Fish Physiology and Biochemistry 28: 113–117, 2003.

© 2004 Kluwer Academic Publishers. Printed in the Netherlands.

113

The role estrogens play in sex differentiation and sex changes of fish

Masaru Nakamura1,2 , Ramji K. Bhandari1 and Mikihiko Higa1

1 Sesoko
Station, Tropical Biosphere Research Center, University of the Ryukyus, 3422 Sesoko, Motoobu, Okinawa
905-0227, Japan; 2 CREST, JST, Shibuyaku, Tokyo, 150-0002, Japan

Key words: aromatase, aromatase inhibitor, bipotency, estrogen, sex change, sex differentiation

Abstract
Using genetically controlled all-female and all-male tilapia (Oreochromis niloticus), the role steroid hormones
play in the sex differentiation was analyzed histologically, ultrastructurally, immunohistichemically and experi-
menntally. The results strongly suggest that endogenous estrogen acts as an ovarian inducer, and that the lack
of steroid hormone including androgen is important for testicular differentiation. Moreover, the roles of steroid
hormones in protogynous sex change of three-spotted wrasse (Halichoeres trimaculatus) and saddleback wrasse
(Tharassoma duperrey) were examined. The results strongly support the hypothesis that the endogenous estrogen
plays an important role in protogynous sex change.

