Fish Physiol Biochem (2018) 44:1561–1576
https://doi.org/10.1007/s10695-018-0507-z
Endocrine-disrupting chemicals in aquatic environment: what
are the risks for fish gametes?
Oliana Carnevali & Stefania Santangeli & Isabel Forner-Piquer & Danilo Basili & Francesca Maradonna
Received: 21 December 2017 / Accepted: 23 April 2018 / Published online: 11 June 2018
# Springer Science+Business Media B.V., part of Springer Nature 2018
Abstract Over the past 25 years, extensive research in
vertebrate species has identified several genomic path-
ways altered by exposures to anthropogenic chemicals
with hormone-like activity mediated by their interaction
with nuclear receptors. In addition, many pollutants have
been shown to interfere with non-genomic (non-classical)
pathways, but this mechanism of endocrine disruption is
still poorly understood. Recently, the number of publica-
tions describing the effects of Endocrine disrupting
chemicals (EDCs) on fish reproduction, focusing on the
deregulation of the hypothalamus-pituitary-gonadal axis
as well as on gamete quality, significantly increased. De-
pending on their ability to mimic endogenous hormones,
the may differently affect male or female reproductive
physiology. Inhibition of gametogenesis, development of
intersex gonads, alteration of the gonadosomatic index,
and decreased fertility rate have been largely documented.
In males, alterations of sperm density, motility, and fertility
have been observed in several wild species. Similar
Oliana Carnevali and Francesca Maradonna contributed equally to
this work.
O. Carnevali (*) : S. Santangeli : I. Forner-Piquer :
D. Basili : F. Maradonna (*)
Dipartimento di Scienze della Vita e dell’Ambiente, Università
Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona,
Italy
e-mail: o.carnevali@univpm.it
e-mail: f.maradonna@univpm.it
O. Carnevali : S. Santangeli : F. Maradonna
INBB Consorzio Interuniversitario di Biostrutture e Biosistemi,
00136 Rome, Italy
detrimental effects were described in females, including
negative outcomes on oocyte growth and maturation plus
the occurrence of apoptotic/autophagic processes. These
pathways may affect gamete viability considered as one of
the major indicators of reproductive endocrine disruption.
Pollutants act also at DNA level producing DNA muta-
tions and changes in epigenetic pathways inducing specif-
ic mechanisms of toxicity and/or aberrant cellular re-
sponses that may affect subsequent generation(s) through
the germline. In conclusion, this review summarizes the
effects caused by EDC exposure on fish reproduction,
focusing on gametogenesis, giving a general overview of
the different aspects dealing with this issue, from morpho-
logical alteration, deregulation of steroidogenesis, hor-
monal synthesis, and occurrence of epigenetic process.
Keywords Ovary . Testis . Environmental pollution .
Sex reversal . Epigenetic
Introduction
The “endocrine disruptor” concept, the interest for spe-
cific studies on their endocrine activity and the link
between human health and environmental exposure
flashed for the first time at the European Workshop on
the Impact of Endocrine Disruptors on Human Health
and Wildlife (Weybridge, UK) in 1996. Nowadays, the
term endocrine-disrupting chemical (EDC) is used to
define a structurally diverse class of synthetic and natu-
ral compounds that possess the ability to interact with
the endocrine system. These anthropogenic chemicals
1562
are continuously released into the environment through
many sources, mainly of anthropogenic origin, e.g.,
wastewater treatment plants, terrestrial runoff and pre-
cipitation, and finally targeting humans and wildlife
health (Illuminati et al. 2010, 2017; Annibaldi et al.
2015; Afifah et al. 2017; Ribeiro et al. 2017). EDCs
are diverse and include herbicides and pesticides, plastic
contaminants, heavy metals, biocides, heat stabilizers,
chemical catalysts, pharmaceuticals and dietary compo-
nents, flame retardants, and others. Given their physico-
chemical differences and distinct biological effects, it is
not surprising that EDCs interact with the endocrine
system through a wide range of mechanisms of action
(Henley and Korach 2006). They interfere with the
neuroendocrine and endocrine functions involved in
embryo development and reproduction (Godfrey et al.
2017). Once released in the environment, they exert
their effects by mimicking, antagonizing, or altering
endogenous steroid levels (androgens or estradiol) by
changing rates of their synthesis or metabolism and/or
expression or action at receptor targets. EDCs, in fact,
share structural homologies with natural hormones, be-
ing able to bind their cognate receptors and triggering
their same biological response. Considering the repro-
ductive pathway, the main gonadal steroids include
estrogens, androgens, and progesterone, which are de-
rivatives of cholesterol. In their biosynthesis, testoster-
one derives from androstenedione, while estrogens are
synthesized by the aromatization of either testosterone
(T) into estrogen (17β-estradiol) or androstenedione
into estrone, until final conversion into estradiol. Two
specific aromatase enzymes, responsible for catalyzing
these reactions are encoded in fish: CYP19a mainly
expressed in the gonads and CYP19b mostly found in
the brain (Zhang et al. 2014c). Estrogens are known to
be involved in vitellogenesis regulation, oogenesis, go-
nadotropin regulation, testicular development, and
many other reproductive processes (Yilmaz et al.
2015). Androgens, including testosterone, working via
the androgen receptor (AR), play a critical role in males’
development and secondary sex characteristics (Schulz
et al. 2010). Despite most of the EDCs have environ-
mental concentrations far below the range established
by environmental agencies, it should be considered that
an environmental health problem regarding EDCs is
related to their bioaccumulation, chronic exposures,
and eventual occurrence of synergistic/antagonistic in-
teraction among them. Furthermore, EDCs are known to
act through non-monotonic dose-response curves, and
Fish Physiol Biochem (2018) 44:1561–1576
therefore it has been argued that effects observed at high
doses cannot be used for the extrapolations into the low
dose range. Endocrine disruption has been demonstrated
to occur in wildlife, particularly in aquatic species or in
species that are connected to the aquatic food chains.
Dietary uptake due to consumption of polluted food
represent an important source of contamination
(Maradonna et al. 2014, 2015; Traversi et al. 2014;
Ruhí et al. 2016; Carnevali et al. 2017). Given the
widespread environmental distribution of the pollutants,
the questions being addressed today, are not whether
endocrine disruption occurs in wildlife but rather in
which concentration it occurs, what mechanisms are
involved, and whether disruption of the endocrine sys-
tem will lead to ecologically relevant effects.
