role of sex hormones in sex change of grouper

Special Issue, pp. 23-27 (2007)
Masaru Nakamura*,**, Mohammad Ashraful Alam*, Yasuhisa Kobayashi*,
Ramji Kumar Bhandari***
Key words: sex steroids, sex change, E. merra, protogynous grouper.
Groupers are commercially important coral reef fishes because
of their excellent flavor and high price. Due to their sex changing
nature, commercial aquaculture operations have not yet been developed for groupers. Sex steroids play a critical role in the initiation
of sex change, but their exact function and mechanism of action are
unknown. We have investigated the role of sex steroids in
protogynous sex change using the honeycomb grouper, Epinephelus
merra, for a number of years. Our studies have shown that sex
steroids play a major role in sex change. In this review, we integrate
our findings to better understand the mechanism of sex change in
Groupers of the genus Epinephelus are widely
distributed throughout tropical and subtropical waters,
and are very important aquaculture species. Most groupers are protogynous hermaphrodites, which change sex
from female to male depending on their size and/or age
[4], or on the basis of their complex social structure
[12]. The process of sex change occurs in a number of
ways, although it is always initiated and regulated by
gonadal steroids [10]. For over 30 years, biologists
have hypothesized about the involvement of steroid
hormones in sex change [10], but the endocrine mechanisms of sex change are not clearly understood.
Our research focuses on analyzing the mechanism
of protogynous sex change in groupers from an endocrinological viewpoint using morphological, physiological,
and molecular techniques. We use the honeycomb
Author for Correspondence: M. Nakamura.
E-mail: [email protected]
*Sesoko Station, Tropical Biosphere Research Center, University of the
Ryukyus, Sesoko 3422, Motobu, Okinawa 905-0227, Japan.
**Solution Oriented Research for Science and Technology (SORST), Japan
Science and Technology Corporation, Saitama 32-0012, Japan.
*** Laboratory of Reproductive Biology, National Institute for Basic Biology,
Nishigonaka, Myodaiji, Okazaki, Aichi, Japan.
grouper, E. merra, as a model organism for protogynous
sex change studies, because it is one of the smallest
grouper species and is readily obtainable in the wild.
Given our studies of the endocrine control of sex change
in E. merra, the aim of this review is to describe the role
of gonadal steroids and their sites of synthesis during
the process of sex change. Additionally, we will focus
on the manipulation of endocrine-controlling mechanisms to develop a method to initiate sex change of
grouper in captivity.
1. Changes of serum sex hormones during sex change
We investigated changes in the gonadal structures
and levels of serum sex steroid hormones during female- to-male sex change. On the basis of histological
changes, entire process of sex change was assigned into
four developmental phases: female, early transition (ET),
late transition (LT), and male phase (Figure 1). At the
female phase, the oocytes of several developmental
stages were observed including gonial germ cells in the
periphery of ovigerous lamellae (Figure 1a). At the
beginning of ET phase, perinucleolar and previtellogenic
oocytes began degenerating, followed by proliferation
of spermatogonia toward the center of lamella (Figure
1b). The LT phase was characterized by further degeneration of oocytes and rapid proliferation of spermatogenic germ cell throughout the gonad (Figure 1c). At
the male phase, no ovarian cells were observed and
testis had germ cells undergoing active spermatogenesis (Figure 1d). Serum levels of estradiol-17β (E2)
were high in females in the breeding season, but low
in the non-breeding female, transitional and male
phase and those of 11-ketotestosterone (11-KT) and
testosterone (T) were low in females and gradually
increased in the transitional and male phase. The
present results suggest that low serum E2 levels and
degeneration of oocytes accompanied by concomitant increase in the 11-KT levels and proliferation of
spermatogenic germ cells are probably the events
mediating protogynous sex change in E. merra (Figure
2) [1, 9].
Special Issue (2007)
Fig. 1. Histological observation of the gonads during natural sex
change in honeycomb grouper. (a) Female phase; (b) Early
transitional phase; (c) Late transitional phase, and (d) Male
Note: PO: primary oocyte; PVO: pre-vitellogenic oocyte; DO: degenerating oocyte; SG: sper-matogonia; SC: spermatocyte; ST:
Theca cells
2. The side of androgen production in the gonads
In order to clarify a role of androgen play in sex
change, we examined the dynamics of steroidogenic
enzymes immunolocalization, viz cholesterol side-chain
cleavage (450scc), biomarker of steroids and cytochrome
P45011β-hydroxylase (P45011β), downstream to 11KT production, throughout the process of sex change in
grouper. In female, P450scc immunoreactivity was
observed in the thecal layer and tunica near blood
vessels (BV) (Figure 3a, b). During the onset of sex
change, P450scc reactive cells were observed in the
remaining follicle layer of degenerated oocyte of the
ovo-testis and tunica in early transitional (ET) and late
transitional (LT) gonads. In male, P450scc was localized in the interstitial Leydig cells of testis. P45011β
reactive cells were observed in the tunica near blood
vessels in female but not in the theca layer (Figure 3c,
d). In ET and LT phases gonads, P45011β appeared in
remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against
P45011β. Moreover, in vivo and in vitro levels of 11KT related with the changes in the nuclei diameter of
P45011β-positive cells in the both tunica near BV and
remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present
results suggest that 11-KT produced in the tunica near
Fig. 2. Changes in plasma profile of E2 and 11-KT during the process
of sex change.
Note: 1. Data are shown as the mean and SEM. Values not sharing a
letter are significantly different by Tukey-Kramer HSD test (P
< 0.05).
