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16. Control of Sex Differentiation

By T. Yamamoto

It thus becomes of great interest to discover the mechanism by which sex is determined, and to find whether by any means we can bring it under our control. Julian Huxley (1938)

Establishment of the d-rR strain

As materials adequate for the experiments on the control sex differentiation, the d-rR strain in which females are white (bb XrXr) and males orange-red (bb XrYR ), has been established. In this strain, white may simply be expressed as r and orange-red as R since both have common bb.

In 1946 a white (r) female (XrXr) was mated with a homozygous orange-red (R) male (XRYR) to obtain F1 heterozygous orange-red males (XrYR) and orange-red females (XrXR ). In the following year, an F1 heterozygous R male (XrYR) was backcrossed to another white (r) female (XrXr) (Fig. 16-1). The father-to-son or the Y linked inheritance is established in this mating type, so that females are ordinary white (r) and males are usually orange-red (R). Hence, the sex genotype, either XX or XY, can be discriminated by the body color, either r or R, respectively. The stock has since been maintained by interbreeding them in each succeeding generation. At present, the stock is of 24th generation.

In the stock, exceptions of two kinds, (1) arising by crossing over between the Xr and the YR chromosomes and (2) appearing by genic imbalance between the sex chromosomes and autosomes (see Chap. 15), are of rare occurrence, the total being less than 0.5 percent. A stock census has been conducted every year before the breeding season and all the exceptions appeared were removed from the stock.


Fig. 16-1. A diagram of establishment of the d-rR strain (XrXrfemale female-XrXRmale male) used in experiments on sex differentiation. Yamamoto, 1953.
XY-females induced by estrogens

Daily hatchings from early July to mid-July were divided each day into a number of groups and reared consistently on powdered diet containing varying dosage-levels of an estrogen. One group of fry was reared on the normal diet. In the first study (1953) in which estrone- and stilbestrol-induction or reversal in sex differentiation of XrYR zygotes was successful in the first place, a commercial nutritious food (Vita-Yoso) was used as the normal diet and the duration of hormone-administration was 8 months.

In later experiments, the normal diet having the following constituents has been used: 60 gm of shrimp powder, 30 gm of parched-barley flour, 6 gm of yeast preparation and 4 gm of powdered green tea. Perhaps the last mentioned ingredient can be ommitted. It was found (Yamamoto, 1959b) that the continuous peroral administration of an estrogen, beginning with the time of hatching and ending at a certain juvenile stages, covering the priod prior to and passing through the stage of the gonadal sex differentiation, is sufficient to induce permanent reversal in sex differentiation of XY zygotes.

Hence, in later experiments, a hormone diet has been administered from newly hatched fry (nearly equal 4.8 mm) to the juvenile fish of 12 mm or longer. Fortunately, the newly hatched larva of the medaka has an indifferent gonad (gonad primordium) and gonadal sex differentiation takes place between 6 and 11 mm stages. This hold entirely true in XY fry. As to XX larva a reference will be given latter. When an indoor experiment begins in early July, the necessary and sufficient period of hormone treatment is usually two months. Thereafter, young can be reared outdoors on the normal diet until they reach maturity, with occasional addition of live foods.

Fig. 16-2 shows a diagram of estrone experiment (Yamamoto, 1953). In controls, fish with XrXr genotype have differentiated into females and those with XrYR genotype have developed into males, as expected. In the experimental group, all estrone-treated fish have differentiated into females regardless of their sex genotypes.

Obviously, estrone-treated XrYR females are induced reversals in sex differentiation. An estrone-induced XrYR female is shown in Figure 16-3. The same result was obtained with a synthetic estrogen, stilbestrol. In this experiment, the dosage-level of estrone was 125 micro g/gm diet and that of stilbestrol was 250 micro g/gm diet with the result of sex-reversal in genetic males of 100 percent. In a further study (Yamamoto, 1959b), the administration of stilbestrol at the dosage level of 62.5 micro g/gm diet was found to be sufficient to induce sex-reversals in 100 percent.


Fig. 16-2. Right: A diagram illustrating reversal of sex differentiation in genetic males (XrYR) by administration of estrone diet to fly. Left: Control. Yamamoto, 1953.

