Fish Physiology and Biochemistry 26: 1329, 2002.

2003 Kluwer Academic Publishers. Printed in the Netherlands.

13

Morphology, endocrinology, and environmental modulation of gonadal sex

differentiation in teleost fishes

Carlos Augusto Strssmann1 & Masaru Nakamura2

1 Department of Aquatic Biosciences, Tokyo University of Fisheries. Konan 4-5-7, Minato, Tokyo 108-8477,
Japan (Phone/Fax: +81-3-5463-0541; E-mail: carlos@tokyo-u-fish.ac.jp); 2 Tropical Biosphere Research Center,
University of the Ryukyus, Sesoko Station. 3422 Sesoko, Motobu, Okinawa 905-0227, Japan

Accepted: October 25, 2002

Key words: environment, fish, sex determination, sex differentiation, steroidogenesis

Abstract
Successful reproduction by an adult depends on the normal ontogenesis of the gonads, a complex process of
cellular and histological differentiation that starts early in life. This process is theoretically predetermined by
genetic factors and includes sensitisation of the bipotential gonads to endogenous endocrine factors prior to, during
and even after commitment to maleness or femaleness. However, young fish are relatively vulnerable to a host of
environmental (physical and chemical) factors that can affect this endogenous endocrine axis, disturbing or even
overriding the putative developmental pathway. This sexually lability can be exploited to our advantage for the
production of monosex fish populations of the most valuable sex for food production or aquarium fish trade. On
the other hand, it represents also a potential path for undesirable influences from endocrine-disrupting chemicals
and climatic factors, particularly environmental temperature. This paper provides a detailed account of the early
histological process of gonadal sex differentiation, with special reference to gonochoristic species, and reviews the
criteria employed to positively identify ovarian and testicular differentiation. It also reviews the development of
endocrine competence and sensitivity of the differentiating gonads to exogenous influences in the context of the
relative stability of genotypic sex determination in various fish species. Sex differentiation in some species seems
to be under strong genetic control and may not require endogenous sex steroid production. Conversely, reliance on
endogenous sex steroids for gonadal differentiation is observed in other species and this phenomenon is apparently
associated with a higher incidence of environment (mainly temperature)-labile sex differentiation.

Introduction effects of raising global temperature and unforeseen

Fish reproduction is central to productive processes
in natural and aquacultural environments. Sexual re-
production, which depends on the presence of males
and females, guarantees not only propagation but also
the mixing together of genetic information from two
individuals. Obviously, successful reproduction by an
individual depends on the normal ontogenesis of the
gonads, a delicate developmental process that starts
early in life. Thus, the critical period of early gonado-
genesis is also likely to hold one key to understand
phenomena of current interest such as reproductive
dysfunctions caused by pesticides and other chemi-
cals with endocrine-disrupting activity as well as the
climatic changes on natural fishery resources (Sumpter
1997; Strssmann and Patio 1999). Further, together
with other modern fish farming practices, such as off-
season induced spawning, prevention and control of
diseases, optimised feeding regimes, stress mitigation,
and artificial selection of superior strains, controlling
the phenotypic sex of farmed fish is potentially one
of the most promising strategies for improving pro-
duction and profitability in aquaculture. Early sexual
maturation or unrestrained reproduction during grow-
out, for instance, diverts energy for gamete production
and spawning behaviour that could otherwise support
faster somatic growth. Moreover, other market traits
such as texture and flavour or, in the case of aquarium
14
fish, external appearance, are often markedly differ-
ent between males and females. In these commercial
situations, and where the introduction of exotic or ge-
netically modified species is considered, monosex or
sterile fish populations may be more profitable and, at
the same time, ecologically safer (Hunter and Donald-
son 1983; Thorgaard 1983; Patio 1997; Pandian et al.
1999; Beardmore et al. 2001; Piferrer 2001; Devlin
and Nagahama 2002; Strssmann et al. 2002).
Several strategies have been developed to control
the phenotypic sex of fish but unfortunately all tech-
nologies currently available for this purpose have ap-
plication, legislation, or simply consumer-acceptance
problems that greatly restrict their practical utilisa-
tion (for details see the recent reviews by Pandian
et al. 1999; Arai 2001; Beardmore et al. 2001; Pi-
ferrer 2001; Devlin and Nagahama 2002; Strssmann
et al. 2002). Treatment of the young with steroid hor-
mones has been the closest to an ideal strategy. Ever
since Yamamoto (1953) first achieved the functional
feminization of medaka Oryzias latipes by treatment
with estrogen, the effects of sex hormones on go-
nadal sex differentiation have been investigated in an
ever-growing number of fish species. Several reviews
have been published on this issue (Yamamoto 1969;
Schreck 1974; Hunter and Donaldson 1983; Yamazaki
1983; Pandian and Sheela 1995; Pandian et al. 1999;
Piferrer 2001) and these indicate that, with few ex-
ceptions, hormones are very effective for routine sex
control in fish. However, even with repeated assur-
ances that hormones can be safely used (Green and
Teichert-Coddington 2000; Piferrer 2001), concern of
consumers over the effects of chemicals, particularly
endocrine disrupters, have accelerated a trend to avoid
the use of chemicals in fish farming regardless of kind
and quantity.
Thus, the development of methods to control the
sex of fish that satisfactorily addresses both production
and consumption issues still poses a great challenge to
scientists. This challenge is compounded by the large
number of extant species and the variability of their
sexuality. For instance, unlike higher vertebrates, in
which gonochorism (having male and female sexes in
separate individuals) and strong genetic channelling
(sex determination) of sexual development are the
rule, fishes include species that reproduce via gono-
choristic, hermaproditic (sequential and even simulta-
neous), and parthenogenetic (unisexual) mechanisms
(Yamamoto 1969). Also, even among gonochorists,
the ultimate fate of the developing gonads is governed
by a delicate balance of genetic and environmen-
tal (physical, chemical, or social) factors, so that
not infrequently the phenotypic (gonadal) sex does
not conform to the genotypic sex (Strssmann and
Patio 1995, 1999; Pandian and Koteeswaran 1999;
Baroiller et al. 1999; Devlin and Nagahama 2002).
Finally, the timing of gonadal development and the
anatomy of the gonads vary greatly even in closely
related species (Hoar 1969; Harder 1975; Patio and
Takashima 1995). In the context of this diversity and
in view of space limitations, the purpose of this re-
view is to present a synopsis of the current knowledge
on the morphological, biochemical and environmental
aspects of the primary sex differentiation of gonocho-
ristic fish. In-depth coverage of topics not included
in the present review (e.g., sex differentiation and sex
reversal of hermaphroditic fish, genetics of sex deter-
mination, examples of genotypic and environmental
sex differentiation, and methods for sex control) can
be found in the reviews by Nakamura et al. (1998),
Baroiller et al. (1999), Piferrer (2001), Devlin and
Nagahama (2002), and Strssmann et al. (2002) (and
other reviews indicated above), to which the reader is
referred.
Anatomical and morphological aspects of gonadal
sex differentiation
Basic gonadal histology and formation of the
indifferent gonads
The gonads are composed of somatic (structural) and
germinal (germ cells) elements. It is generally be-
lieved that the somatic elements of the teleost gonads
derive only from cells of the peritoneal wall and thus
have a monistic origin (Hoar 1969). This is in con-
trast to amphibians and most other vertebrates, where
stromal cells in the developing gonads derive from
two different sources: the peritoneal wall and the
mesonephric or interrenal blastema. This dual origin
forms the basis for the process of sex differentiation
in these taxa known as cortico-medullary antagonism,
under which the predominance of either portion during
development gives rise to an ovary or testis (Witschi
1957). Among fish, only elasmobranchs have been
shown to posses an analogous dualistic structure dur-
ing gonadal sex differentiation (Chieffi 1975). Teleost
fish characteristically lack this dual structure (Fig-
ure 1), a fact confirmed by Nakamura (1978) in a
detailed study of the sex differentiation of 7 species
from 5 families (Mozambique tilapia, Oreochromis

