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JOURNAL OF EXPERIMENTAL ZOOLOGY 283:295–306 (1999)
Sex-Determining Mechanism in Buergeria buergeri
(Anura, Rhacophoridae). III. Does the ZZW Triploid
Frog Become Female or Male?
S. OHTA,1* M. SUMIDA,2 AND M. NISHIOKA2
High School attached to Hiroshima-Denki Institute of Technology,
Hiroshima 736-0022, Japan
2
Laboratory for Amphibian Biology, Faculty of Science, Hiroshima
University, Higashihiroshima 739-8526, Japan
1
ABSTRACT
Both triploids and gynogenetic diploids (GDs) were produced to clarify the relationship between the sex-chromosome constitution and the expression of sex in the common bellring frog, Buergeria buergeri. The sex differentiation of triploids in B. buergeri is quite remarkable.
Triploid frogs consisted of three sex genotypes, ZZZ, ZWW and ZZW. All ZZZ triploids were males,
and all ZWW triploids were females. It is very interesting that half of the ZZW triploids became
female, and the other half became male. The GD frogs consisted of two sex genotypes, ZW and ZZ,
which did not differ from the controls in sex differentiation. Since the ratios of ZZ and ZW eggs
were significantly different among female parents, it is assumed that most (approximately 80–
90%) of the eggs made pre-reductional division in some females and post-reductional division in
others during meiosis. It seems that ZW eggs were produced by the occurrence of recombination
between the centromere and the sex-determining genes in B. buergeri. It was also found that the
number of Z chromosomes in each cell of these triploids and GDs agreed with that of the nucleoli
in each cell. J. Exp. Zool. 283:295–306, 1999. © 1999 Wiley-Liss, Inc.
Buergeria buergeri, which is endemic in Japan,
is female heterozygous in sex determination, and
chromosome pair no. 7 of this species is sex chromosomes of the ZZ/ZW-type (Ohta, ’82, ’86). The Z chromosome has a nucleolus organizer recognized as a
secondary constriction and a satellite attached to
the end of the long arm. On the other hand, the W
chromosome does not have either the secondary constriction nor the satellite, making the distinction of
the W chromosome from the Z chromosome easy.
Schmid et al. (’93) showed that the nucleolus organizer region (NOR) and its associated constitutive
heterochromatin are located exclusively in the long
arm telomeric region of the Z chromosome, and not
in the W chromosome or in any autosomes. Ohta
(’87) obtained two mature sex-reversed genetic females from tadpoles administered with estrogen.
Of the offspring from sex-reversed genetic females
mated with normal females, 28% were ZZ zygotes,
49% were ZW zygotes, and the remaining 23% were
WW zygotes which were mortal because they could
not produce nucleoli. Recently, Atsumi et al. (’98)
elucidated that the AAT-1 locus is sex-linked and
that the codominant alleles are expressed on the Z
chromosome, but not on the W chromosome in the
Hiroshima population of B. buergeri.
© 1999 WILEY-LISS, INC.
Since triploid amphibians were first produced
in an American newt Notophthalmus viridescens
by Fankhauser and Griffiths (’39), numerous triploids have been obtained in many amphibian species by cold or heat-shock treatment of the eggs
shortly after insemination in order to suppress
extrusion of the second polar body (reviewed by
Kawamura, ’84). The sex differentiation of artificially produced triploids has been also reported
in several amphibian species. Fankhauser (’45)
reported that most triploids produced by refrigeration in Notophthalmus viridescens were females. According to Humphrey and Fankhauser
(’46), the triploids produced in Ambystoma mexicanum (axolotl) consisted of 19 females and seven
males. When Ueda (’80) produced a large number
of triploids in the toad Bombina orientalis, there
was a nearly equal number of males and females
among triploids. Triploids produced by cold or
heat-shock treatment of fertilized eggs in Rana
japonica and R. tsushimensis were all males
(Kawamura and Tokunaga, ’52; Sumida and
*Correspondence to: S. Ohta, High School attached to HiroshimaDenki Institute of Technology, Hiroshima 736-0022, Japan.