Fig. 16-3. Above: A control orange-red male (XrYR), Below: An estrone-induced sex-reversal of male genotype (XrYR) carrying a cluster of fertilized eggs. Yamamoto, 1959.
Effects of estrone dosage level upon the percentage of XrYR females have been studied with varying doses of estrone (Yamamoto, 1959a). Percentage of sex-reversals in genetic males (XY) increases in proportion to dosage level of estrone from 10 micro g to 25 micro g/gm diet with 100 percent sex-reversals being secured at dosage level of 50 micro g/gm diet. The dosage level of estrone used in the previous study was more than twice as high as the minimum dosage level at which 100 percent sex-reversals can be obtained. Adequate dosage levels vary greatly with kinds of estrogens. For the accurate comparison of potencies of estrogens in inducing reversal of sex differentiation in genetic males the symbol GD50 was suggested as the designation of the dosage level at which there are 50 percent induced XY females (Yamamoto and Matsuda, 1963). Potencies of naturally occuring estrogens, viz. estradiol-17beta, estrone and estriol are shown in Figure 16-4 (Yamamoto, 1965). The writer and his co-workers have performed experiments to estimate GD50 values of a number of natural and synthetic estrogens (Table 16-1) (Yamamoto, 1969).


Fig. 16-4. Potencies of estradiol, estrone and estriol in inducing XY females. Yamamoto, 1965.
Using our d-rR strain as material, Hishida (1964) performed micro-injections of estrone-16-C14 (1.3 x l0exp-3 micro g/egg) and diethylstilbestrol-(Monoethyl-1-C14 ) (1.7 x 10exp-3 micro g/egg) into the egg just after insemination. Injected eggs were successfully reared until they reach young and adulthood. In the case of estrone, all zygotes with the male genotype (XrYR ) differentiated into females. In the case of stilbestrol, sex-reversals in genetic males were 90 percent.


Table 16-1. GD50 dosage levels of estrogens which induce 50% XY females in medaka, Oryzias latipes. Yamamoto, 1969.
XX-males induced by androgens

Attempts to inverse sex-differentiation in the reverse direction, viz. to obtain functional XX-males by administration of an androgen, methyltestosterone, did not go on smoothly by some reasons to which we will touch on later. Above all things, dosage levels used in the preliminary experiments have been too high. The gonad of intensively androgenized fish irrespective of their sex genotype, either became completely degenerated in juvenile stage or first differentiated into rudimentary testis through a drastic inhibition and later became completely atrophied with the result that when the fish were fully grown, they have no gonad, viz. they are sexless (neuter).


Fig. 16-5. Right: A diagram illustrating reversal of sex differentiation in genetic female (XrXr) by administration of methyltestosterone diet to fly. Left: Control. Yamamoto, 1958.
Eventually, however, functional sex-reversals of genetic females (XrXr) were successfully induced by administration of adequate dosage levels of methyltestosterone (Yamamoto, 1958). Methods used were the same as those employed in estrogen experiments. Daily hatchings from early July to mid-July were divided each day into a number of groups and reared indoors consistently on adequate dosage levels of methyltestosterone diet during 7-8 weeks until they reach 12 mm or larger. One group of fry was reared on normal diet as controls. Thereafter, they were reared outdoors in separate concrete pools until they reached maturity. Diet for outdoor rearing is a mixture of powdered shrimp and parched-barley flour with water fleas and tubificid worms occasionally added.


Fig. 16-6. Above: A control white female (XrXr. Below: A methytestosterne-induced functional male with female genotype (XrXr). Yamamoto, 1958.
The experiment on the production of methyltestosterone-induced XrXr females is diagramatically illustrated in Fig. 16-5. A methyltestosterone-induced XrXr male is shows in Figure 16-6. Figure 16-7 shows relationship between percentage of sex-reversals in genetic females (XrXr) and dosage levels of methyltestosterone. The threshold dosage level for induction of sex-reversals in a few XrXr fish is found to be 5 micro g/gm of diet. The percentage increases with dosage until it reach the maximum level at 25-50 micro g/gm of diet. The fact that the curve does not reach 100 percent is because of production of a few neuters (sexless) at 50 micro g/gm of diet. Beyond this level neuters increase. At the dosage level of 300 micro g/gm of diet or higher, all the full-grown fish, irrespective of their sex-genotype, become neuters.

