Secondary Sexual Characters
          Nobuo EGAMI
          In: "MEDAKA(killifish) : Biology and Strains"
          Yamamoto, T. (ed.) , Keigaku Pub. Co., Tokyo, 1975, pp. 109-125. 

8. Secondary Sexual Characters

By N. Egami

Secondary sexual characters of the Medaka and experimental modification of the characters have been reported by many investigators. In early years this species was used for genetics and embryology and the sex of the material was identified by the size and shape of the dorsal and the anal fins (Ishihara, 1919; Aida, 1923; Ono, 1927; Goodrich, 1929 and Kamito, 1929). For the practical purpose, the sex of adult fish can be determined easily by the size of the anal fin from lateral side of swimming fish by naked eyes (Fig. 8-1). However, in order to determine the sex of material it is safe to observe an anesthetized fish under a low-power microscope. Among various sexual characters, the most prominent feature of the male sexual character is the presence of small papillar processes on fin rays of the posterior region of the anal fin. The secondary sexual characters of this fish will be described:

Body size and body shape

Body length of many specimens of Oryzias latipes collected at 30 different localities were measured. However, no significant differences in adult body-length were recognizable between the both sexes. No sexual difference in body size is noticed by careful observation of living fish in aquarium.

Slight and statistically significant difference in the body shape of this species has been pointed out in wild materials (Egami, 1959). The adult males are relatively larger in depth of the body than the adult females and young of both sexes. This difference is caused by an elongation of the interhemal and interneural spines in male fish (Fig. 8-2). Some measurements are given in Table 8-1 (Egami, 1959).

Dorsal fin

In the male the rays of the dorsal fin are longer and much thicker than in the female (Oka, 1931; Okada and Yamashita, 1944, and others). The dorsal fin of the male is longer and has a saw-toothed distal edge (Fig. 8-3, Oka, 1931). The posterior margin has a shallow notch, the hindmost ray being separated from the rest. The interneural spines supporting the anal fin in the male are better developed than in the female, the bony wings along the anterior and posterior sides of the spines are well developed in the male (Fig. 8-4, Egami and Ishii, 1956). Administration of androgen induces development of the dorsal fin.

Pectoral fin

As Oka (1931) has already reported, no significant differences are found in the size and shape of the pectral fin between the male and the female fish except for the presence of small papillar process on some joints near the distal end of the second fin ray in the male fish during the breeding season (Fig. 8-5, Egami, 1956). The fin rays of the second ray in the male are slightly thick. Development of the second ray and the processes are controlled by androgen. The nature of the papillar processes found on the fin ray is same to that found on the male anal fin rays but the size is very poor in development in the pectoral fin.

Ventral fins

In the male fish the ventral fin barely reaches the anterior margin of the anal opening. In the female, on the contrary, it is larger and goes beyond the anus and reaches the anterior base of the anal fin the shape of the fins being shown in Figure 6 (Oka, 1931; Egami, 1956). The pelvic girdle supporting the ventral fin in the male is well developed (Egami, 1956).

Gonadectomy in the male induced noticeable growth of the intact ventral fin and a rapid regeneration of the amputated fin, whereas in the female it did not result in any increase of the fin length. Administration of androgenic substances depressed both the regeneration of the fin in the two sexes and the potential growth of the intact fin. From these results it is concluded that the shorter ventral fin of the male is a result of inhibitory effect of androgen (Suzuki Niwa, 1959).

Caudal fin

No morphological differences between the male and the female caudal fins are observable. However, the leucophores, which become distributed along the distal border of the caudal fin of the male in the breeding season are not apparent in the female (Okada and Yamashita, 1944). The sexual difference in the degree of development of the caudal-fin leucophores has been found by several investigators. Arai and Egami (1961) studied this sexual character and role of androgen in the development of the character was demonstrated by a series of experiments.