Received 3 April 1998; Accepted 25 June 1998.
296
S. OHTA ET AL.
Nishioka, ’93). A similar observation was made
on triploid frogs of Rana rugosa from the Hiroshima population (Kashiwagi, ’93). Mature triploid frogs of Hyla japonica included an excessive
number of males (Nishioka and Ueda, ’83). A nearly
equal number of males and females was reported
in triploids of Rana nigromaculata and R. brevipoda
(Kawamura and Nishioka, ’67). Nevertheless, the
relationship between the sex-chromosome constitution and the sex differentiation has never been reported in amphibian triploids except axolotl’s
(Humphrey and Fankhauser, ’46).
The present authors produced triploids of various sex-chromosome constitution using B. buergeri
and examined their sexes in order to elucidate the
relationship between the sex-chromosome constitution and the expression of sex in this species.
Preliminary report of this research was made previously by Ohta (’88).
MATERIALS AND METHODS
Female and male B. buergeri (SCHLEGEL)
were collected from Sandankyo, Togouchi, Hiroshima Prefecture. Triploids were produced principally according to the method of Nishioka (’72)
by refrigerating fertilized eggs and suppressing
extrusion of the second polar body. Refrigerating
treatments were performed at 1–2°C for 3 hr,
about 10 min after fertilization. Gynogenetic diploids were produced by refrigeration after insemination with ultraviolet irradiated sperm as the
controls of the triploid production principally according to the method of Kawamura and Nishioka
(’81). Toshiba GUL-5-J UV-lamp was used to inactivate the sperm nucleus. This lamp was operated at 68 V, 125 mA with a highest intensity of
254 nm. The effective time for inactivation of
sperm nuclei exposed to UV-rays was 2 min.
Somatic chromosomes were observed in the epidermal cells of the tail-tips of tadpoles by the techniques described previously (Ohta, ’86). The number
of nucleoli of embryos or tadpoles was counted in
each cell nucleus stained with Heidenhain’s iron
hematoxylin or Giemsa solution (Ohta, ’86).
Gonads were fixed in Navashin’s fluid, sectioned
at 12 µm and stained with Heidenhain’s iron hematoxylin.
RESULTS
Production of triploids and
gynogenetic diploids
The mating experiments were carried out to produce triploids and GDs in the breeding season of
the year of 1984. The developmental capacity of
the offspring obtained from these matings was examined at six developmental stages from cleavage to completion of metamorphosis (Table 1). In
order to determine the ploidy and sex-chromosome
constitution of the offspring, karyotype and number of nucleoli were examined in each cell nucleus
of the embryos or tadpoles (Tables 2 and 3).
Controls
Control matings were also made between nine
females (84F, nos. 1–9) and nine males (84M, nos.
1–9) (Table 1). In these matings, 238 (56.9%) became normally feeding tadpoles. When the number of chromosomes was examined in 219 of these
tadpoles, 218 were normal diploids having 26 chromosomes (Table 2). Of these diploid tadpoles, 115
(52.8%) were of ZW-type in sex-chromosome constitution and contained a single nucleolus in each
cell (Table 3, Figs. 1b and 5a), while the other 103
(47.2%) were of ZZ-type and possessed a maximum
of two nucleoli (Table 3, Figs. 1a and 5b). The remainder was a triploid having 39 chromosomes,
TABLE 1. Developmental capacity of the eggs in two experimental series and the controls
Parents
Female
84F, nos.
1–9
84F, nos.
1–9
84F, nos.
1, 2,
4–7
1
Male
84M, nos.
1–9
84M, nos.
1–9
84M, nos.
