Vol.3, No.3, 227-233 (2011) Natural Science
Copyright © 2011 SciRes. OPEN ACCESS
Induction of testis-ova in nile tilapia (Oreochromis
niloticus) exposed to 17-estradiol
Piya Kosai1, Wannee Jiraungkoorskul1*, Chaivira Sachamahithinant2,
Kanitta Jiraungkoorskul3
1Department of Pathobiology, Faculty of Science, Mahidol University, Bangkok, Thailand;
*Corresponding Author: tewjr@mahidol.ac.th
2Mahidol University International College, Mahidol University, Salaya Campus, Nakhonpathom, Thailand
3Department of Occupational Health and Safety, Faculty of Public Health, Mahidol University, Bangkok, Thailand
Received 12 November 2010; revised 27 December 2010; accepted 28 December 2010.
The efficacy of Endocrine Disrupting Com-
pounds (EDCs), 17β-estradiol was tested on the
fish Oreochromis niloticus in order to under-
stand the intersex relationship of fish, in which
sequential hermaphrodism can consist of a
male changing into a female (protandry) or a
female changing into a male (protogyny). The
fish were equally divided into 3 groups. The first
group was the control group; the second and
third groups were treated with 10 and 100 g L-1
of 17β-estradiol, respectively, for 30 days. The
overall result in this experiment had no signifi-
cant effect on the growth parameters. Among
the two treated groups, the low concentration
group shows results similar to those of the
control groups. The high concentration group
shows changes to the male reproductive sys-
tem with the appearance of the testis-ova pre-
sent resulting in an intersex condition of the
male gonads. With this experiment, it can be
concluded that 17β-estradiol at high concentra-
tion reveals positive changes towards the male
reproductive system of the fish, Oreochromis
Keywords: Oreochromis niloticus; Nile Tilapia;
Endocrine Disruption; Xenoestrogens; Testis-Ova;
“Endocrine Disruption” has been defined as exoge-
nous chemicals that alter the functions of the endocrine
system and consequently cause adverse health effects in
the intact organisms [1]. Endocrine Disruptor Screening
and Testing Advisory Committee were assigned the task
of making recommendations for the development of test-
ing and screening programs for endocrine disrupters [2].
Likewise, the Organization for Economic Co-operation
and Development also established a special activity for
endocrine disrupter testing and assessment [3]. Subse-
quently, the World Health Organization tasked the inter-
national program on chemical safety with preparing a
report describing the global assessment of the scientific
literature on endocrine disrupting chemicals [4].
There is increasing concern about male reproductive
disorders in humans, and widespread sexual disruption
among wildlife. Numerous studies have documented
generation rises in testicular cancer, declines in sperm
quality and increases in infertility [5]; increases in rates
of male infants born with incompletely-formed penises
and undescended testicles [6,7]. The ratio of baby boys
to baby girls is declining in the U.S. and other industri-
alized countries [8]. The causes of these worrying trends
are largely unknown, but one possibility is that they
might be linked to endocrine disrupting chemicals
Among EDCs found in the aquatic environment are
the steroid estrogens such as 17β-estradiol, estrone and
estriol [9]. In oviparous fish species, the production of
vitellogenin, an egg yolk protein precursor, is a critical
step for successful reproduction in females. Normally,
only mature females produce enough endogenous estro-
gen to induce vitellogenesis in fish, but exposure to es-
trogenic chemicals in the external environment can trig-
ger this response in male and juvenile fish as well. Many
wildlife species, especially fish, show signs of feminisa-
tion, with male fish producing large quantities of vitel-
logenin and developing sexual organs that are interme-
diate between male and female features, resulting in
so-called intersex fish [10]. In the aquatic environment,
EDCs are easily bioavailability to fish through a variety
P. Kosai et al. / Natural Science 3 (2011) 227-233
Copyright © 2011 SciRes. OPEN ACCESS
of routes, including aquatic respiration, osmoregulation
and maternal transfer of contaminants in lipid reserves of
eggs [11]. Dermal contacts with contaminated sediments
or ingestion of contaminated food are additional expo-
sure routes. EDCs that are apt to cause endocrine modu-
lation in vivo have one of three characteristics: they are
present in the environment at high concentrations, they
are persistent and bioaccumulative, or they are con-
stantly entering the environment [12]. EDCs that are
resistant to biodegradation and are lipophilic may bio-
accumulation in exposed organisms. Such EDCs include
organochlorine pesticides, polychlorinated biphenyls and
alkylphenols, all of which are found in aquatic environ-
ments. Other EDCs i.e., bisphenol A and 17β-estradiol,
do not bioaccumulation to any extent but are constantly
entering the aquatic environment through operations
such as effluent from sewage treatment works and run-off
from concentrated animal feeding operations [13].
