial response, i.e., the cortical alveoli in the animal hemisphere break down while those in the vegetal hemisphere remain unchanged [18,21]. As has been mentioned before, there exists an excitation-conduction gradient in the unfertilized egg. Since the fertilization-wave propagates with decrement in such a heterobolic system, its velocity in a certain region varies with the direction of the wave. When the animal pole is pricked with a 15-20~ needle, the velocity at the middle region (AE) between the animal pole and the equator is 26,+ec., while that at the middle region (VE) between the vegetal pole and the equator is 15&ec. (29-33°C). When, however, the vegetal pole is pricked with a needle of the same size, the velocity at VE is 22p/set. while that at AE 19p/set. At the equator the conduction velocity is about 20,&ec. in both directions (unpublished). It is worth while noting that the decrement conduction of the fertilizationwave (impulse) was also confirmed by Allen [2] in sea urchin eggs.

Chemical Nature of Cortical Alveoli It is now possible to say that all the teleostean eggs so far examined have the cortical alveoli in unfertilized, non-activated ova [cf. 61, Their presence was demonstrated also in ova of salmonid fishes in which it has been difficult to observe them because of the opacity of the egg membrane [4,12]. It is possible to isolate the cortical alveoli from eggs of the medaka by crushing the unfertilized egg with a pair of glass needles in Ringer’s solution. This may mean that the fertilization-wave is not instantly established. It may be remarked that the reaction time of the alveolar breakdown is lo-20 seconds. The cortical alveoli isolated in Ringer’s solution are relatively stable. The cortical alveolus of the medaka consists of two parts, a thin wall and the interior content. Solubility tests on isolated alveoli indicate that their wall is soluble in such lipoid solvents as chloroform, ether, benzene, toluene and iso-amyl alcohol but insoluble in acetone which is a neutral fat solvent. This indicates that the wall of the cortical alveolus consists of lipoid [21]. That fact was subsequently confirmed by histological method by Aketa [I] in our laboratory. Konopacka [5] seems to be the first to demonstrate histologically that the cortical alveoli (gouttes claires) of the goby and the carp contain a mucoprotein. The polysaccharide nature of the cortical alveoli of some Experimental Cell Research 10

T. Yamamofo teleosts has been demonstrated by Kusa [7] and Thomopoulos, [ll]. same is true of the interior content of the alveoli in the medaka [l].

The

Chain Reactions in Cortical Changes It is inferred that the cortical changes involve a series of reactions which are connected in a catenary way. When unfertilized eggs are treated with isotonic oxalate solution, they reversibly lose the capacity for cortical changes on stimulation by pricking. These eggs, however, show the cortical response without further stimulation when transferred to Ringer’s solution. In this respect oxalated eggs differ fundamentally from completely anesthetized eggs which also fail to respond to stimulation, but no cortical response takes place on return to Ringer’s solution. It requires a further stimulation in order to induce the cortical response. Obviously some event occurs in oxalated eggs when they are pricked in the oxalate solution. To account for the stimulation in oxalated eggs, it is inferred that calcium ions are not necessary in the primary phase but that they are necessary in secondary or later phases of the cortical response. Not only are calcium ions unnecessary in the primary phase but release of Ca ions from the cortical protoplasm at the time of stimulation was demonstrated by a reaction of an exudate of protoplasm with sodium alizarin sulfonate. It was postulated that the first phase (stimulation) of activation takes place even in Ca-free condition, but the second phase (the fertilization-wave) is inhibited in oxalate solution. The cortical changes in Oryzias eggsinduced by activating agents may be expressed in the following chain reactions: Stimulants -+ stimulation -+ fertilization-wave --f breakdown of cortical alveoli --f release of alvoelar colloid -+ membrane separation [24]. For the establishment of stimulation, no calcium ions are necessary. In this phase free Ca ions are released from the cortical protoplasm. Calcium ions may be necessary for the establishment of the fertilization-wave or for the breakdown of the cortical alveoli or for both. As has been pointed out previously [ 13, 171 calcium ions are necessary in fertilization. In fertilization, union of the sperm with the cortical protoplasm is necessary prior to the establishment of stimulation. Calcium ions seem to be necessary both for the union of gametes and for the later phase or phases in the activation process followed by the establishment of stimulation by the sperm.

Mechanism of Breakdown of Cortical alveoli As mentioned before, the wall of the cortical alveoli of Oryzias egg consists of lipoid, and the isolated alveoli are relatively stable in Ringer’s solution. Experimental

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Fertilization in fish eggs

393

It was found that their wall is easily dissolved in dilute solutions of venoms from poisonous snakes and bees which contain lecithinase A. When isolation of alveoli is performed with a pair of polished steel needles instead of glass needles, the alveolar wall is dissolved. It was also found that the wall of the cortical alveoli suspended in crushed egg materials is dissolved in the presence of a trace of divalent Fe ions but not of trivalent Fe ions. These experiments suggest that the alveolar breakdown is brought about enzymatically at the time of fertilization and artificial activation. It may be postulated that an esterase is present in inactivated state in the unfertilized egg and the enzyme is activated as a consequence of the fertilization-wave. REFERENCES 1. AKETA, ALLEN, KAGAN,

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

K., Embryologia 2, 63 (1954). R. D., Expfl. Cell Research 6, 403 (1954). B. M., Biof. Bull. 69, 185 (1935).

KANOH, Y., Annof. Zool. Japon. 24, 13 (1950). KONOPACKA, B., Bulf. Acad. PoZonaise, Sci. et Let. (Ser. B. II) 62, 193 (1935). KUSA, M., Annot. Zool. Japon. 26, 73 (1953). Annot. Zool. Japon. 26, 138 (1953). RUNNSTRGM, J., Acfa Zool. 1, 321 (1920). SUGIYAMA, M., Biol.. Ball. 104, 216 (1953). TCHOU, S. and CHEN, C. H., Confr. Inst. Zool. Nat. Acad. Peiping 3, 35 (1936). THOMOPOULOS, A., Bull. Sot. Zool. France 78, 106 (1953). YAMAMOTO, K., J. Fat. Sci., Hokkaido Univ. Ser. VI (Zool.) 10, 253 (1951). YAMAMOTO, T., Proc. Imp. Acad. (Tokyo) 15, 269 (1939). Proc. Imp. Acad. (Tokyo), 15, 272 (1939). Proc. Imp. Acad. (Tokyo) 16, 482 (1940). J. Fat. Sci., Imp. Univ. Tokyo Sec. IV (Zoot.), 5, 461 (1941). Annof. Zool. Japon. 22, 109 (1944). Annof. Zool. Japon. 22, 126 (1944). __ Proc. Japan Acad. (Tokyo) 21, 197 (1945). Cyfologia 14, 219 (1949). Cyfofogia 15, 1 (1949). Annof. Zool. Japon. 24, 74 (1951). __ Expfl. Morphol. (Tokyo) (in Japanese) 7, 61 (1951). Expfl. Cell Research 6, 56 (1954). ADDENDUM

On the basis of observation carried out on sea urchin eggs stuck to a glass surface, Runnstrom and Kriszat (Exptl. CelZ Research 3, 419 (1952)) postulated that the impulse caused by the sperm attachment is propagated in the cortical layer by a chain reaction. In addition to the work of Allen (1954, lot. cit.) this conclusion was also proved beyond doubt for the sea urchin egg by the work of Runnstrom’s colleagues, Allen, and Hagstrom (Exptl. Cell Research 9, 157 (1955)). It is most interesting to find that there is such an agreement among recent works on sea urchin eggs with our results on a teleostean egg. Experimental

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