CHROMOSOMA
Chromosoma (Bet1) (1984) 90:261-264
9 Springer-Verlag 1984
Cytogenetic and genetic evidence of male sexual inversion
by heat treatment in the newt Pleurodeles poireti
C. Dournon 1, F. Guillet 1, D. Boucher 2, and J.C. Lacroix 2
1 Laboratoire de Biologic Animale;
2Laboratoire de G6n~tique du D~veloppement, Universit~ P. et M. Curie, 9 quai St Bernard 75230 Paris Cedex 05, France
Abstract. Larvae of Pleurodeles poireti were maintained
during their development at a high temperature (31 ~ C).
In several species of amphibians, such a treatment is known
to change the sex ratio through the inversion of genotypic
females into phenotypic males. Pleurodeles poireti is an exception. It is the first reported amphibian in which heat
induces an inversion of genotypic males into functional phenotypic females. The sexual genotype of standard and experimental phenotypic females was determined through heterochromosomes in lampbrush stage. In the present study,
we have utilised another technique for identification of sexual genotype, applicable to both phenotypic males and females. It is based on the differential expression of a sexlinked gene, the peptidase 1.
the lengthy reproductive cycle of the amphibians. It should
be interesting therefore to identify the genotypic sex of an
experimental animal by cytogenetic or other genetic criteria,
i.e. through sex chromosomes or sex-linked characters respectively.
In a geographical race of P. poireti, the sex chromosomes can be distinguished in preparations of lampbrush
chromosomes of the oocytes (Lacroix 1970). Moreover,
Ferrier et al. (1980, 1983), have shown that in P. waltlii
the enzyme peptidase I shows a sex-linked polymorphism.
The effects of heat treatment on sexual differentiation
of larvae of P. poireti are presented here. We show that,
contrary to the results obtained under similar conditions
in P. waltlii, the sex ratio is biased in favour of females.
Demonstration of sexual inversion of genotypic males is
based on cytogenetic and genetic analysis.
Introduction
Materials and methods
The amphibians, urodeles as well as anurans, constitute
a particularly favourable material for studies on phenotypic
functional sexual inversion. Obtaining unisexual offspring
from animals having undergone gynogenic or androgenic
treatment demonstrates the phenotypic sexual inversion of
one of the parents. Crosses between normal and sex-reversed individuals allow one to define the homogametic
and heterogametic sex for each of the studied species when
no sex chromosome can be cytologically detected, as is often
the case in amphibians (for review see Foo~e 1964).
The two species of Pleurodeles, P. waltlii and P. poireti
(urodeles) have often been utilised for such studies. It was
thus demonstrated that in P. waltlii, the males are homogatactic ZZ: crosses between a normal male and a neo-female
(genotypic male inversed into phenotypic female by estradiol benzoate treatment) give an exclusively male progeny
(Gallien 1951). The homogametic nature of males in P.
poireti was demonstrated by a similar experimental procedure (Lacroix 1970). Conversely, the phenotypic inversion
of heterogametic ZW female gonads to functional male ones
obtained in P. waltlii by grafts of embryonic gonads produced females of the sexual genotype WW. Theses females
gave rise to only female offspring when mated with standard males (Collenot 1973, 1975). Sexual inversion of genotypic females into males has also been induced in this species by heat treatment (Houillon and Dournon 1978 ; Dournon and Houillon 1983).
In these studies, genetic identification of the sex-reversed
individuals was done by an analysis of the sex ratio of
the progeny. Such a procedure is necessarily long due to
Heat treatment. The procedure followed was the one that
was previously developed for P. waltlii (Dournon 1981).
The experimental embryos developed at room temperature
(20~ 3 ~ C) from the egg until the stage immediately preceding hatching (stage 33a of the developmental table of
P. waltlii, GaUien and Durocher 1957). From hatching until
the end of metamorphosis, the larvae were placed at a temperature of 31~ 1~ C, and from the end of metamorphosis
until sexual maturity (6-8 months) the animals were maintained at a temperature of 230-27 ~ C and thereafter at room
temperature.
Control individuals developed from the egg to the adult
stage at room temperature.
Early identification of sexualphenotype. The sexual phenotype of the gonads was identified on living animals about
3 months after metamorphosis, a stage at which a simple
examination of gonad morphology under a dissecting microscope provides an unambiguous diagnosis. This examination was performed on anaesthetized animals through
a lateral opening of the abdomen.
Analysis of sex chromosomes of phenotypic females. In the
race of P. poireti utilized here, the W chromosome carries
a specific morphological differentiation in the lampbrush
stage. In the oocyte nuclei, the sexual bivalent (IV) is heterozygotic or heteromorphic ( - / + ) for this differentiation in
the heterogametic ZW individuals, and homozygotic
( - / - ) in the homogametic ZZ individuals (Lacroix 1970).