The aim of this review is to summarize the effects
induced by the exposure to EDCs on reproduction,
focusing on gametogenesis, giving a general overview
of all aspects dealing with this issue. This will provide
the knowledge to build an integrated model, using mor-
phological, biochemical, and molecular results, to be
used in further studies in an environmentally relevant
contest. Results shown will provide evidence to sustain
that many of the molecular changes are associated with
ecologically relevant effects in terms of their impact on
population and further generations.
Effects of EDC exposure on ovarian physiology
Most of studies described so far documented the abil-
ity of EDCs to affect ovarian morphology, oocyte
maturation and steroidogenesis, altering the normal
functioning of the endocrine and reproductive systems
by mimicking or inhibiting endogenous hormone ac-
tion, modulating the production of endogenous hor-
mones or altering hormone receptor populations
(Sonnenschein and Soto 1998). A major mechanism
of endocrine disruption is the EDC binding to hor-
mone receptors. Other mechanisms, besides receptor-
mediated events, may include mechanisms of inhibi-
tion or stimulation of hormone metabolism or alter-
ations in serum hormone-binding proteins.
EDCs affects ovarian morphology: oocyte growth,
maturation, and fertility
Gobious rarus exposure to bisphenol A (BPA) treat-
ments (14 and 35 days) induced ovarian morphological
Fish Physiol Biochem (2018) 44:1561–1576
alterations consisting on the presence of a relatively
higher proportion of pre-mature oocytes and the occur-
rence of several atretic follicles (Zhang et al. 2014a).
Moreover, in the same species, the exposure to 2,4-
Dichloro-6-nitrophenol, a chloride-based herbicide, in-
duced detrimental effects both in male and female fish.
In female gonads, an increase in the number of de-
formed follicles along with the presence of degenerating
vitellogenic oocytes were observed, suggesting a defect
in follicle recruitment (Chen et al. 2016). Effects on
male are described in the section below. When catfish,
Clarias gariepinus, were exposed, from hatching to
50 dph, to ethinylestradiol (EE2) (50 ng/l) and diethyl-
stilbestrol (DES) (10 ng/l), and development was mon-
itored until adulthood, morphological deformities, such
as stunted growth, spinal curvature, and yolk-sac fluid
retention were observed. In addition, histological studies
revealed rudimentary or malformed ovaries with preco-
cious oocyte development as well as follicular atresia
(Sridevi et al. 2015). In zebrafish female, Danio rerio,
the chronic exposure to 25 ng/l EE2 and to 0.02, 0.2, 2,
20, and 40 μg/l Di-(2-ethylhexyl)-phthalate (DEHP),
severely impaired ovulation and embryo production.
Ovarian observation suggested the ability of both EE2
and DEHP to modulate vitellogenesis inducing a reduc-
tion of previtellogenic oocytes associated with an in-
crease of vitellogenic ones. After the exposure, fish were
crossed and the number of embryos obtained was about
1% of the embryos produced by the control, showing a
clear impairment of fecundity (Carnevali et al. 2010).
Recently, in the same species, the effects of the chronic
exposure to five different concentrations (0.42, 4.2, 42,
Fig. 1 Histological analysis of ovaries from control fish and fish
exposed to five DINP concentration. a Percentage of
previtellogenic, vitellogenic, and mature oocytes in the ovary of
1563
420, and 4200 μg/l) of di-isononyl phthalate (DiNP), a
substance which was introduced to replace DEHP, were
analyzed focusing on a number of reproductive param-
eters, including follicles number and size frequency and
macromolecular composition of vitellogenic oocytes.
Histological analysis revealed a significant reduction
in the number of vitellogenic oocytes in gonad of fish
exposed to the lower DiNP concentrations (0.42, 4.2,
and 42 μg/l) as well as a reduction in the number of
mature oocytes in those of fish exposed to higher doses
(420 and 4200 μg/l) (Fig. 1). In vitellogenic oocyte
sampled from exposed fish, a decrease of lipids, phos-
phates, and proteins components was measured by FT-
IR, suggesting that DiNP might affect vitellogenin up-
take and induce changes of lipid composition. Further-
more, results demonstrated that DiNP adversely affects
female reproductive physiology acting in a non-
monotonic fashion. In the same experimental model,
exposure from 2 h post fertilization (hpf) to sexual
maturity, to the flame retardant tris (1,3-dichloro-2-pro-
pyl) phosphate (TDCPP) promotes oocyte maturation as
documented by an increase in the percentage of late
vitellogenic/mature oocyte (Wang et al. 2015). More-
over, in the F1 generation, high malformation rates were
observed demonstrating that parental exposure to
TDCPP causes developmental toxicity in offsprings
(Wang et al. 2015). In marine medaka, Oryzias latipes,
ovary, the exposure to 8.5 μg/l of 3,3′-diindolylmethane
(DIM), induced an increase of VTG storage, but a
reduction of cathepsin enzyme activities, suggesting a
blockage of VTG uptake into growing oocytes. This
reduction occurred together with lower levels of eggshell
control (C) and fish exposed to DINP; b representative ovarian
section showing previtellogenic (PreV), vitellogenic (Vit), and
mature (M) oocytes
1564
proteins leading to an inhibition of primary oocyte mat-
uration and contributing to a decreased fecundity (Chen
et al. 2017). In trout (Salmo trutta f. fario), a 3-month
exposure to 5 μg/l BPA, starting at the beginning of the
spawning season, affected the percentage of ovulated
eggs and shifted ovulation after 3 weeks, without affect-
ing the quality of the eggs (Lahnsteiner et al. 2005).
Occurrence of atretic oocytes characterized by disorgani-
zation of the ooplasm was documented in carp treated
with 2 mg/l Perfluorooctanoic acid (PFOA) for 56 days
(Giari et al. 2016). In a 21-day reproduction study on
fathead minnow exposed to the pharmaceutical
dutasteride (100 μg/l), several ovarian histopathological
conditions including presence of atretic follicles and
mitotically dividing oogonia, proliferation of interstitial
connective tissue containing different types of somatic
cell, and macrophage aggregates were detected. Accu-
mulation of adipose tissue and interstitial proteinaceous
fluid was also observed (Margiotta-Casaluci et al. 2013).