Introduction mones play an important role in protogynous sex

change.
In teleost fish as well as in other vertebrates, gon- In this paper, we will review the overall importance
adal primordia are formed during ontogenesis. Sub- of estrogens in the process of gonadal sex differ-
sequently, they differentiate into either ovaries or entiation in gonochoristic tilapia and sex change in
testes. Morphological differences between ovaries and protogynous wrasse.
testes become apparent during this process. This pro-
cess is so-called gonadal sex differentiation. Recent Differentiation of steroid-producing cells during sex
experiments with genetically controlled all-female and differentiation
all-male tilapia such as Oreochromis niloticus have
The results of experiments with a wide variety of
provided important information about the role endo-
fish species (see Yamamoto 1969; Schreck 1974;
genous sex hormones, especially estrogen, play in
Yamazaki 1983; Hunter and Donaldson 1987; Pan-
sex differentiation. In addition to gonochorism, vari-
dian and Sheela 1995; Peferrer 2001) suggest that, in
ous types of hermaphroditsm are known: protandrous
general, exogenous estrogens and androgens induce
hermaphrodites, protogynous hermaphrodites, and
sex reversal from the genetic gender to the pheno-
multiple-sex-changing fish. These phenomena well
typic gender. The accumulated evidence strongly sug-
show how the sexual characteristics of fish contrast
gests that endogenous sex hormones play an important
with amphibians and even more strikingly with those
role in natural sex differentiation, as hypothesized by
of higher vertebrates. We have studied the role sex
Yamamoto (1969). However, the point in time dur-
hormones play in protogynous sex changes in the
ing the process of differentiation and development of
Hawaiian wrasse Tharassoma duperrey and the three-
gonads at which steroid hormone biosynthesis begins
spotted wrasse Halichoeres trimaculatus using the
is as yet unknown, because at this time the gonads
methods of histology, immunohistochemstry, and mo-
are still too small to detect chemically. In general,
lecular biology (Nakamura et al. 1989; Morrey et al.
sex hormones are produced by two types of special
1998). Our results show that endogenous sex hor-
cells: (1) the steroid-producing cells (SPCs) local-
ized in the thecal layers surrounding oocytes in the
114
ovary, and (2) Leydig cells in the interstices among
cysts of spermatogenic germ cells in the testis. The
ultrastructural characteristics of SPCs include mito-
chondria with tubular cristae, well-developed smooth
endoplasmic reticulum, and free ribosomes (Lofts and
Bern 1972). The differentiation sequence that takes
place in SPCs during the process of gonadal sex dif-
ferentiation was determined ultrastructurally in the
tilapia O. niloticus (Nakamura and Nagahama 1985
and 1989). Recent observation using genetically con-
trolled female and male tilapia showed that SPCs
differentiate in the area around blood vessels in the
undifferentiated gonads in genetic female, but not in
genetic male. This research showed that SPCs ap-
pear initially around the time of sex differentiation.
In addition, it showed that in amago salmon SPCs
differentiate in the ovary at the onset of ovarian dif-
ferentiation (Nakamura and Nagahama 1993). These
ultrastructural observations support the hypothesis that
endogenous steroid hormones play an important role
in sex differentiation.
Expression of steroidogenic enzymes
It is impossible to determine from ultrastructural study
what steroid hormones are produced by SPCs in the
gonads around the time of sex differentiation. Steroid
hormones (estrogens and androgens) are synthesized
by several steroidogenic enzymes from cholesterol in
the gonads. In the past few years, cDNAs encod-
ing some steroidogenic enzymes have been cloned
and their sequences were determined (Nagahama
et al. 1995). Based on these sequences, researchers
synthesized peptides and then generated four spe-
cific antibodies against steroidogenic enzymes (cho-
lesterol side-chain cleavage cytochrome P450, 3β-
hydroxysteroid dehydrogenase, cytochrome P45017α-
hydroxylase/17,20 lyase, and cytochrome P450 aro-
matase) that are essential for the biosynthesis of
all major sex steroid hormones, including estrogen
and androgen (Kobayashi et al. 1996; Morrey et al.
1998). Aromatase is the critical enzyme that must be
present in order to biosynthesize estradiol-17β (E2)
from testosterone. Using these antibodies, differenti-
ating gonads of genetically controlled all-female and
all-male tilapia O. niloticus were stained immunohis-
tochemically. Immunopositive cells against all anti-
bodies appeared first in the undifferentiated gonads
of genetic females. A further increase in the number
of immunopositive cells accompanied ovarian devel-
opment. This evidence suggests that E2 is produced
in the gonads around the time of ovarian differenti-
ation and that this estrogen plays an important role
in ovarian differentiation. In contrast, immunopositive
reactions were not observed in the gonads of undif-
ferentiated and differentiating testes of genetic males.
Weakly positive cells first appeared after testicular dif-
ferentiation, except for aromatase. Stronger positive
reactions were seen in the testes just before the onset
of spermatogenesis. Thus, it is less likely that ster-
oid hormones, including androgen, have significant
function in testicular differentiation.
Effects of aromatase inhibitor and estrogen
antagonist on sex differentiation
To further clarify the role of endogenous estrogen in
sex differentiation, the effects of aromatase inhibitor
(AI, Fadrozole) and estrogen antagonist (Tamoxifen)
on genetically controlled all-female tilapia was ex-
amined. All fish treated with AI at 200 and 500 µg/g
diet from 8 DAH to 22 DAH had well developed testes,
suggesting that AI masculinizes genetic females by in-
hibiting aromatase activity (Nakamura et al. 1999). AI
blocks the conversion from testosterone to E2 in fish
ovaries (Afonso et al. 1999), and it effectively induces
masculinization of genetic females of the chinook sal-
mon Oncorhynchus tshawitisha (Piferrer et al. 1994).
In contrast, when fish were treated with both AI and
E2 they had normal ovaries. Tamoxifen at doses of
1–10 mg/g diets were geven to fry from 8 DAH for
150 days. Although most fish treated with Tamox-
ifen at 1 mg/g diet had ovaries, apparent delay of
development of oocytes and delay of ovarian cavity
formation were observed. All fish treated with Tamox-
ifen at 2 mg/g diet had gonads both with testicular
tissue and ovarian tissue. All fish treated with 5 and
10 mg/g diets were died by the end of treatment. From
these results, it is concluded that estrogen antagon-
ist, Tamoxifen, had potency to induce masculinization
in the gonads of genetic female. Taken together, the
above experiments support the hypothesis that endo-
genous estrogen induces ovarian differentiation, and
that the lack of estrogen biosynthesis during ovarian
differentiation may lead to testicular differentiation.
Effects of androgen on sex differentiation
On the basis of the absence of steroidogenic enzymes
at the time of testicular differentiation, it has been as-
sumed that endogenous androgen does not play an im-
portant role in natural testicular differentiation. Des-
pite this fact, it is well known that exogenous androgen

