Comparative Biochemistry and Physiology Part C 130 2001.

399409

Environment and sex determination in farmed fish

J.F. Baroiller U , H. DCotta
CIRAD-EMVT, Station Commune de Recherches en Ichtyophysiologie, Biodi ersite
et En ironnement, INRA-SCRIBE,
Campus de Beaulieu, 35042 Rennes Cedex, France
Received 25 January 2001; received in revised form 3 July 2001; accepted 30 July 2001

Abstract
A plasticity of gonadal sex differentiation was reported in the 1930s following exogenous steroid treatments in fish,
but demonstration that environmental factors temperature, pH, density and social interactions . could influence the sex
ratio in gonochoristic species has been relatively recent. In fish, as in reptiles and amphibians displaying environmental
sex determination, the main environmental factor influencing sex seems to be temperature TSDs Temperature Sex
Determination.. In most thermosensitive species some Atherinids, Poecilids, Cichlids: tilapias, goldfish, a Siluriform, a
flatfish . . . . male to female ratio increases with temperature andror ovarian differentiation is induced by low temperatures. Conversely, in some rare species Dicentrarchus labrax, Ictalurus punctatus., high temperatures may produce
female-biased sex ratios andror low temperatures promote male-biased sex ratios. In the hirame Paralichthys oli aceus,
both high and low temperatures induce monosex male populations while intermediate temperatures yield a 1:1 sex ratio
U-shape curve.. Fish show particularities in their TSD patterns since mono-sex populations are generally not produced
at extreme temperatures, suggesting the existence of strong temperaturergenotype interactions. In reptiles, amphibians
and fish displaying TSD, temperature treatments must be applied at a critical sensitive period, relatively similar to the
hormone sensitive period. In gonochoristic fish, steroid hormones with estrogens in females and 11-oxygenated
androgens in males, are probably key physiological steps in the regulation of gonadal sex differentiation. Cytochrome
P450-aromatase, enzyme catalysing conversion of androgens to estrogens, seems to be a critical enzyme for ovarian
differentiation. Molecular mechanisms of thermosensitivity have been addressed in two species tilapia Oreochromis
niloticus and the hirame, where aromatase gene expression is down-regulated by masculinizing temperature treatments.
Furthermore, in tilapia the gene expression of 11-hydroxylase a key enzyme involved in the synthesis of 11-oxygenated
androgens. does not appear to be affected by temperature treatments. 2001 Elsevier Science Inc. All rights reserved.
Keywords: Environmental factors; Fish; Sex differentiation; Gene expression

This paper was originally presented at a symposium dedicated to the memory of Marcel Florkin, held within the ESCPB 21st
International Congress, Liege,
` Belgium, July 2428, 2000.
U
Corresponding author. Tel.: q33-2-2348-5012; fax: q33-2-2348-5020.
E-mail address: jfb@beaulieu.rennes.inra.fr J.F. Baroiller..
1532-0456r01r$ - see front matter 2001 Elsevier Science Inc. All rights reserved.
PII: S 1 5 3 2 - 0 4 5 6 0 1 . 0 0 2 6 7 - 8

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J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

