Vessels Were Made on July 2, 1953. Oviposition by These Fish Was

Endocrinol. Japon., Vol.2, No.2(1955).

EFFECT OF ESTROGEN ADMINISTRATION ON OVIPOSITION OF THE FISH, ORYZIAS LATIPES

NOBUO EGAMI

Zoological Institute, Faculty of Science, Tokyo University

SEVEN FIGURES

During the breeding season extending from spring to summer, the ovaries of the fish, Oryzias latipes, contain hundreds of follicles, each with an egg at various stages of maturation. Usually, five to fifty eggs are laid by a single female almost every morning(Egami, 1954c). On the other hand, ovaries are small and inactive in winter months. The present author has already reported that small winter ovaries of Oryzias females are caused to develop when fish are kept at 22-26•Ž, and that development of winter ovaries following exposure to higher temperature is inhibited by administration of large doses of estrogens(Egami, 1954b, d). In the present paper, the results of experiments designed to study the effects of estrogen administration on the ovaries of Oryzias latipes in the breeding season will be presented. The author wishes to thank Profs. Yo K. Okada and Kiyoshi Takewaki for their kind criticism and encouragement.

EXPERIMENTS

Adult males and laying females of the red variety of Oryzias latipes, 30 to 33mm in total length, were used. A group of several fish was kept in a separate glass vessel containing about two liters of water and some water plants. Several such groups were used for each experiment. The fish were fed with living fresh- water oligochaetes almost every day. All experiments were carried out in the spring and summer months of 1953 and 1954 at room temperature under natural daylight conditions. In each group, number of females which had laid eggs was examined at 8 a. m. and at noon every day during the course of experiments. At autopsy, gonads were weighed and fixed in Bouin's fluid.

(1) Injection of suspension of estrone benzoate microcrystals Nine groups consisting of two males and two laying females kept in separate vesselswere made on July 2, 1953. Oviposition by these fish was observed for fivesuccessive days. At 9 a.m. on July 7, ten femalcs in five groups were in- traperitoneally injected with about 1/50mg. of microcrystals of estrone benzoate suspended in 1/50cc. of Ringer's solution, and eight females in the remaining four

Received for publication March 16, 1955. 90 EGAMI Vol.2, No.2 groups with about 1/50cc. of Ringer's solution. After the injection, they were kept separately and numbers of females having laid eggs were examined every day.

Table 1. Oviposition of the females injected with suspension of microcrystals of estrone benzoate

Fig. 1 Effect on oviposition of injection of estrone benzoate-

microcrystal suspension. •œ: estrone benzoate-group,

○: control-group. Results are given in Table 1 and Figure 1. Before the injection, more than 60 per cent of the females laid eggs every day. After the injection, however, no females laid eggs for the first two days, both in the estrogen-and in the Ringer- groups. Afterward, some females began to lay eggs, but the percentages of females laying eggs were low, especially in the estrogen-groups. It was not clear, however, whether the estrogen administration inhibited oviposition or not, since the oviposition was also inhibited in the Ringer-control groups. (2) Single implant of estronepellet In the first series, three groups comprising two males and four laying females kept in separate vessels were used. Each of the female was given a single sub- June 1955 EFFECT OF ESTROGENS ON OVIPOSITION 91

cutaneous implant of a small estrone pellet on June 23, 1953. Another group containing intact four females and two males served as controls(Table 2, Fig. 2A).

Table 2. Oviposition of the females receiving a single implant of estrone pellet(I) (preliminary experiment)

Fig. 2 Effect on oviposition of single implant of estrone pellet. Vertical dotted lines indicate additions of new males.