2. ND: non-detectable; ET: Early transitional; LT: Late
Tunica ovary
Tunica ovary
Fig. 3. Immunohistochemistry of P450scc and P45011β in gonads of
E. merra. P450scc immunoreactivity in tunica and theca cells
(a, b). P45011β like immunoreactivity only in tunica not in
theca cells (c, d). Arrowhead indicates positive reaction in
theca cells (b). Scale bar 10 µm.
M. Nakamura et al.: Role of Sex Hormones in Sex Change of Grouper
BV may provide the stimulus for female to degenerate
oocyte and initiate sex change. Moreover, 11-KT
produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly play critical role during testicular differentiation as well as
gonadal restructuring at mid to late phases of sex
change [1, 3].
3. Artificial sex change by the treatment of 11-ketotestosterone
To elucidate further the role of an androgen in sex
change, we attempted to induce female to male sex
change by exogenous 11-KT treatments. The 75days
11-KT treatment caused 100 % masculinization of
spawning females. Ovaries of the control fish had
oocytes at various stages of oogenesis, while the gonads of the 11-KT treated fish had transformed into
testes. In the sex-changed fish, plasma levels of E2
were significantly low, while both testosterone and
11-KT were significantly increased. Our results suggest that 11-KT plays an important role in sex change
in the honeycomb grouper. Whether the mechanism of
11-KT induced female to male sex change acts through
direct stimulation of spermatogenesis in the ovary or
via the inhibition of estrogen synthesis remains to be
clarified [5].
5. Quickest sex change by aromatase inhibitor in the
breeding season
Artificial sex change was induced within two full
moons by aromatase inhibitor during the breeding season to establish the quickest method of sex change and
natural spawning of the honeycomb grouper. The sex
change from female-to-male occurred during the time
between the two full moons (3 weeks) following AI
implantation in females just after spawning, and the
efferent ducts of sex-changed males were filled with
sperm (Figure 4b). In contrast all fish in control group
had ovaries with matured oocytes (Figure 4a). To
4. Role of estrogen in sex change
Circulating E2 levels decrease precipitously during female-to-male sex change. Whether this drop in E2
is a cause or consequence of sex change is still largely
unknown. We treated adult female with aromatase
inhibitor (AI, fadrozole), either alone or in combination
with E2, to investigate the role of estrogen in protogynous
sex change. Control fish had ovaries undergoing active
vitellogenesis; the gonads of AI-treated fish had already
developed into testes, which produced sperm capable of
fertilization. In contrast, co-treatment of fish with E2
completely blocked AI-induced sex reversal. AI treatment significantly reduced circulating levels of E2,
whereas the addition of E2 to AI prevented the loss. The
plasma androgen (T and 11-KT) levels were increased
in the AI-treated fish, while the levels in the E2 supplemented fish were low compared to controls. Present
results show that E2 plays an important role in maintaining female sex of hermaphrodite fishes, and that the
inhibition of E2 synthesis causes oocyte degeneration
leading to testicular differentiation in the ovary. A
significant drop in endogenous E2 levels alone triggers
female to male sex change, and the subsequent elevation
of 11-KT levels correlates with progression of spermatogenesis [6, 7, 8, 11].
Fig. 4. The characteristics of gonadal histology in the sex change of
E. merra by implantation with AI. (a) an ovary of control female
mostly consisted with pre-vitellogenic oocyte (PVO) and
vitellogenic oocyte (VO) and; (b) a testis of sex changed male
filled with spermatozoa. Scale bar 50 µm.
Special Issue (2007)
titled “The Comprehensive Analyses on Biodiversity in
Coral Reef and Island Ecosystems in Asian and Pacific
Regions” of the University of the Ryukyus, Okinawa,
Japan, These studies were also supported by research
project entitled “Utilizing advanced technologies in
agriculture, forestry and fisheries”, Japan and a Japanese Government (Monbukagaku-sho) Scholarship.
Fig. 5. Embronic and larval development of E. merra naturally spawned
in the laboratory condition. (a) embryo after 7 hrs of fertilization;
(b) hatched larvae (1day after fertilization); (c) 2-day-old
larvae; (d) 6-day-old larvae.
examine sperm fertility, sex-changed males were mated
with natural, normal females and produced fertilized
eggs (Figure 5a). Most of the hatched larvae grew
normally without any morphological deformities (Figure
5b, c, d). Therefore, the use of this method which is the
quickest known sex change method using AI, may contribute to quality sperm production for grouper aquaculture [2].
The role of sex steroids in sex change is important
both for commercial aquaculture development and for
understanding the endocrine mechanisms of sex change.
In E. merra, E2 and 11-KT are the predominant steroids
regulating sex change. According to our current
understanding, two possible manipulations are doable
to initiate female-to-male sex change in protogynous
grouper. One is the direct suppression of E2 production
by blocking aromatase, thereby creating a suitable environment for the proliferation of gonial germ cells into
spermatogonia, followed by spermatogenesis. Additionally, the use of exogenous 11-KT can directly control oocyte degeneration or increase plasma androgen
levels, allowing male germ cell proliferation, spermatogenesis, and sex change.
These researches were supported by the Takeda
Science Foundation and 21st Century COE project en-
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Grouper, Epinephelus merra,” Molecular Reproduction
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K., and Nakamura, M., “Induction of Sex Change Within
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M. Nakamura et al.: Role of Sex Hormones in Sex Change of Grouper
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