1. Introduction
An array of sex determining mechanisms have
been reported in fish. In the majority of fish, sex
chromosomes are not sufficiently divergent morphologically to be identified by classical karyotype. Indirect approaches have demonstrated that
under normal conditions sex is considered to be
strongly determined by genotype. Nevertheless,
influence of environmental factors on sex differentiation in fish has long been suggested due to
intersexual features detected in the gonads of
some gonochoristic individuals and by the presence of skewed sex ratios in wild populations. In
these early descriptive studies, evidence was not
conclusive as to the possible involvement or the
respective role of various environmental factors.
The first experimental demonstration that some
exogenous factors could influence the sex ratio in
gonochoristic species was through the manipulation of gonadal sex differentiation by steroid
treatments reviewed by Yamamoto, 1969.. The
definitive evidence that an environmental factor
affected sex differentiation in a gonochoristic fish
has been very recent Conover and Kynard, 1981.,
and concerned only one species Menidia menidia.
The last few years have clearly established that
sensitivity of gonadal sex differentiation to environmental factors Environmental Sex Determination: ESD. is more widespread than previously
expected reviewed by Baroiller et al., 1999.. Until the first evidence that temperature had effects
on sex differentiation in M. menidia by Conover
and Kynard 1981., most of the studies had been
focused on reptile and amphibian models. In these
species, the term ESD was used to characterise
the global effect, concerning the cascade from sex
determination to gonadal sex differentiation.
Hayes 1998. described sex determination as the
mechanisms directing sex differentiation whereas
sex differentiation is the development of testis or
ovaries from the undifferentiated gonad. In vertebrates displaying an environmental sensitivity,
the genetic sex determination takes place during
fertilisation by the combination of genetic factors
brought by the male and female breeders. However, are the environmental effects only acting on
the directing genes andror on the sex differentiating genes? The term ESD has also been applied in fish even by Conover and Kynard, 1981.,
without knowing exactly if temperature was affecting the sex determination or the process of

sex differentiation. Recent data on both reptiles

and fish suggest that temperature could be mainly
affecting sex differentiation rather than sex determination. Nevertheless, an interesting aspect
to consider is the important interactions which
have been demonstrated between temperature
and genotype Baroiller and Clota, 1998.. Are
these interactions related to sex determinism linked to the sex chromosomes. or to the sex differentiation? This point is still unclear and therefore, in the present review, we will also use the
terms ESD and TSD as initially applied in fish. In
addition, the terms masculinization or feminization refer to the functional gonadal sex inversions, of at least part of the individuals of a
progeny.
Studies focused on two families, the
Atherinidae Menidia menidia, M. peninsulae,
Odontesthes bonariensis, Odontesthes argentinensis
and Patagonina hatcheri . and the Cichlidae
Apistogramma sp., Oreochromis sp., . . . ., as well
as a marine flatfish, Paralichthys oli aceus, have
provided a better knowledge on: 1. the main
environmental determinants of sex; 2. the critical
period of gonadal sensitivity to these factors; 3.
the interactions between genotype and the environmental effects; and 4. the molecular mechanisms involved in the modulation of sex differentiation by external factors. This review will cover
these four aspects of the environmental sex determination ESD. in gonochoristic fish.

2. The main environmental determinants of sex

Among the main demonstrative studies Table
1., very few environmental factors have been
analysed and therefore, it is probably premature
to generalise. However, among the factors studied
up till now, temperature appears to be the main
environmental determinant of sex in most sensitive species. Interestingly, in these species sensitive to temperature andror pH, there appears to
be no effect of other factors such as photoperiod,
density, or salinity, and thus suggests a certain
specificity for the type of sensitivity to external
factors. For instance in the genus Apistogramma,
temperature is the major environmental determinant of sex. However, progenies of A. caetei are
not sensitive to temperature but their sex ratios
are strongly influenced by pH. Similarly, tilapias
are sensitive to temperature variations but salin-

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

401

Table 1
Main factors and their influence on the sex ratio in fish
Species

Environmental
determinant

Inefficient
factors

Apistogramma sp.
33 sp.
Carassius auratus
Cichlasoma
citrinellum
Clarias gariepinus

T, pH
T
d, sf

Dicentrarchus labrax

Hoplosternum litorale
Ictalurus punctatus
Macropodus
opercularis
Menidia menidia

T
T
sf, d

Menidia pensinsulae
Misgurnu
anguillicaudatus
Odonthestes
argentinensis
Odontesthes
bonariensis
Oreochromis aureus

T
T

Conover and Kynard, 1981

Conover, 1984
Conover and Fleisher, 1986
Conover and Heins, 1987a
Conover and Heins, 1987b
Conover and De Mond, 1991
Middaugh and Hemmer, 1987
Nomura et al., 1998

Strussmann
et al., 1996

Strussmann
et al., 1996

Oreochromis niloticus

Paralichthys oli aceus

Patagonina hatcheri

Pel icachromis sp.