induces the masculinization of genetic females. To ex-
plain this discrepancy, the effect of methyltestosterone
(MT) on the expression of steroidogenic enzymes dur-
ing sex reversal was examined (Nakamura et al. 1999).
MT at 50 µg/g diet was administered all-female fish,
starting from 8 DAH, for 22 days. All treated fish
were found to be male with normal testes, indicat-
ing sex reversal. During sex reversal, the expression
of steroidogenic enzymes was examined immunohis-
tochemically using antibodies against steroidogenic
enzymes. Immunopositive cells against steroidogenic
anti-bodies including aromatase were not observed in
the gonads (testes) of fish treated with MT during
sex reversal. It may be concluded that the exogen-
ous androgen, MT, directly or indirectly suppresses
the expression of steroidogenic enzymes at the time
of ovarian differentiation. The decrease in estrogen
biosynthesis due to the inhibition of the expression
of steroidogenic enzymes may induce testicular dif-
ferentiation of genetic females. In the experiments
in which fish were treated with AI and MT, appar-
ent differences were observed in the expression of
steroidogenic enzymes during sex reversal. These dif-
ferences suggest different mechanisms by which AI
and MT lead to a similar outcome, that is, sex reversal
of genetic female. In either case, estrogen cannot be
produced in the both treated gonads of genetic fe-
males at the stage of ovarian differentiation; testicular
differentiation occurs instead (Nakamura et al. 1999).
The role of estrogen in the process of sex change in
fish
It is well known that considerable numbers of tele-
ost species change sex from male to female and vice
versa in the adult phase. However, the physiological
mechanisms underlying the sequential hermaphrodit-
ism are still unclear. The saddleback wrasse, Thalas-
soma duperrey, is a protogynous hermaphrodite which
change sex from female to male. During this sex
change, they show complete transformation of gonad
from ovary to testis, in which no testicicular tis-
sues are present in the female ovary (Nakamura et al.
1989). We examined the role sex hormones play in sex
changes of the wrasse with a variety of methods.
In the first method, we immunohistochemically
characterized changes in the amount of expression of
three steroidogenic enzymes during sex change. The
three enzymes were cholesterol side-chain cleavage
cytochrome P450 (SCC), P450 Aromatase (Arom),
and P450 11β-hydroxylase (11β-H). In mature fe-
115
males, arom immunopositive reactions were observed
in the follicle cells around yolky oocytes in developed
ovaries. This positive reaction disappeared in the gon-
ads just after the onset of sex change, indicating sharp
decrease of arom in the follicle cells. Interestingly, the
follicle cells also showed 11β-H immunopositive re-
actions, which again disappeared in the ovary during
the early phase of sex change. In the mid-transitional
stages of sex change, 11β-H positive reactions were
observed in the interstitial cells which increased in
number gradually with progress in sex change. SCC-
immunopositive cells were observed in the gonads at
all stages of sex change.
Changes in the expression of Arom and 11β-H dur-
ing sex change correlated with the change in serum
level of estradiol-17β (E2) and 11-ketotestosterone
(11-KT) (Nakamura et al. 1989). The E2 levels were
high in the female phase, but suddenly dropped sim-
ultaneously with the onset of sex change. It then
maintained low levels during the transitional and male
phases. The 11-KT levels increased gradually from
the mid-transitional stage of sex change, concomit-
antly with the expression of 11β-H. In order to confirm
whether the rapid drop of E2 at the time of onset of sex
change causes sex change or is simply a coincidental
event, we examined the effects of AI on females. AI
treatment resulted in a decrease of serum E2 levels in
females.
Finally, all female fish treated with AI changed sex
to male which had developed testes and produced act-
ive spermatogenic germ cells within 6 weeks, while
fish in the control group showed no changes. E2
compensation in AI treatment suppressed sex change,
indicating that AI induces sex change through estro-
gen depletion (Higa et al. this issue). These results
strongly suggest that the E2 drop at the onset of sex
change is a causative factor for sex change. Moreover,
in the protogynous grouper Epinephelus merra, com-
mencement of sex change was observed when serum
E2 levels were below the threshold levels (Bhandari
et al. 2003). One mg AI implanted into the female
groupers induced complete sex change in two and a
half months, in which mature testes developed with ac-
cumulation of spermatozoa in the seminiferous tubules
(Bhandari et al. 2004).
Bipotency of germ cells and somatic cells after sex
differentiation