In tilapia, Oreochromis niloticus, the action of diuron, a
substituted urea herbicide with a documented anti-
androgenic action, and three of its metabolites were
evaluated. Pollutant exposure induced an increase of
gonadosomatic index and the ovary presented a higher
number of vitellogenic oocytes and a decrease of germi-
native cells, suggesting that diuron metabolites have
estrogenic action and accelerate this species ovarian de-
velopment (Boscolo Pereira et al. 2016).
EDC effects on molecular markers linked
to steroidogenesis, ovarian growth, and maturation
Exposure of G. rarus to different BPA concentrations
differently affected the expression of steroidogenic en-
zymes star, cyp11a1, cyp17a1, hsd3b, hsd11b2, and
cyp19 expression at lower (5 and 15 μg/l) concentra-
tions; BPA induced the increase of ovarian steroidogen-
ic gene transcription and sexual steroid receptor genes,
while at higher concentration (50 μg/l), the increase of
nr5a1s. Variation of the steroidogenic gene expression
was probably linked to changes in er and ar transcripts
levels able to affect both the steroid hormone signaling
and nr5a1s expression, suggesting a possible epigenetic
regulation (Zhang et al. 2014a). Moreover, in the same
fish, alteration of gdf9 and bmp15 mRNA expression
and their levels may play a key role in the weight gain
and in the abnormal ovarian development, above de-
scribed (Zhang et al. 2014b). In catfish, transcript levels
and activity of aromatase, the rate-limiting enzyme of
Fish Physiol Biochem (2018) 44:1561–1576
estrogen biosynthesis were significantly altered by EE2
and DES exposure (Sridevi et al. 2015). More specifi-
cally, upon exposure to EE2, at 50 and 150 dph, the
transcript levels of star and P450scc exhibited signifi-
cant increase. Regarding cyp17, 3b-hsd, and 17b-hsd1,
a gradual increase from 50 to 350 dph following EE2
treatment was observed. Conversely, exposure to DES
decreased cyp17 transcript levels, while 3b-hsd and 17b-
hsd1 transcripts were unaltered. Neither EE2 nor DES
exposures affected ovarian aromatase (cyp19a1a) levels
(Sridevi et al. 2015).
The decrease of fecundity observed in zebrafish
chronically exposed to EE2 or DEHP, was associated
with the increase of BMP15 levels and the reduction of
lhr, mprb, and ptgs2 expression, the final triggers of
ovulation. By an in vitro maturation assay, the inhibitory
effect of DEHP on germinal vesicle breakdown was
further confirmed (Carnevali et al. 2010). In the same
species, exposure to DiNP resulted in modulation of
genes involved in steroidogenesis in a dose-related, but
non-monotonic manner. Greater differences were ob-
served for star and cyp11a1 transcript levels among
the different tested concentrations (0.42, 420, and
420 μg/l). Only the lowest dose affected fshr transcript.
Esr1 and esr2b levels were affected only in the 420 μg/l
trial. Exposure to DiNP reduced esr2a transcript level in
a non-monotonic manner, with great differences ob-
served among the lowest (0.42 mg/l) and the higher
concentrations (420 μg/l; 4200 μg/l). Exposure to DiNP
did not change pgrmc1 transcript level in any of the
treatment groups, but in the 420 and 4200 μg/l expo-
sures, a reduction of lhcgr and pgrmc2 transcript levels
was measured. Conversely, bmp15 transcript level was
significantly increased only in fish exposed to the
0.42 μg/l concentration. The results described suggested
that low DINP concentrations mainly interfere with
steroidogenesis and oocyte growth while higher concen-
trations impair oocyte maturation (Santangeli et al.
2017b), suggesting that there is still the need to find a
safer DEHP substitute (Forner-Piquer et al. 2017).
In addition to the estrogenic effects, EDCs may also
exert and anti-androgenic action as evidenced in fathead
minnow (Pimephales promelas) treated with the
Dutasteride, a pharmaceutical product acting as a 5α-
reductase (5αR) inhibitor, the enzyme that converts
testosterone into dihydrotestosterone (DHT).
Dutasteride impairs reproductive functions in fish, con-
sistent with an anti-androgenic mode of action
(Margiotta-Casaluci et al. 2013).
Fish Physiol Biochem (2018) 44:1561–1576
In vitro exposure of trout post-vitellogenic oocytes to
prochloraz, an imidazole fungicide, widely used in ag-
riculture, affected oocyte maturation process. It is well-
known that prochloraz antagonizes both androgen and
estrogen receptors and agonizes the aryl hydrocarbon
receptor. Prochloraz (10−5, 10−6, or 10−7 M), in combi-
nation or not with LH, was able to alter the GVBD in
Rainbow trout post-vitellogenic follicles. Prochloraz ad-
ministered alone is able to trigger oocyte maturation
stimulating the transcription of a set of genes responsi-
ble for pre-ovulatory follicular recruitment and differen-
tiation, such as igf1, igf2, connexin 43, and hsbd20.
Some of them were also triggered by LH, which seems
to synergically cooperate with prochloraz in inducing
GVBD (Rime et al. 2010).
Most of the studies described so far documented the
ability of many xenobiotics to affect ovarian morpholo-
gy, oocyte maturation, and steroidogenesis following
the classical genomic pathway (Blair et al. 2000). More
recently, interest has moved to the investigation of the
non-genomic mechanisms activated by xenobiotics
(Thomas and Doughty 2004). The key role of a novel
seven pass-transmembrane receptor, GPR30, in mediat-
ing several rapid estrogenic responses was demonstrat-
ed. In zebrafish, it was demonstrated that low BPA
concentrations (10–200 nM) and three alkylphenols,
e.g. tetrabromobisphenol A, 4-nonylphenol, and
tetrachlorobisphenol A (5–100 nM) disrupted oocyte
maturation by a non-genomic mechanism of action in-
volving the activation of the Gper/Egfr/Mapk3/1 path-
way (Fitzgerald et al. 2015).
