Sex differentiation of germ cells in the teleost, Oryzias latipes, during normal embryonic development

Transcription

1 J. Embryol. exp. Morph. Vol. 2, 2, pp. -, 2 Printed in Great Britain Sex differentiation of germ cells in the teleost, Oryzias latipes, during normal embryonic development ByNORIYUKI SATOH AND NOBUO EGAMI From the Zoological Institute, University of Tokyo SUMMARY Mitotic and meiotic activities of germ cells during early development in the medaka, Oryzias latipes, are dealt with in this report. Primordial germ cells were obviously distinguishable from somatic cells days after fertilization and began to proliferate about days after fertilization. The mean number of primordial germ cells increased during a period of - days after fertilization, reaching about immediately before hatching. Newly hatched fry could be classified into two types according to the number and the nucleic activity of germ cells in the gonadal rudiment. One type consisted of fry containing about germ cells and no cells in the meiotic prophase. In the other type of fry the number of germ cells increased by mitotic divisions and some of the cells began to enter into the meiotic prophase. During the course of further development the fry of the former type differentiated into males and the latter into females. Therefore it can be concluded that the morphological sex differentiation of germ cells occurs at the time of hatching. However, no sexual differences in the histological structure of somatic elements in the gonad are observable at that time. INTRODUCTION The origin of primordial germ cells (PGCs) and the formation of the gonad during early development in fish has been investigated by many authors (cf. Johnston, ). In the medaka, Oryzias latipes, Gamo () has demonstrated the mesentodermal origin of primordial germ cells by histological observation. Yamamoto () has reported, in his first report on artificially induced sexreversal in this species, that the gonad of the newly hatched fry was sexually indifferent and that morphological differentiation of the gonad became apparent in - mm young. In Tsuzuki, Egami & Hyodo, and Hyodo, Tsuzuki & Egami showed preliminarily that the mitosis and meiosis of newly hatched fry are more active in females than in males. The present paper will deal with the proliferation kinetics of germ cells during the course of normal embryonic development in the medaka, and the sex differentiation of germ cells will be compared with that in other vertebrate animals. Authors' address: Zoological Institute, Faculty of Science, University of Tokyo, Tokyo, Japan.

2 N. SATOH AND N. EGAMI Table. Changes in the number of primordial germ cells in the gonadal region of embryos in the medaka, Oryzias latipes Developmental stage* Days after fertilization 4 * By Matsui embryos examined (4). PGCs means ±s.e.f 2 ±-4 4 ±4- ±- ±2- ±- ± - ±-2 ±- embryos with mitotic PGCs t Mean ± standard error. Total no. of mitotic PGCs 2 MATERIALS AND METHODS Fertilized eggs, embryos and fry of the orange-red variety of the medaka, Oryzias latipes, were kept in vessels placed indoors at 22- ± 2 C. Eleven days after hatching the young were transferred outdoors. All the fry were reared on a usual diet consisting of the following ingredients: g shrimp powder, g toasted whole-barley flour, g yeast preparation (Wakamoto) and 4 g powdered green tea. Embryos at various stages (Table ) and fry at various intervals after hatching (Tables 2-4) were fixed in Bouin's solution in toto. In order to define the stage of development, the illustration of normal development reported by Matsui (4) was used. For histological observations, all the materials were sectioned transversally in paraffin at /an and were stained with Delafield's haematoxylin and eosin. The total number of germ cells at each stage of development was counted by a careful examination of all the serial sections. The mitotic and meiotic figures of the germ cells were also observed. RESULTS Primordial germ cells were clearly distinguishable from somatic cells in the embryonic stage at the appearance of granules of melanin pigment in the eyes (stage 2, about days after fertilization). The cells characteristically had a large size of the cell and of the nucleus (about and /an in diameter respectively), a distinct round outline, a less stained cytoplasm, and a single, clear nucleolus (Fig. 2). The exact number of germ cells in the gonadal region of each individual was determined by the examination of /on serial sections. In order to check any error in the counting of the cell number, the same preparation was observed twice. For instance, the results of the first cell count of five fry one day after