In general, it is easy to induce sex reversal by treat-
ing fish with sex hormones around the time of sex
116
differentiation, but it is difficult or impossible to in-
duce sex reversal after sex differentiation. The main
reason for such a loss of capability is thought to be
the loss of bipotency of germ cells and somatic cells
after sex differentiation. In protogynous sex change
of wrasse, testicular structure could not be identified
in female ovaries. Thus, it is not yet known whether
testicular tissue originates from cells with bipotency.
Alternatively, differentiated testicular tissue, which is
difficult to distinguish from ovarian tissue, is present
in the ovary. Thus, it is essential to know the bipotency
of germ cells and somatic cells in order to analyze the
mechanism of sex differentiation and sex change. In an
attempt to clarify the mechanism of sexual plasticity
in fish, the effects of MT and AI on the differentiated
ovary were examined in tilapia. AI at 200 µg/g diet
and MT at 50 µg/g diet were given to all-female, 4–
5 cm fish for two and half months. In fish treated with
MT, we observed ovaries with degenerated oocytes but
with no testicular tissue. In contrast, all fish treated
with AI had hermaphroditic gonads with both ovarian
tissue and testicular tissue. Testicular tissue with sper-
matogenic germ cells at various stages was distributed
around the outer periphery of the ovigerous lamellae.
Moreover, clusters of Leydig cells, which are the site
of androgen production, were seen in the testicular
tissue. Sperm ducts were also differentiated in the tis-
sue near blood vessels and in the wall of the ovarian
cavity. At four months after the end of the treatment,
AI-treated fish showed the nuptial coloration of male
secondary characteristics on the body surface and had
male territorial behavior. They mated with normal
females and produced offspring (Nakamura M., un-
published data). This shows that it is possible to induce
functional sex reversal of the gonads after sex differ-
entiation, and that germ cells and somatic cells in the
ovary after sex differentiation retain bipotency for long
time.
Discussion
On the basis of ultrastructural and immunohistochem-
ical observations, and experiments with AI, Tamox-
ifen, and MT, there is no doubt that estrogen biosyn-
thesis occurs in the undifferentiated gonads of genetic
females and that estrogen acts as an ovarian inducer.
In contrast, the lack of steroid hormones, includ-
ing androgen, plays an important role in testicular
differentiation.
The present study demonstrated that treatment with
an aromatase inhibitor was sufficient to induce func-
tional sex reversal of fish after completion of ovarian
differentiation. This result shows that both germ cells
and somatic cells in developed ovaries still have sexual
bipotency long after the period of sex differentiation.
Artificial sex reversal of gonads after sex differenti-
ation was only possible with AI treatments, but not
MT. These results show that the low levels of estro-
gen, which are less than the threshold value in the
ovary after sex differentiation, induced masculiniza-
tion of germ cells and somatic cells. It was not a direct
androgenic effect on the gonadal differentiation.
There is no visible testicular tissue in the ovary
of the female phase of two protogynous species of
wrasse. Thus, the origin of testicular tissue which
appears during sex change is not yet known. In ex-
periments in which individuals of gonochoristic tilapia
were artificially reversed after ovarian differentiation
by treating them with AI, it was shown that bipoten-
tial germ cells and somatic cells are present in the
ovary after sex differentiation. Low levels of estrogen
resulting from AI treatment stimulated testicular dif-
ferentiation in the ovary of tilapia. Likewise, a rapid
drop of E2 was found at the time of onset of sex
change in the wrasse, and sex change was induced
by AI treatment. These results support the hypothesis
that testicular tissue, which appears at the time of sex
change, originates from germ cells and somatic cells
with bipotency are present in the ovary of the wrasse.
However, the mechanism by which low levels of estro-
gen induce masculinization of germ cells and somatic
cells in the gonad after sex differentiation and during
sex change are still largely unknown.
Acknowledgements
This work was supported in part by CREST, JST
(Japan Science Technology Corporation), the Min-
istry of Education, Culture, Sports, and Technology
(14042103), and the Japan Society for the Projec-
tion of Science (14360114). Special thanks are due
to Drs Y. Nagahama and T. Kobayashi, National In-
stitute for Basic Biology, for providing antibodies for
the immunochemical identification of steroidogenic
enzymes.

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