Poecilia melanogaster
Poeciliopsis lucida
Red tilapia 4-ways
hybrid, red Florida.
Verasper moseri
Xiphophorus helleri

pH
T, pH
T
T

Desprez and Melard,

1998

Baras et al., 2000

Abucay et al., 1999
Baroiller et al., 1995a
Baroiller et al., 1995b
Baroiller et al., 1996a
Baroiller et al., 1996b
Baroiller et al., 1999
Baroiller and DCotta, 2000
Baroiller and Clota, 1998
DCotta et al., 2001a
Tabata, 1995
Yamamoto, 1999
Kitano et al., 2000
Strussmann
et al., 1996

Strussmann
et al., 1997

Rubin, 1985
Romer
and Beisenherz, 1996

Sullivan and Schultz, 1986

Desprez et al., 1997

T
pH

Goto et al., 1999

Rubin, 1985

Romer
and Beisenherz, 1996

P, d

References

S, P

d, FR, S

Goto et al., 2000

Francis and Barlow, 1993
Van den Hurk and Richter,
unpublished data cited in
Van den Hurk and Lambert, 1982
Blazquez et al., 1998
Pavlidis et al., 2000
Hostache et al., 1995
Patino et al., 1996
Francis, 1984

Inefficient factors were those tested but shown to have no effect in thermosensitive species. d: density; FR: feeding rate; P:
photoperiod; pH: pH; S: salinity; sf: social factors; T: temperature.

402

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

ity Abucay et al., 1999., density, or confinement

Baroiller, unpublished data. do not seem to influence their sex ratios.

3. Critical sensitive period

As already demonstrated in reptiles Pieau et
al., 1999. and amphibians Chardard et al., 1995.,
fry or eggs have to be exposed to environmental
treatments before and during the onset of the
histological gonadal sex differentiation, for temperature to affect sex ratios. In the tilapia, O.
niloticus, it has been demonstrated that the increase of male proportions in the high temperature-treated groups corresponded to the functional masculinization of genetic females Baroiller et
al., 1995a.. In this study XX males which sire all
female populations. have been only detected in
the temperature treated group. In order for hormonal treatments of sex inversion to be efficient
in fish, they also have to be applied at these very
similar critical stages, as has been demonstrated
in: tilapias Baroiller and Toguyeni, 1996.; pejerrey, Odontesthes bonariensis Strussmann
et al.,

1997.; channel catfish Patino et al., 1996.; or

Japanese flounder Kitano et al., 1999.. Even
though a short temperature treatment 10 days.
Baroiller et al., 1995a. can be just as efficient as
a longer 21 days. hormonal treatment Baroiller
and Toguyeni, 1996., both of them cover the same
initial critical period comprising the 1424 days
after fertilisation.
Nevertheless, recent results in both tilapia and
European sea bass Dicentrarchus labrax suggest
that different thermosensitive windows may exist.
In two tilapia species, O. niloticus and O. aureus,
and in a four-way hybrid of tilapia red Florida
strain., low temperatures had no effect on sex
ratios even when applied during the classical
period of sensitivity to hormonal treatment
Baroiller et al., 1995b; Desprez et al., 1997;
Desprez and Melard,
1998; Abucay et al., 1999..

Similar results have been recently reported in O.

mossambicus: when treatments were applied from
10 days post-hatching, high proportions of males
were induced by elevated temperatures, but low
temperatures did not affect the sex ratios. However, Wang and Tsai 2000. exposed the same
species to low temperatures 20C. at earlier
stages from hatching or 5 days post-hatching.