It was found that percentage of females laying eggs fell after estrone implantation. It was also observed that the number of eggs laid per single female was reduced in the estrone-groups in comparison with the control-groups. In estrone-groups of the second series, females were subcutaneously implanted with small estrone pellets on August 11, 1954. Females of control groups received no implants. At the beginning of experiment, each of the groups consisted of two males and two or three females. Both on August 17 and 27, however, two more new males were put in each vessel to exclude the possibility that the males which had been kept in the same vessel together with estrone-implanted females might in some way disturb the outcome. At the end of the experiment, gonads of the fish were weighed and fixed in Bouin's fluid. Numbers of females having laid eggs in the estrone-groups were less than those in the control-groups. Especially at two days after estrone-implant, most of the females did not lay eggs. In the estrone-groups, some eggs remained unfer - tilized before new males were put, but after addition of new males all eggs laid became fertilized(Table 3, Fig. 2B), although percentage of females laying eggs was not definitely increased. It was also observed that, in the estrone-groups, oviposition took place sometimes between 9a. m. and 2p.m.; i. e., it was retarded 92 EGAMI Vol.2, No.2

Table 3. Oviposition of the females receiving a single implant of estrone pellet (II)

*new males were added **()unfertilized eggs

Table 4. Weight of ovaries of the fish receiving a single implant of estrone pellet

*standard error a few hours when compared with that in the intact fish(see Egami, 1954 c). Studies of the weight(Table 4)and histology of ovaries indicated that the development of oocytes was generally suppressed in estrone-fish in comparison with that in control-fish. Also histologic studies of testes revealed that the testes of the males which had been kept in the same vessel together with the estrone-implanted females became degenerated showing a few or no mature spermatozoa. (3) Repeatedimplants of estronepellets Four groups of four females were used in this series. Each female was given the first subcutaneous implant of a small estrone pellet on June 14, 1954. They received the second, third and fourth implants of estrone pellet on June 21, July 2 and July 9 respectively, and were sacrificed on July 17. Two other groups of four intact females served as controls. Two intact males were kept together with each group of females. From Table 5 summarizing the results, it was evident that the oviposition was completely inhibited by repeated implants of estrone pellets(Table 5, Fig. 3). Histologically, ovaries of all estrone-fish became atrophic at the end of the experiment, and most of them(seven out of nine)contained a large cavity filled with fluid(Fig. 4). 94 EGAMI Vol.2, No.2

(4) Culture of fish in aqueous solution of estradiol The first series was started on May 14, 1954. Six groups, each containing two males and three females, were kept separately in vessels containing tap water. On May 27, two of three groups were transferred to new vessels each containing two liters of water in which 400ƒÊg. of estradiol was dissolved, and the other two groups to vessels with water containing 200ƒÊg. of eastradiol. The remaining two groups were kept in normal tap water. On June 8, the media were renewed, but the concentrations of estradiol in respective vessels were not changed. On

June 17, two new males were added to each group. On June 19, the fish were sacrificed and the gonads were weighed and fixed.

Table 6. Oviposition of the females kept in aqueous solution of estradiol(I)

*new males were added **medium was renewed ***()unfertilized eggs 96 EGAMI Vol.2, No.2

were few in number in the ovaries of the estrogen-fish, and that some ovaries contained a large cavity filled with fluid(Fig. 6). In all males kept in estradiol- water for a period of 23days spermatogenetic activity was interfered with, and two out of five testes contained very small testis-ova. The second series of experiment was carried out with six groups each com-

prising two males and three females. Concentrations of estradiol in this series were 40 and 20ƒÊg. per liter of water. Culture of fish in estradiol-water was begun on July 6, 1954, and ended on July 26. Each culture medium was renewed on July 15, and two new males were added to each group on July 22. In this series, inhibitory effect of estradiol on oviposition was slighter than in the preceding series(Table 8, Fig. 7). The percentages of females laying eggs in

Table 8. Oviposition of the females kept in aqueous solution of estradiol(II)

*new males were added **medium was renewed ***()unfertilized eggs

the estradiol-groups(especially in the higher concentration groups)were lower than those in the control-groups during 10 to 16days after the commencement of estrogen treatment. But the percentages rose after the addition of new males (July 23 to 26). No significant differences were found in weight(Table 9)and histology of the ovaries between the estrogen-and control-groups. On the other hand, testes of the males kept in estradiol solution for 20days were histologically different from normal ones, showing a definite reduction in amount of spermatozoa. June 1955 EFFECT OF ESTROGENS ON OVIPOSITION 97

Fig. 7 Effect on oviposition of culture of fish in solution of estradiol.

●: 40μg. of estradiol per liter, ◎: 20μg. of estradiol per liter,

○: control.