3 Sex differentiation in a teleost : H i j! y. 2 i i i i i i i i I I i I i i I i i i i i i i i i Days after fertilization I Davs after hatching Hatching Fig.. Changes in the number of germ cells in the medaka, Oryzias latipes, during normal embryonic development. The open circles show embryos and fry containing no germ cells in meiotic prophase, the solid ones fry containing germ cells in meiotic prophase. hatching were,,, and respectively. One week after the first count a second count was done; we then obtained the numbers of 4, 4,, and respectively. The differences between the counts were only cells or less. () Embryos The numbers of primordial germ cells in embryos at each developmental stage are given in Table and are plotted in Fig.. The average number of primordial germ cells of embryos at stage 2, when primordial germ cells were found on both sides of the gut, was about 2, and no mitotic figure was observed at all. At stage 2, about 4 days after fertilization, primordial germ cells were found to move to the presumptive gonadal area between the pronephric ducts and the gut, and the mean number of the germ cells was 4. Among 4 embryos at stages 2- only one embryo contained a germ cell in the mitotic metaphase. The increase in the germ cell number during these stages may be mainly attributable to the migration of the cells into the gonadal region. At stage, however, seven of embryos had primordial germ cells in mitosis. In embryos at later stages, mitotic figures were found in their primordial germ cells (Fig. ). Concomitantly with mitotic division, the number of primor-

4 N. SATOH AND N. EGAMI Fig. 2. Transverse section of embryo days after fertilization. Primordial germ cells located beside the gut (arrow). G, gut; P, pronephric duct. Fig.. Embryo days after fertilization, immediately before hatching. Primordial germ cell in the mitotic metaphase (arrow). Bottom bar: 2/tm. dial germ cells increased, reaching about immediately before hatching. No sign of meiosis in primordial germ cells was, however, observed in any embryos. The number of somatic cells in the gonadal rudiment was very small (Fig. ). No indications of sexual differences in the gonad primordia were observable before hatching by histological observation. (2) Fry (- days after hatching) Table 2 shows the changes in the number of germ cells in the fry of the fish, while the total numbers of germ cells in each fry are plotted in Fig.. The newly hatched fry of the medaka were about mm in total length. The gonads of the newly hatched fry were suspended in the coelom between the pronephric ducts and the gut in the posterior trunk region. They were bilaterally distended lobes constricted in the median part. Of 4 newly-hatched fry within 24 h after hatching, six were observed to have germ cells in meiosis (solid circles in Fig. ). In the cells entering the meiotic prophase the appearance of dispersing chromonema in a nucleus, which was characteristic of preleptotene, or the formation of a lump of chromonema in the centre of the nucleus (leptotene stage), was observed (Fig. 4b). Many mitotic germ cells were also detected in fry having cells in the meiotic prophase. The average number of total germ cells of these fry was more than 2 (Table 2). On the contrary, in fry containing no germ cells in the meiotic prophase, the average number of germ cells was only 2 (open circles in Fig. ), although small number of the cells were in mitosis (Fig. 4a). One day after hatching, out of fry were found to have germ cells in the meiotic prophase, a few of them with zygonema which were concentrated on the side opposite a definite nucleolus (Fig. b). However, in fry no meiotic

5 Days after ing 2 4 Total no. of fry Table 2. Changes in the number of germ cells in fry of the medaka, Oryzias latipes Fry not containing GCs in the meiotic prophase Fry containing GCs in the meiotic prophase No. of Ul fry fife mean + S.E. 2 + ± ± 2 ±2 ± 2 ± 2 + ± ±4 ± 2 ± fry with mi tr^ttr ILJUL GCs 24 * IP, t L,; Total no. of ' ULUIH tniti^tir* GCs 4 2 interphase. Leptotene; No. of ui fry n Total no. of mean + S.E. in IP* 2 ±2 ± 22 ±4 ± ± 2 ±4 ± ±2 4 ± 4 ± Z, zygotene; P, pachytene; L D, GCs in i meiotic Z diplotene. the prophase f P 4 D fry with ryiit r\f\c* JIUlULll' GCs Total no. of mitotif GCs X ft» * oo

6 N. SATOH AND N. EGAMI Fig. 4. Newly hatched fry within 24 h after hatching, (a) Germ cells in fry not containing cells in meiosis (arrow). G, gut; P, pronephric duct, (b) Germ cells in leptotene of the meiotic prophase (arrow). Fig.. Fry days after hatching, (a) Fry not containing germ cells in the meiotic prophase. (b) Germ cells in zygotene of the meiotic prophase (arrow). Fig.. Fry days after hatching, (a) Fry not containing germ cells in the meiotic prophase. (b) Germ cells in pachytene of the meiotic prophase (arrow). Fig.. Fry days after hatching, (a) Fry not containing germ cells in the meiotic prophase. (b) Germ cells in diplotene of the meiotic prophase (arrow). Bottom bar: 2 [im. b