In the present study, we describe the normal histology
of Nile tilapia (Oreochromis niloticus) reproductive or-
gan, and illustrate histopathological alterations in this
organ associated with endocrine disrupting chemicals in
the laboratory studies.
2.1. Experimental Fish
This study was performed at the Department of
Pathobiology, Faculty of Science, Mahidol University,
Bangkok, Thailand. Nile tilapia, O. niloticus, was juve-
nile and young adults with 1.22 ± 0.45 g, and 4.25 ±
0.58 g body weight, respectively. Tap water was filtered
to eliminate chemical contamination. The physicochemi-
cal characteristics of water were measured daily, ac-
cording to the experimental procedures described in
Standard Methods for the Examination of Water and
Wastewater [14]. Under laboratory condition, fish were
acclimated for 30 days at 29.0 ± 1.0˚C, pH = 6.6 – 7.0,
total hardness = 68 – 80 mg L-1 (as CaCO3), alkalinity =
75 - 80 mg L-1 and conductivity = 190 – 220 µmhos cm-1.
A 16:8 hour light-dark cycle was maintained throughout.
Chlorine residual and ammonia were below detection
limits. Fish were fed twice a day with 37%-protein
commercial fish food (Charoen Pokphand Group,
Bangkok, Thailand). The quantity of food was 2% of the
initial body weight per day. The animal care and han-
dling in this research was performed following the in-
struction of the Mahidol University-Institutional Animal
Care and Use Committee (MU-IACUC). Therefore, this
research was followed the mammal animal care and use
i.e., 1) Use, care and transportation of fish for toxicopa-
thological testing was complied with all applicable ani-
mal welfare laws. 2) Number of fish was kept to the
minimum requirement for achieve scientifically valid
results. 3) All protocols were taken to avoid the discom-
fort, distress or pain in the fish. 4) The appropriate dos-
age of the anesthesia was 200 mg L-1 ethyl-3-amino-
benzoate methanesulfonate salt (MS222, Sigma) and the
euthanasia was overdose of this chemical.
2.2. Experimental Design
The experiments were carried out in three glass
aquaria containing of the same physicochemical charac-
teristics of water, with continuous aeration. Fish (n = 30)
were randomly assigned to the three groups: Group 1
was the control group; Group 2 and 3 were the treated
groups with 10 and 100 g L-1 of 17β-estradiol, respec-
tively. After 30 days exposure, fish from each aquarium
were anesthetized. Dissection of fish was performed
after completing the external examination. The repro-
ductive organ was removed and prepared for histopa-
thological studies.
2.3. Specimen Preparation for Light
Microscopic Studies
The procedures for light microscopy were modified
Humason’s method [15]. Briefly, small pieces of tissues
were fixed in the 10% buffered formaldehyde for 24 h,
dehydrate through a graded series of ethanol and clear
with xylene solutions. They were embedded in a block
using melted paraffin at the embedding station. The par-
affin blocks were sectioned at 4 m thickness using a
rotary microtome, and stained with hematoxylin and
eosin. The tissue glass slides were examined for abnor-
malities by a Nikon E600 light microscope and photo-
graphed by a Nikon DXM 1200 digital camera (Tokyo,
Female gonads were classified of being in one of six
maturation stages according to a classification scale
proposed by Gupta [16]. Stage I corresponded to imma-
ture ovaries, with youngest oocytes diameter 0.05 - 0.15
mm. Stage II showed maturing oocytes, characteristi-
cally containing small vacuoles in the cytoplasm diame-
ter 0.15 - 0.3 mm. Stage III was characterized by ad-
vanced maturing oocytes diameter 0.3 - 0.7 mm. Then,
as the ovaries mature, from Stage III onward a number
of ova undergo a process of resorption. At this stage the
whole follicle loosed its shape and was described as an
atretic follicle. Stage IV represented mature oocytes di-
ameter 0.7 - 0.8 mm, filled with chromophilic yolk. At
Stage V, the mature ova increased in size with yolk ac-
cumulation, and at this stage they were ripe. At Stage VI,
the ovary did not differ greatly from the previous stage,
P. Kosai et al. / Natural Science 3 (2011) 227-233
Copyright © 2011 SciRes. OPEN ACCESS
with the remaining ova not as closely packed together
since some had been extruded.
