By T. Yamamoto
In: "MEDAKA(killifish) : Biology and Strains"
Yamamoto, T. (ed.) , Keigaku Pub. Co., Tokyo, 1975, pp. 236-242.

Successful induction of complete reversal in sex differentiation in the medaka (Oryzias latipes), where the normal sex differentiation is XX for female and XY for male, makes it possible to attack a number of problems otherwise difficult to approach. The viability of YY zygotes is one of challenging problems. Since the time of Bridges (1916) the YY zygote in Drosophila, arising from nondisjunction, has been known to be invariably nonviable. This is because the Y in Drosophila is mostly, if not entirely, inert.

Exceptional fishes that have arisen by genic outbalance between allosomal and autosomal sex-genes can be and have been used for such a purpose. For instance, Winge (1934) and Winge and Ditlevsen (1938, 1947) in the guppy, Poecilia reticulata (Lebistes reticulatus) demonstrated viability of YMaYPa (Ma = Maculatus, Pa = Pauper) versus lethality of YMaYMa. Aida (1936) showed that YrYr males in Aplocheilus (now Oryzias) latipes are viable. They used exceptional XY females, which sporadically appeared in their breeds. The appearance of such exceptions, however, is often too infrequent and sporadic to be used satisfactorily to attack the problem under consideration. Aida for instance, used only two exceptional XY females, which were inappropriately called sex-reversals.

As stated before viable YRYR zygotes are extremely rare. A lethal action of some sort seems to be operative in the majority of the zygotes having the constitution YRYR. In contrast to the rarity of YRYR males, the majority of YRYr and YrYr males are found to be viable (Yamamoto, 1961, 1964). Furthermore, it was possible to invert sex differentiation of YRYr (Yamamoto, 1963a) and YrYr zygotes (Yamamoto, 1967) and actually to detect them as functional females.

The hypothesis that the writer has been led to adopt (Yamamoto 1964a) is the presence of an inert segment (-) in the regular YR chromosome, which in the duplex condition results in nonviavility, and the presence of a "viability" section (+) at the corresponding region in the X chromosome (Fig. 19-1). It is true that our results could be interpreted as well on the basis of the presence of a recessive lethal factor (l) in the regular YR chromosome. The asymmetry of crossing over occurring in XrYR and XRYr males (Aida, 1930) favors the inertsection hypothesis rather than a single lethal factor, since a point lethal does not, as a rule, affect the frequency of crossing over.

If our hypothesis be correct, we would expect the following three corollaries: (1) the F1 orange-red sons from XrXr(female) X YRYR(male) may comprise the two genotypes, Xr,+YR,- and Xr,+YcR,+ (2) non viability of the majority of XcRYR males (XcR,-YR,-), where XcR is a recombinant XR and (3) viability of YrYr zygotes, which would be of the constitution Yr,+Yr,+. All these consequences of the hypothesis are actually found to be true. The presence of the inert section (-) in a region of the regular YR chromosome, therefore, has become more than just a hypothesis.

Genetic analysis of F1 sons of YRYR male If our hypothesis were correct, it is legitimate to suppose that surviving YRYR males would be different from nonviable YRYR zygotes in their genetic constitution, the former would be YR,-YcR,+ and the latter YR,-YR,-. We would then expect the F1 of the YR,-YcR,+ male mated with Xr,+Xr,+ females to comprise two genotypes, Xr,+YR,-and Xr,+YcR,+. It is impossible to distinguish the two genotypes by testing with normal Xr,+Xr,+ females, but the estrogen-induced XrYR female (presumably Xr,+YR,-) using as tester would provide information about their genotypes.

To follow up this expectation, 12 orange-red sons (XrYR) of the YRYR male (the second YRYR reported in 1959) by normal females (XrXr) were selected at random and mated singly to estrone-induced females of male genotype (XrYR, ER females). Of 12 matings, seven produced offspring in a reasonable numbers. Of seven male parents, four fathered offspring in a ratio of 1r(female) : 2R(male) and two sired to conform to a 1r(female) : 3R(male) ratio. The remaining one produced offspring which are in agreement with both 1r(female) : 2R(male) and 1r(female) : 3R(male) expections, but closer to the former. It appears that matings of five out of seven were induced Xr,+YR,-(female) X Xr,+YR,-(male), and the remaining two were induced Xr,+YR,-(female) X Xr,+YcR,+(male). Among the offspring of the former, the genotype YR,-YR,- is considered to be lacking, although rare YR,-YcR,+ could be present.