Effects of EDC exposure on male gonadal physiology
and functionality
The main determinant of male fertility includes the
evaluation of sperm motility once released into the
environment, spermatozoa velocity, and motility dura-
tion (Linhart et al. 2008). In vertebrates, androgens
regulate testicular functioning, spermatogenesis, sec-
ondary sexual characteristics and behavior, and their
effects are mediated by androgen receptors (Martyniuk
and Denslow 2012). EDC exposures caused testes
growth inhibition and maturation delay, changes of male
sex cell types, low semen quality, occurrence of ova-
testis, and changes in estrogen and testosterone levels in
several teleost models (Kime 1999). The adverse im-
pacts of ECDs occur at lower concentrations as duration
1565
of exposure increases (Kumar 2013). Although techni-
cal and biological differences among studies make it
difficult to classify toxicity of EDCs, it seems that heavy
metals have greater potential to impair spermatozoa
functioning and exhibit higher toxicity than organic
EDCs do (Popek et al. 2006).
Effects of EDCs on testis morphology
and spermatogenesis
In marine medaka, Oryzias melastima, exposure to rel-
atively low doses (0 and 8.5 μg/l) of 3, 3″
diindolylmethane (DIM), an antifouling agent, led to
anomalous production of vitellogenin and eggshell pro-
teins in the testis, clearly highlighting its estrogenic
potency (Chen et al. 2017).
In zebrafish, the effects of a chronic exposure to
environmentally relevant concentrations of DEHP
(0.2 and 20 μg/l) were analyzed, highlighting DEHP
ability to impair reproduction inducing a mitotic
arrest during spermatogenesis and decreasing em-
bryo production (up to 90%). These changes were
associated with the increase of spermatozoa DNA
fragmentation (Corradetti et al. 2013). Similarly,
zebrafish exposure to a mixture of environmentally
relevant concentrations of triclocarban and inorganic
mercury, induced severe histological lesions in the
testis, which size and mature sperm abundance was
decreased if compared to those of fish exposed to
the compounds individually (Wang et al. 2016).
Similarly, the exposure to DES, flutamide (FLU)
and their combination induced an impairment of
spermatogenesis, decreasing sperm concentration,
and affecting both meiotic and apoptotic processes
(Yin et al. 2017).
A possible delay of spermatogenesis was hypothe-
sized in male carp, Cyprinus carpio, exposed to 2 mg/l
PFOA for 56 days, where spermatogonia and spermato-
cytes were the dominant germ cells. Moreover, an in-
creased amount of interstitial tissue and proliferation of
Sertoli cells was observed in the testis (Giari et al. 2016).
In the same species, a 14-day exposure to graded-BPA
concentrations, from 1 to 1000 μg/l, caused severe
alterations of the lobular structure and spermatogenic
cysts, with free spermatozoa often degenerating into the
lumen (Mandich et al. 2007). In goldfish exposed to
BPA, an increase of hepatic vitellogenin was induced,
but gonadosomatic index (GSI), hepatosomatic index
(HSI), and E2 levels were not affected, suggesting that
1566
BPA might act on testicular spermatogenesis causing an
alteration of sperm maturation (Hatef et al. 2012a, c).
Spermatozoa production, morphology motility,
and velocity
Morphological sperm alteration, including damage to
the flagellum and its beating, can be revealed using a
dark-field microscope equipped with stroboscopic illu-
mination, or a phase contrast microscope with high-
speed video camera (Alavi et al. 2012a, b). Computer-
assisted sperm analysis (CASA) systems, which are
based on video recording approaches, are employed
for analysis of sperm motility and velocity (Alavi et al.
2012a). Valuable parameters of sperm velocity are cur-
vilinear velocity (VCL), straight line velocity (VSL),
and the angular path velocity (VAP), being especially
this last parameter, differently affected by ECDs.
Exposure to cadmium, mercury, BPA, zinc, and tri-
butyltin decreased VCL and VSL of spermatozoa in
different fish species. As reported by Hatef et al.
(2013), specific spermatozoa responses can be supposed
when different species are exposed to a given EDCs. In
African catfish—Clarias gariepinus, burbot—Lota lo-
ta, European chub—Leuciscus cephalus, a decrease of
sperm motility was observed immediately after copper
or cadmium exposure of spermatozoa, while no evident
effects were induced in brown trout, S. trutta f. fario,
spermatozoa exposed to the same metals (Lahnsteiner
et al. 2004). Different results were also evidenced when
the effects of exposure to mercury, zinc, lead, nickel,
cyclohexane, and 2,4-diclorophenol were compared
among seabass (Abascal et al. 2007), African catfish,
burbot, and European chub spermatozoa motility
(Lahnsteiner et al. 2004). Exposure to cadmium nega-
tively affect sperm quality also in D. rerio, including
motility, time of motility, curvilinear velocity, average
path velocity, and straight line velocity, which alteration
may reduce the fertility rate of these animals (Acosta
et al. 2016). No effects on motility were observed in
medaka, exposed to scalar concentration of nonylphenol
(Hara et al. 2007). In croaker, many estrogenic and non-
estrogenic pollutants, at environmentally relevant con-
centrations, blocked the ability of 17,20β,21-trihy-
droxy-4-pregnen-3-one to induce sperm motility, a pro-
cess physiologically mediated by a rapid, non-genomic
steroid action (Thomas and Doughty 2004).
These evidences suggest that despite the recognized
mode of action of the EDCs, the variability in the
Fish Physiol Biochem (2018) 44:1561–1576
responses may be related to differences in spermatozoa
and seminal plasma physiology and biochemistry.
Sperm velocity was decreased in Perca fluviatilis sperm
incubated for 3 h with mercury. A trial was set up to gain
information on the effects of mercury chloride (HgCl2)
on both sperm physiology and structure, to identify sites
of action of HgCl2 and to investigate the mechanism of
action of HgCl2 on spermatozoa. Results demonstrated
that HgCl2 acts on different sites of sperm, such as the
plasma membrane, the axoneme, and the midpiece.
