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18. YY Males and Induced YY Females

By T. Yamamoto
In: "MEDAKA(killifish) : Biology and Strains"
Yamamoto, T. (ed.) , Keigaku Pub. Co., Tokyo, 1975, pp. 223-235.

Aida (1936) reported appearance of a single sporadic XrYr female. He regarded this exception as a naturally occurring sex reversal. As stated before, however, no sporadic sex reversal in the medaka may occur since it is a "differentiated" species and its sexuality is fairly stable. So that this exception may rightly be regarded as an individual in which the totality of sex genes is in the state of ·F > ·M despite the XY constitution by ovarly accumulation of autosomal polygenic F-genes (see Fig. 15-1).

Setting it aside, Aida mated the exceptional XrYr female with a normal XrYr male and obtained F1 r (white) daughters and r sons in a l : 3 ratio. Of three F1 r sons tested singly by XrXr females, two proved to be XrYr and one YrYr which fathered all males.

From the outset of our series of experiments, a fascinating interest has been focussed on the problem of viability of YY zygotes. Figure 18-1 shows a diagram of the expected result from mating an induced XrYR female with a normal XrYR male in our case (Yamamoto, 1955). If all YRYR males were viable, the offspring would be in a ratio of 1 r daughter (XrXr) : 3 R sons (2 XrYR plus 1 YRYR ). Actually, however, observed ratio was 1 r female :³2 R males, suggesting that YRYR males would be rare even if they were viable. As a matter of fact, only two (one in 1955, the other in 1959) out of 57 F1 R sons viz. only one out of every 28.5 instead of one out of every three proved to be YRYR sons. The first YRYR son is shown in Fig. 18-2. The rarity of viable YRYR males is beyond question. This fact was in strong contrast to Aida's result on the viability of white YrYr males. However, the discrepancy is cogently rationalized on the basis of the inductive reasoning and experiments described in this chapter. In short, both of us are right.


Fig. 18-1. A diagram illustrating the cross between an estrone-induced XrYR female and a normal XrYR male and the expected Fl classes. Yamamoto, 1955. See text for further discussion.

Fig. 18-2. A YRYR male detected among F1 R sons of estrone-sex-reversal females (XrYR) mated with normal XrYR males. This YRYR male in cross with XrXr females sired all male progeny, a total of 72 R sons. Formalin fixed specimen. Yamamoto, 1955.
The scarcity of surviving YRYR zygotes is reflected by failure to detect induced YRYR females among estrone-induced XrYR plus YRYR females produced by administration of estrone in two consecutive generations (Fig. 18-3). Theoretically, we would expect that one-third of these induced females of male genotype would have the constitution YRYR. Of 21 fish tested, all were induced XrYR females and no induced YRYR ones were detected (Yamamoto, 1959). This implies that if induced YRYR females were ever viable, they would also be rare as YRYR males.


Fig. 18-3. A diagram illustrating an attempt to generate estrone-induced YRYR females by administration of estrone to both Fl and F2 generations. See text for the result. Yamamoto, 1959a.
Assuming that the ratio of induced YRYR females to induced XrYR plus YRYR ones is 1 : 28.5 as that of YRYR males to XrYR plus YRYR ones, we can estimate the minimum number of fish to be tested in order to obtain at least one disirable type (YRYR) at the 99 percent level of probability. Applying Muller's (1923) formula it is n=4.6 x (28.5-0.5)=129. The failure in detecting YRYR females was entirely due to the small number (2l) of test matings.

Contrary to the rarity of viable YRYR males, surviving YRYr males are found to be by no means rare, and as viable as XrYR and XRYR males. This was first suggested from the numerical proportions of phenotypic classes among the F1 offspring of sex-reversal XrYR females mated with normal white males (XrYr) (Yamamoto, 1959b). Validity of this fact was convincingly demonstrated in the later study (Yamamoto, 1961) in which three XrYR and eight YRYr males were actually detected among orange-red sons of sex-reversal females (XrYR) mated with normal XrYr males. These facts led to the conviction that sex reversal in YRYr zygotes would be induced by administration of an estrogen in two consecutive generations.

Diagrams of experiments (Yamamoto, 1963) in obtaining YRYr males (control) and induced YRYr females are illustrated in Fig. 18-4 and Fig. 18-5, respectively. In the first generation offspring were used of our d-rR strain, in which female parents were XrXr and male parents XrYR. Twelve estrone-treated female (symbolized ER(female+female)) were mated with six normal white males (XrYr), denoted Nr(male+male). In the control group (ER(female+female) X Nr(male+male) > N), the ratio of 1 r female (XrXr) : 1 r male (XrYr) : 2R males (XrYR + XRYr) would be expected if all 12 female parents were of the genotype XrYR. Actually, however, R females appeared also. This has been resulted by presence of one or more crossover XRXr females among 12 estronized mothers (ER) (see original paper of Yamamoto, 1963). Setting it aside and ommiting resultant unexpected offspring, the following results of test crosses were obtained. Of nine R sons singly tested by r (XrXr) females, five were XrYR and four were YRYr males. The latter ones produced all male progeny.


