By H. Tomita
There are a number of color types of the medaka. The chromatophores of this fish are melanophores, xanthophores and leucophores. The inheritance of body colors has been studied by Aida (1921). The various body colors responsible to the genic action and the genic symbols of color types are determined phenotypically as follows: brown (wild type) BR; blue Br; orange-red bR ; white br; variegated orange-red B'R and variegated white B'r. The multiple alleles B, B' and b control the formation of melanin in melanophores and are autosomal. The gene B which is dominant to both the B' and b genes, makes the full formation of melanin. The B' gene which is dominant to the b gene causes variegations having pigmented (black melanophores) and non-pigmented areas (colorless melanophores) (Aida, 192l). The black pigmented area of the B' type are irregularly distributed. In the light color types (bR and br), the formation of melanin is so slight that melanophores usually invisible in the dispersed condition. Goodrich (1927) showed that a small amount of melanin in colorless melanophores can be demonstrated by concentrating melanin granules with adrenaline, and the number of melanophores is the same in all, these phenotypes controlled by the multiple alleles B, B' and b, but they differ in capacity of the melanin formation. Oka (1931) stated that the multiple alleles B, B' and b cause the difference in the development of melanophores with respect to the cell size and the degree of melanin content in embryos.
The genes R and r govern the deposition of an orange-red pigment in xanthophores and are sex-linked. According to Goodrich, Hill and Arrick (1941) the carotenoid in this fish is lutein. But Hirao, Kikuchi and Hama (1969) reported that lutein is only 4 per cent of total carotenoid, and tunaxanthin is a main component (about 40 per cent).
Aida (unpublished) analyzed a color interferer (ci) which in the duplex condition results in paleness of the body color. Thus the phenotype BciR is gray. This character had been analyzed by Takeuchi (1969) in details.
For long time, only four mutant genes have been known to be concerned with body colors. Recently many mutants for body colors have been found and established in our laboratory. The new gene bd belonging to the multiple alleles B,B' and b (B>bd>B'>b) was found. In this mutant the colorless melanophores of fry differentiate into pigmented melanophores like those of the wild type at about 10 mm body length. At least 25 genes seem to be concerned with the manifestation of body colors. The differentiation of melanophores and the formation of melanin may be controlled by at least 12 genes. However, genetic analyses of these color genes have not yet been completed.
Biochemical studies of the melanin formation of the medaka have not been advanced so far since Goodrich (1933) first demonstrated the presence of dopa oxidase in colorless melanophores. Rothman, Frysa and Smiljanic (1946) demonstrated the presence of an inhibitor of tyrosinase in the human skin and the removal of this by iodoacetamide. The studies in vertebrates have shown that the conversion of tyrosine into melanin is also catalyzed by tyrosinase (Hogeboom and Adams, 1942; Lerner and Fitzpatric, 1949; Foster 1951). Only a few studies have been performed along this line in the medaka. Oikawa (1972) studied biochemical nature of colorless melanophores in the medaka. Hama (1969) and Ide and Hama (1969) studied the difference among the color types in the medaka biochemically and morphologically.
Histochemical detection of dopa oxidase and tyrosinase in light color types
In colorless melanophores of the light color types (bR and br) Hishida, Tomita and Yamamoto (1961) have not only detected dopa oxidase but also succeeded to detect tyrosinase.
1. Dopa oxidase
Goodrich (1933) first demonstrated dopa oxidase in the skin of the medaka. He found that colorless melanophores of the light color types become to pigmented ones with incubation in dopa solution and suggested that colorless melanophores may not produce the normal amount of chromogen although sufficient oxidase is present and that pigmented melanophores possess both chromogen and oxidase in sufficient amount.
In our experiment, the skin of the light color types are incubated in dopa solution at pH 6.8 for detection of dopa oxidase. Melanophores with dopa melanin appear in stellate form. As dopa solution is autoxidizable, dopa melanin often accumulates on the surface of skin. Short treatment of skin with cold formalin is effective for obtaining clear results of pigmentation of colorless melanophores.