HgCl2 exposure decreased stored ATP and increased
morphological alterations (Hatef et al. 2011). In the
same species, in vitro treatment of sperm with BPA,
decreased both motility and velocity and its capacity to
damage the flagella structure changing its shape from
the straight position to the C-shaped one was demon-
strated (Hatef et al. 2010). In sea bass, a 5-min incuba-
tion with 1 mg/l mercury or an instant exposure to
100 mg/l, significantly affected sperm velocity, while
no effect was induced by exposure to lead or copper
(Abascal et al. 2007). Velocity was also affected in
common carp, after a 24-h incubation with TBT
(Rurangwa et al. 2002) and to graded concentration of
mercury (Chyb et al. 2001a,b) and zinc (Chyb et al.
2000). Impairment of spermatogenesis was observed
in trout, S. trutta f. fario, exposed to environmentally
relevant concentrations of BPA (1.75, 2.4, and 5 μg/l)
during pre-spawning and spawning periods. Results
showed that at the beginning of spawning, the two
lowest BPA concentrations, reduced sperm density, mo-
tility rate, and swimming velocity. In the middle of
spawning season, BPA reduced swimming velocity
and only the 2.4 μg/l concentration, reduced motility
rate. The highest concentration completely impaired
spermatogenesis and only one out of eight fish gave
low-quality semen (Lahnsteiner et al. 2005). In goldfish,
a 20- or 30-day exposure to BPA decreased sperm
motility at 15-, 30-, 60-, and 90-s post-activation; a
shorter exposure, 10 days, affected sperm velocity at
30-, 60-, and 90-s post-activation. These alterations
were possibly caused by variation of hormonal levels,
as described below (Hatef et al. 2012a). In the same
species, using a phase contrast microscopy, a shorter
flagella length was measured in spermatozoa in pres-
ence of high mercury concentration (Van Look and
Kime 2003).
In rainbow trout, seminal plasma was used as incu-
bation medium for monitoring short-time exposure to
mercury and cadmium ions on sperm. Although
Fish Physiol Biochem (2018) 44:1561–1576
mercury exposure affected sperm motility immediately
after dilution with milt as well as at 4 h of exposure, no
differences were found respect to control sperm after a
24-h treatment, suggesting that in trout, seminal plasma
has a protective role against the toxic effects of mercury
ions on motility (Dietrich et al. 2010).
In Gobius niger, exposed for 48 h to 1 or 10 mg/l Cd
acetate, hsp70, mtt and casp3 mRNA levels measured in
the testis, significantly increased, highlighting the tox-
icity of this metal on testis physiology (Migliarini et al.
2005). Similarly, in starlet was recently demonstrated
that exposure of sperm to NP and DES can induce
reactive oxygen species in spermatozoa, leading to an
impairment of sperm quality, e.g. decrease the percent-
age of intact sperm cells (Shaliutina et al. 2017).
Recently, the effect of seven heavy metals on the
motility parameter of zebrafish sperm was tested in
order to develop an in vitro toxicological test system
as an alternative to live animal testing. The endpoints
analyzed were progressive motility, VCL, and linearity.
Results demonstrated that progressive motility was the
most sensitive of the three investigated parameters, sug-
gesting its use as an accurate and fast bioindicator of
heavy metal load (Kollár et al. 2018).
Effects of EDCs on steroidogenesis and hormone
synthesis
The alteration of testis morphology described in
zebrafish exposed to triclocarban and inorganic mercury
was associated to variation of the transcription of a set of
steroidogenic genes, such as cyp19a, 3β-HSD, cyp17,
and 17β-HSD (Wang et al. 2016). In G. rarus, the
exposure to BPA evidenced non-monotonic effects on
the mRNA expression of StAR, cyp11a1, 3β-HSD,
cyp17a1, and cyp19a1a resulting the medium concen-
tration, 15 μg/l, to be the most detrimental. More spe-
cifically, the increase of the expression of several
mRNAs codifying for testicular steroidogenic enzymes
suggested an increase of androgens, which effect could
be counteracted by the non-significant downregulation
of cyp17a1 mRNA expression (Liu et al. 2014a). The
correlation analysis at mRNA level demonstrates that
the BPA-mediated actions on testicular steroidogenesis
might involve sex steroid hormone receptor signaling,
gonadotropin/gonadotropin receptor pathway, and tran-
scription factors, such as the nuclear receptor subfamily
5, group A (Nr5a), and the fork head box protein L2
(Foxl2) (Wang et al. 2012; Zhang et al. 2013). Similarly,
1567
the exposure to 25 ng/l EE2, modulated the expression
of the same steroidogenic genes StAR, cyp11a1, 3β-
HSD, cyp17a1, and cyp19a1a affected by BPA. The
authors hypothesized that this inhibition might occur
in response to a negative feedback of EE2 on pituitary
follicle stimulating hormone (FSH) or could be mediat-
ed by a direct EE2 effect on the testis (Liu et al. 2012).
The endocrine disruptive activity of DEHP was doc-
umented in goldfish, where a 30-day exposure to this
contaminant reduced the levels of 11- KT and LH,
suggesting that the decreased sperm quality could be
caused by the contaminant effects on testicular hormone
levels. However, DEHP does not affect GnRH and Kiss-
1/Gpr54 system. In addition, DEHP does not exhibit
estrogenic activity and does not act through sex steroid
receptor (Golshan et al. 2015). In a similar trial, goldfish
were exposed to three nominal vinclozolin (VZ) con-
centrations (100, 400, and 800 μg/l). Dose-dependent
VZ effects were observed, resulting in an impairment of
sperm quality via disruption of steroidogenesis. Aside
from VZ effects mediated by a competitive binding to
AR, potential effects of VZ by direct inhibition of 11-
KT biosynthesis were also demonstrated (Hatef et al.
2012b). In the same species, a chronic exposure to BPA
(0.6, 4.5 and 11 μg/l) significantly decreased testoster-
one (T) and 11-KT levels and impaired sperm motility
and velocity (Hatef et al. 2012a).
In zebrafish exposed to DES and FLU, the impairment
of spermatogenesis was associated to a reduction of
plasmatic 11-KT and a deregulation of aldh1a2, cyp26a1,
nonos1, sycp3, dmc1, bax, and bcl2 genes codifying for
signals regulating meiosis and apoptosis and dmrt1, sf1,
cyp11b2, cyp17a1, ar, fshr, and lhr genes orchestrating
gonadal steroidogenesis (Yin et al. 2017).