1, 2,
4–7
Experimental
series1
No. of
eggs
Control
418
Exp (3n)
1098
Exp (GD)
1451
No. of
normally
cleaved
eggs
(%)
No. of
normal
neurulae
(%)
No. of
normal
tail-bud
embryos
(%)
No. of
normally
hatched
tadpoles
(%)
No. of
normally
feeding
tadpoles
(%)
No. of
metamorphosed
frogs
(%)
387
(92.6)
1014
(92.3)
1144
(78.8)
340
(81.3)
883
(80.4)
855
(58.9)
288
(68.9)
802
(73.0)
719
(49.6)
260
(62.2)
742
(67.6)
589
(40.6)
238
(56.9)
628
(57.2)
448
(30.9)
138
(33.0)
214
(19.5)
48
(3.3)
Exp (3n), producing triploids by refrigeration after insemination with normal sperm; Exp (GD), producing gynogenetic diploids by refrigeration after insemination with UV-irradiated sperm.
SEX OF ZZW TRIPLOIDS IN BELL-RING FROG
297
TABLE 2. Production of triploids and gynogenetic diploids
Parents
Female
Male
84F, nos. 1–9
84F, nos. 1–9
84F, nos. 1, 2,
4–7
84M, nos. 1–9
84M, nos. 1–9
84M, nos. 1, 2,
4–7
Ploidy (%)
Experimental
series1
No. of tadpoles
examined
2n
3n
Mosaics
Control
Exp (3n)
Exp (GD)
219
410
163
218 (99.5)
66 (16.1)
138 (84.7)
1 (0.5)
329 (80.2)
—
—
15 (3.7)
25 (15.3)
1
Exp (3n), producing triploids by refrigeration after insemination with normal sperm; Exp (GD), producing gynogenetic diploids by refrigeration after insemination with UV-irradiated sperm.
and was ZZZ-type having a maximum of three
nucleoli in each cell. It seems that this triploid
was produced by extrusion failure of the second
polar body nucleus from the egg.
Triploids
In order to produce triploids, a total of 1098 eggs
obtained from nine females (84F, nos. 1–9) were
refrigerated after insemination with the sperm of
nine males (84M, nos. 1–9). There was no difference in the developmental capacity between the offspring from refrigerated eggs and the controls
(Table 1). When the numbers of chromosomes and
nucleoli in each cell were examined in 410 normally
feeding tadpoles, 329 (80.2%) were triploids having 39 chromosomes, and 66 (16.1%) were diploids.
The remaining 15 (3.7%) were diploid-triploid mosaics and diploid-tetraploid mosaics (Table 2).
The examination of sex-chromosome constitution revealed that there were three sex genotypes,
ZZW, ZWW and ZZZ, in these triploid tadpoles.
Among the 329 triploid tadpoles, 84 (25.5%) were
ZWW-type having a single nucleolus in each cell
(Table 3, Figs. 2 and 5e), and 80 (24.3%) were ZZZtype having a maximum of three nucleoli (Table
3, Figs. 4 and 5g). The remaining 165 (50.2%) were
ZZW-type having a maximum of two nucleoli in
each cell nucleus (Table 3, Figs. 3 and 5f).
ZWW and ZZZ zygotes predominated in the
three, 84F, nos. 1–3 × 84 M, nos. 1–3, of the nine
matings. Among the 170 triploids of these three
matings, 78 (45.9%) were ZWW-type, 66 (38.3%)
were ZZZ-type, and the remaining 26 (15.3%) were
ZZW-type (Table 3).
On the other hand, ZZW zygotes predominated
in the other six matings, 84F, nos. 4–9 × 84M,
nos. 4–9. Among the 159 triploids of these six
matings, 139 (87.4%) were ZZW-type, six (3.8%)
were ZWW-type, and 14 (8.8%) were ZZZ-type.
Especially, among 73, 22 and 38 triploids obtained
from three female parents (84F, nos. 5, 8 and 9),
62 (84.9%), 22 (100%) and 36 (94.7%) were ZZWtype, respectively (Table 3).