No recognizable changes were observed in the female
reproductive system of the control (Figures 1-2) and low
(Figure 3) exposure groups throughout this experiment.
The majority of females were classified as being at Stage
I, characteristically showing immature ovaries, with the
presence of young oocytes at different stages of devel-
opment. Female exposed to high 17-estradiol (Figure 4)
was classified as being at Stage II with maturing oocytes,
characteristically containing small vacuoles in the cyto-
Figure 1. Low and high magnification light micrographs of
ovary in the control group of juvenile O. niloticus. The ovary
was filled with primary follicles (F) containing oocytes which
had a large, light staining nucleus and strongly basophilic cy-
toplasm. Note tubnica albuginea (T) surrounding the ovary was
very thin.
Figure 2. Low and high magnification light micrographs of
ovary in the control group of young adult O. niloticus, simi-
larly with the juvenile ovary.
Figure 3. Low and high magnification light micrographs of
ovary in the low concentration 17β-estradiol group of O.
niloticus showing the internal structure similarly with those of
control group.
Figure 4. Low and high magnification light micrographs of
ovary in the high concentration 17β-estradiol group of O.
niloticus were classified as being at Stage II with maturing
oocytes, characteristically containing small vacuoles in the
Light microscopic studied of the longitudinal sections
showed that the control testes of O. niloticus consist of
lobules separated from each other by interstitial tissue.
The interstitial tissue became very thin and compressed
in the maturing testis. The testes consisted of lobules
with cysts in different stages of development. Sper-
matozoa could be observed in the lumen of the lobule.
The lobules were lined in the inside by Sertoli cells,
which were important for the support and nutrition of
developing spermatozoa. In order to describe the tes-
ticular development, and to use the testis as a reproduc-
tive biomarker, it was necessary to determine the repro-
P. Kosai et al. / Natural Science 3 (2011) 227-233
Copyright © 2011 SciRes. OPEN ACCESS
ductive maturity of each fish testis that was examined.
This was done by using the classification system that
recognized four developmental gonad stages (stages I-IV)
for males.
The histological characteristic of the gonad stages that
were used during this investigation were adapted from
Gupta [16], Nagahama [17], and Goodbred et al. [18].
The stages were based on the maturity of the predomi-
nant stage of spermatogenesis. Stage I: Immature testes
were recognized by the absence of spermatogenic activ-
ity in the interstitial tissue and the presence of primarily
spermatocytes. No spermatozoa are present in the lob-
ules. Stage II: Early spermatogenesis was characterized
by mostly thin interstitial tissue and the presence of pri-
marily immature cells (spermatocytes to spermatids);
however, some spermatozoa were also present. Stage III:
Mid-spermatogenesis, the interstitial tissue was moder-
ately thick and some proliferation and maturation of the
sperm could be observed; spermatocytes, spermatids and
spermatozoa were present in roughly equal proportion.
Stage IV: Late spermatogenesis, the interstitial tissue
was thick. Although all cell types were represented,
spermatozoa predominate in this stage. Stages II through
IV were characteristic of sexually mature fish, with the
least activity occurring in off-season (stage II) and the
most activity taking place immediately prior to and dur-
ing the spawning season (stage IV).
No recognizable changes were observed in the male
reproductive system of the control (Figure 5) and low
(Figure 6) exposure groups throughout this experiment.
The testes had normal appearance; containing cells at all
spermatogenic stages, and was classified as maturing
testes (Stage II). Histological the testis of the fish con-
sisted of lobules of various shapes, which were con-
nected with each other by thin connective tissues. The
interstitial cells were observed in between the testis lob-
During the spermatogenic process, the sperm mother
cell in the germinal epithelium multiplied and produced
the primary and secondary spermatocytes, spermatids,
and sperms. The lumina were filled with spermatozoa
and the lobules contained numerous spermatogenic cysts.