Fig. 18-4. A diagram illustrating the crosses used to generate YRYr males. Estrone was fed to the Fl generation whereas normal diet was given to the F2 generation. Yamamoto, 1963a.

Fig. 18-5. A diagram illustrating crosses used to generate estrone-induced YRYr females. Estrone was fed to both the F1 and the F2 generations. Yamamoto, 1963a.
In the experimental group (Fig. 18-5), estrone has been administered to fry of the second generation again. All white and all orange-red ones differentiated into females.

Of estrone-induced R daughters (ER(female+female) X Nr(male+male) > ER females) successfully tested singly by XrYR males, seven were identified as XrYR three as YRYr. Three sex reversals among (ER(female+female) X Nr(male+male) > E) R females, being of YRYr genotype, produced a total of 381 all-male progeny with a single exceptional female in one family. Theoretically, we would expect that the ratio of induced XrYR females to induced YRYr ones in experimental series would be 1 : 1 if all of the latter genotype were viable. The observed numbers (7 XrYR :3 YRYr) do not deviate significantly from 5 : 5 (P = 0.34) using the estimate by binomial expansion (see Mather, 1951, 1960). These findings convincingly demonstrate the accomplishment of induction of sex reversal in the extraordinary genotype YRYr of the medaka under the influence of estrone administered during larval stage. Thus, estrone is effective in permitting the indifferent gonad not only of XY but also YY zygotes of the medaka to develop as ovary. What is noteworthy is that induced sex reversals of the YY genotype can retain female equipment and functioning despite the absence of the X chromosome.

The fact that practically all YRYr zygotes are viable whereas surving YRYR ones are rare led us to the inquiry into the viability of YrYr males. A diagram of the experiment performed (1964) is given in Figure 18-6. Parents used are XrXr females and XRYr males produced by mating XRXR females with XrYr males. By administration of estrone (50 micro g/gm diet) to F1 fry, estrone-induced XrYr females were obtained. By mating these females with XrYr males 24 r daughters (XrXr) and 58 r sons (XrYr + YrYr) are obtained. Twelve out of these r sons, presumably either XrYr or YrYr,were individually tested by normal XrXr females. Of 12 males tested, 10 sired white females and white males in a 1 : 1 ratio demonstrating their genotype to be XrYr. The remaining two fathered only males, proving their sex genotype to be YrYr. In this type of matings, the probability of picking a YrYr son is 0.33 if all YrYr survive. The observed ratio of 10 : 2 does not deviate significantly from the theoretical 8 : 4. Thus, both numerical relations of the ratio of phenotypic classes segregating among the F1 of estrone-induced XrYr females mated with XrYr males, and the ratio of the genotypes XrYr to YrYr actually detected by testcrosses, demonstrate the viability of all YrYr males. Aida's (1936) result stated before is confirmed herein by induced XrYr females mated with normal XrYr males. Viability of practically all YrYr zygotes versus rarity of YRYR ones is rationalized by a theory presented in the next chapter. This being so, it is not surprizing that estrone induction of sex reversal in YrYr zygotes has successfully been accomplished (Yamamoto, 1967). Diagrams of control and experimental groups in obtaining induced YrYr females are shown in Figures 18-7 and 18-8, respectively. Parents of the first generation were 12 white females (XrXr) and 12 orange-red (XRYr) males. By administration of estrone to fry of the first generation, all 38 orange-red (XRXr) and 24 white (XrYr) fish developed into females, with only two non-reversed white males. Obviously, 92 percent (24/26) of white fish were estrone-induced XrYr females.


Fig. 18-6. A diagram illustrating crosses used to generate YrYr (white) males. Estrone was fed to the F1 generation whereas normal diet was given to the F2 generation. Yamamoto, 1964a.
As parents of the second generation, 24 estrone-induced white females, Er(XrYr) females, and 12 normal orange-red males (XRYr) were used. In the control (see Fig. 18-7), 11 R females (XrXR), 6 R males (XRYr) and 17 r males (XrYr + YrYr) were produced, which approximate a ratio of 1 : 1 : 2 (Xexp2 2 = 1.5, P = 0.5-0.3). The numerical relation of the classes in segregating progeny seems to indicate that YrYr males are all viable, reconfirming the earlier conclusion (Yamamoto, 1964). This is verified by actual detection of YrYr males in the following progeny tests.