Shape and size of the anal fin

Many investigators reported the marked sexual difference in shape and size of the anal fin. The anal fin of the male takes the shape of a parallelogram and its pointed posterior and reaches as far as the base of the caudal fin. In the female, on the contrary, the small anal fin is right-angled triangle in shape and the narrowed posterior edge ends anterior to the base of the caudal fin. The anal fin rays of the male is longer and thicker than those of the female. Ray joints in the male are much more in number than those in the female. In the male the fin-rays rarely branch dichotomously, in the adult female, on the contrary all the fin-rays are bifurcated dichotomously at their distal ends (Oka, 1931; Okada and Yamashita, 1944). The distal edge of the male fin is a saw-toothed in the male (Fig. 8-7).

During the mating behavior, the male embrances the posterior part of the female body by means of the long dorsal and anal fins (Ono and Uematsu, 1957; Egami and Nambu, 1961). During the breeding season, many lencophores distribute along the distal border of the anal fin in the male (Okada and Yamashita, 1944).

Besides, the male possesses small papillar processes on most of the joints of the second to the seventh to nineth ray from the posterior end of the anal fin.

Papillar processes on the anal fin

The processes on the male anal fin rays were first reported in 1931 by Oka. These processes appear only in adult male and are found on every joint of the five to seven rays; from the second to the sixth, seventh or eighth counting from the posterior end of the anal fin. The processes look opaque by reflected light while they present a somewhat mottled appearance with a clear axial area along the whole length by the transmitted light and the joint of the fin-rays from which the process arises is transparent (Oka, 1931).

Appearance and histological structure and development of these processes was studied (Oka, 1931; Okada and Yamashita, 1944; Egami et al, 1965; Figs. 8-7, 8-8). Even in winter the processes do not disappear, however, they were better developed during the breeding season.

Fine structure of the surface of the fin-ray process has recently been examined (Yamamoto and Egami, 1974) (Fig. 8-9). The fin is covered with epidermal cells and finger print like patterns composed of discontinuous lines were observed. Some of the epidermal cells near the base of the papillar process become remarkably enlarged in the surface area. Cells on the surface of the protruding part of each process, especially near the apex, are small in area and exhibit a pleated appearance with compactly arranged wavy ridges. An amorphones material presumably mucous substance, usually covers some areas near the apex of the process.

If the female fish were treated with androgen, the papillar processes were induced on the comparable area of female anal fins. This process was studied by several works and interesting results were obtained. For instance, competence of female tissue to androgen (Okada and Yamashita, 1944; Egami, 1954), antagonistic and synergistic action of various steroids and other substances to androgen (Egami, 1954), effect of environmental conditions (temperature, ionizing radiations, light conditions, salinity of medium etc.) on the androgen induced process formation (Egami, 1954, 1955; Egami, et al, 1965; Masuda, 1953; etc.) were investigated.

Cytological and cytochemical processes during the process formation after androgen treatment were also studied (Egami, et al, 1965; Uwa, 1969, 1971). Mitotic figures begin to appear in the process forming area within 24 hours after beginning of the androgen treatment (Egami, et al., 1965). The increased incorporation of 3H-uridine was detected between 12 and 24 hours after androgen administration. The percentage of 3H-thymidine labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of 3H-leucine was found to increase during the first 12 hours, attaining a constant level at 24 hours. These results are correspond to the histological changes in the process forming area (Uwa, 1969). The synthesis of collagen during the process formation was also studied by the autoradiographies with 3H-proline and 3H-hydroxyproline. The rapid increase in the incorporation was observed and it was concluded that collagen synthesis is initiated between 48-72 hours after the treatment (Uwa, 1971).

Urinogenital papillae

The urinogenital papillae in the female are a pair of protuberances from the ventral surface between the anus and the oviduct opening and the papillae have a thick cortex of stratified epithelium as well as a highly vascularized medulla (Fig. 8-10) (Yamamoto, 1953; Yamamoto and Suzuki, 1955; Robinson and Rugh, 1934). In the female, their anterior end is single and their posterior end is bi-lobed (rarely tri-lobed). They are persistent the year around although they seem to grow in size during the breeding season. The corresponding structure in the male is a small protuberance between the anus and the urinogenital pore. In the male it is uni-lobed in both anterior and posterior ends and its medulla is very small.