By a neuter we define individual in which there is no gonad. The original gonad in a neuter is represented by a thread-like tissue without any germ cells. The neuter should not be confused with the intersex which has a gonad with both male-and female germ cells. Because of the fact that the size and shape of the anal and the dorsal fins of a neuter are exactly the same as those of a female, beginners are liable to confuse it with a female. However, a neuter has a poor urogenital papilla (UGP) without the medulla by which it can be distinguished, from a female. Secondary sexual characters of female, male, intersex and neuter are illustrated in Figure 16-8.

The most characteristic effect of methyltestosterone on juvenile gonads are (1) suppression of gonadogenesis in both sex genotypes and (2) induction of reversal of gonadal sex-differentiation in genetic females (XX). With increasing dosages of the androgen, the inhibitory effect on gonadogenesis is intensified.

In juvenile fish (12-13 mm TL) treated in a routine experiment with a high dosage of 300 micro g/gm of diet, the gonad of either sex genotype is reduced in size but definitely testicular with germ cells showing signs of degeneration. In all fish treated during larval stage at this dosage, the testis is completely degenerated by the time fish reach adulthood so that they can be classified as neuters. In an adult neuter, the original testis is reduced to a melanotic strand deprived of germ cells (Fig. 16-9A).

Testes of adult sex-reversals of genetic females (XrXr) and androgenized genetic males (XrYR) treated with moderate dosages in larval stage also, are by no means normal. They are atypical in form in varying grades. Some of them are illustrated in Fig. 16-9. Usually it is the more anterior region of the gonad that is most drastically reduced. In most cases, an irregular testicular lobe is attached to a melanotic sac of non-germinal elements.


Fig. 16-7. Relationship between percentage of induced sex-reversals of genetic females (XrXr) and dosage levels of methyltestosterone. i. u. =International unit. Yamamoto, 1958.

Fig. 16-8. Secondary sexual characters of female, male, intersex and neuter (sexless) of the medaka. Yamamoto, 1958.
Histological examination of the melanotic sac indicates that it is the non-germinal part of the testis resulting from complete destruction of germ cells. A melanotic peritoneal covering is present in the periphery, connective stromata are highly developed in the core and epithelial cells of the sperm ducts are columnar. No germ cells is found within the sac. Attached irregular testicular lobes, however, have essentially normal structures and contain mature sperms.


Fig. 16-9. Gonads of mature fish treated with methylteststerone (M) during larval stages. r=XrXr, R=XrYR. Dosage levels in micro g/gm-diet or ppm are indicated as subscripts following M. A, A thread-like tissue of a r neuter (M300), B, A degenerated testis of a R male (M50). C, A testis of a R male (M75). D, A fecund testis of a R male (M25). E, Free testicular lobes of r sex-reversal (M50). G. and H, Fecund testes of r sex-reversals (M12.5). I, A reduced ovary of a r non-reversal (M20). Yamamoto, 1958a.
Males having testicular lobes closely attached to the melanotic gonadal sac usually prove fertile in mating while those having lobes with connecting tubular peduncle (Fig. 16-9C) are generally impotent. Usually the frequency of impotent males increases with increase of dosage level. In some cases, free testicular lobes are found in the body cavity (Fig. 16-9E). With decrease of dosage level, the form of testis gradually approaches normal one. But even at a dosage level as low as 12.5 micro g/gm of diet the form of the testis of induced XrXr males is somewhat atypical and asymmetric (Fig. 16-9H).

A diagram of progeny of methyltestosterone -induced XrXr males mated with normal XrXr females is shown in Figure 16-10. Results of 9 sets of crosses using 27 induced XrXr males were none but daughters, a total of 1, 376. Sperms in testicular lobes of induced 'impotent' XrXr males were also functional.

To sum up, methyltestosterone in moderate and higher dosages alters the direction of sex-differentiation in genetic females and also exerts definite suppressive effect on gonadogenesis in both sex genotypes, degree of which varies with dosage levels. The regression of primitive germ cells is more intensive in the anterior region than in the posterior part of the testis and the left side is more suppressed than the right side. The situation is well realized in cases of intersexes (see next chapter).