Figure 1. Electron micrograph of the undifferentiated gonad of am-
ago salmon, Oncorhynchus rhodurus just after hatching. Germ cells
(GC) at the gonial stage are enclosed by somatic cells. Note the ab-
sence of a dual structure (cortex and medulla) in the somatic portion.
Bar indicates 5 m.
mossambicus; masu salmon, Oncorhynchus masou;
chum salmon, Oncorhynchus keta; whitespotted char,
Salvelinus leucomaenis; goldfish, Carassius auratus;
medaka, and top minnow, Gambusia affinis). Another
distinguishing developmental feature of the teleost
gonads in relation to that of amphibians and higher
vertebrates occurs in the development of the gonod-
uct system, essential for releasing ovulated eggs and
mature sperm from the gonads during spawning. In
non-teleostean vertebrates, the Mllerian and Wolf-
fian ducts play embryologically important roles in the
formation of the gonoducts in females and males, re-
spectively. In contrast, the gonoduct system of most
teleosts, which includes the ovarian cavity in females
and the efferent ducts in males, is formed by differ-
ent morphogenetic processes, as explained later (see
Figure 2).
The first steps towards the formation of functional
ovaries and testes are the differentiation of the future
15
Figure 2. Light micrograph of the undifferentiated gonads (arrows)
of amago salmon O. rhodurus just after hatching. Note that the
mesonephric ducts (MD), which in non-teleostean vertebrates par-
ticipate in gonoduct formation (Wolffian and Mllerian ducts), are
completely unrelated to the gonads in teleost fish. Bar indicates
25 m.
germinal elements (primordial germ cells) from the
totipotent blastomeres early during embryogenesis,
the formation of the germinal ridge, and the migration
of the primordial germ cells into these ridges. Detailed
histological description of early gonadogenesis was
provided by Patio and Takashima (1995) whereas
the current knowledge on the origin, characteristics,
proliferation and migration of primordial germ cells
was reviewed by Devlin and Nagahama (2002) and
more recently by Yoshizaki et al. (2002, this issue),
and therefore is omitted here. The rudimentary gonads
then undergo a period of slow somatic growth with no
apparent histological differentiation or germinal activ-
ity, which can last for days, months, or even years
depending on the species. This period of relative in-
activity is followed by the appearance of cellular or
histological characteristics that in some species signal
the onset of sex differentiation whereas in others mark
the beginning of an all-female or intersex phase prior
to the actual differentiation into ovaries and testes
(see subsequent sections). Thus, in the former situa-
16
Figure 3. Light micrograph of the ovary of Nile tilapia, Ore-
ochromis niloticus just after ovarian differentiation. Large arrows
show elongation of two somatic ridges for formation of the ovarian
cavity. Note cysts of meiotic prophase germ cells (O) and clusters of
steroid-producing cells (SPC, small arrows) in the vicinity of blood
vessels (BV). Bar indicates 30 m.
tion, which is commonly referred to as differentiated
gonochorism (Yamamoto 1969), ovarian and testicular
differentiation proceeds directly from the undifferen-
tiated gonads even though both events may not occur
simultaneously. By contrast, the latter condition is des-
ignated as undifferentiated gonochorism (Yamamoto
1969), juvenile ambisexuality (Colombo and Grandi
1996) or juvenile hermaphroditism.
Ovarian differentiation
Sex differentiation has been shown to begin first in the
gonads of females than in males in the majority of the
gonochoristic teleosts examined to date. A few species
show simultaneous differentiation of both sexes (e.g.,
Figure 4. Light micrograph of the ovary of goldfish, Carassius
auratus just after ovarian differentiation. Note the newly-formed
ovarian cavity (OC) between ovary on left and peritoneum on right.
Bar indicates 15 m.
as in Mozambique tilapia (Nakamura and Takahashi
1973) and Nile tilapia, Oreochromis niloticus (Naka-
mura and Nagahama, 1985, 1989)) but no case of
male precedence has been reported to date. The be-
ginning of ovarian differentiation can be judged from
characteristic changes in the gonads such as the on-
set of intensive germ cell proliferation (mitosis), their
entry into meiosis, and peculiar arrangements of so-
matic cells that eventually lead to the formation of
the ovarian cavity. For instance, it is known that the
early gonads of putative females have more germ cells
than those of males in medaka (Hyodo et al. 1966;
Tuzuki et al. 1966; Yamamoto 1969; Satoh and Egami
1972; Onitake 1972), stickleback, Gasterosteus ac-
uleatus (Shimizu and Takahashi 1980), rainbow trout,
Oncorhynchus mykiss (Lebrun et al. 1982), pejerrey,

