J. Embryol. exp. Morph. Vol. 28, 2, pp. 385-395, 1972
Printed in Great Britain
385
Sex differentiation of germ cells
in the teleost, Oryzias latipes, during normal
embryonic development
ByNORIYUKI SATOH 1 AND NOBUO EGAMI 1
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 3 days after
fertilization and began to proliferate about 7 days after fertilization. The mean number of
primordial germ cells increased during a period of 7-10 days after fertilization, reaching
about 90 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 100 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, 1951). In the medaka, Oryzias latipes, Gamo (1961) has demonstrated
the mesentodermal origin of primordial germ cells by histological observation.
Yamamoto (1953) 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 6-5 mm young. In 1966 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.
1
Authors' address: Zoological Institute, Faculty of Science, University of Tokyo, Tokyo
113, Japan.
386
N. SATOH AND N. EGAMI
Table 1. Changes in the number of primordial germ cells in the gonadal region of
embryos in the medaka, Oryzias latipes
Developmental
stage*
Days
after
fertilization
No. of
embryos
examined
26
27
28
29
30
31
32
33
3
4
5
6
7
8
9
10
17
24
28
21
11
19
16
21
* By Matsui (1949).
No. of PGCs
means ±s.E.f
No. of
Total no.
embryos
with mitotic of mitotic
PGCs
PGCs
32 ±3-4
46 ±4-0
59 ±3-6
56 ±2-7
71 ±5-0
69 ± 5-3
63 ±5-2
89 ±7-6
t Mean ± standard
0
1
0
0
0
7
7
9
0
1
0
0
0
11
8
12
error.
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 22-5 ± 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: 60 g shrimp powder, 30 g
toasted whole-barley flour, 6 g yeast preparation (Wakamoto) and 4 g powdered
green tea.
Embryos at various stages (Table 1) 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
(1949) was used. For histological observations, all the materials were sectioned
transversally in paraffin at 5 /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 26, about 3 days after fertilization). The cells characteristically had a large
size of the cell and of the nucleus (about 15 and 8 /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 5 /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
Sex differentiation in a teleost
387
1000
:
H
• i • j
!
y.
20
i
3
i
i
i
i
i
i i I
4 5 6 7 8 9 10| 0
Days after fertilization
I
Hatching
I
i
I
i
2
3
4
i
I
i
i
i
i
5 6 7 8 9 10
Davs after hatching
i
i
i
i
i
12
13
14
15
Fig. 1. 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 66, 70, 96, 98 and 160 respectively. One week after the first count
a second count was done; we then obtained the numbers of 64, 74, 93, 103 and
163 respectively. The differences between the counts were only 5 cells or less.
(1) Embryos
The numbers of primordial germ cells in embryos at each developmental stage
are given in Table 1 and are plotted in Fig. 1.
The average number of primordial germ cells of embryos at stage 26, when
primordial germ cells were found on both sides of the gut, was about 32, and no
mitotic figure was observed at all. At stage 27, 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 46. Among 84 embryos at stages 27-30 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 31, however, seven of 19 embryos had primordial germ cells in
mitosis. In embryos at later stages, mitotic figures were found in their primordial
germ cells (Fig. 3). Concomitantly with mitotic division, the number of primor-
388
N. SATOH AND N. EGAMI
Fig. 2. Transverse section of embryo 3 days after fertilization. Primordial germ cells
located beside the gut (arrow). G, gut; P, pronephric duct.
Fig. 3. Embryo 10 days after fertilization, immediately before hatching. Primordial
germ cell in the mitotic metaphase (arrow). Bottom bar: 20/tm.
dial germ cells increased, reaching about 90 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. 3). No
indications of sexual differences in the gonad primordia were observable before
hatching by histological observation.
(2) Fry (0-10 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. 1.
The newly hatched fry of the medaka were about 5 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 43 newly-hatched fry within 24 h after hatching, six were observed to have
germ cells in meiosis (solid circles in Fig. 1). 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 200 (Table 2).
On the contrary, in fry containing no germ cells in the meiotic prophase, the
average number of germ cells was only 112 (open circles in Fig. 1), although
small number of the cells were in mitosis (Fig. 4a).
One day after hatching, 6 out of 36 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. 5b). However, in 30 fry no meiotic
43
36
43
24
21
26
28
30
25
27
29
0
1
2
3
4
5
6
7
8
9
10
37
30
16
10
8
5
8
15
15
13
18
fry
112 + 5
95 ± 6
107 ±8
121 ±12
103 ±6
72 ±5
112 + 8
89±8
106 ±4
98 ±10
102 ± 5
mean + S.E.
fife
Ul
of
of
fry
ing
No. of
Days Total
No.
after no.
mi tr^ttr•
1111ILJUL'
49
21
11
1
1
0
0
1
0
0
1
n
6
6
27
14
13
21
20
15
10
14
fry
ui
of
No.
No. of
219 ±29
197±13
226 ±14
333 ±39
401 + 44
271 ± 26
380 ±41
376 ±59
355 ±28
345 ± 36
435 ±57
210
185
203
224
221
148
220
164
120
151
189
mean + S.E. in IP*
Total
no. of
5
5
11
52
56
16
21
20
26
6
9
L
0
1
4
33
71
33
65
46
64
45
68
Z
0
0
0
0
31
50
98
181
116
146
151
P
D
7
6
68
1
0
0
0
0
0
0
0
No. of GCs in the
i
meiotic prophase f
Total
no. of
3
5
16
9
7
2
4
2
6
3
4
GCs
19
26
57
34
25
14
9
7
17
3
4
GCs
rYiit r\f\c* mitotif
JIUlULll'
with
fry
No. of
Fry containing GCs in the meiotic prophase
* IP, interphase.
t L,;Leptotene; Z, zygotene; P, pachytene; D, diplotene.