Table 9. Weight of ovaries of the fish kept in aqueous solution of estradiol(II)

*standard error

DISCUSSION

Inhibitory effect of estrogens on the development of fish ovaries in sexually inactive seasons has been reported by the present author both in the loach

(Egami, 1954a)and in Oryzias latipes(Egami, 1454b, d). From the results presented in this paper, it is evident that also in Oryzias females in the breeding season oogenesis is inhibited and the rate of oviposition is reduced by administration of large doses of estrogens. Under the circumstances of the author's experiment, threshold concentration of estradiol for inhibiting the ovarian activity is about 100ƒÊg. per liter of water. No evidence for the promotion of oviposition by estrogen has been obtained. In the previous paper(1954d), the present author descrived ovaries containing a large cavity filled with fluid found in estradiol- treated female fish. Similar changes in ovaries of the fish in the breeding season were observed in the present experiment with large doses of estrone or estradiol. Tavolga(1949)reported an inhibition of the ovaries of platyfish by estradiol benzoate, and attributed this to the androgenic effect of the estrogen. However, the present author demonstrated in winter females of Oryzias that simultaneous administration of pituitary substance attenuated the inhibitory effect of estrogen on the ovaies(Egami, 1954b). It seems highly probable that the inhibitory effect of estrogen on ovarian activities in breeding fish as in non-breeding fish is due to the suppression of gonadotrophin secretion from the pituitary body. Suppression of the testis of the males kept in the same vessel together with estrone-implanted females or in that containing estradiol-water accords well with the author's previous observations on male Oryzias treated with estrogen(Egami, 98 EGAMI Vol.2, No.2

1955). Spermatozoa in the testis were definitely reduced in amount in estradiol- water, even at the concentrations as low as 40ƒÊg. or 20ƒÊg. per liter water. In males kept in the estrogen-water, sexual behavior was also inhibited. On the other hand, oogenesis was not inhibited at these concentrations. The threshold concentration of estradiol for decreasing amount of spermatozoa in the testis, therefore, seems to be lower than that for inhibiting oogenesis. In females kept together with males in water containing estradiol at subthreshold concentrations

(40ƒÊg. or 20ƒÊg. per liter)for a period of ten or more days, the rate of oviposition dropped. But if new males were then put to the estrogen-females, the rate rose again. It seems probable, therefore, that the decrease in oviposition rate in the females is due to the decrease of sexual activity or a reduction in amount of spermatozoa in the male fish. Usually, the fertilization of Oryzias eggs takes place during or immediately after oviposition. Copulation is preceded by a brief court- ship and accomplished by the male holding the female with its dorsal and anal fins while spreading the milt over the eggs with its pectoral fin. If laying females are isolated from males, the females cease to lay eggs for a few days and then begin to deposit unfertilized eggs at irregular intervals. It seems highly probable that the presence of active male fish is one of the important factors stimulating the oviposition. It was not yet been determined whether the delay of oviposition in estradiol-treated fish is due to some physiological changes in either sex.

SUMMARY Females of Oryzias latipes lay eggs at dawn almost every day during the breeding season. Injection of estrone benzoate or implantation of small pellets of estrone inhibits the oviposition of females. If females are kept in estradiol-water, the development of oocytes in the ovaries is more or less suppressed, the rate of oviposition is reduced and the time of oviposition is delayed. Sometimes, in estrogen treated females, oocytes undergo degeneration and a large cavity filled with fluid is formed in the ovaries. Administration of estrogens also interferes with spermatogenesis and brings about a reduction in amount of spermatozoa in the testis of male fish. The threshold concentration of estradiol for inhibiting spermatogenesis is, however, lower than that for suppressing oogenesis.

REFERENCES

Egami, N.: Jour. Fac. Sci. Tokyo Univ. Sec. IV, 7: 113, 1954 a. Egami, N.: Annot. Zool. Japon. 27: 13, 1954 b. Egami, N.: Ibid. 27: 57, 1954 c. Egami, N.: Endocrinol. Jap. 1: 75, 1954 d. Egami, N.: Jap. Jour. Zool. 11: 353, 1955. Tavolga, M. C.: Zoologica 34: 215, 1949.