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Mercury-induced reproductive impairment in fish
Article in Environmental Toxicology and Chemistry · May 2009
DOI: 10.1897/08-151.1 · Source: PubMed
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Environmental Toxicology and Chemistry, Vol. 28, No. 5, pp. 895–907, 2009
䉷 2009 SETAC
Printed in the USA
0730-7268/09 $12.00 ⫹ .00
Critical Review
MERCURY-INDUCED REPRODUCTIVE IMPAIRMENT IN FISH
KATE L. CRUMP and VANCE L. TRUDEAU*
Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ontario K1N 6N5, Canada
( Received 2 April 2008; Accepted 24 November 2008)
Abstract—Mercury is a potent neurotoxin, and increasing levels have led to concern for human and wildlife health in many regions
of the world. During the past three decades, studies in fish have examined the effects of sublethal mercury exposure on a range
of endpoints within the reproductive axis. Mercury studies have varied from highly concentrated aqueous exposures to ecologically
relevant dietary exposures using levels comparable to those currently found in the environment. This review summarizes data from
both laboratory and field studies supporting the hypothesis that mercury in the aquatic environment impacts the reproductive health
of fish. The evidence presented suggests that the inhibitory effects of mercury on reproduction occur at multiple sites within the
reproductive axis, including the hypothalamus, pituitary, and gonads. Accumulation of mercury in the fish brain has resulted in
reduced neurosecretory material, hypothalamic neuron degeneration, and alterations in parameters of monoaminergic neurotransmission. At the level of the pituitary, mercury exposure has reduced and/or inactivated gonadotropin-secreting cells. Finally, studies
have examined the effects of mercury on the reproductive organs and demonstrated a range of effects, including reductions in
gonad size, circulating reproductive steroids, gamete production, and spawning success. Despite some variation between studies,
there appears to be sufficient evidence from laboratory studies to link exposure to mercury with reproductive impairment in many
fish species. Currently, the mechanisms underlying these effects are unknown; however, several physiological and cellular mechanisms are proposed within this review.
Keywords—Mercury
Endocrine disruption
Fish
Reproduction
bioaccumulation of methylmercury is evident, because the average proportion increases from approximately 5% methylmercury in the aqueous environment to 15% in phytoplankton,
30% in zooplankton, and more than 90% in fish [9]. Total
mercury levels in freshwaters of North America generally do
not exceed the ng/L range [8,10]. Methylmercury in surface
water normally comprises 0.1 to 5% of the total mercury content, but in anoxic waters, it can be one of the dominant mercury species [8]. Concentrations of methylmercury in aquatic
organisms increase because of trophic transfer. Methylmercury
concentrations in phytoplankton range from 0.003 to 0.005
␮g/g wet weight [9], whereas the range of methylmercury
concentration increases to from 0.03 to 0.22 ␮g/g wet weight
in muscle of lower-trophic-level fish, such as bluegill (Lepomis
macrochirus) and black crappie (Pomoxis nigromaculatus)
[11]. In higher-trophic-level fish, including largemouth bass
(Micropterus salmoides ), methylmercury concentrations
range from 0.10 to 1.0 ␮g/g wet weight [11]. Health Canada
has established a human consumption guideline level of 0.5
␮g/g wet weight of total mercury in commercial marine and
freshwater fish. Even in remote regions, methylmercury in fish
sometimes exceeds the concentration deemed to be safe for
human consumption [3]. A study of 775 water bodies in the
province of Québec, Canada, reported maximum muscle mercury levels of 2.30, 4.90, and 5.60 ␮g/g wet weight for northern
pike (Esox lucius), walleye (Stizostedion vitreum), and lake
trout (Salvelinus namaycush), respectively [12]. The adverse
effects of inorganic mercury and methylmercury on humans
is well documented [13,14]; however, relatively little is known
about the effects on the health or condition of wild-fish populations.
Methylmercury exposure can affect behavior, biochemistry,
growth, reproduction, development, and survival in fish
[10,15]. Long-term laboratory studies of fish exposed to die-
INTRODUCTION
The presence of industrial contaminants in the aquatic environment has been of increasing concern for human and wildlife health. Mercury is of particular concern because of the
bioaccumulation and potent neurotoxicity of some of its forms.