Dopa melanin formation in colorless melanophores is completely inhibited by the addition of KCN (M/1,000) and diethylthiocarbamate (M/l,000). Pretreatment of heating for five minutes at 80 oC also inhibits the formation of melanin.
Goodrich (1927) showed that colorless melanophores in the light color types (bR and br) of the medaka owe to the reduction in the amount of melanin, and colorless melanophores are incapable to produce the normal amount of chromogen although sufficient oxidase is present. This conclusion however is based on the dopa oxidase reaction. But the dopa oxidase reaction is not so excellent as a practical method for the detection of colorless melanophores. Dopa melanin usually deposits itself uniformly on the surface of isolated skin owing to the autoxidation of dopa. Moreover, a positive dopa oxidase reaction seems to be not so reliable as proof for the presence of dopa oxidase because dopa is too easily oxidized by nonspecific substances.
2. Tyrosinase
It is difficult in the medaka to detect the presence of tyrosinase in the incubation method used in the detection of dopa oxidase. The skin of light color types are incubated in tyrosine solution buffered to pH 7.3 for 48 hours at 30 oC. The results of them are usually negative. Foster (1951) showed that tyrosinase activity in light color mice was demonstrated by adding a sulfhydryl blocking reagent such as iodo-acetamide. In the medaka, the demonstration of tyrosinase in the light color types also is succeeded by adding iodoacetamide. Monoiodoacetic acid also are effective. By the addition of guanofuracin (0.05%) (antibiotics) to incubation media, tyrosinase is detected in place of sulfhydryl blocking reagents. After the pretreatment (one minute) of skin with chloretone (0.35%), chloroform (saturated solution) and octyle alcohol in the cold condition, the formation of melanin is induced in colorless melanophores. In some case, the melanin formation occurs in colorless melanophores incubated in tyrosine solution with antibiotics (penicilline or streptomycine). In any cases, the melanin formation does not occur without the substrate (tyrosine). Tyrosine is essential for the melanin formation in colorless melanophores of the light color types. The melanin formation is completely inhibited by the presence of KCN (M/500), phenylthiourea (M/1,000) and diethylthiocarbamate (M/1,000) in vitro. The skin fixed with cold formalin (10%) for an hour or heated for a few minutes results in inhibition of melanin formation.
Tyrosinase reaction is more useful for the detection of colorless melanophores as well as the evidence for the presence of tyrosinase, because the dopa oxidase reaction is not reliable because of the autoxidation of dopa. The colorless melanophores of light color types (bR and br) of the medaka are the well developed state with the dendritic processes and are capable to form black melanin with tyrosine and certain treatments. The colorless melanophores form full amounts of melanin in the presence of tyrosine and sulfhydryl specific poisons. The formation of melanin in colorless melanophores, however, is induced not only by guanofuracin (bacteriocide) but also by a short treatment with such agents as chloroform, octyl alcohol, acetone, urea and heat which have been used by Bodine and Allen (1938) in the study of melanin formation in insects.
It is difficult to draw any conclusion as to the nature of the inhibitor or the inhibitory state. It may be true that all the agents induced the pigmentation kill melanophores, while tyrosinase has not yet been denatured, and tyrosine penetrates to them freely. In some case, those cells incubated in tyrosine solution with an antibiotic (penicillin or streptomycin) without any pretreatment induce the pigmentation, when the cells die in these incubation media. There is a possibility that the inhibitor or inhibitory state is removed with the killing the cells caused by various agents. The colorless melanophores in the light color types (bR and br) of the medaka are in the state of full differentiation and tyrosinase may be in an inactive state within them. The inhibitory substance or state can be removed not only by sulfhydryl poisons but by a various agents which kill cells but not denature tyrosinese.
A quantitative study of dopa oxidase and tyrosinase
Tomita and Hishida (196la) have performed a quantitative study of dopa oxidase and tyrosinase.