Gynogenetic diploids
Eggs of seven females (84F, nos. 1–7) were refrigerated after insemination with UV-irradiated
sperm to produce GDs. None of the offspring raised
from one female (84F, no. 3) developed beyond the
neurula stage. Of 1451 eggs obtained from the re-
TABLE 3. Sex chromosomes and nucleoli of triploids, gynogenetic diploids and the control tadpoles1
Control
Parents
Male
Total
ZW
(1-nu)
84M, no. 1
84M, no. 2
84M, no. 3
84M, no. 4
84M, no. 5
84M, no. 6
84M, no. 7
84M, no. 8
84M, no. 9
32
23
5
4
59
7
20
40
28
21
12
2
1
26
4
12
22
15
Female
84F, no. 1
84F, no. 2
84F, no. 3
84F, no. 4
84F, no. 5
84F, no. 6
84F, no. 7
84F, no. 8
84F, no. 9
Total
(%)
1
218
Triploids
ZZ
(2-nu)
11
11
3
3
33
3
8
18
13
115
103
(52.8) (47.2)
The numbers in parentheses represent percentages.
Gynogenetic diploids
Total
ZWW
(1-nu)
ZZZ
(3-nu)
ZZW
(2-nu)
Total
ZW
(1-nu)
ZZ
(2-nu)
69
69
32
12
73
6
8
22
38
31
30
17
0
3
2
1
0
0
24
33
9
0
8
0
4
0
2
14
6
6
12
62
4
3
22
36
1
12
—
38
70
7
10
—
—
0
2
—
38
62
5
8
—
—
1
10
—
0
8
2
2
—
—
84
(25.5)
80
165
(24.3) (50.2)
329
138
115
(83.3)
23
(16.7)
298
S. OHTA ET AL.
Fig. 1. Metaphase plates and karyotypes of a male and a
female Buergeria buergeri. Scale bars equal 5 µm. a: Male.
The pair of no. 7 chromosomes was homomorphic (ZZ-type).
b: Female. The pair of no. 7 chromosomes was heteromorphic (ZW-type).
maining matings between six females (84F, nos. 1,
2, 4–7) and six males (84M, nos. 1, 2, 4–7), 1144
(78.8%) cleaved normally (Table 1). At the neurula
stage, 855 (58.9%) became normal embryos, but 289
(20.0%) were abnormal. Although 719 (49.6%) developed into normal tail-bud embryos, 136 (10.9%)
were abnormal. The number of nucleoli in each cell
nucleus was examined in the 289 abnormal neurulae and 136 abnormal tail-bud embryos. The results show that 124 neurulae and 94 tail-bud
embryos, 218 in total, were WW zygotes having no
nucleolus (Fig. 6c, d). WW individuals are mortal
(Ohta, ’87). The remaining 207 neurulae and tailbud embryos had a single or a maximum of two
nucleoli in each cell (Figs. 5c,d and 6a,b). Thereafter, 448 (30.9%) became normally feeding tadpoles
(Table 1). When the chromosomes and nucleoli were
examined in 163 normally feeding tadpoles, 138
were diploids, and the remaining 25 were diploidtetraploid mosaics and diploid-tetraploid-octoploid
mosaics (Table 2). Among 138 diploids, 115 (83.3%)
were ZW-type having a single nucleolus in each
cell and the other 23 (16.7%) were ZZ-type having
a maximum of two nucleoli in each cell in sex-chromosome constitution (Fig. 5c, d). Especially, among
12 diploids obtained from the female parent 84F,
no. 2, ten (83.3%) were ZZ-type. On the other hand,
among 38, 70 and 10 diploids obtained from three
other female parents (84F, nos. 4, 5 and 7), 38
(100%), 62 (88.6%) and 8 (80.0%) were ZW-type,
respectively (Table 3).
Sex of triploids and gynogenetic diploids
The gonads of frogs at ages of more than 3
months after metamorphosis were examined histologically (Table 4). The 17 ZW-type and 11 ZZtype GDs examined had similar ovaries and testes
in terms of inner structure as those of the controls, respectively (Fig. 7a, b). These findings show
that the sex differentiation in GDs is the same as
that in the controls.