The histological alteration of the testes of high (Figures
7-8) exposure groups showed normal appearance of the
testis lobules, however, there were found testis-ova in
some areas.
Several endocrine disrupting chemicals including xeno-
estrogens or estrogenic chemicals released into the aquatic
environment have the potential to disrupt the endocrine
systems, especially of fish and subsequently cause adverse
effects on the sexual development and reproduction [13].
Figure 5. Low and high magnification light micrographs of
testis in the control group of O. niloticus showing the exter-
nally testis is covered by the tunica albuginea (TA). Note the
position of the Sertoli cell (S), which lines the lobule; SG =
spermatogonia; SP = spermatozoa.
P. Kosai et al. / Natural Science 3 (2011) 227-233
Copyright © 2011 SciRes. OPEN ACCESS
Figure 6. Low and high magnification light micrographs of
testis in the low concentration 17β-estradiol group of O.
niloticus showing the internal structure similarly with those of
control group.
Figure 7. Low and high magnification light micrographs of
testis in the high concentration group of O. niloticus. Note
arrows = testis-ova.
Figure 8. Light micrographs of testis in the high concentration
group of O. niloticus. Note arrows = testis-ova.
Xenoestrogens can interfere with natural estrogens by
binding to the physiological estrogen receptor and thus
mimicking or degradation natural estrogen [19]. The
consequences of exposure to xenoestrogens may be re-
productive disorders such as reduced fertility, reduced
hatchability, reduced viability of off-spring, impaired
hormone activity, structural abnormalities of the repro-
ductive tract, and altered adult sexual behavior [20-22].
Studies regarding 17β-estradiol effects on fish have
been focused on endocrine aspects, mainly reproduction.
It is known that it may alter gonadosomatic index in
males, reduce egg production in females, induce vitel-
logenesis in males and juveniles as well as decrease fer-
tility [23-25]. The mechanisms whereby these effects
take place are unclear. The sex steroids exert both posi-
P. Kosai et al. / Natural Science 3 (2011) 227-233
Copyright © 2011 SciRes. OPEN ACCESS
tive and negative feedback control on the hypothalamus-
pituitary system to regulate the release of gonadotropins.
The 17-estradiol has been shown to inhibit gonadotro-
pin secretion in fish. This may indicate that 17-estradiol
cause its effects on the testes through alterations in go-
nadotropin secretion.
Mills [26] reported that 17-estradiol treated fish ex-
hibited decreased gonadosomatic index (GSI), altered
hepatosomatic index, elevated plasma estradiol, reduced
plasma testosterone, and high levels of plasma vitel-
logenin. Reduced GSI always corresponded with ob-
servable histopathological changes indicative of re-
gressed gonads. The present study, Nile tilapia was ex-
posed to 100 g L-1 of 17β-estradiol for 30 days showed
this intersex (testes-ova). These obtained results agree
with the previously study, exposure to estrogenic
chemicals during the critical periods of sex differentia-
tion has induced testis-ova in several fish species [27,
As suggested by Arcand-Hoy and Benson [29], 17β-
estradiol given to the male fish can disturb the hypo-
thalamus–pituitary-gonadal axis in which several hor-
mones, including androgens, normally regulates sexual
development, sexual physiology, and sexual behavior.
Since androgens, also in male regulate secondary sexual
characters and reproductive behavior, a disruption of the
androgen synthesis or the processes in which androgens
participate can cause severe effects [21].
Gimeno and colleague [10] indicated the role of es-
trogens in the induction of intersex gonads in adult
gonochoristic species seems similar to that of the normal
process of sex reversal in hermaphrodite species. Indeed,
the treatment of juvenile male black porgy, Acanthopa-
grus schlegeli, a protandrous hermaphrodite marine fish,
with 17β-estradiol caused the regression of the testes and
the lack of spermiation, as well as the induction of pre-
cocious sex change, as shown by the appearance of oo-
cytes [30].
In conclusion, the present study shows that water-
borne exposure to the xenoestrogens 17β-estradiol
cause’s severe effects on the reproductive system of
male Nile tilapia.
This study was funded by the Thailand Research Fund and the Com-
mission on Higher Education: Research Grant for Mid-Career Univer-
sity Faculty 2008 (RMU5180001) and in part by Mahidol University
International College, and Faculty of Science, Mahidol University.
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