Figure 5. Light micrograph of the ovary of medaka, Oryzias latipes showing the formation of the ovarian cavity (arrows indicate sites of
somatic cell outgrowth). The ovary (O) and the intestine (I) are also indicated. Bar indicates 30 m.
Odontesthes bonariensis (Strssmann et al. 1996a),
and in many other species (see Hunter and Donaldson
1983). This sexual dimorphism in germ cell number is
brought about by the rapid rate of germ cell mitosis in
females, forming characteristic clusters, and is gener-
ally followed shortly thereafter by the onset of meiosis
(Figure 3; see also Patio and Takashima 1995; Strss-
mann et al. 1996a for additional illustrations). While
germ cell mitosis necessarily precedes meiosis, ger-
minal and somatic (onset of formation of the ovarian
cavity) differentiations generally occur simultaneously
in females. Somatic differentiation clearly precedes
germinal differentiation only in a few species (e.g.,
as in cyprinids; Jensen and Shelton 1983) and may
not be recognised at all or only in part of the go-
nads of species that lack a true ovarian cavity (e.g.,
as in eels (Colombo and Grandi 1996) and salmonids
(Nakamura et al. 1974, Nakamura 1982)).
Three distinct processes by which the ovarian cav-
ity is formed have been described in detail. In mottled
sculpin, Cottus bairdii (Hann 1927), Mozambique
tilapia (Nakamura 1978), and pejerrey (Strssmann
et al. 1996a), as well as in many other species, somatic
tissue outgrowths elongate laterally from both the ven-
tral and dorsal edges of the gonads toward the lateral
body wall. The two elongating sheets of somatic tis-
sue develop, one upward and one downward, to form
a groove lateral to the gonad proper (Figure 3). The
outgrowths eventually fuse with each other along their
coincident edges, thus constructing a wide, flat cavity
17
lying along the lateral side of the gonads. In C. auratus
and other cyprinids, putative ovaries become extended
at their ventral edges, where stromal cells increase in
number and spread laterally to the dorsal peritoneal
wall (Nakamura 1978). Concomitantly, small aggre-
gations of cells appear on the peritoneal walls near
the extended edges of the gonads and eventually fuse
with these extensions to form a narrow ovarian cavity
in the region between the gonads and the peritoneal
wall (Figure 4). In O. latipes and G. affinis, the lateral
edges of the fused ovary begin to elongate dorsally
and the tips of the ovarian elongation eventually fuse
with the dorsal coelomic wall (Nakamura 1978). As
a result, an ovarian cavity forms dorsal to the ovary
proper (Figure 5).
Other early signs of sex differentiation that may be
encountered are the relative size of the gonads and of
the blood vessels. For example, the cross section area
of the gonads in presumptive females was larger than
that of males in pejerrey (Strssmann et al. 1996a)
and channel catfish, Ictalurus punctatus (Patio et al.
1996) at the time that other signs of differentiation
(e.g., germ cell mitosis and somatic cell rearrange-
ment) became evident. This increase in gonadal size
in females is consistent with the proliferation of germ
cells and possibly also of somatic cells. Blood vessels
are also larger in putative females of pejerrey at the
time of appearance of somatic cell outgrowths, germ
cell mitosis and meiosis (Strssmann et al. 1996a; Ku-
rita 1999). Nonetheless, caution is needed in that the
18
Figure 6. Light micrograph of the testis of Nile tilapia O. niloticus
just after testicular differentiation. A slit-like structure indicating the
formation of the efferent duct (ED) is visible in the stromal tissue.
Note the germ cells (large arrows), which remain at the gonial stage,
and a steroid-producing cell (small arrow). Bar indicates 20 m.
appearance of any kind of somatic or germinal female
elements in the gonads of all individuals, including the
presumptive males, could be an indication of the un-
differentiated form of gonochorism mentioned above.
In such cases, definitive ovarian differentiation can
be inferred directly from the regression of male ele-
ments (e.g., as in intersex juveniles of the European eel
(Colombo and Grandi, 1996)) or indirectly from the
differentiation of testicular elements in the genotypic
males (see below).
Testicular differentiation
In the undifferentiated type of gonochorists, the go-
nads of genotypic males may develop female charac-
teristics up to the appearance of a rudimentary ovarian
cavity and meiotic oocytes as in zebrafish (Takahashi
1977; Uchida et al. 2002), Sumatra barb Barbus tetra-
zona tetrazona (Takahashi and Shimizu 1983), and
masu salmon (Nakamura 1984). In the European eel,
presumptive males as well as some of the females
temporarily posses an intersex gonad called Syrski or-
gan, that resembles a testis but contains oogonia and
oocytes in addition to spermatogonia (Colombo and
Grandi 1996). In these species, the first sign of com-
mitment to maleness is the regression of the female
elements. In zebrafish, the disappearance of the early
developing oocytes and the ovarian cavity has been
recently shown to occur through apoptosis (Uchida
et al. 2002). The gonad is then readily taken over by
the proliferation of spermatogonia and the subsequent
initiation of spermatogenesis.
Even in differentiated gonochorists, however, the
process of testicular differentiation may not be clearly
recognised until much later than in females because
the germinal and somatic elements in the putative
testes usually remain quiescent for an extended period
after ovarian differentiation (Nakamura 1978). Thus,
in such cases it can be presumed, but not conclu-
sively, that the individuals that did not form an ovary
are destined to become males. In the majority of the
species examined to date the first clear sign of testic-
ular differentiation is the appearance of the anlagen of
the efferent duct. In Mozambique tilapia (Nakamura
and Takahashi 1973) as well as in many other species
(Nakamura 1978; Strssmann et al. 1996a), the effer-
ent duct appears first as a slit-like space in the stromal
tissue (Figure 6). In pejerrey, the appearance of this
anlagen is actually preceded by a distinct maze-like
arrangement and increased eosinophily of the stromal
cells in the area (Strssmann et al. 1996a). The on-
set of spermatogenesis, signalled by intensive germ
cell mitosis and formation of germ cell cysts or lob-
ules in the periphery of the gonad, is another possible
sign of testicular differentiation. This process is also
greatly delayed in relation to the analogous process
in females. As examples, intensive germ cell mito-
sis (followed by meiosis) in females and males were
first observed at 35 and 70 days after hatching, re-
spectively, in Nile tilapia (Nakamura and Nagahama
1985, 1989) and at 49 and 98119 days, respectively,
in pejerrey (Strssmann et al. 1996a). Other reported
signs of testicular differentiation include the early for-
mation of the lobed structure that characterises the
adult testis as in channel catfish (Patio et al. 1996)
and the appearance of blood vessels in the hilar region
of the gonad as in stickleback (Shimizu and Takahashi
1980).
 19