In this connection it is interesting to refer to early differentiation of germ cells in the genetic male and female.

In the first paper (Yamamoto, 1953) in our sex-reversal series, the writer stated that newly hatched fry of both genetic males and females have indifferent gonads and that gonadal sex differentiation takes place during post-embryonic stages. This is virtually true in the genetic male even now.

Recent studies by Onitake (1972) and Satoh and Egami (1972) revealed that sex differentiation has already started before hatching in the genetic females. Some germ cells enter into meiotic prophase.

This fact may be a clue to interprete the production of non-germinal melanotic sac in genetic females treated with an androgen during larval stage. Already differentiated germ cells (oogonia and oocytes) might be completely degenerated by the action of androgen which is heterotypic to genetic females.

Taking into consideration of the production of a testicular lobe their germ cells at the posterior region seems to be still in indifferent state. This is possible by the presence of the anterior-posterior gradient in gonadal sex-differentiation as pointed out earlier (Yamamoto, 1953). The differentiated female germ cells might be destroyed by the effect of the androgen whereas indifferent germ cells (protogonia) at the posterior region have inverted their differentiation and differentiated into a testicular cells.


Fig. 16-10. A diagram of progenies of sex-reversal of female genotype (XrXr) and of treated genotypic males (XrXR). Yamamoto, 1958.
The fact that administration of estrogens to genetic males (XY) during larval stage induce a typical ovary may be ascribed to their still indifferent state of their gonad. Probably all germ cells of genetic males at hatching are either primordial germ cells or protogonia having the ambisexual potency of differentiation. So that entire germ cells differentiated into oocytes by administration of estrogens which are heterotypic.

The writer and his co-workers have studied the effects of several natural and synthetic androgens on reversal of sex differentiation in genetic females (XrXr). All the androgens tested were found to have similar mode of action as methyltestosterone excepting their potencies. The AD50 dosage levels of androgens which induce 50 percent of XrXr males are listed in Table 16-2.


Table 16-2. AD50 dosage levels of androgens which induce 50% XX males in the medaka, Oryzias latipes. Yamamoto, 1969.
Induced XY females mated with induced XX males

Success in reversing sex differentiation in both directions in the medaka rendered it possible to mate estrone-induced XY females with methytestosterone-induced XX males (Yamamoto, 1961, Fig. 16-11).

Twelve successful matings of sex-reversal females (XrYR ) by sex-reversal males (XrXr) resulted in 615 r females and 664 R males with 7 exceptional r males and 12 exceptional R females. Of these exceptions those successfully tested proved to be crossovers (XrYr males and XrXR females). Crossing over between the Xr and YR chromosomes does take place in induced sex-reversal females with the obligatory heterogametic constitution XrYR. The recombination frequency between the X and Y in the region delimited by the r and the sex-differential loci in induced heterogametic females XrYR is estimated to be 1.0+-0.4 crossover unit (%) which is 5 times as large as that (0.2+-0.01) in normal heterogametic XrYR males. Possible causes of sex difference in crossover value are discussed (Yamamoto, 1961). Hence, estrogen-induced XrYR females have a practical merit in obtaining otherwise rare white males (XrYr) by mating induced XrYR females with either normal XrYR or androgen-induced XrXr males.

Permanency of induced reversal in sex-differentiation

To achieve induction of complete reversal in sex differentiation in such a fish as the medaka whose sexuality is relatively stable among fishes, two conditions must be fulfilled. First, it is of paramount importance to administer a heterotypic sex hormone to fry continuously prior to and passing through the critical stage of gonadal sex differentiation. Second, adequate dosage-levels of heterologous sex hormone must be used. When these conditions are fulfilled, complete reversal in sex differentiation can be achieved.

In short, a heterologous sex hormone can cause zygotes which otherwise would become one sex to develop into the other. Resulted reversal in sex differentiation is not a temporary modification but a permanent one. Indeed we have no cases that the gonad produced by complete reversal fish ever reverts to that appropriate to its sex-genotype. Four of 4-year old methytestosterone-induced males of the female genotype (XX), induced during larval stage, have retained male capacities. In crossing with normal females (XX), they sired all daughters. An estrone-induced female of the male genotype (XY) at the age of 3 years has maintained female capacities (Yamamoto, 1968).