7 Sex differentiation in a teleost germ cells were found, the average number of germ cells being in these fry. Twenty-seven out of 4 fry 2 days after hatching and 4 out of 24 fry days after hatching were found to have germ cells in leptotene or in zygotene. A rapid increase in the total number of germ cells took place in these fry. Four days after hatching out of 2 fry contained germ cells in the meiotic prophase and most advanced germ cells entered into the pachytene stage (Fig. b). Eight other fry had only about germ cells, on the average, and the mitotic rate was very low (Table 2). Similar differences in the mitotic and meiotic activities of germ cells were detectable among fry fixed - days after hatching. The thick chromonema with abundant cytoplasm making the form of the diplotene stage appeared days after hatching. A few of the germ cells, however, degenerated under pycnotic conditions. Germ cells with a nucleus at dictyate stage were found days after hatching (Fig. b). From the histological observations, the female germ cells were recognizable as oocytes in the fry fixed at days. Moreover, it became clear that the total number of germ cells in the gonad containing oocytes was always larger. From these results the fry may be classified into two types (type I and II) according to the presence or absence of germ cells in the meiotic prophase and according to the number of germ cells per individual. Fry of type I contain a smaller number of germ cells and no cells in the meiotic prophase, while those of type II contain a larger number of germ cells both in mitosis and in the meiotic prophase. Judging from the present observations, it is highly probable that type I fry differentiate into males and II into females. No sexual dimorphism was observable in the structure or arrangement of somatic cells in the gonad, at least within days of hatching. () Male fry Table shows the changes in the number of germ cells in male fry; the total numbers of individual are plotted in Fig.. From the th day to the th day after hatching some of the germ cells were in the pycnotic condition and the average number of germ cells were between and in the male fry (Fig. <z). On the 2th day after hatching (- mm in mean body length) the average number of germ cells was about 2. In some fry, supporting tissue enclosing germ cells developed. On the th day, out of fry (about - mm long) contained some spermatogonia in mitosis. A testicular structure with some acini containing spermatogonia was found in young fixed on the 4th day after hatching (Fig. a). In such male fish the proliferation of spermatogonia was very active, but no meiotic figures at all of male germ cells could be detected in the present observations.

8 2 N. SATOH AND N. EGAMI Table. Changes in the number of germ cells in male fry of the medaka, Oryzias latipes Days after hatching fry examined germ cells mean + S.E. ± ± ±ll 2± 4+ 2±ll 24± ± ± 4 ±4 fry with mitotic germ cells Total no. of mitotic germ cells Fig.. Fry days after hatching, (a) Testis in male fry; P, pronephric duct; T, testis. (b) Ovary in female fry; O, ovary. Fig.. Fry 4 days after hatching, (a) Testis in male fry; G, gut. (b) Ovary in female fry. Bottom bar: fim.

9 Sex differentiation in a teleost Table 4. Changes in the number of germ cells in female fry of the medaka, Oryzias latipes Days. after fry hatching examined Total no. of germ cells mean ± S.E. 44 ±4 422 ± 4 ±4 4 ± 24 ± * 4* * * 2* oogonia L oocytes * Rough estimates based on a sample of every five sections. Z P D fry Total with no. of mitotic mitotic oogonia oogonia 2 2 (4) Female fry In the female fry, during the course of further development, oocytes at each stage of the meiotic prophase increased in number; the average number of total germ cells became beyond on the th day. On the 2th day after hatching many oocytes were in the resting stage, the average number being about. The ovarian structure was established within 2 days after hatching. The changes in the number of germ cells in female fry are summarized in Table 4. DISCUSSION From the present observations of germ cells in the embryos and fry of Oryzias latipes it is clear that the morphological sex differentiation of germ cells occurs at the time of hatching. At this time no sexual differences in the morphology of somatic cells in the gonad were observable. Although primordial germ cells proliferate both in male and female embryos during the few days before hatching, the cells cease to divide in male fry immediately after hatching. However, in female fry the cells continue to increase in number. Furthermore, some of the female germ cells enter into the meiotic prophase within a day of hatching. In other words, the sex differentiation of germ cells takes place immediately after hatching. In the medaka, Gamo (), Tsuzuki et al. () and Hyodo et al. () have counted the number of germ cells during early development. The purpose of Gamo's observation, however, was to detect the origin of the primordial germ cells; he concluded that there is a mesentodermal origin of primordial