Figure 7. Electron micrograph of part of a steroid-producing cell in the differentiating ovary of Nile tilapia O. niloticus at 23 days after hatching.
Note the presence of a moderate number of mitochondria (M) with tubular cristae, the smooth endoplasmic reticulum, and free ribosomes. The
nucleus (N) is also indicated. Bar indicates 1 m.

Role of endogenous sex hormones during gonadal
sex differentiation
While the morphological processes of gonadal sex dif-
ferentiation are relatively well identified, the genetic
and biochemical bases for sex determination and their
expression into functional gonads are now just begin-
ning to be revealed. The results of experiments with a
wide variety of fish species (see reviews of Yamamoto
1969; Schreck 1974; Hunter and Donaldson 1983; Ya-
mazaki 1983; Pandian and Sheela 1995; Piferrer 2001;
Devlin and Nagahama 2002) indicate that, in general,
administration of exogenous estrogens and androgens
effectively direct (or redirect) gonadal sex differenti-
ation into an ovary or testis, respectively. Thus, the
evidence accumulated in numerous experiments with
medaka led Yamamoto (1969) to suggest that natural,
endogenous sex hormones are the inducers of gonadal
sex differentiation, a notion that was broadly accepted
but not adequately scrutinised over the years. Recent
experiments with a broader pool of species, combined
with analytical advances, indicate that sex steroid hor-
mones may be indeed the ultimate factors for sex
differentiation in some, albeit not all, species (see later
discussion).
20