Lampbrush chromosomes from oocytes of 11 phenotypic females from the experimental group, were analysed to
262
define the homo- or heterozygotic nature of the sexual bivalent. The chromosomal preparations were obtained following routine procedures from ovarian biopsy of sexually mature animals (Lacroix and Loones 1974). These individuals
were then appropriately catalogued and maintained to
study their progeny.
Genetic study on the offsprings. The experimental phenotypic females having obtained sexual maturity were bred with
standard ZZ males and the sex ratio of the progeny of
each of them was analysed.
Table l. Sex ratio of descendents of standard couples of Pleurodeles
poireti as a function of rearing temperature
No. of ~
No. of 9"
No. of 9
Total
Control
(20~ 3~ C)
47 (52.8)"
0 (0)
42 (47.2)
89
Heat treatment
12 (20.7)
4 (6.9)
42 (72.4)
58
(31~
1~
c)
a Percentages are given in parentheses
Enzymatic analysis. A sex-linked polymorphism of the enzyme peptidase 1 has been demonstrated by electrophoresis
in P. waltIii. This polymorphism depends on a pair of codominant alleles Pep-lA and Pep-lB. It allows the identification of the genotypic sex of different individuals (Ferrier
et al. 1980, 1983). In this study, the method has been
adapted to P. poireti.
Erythrocyte haemolysates were subjected to electrophoresis in horizontal starch gels according to the technique
of Wright et al. (1976) using Tris-citrate for 16 h at 4 ~ C.
The peptidase was revealed in the presence of valyl-leucine
by incubating the lower part of the starch gels in the medium described by Lewis and Harris (1967) for 1 h at 37 ~ C.
These results clearly show an influence of the high temperature on the sexual differentiation of gonads. This influence is shown firstly by the presence of a high percentage
of intersexual individuals. As a matter of fact, no case of
spontaneous intersexuality in normal stocks of Pleurodeles
have been reported. This intersexuality thus corresponds
to a partial modification of the sexual phenotype of the
gonads. Secondly, the influence of heat treatment is corroborated by the significant deviation of the sex ratio in favour
of the female phenotype, suggesting that some genotypic
ZZ males are sex-reversed (heat-induced neo-females).
Results
Cytogenetic analysis. The proposed interpretation was
checked by karyological analysis of the lampbrush chromosomes of experimental females once they had reached adult
stage. This analysis was done on 11 females of the second
experimental batch (Table 2). Of these, 5 are heteromorphic
for the sexual bivalent IV and 6 are homomorphic (Fig. 1).
The 6 homomorphic individuals thus really have a male
ZZ genotype.
Influence of heat treatment on sexual differentiation
Three lots of animals derived from different couples have
been utilised. The experimental animals were taken from
all three batches while the controls animals were taken from
only two of them. Of 110 control animals 89 reached a
stage when the sexual phenotype could be identified: 22
from the first batch, 67 from the second. Of the 89 individuals, 47 (53%) were males and 42 (47%) females (Table 1).
Amongst the 125 experimental heat-treated animals, 58
reached the stage of sexual phenotype identification, 15 of
50 individuals for the first, 20 of 25 for the second, and
23 of 50 for the third batch. Of these 58 animals, 12 (21%)
had the male phenotype, 42 (72%) the female phenotype,
and 4 (7%) were intersexual (Table 1). In the intersexual
animals, the gonads were differentiated into testes in their
anterior parts and into ovaries in their posterior parts. The
stage of development of the two juxtaposed territories of
the intersexual gonads was comparable to that of unisexual
gonads of other experimental animals of the same age.
Proof of sexual inversion of genotypic Z Z males
Genetic analysis of the progeny. Three of the ZZ females
were crossed with standard male and gave rise to descendents. The progeny of two of these females were almost
completely decimated by a parasitic infection, the third
batch developed normally and gave 66 animals. Raised at
room temperature, all these individuals were males (Table 2). The unisexual nature of this progeny confirms the
sex inversion of males into functional females as well as
the homogametic nature of males in this species.