1. Substrate specificity
Tyrosinase in the skin of the medaka can oxidize tyrosine and dopa but it does not oxidize p-, m- and o-cresols, p-chlorophenol, p-bromophenol, 3,5-dimethylphenol, 3,5-dimethylphenol, 3,5-dihydroxybenzoic acid, phenol and catechol. Tyrosinase activity is inhibited by M/1,000 phenylthiourea, M/1,000 sodium cyanide and M/1,000 diethylthiocarbamate, and is lost by heating for 10 minutes.
2. Dopa oxidase and tyrosinase
Crude enzyme extracted from skins of the light color types (bR and br) show dopa oxidase activity which is about ten times higher than that of the brown and gray types. Tyrosinase activity also is higher in the orange-red type than in the brown type. In the brown type, tyrosinase and dopa oxidase are extremely low. The variegated orange-red type shows almost the same dopa oxidase activity as the orange-red and white types. There is no clear relation between dopa oxidase activity and region of unspotted skin in the variegated type.
The colorless melanophore remains in the state of incomplete pigmentation. The possible explanation of this feature are as follows; (1) colorless melanophores are deficient in substrate (tyrosine), (2) they contain an inactive enzyme precursor, (3) in these cells, intermediate products in the processes of the formation of melanin are carried off to other metabolic pathways. It is difficult to analyze the control mechanisms which play a role in skins of the various color types. The activities of dopa oxidase and tyrosinase in the skin are higher in the orange-red and white types than in the brown and gray types. This fact is quite contrary to the first expectation. The results indicate that the activity of tyrosinase is very low when melanin is fully formed, and the action of gene B is restricted only to the stage during the time of formation of melanin. As granules having tyrosinase activity become more fully melanized, perhaps tanning in a chemical sense occurs. Some of active sites on the enzyme may be blocked so that apparent activity of the enzyme is no longer noticed in more fully melanized melanophores.
Dopa oxidase and tyrosinase in embryos and larvae
Tomita and Hishida (196lb) have examined dopa oxidase and tyrosinase in embryos and larvae.
1. Substrate specificity
Crude enzyme extracted from embryos is capable of oxidizing tyrosine and dopa, but it does not oxidize other phenols, such as p-, m-, and o-cresols, phenol, catechol, p-chlorophenol, p-bromophenol, p- and m-nitrophenols, 3,4- and 3,5-dimethylphenols. The substrate specificity of tyrosinase in embryos is restricted to tyrosine and dopa, as found in that of adult fish.
2. Dopa oxidase and tyrosinase
In the wild type, embryonic black melanophores first appear on the yolk sac when several somites are formed in the embryonic body. In the light color types only a few slightly pigmented melanophores are found on the yolk sac even in later stages. In both the brown and light color types the amount of melanin increase considerably a few days before hatching. Tyrosinase and dopa oxidase are first detected at stage 26 (melanin begins to accumulate in the eyes). Then, as the pigmentation proceeds in eyes and peritoneal covering, the activities of both enzymes increase linearly until hatching and the activities of dopa oxidase are nearly ten times higher than that of tyrosinase. It should be noted that the ratio of both activities remains almost constant throught the development. The difference in dopa oxidase activity between the brown and orange-red types is shown to be negligible. This may be due to the fact that the embryos are used as a whole including the eye and peritoneal covering, melanin synthesis of which is stronger than those of the yolk sac and the surface of the embryonic body. Activities of dopa oxidase and tyrosinase in adult skins of the orange-red type are higher than those in the brown type, contrary to the first expectation.
3. Inhibitor
Embryos cultured in phenylthiourea solution higher than M/3,000 from an early stage are wholly devoid of the melanin pigment, and in M/8,000 a trace of pigment appear only in the eye and head regions. When the pigment-free embryos, which are cultured in phenylthiourea solution till stage 26, are transferred into the isotonic balanced salt solution, the melanin pigment soon develops to the same extent as normal embryos. Non-pigmented larvae induced by phenylthiourea, gradually produce pigment in the isotonic balanced salt solution, and after three weeks, a considerable amount of pigment appears in the eye and head regions.