Di-n-butyl phthalate exposure significantly increased
plasma testosterone concentrations and significantly re-
duced spiggin levels in male three-spined sticklebacks,
Gasterosteus aculeatus, exposed to 35 μg/l of DBP after
22 days. Thus, DBP appears to be able to act as an
endocrine disruptor at environmentally relevant concen-
trations in sexually mature three-spined sticklebacks,
suggesting that, similarly to mammals, DBP acts as
anti-androgenic also in fish (Aoki et al. 2011).
In newborn guppies, Poecilia reticulata, exposed for
3 months to a sub-lethal dose of nonylphenol (NP), an
anomalous transcription of hepatic vitellogenin correlat-
ed with significant reduction of the gonadosomatic in-
dex (GSI) was described. Males did not approach fe-
males and typical posturing and sigmoid courtship
1568
behaviors were impaired. Normal courtship behavior
had a 3-month delay, suggesting that NP has an estro-
genic potency sufficient to temporary disrupt reproduc-
tion (Cardinali et al. 2004).
Effects of EDCs on sex reversal
In fish, EDCs exposures during critical developmental
stages can alter sex phenotypes (Fenske and Segner
2004; Scholz and Klüver 2009), impair gonad develop-
ment (Zhu et al. 2016), and disrupt the progress of the
reproductive process (Fenske et al. 2005; Schäfers et al.
2007). In teleosts, indeed, the tight interplay between
genetic and environmental mechanisms of sex determi-
nation is responsible for the final gonadal differentia-
tion, making teleosts excellent experimental models to
study this process (Devlin and Nagahama 2002;
Guiguen et al. 2010). Estrogens play a crucial role in
differentiation of fish ovaries, resulting susceptible to
several estrogenic mediators and endocrine disruptors.
In a study exposing medaka (Oryzias latipes) embryos
to a potent nonsteroidal estrogen, the diethylstilbestrol
(DES), molecular and cellular mechanisms involved in
ovarian differentiation were analyzed (Paul-Prasanth
et al. 2011). A short-term exposure, from 0 to 8 dpf, of
XY fish to DES, induced a reduction of the expression
of genes codifying for the male-dominant somatic cell
markers, such as GSDF, SCP3, and 42SP43, while a
longer period, 28 days, caused a strong reduction of
their gene expression, and the gonads developed as
ovaries clearly highlighting the estrogenic properties of
this pollutant.
In general, the presence of oocytes in testis of
gonochoristic species is considered a pathological con-
dition, and in the roach, Rutilus rutilus, a higher preva-
lence of intersex has generally been observed down-
stream of major wastewater treatment plants in English
rivers (Jobling et al. 1998, 2002). Detection of intersex
individuals and upregulation of aromatases,
vitellogenins, and zona radiata proteins in juvenile and
male fish have been also described in a wide array of
mullet species worldwide, clearly evidencing a
xenoestrogenic contamination. Much effort has been
focused on the identification of new molecular markers
for a precocious identification of feminization responses
and intersex conditions in fish populations. The ovarian
RNA commonly shows a strong expression of 5S rRNA
in oocytes, supporting the use of such RNA as
Fish Physiol Biochem (2018) 44:1561–1576
molecular marker of oocyte presence in the testis to
assess xenoestrogenicity in field conditions (Ortiz-
Zarragoitia et al. 2014). Intersex in the form of testicular
oocytes was described in largemouth bass (Micropterus
salmoides) collected over a 5-year period survey in
surface waters on the Delmarva Peninsula, USA, a
region dominated by agricultural land use and poultry
production (Yonkos et al. 2014). Disruption by EDCs
can be transitory or permanent, mainly depending on the
nature, the concentration and the windows of exposure
to the chemical. However, despite an increase of vtg
mRNA transcripts and protein synthesis was document-
ed in G. niger exposed for 48 h to alkylphenols, no
histological changes were observed in the testis
(Maradonna et al. 2004). Nevertheless, a number of
studies described the ability of zebrafish to recover from
estrogenic exposures (Larsen et al. 2009; Baumann et al.
2014a, b). An exposure to the estrogen-antagonist
fadrozole, alone or in combination with EE2, perma-
nently disrupts zebrafish sexual development, inducing
masculinization and severe pathological testicular alter-
ations. Gonad histopathology revealed interstitial pro-
teinaceous fluid deposits, ovarian atresia, and presum-
ably degenerative mineralization. In addition, the go-
nadal changes induced by EE2 alone seem to be partial-
ly reversible after a recovery period (Luzio et al. 2016).
BPA exposure induced an intersex phenotype in carp: in
testes of males exposed to 1000 μg/l BPA, in addition to
several inflammatory cells into the residual lobules, few
previtellogenic oocytes were scattered within the testic-
ular tissue (Mandich et al. 2007).
Effects of EDCs on epigenetic process
The scientific community started to talk about epige-
netics in 1942 when Conrad Waddington described it as
“…the interaction of genes with their environment
which brings the phenotype into being” (Waddington
1942). Epigenetic modifications are now defined as
reversible and heritable chromatin chemical modifica-
tions resulting in adjustment of its activity without
changes in the underlying DNA sequence. Epigenetic
plays an important role in many cellular processes from
differentiation, growth, metabolism, and regulation of
gene expression by silencing or enhancing specific
genes. In addition, it was shown that epigenetic mech-
anisms enable the communication between gene tran-
scription and environment (Esteller 2007). Many
Fish Physiol Biochem (2018) 44:1561–1576
epigenetic modifications include DNA methylation and
histone modifications (Labbé et al. 2017). Recently, the
implication of microRNA deregulation in response to
EDCs has been noted in many vertebrate species
(Cameron et al. 2016). In this context, data obtained in
D. rerio and in Carassius auratus demonstrated that the
exposure to fluoxetine, except from a deregulation of the
hormone-dependent processes, causes changes in
miRNA expression, potentially representing a new group
of biomarkers of the exposure to toxicants. Noteworthy
would be the increasing knowledge on the transcriptional
variation of miRNA after chronic or low-level EDCs
exposure, providing an epigenetic fingerprint of environ-
mental exposure. The detrimental effects of EDCs on
epigenetic mechanisms were evidenced a long time ago;
for example, the hexabromocyclododecane (HBCD) and
the 17-β oestradiol (E2) were evaluated on global DNA
methylation levels in the gonads of the three-spine stick-
leback (Gasterosteus aculeatus). Results demonstrated an
increase of global genomic hypermethylation in both
ovaries and testis although the increase seen in the female
gonads was not statistically significant (Aniagu et al.