Atmospheric emissions of mercury are the result of anthropogenic activities (metal production and coal-fired power generation) and natural geochemical processes (volcanic eruptions
and forest fires). Based on lake sediment records from midcontinental North America, atmospheric deposition of mercury
is estimated to have tripled over the past 140 years [1]. Once
in the atmosphere, mercury can exist for up to a year, allowing
widespread distribution via long-range atmospheric transport.
Mercury pollution is a ubiquitous problem, with atmospheric
deposition contaminating watersheds in areas far from anthropogenic or natural atmospheric point sources [1,2].
Several forms of mercury are present in the aquatic environment, including elemental, ionic, and organic [3]. Methylmercury, one of the most toxic forms, bioaccumulates in fish
primarily through dietary uptake [4,5]. The level of bioaccumulation is a function of age, species, and trophic transfer [4].
Preferential bioaccumulation of methylmercury over inorganic
mercury results from the greater assimilation efficiency [6]
and trophic transfer efficiency [7] of methylmercury. Inorganic
mercury is found in piscivorous birds and mammals, but its
presence generally is attributed to the ability of these species
to convert methylmercury to the less toxic inorganic form
rather than to dietary uptake or bioaccumulation [8]. Specific
* To whom correspondence may be addressed
(trudeauv@uottawa.ca).
Published on the Web 12/10/2008.
Presented at the SETAC 28th Annual North American Meeting,
Milwaukee, WI, USA, November 10–15, 2007.
895
896
Environ. Toxicol. Chem. 28, 2009
tary methylmercury have noted loss of coordination, diminished swimming activity, starvation, and mortality [8]. Industrial activities have resulted in highly contaminated local fish
populations, with muscle mercury concentrations ranging from
8.4 to 24 ␮g/g wet weight in Minamata Bay, Japan [16], and
from 6.3 to 16 ␮g/g wet weight in Clay Lake, Ontario, Canada
[17]. At lower concentrations, mercury may affect fish populations indirectly, through impairment of physiological processes, rather than through the severe neurological impairment
and death observed in extreme contamination incidents. During the past three decades, studies using sublethal concentrations of mercurials have demonstrated negative impacts on
reproduction in numerous freshwater fish species [18]. Weiner
and Spry [10] concluded that reduced reproductive success
was the most plausible effect of mercury on wild-fish populations at current exposure levels in aquatic ecosystems. The
mechanistic effects of methylmercury on reproduction, however, remain unclear.
The goal of the present paper is to present a comprehensive
review of the data demonstrating effects of sublethal mercurial
pollution on the reproductive axis of teleost fish, from neuroendocrine control through to gonadal development and
spawning. In addition to providing a review, the present paper
also provides an interpretive discussion of possible mechanisms underlying mercury-induced reproductive impairment
and future directions for studies. Evidence from aqueous exposures, dietary exposures, and correlative studies using wildfish is discussed. The concentrations of mercury used in the
aqueous exposures generally are much higher (␮g/L) than
those encountered in the environment (ng/L), and as such, the
interpretations from these studies are limited to discussions of
possible mechanistic effects. Interpretations from wild-fish
studies also are limited because of the confounding factor of
multiple contaminants; the potential synergistic effects of natural and anthropogenic endocrine active contaminants make it
challenging to elucidate mercury-specific effects. Although
particular emphasis is placed on methylmercury in fish, when
appropriate, data from fish exposures to other forms of mercury
and pertinent examples from the rodent literature are drawn
on to further our understanding of the mechanisms underlying
reproductive impairment in fish.