Of triploids, all 33 ZWW-type frogs were females
SEX OF ZZW TRIPLOIDS IN BELL-RING FROG
299
Fig. 2. Metaphase plate and karyotype of a ZWW-type
triploid Buergeria buergeri. Scale bar equals 5 µm. The trip-
let of no. 7 chromosomes consisted of one Z chromosome having a satellite and two W chromosomes having no satellite.
having ovaries that were smaller than those of
the controls at the same ages. Growing auxocytes
were scarce and small, but there was little difference in the number of germ cells (Fig. 7c). All 53
ZZZ-type triploid frogs examined were males having testes. In these testes, the heads of the spermatozoa seemed to be somewhat long and thick
compared with those of the controls (Fig. 7d).
It was astonishing that there were both female
and male ZZW zygotes, but no hermaphrodites
were observed. Out of 80 ZZW-type triploid frogs
examined, 43 (53.8%) were females having ovaries, and the remaining 37 (46.3%) were males
having testes. This result shows that ZZW zygotes
consisted of females and males at the rate of
nearly 1:1 (Table 4). The ovaries of 43 ZZW females were small and underdeveloped compared
with those of the controls, and were very similar
300
S. OHTA ET AL.
Fig. 3. Metaphase plate and karyotype of a ZZW-type triploid Buergeria buergeri. Scale bar equals 5 µm. The triplet of
no. 7 chromosomes consisted of two Z chromosomes having a
satellite and one W chromosome having no satellite.
to those of ZWW females in inner and outer structures (Fig. 7e). On the other hand, the testes of
37 ZZW males were somewhat larger than those
of the controls at the corresponding age. Similar to
the testes of ZZZ-type triploids, the heads of these
spermatozoa were somewhat long and thick as compared with those of the controls (Fig. 7f). These findings show that triploids having two W chromosomes
differentiate into females without fail, triploids having no W chromosomes differentiate into males, and
triploids having a single W chromosome differentiate into either females or males.
DISCUSSION
Triploidy is established by a union of female and
male pronuclei when the extrusion of the nucleus
of the second polar body is suppressed by extremes
of temperature. The sex differentiation of triploids
has been reported in several amphibian species
by many investigators. Fankhauser (’45) reported
that most triploids produced by refrigeration in
Notophthalmus viridescens were females. He explained this result with two hypotheses: the female is heterogametic, and post-reductional
division for sex-determining segments of chromo-
SEX OF ZZW TRIPLOIDS IN BELL-RING FROG
301
Fig. 4. Metaphase plate and karyotype of a ZZZ-type triploid Buergeria buergeri. Scale bar equals 5 µm. The triplet of
no. 7 chromosomes consisted of three Z chromosomes having
a satellite.
somes occurred in most of the eggs, which were
then expected to become ZZW-type female. Humphrey and Fankhauser (’46) produced triploids in
Ambystoma mexicanum (axolotl) by using normal
females (ZW), normal males (ZZ) and sex-reversed
genetic females obtained by Humphrey (’42a,b,
’45). When the eggs of ZW females were refrigerated after insemination with sperm of ZZ males,
the triploids consisted of 19 females and 7 males.
These results have verified the correctness of the
hypothesis that there are two types of reduction
division, pre- and post-reductional divisions, for
sex-determining segments of chromosomes during
oogenesis. Cayrol et al. (’83) produced triploids
from fertilized eggs by cold-shock treatment in
Pleurodeles waltl, in which the female is heterogametic (Gallien, ’54; Collenot, ’73). They did not
observe any significant modifications in the sex
ratios of offspring of various crosses between the
triploids. This result suggested pre-reduction at
302
S. OHTA ET AL.
Fig. 5. Epidermal cells of triploid, GD and control tadpoles. Scale bars equal 5 µm. a: ZW-type control. b: ZZ-type
control. c: ZW-type GD. d: ZZ-type GD. e: ZWW-type triploid. f: ZZW-type triploid. g: ZZZ-type triploid.
meiosis leading to diploid eggs of the ZZ- or WWtype, and fertilization with Z sperm resulting ZZZ
triploid females or ZWW triploid males. The
present study clearly showed the relationship be-
tween the sex-chromosome constitution and the
expression of sex in triploid Buergeria buergeri.