Evidence in support of Yamamotos (1969) hy-

pothesis was obtained in ultrastructural studies of the
ontogeny of steroid-producing cells in relation to the
time of sex differentiation. Sex steroids are produced
primarily by the follicle cells surrounding the oocytes
in the ovary and the Leydig cells found in the inter-
stices among cysts of spermatogenic germ cells in the
testis of adult teleosts (Nagahama et al. 1982). Naka-
mura and Nagahama (1985, 1989) showed that the pre-
cursors of these cells, which are ultrastructurally char-
acterised by their well-developed smooth endoplasmic
reticulum, mitochondria with tubular cristae, and free
ribosomes (Lofts and Bern 1972), appeared initially
before or around the time of sex differentiation in
O. niloticus (Figure 7). Similar results were obtained
in other species such as amago salmon, Oncorhynchus
rhodurus (Nakamura and Nagahama 1993), rainbow
trout (Van den Hurk et al. 1982), and sterlet sturgeon,
Acipenser ruthenus (Fedorov et al. 1990). Further evi-
dence of steroid biosynthesis during sex differentiation
was provided by immunohistochemical analysis of the
gonads using antibodies developed against some of the
enzymes (cholesterol side-chain cleavage cytochrome
P450, 3-hydroxysteroid dehydrogenase, cytochrome
P450 17-hydroxylase/17,20 lyase, and cytochrome
P450 aromatase) that are essential for the synthesis of
the major sex steroids (Nagahama 2000). Aromatase, Figure 8. Light micrograph of the undifferentiated gonad of a
in particular, is the critical enzyme for the biosynthesis genotypic female Nile tilapia O. niloticus showing aromatase im-
munopositive cells (arrows). Note presence of germ cells (G). Bar
of estradiol-17 from testosterone. This experimental indicates 15 m.
approach revealed that only the gonads of genotypic
females had immunopositive cells for all antibodies
before sex differentiation in O. niloticus (Figures 8 Thus, the combined results of ultrastructural, immuno-
and 9) (Nakamura et al. 2000). Molecular biological histochemical and pharmacological studies suggest
studies also showed that aromatase gene expression that the presence of endogenous estrogens in these
was much higher in differentiating ovaries than in species is necessary for ovarian differentiation, and
testes of Japanese flounder, Paralichthys olivaceus therefore support Yamamotos (1969) hypothesis at
(Kitano et al. 1999), rainbow trout (Guiguen et al. least concerning the role of estrogens.
1999), and pejerrey (Karube 2001; Strssmann et al. Conversely, there is also evidence from ultrastruc-
2002). Finally, pharmacological inhibition of aro- tural, histochemical and pharmacological studies of
matase activity by treatment with specific inhibitors other species that does not support the hypothesis
(Fadrozole or 1,4,6-androstratriene-3-17-dione) prior of sex steroid hormones, in particular of estrogens,
to and during the expected period of sex differentia- as physiological inducers of gonadal sex differenti-
tion caused masculinisation of genotypic females in ation. For example, steroid-producing cells or signs
Nile tilapia (Figure 10) (Guiguen et al. 1999; Kwon of steroidogenic enzyme activity were found in the
et al. 2000; Nakamura et al. 2000; Afonso et al. 2001), gonads only after histological sex differentiation in
chinook salmon Oncorhynchus tshawytscha (Pifferer the medaka (Satoh 1974; Kanamori et al. 1985),
et al. 1994), rainbow trout (Guiguen et al. 1999), and guppy, Poecilia reticulata (Takahashi and Iwasaki
the Japanese flounder (Kitano et al. 2000). Inciden- 1973), black molly, Mollienesia latipinna (Van den
tally, genotypic females of Nile tilapia had normal Hurk 1974), and platyfish, Xiphophorus maculatus
ovaries when they were treated simultaneously with (Schreibman et al. 1982). The lack of steroidogenic
Fadrozole and estradiol-17 (Nakamura et al. 2000). cells prior to sex differentiation in medaka, which