and could induce high proportions of females. In

the European sea bass, similar results have been
found since in one case, low temperature treatments 15C. applied during the sensitive period
to hormonal treatments 57137 days postfertilisation . could induce a high proportion of
males Blazquez et al., 1998.. On the other hand,
high proportions 7073%. of females could be
induced when sea bass were exposed to low temperature 1315C. at very early stages stage of
half-epiboly s 30 h post-fertilisation ., lasting until
the middle of metamorphosis with the following
on-growing phase performed under ambient temperature condition Pavlidis et al., 2000..
3.1. Different patterns of ESD
3.1.1. Influence of temperature
Similarly to what has been encountered in reptiles and amphibians, three main types of response to temperature have been reported in fish
Baroiller et al., 1999.. In most of the thermosensitive species, the male to female ratio increases
with temperature, andror ovarian differentiation
is induced by low temperatures. This type of
response concerns species from various freshwater or marine families such as: the Atherinids
Menidia menidia, Odonthestes bonariensis, O.
argentinensis, Patagonina hatcheri .; Poecilids
Poeciliopsis lucida, Poecilia melanogaster .; Cichlids various Apistogramma sp. and tilapias.;
Cyprinids (Misgurnus anguillicaudatus, Carassius
auratus.; Callichthyidae Hoplosternum littorale.,
Pleuronectidae Verasper moseri . . . . reviewed by
Baroiller et al., 1999.. Conversely, in some rare
species i.e. the channel catfish Ictalurus punctatus ., high temperatures may produce femalebiased sex ratios andror low temperatures promote male-biased sex ratios Patino et al., 1996..
In the case of the Japanese hirame, Paralichthys
oli aceus, both high and low temperatures induce
monosex male populations Yamamoto, 1999.
while intermediate temperatures yield a 1:1 sex
ratio U-shape curve..
This classification is probably premature because the same range of temperatures can either
masculinize or feminize progenies belonging to a
same species depending, on the period when
treatment is applied i.e. the European sea bass
or O. niloticus. or depending on the sexual genotype in O. niloticus see Section 4..

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

3.1.2. Influence of pH
In all the pH sensitive Apistogramma species
Romer
and Beisenherz, 1996., the proportion of

males is higher at an acid pH 4.5. than at a more

neutral value 6.5..
3.1.3. Influence of density
In the paradise fish, Macropodus opercularis,
the proportion of females could be directly proportional to the density Francis, 1984..
3.2. Validity of the experimental results with regard
to the natural conditions and adapti e role of ESD?
With the exception of the study by Conover
and Kynard 1981. on M. menidia, demonstrating
an adaptive role of TSD, conclusive studies
concerning ESD have been addressed either under controlled or semi-controlled conditions.
Hence, in most of these studies extreme conditions of temperature, pH, density . . . have been
often chosen in order to maximise a putative
effect on sex ratio. Even if physiological temperatures or pH have been also chosen in some cases,
constant regimes were mainly applied. These conditions do not truly mimic the natural regimes,
and the demonstration that ESD exists in the wild
can be a very difficult issue for some species
sexrage-specific migration, mixing of different
progenies in a same shoal . . . .. It has been shown
to exist in the case of M. menidia, where skewed
sex ratios were both obtained in the wild during
early or late breeding seasons resulting in high
proportion of females and males, respectively, but
also under constant laboratory conditions. In another recent case, in the blue tilapia, O. aureus,
fluctuating temperatures have been demonstrated
to have less masculinizing potentials than constant high temperatures but can still produce
significant deviations of sex ratios Baras et al.,
2000..
Female tilapias after fertilisation collect in their
mouth first their eggs and then the vesiculated fry
for mouth brooding, fry which will swim into
shallow terrace waters or flooded eulitoral grassland Keenleyside, 1991.. A first release of the
progeny occurs at approximately day 1012 postfertilisation and consist mainly of fry occupying
shoals, present in shallow waters during the day,
but that take refuge into the mothers mouth in

403

times of danger and during the night guarding

period.. The critical period for gonadal sex differentiation occurs during this guarding sequence; temperatures in the shallow waters occupied by the shoals can reach more than 3435C.
Under controlled conditions, these high temperatures can strongly masculinize a progeny Baroiller
et al., 1995a..
Likewise, Apistogramma species in the wild live
in habitats where a temperature gradient exists
with respect to the temperature depth which
ranges from approximately 23 to 30C. Under
laboratory conditions strong masculinization is induced by 29C treatments Romer
and Beisen
herz, 1996.. Therefore putative masculinizing situations can be encountered by tilapias and Apistogramma species in the wild.