DISRUPTION OF HYPOTHALAMIC AND
PITUITARY FUNCTION
The hypothalamus
Multiple hypothalamic neuropeptides and neurotransmitters
exert a dual stimulatory and inhibitory control over pituitary
gland release of gonadotropic hormones that regulate gonadal
function in fish [19,20]. The inhibitory effects of pollutants
on reproduction may be mediated via changes in regulation at
the level of the hypothalamus and pituitary. Methylmercury
is a known neurotoxin, the effects of which in fish after longterm dietary exposure are characterized by brain lesions, oxidative stress, and decreased activity [21]. Various molecular
targets within the nervous system have been implicated in
methylmercury neurotoxicity, including the cytoskeleton, axonal transport, neurotransmission parameters, protein synthesis, and mitochondrial energy-generating systems [22]. Although methylmercury will readily react with any sulfhydryl
group, it shows brain region specificity, with neuron loss only
in certain areas. Methylmercury exposure in Atlantic salmon
(Salmo salar; 0.68 ␮g Hg/g wet wt brain tissue) caused widespread vacuolation, with the most severe effects noted in the
K.L. Crump and V.L. Trudeau
medulla and cerebellum and the cerebrum and other regions
being less affected [21]. Furthermore, mitochondria from cortical astrocytes are more sensitive to methylmercury toxicity
than are those in cerebellar astrocytes [23]. Thus, it is important to examine the effects of mercury exposure on isolated
brain regions. In addition, the endocrine system amplifies signals such that minor changes in neuronal function may cause
major disturbances in reproduction [18]. Consequently, mercury may affect reproduction via the hypothalamus at levels
lower than those that induce general neuronal damage.
In teleosts, gonadotropin-releasing hormone (GnRH) neurons, originating in the ventral telencephalic–preoptic–anterior
region of the forebrain, project into the anterior pituitary gland
[24]. Gonadotropin-releasing hormone regulates the synthesis
and release of the gonadotropins (luteinizing hormone [LH]
and follicle-stimulating hormone [FSH]), making it a crucial
neuroendocrine mediator of reproductive function. Histological analysis of neurons in the hypothalamic preoptic nuclei
of the murrel (Channa punctatus) has provided evidence for
potential inhibitory effects of mercury on neurosecretion. The
magnocellular isotocin and vasotocin-secreting neurons of the
preoptic nuclei were smaller and inactive (characterized by
reduced staining of neurosecretory material) in murrels treated
for six months with 10 ␮g/L of inorganic mercury [25]. This
may be important, because isotocin and vasotocin control sexual behavior in teleosts [26]. Many neurons also exhibited
various degrees of degeneration, from apoptotic chromatin
condensation to necrotic nuclear dropout. Some of the affected
neurons were uncharacterized and may have included GnRH
cells, although this was not proven. In rats, dietary methylmercury decreased GnRH content in the medial hypothalamus
[27]. The paucity of information on the effects of mercury on
GnRH neurons in vertebrates may be a reflection of the difficulty in studying them, because they do not form a compact
nucleus but, rather, a diffuse, interconnected network (i.e., hypothalamus and telencephalon) that is specifically involved in
reproductive control [28].
Genomic techniques, such as real-time quantitative polymerase chain reaction and microarray, allow the simultaneous
measurement of genes involved in multiple biochemical pathways. Assessment of methylmercury-induced changes in hypothalamic gene expression could provide mechanistic information relating methylmercury exposure to reproductive
changes. Genetic responses to dietary methylmercury exposure
were assessed in whole brain of zebrafish (Danio rerio) using
real-time quantitative polymerase chain reaction after 7, 21,
and 63 d [29]. No changes in transcript levels were measured
for any of the 13 genes assayed in fish with mean muscle
mercury concentrations of 3.0 and 6.54 ␮g/g wet weight (assuming 80% water content) [29]. None of the genes assessed
are specifically involved in regulating reproduction, and the
results of the study were limited by the use of whole brain
rather than isolated brain regions relevant to neuroendocrine
control. A rainbow trout cDNA microarray recently was used
to identify differences in liver gene expression patterns between wild-caught cutthroat trout (Oncorhynchus clarki) from
two lakes with significantly different mercury concentrations
(0.016 and 0.054 ␮g/g wet wt whole fish) in their respective
fish [30]. Surprisingly, several reproductive neuroendocrine
genes (GnRH-1, GnRH-2, and GnRH receptor) were expressed
in the liver and were affected by mercury. Expression levels
of GnRH-1 and -2 were significantly lower (approximately
eightfold and approximately threefold, respectively), and
Environ. Toxicol. Chem. 28, 2009
Mercury-induced reproductive impairment in fish
GnRH receptor was significantly higher (⬃13-fold) in cutthroat trout with elevated mercury levels. To our knowledge,
a comprehensive assessment of gene expression changes in
the hypothalamus in relation to methylmercury-induced reproductive impairment in any species remains to be done.