suggests that endogenous steroids are not required for
initial gonadal sex differentiation, was substantiated
by the observation that aromatase inhibition by Fadro-
zole did not impair ovarian formation in this species
(Kawahara and Yamashita 2000).
Studies of the role of androgens in testicular for-
mation seem to have yielded equally inconsistent ob-
servations among different species. In rainbow trout,
for instance, gene expression of 11-hydroxylase, an
enzyme necessary for the synthesis of the potent an-
drogen 11-ketotestosterone, was found prior to sex
differentiation only in genotypic males, reinforcing an
earlier view that this androgen might be implicated in
testicular formation (Guiguen et al. 2000; Liu et al.
2000). In an immunohistochemical study with Nile
tilapia, on the other hand, genotypic males showed
weakly immunopositive cells for three steroidogenic
enzymes only after somatic differentiation of the testis
has taken place and aromatase-positive cells, in par-
ticular, could not be identified even after the onset of
spermatogenesis (Figure 9) (Nakamura et al. 2000).
Hence, in Nile tilapia, the absence of endogenous es-
trogens, rather than the presence of androgens, seems
to correlate with testicular differentiation. Further, the
recent discovery of DMY in medaka (Matsuda et al.
2002) seems to lend credit to the notion that endoge-
nous androgens are not involved in testicular differ-
entiation in this species. DMY is a testis-determining
gene considered to be the equivalent of the mammalian
SRY gene, whose expression in mammals triggers tes-
ticular differentiation prior to the production of steroid
hormones. Although preliminary, these findings with
medaka are particularly exciting as they suggest the
possibility of the presence in fish of a mode of pri-
mary gonadal sex differentiation which is inherently
similar to that in mammals; namely, where gonadal
development proceeds on a female direction (default)
unless diverted to a male pathway by the expression
of specific testis-determining gene(s). In such cases,
as in mammals, steroids are though to play no part
in deciding the initial direction of development but
rather are responsible for shaping sexually dimorphic
structures during subsequent sexual development. It
remains to be seen if DMY (or DMRT1 in rainbow
trout; Marchand et al. 2000) is also responsible for
testicular formation in other species including those
like Nile tilapia, in which testicular differentiation oc-
curs after ovarian differentiation and in the apparent
absence of endogenous steroid production.
The preceding observations, in conjunction with
the fact that exogenous sex hormones are unable to
21
induce sex reversal in some species (see Hunter and
Donaldson 1983; Piferrer 2001), suggest the pres-
ence of a varying degree of steroid involvement in
primary gonadal sex differentiation (not to confound
with the importance of steroids for the maintenance
of post-differentiation gonadogenesis) in teleost fish.
Thus, in some species sex differentiation seems to
require the endogenous production of both estrogens
and androgens for the formation of ovaries and testes,
respectively (e.g., rainbow trout) whereas in others,
one sex (e.g., Nile tilapia males), if not both (e.g.,
medaka), has a developmental mechanism which does
not depend on steroid hormones as the initial trigger of
primary (gonadal) sex differentiation. In this context,
much research needs to be conducted to clarify the true
extent of steroid dependence (or independence) in the
primary sex differentiation of teleost species. Because
the teleost gonads retain their sexual bipotentiality into
adulthood and because exogenous hormones can dis-
rupt natural biochemical processes in the gonads and
override normal developmental patterns to the extent
of causing sex reversal even after gonadal differen-
tiation has taken place (Devlin and Nagahama 2002;
and below), future research should avoid the use of
exogenous hormone administration as primary exper-
imental tool. It will be also important to keep in mind
the possibility of extragonadal sources of steroid hor-
mones and their role during sex differentiation (e.g.,
maternally inherited (Rothbard et al. 1987; Feist et al.
1990) and interrenal synthesis (Fitzpatrick et al. 1993;
Govoroun et al. 2001b)).
Environmental modulation of gonadal
steroidogenesis and sex differentiation
As discussed in the preceding section, exogenous
steroid hormones can affect gonadogenesis in fish re-
gardless of the level of dependence on endogenous
steroids for natural gonadal sex differentiation. In
most species, if administered at the right time and
dosage during early gonadal development, exoge-
nous sex steroids induce functional sex reversal of
the gonads that in adults will be no different from
gonads differentiated naturally (Yamamoto 1969; Pi-
ferrer 2001). The mechanisms of action of exogenous
steroids during sex differentiation are not sufficiently
clear, but it is assumed that they bind to specific recep-
tors and induce sexually dimorphic gene transcription
and/or expression. Interestingly, estrogen and andro-
gen receptors are present in both ovaries and testes
22
Figure 9. Schematic representation of the temporal changes in the expression of steroidogenic enzymes and production of estrogen and
androgen during sex differentiation and early gametogenesis in males and females of Nile tilapia O. niloticus. P450 scc: cholesterol side-chain
cleavage cytochrome P450; 3-HSD: 3-hydroxysteroid dehydrogenase; P450 c17: cytochrome P450 17-hydroxylase/17,20 lyase; P450
arom: cytochrome P450 aromatase.
Figure 10. Light micrograph of the sex-reversed testis of a geno-
typic female Nile tilapia O. niloticus treated with aromatase in-
hibitor. Spermatogenesis has progressed normally, indicating nor-
mal testicular differentiation. Bar indicates 25 m.
and they can be induced by the presence of estrogens
and androgens, respectively (reviewed by Devlin and
Nagahama 2002), thereby providing the basis for the
effects of exogenous steroids in both sexes. Steroid
hormones also exert a great influence on subsequent
steroidogenesis. For instance, administration of es-
trogens during gonadal differentiation stimulated and
inhibited production of estrogenic and androgenic en-
zymes, respectively (Chang et al. 1999; Scholz and
Gutzeit 2000; Govoroun et al. 2001b) whereas admin-
istration of androgens suppressed aromatase activity
(Chang et al. 1999; Kitano et al. 2000; Nakamura et al.
2000; Govoroun et al. 2001a). These results point to