All 53 ZZZ triploids were males, and all 33 ZWW
triploids were females. It is very interesting that
Fig. 6. GD embryos at the tail-bud stage and their epidermal cells. Scale bars equal 2 mm (a, c) and 5 µm (b, d). a:
ZZ- or ZW-type GD embryos. b: Epidermal cells of a ZZ-type
GD embryo having a maximum of two nucleoli. c: WW-type
GD embryos. d: Epidermal cells of a WW-type GD embryo
having no nucleolus. The large black-spots stained vaguely
with Giemsa solution were nuclei having no nucleolus.
SEX OF ZZW TRIPLOIDS IN BELL-RING FROG
303
TABLE 4. Sex of triploids (3n), gynogenetic diploids (GD) and the control frogs
Control
Parents
Gynogenetic diploids
Male
Total
ZW
X
ZZ
Y
Total
ZWW
X
ZZZ
Y
84M, no. 1
84M, no. 2
84M, no. 3
84M, no. 4
84M, no. 5
84M, no. 6
84M, no. 7
84M, no. 8
84M, no. 9
31
22
4
—
9
—
6
36
18
19
11
2
—
4
—
2
20
8
12
11
2
—
5
—
4
16
10
41
42
9
4
14
6
8
10
32
13
15
2
0
1
1
1
0
0
17
24
4
0
4
0
4
0
0
3
1
3
4
8
4
3
7
10
126
66
60
166
33
53
43
Female
84F, no. 1
84F, no. 2
84F, no. 3
84F, no. 4
84F, no. 5
84F, no. 6
84F, no. 7
84F, no. 8
84F, no. 9
Triploids
Total
of 80 ZZW triploids, about half of them (43) became female, and the other half (37) became male.
These results suggest that ZZZ and ZWW triploids were rigidly determined in sex differentiation, whereas ZZW triploids could differentiate
into either males or females.
The method for producing gynogenetic diploids
has been applied to investigate various recessive
mutations (Volpe and Dasgupta, ’62; Kawamura
and Nishioka, ’77, ’81; Nishioka, ’77; Nishioka and
Ueda, ’77, ’85a,b,c), and also to elucidate sex-determining mechanisms (Kawamura and Nishioka,
’77; Schmid et al., ’91). When Volpe and Dasgupta
(’62) produced GDs by suppressing extrusion of
the second polar body from eggs of burnsi mutants of Rana pipiens, 82.4% were burnsi mutants
and only 17.6% were wild-type frogs. From this
result, they assumed that recombination occurred
with high frequency between the major burnsi locus and the centromere, and that the major burnsi
gene made post-reductional division in most of the
eggs. When Colombelli et al. (’84) obtained Xenopus laevis GDs by refrigerating eggs after insemination with UV-irradiated sperm, about 80% were
females. To explain the predominance of females,
they considered that many ZW zygotes were produced in addition to ZZ and WW zygotes in GDs
due to the high frequency of recombination between the centromere and the sex-determining
segments. The present authors obtained triploids
by refrigerating eggs after fertilization with normal sperm and GDs by refrigerating eggs after
insemination with UV-irradiated sperm in B.
buergeri. As a result, ZZZ, ZWW and ZZW zygotes
were obtained in triploids and ZZ, ZW and WW
zygotes in GDs. These results suggest that the
differential segments of the sex chromosomes
make pre- or post-reductional division depending
ZZW
X
Y
Total
ZW
X
ZZ
Y
8
2
0
0
1
1
0
3
22
—
5
—
8
4
7
4
—
—
—
0
—
8
3
5
1
—
—
—
5
—
0
1
2
3
—
—
37
28
17
11
on the eggs. Since the spermatozoa of the males
of the present species have only Z chromosomes,
it is clear that the ZZZ and the ZWW zygotes of
triploids and the ZZ and the WW zygotes of GDs
were produced from eggs in which sex chromosomes made pre-reductional division, and that the
ZZW zygotes in triploids and the ZW zygotes in
GDs were obtained from eggs in which sex chromosomes made post-reductional division.