Neurotransmission
Neurotransmitters are released by presynaptic nerve cells
into the synapse, where they stimulate receptors on postsynaptic nerve cells [31]. The release of hypothalamic and pituitary hormones is regulated by neurotransmitters, and in turn,
neurotransmitter release is modulated by hormones. The effects of mercury on neurotransmission may be mediated by
disruption of synthesis, storage, release, and/or reuptake by
the presynaptic cell; by changes in activity of the deactivating
enzyme; or by mimicking/blocking the neurotransmitter at the
synapse [31]. Individual neurotransmitters may be stimulatory
and/or inhibitory in terms of reproduction and may modulate
the effects of other neurotransmitter systems, resulting in a
multitude of effects from alterations in one neurotransmitter
[20]. Furthermore, because each neurotransmitter may have
more than one type of receptor, and because these various
receptors may be on different types of cells, alterations in the
level of a single neurotransmitter may affect multiple cells in
different ways. In fish, mercury affects the levels of several
neurotransmitters involved in regulating the synthesis and release of GnRH and the gonadotropins.
Mercury exposure decreased serotonin (5-HT) concentrations in the brains of several fish species. Serotonin is synthesized from the amino acid tryptophan and activates GnRH
neurons to stimulate release of LH from the pituitary. In a
laboratory study with Nile tilapia (Oreochromis mossambicus), 5-HT levels decreased significantly in the hypothalamus
of males exposed for six months to 15 and 30 ␮g/L of inorganic
mercury [32]. No change in 5-HT levels was observed in the
telencephalon or optic lobe. Levels of 5-HT also decreased in
whole brain of walking catfish (Clarias batrachus) exposed
for 90 to 180 d to 40 ␮g/L of methylmercury [33]. A field
study with mummichog (Fundulus heteroclitus) from an unpolluted site (0.029 ␮g Hg/g wet wt brain tissue) and a polluted
site (0.066 ␮g Hg/g wet wt brain tissue) observed lower
5-HT levels in the medulla, but not in the cerebellum, of fish
from a mercury-contaminated site [34]. Those authors also
noted lower levels of the 5-HT metabolite, 5-hydroxyindole
acetic acid (5-HIAA), in contaminated fish, suggesting that the
low 5-HT levels may be caused by reduced synthesis rather
than by increased degradation. These results agree with those
of Tsuzuki [35], who observed a decrease in 5-HT and
5-HIAA in the hypothalamus of rats exposed orally to 4.0
␮g/g of methylmercury for 50 d. Further support in rats comes
from the methylmercury-induced decrease in activity of tryptophan hydroxylase, the rate-limiting biosynthetic enzyme in
the 5-HT pathway [36]. The inhibitory effects of mercury on
the 5-HT pathway may depend on the developmental stage of
fish. For example, methylmercury exposure (10 ␮g/L) increased 5-HT in the heads of 7- and 14-d posthatch mummichog larvae with no concurrent change in 5-HIAA [37].
Collectively, these studies indicate that in sexually mature fish,
mercury exposure can decrease 5-HT levels, which in turn
could be linked to decreased reproduction.
Levels of the catecholamines norepinephrine and dopamine
also have been measured after mercury exposure in fish. Dopamine is the single most potent inhibitor of LH secretion in
897
fish, and it acts by binding directly to D2 receptors on the
gonadotrophs and by altering GnRH signaling [24]. Dopamine
was significantly increased in brains of walking catfish after
a 90-d exposure to 40 ␮g/L of methylmercury [33]. Levels of
norepinephrine, a stimulatory monoamine synthesized from
dopamine, also were increased significantly in whole brain of
walking catfish after methylmercury exposure [33]. Aqueous
methylmercury (10 ␮g/L) elevated dopamine levels in heads
of 7- and 14-d posthatch mummichog larvae without any
change in its major metabolites, 3,4-dihydroxyphenylacetic
acid or 3-methoxy-4-hydroxyphenylacetic acid [37]. No significant differences in dopamine, norepinephrine, or 5-HT were
observed in fathead minnows (Pimephales promelas) after a
10-d aqueous exposure to inorganic mercury (1.69–13.57
␮g/L); however, whole-brain measurements were made that
would have obscured any brain region–specific changes [38].