Electrophoretic pattern of peptidase 1. The procedure for
the detection of peptidase 1, adapted to P. poireti, allowed
us to obtain distinct electrophoretic patterns for the two
Table 2. Cytogenetic, enzymatic, and genetic (sex ratio) analyses of 11 experimental females of Pleurodelespoireti
No. of
females
1-81
2-81
3-81
4-81
5-81
6-81
7-81
8-81
9-81
10-81
11-81
Structure of
bivalent IV
-- / -- /+
- / -- /+
- /-- / +
-- / +
--/-- / -- / +
- /-
Sexual
genotype
ZZ
ZW
ZZ
ZW
ZZ
ZW
ZW
ZZ
ZZ
ZW
ZZ
Spectrum of
peptidase i
fast
slow
fast
slow
fast
slow
slow
fast
fast
slow
fast
Descendents
No. of d'
No. of ~
Total
66
0
66
2
263
Fig. 1. Phase-contrast micrograph of heterochromosomes of Pleurodeles poireti in the lampbrush stage. The W chromosome carries
a sequence of loops characterizing its differential segments (bracket); this sequence is absent in the Z chromosome. The bivalent IV
from an oocyte of a standard Z W female (lower) is heterozygotic ( + / - ) for this sequence. The bivalent IV from an oocyte of the
ZZ neo-female 1-81 (upper), is homozygotic ( - / - ) . Bar represents 30 gm
sexes. Furthermore, these patterns are different from those
obtained for P. watdii (Ferrier et al. 1980, 1983). For the
controls, the analysis was done on a sample of 10 animals
of each sex. In the males, the enzyme is located in a fast
moving band, whereas in the females the band containing
the enzyme moves more slowly (Fig. 2). These results show
that there is a correlation between the enzyme expression
and the sex of the individuals; however, they do not distinguish whether the correlation is due to the sexual genotype
or to the phenotype, as the two are the same in these control
animals.
Of the 20 experimental animals examined by this procedure, 8 were phenotypic males and 12 were phenotypic females, including the 11 utilised for cytogenetic analysis. For
the 8 males, the electrophoretic pattern was identical to
that of standard males. Of 12 females, 5 showed a spectrum
identical to that of standard females (a slower moving band)
whereas 7 females showed a spectrum comparable to that
of standard males (a faster moving band). There is a perfect
correlation between the karyological and electrophoretic
analyses for the 11 females studied by the two techniques
(Table 2). The heat-induced neo-females thus have an electrophoretic pattern for peptidase 1 of the male type, which
indicates that the expression of this enzyme depends on
the genotypic sex. These observations also suggest that peptidase 1 gene is sex-linked in P. poireti as in P. waltlii.
Discussion
In all the amphibians previously studied, the anurans Rana
temporaria and R. sylvatica (Witschi 1914, 1929), R. temporaria and Bufo vulgaris (Piquet 1930), R..japonica (Yoshikura
1959), R. catesbeiana (HSii et al. 1971) and urodeles Hynobius retardatus (Uchida 1937), Pleurodeles waltlii (Houillon
and Dournon 1978), an unusual high temperature orients
the sexual differentiation of the gonads towards testes.
Pleurodeles poireti is thus an exception: it is the first amphibian in which heat treatment has been found to orient
the gonads of genot.ypic males towards an ovarian differentiation. This result is all the more surprising because the
sexual inversion is obtained under conditions comparable
to those utilised for P. waltlii. Actually, the two species,
besides being homogametic for the mate sex, are closely
allied giving rise to hybrids fertile up to the third generation
(Lacroix 1968).
In our experiments, we did not obtain sexual inversion
of all the genotypic males. It is probable that application
of a temperature slightly higher than 31 ~ C during the heatsensitive period, which remains yet to be determined precisely for P. poireti, could ameliorate the efficiency of sexual
inversion as has been observed for P. wahlii (Dournon,
in preparation). However, it should be noted that all the
heat-induced neo-females present complete and regular
264
Fig. 2. Electrophoretic patterns of peptidase 1 in five experimental
and two control animals. Fast moving bands are from a control
ZZ male (a), female 3-81 (c), and female 5-81 (e). Slow moving
bands are from a control ZW female (b), female 4-81 (d), female
6-81 (f), and female 7-81 (g). Note the presence of two types of
patterns in experimental females
Mfillerian ducts as in standard females. Treatment of the
larvae of P. poireti with steroid hormones (estradiol benzoate) can result in 100% sexual inversion of gonads in
genotypic males (Lacroix 1970). However, under these conditions Mfillerian ducts often display abnormalities.
The differentiation of sex chromosomes in the lampbrush stage allows determination of the genotypic sex. This
procedure is unequivocal b u t is applicable only to phenotypic females with oocytes in vitellogenesis. D e m o n s t r a t i o n
of a sex-linked genetic marker permits us to extend the
analysis of sexual genotype to individuals of either sexual
phenotype. This procedure should also be applicable to larvae prior to metamorphosis, as has been shown for P. waltlii
(Ferrier et al. 1983).
To conclude, we have obtained a functional feminization of genotypic males by increasing the temperature during development of embryos and larvae in the urodela
Pleurodeles poireti. This observation is confirmed by cytogenetic and genetic analysis. D e m o n s t r a t i o n of a sex-linked
gene of a peptidase extends the analysis of sexual genotype
to adults of both sexual phenotypes.
Acknowledgements. This work was supported by grants from the
Centre National de la Recherche Scientifique (A.T.P. "Biologic
Mol6culaire du g6ne" 1982-]983) and the Minist6re de l'Education
Nationale (Direction de la Recherche A R U 1983).
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Received May 8, 1984
Accepted by W. Hennig