These results can be explained if we assume that the viable YRYR males had the constitution YR,-YcR,+, where the (+) has been transferred from the Xr,+ by crossing over. The ratio of Xr,+YR,- (5) to Xr,+YcR,+ (2) agrees statistically with the expected 1 : 1 ratio. Although the F1 sons (XrYR) of the viable YRYR male are phenotypically alike, they are of the two different types with respect to the nature of their offspring, in agreement with our hypothesis.

Curious genetic behavior of crossover XrXcR Another evidence for the presence of an inert section in the regular YR chromosome comes from a curious genetic behavior of crossover XrXcR females, where XcR stands for the XR chromosome, the R of which is known to have been drived from the YR by crossing over. On several occasions, the writer has met with the fact that a crossover XrXcR female in mating with a normal XrYR male produced offspring in the ratio lr(female):1R(female):1R(male) instead of the theoretical lr(female) : 1R(female) : 2R(male). This fact is particularly evident when enough offspring are produced for statistical tests of significance. In a previous paper (Yamamoto, 1959a, p. 751), the writer commented that "Although the observed proportion showed a significant departure from the expected, we can not label these exceptions other than XrXR viz. crossovers." Further data relevant to this curious fact were given later (Yamamoto, 1961 p. 173). Now we can conclude convincingly that it is the XcR,-YR,- class that is lacking among progeny of crossover Xr,+XcR,- females X Xr,+YR,- males. There is a further evidence to prove nonviability of the genotype XcRYR. The F1 orange-red sons (XrYR plus XcRYR) from mating XrXcR female with XrYR male are submitted to progeny tests in order to ascertain whether or not the genotype XcRYR could be detected (Yamamoto, 1964a). If the genotype XcRYR were viable we would expect to find XrYR and XcRYR males in a 1:1 ratio among males tested. Of 26 males tested all proved to be XrYR and no XcRYR was detected. Therefore, it seems inevitable to suppose that the majority of XcRYR would be nonviable since they are duplex for the (-) segments (XcR,-YR,-). Since both XcR,- and YcR,+ could only be produced by crossing over between the Xr,+ and YR,-, the XcR,-YR,+ combination would be extremely rare.

Viability of all YrYr zygotes Practically all YrYr zygotes are viable because they are Yr,+Yr,+ in constitution.

The problem of viability of YY zygotes is considered on the basis of an inert segment in the ordinary YR chromosome and of a viability (positive) segment in the corresponding section of its mate (regular Xr). For our symbols the ordinary XR and Xr are XR,+ and Xr,+, while the regular YR and Yr are YR,- and Yr,+, respectively. At this point it is necessary to refer to the origin of the Yr chromosome in our fish. We have as yet no evidence to indicate that the Yr chromosome has arisen from the YR chromosome by mutation. Originally, the r gene seems to have arisen from the R in the X chromosome by mutation in some time in Japan. The Yr chromosome in both Aida's and our breeds is known to have originated through transfer of the r from the Xr chromosome by crossing over. Hence, the Yr is a recombinant in reality. It is natural to infer that the r gene together with the (+) section must have transferred from the Xr,+ to the Y by crossing over, resulting in the formation of the Yr,+ chromosome. From this we can plainly understand why the Yr chromosome contains the viability segment (+) whereas the regular YR involves an inert section (-).

While we can detect crossovers occurring between the differential segment (x) and the r locus by their color phenotypes and sex, crossovers occurring in the region delimited by the r and (+) segment cannot be identified by any visible traits. They can only be detected by the sex-ratio among the progeny of suitable testcrosses.

The (+) segment is referred to as a "viability" section which must include genes essential for viability. In the medaka, the regular Y chromosome still retains the homologous section containing the R or r and perhaps other genes in addition to the (-) segment. From the evolutional viewpoint, it is interesting to find that even among fishes there is such a species as the medaka in which the Y chromosome already showing a sign of deterioration, perhaps representing a transitional stage in the gradual deterioration of the Y chromosome in the animal kingdom (Yamamoto, 1963b, 1964a).