Cross section through the urinogenital papilla of a full-grown female and a full-grown male are examined by Yamamoto and Suzuki (1955). The papilla consists of two tissues, the cortex and the medulla. The cortex is non-vascularized and is nothing but a thickened epidermis which composed of stratum coreum and stractum germinativum. At the base of the cortex is a basement membrane. The medulla is highly vascularized and it appears to be a modified mass of the corium. In the female a large medulla is surrounded by a thick epidermis which invade the medulla in places. In the male, on the other hand, the medulla is very small and the infiltrative process of the epidermis into the medulla is more pronounced than that in the female. In passing it may be remarked that in the male the genital duct (vas deferens) and the short urinary duct from the "urinary bladder" are fused at the end of the protuberance and open as the urinogenital pore, while in the female the oviduct and the urinary duct open separately. In fully-grown females the papilla cover both genital and urinary pores. It becomes obvious from a sense of experiments that the papillae of the Medaka are female-positive sex-characters in the sense that they are more developed in the female than the male (Yamamoto and Suzuki, 1955).

Both ovariectomy and castration induces an atrophy of the medulla of the papilla in both sexes. Implantation of the ovary into castrated male causes development of the organ. In both sexes administration of estrogen induces hypertrophy of the papillae, and that of androgen stimulates a slight enlargement. As conclusion, the medulla of the urinogenital papilla is manifested by both estrogen and androgen but its development is far more sensitive to estrogen than androgen (Yamamoto and Suzuki, 1955) (Fig. 8- 11).

Body color

In the Medaka the nuptial coloration is not prominent, but sexual differences in the development of the leucophores (Oka, 1938) and melanophores of wild fish (Suzuki-Niwa, 1965) have been pointed out. Melanophores in the ventral fins, caudal fin and body side of the male are more numerous than those of the female during the breeding season. Suzuki- Niwa has concluded that this character is also manifested by stimulative action of androgen (1965).

Teeth

Sexual dimorphism in the teeth has been pointed out in 1957 (Egami), and further detailed studies were carried out by Takeuchi (1966, 1967a, b, 1968). In the male, large distal teeth on the maxillae and mandible appear when the fish is young (Fig. 8-12). The number of the large teeth increases to about 6 in each jaw by the fish total length reaches 30 mm. The female have no large teeth during the growth stage at 22-30 mm total length. However the large distal teeth were formed in the females by giving androgen (Takeuchi, 1968).

The liver and other organs

Besides external sexual characters, sexual dimorphism in some organ or tissues is found in the Medaka. For instance marked histological difference between both sexes in the liver is observable during breeding season (Egami, 1955). The difference is confirmed by electron microscopic examination. These sexual differences are, however, not a usual secondary sexual character, and details are not given here.

Development of the characters

The secondary sex characters first appear when the fish have reached a length of about 16-18 mm. The urinogenital papillae of the female become larger than that of the male and the anal fin of the male becomes slightly larger than that of the female. The urinogenital papillae may be used as the criterion for sex-discrimination when fish become more than 16-18 mm in length. The differences in the anal and dorsal fins become prominent when the fish reach 20 mm in length. The papillar processes in the male anal fin begin to appear in 26 mm fish, and sexual maturity is attained in both sexes when the fish reach 26-28 mm in length. Fish whose body length is more than 28 mm may be regarded as fully grown (Okada and Yamashita, 1944; Yamamoto, 1953; Yamamoto and Suzuki, 1955). Patterns of regulation of the development of the secondary sexual characters by hormonal conditions are different among different characters as described above and not simple.

In some report, development of the secondary sexual characters is different among wild specimens collected at different localities (Yamamoto, 1949; Egami, 1954), however, further studies on the basis of many materials collected at different seasons are needed before final conclusions (Uchida, 1951).

References

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Egami, N., 1954a Effects of hormonic steroids on the formation of male characters in females of the fish, Oryzias latipes, kept in water containing testosterone propionate. Annot. Zool. Japon. 27: 122- 127.

Egami, N., 1954b Geographical variation in the male characters of the fish Oryzias latipes. Annot. Zool. Japon. 27: 7-12.

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