Fig. 18-7. A diagram illustrating another crosses (control ones of those shown in the next Figure) used to generate YrYr males. Estrone was fed to the F1 generation whereas normal diet was given to the F2 generation. Based on the experiments of Yamamoto, 1967a.
Of 17 r males in the control group of the second generation (Fig. 18-7), nine were tested singly by XRXr females to identify their genotype either as XrYr or YrYr. They are clearly divided into two categories in respect of numerical relations of classes in their progenies. Five families comprise R(female), R(male), r(female) and r(male) in a 1 : 1 : 1 : 1 ratio showing that the genotype of the parental r males is XrYr. The remaining four r males are identified as YrYr males since they sired all-male progenies (1,158 in number) with R and r males in a 1 : 1 ratio with only one exceptional R and one r females.

Testcrosses showed that exceptional R and r females are of the genotypes XRYr and XrYr, respectively. Hence, these exceptions may be regarded as rare individuals in which autosomal F-gene so overly accumulated that they differentiated into females in spite of their XY constitution.

In experimental group (Fig. 18-8), estrone was administered to the second generation again and obtained 28 R females (XrXR + XRYr) and 13 induced r females (XrYr + YrYr). Of R 28 ER females, 12 were submitted to testcrosses. Of these, eight successfully produced offspring sufficient for identification of their genotype. The remaining four either died before reproducing or produced only a few fry which were not raised. Of eight ER females tested individually by XRYr males, three produced R(female), R(male) and r(male) in a ratio of 2 : 1 : 1 and were identified as XRXr, while five did so in a ratio of 1 : 2 : 1 and were inferred as XRYr. None of these families deviate significantly from expectations, nor does the total.


Fig. 18-8. A diagram illustrating crosses used to generate estrone-induced YrYr (white) females of the medaka. Estrone was fed to both the F1 and the F2 generations. Based on the experiments of Yamamoto, 1967a.
Of 13 Er females tested, three died before reproducing, two were barren owing to extreme plump abdomen, and two were lost, so that only six produced offspring. The six families are divided into distinct two categories, as expected. In mating with XRYr males, three produced offspring comprizing R females, R males and r males in a ratio of 1 : 1 : 2 showing that their genotype to be XrYr. The remaining three Er females produced all-male offspring, R males and r ones in a 1 : l ratio, with a single exceptional R female. Obviously the genotype of these three Er females must be YrYr. None of these families deviates significantly from expectation. Thus, production of estrone-induced YrYr females was successfully achieved.

The single exceptional R female was tested by an XRYr male and found to be an XRYr female arising from genic imbalance between allsomal and autosomal sex genes.

In addition to 4 YrYr males detected among Nr males (Fig. 18-7), 324 YrYr ones were detected among offspring of testcrosses of ER and Er females (Fig. 18-8) and exceptions in the writer's experiment (Yamamoto, 1967).

Artificial production estrone-induced white YrYr females and mass production of YrYr males corroborate the conclusion that practically all YrYr zygotes are viable in contrast to rarity of viable YRYR ones.

References

Aida, T., 1936 Sex reversal in Aplocheilus latipes and a new explanation of sex differentiation. Genetics 21 : 136-156.

Mather, K., 1951 The Measurements of Linkage in Heredity. Methuen & Co. Ltd., London, John Wiley & Sons, Inc., New York.

Mather, K., 1960 Statistical Analysis in Biology. Methuen & Co., London.

Muller, H. J., 1923 A simple formula giving the number of individuals required for obtaining one of a given frequency. Am. Natural. 57 : 66-73.

Yamamoto, T., 1955 Progeny of artificially induced sex reversals of male genotype (XY) in the medaka (Oryzias latipes) with special reference to YY-male. Genetics 40 : 406-419.

Yamamoto, T., 1959a A further study of induction of functional sex reversal in genotypic males of the medaka (Oryzias latipes). Genetics 44 : 739-757.

Yamamoto, T., 1959b The effects of estrone dosage level upon the percentage of sex reversals in genetic male (XY) of the medaka (Oryzias latipes). J. Exp. Zool. 141 : 133-154.

Yamamoto, T., 1961 Progenies of induced sex-reversal females mated with induced sex-reversal males in the medaka, Oryzias latipes. J. Exp. Zool., 146 : 163-179.

Yamamoto, T., 1963 Induction of reversal in sex differentiation of YY zygotes in the medaka, Oryzias latipes. Genetics 48 : 239-306.

Yamamoto, T., 1964 The problem of viability of YY zygotes in the medaka, Oryzias latipes. Genetics 50 : 45-58.

Yamamoto, T., 1967 Estrone-induced white YY females and mass production of white YY males in the medaka, Oryzias latipes. Genetics 55 : 329-336.