the presence in the developing gonads of a feedback
mechanism of estrogen and androgen production by
which the timely presence of even minimal amounts of
steroids (or steroid-like substances) could effectively
direct subsequent steroidogenesis. Such a mechanism
might explain the high sensitivity of the differentiating
fish gonads to exogenous steroids, xenoestrogens and
androgens, and perhaps maternally inherited steroids.
In species whose sex differentiation depends on the
endogenous production of steroid hormones, it might
be important also to ensure that the entire differentiat-
ing gonad responds uniformly to the very first stimulus
(see further discussion below).
Figure 11. Graphical representation of the reviewed information on the effects of various factors on the process of sex differentiation in teleost fishes. The representation is a compilation of
literature information for various species; it is not intended to represent any species in particular and is not all-inclusive. Little is know on the mode of action of genetic factors, which together
with other hypothetical factors are indicated with question marks. Abbreviations: T, testosterone; 11KT, 11-Ketotestosterone.
23
24
There are also a number of physical, chemical and
social environmental factors that can affect sex differ-
entiation in fishes (for reviews see Korpelainen 1990;
Francis 1992; Strssmann and Patio 1995, 1999;
Pandian and Koteeswaran 1999; Baroiller et al. 1999;
Baroiller and DCotta 2001), although the feminizing
or masculinizing potencies of these factors typically
are less than those of exogenous steroids. Tempera-
ture seems to be a particularly prominent determinant
of sex in fishes. This phenomenon has been termed
temperature-dependent sex determination, or TSD. As
in reptiles (Crews 1996; Pieau 1996) and amphib-
ians (Hayes 1998), it has been shown that temperature
affects early steroidogenesis in fish gonads through
the modulation of aromatase gene expression. For in-
stance, high temperatures impaired aromatase activity
and caused masculinisation of genotypic females in
Japanese flounder (Kitano et al. 1999) and Nile tilapia
(DCotta et al. 2000; Kwon et al. 2000). Interestingly,
Kwon et al. (2000) also described the paradoxical
feminization of genotypic males (YY genotype) of
Nile tilapia by high temperature and its prevention
by simultaneous Fadrozole-induced aromatase inhibi-
tion (a result which is difficult to reconcile with the
effects observed in genotypic females of the same
species). Nevertheless, several other species or strains
also show more females at high temperatures and/or
more males at low temperatures (reviewed by Strss-
mann and Patio 1995, 1999). In addition, preliminary
results for pejerrey, a species with truly remarkable
thermolabile sex differentiation (see below) indicate
that, while aromatase gene expression was sexually
and thermally dimorphic, it was not temporally con-
sistent with a possible role of this enzyme in the sex
determination of this species (Karube 2001; Strss-
mann et al. 2002). Thus, it is highly probable that, in
addition to gonadal aromatase, temperature is differ-
entially affecting the expression of other genes related
to sex differentiation and/or steroidogenesis such as
those for steroid receptors, other steroidogenic en-
zymes, and non-steroid hormones in the gonads as
well as perhaps in the brain and pituitary (see below)
(Crews 1996; Baroiller et al. 1999; DCotta et al.
2001; Trant et al. 2001).
Considering the effects of temperature on gonadal
steroidogenesis, it could be argued that species that
rely on endogenous sex steroid production for primary
sex differentiation are conceivably at greater risk of
environmental disturbances of this process than those
that do not. Interestingly, the species in which gonadal
steroidogenesis was detected or presumed to occur be-
fore sex differentiation belong to genera and families
(Atherinidae, Salmonidae, Cichlidae, Paralichthyidae
and Acipenseridae, with the possible inclusion of Mo-
ronidae (European sea bass, Dicentrarchus labrax;
Zanuy et al. 2001)) with abundant examples of envi-
ronmentally labile sex determination, particularly as
a function of ambient temperature (see reviews by
Strssmann and Patio 1995, 1999; Pandian and Ko-
teeswaran 1999; Baroiller and DCotta 2001; Akhun-
dov 2000). Conversely, the families of species in
which steroidogenesis could not be detected prior to
sex differentiation (Adrianichthyidae and Poecilidae)
seem to have strong genotypic sex determination (see
reviews by Kallman 1984; Devlin and Nagahama
2002). Occasional claims of environmental sex deter-
mination in poeciliids (see reviews above), which are
viviparous species and whose sex is determined during
gestation, need to be further scrutinised to rule out an
indirect effect of temperature on the mothers ovarian
steroid hormone profile. Thus, it is tempting to classify
teleost species according to the relative importance of
endogenous steroid production for sex differentiation
and to inversely correlate this importance with the de-
gree of stability of sex determination. As stated earlier,
however, a more systematic research approach is re-
quired before generalizations about the involvement
of endogenous steroids can be made. This approach
should include a wider range of species, the exami-
nation of sources of endogenous steroids other than
the gonads, and the ability to discriminate between the
effects of environmental variables on steroidogenesis
and primary sex differentiation versus those affecting
post-differentiation gonadal development.
One intriguing aspect of the plasticity of sex in
teleosts in relation to environmental factors is that
sexually thermolabile species do not seem to present
abnormal incidences of intersex individuals at inter-
mediate temperatures (i.e., temperatures that allow
the formation of both sexes). For example, in pejer-
rey, the proportion of females changes gradually from
100% at 1519 C to 0% at 29 C, and at 2425 C
there is an equal chance of becoming a male or a fe-
male (Strssmann et al. 1996b, 1997). Regardless of
the rearing conditions, however, pejerrey intersexes
are exceptionally rare (less than 1:1,000) (Strssmann
et al. 1996a). This observation suggests that environ-
mentally labile species such as pejerrey must have
mechanisms to coordinate the direction of sex differ-
entiation throughout the entire length of the gonad
(as well as in both gonads). One such mechanism
could be the feedback system of steroid production