4. Interactions between temperature and genotype

In tilapia, as in M. menidia, a high variability in
thermosensitivity has been reported depending on
the progenies from a same strain stock Baroiller
et al., 1995b, 1996a; Conover and Heins, 1987b.,
suggesting strong interactions between temperature and genotype. In the tilapia, O. niloticus,
parental effects have been first demonstrated at
basal temperatures Fig. 1.. Within a defined temperature, sex ratios of successive progenies generated by a given couple of breeders are very similar. Moreover, the masculinizing effect of a same
high temperature treatment on these successive
progenies is also very stable Fig. 2.. Therefore,
both sex ratios and thermosensitivity are very
stable for a given couple of breeders Baroiller et
al., 1995b.. Finally, parental effects on thermosensitivity have been also reported: a same
male or female breeder can generate both sensitive and insensitive progenies. However, only male
breeders can sire either all sensitive or all insensitive progenies Fig. 3..
In the same tilapia species, similar high temperature treatments have been applied on all
male, all female, or mixed populations Fig. 4..
All female populations XX, all male populations
XY and mixed populations XXq XY. were obtained by mating a classic female breeder, respectively, with a XX, XY, or a YY male Abucay et
al., 1999; Baroiller and DCotta, 2000; Kwon et

404

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

Fig. 1. Parental Effects on Oreochromis niloticus sex ratios,

male proportion % of individual progenies obtained through a
diallel mating table between four males ME, MA, MD, MC.
crossed with four females FA, FB, FC, FD.. A minimum of
100 fry per group were sexed by microscopic analysis of
gonadal squash from Baroiller and Clota, 1998..

al., 2000a.. All male populations YY were obtained by mating a YY male with a YY female
Abucay et al., 1999.. In all these experimentations, all female and mixed populations were masculinized by high temperature 3536C. treatments Abucay et al., 1999; Baroiller and DCotta,
2000; Kwon et al., 2000a.. Conversely, YY all
male populations were feminized by a similar
high temperature treatment Abucay et al., 1999;
Kwon et al., 2000a.. The occurrence of a similar
feminizing effect on XY genotypes remains controversial Abucay et al., 1999; Baroiller and
DCotta, 2000; Kwon et al., 2000a.. Therefore,
depending on the genotype and more particularly
on the sexual genotype, a same high temperature
treatment may either masculinize or feminize a
same species.

Fig. 2. Stability of sex ratios and thermosensitivity in three

successive progenies, obtained from the same couple of Oreochromis niloticus breeders reared at the basal temperature of
27C and the masculinizing temperature of 36C from
Baroiller et al., 1995b..

Fig. 3. Parental Effects on Oreochromis niloticus thermosensitivity: diallel mating table between four males ME, MA, MB,
MC. crossed with four females FA, FB, FC, FD.. Each
progeny was divided into two parts, one of them reared at the
basal temperatures of 27C while the other part was masculinized using high temperatures of 36C. The figure represents
the deviation of the sex ratio male proportion at 35C minus
the male proportion at 27C. from Baroiller and Clota, 1998..

5. Molecular mechanisms involved in the

modulation of sex differentiation by external
factors
Vertebrates have different mechanisms of sex
determination but they all seem to undergo a
histological neutral stage during embryonic devel-

Fig. 4. Influence of sexual genotypes on thermosensitivity in

O. niloticus. Schematic representing of data from Abucay et al.
1999., Baroiller and DCotta 2000., Kwon et al. 2000a.. Two
different results have been reported regarding the experiments on the XY genotype: the first by Baroiller and DCotta
2000. and Kwon et al. 2000a. shows no effect of high
temperatures on the sex ratios; whereas in the second report,
Abucay et al. 1999. evidenced a feminising effect of high
temperatures on XY progenies.