The distribution of chiasmata between the centromere and the nucleolus organizer was observed
in the lampbrush bivalent chromosome no. VII in
order to examine whether Z and W chromosomes
segregate at pre- or post-reductional division in the
oocytes of B. buergeri (Ohta, unpublished). These
chiasmata could be interpreted as recombination
sites between Z and W chromosomes. The nucleolus organizer was supposed to be closely linked with
the sex-determining genes in this species (Ohta, ’86;
Atsumi et al., ’98). The number of chiasmata was
observed to be zero or one in the lampbrush bivalent no. VII by Ohta (unpublished). From this observation, the present authors assume that bivalent
no. VII, which was converted into tetrad ZZWW,
segregated into ZZ and WW in the case of no chiasmata between the centromere and the nucleolus organizer, whereas bivalent no. VII segregated into
ZW and ZW in the case of one chiasma. It is supposed that ZZ and WW eggs were produced by
pre-reductional division, whereas ZW eggs were produced by post-reductional division. In the present
study, three kinds of triploids, ZZZ, ZWW and ZZW,
were produced. Of these, ZZZ and ZWW triploids
were considered to be derived from ZZ and WW
eggs, respectively, having no chiasmata between the
centromere and the nucleolus organizer in the bivalent no. VII. That is, ZZZ and ZWW triploids were
produced by refrigerating these eggs and suppress-
304
S. OHTA ET AL.
Fig. 7. Cross-sections of the gonads of triploid, GD and
control Buergeria buergeri. Scale bars equal 50 µm. a: Ovary
of a ZW-type GD frog. b: Testis of a ZZ-type GD frog. c: Ovary
of a ZWW-type triploid frog. d: Testis of a ZZZ-type triploid
frog. e: Ovary of a female ZZW-type triploid frog. f: Testis of
a male ZZW-type triploid frog.
ing extrusion of the second polar body after insemination with Z sperm. On the contrary, ZZW triploids were considered to be obtained by refrigerating
ZW eggs, in which a recombination occurred between the centromere and the nucleolus organizer
in the bivalent no. VII, inseminated with Z sperm.
Moreover, it is certain that approximately 80–
90% of the eggs obtained from three females (84F,
nos. 1, 2 and 3) made pre-reductional division,
whereas the predominant majority of eggs (approximately 85–100%) laid by another four females (84F,
nos. 4, 5, 8 and 9) made post-reductional division
(Table 3). From these lines of evidence, it seems
probable that there are great differences between
SEX OF ZZW TRIPLOIDS IN BELL-RING FROG
female parents in the rate of eggs made by prereductional and post-reductional division. Ohta
(unpublished) examined the genotypes of triploid tadpoles obtained from one female frog (88F,
no. 1) of the Hiroshima population and revealed
that of 156 triploids, 127 (81.4%) were ZZZ or
ZWW in sex genotype, and the other 29 (18.6%)
were ZZW in sex genotype. When the bivalent
no. VII was observed 4 months later in the number of chiasmata between the centromere and
the nucleolus organizer, using the oocytes of the
female frog (88 F, no. 1), the number of chiasmata was zero in 251 oocytes (88.1%) and one
in 34 oocytes (11.9%). These results suggest that
the number of chiasmata tends to inherit in this
species. It is certain that the recombination was
recognized as the chiasma, and that ZW eggs
were produced by the occurrence of recombination between the centromere and the nucleolus
organizer linked closely with the sex-determining genes in B. buergeri.
Humphrey and Fankhauser (’46) conducted an
interesting study on the relation between the sexchromosome constitution and the expression of sex
in diploids and triploids of Ambystoma mexicanum
(axolotl). According to them, triploids having only
one W chromosome became female without exception. Namely, ZZW zygotes differentiated into females as did ZWW and WWW zygotes, but ZZZ
zygotes differentiated into males. In the present
study, all ZWW zygotes became female and all ZZZ
zygotes became male. Although 80 ZZW triploid
frogs were obtained, 43 of them were females and
the remaining 37 were males. From these facts,
it may be probable that female determining factors on the W chromosome of Buergeria buergeri
are not always dominant for the Z chromosome,
being different from axolotl’s.
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