Fig. 16-11. A diagram illustrating progenies of estrone-sex-reversal female of male genotype (XrXR) mated with methyl-testosterone-sex-reversal of female genotype (XrXr). Yamamoto, 1961.
The medaka belongs to the type of short-lived species which undergo prominent senile changes. Its logevity under protected conditions is 4 years in males and 3 years in females. Aforementioned facts show that induced alteration of sex differentiation persists during the full life span of the medaka.

Ultimate sex-factors and proximate sex inducers

Indiscussing the problem of sex determination it seems necessary to separate the ultimate sex-factors and proximate causes which lead to the production of one or the other sex. As to the former, the theory of polygenic sex factors with superior or epistatic sex-genes in the sex chromosomes are cogently applicable in the medaka. The balance between the sum of female genes (·F) and that of male genes (·M) has ultimate bearing on sex determination. Proximately, however, sex differentiation seems to be caused by sex-gene-controlled sex inducers. Substances which induce or determine female (ovary bearer) and male (testis bearer) are called as gynotermone (termone < terminis) and androtermone, respectively (Hartmann, 1951).

A diagram of normal sex differentiation and induction of reversal of sex differentiation (boken line) is illustrated in Figure 16-12. An individual with the AAXX constitution, the balance in which is usually ·F > ·M, will produce more gyno-inductor (gynotermone) than andro-inductor (androtermone) and will induce ovary in acting on the indifferent gonad. An individual with the AAXY constitution, the balance in which is ordinary ·M > ·F, will produce more andro-inductor (androtermone) and induce testis in acting on the indifferent gonad (solid line). If, however, gyno-inductor were administered to the indifferent gonad of AAXY, it would differentiate into the ovary. On the contrary, if andro-inductor were administered to the indifferent gonad of AAXX, it would develop into the testis (broken line). From this scheme it is seen that the sex-genes are not direct factors for sex determination but they act indirectly through controlling the production of sex-inductors which are direct factors for gonadal sex differentiation.


Fig. 16-12. A diagram illustrating normal sex differentiation (solid line) and the induced reversal of sex differentiation (broken line). Yamamoto, 1962. See text for details.
Artificial direction of sex differentiation

Estrogens and androgens are defined as substances of hormonal nature which are responsible for expression of secondary sexual characters and for maintenance of sexual capacities. However, we are concerned with artificial induction of XX males (sperm producer) and XY females (egg producer).

Our experiments provide us with a conclusive evidence that the indifferent gonad, irrespective of its sex-genotype, is ambivalent as regard to its future mode of differentiation, viz. having potentiality in differentiating either the ovary or the testis. Whether or not natural sex inductors are estrogens and androgens or allied substances or entirely different other substances is still to be elucidated.

However our experiments show that estrogens can act as the gyno-inductor and androgens can act as the andro-inductor. It is noteworthy that progesterone and 17a-hydroxy-progesterone, and corticoids have no action while androstenedione, the precursor of testosterone, acts as a male-inductor although its poency is weak (Yamamoto and Matsuda, 1963; Yamamoto, 1968).

It has been commented even by outstanding geneticists in 1910's that the sex is irrevocally fixed at fertilization although its visible signs do not appear until later, so that it is not altered by events which may take place later (see Castle et al. 1912, p. 79; Correns, 1913, p. 72; Doncaster, 1914, p. 49; Morgan, 1919, pp. 197-198). For example, Correns (l.c.) stated that the determination of sex at will is as impossible as "squaring of circle" or "Perpetuun mobile".

In the case of vertebrates, there is a stage at which the individual has the indifferent gonad. In strict gonochorist such as the medaka, the indifferent gonad differentiates into either ovary or testis at the critical stage of sex differentiation. The sex is determined at this stage in reality.

Artificial production of one or the other sex at will has been accomplished in the medaka, Oryzias latipes. In passing it may be stated that induction of reversal of sex differentiation in both directions has also been successful in the goldfish (Yamamoto and Kajishima, 1968).

References

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Doncaster, L. 1914 The determination of Sex. Cambridge University Press.

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