Thiourea also induces artificial albinism in concentrations higher than M/4, but embryos die before hatching. In concentrations lower than M/20, thiourea has no effect on the melanin formation. Thiouracil and methylthiouracil in nearly saturated solutions (M/200 and M/400 respectively) show no effect on the melanin formation.
In embryos in which the synthesis of melanin pigment has already begun it inhibits further pigmentation and phenol oxidase synthesis in M/3,000 phenylthiourea.
The inhibitory effect of phenylthiourea and thiourea on phenol oxidase may be resulted by chleating copper ion which is essential for phenol oxidase action. It is known these agents depress the function of thyroid hormone regulating the pigmentation of feather in bird, but the concentration in which these are effective as such are much lower. It may, therefore, be that these agents directly inhibit the process of melanin formation but not indirectly through thyroid function. In fact, these agents inhibit completely both activities of dopa oxidase and tyrosinase in extracts of embryos of the medaka, probably combinating with copper as described in other sources (Lerner, 1953).
Hydroquinone is known to accelerate the oxidation of dopa, but to inhibit tyrosinase activity in vitro (Lerner and Fitzpatrick, 1953). Monobenzylether of hydroquinone has no effect on the oxidation of dopa and tyrosine in vitro. Both substances are harmful to embryos and larvae, at least in higher concentrations. The most suitable con-centrations for the production of melanin-free larvae are 5 ´ 10 exp-4 M for hydroquinone, and 2.5 ´ 10 exp-6 M for its monobenzylether. The hydroquinone solution has to be renewed frequently, because it is autoxidizable. The melanin-free embryos obtained show low dopa oxidase activity, not withstanding the fact that 5 ´ 10 exp-4 M hydroquinone is shown to accelerate dopa oxidase in vitro. These agents which in vitro do not inhibit dopa oxidase activity, causes in vivo a great decrease in the activity. To account for this fact, there may be two possibilities either (1) the two enzymes are synthesized in the same process, which is inhibited by hydroquinone or its benzylether or (2) tyrosinase and dopa oxidase are in reality one and the same enzyme, at least in the medaka.
Embryos cultured in p- and m-cresol solutions in concentrations ranging from 0.005 to 0.01 per cent often lost their melanin pigment. They also show malformations and their developments are retarded. All embryos die before hatching in concentrations above 0.01 per cent. These weakly pigmented embryos have only very low phenol oxidase activity. Ortho-cresolindophenol (1 : 5,000) induces weakly pigmentation. Meta-cresolindophenol at the same concentration which is nearly saturation shows no effect. Meta-aminophenol (M/500) partially inhibits the melanin formation, but true albinism occurs only a few cases. Para-aminophenol has not been tested because of its toxicity. Meta-phenylenediamine (M/1,000) induces low pigmentation. Para-and ortho-phenylenediamines are autoxidizable and hence unsuitable.
4. Histochemical study
Hatched fry are not tested for enzymatic activities quantitatively, since it is not easy to collect sufficient materials. Instead, tyrosinase and dopa oxidase activities are checked histochemically. It is found that on the incubation with dopa or tyrosine solution with iodo-acetamide, colorless melanophores of the orange-red type turn black and assume the same shape as the black melanophores of the brown type.
Oka (1931) studied the development of melanophores in embryonic and larval stages of the medaka, and stated that normal cell size and melanin formation are controlled by the dominant gene B, whereas the recessive gene b governs the development of melanophores of small size and the production of smaller amount of melanin. In our experiment, presence of tyrosinase and dopa oxidase in larvae of the orange-red type is histologically demonstrated. However, the size and shape of colorless melanophores of the orange-red type are found to be nearly the same as those of fully developed pigmented melanophores of the brown type. It may be remarked that in larvae of the orange-red type, tyrosinase and dopa oxidase in vivo are present in an inactivated state.
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