2008). In addition, recent studies demonstrated the ability
of EDCs to induce epigenetic transgenerational inheri-
tance (Skinner 2014).
Many compounds, including dioxins, phthalates, and
polychlorinated biphenyls can directly affect the
germline. Oocytes and spermatozoa are specialized cells
which possess the ability to fuse and to generate an
embryo. At puberty, this embryo will develop into a
mature organism producing gametes. At this point, epi-
genetic modifications are major actors of cell differenti-
ation and reprogramming processes, both during game-
togenesis and embryo development (Labbé et al. 2017).
In this context, it was recently demonstrated that repro-
ductive physiology was significantly impaired in
zebrafish females chronically exposed to 5 ul/l BPA.
In mature follicles, a downregulation of signals involved
in oocyte growth and maturation, associated with a
promotion of apoptosis was reported (Santangeli et al.
2016). This likely occurred through changes in the
chromatin structure mediated by histone modifications,
indicating that the negative effects of BPA on the female
reproductive system may be due to its upstream ability
to deregulate epigenetic mechanisms. In the zebrafish
ovary, BPA interferes with histone modification, leading
to the downregulation of lhcgr mRNA levels, potential-
ly affecting also global methylation and interfering with
the dnmt expression (Santangeli et al. 2016, 2017a)
1569
(Fig. 2). In rare minnow, G. rarus, it was demonstrated
that a 7-day exposure to 15 μg/l BPA significantly
upregulates the expression of gonadal cyp19a1a mRNA
levels, while a longer exposure, 35 days, resulted in a
downregulation of aromatase mRNA. Only in the ovary,
an inverse correlation was demonstrated between
cyp19a1 DNA methylation status and its mRNA levels,
suggesting that in the ovary, DNA methylation could be
responsible for the transcriptional control of cyp191a1
(Liu et al. 2014b). In both gonads, BPA could change
the DNA methylation in the 5′ flanking region of
cyp17a1 and cyp11a1 at specific CpG loci. While a
direct correlation was found between CpG methylation
at S4 loci and cyp11a1 gonadal mRNA levels, an in-
verse correlation was found between CpG methylation
at P4 and P6 loci and cyp17a1 mRNA levels (Zhang
et al. 2017). In the testis, BPA caused a hypermethyla-
tion of global DNA, inducing a decrease of ten-eleven
translocation proteins (TETs) after a 7 day exposure to
15 ul/l. The exposure to a higher (225 μg/l) BPA con-
centration significantly increased the global DNA meth-
ylation due to DNMTs upregulation, which was due to
an increased de novo synthesis of glutathione (Yuan
et al. 2016). On the contrary, in zebrafish ovary, expo-
sure to 1 mg/l BPA induced a decrease of the expression
of dnmt1, resulting in a downregulation of the global
methylation (Laing et al. 2016). These contrasting re-
sults suggest that the epigenetic effects induced by the
single pollutants can be concentration- and time-
dependent and vary among vertebrates. In a field survey,
juvenile silver eels, Anguilla anguilla, were sampled in
two locations in the southwest of France, presenting a
different level of contamination. Results evidenced that
the DNA methylation levels of the genes encoding for
the aromatase and fshr were higher in contaminated fish
than in fish from the clean site, suggesting that pollution
could be responsible for an increase in the methylation
level of gonadal genes involved in oocyte growth and
differentiation. Chronic pollution experienced by ani-
mals throughout their life can affect their reproductive
capacities and possibly, their offspring, inheriting such
epigenetic marks (Pierron et al. 2014).
SOS environment: evidence from the wild
Laboratory trials are essential to understand and focus
on the effects of the single pollutants on fish physiology.
In the wild, fish are exposed to many xenobiotics,
1570
Fig. 2 Effects of BPA on histone modification. a Histograms
bar represents the enrichment of H3K4me3 and
H3K27me—trimethylation of lysine 4 (K4) and 27 (K27) in the
amino terminal of histone 3 (H3). Results are analyzed with the
method of percentage of input. b Table represents the gene
potentially xenoestrogens, whereby understanding the
impacts of mixtures could be of great importance. In this
contest, zebrafish were exposed to a mixture of the same
contaminants widespread in the Douro River (Portugal).
Results clearly demonstrated a reduction of germ cell
volume in both male and female gonads, but gameto-
genesis was significantly disrupted only in males. In
testis, the presence of abundant interstitial tissues and
the presence of a vitellogenin-derived proteinaceous
fluid was evidenced (Silva et al. 2012). Hypotheses have
been formulated to explain the deleterious effect exerted
by these environmental xenoestrogen mixes in males; a
direct action on testis leading to an inhibition of andro-
gen production or a different-level interference with the
hormonal cascade that regulates maturation, finally
inhibiting spermatogenesis, have been hypothesized.