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

opment where the gonad is bipotential, and subsequently follow a sex differentiating pathway oriented towards either ovary or testes development.
Biochemical and molecular approaches have been
used to analyse the cascading events and the
genes involved in the sex determination and differentiation of lower vertebrates. To date no testis
sex determinant, an SRY Sinclair et al., 1990.
equivalent has been identified in non-mammalian
vertebrates. In these vertebrates the DMRT1 gene
has been recently suggested to have an important
role in maleness due to the evolutionary conservation, being testis-specific and because it has
been found during testis sex differentiation of fish
Marchand et al., 2000. and chickens, as well as in
mammals Raymond et al., 1999; Smith et al.,
1999.. Fish have a certain plasticity during sex
differentiation since several functional sex phenotypes can be generated by diverse mechanisms.
Most of our knowledge in fish has been initially
due to the sex inversion effects of steroid hormone treatments in natural genetic. sex differentiating species. It is possible that at some point,
similar sex differentiating steps may exist between
the pathway followed by the temperature-induced
sex phenotypes to that of genetic or default. sex
differentiating fish. We consider that both hypotheses by Yamamoto 1969. and Bogart 1987.
are being slowly confirmed in fish, since it appears
that gonadal sex differentiation is oriented towards one of the sexes largely dependent on the
levels of endogenous sex steroids, but a change in
this steroid balance can shift the differentiating
pathway.
Administration of androgens to fish larvae at a
stage when gonads are still undifferentiated, can
generate partial or complete masculinization in a
number of fish species Hunter and Donaldson,
1983.. Likewise, functional female phenotypes can
also be induced at this same period with estrogens. Steroid assays performed in the gonads of
natural differentiating tilapia larvae revealed high
levels of 17-estradiol in genetically XX. all
female individuals and low levels found in genetically XY. all male fish Fig. 5.. In these fish, low
levels of 11 ketotestosterone were found in both
genotypes with slightly higher amounts in females
than in males at this same period DCotta et al.,
2001b.. Gonads in these differentiating tilapia
males were in a period of semi-quiescent state
but yet levels of both hormones could be found.
In the case of rainbow trout, elevated levels were

405

Fig. 5. Oestradiol-17 and 11-ketotestosterone concentrations

measured in dissected gonads of genetic all females and all
males sampled between 486 and 702 degreerdays corresponding to 1826 days post-fertilisation . during normal sex
differentiation at 27C. of tilapia Oreochromis niloticus. Analysis was performed on separate pools of gonads 6001100.
from DCotta et al., 2001a,b..

found of androstenedione and testosterone in

whole body mono-sex individuals Fitzpatrick et
al., 1993.. Steroid in vitro metabolism evidenced a
specificity of 11-oxygenated androgens during
tilapia Baroiller, 1988., carp Komen et al., 1995.
and catfish Clarias gariepinus testis differentiation
Van den Hurk et al., 1989.. Because estrogens
are converted from androgens, several studies
have been focussed on the cytochrome P450 aromatase enzyme which catalyses this reaction. Aromatase activity has been shown to be elevated in
the natural sex differentiation of tilapia Guiguen
et al., 1999.. The blocking of estrogen biosynthesis using aromatase inhibitors, results in partial or
complete masculinization associated with a functional testis development in tilapia Guiguen et
al., 1999; Kwon et al., 2000b. as well as in salmonids Piferrer et al., 1994.. Furthermore, strong
aromatase gene expression was revealed during
sex differentiation in genetic female trout
Guiguen et al., 1999. and in female tilapia
DCotta et al., 2001a..
Few studies have been dedicated to the molecular mechanisms of fish sex differentiation and
even less have dealt with ESD. The type of ESD
system involved in these studies is exclusively the
influence of temperature on sex differentiation
and basically concerns two species: the tilapia O.
niloticus; and the Japanese hirame flounder P.
oli aceus. Certain temperatures can induce sex