discussed earlier where estrogens or androgens respec-
tively induce their increased production as well as that
of their specific receptors. However, to be effective,
this system would still require temporal and spatial
coordination perhaps in the form of a propagating
wave of sex differentiation. In fact, a gradient of
differentiation is apparent in pejerrey where the first
signs of histological sex differentiation are observed
more frequently in the anterior portion of the left lobe
of the gonads (Kurita 1999). An alternative mecha-
nism to guarantee conformity of sex differentiation
along the entire length of both gonads could involve
the brain and pituitary. This possibility is particularly
plausible in species with environmentally labile sex
determination that respond to social cues, as the brain
is the likely site of recognition of social events (Fran-
cis 1992). Studies in several species have shown that
the ontogeny of hormones known to regulate gonadal
function and steroidogenesis (mainly gonadotropin
releasing hormone and gonadotropins) actually pre-
ceded gonadal sex differentiation (reviewed in Parhar
1997; Strssmann et al. 2002; Devlin and Nagahama
2002). Recently, Miranda et al. (2001) showed by
immunocytochemistry the occurrence of temperature-
dependent peaks of gonadotropins I and II (analogous
to mammalian FSH and LH, respectively) in pejerrey
pituitaries concomitant with the temperature-sensitive
period of sex determination. Thus, while the levels
of steroids in the gonads may be ultimately the di-
rect factors responsible for gonadal sex differentiation
(at least in thermolabile and some other species), the
regulation of steroidogenesis in environmentally la-
bile sex determination systems may not necessarily be
based in the gonads.
Concluding remarks
The developmental patterns leading to the expres-
sion of gonadal sex in fish are tremendously diverse,
suggesting the presence of an equally wide array of
differentiation mechanisms. The relatively long pe-
riod of time that separates commitment to maleness
(generally the time of ovarian differentiation) and ac-
tual testicular differentiation in many species suggests
that the two are different developmental events con-
trolled by different mechanisms, as pointed out by
Patio et al. (1996). Also, the fact that germinal mi-
tosis (and meiosis in females but not in males) and
somatic differentiation (formation of the ovarian cav-
ity in females and efferent duct in males) generally
25
occur in close temporal proximity may indicate that
both are under control by the same gene(s) or that
they depend on the interaction between germ and so-
matic cells. However, like in mammals, ovarian and
testicular formation in medaka seem to occur indepen-
dent of germ cell-somatic cell interactions (Shinomiya
et al. 2001). Interestingly, high temperature-induced
male sex determination in pejerrey is preceded by the
degeneration and partial loss of some germ cells, a
phenomenon which is not observed at female-forming
temperatures until after sex differentiation (Ito et al.
2002). The mechanism of germ cell death in this case
is unknown but could be apoptosis, as observed in
differentiating gonads of zebrafish (Strssmann et al.
2002; Uchida et al. 2002). Thus, further studies should
examine the role of apoptosis in gonadal sex dif-
ferentiation, particularly in species that change sex
according to the environmental conditions, and de-
termine if germ cell-somatic cell interactions affect
endogenous gonadal steroidogenesis.
The brain-pituitary axis interacts with the gonads
in adults and, considering that it is active during the
period of sex differentiation, there is no reason to rule
out a priori an involvement of hypothalamic and hy-
pophysial hormones such as gonadotropin releasing
hormone and gonadotropins, among others, on sex
differentiation. Even by limiting the search for mech-
anisms of sex differentiation to steroidogenesis and
the gonads, however, still there are numerous mecha-
nisms by which genetic and environmental factors can
affect the process and outcome of sex differentiation.
A graphical summary of the information presented in
this review is provided in Figure 11 as an attempt to
visualise the diversity of such possible turning points
during sex differentiation. As our understanding of the
mechanisms of sex differentiation and the effects of
environmental factors on these processes increase, we
may be able to develop methods of sex control that are
less dependent on the use of chemicals. For instance,
it was shown that temperature and exogenous steroids
share a common mechanism of action in that both af-
fect aromatase activity and hence steroidogenesis. Not
surprisingly, there seems to be a synergism between
both during endocrine manipulation of sex differentia-
tion (reviewed by Strssmann and Patio 1995; Strss-
mann et al., unpublished observations). Manipulation
of the rearing temperature or other environmental fac-
tors is therefore a potential means to reduce, if not
avoid, the use of chemicals for sex control in aqua-
culture (Strssmann et al. 2002). Information on the
degree of environmental influences on sex differenti-
26
ation is also relevant for fisheries biologists working
to predict the effects of climatic phenomena and pol-
lution on the reproduction and demography of natural
fish populations.
Acknowledgements
The authors would like to express their sincere grat-
itude to J. Michael Redding and an anonymous re-
viewer for their constructive comments and helpful
suggestions to improve this paper. This work was
supported in part by grants-in-aid from the Ministry
of Education, Culture, Sports, Science and Technol-
ogy (#12460085 to CAS and #14042103 to MN),
Tokyo University of Fisheries (CAS), the Japan Soci-
ety for the Promotion of Science (#14360114 to MN),
and CREST-JST (Japan Science Technology Corpora-
tion) Grant-in-Aid for Research for the Future (JSPS#
RFTF96L0041).
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