A major source of contamination for wild species is
represented by wastewaters treatment plant (WWTP)
discharges in the ecosystem. Fish inhabiting highly in-
dustrialized or the downstream areas, present severe
reproductive problems from impaired gametogenesis
to intersex gonads. A field study conducted in two
Fish Physiol Biochem (2018) 44:1561–1576
expression level of several genes involved in oogenesis process. In
red is pointed out the lhcgr gene expression which downregulation
was correlated with the specific profile of the abovementioned
histone modifications
different sites of the Bay of Biscay clearly evidenced
the presence of intersex gonads in several mullet species
associated with vtga gene transcription in the liver of
both intersex and male fish. In females, transcription
levels of cyp19a1a and gtf3a suggested an alteration of
gametogenesis (Valencia et al. 2017). In a USA field
survey, male largemouth bass, Micropterus salmoides,
were sampled from two locations in Lake Mead, a site
influenced by treated municipal wastewater effluent and
urban runoff (Las Vegas Bay), and a reference site
(Overton Arm). GSI and sperm motility did not differ
between sites, but sperm count was lower by nearly 50%
in fish from Las Vegas Bay. A non-linear positive asso-
ciation between ketotestosterone (KT) and GSI was
identified (Goodbred et al. 2015). In conclusion, the
higher concentration of contaminant body burdens
coupled with the reduced levels of KT and sperm count
in fish from Las Vegas Bay suggest that male reproduc-
tive condition was influenced by contaminant expo-
sures. Plasma KT and GSI were associated in a non-
linear fashion. This observation provides a framework
to understand why GSI was similar between male bass
Fish Physiol Biochem (2018) 44:1561–1576
from both sites despite their large difference in plasma
KT. The non-linear model also suggested the existence
of post-gonadal growth functions of KT at high concen-
trations (Goodbred et al. 2015). In another monitoring
study, adult females Hoplias malabaricus were sampled
at two locations in São Paulo State (Brazil), one refer-
ence site, Ponte Nova (PN) reservoir, and the polluted
Billings (BIL) reservoir. The GSI, including the pre-
dominance of vitellogenic oocytes, was higher in spring
and summer in both locations, but the oocyte recruit-
ment dynamics were different. In winter, females from
BIL presented vitellogenic oocytes and high 11-
ketotestosterone levels suggesting precocity in the
vitellogenic phase respect to the females from PN. In
animals from PN, high deposition of lipids occurred in
the ovaries. However, plasma estradiol levels did not
vary throughout the annual cycle. In animals from BIL,
plasma estradiol levels peaked during the summer, but
the ovarian lipid content remained unchanged through-
out the year. Fish of both sites regularly reproduce
(Gomes et al. 2015). Summarizing, results show that
in the polluted area, H. malabaricus developed an effi-
cient reproductive plasticity with a longer reproductive
period and an adjustment in hormonal profiles. Recent-
ly, 21 PCBs and 5 organochlorines were detected on the
order of ng/g in anchovy, Engraulis encrasicolus,
caught during a field survey in the western Adriatic
Sea. Female sex-specific reproductive biomarkers, vitel-
logenin, vitellogenin receptor, and genes encoding for
the zona radiata proteins were found transcribed also in
male tissues; in addition, intersex was histologically
identified in the 13% of the testis (Miccoli et al. 2017).
These findings contribute to build a novel scenario; to
date, in fact, variations of the European anchovy stocks
have been linked to uncontrolled fisheries and climatic
changes, without considering the negative effects of
environmental pollution on fertility and reproduction.
Omics technologies: new paths forward
In the last decades, the development of omics technol-
ogies (transcriptomics, proteomics, and metabolomics)
has provided the scientific community with new inte-
grative approaches to explore new aspects of biology.
These technologies enable researchers to assess thou-
sands of genes, proteins, or metabolites in a single
sample offering the potential to investigate responses
to chemical stressor by identifying molecular-level
1571
evidence indicative of a specific mechanism of action
and to improve our understanding of molecular toxicity
pathways (van Ravenzwaay et al. 2012; Ellinger-
Ziegelbauer and Ahr 2014; Baker and Hardiman
2014). Moreover, they offer great potential to study
EDCs. While the studies reviewed in the previous sec-
tions provide essential knowledge about gametes ad-
verse outcomes triggered by EDCS (i.e., gametes qual-
ity and intersex gonads), they only partially investigate
the molecular mechanisms eliciting the observed effect.
Although, many studies have already applied omics
approaches to investigate the molecular mechanisms
involved in ovary and testis maturation (Tingaud-
Sequeira et al. 2009; Groh et al. 2011; Martyniuk et al.
2013; Xu et al. 2016), only few of them have focused on
elucidating the factors involved in the control of egg
(Kohn et al. 2015; Yilmaz et al. 2017; Żarski et al. 2017)
and sperm (Li et al. 2017) quality in fish and even less
are those investigating how EDCs affect fish gametes
quality. Santos and collaborators (Santos et al. 2007)
investigated the gonadal transcriptome responses fol-
lowing exposure to EE2 in both male and female
zebrafish and identified genes involved in the regulation
of cell cycle, ubiquitin system, and glutathione peroxi-
dase to be affected and associated with the changes
observed in gametes quality. Gao et al. (Gao et al.
2017) identified EE2 to disrupt oocyte development
and spermatogenesis in adult rare minnow (Gobiocypris
rarus) by applying gonadal transcriptome analysis.
These studies highlight the ability of omics technologies
to unravel mechanisms of toxicity of EDCs underlying
gametes disruption.
Conclusion
Environmental EDCs have a negative impact on both
male and female gametogenesis. These impacts not only
influence the number of gametes but also their quality
on a genetic and epigenetic level, concealing a potential
transgenerational effect. The physiopathology leading
to altered fertility is based on hormonal disturbances.
The appearance of intersex gonads, gamete quality, and
epigenetic alterations, probably work in combination, to
explain this negative impact. The main concern is the
difficulty to identify the individual role of specific pol-
lutants in the environment, since fish in the wild are
exposed to several pollutants simultaneously. Under-
standing the mechanisms of EDCs action is a priority
1572
due to the growing number of reports describing the
negative effects of such compounds on aquatic organ-
ism reproduction. This is a highly challenging task,
because most of the time, these pollutants occur in
water bodies at very low concentrations. The evi-
dence obtained in the diverse experimental models
could be of great importance to formulate recommen-
dations for water quality and to set the maximum
level of pollutants allowed into the environment.
Studies need to be carried out, and the necessary
policy changes should be done in a definite time-
frame in order to have a proper assessment of endo-
crine disruptors. The damage that has already been
done needs to be assessed and dealt with. Regulatory
decision and research on EDCs should be based on
principles of endocrinology. We envisage that appli-
cation of omics technologies for the investigation of
EDCs effects on fish gametes have the potential to
address these challenges and provide essential
knowledge to be integrated into regulatory decisions.
Funding information Supported by the Ministry of
Health—RICERCA FINALIZZATA 2009 “Food and environ-
mental safety: the problem of the endocrine disruptors” to OC
and AM; 2012 2015 COST European Cooperation in the field of
Scientific and Technical Research “AQUAGAMETE” to OC and
by Progetti di Rilevante Interesse Nazionale (PRIN) 2010–2011
prot 2010W87LBJ to OC.
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