406

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

inversions with the development of either testes

or ovaries, in a similar manner to hormone treatments. The pathway involved in the natural ovarian differentiation seems to be the activation of
estrogen synthesis. The role of estrogen and
whether this pathway is also implicated in temperature sex differentiation, similar to what has
been demonstrated in TSD reptiles Pieau et al.,
1999., has been addressed in the two thermosensitive fish species mentioned.
In tilapia, functional phenotypic males can be
induced by rearing larvae at 35C during sex
differentiation. In these masculinized XX females, a repression of aromatase gene expression
has been observed Fig. 6., and interestingly a
repression was also seen when genetic males were
treated at 35C DCotta et al., 2001a.. In the
Japanese flounder genetic females are obtained
at normal temperatures 18C. while elevated
temperatures 27C. generate male phenotypes. A
suppression of aromatase was also shown in male
groups, whereas females presented a strong aromatase expression Kitano et al., 1999.. The authors detected only a significant amount of estradiol in females at a late period of differentiation.
Both these studies indicate that temperature acts
either directly repressing the aromatase gene or
indirectly through one or more transcription factors. A putative upstream repressor gene could
be, for instance, SOX9. Treatment of 17-methyltestosterone and the use of fadrozole an inhibitor
of the aromatase enzyme, both caused masculinization of genetic female flounders Kitano et al.,
2000.. In both these sex-inversed cases a suppression of aromatase gene expression was observed.
However, it is unclear if the regulation of the
aromatase gene in these two sex-inversed phenotypes of flounder, is similar to that shown in the
temperaturemale phenotype previously described. Nevertheless, in all these cases it appears
that low levels of aromatase gene expression can
redirect sex differentiation towards testis development.
Another important aspect is whether in temperature-induced masculinization there is an increase in the expression of 11-hydroxylase gene,
which is a key enzyme for the production of
11-oxygenated androgens. In tilapia, no marked
differences were noticed for 11-hydroxylase in
phenotypic males, although unexpectedly temperature appeared to up-regulate the expression
when genetic males were treated to high tempera-

Fig. 6. Relative aromatase gene expression levels analysed by

semi-quantitative PCR of genetic all females a, b, c. and all
males gonads d. dissected between 486 and 702 degreerdays
corresponding to 1622 days post-fertilisation for the 35C
and 1826 days post-fertilisation for the 27C. during normal
and temperature-induced tilapia sex differentiation. Aromatase expression was also analysed from the gonads of
genetic all-male fish treated at 35C considered negative
controls. from DCotta et al., 2001a..

tures DCotta et al., 2001b.. In these temperature masculinized females, an increase in expression was also revealed for a differentially expressed gene still unidentified called MM20C
DCotta et al., 2001b..
An important aspect to consider is the role of
the central nervous system in ESD fish. Recent
data have evidenced that sex differences already
exist in the tilapia brain when the gonads are still
undifferentiated DCotta et al., 2001a.. The key
question remains to be answered: are these
brainrpituitary differences a cause or a consequence of gonadal sex differentiation in the thermosensitive species?

6. Conclusion
It is now clearly shown that sex ratios and sex
differentiation of various freshwater or marine
species, from temperate or tropical habitats can
be influenced by some environmental factors.
Among them, temperature seems to be the main
environmental determinant of sex. The characteristics of thermosensitivity in fish strongly differ
from those observed in reptiles, in particular,
because very few true monosex populations are
induced by the modification of an environmental
factor even at extreme values. This seems to be
mainly associated with strong temperature
genotype interactions. Therefore, at least in sensitive fish species, sex is probably determined by
genetic factors both major and minor factors. by

J.F. Baroiller, H. DCotta r Comparati e Biochemistry and Physiology Part C 130 (2001) 399409

environmental factors and their interactions. In

temperature sex differentiation of fish 1. there
are both up- and down-regulated genes and 2.
there is a similarity in the mechanisms or pathways to that of natural fish sex differentiation
regarding ovarian development, where estrogen
synthesis appears a pre-requisite.
References
Abucay, J.S., Mair, G.C., Skibinsky, D.O.F., Beardmore, J.A., 1999. Environmental sex determination:
the effect of temperature and salinity on sex ratio in
Oreochromis niloticus L. Aquaculture 173, 219234.
Baras, E., Prognon, C., Gohoungo, G., Melard,
C.,

2000. Phenotypic sex differentiation of blue tilapia

under constant and fluctuating thermal regimes and
its adaptive and evolutionary implications. J. Fish
Biol. 57, 210223.
Baroiller, J.F., 1988. Etude correlee
de lapparition des
criteres
de la
` morphologiques de la differenciation

gonade et de ses potentialites

chez
steroidogenes

`
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