10 4 N. SATOH AND N. EGAMI germ cells. Preliminary reports by Tsuzuki et al. () and Hyodo et ah () postulated that the mitosis and meiosis of germ cells of newly hatched fry are more active in females than in males. The present results are in agreement with their observations in all important points. Their preliminary observations, however, were based on serial sections at jltm, and the number of fry samples was smaller than in the present study. In his report on artificially induced sexreversal in genotypic males of the medaka, Yamamoto () described that the gonad of the newly hatched fry may be regarded as indifferent, and that the morphological differentiation of the gonad became apparent in - mm young about days after hatching. However, our results indicate that the morphological sex differentiation of germ cells takes place considerably earlier than formation of testis or ovary. Quantitative analyses of germ-cell population kinetics in rats have been done by Beaumont & Mandl (2, ). According to their reports the mitosis of primordial germ cells is more active in a female foetus than in a male, and a few of the germ cells enter into the meiotic prophase within - days only in a female foetus, although the gonadal volume increases earlier in a male foetus than in a female. Recently Mittwoch, Delhanty & Beck () calculated the volume of the gonadal rudiment during normal development in rats and concluded that the gonadal volume increases faster in a male foetus than in a female. Furthermore, Mittwoch, Narayanan, Delhanty & Smith () observed a faster increase of the gonadal volume in female chick embryos than in male, contrary to the case in rats. From these facts Mittwoch () concluded that the Y chromosome might exert an influence by regulating the growth rate of the gonadal rudiment, and that the faster-growing rudiment is differentiated into a testis in mammals and into an ovary in birds. It is thought that the differentiation of the gonad occurs independently of and/or in parallel to that of germ cells. Yamamoto and his co-workers have demonstrated that the sex differentiation of the medaka can be completely controlled by the oral administration of oestrogenic or androgenic steroids after hatching (Yamamoto,, ). In addition, Hishida (2, 4) indicated the selective incorporation of sex steroids into the differentiating gonad. On the basis of these experiments they suggested that the natural sex inducers may be steroid hormones (Yamamoto, ). However, it is worth mentioning the fact found in the present observations that the sex difference in the nuclear behaviour of germ cells has already been expressed at the time of hatching. Therefore the mechanism involved in the sex differentiation of germ cells under natural conditions is not always the same as that under the experimental conditions which held following treatment with exogenous sex steroids.

11 Sex differentiation in a teleost REFERENCES BEAUMONT, H. M. & MANDL, A. M. (2). A quantitative and cytological study of oogonia and oocytes in the foetal and neonatal rat. Proc. R. Soc. B, -. BEAUMONT, H. M. & MANDL, A. M. (). A quantitative study of primordial germ cells in the male rat. /. Embryol. exp. Morph., -4. GAMO, H. (). On the origin of germ cells and formation of gonad primordia in the medaka, Oryzias latipes. Jap. J. Zool., -. HISHIDA, T. (2). Accumulation of testosterone-4-c 4 propionate in larval gonad of the medaka, Oryzias latipes. Embryologia, -. HISHIDA, T. (4). Reversal of sex differentiation in genetic male of the medaka {Oryzias latipes) by injecting estrone--c 4 and diethylstilbestrol (monoethyl--c 4 ) into the egg. Embryologia, HYODO, Y., TSUZUKI, E. & EGAMI, N. (). Multiplication of primordial germ cells and initiation of meiosis in the medaka, Oryzias latipes. Jap. J. exp. Morph. 2,. JOHNSTON, P. M. (). The embryonic history of the germ cells of the largemouth black bass, Micropterus salmoides salmoides. J. Morph., MATSUI, K. (4). Illustration of the normal course of development in the fish, Oryzias latipes. Jap. J. exp. Morph., -42. MITTWOCH, U. (). Sex determination in birds and mammals. Nature, Lond. 2, MITTWOCH, U., DELHANTY, J. D. A. & BECK, F. (). Growth of differentiating testes and ovaries. Nature, Lond. 224, 2-2. MITTWOCH, U., NARAYANAN, T. L., DELHANTY, J. D. A. & SMITH, C. A. B. (). Gonadal growth in chick embryos. Nature New Biol. 2, -2. TSUZUKI, E., EGAMI, N. & HYODO, Y. (). Multiplication and sex-differentiation of germ cells during development in the medaka, Oryzias latipes. Jap. J. Jchthyol., -2. YAMAMOTO, T. (). Artificially induced sex-reversal in genotypic males of the medaka (Oryzias latipes). J. exp. Zool. 2, -4. YAMAMOTO, T. (). Artificial induction of functional sex-reversal in genotypic female of the medaka (Oryzias latipes). J. exp. Zool., YAMAMOTO, T. (). Sex differentiation. In Fish Physiology, vol. m (ed. W. S. Hoar & D. J. Randalls), pp. -. New York and London: Academic Press. (Manuscript received February 2) 2 EMB 2

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