24
18
9
1
1
0
0
1
0
0
1
GCs
tniti^tir*
11ULUIH
with
GCs
Total
no. of
fry
No. of
Fry not containing GCs
in the meiotic prophase
Table 2. Changes in the number of germ cells in fry of the medaka, Oryzias latipes
X
oo
»•*
1
ft
390
N. SATOH AND N. EGAMI
1b
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. 5. Fry 3 days after hatching, (a) Fry not containing germ cells in the meiotic
prophase. (b) Germ cells in zygotene of the meiotic prophase (arrow).
Fig. 6. Fry 5 days after hatching, (a) Fry not containing germ cells in the meiotic
prophase. (b) Germ cells in pachytene of the meiotic prophase (arrow).
Fig. 7. Fry 10 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:
20 [im.
Sex differentiation in a teleost
391
germ cells were found, the average number of germ cells being 95 in these
fry.
Twenty-seven out of 43 fry 2 days after hatching and 14 out of 24 fry 3 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 13 out of 21 fry contained germ cells in the meiotic
prophase and most advanced germ cells entered into the pachytene stage
(Fig. 6 b). Eight other fry had only about 100 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 5-10 days after hatching.
The thick chromonema with abundant cytoplasm making the form of the diplotene stage appeared 7 days after hatching. A few of the germ cells, however,
degenerated under pycnotic conditions.
Germ cells with a nucleus at dictyate stage were found 10 days after hatching
(Fig. 1b). From the histological observations, the female germ cells were
recognizable as oocytes in the fry fixed at 10 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 10 days of hatching.
(3) Male fry
Table 3 shows the changes in the number of germ cells in male fry; the total
numbers of individual are plotted in Fig. 1.
From the 11th day to the 15th day after hatching some of the germ cells were
in the pycnotic condition and the average number of germ cells were between
60 and 80 in the male fry (Fig. 8<z). On the 20th day after hatching (7-5 mm in
mean body length) the average number of germ cells was about 200. In some fry,
supporting tissue enclosing germ cells developed. On the 35th day, 6 out of
7 fry (about 8-5 mm long) contained some spermatogonia in mitosis. A testicular
structure with some acini containing spermatogonia was found in young fixed
on the 40th day after hatching (Fig. 9 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.
392
N. SATOH AND N. EGAMI
Table 3. Changes in the number of germ cells in male fry of the medaka,
Oryzias latipes
Days
after
hatching
11
12
13
14
15
20
25
30
35
40
No. of
examined
No. of
germ cells
mean + S.E.
No. of fry
with mitotic
germ cells
Total no.
of mitotic
germ cells
13
7
12
13
14
10
3
9
7
10
73 ±7
63 ±11
70±ll
72±9
84+11
209±ll
214±86
110±6
181 ±15
314 ±84
0
0
0
1
1
1
0
0
6
—
0
0
0
1
1
1
0
0
18
—
fry
Fig. 8. Fry 15 days after hatching, (a) Testis in male fry; P, pronephric duct; T, testis.
(b) Ovary in female fry; O, ovary.
Fig. 9. Fry 40 days after hatching, (a) Testis in male fry; G, gut. (b) Ovary in
female fry. Bottom bar: 50 fim.
Sex differentiation in a teleost
393
Table 4. Changes in the number of germ cells in female fry of the medaka,
Oryzias latipes
No. of
Days .No. of
after fry Total no. of
hatch- exam- germ cells No. of
ing ined mean ± S.E. oogonia
11
12
13
14
15
20
25
30
35
40
12
11
8
11
10
13
6
12
3
8
464 ±64
422 ±70
704 ±34
734 ±93
524 ±73
985*
1643*
906*
1075*
1278*
223
189
280
286
252
397
428
251
307
432
No. of oocytes
L
Z
P
D
12
14
23
34
30
13
33
8
3
10
49
32
131
131
77
77
274
105
45
101
115
111
158
129
119
155
452
141
178
116
56
75
105
208
119
343
458
401
567
618
fry
Total
with no. of
mitotic mitotic
oogonia oogonia
8
7
5
8
6
—
—
—
—
—
20
8
7
12
17
—
—
—
—
—
* Rough estimates based on a sample of every five sections.
(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 700 on the 13th day. On the 20th day after hatching
many oocytes were in the resting stage, the average number being about 1000.
The ovarian structure was established within 20 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 (1961), Tsuzuki et al. (1966) and Hyodo et al. (1966)
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
394
N. SATOH AND N. EGAMI
germ cells. Preliminary reports by Tsuzuki et al. (1966) and Hyodo et ah (1966)
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 8 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 (1953) 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 6-5 mm young
about 10 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 (1962, 1963). 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 17-5 days only in a female
foetus, although the gonadal volume increases earlier in a male foetus than in
a female. Recently Mittwoch, Delhanty & Beck (1969) 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 (1971) 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 (1971) 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, 1953, 1958). In
addition, Hishida (1962, 1964) 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,
1969). 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.
Sex differentiation in a teleost
395
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14
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(Manuscript received 8 February 1972)
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