In rats, methylmercury also has been shown to increase dopamine in the hypothalamus while decreasing the levels of
3,4-dihydroxyphenylacetic acid [35]. Total mercury concentrations in the brain of wild mink correlated negatively with
D2 receptor density and binding affinity, suggesting a potential
adaptive mechanism to counter the mercury-induced increase
in dopamine [39]. Whether this adaptive mechanism exists in
fish is not known. If mercury-exposed fish are unable to counter the increases in dopamine, then LH release may be reduced
and reproduction impaired.
As discussed above, LH release in fish is affected by several
monoamine neurotransmitters (dopamine, norepinephrine, and
5-HT), suggesting that alterations in their rate of synthesis or
degradation could impact reproduction. The degradation of
monoamines involves monoamine oxidase (MAO), which is
an intraneuronal enzyme complex responsible for oxidative
deamination [28]. Inorganic mercury decreased MAO activity
in murrel after a six-month aqueous exposure (10 ␮g/L) [25]
and in Atlantic salmon after a four-month dietary exposure
(0.13 ␮g Hg/g wet wt brain tissue) [21]. Methylmercury induced a much more potent inhibitory effect compared with
inorganic mercury on MAO activity in Atlantic salmon [21].
Similarly, organic mercury (40 ␮g/L) was a stronger inhibitor
of MAO compared with inorganic mercury (50 ␮g/L) in walking catfish after 45 d of aqueous exposure [33]. Furthermore,
methylmercury significantly inhibited the rat synaptosomal
MAO activity in all brain regions tested, including the hypothalamus [40]. These results indicate that mercury in both
its inorganic and organic forms is capable of impairing the
monoaminergic systems known to be key modulators of hypothalamic–pituitary control of gonadotropic function and,
thus, reproduction. A direct, causal link between monoaminergic disruption and decreased reproduction is required for
substantiation of this hypothesis; however, this link has yet to
be clearly established.
The pituitary
The gonadotropic hormones LH and FSH are released from
the pituitary in fish and control the annual cycle of gonadal
growth, ovulation in females, sperm release in males, and production of sex steroids in both sexes [41–43]. Therefore, disruption of gonadotropin secretion can have a major impact on
fertility. The effects of mercury on the pituitary have been
limited to morphological analysis and measurements of pituitary-derived hormone levels in plasma. Critically missing are
studies regarding the direct effects of mercury on pituitary
hormone release in vitro.
898
Environ. Toxicol. Chem. 28, 2009
The morphological effects of mercurials on the pituitary
gonadotroph in murrel and walking catfish have been studied
by several groups. Histological analyses demonstrated that
compared to controls, gonadotrophs were smaller, appeared to
be inactive, and were fewer in number in both species of fish
exposed to 10 to 50 ␮g/L of methylmercury, inorganic mercury, or a mercurial fungicide for six months [25,44–46]. The
mercury-induced inhibition of gonadotropic activity was correlated with impaired gonadal development. Whether the decrease in sex steroid levels and, thus, an alteration of gonadal
feedback signals is causing the inhibition of gonadotropic
function is not known. Conversely, if the action of mercurials
is directed primarily at the hormone synthesis and release
mechanisms of the pituitary, the resulting decrease in gonadotropin could lead to the inhibition of gonadal development.
A thorough assessment of serum LH and FSH and associated
pituitary mRNA levels would help to determine whether the
pituitary is directly affected by methylmercury exposure. A
recent study in ovariectomized rats observed a decrease in
plasma LH after a 3-d exposure to dietary methylmercury [27].
Moreover, basal LH and sex steroid levels are significantly
reduced in postspawning female goldfish (Carassius auratus)
after a 28-d dietary exposure to 0.88 ␮g/g of methylmercury
(Crump and Trudeau, unpublished data). Further studies in fish
are required to determine the mechanisms of mercury-induced
inactivation of the gonadotrophs.
DISRUPTION OF MALE REPRODUCTIVE FUNCTION
The cycle of spermatogenesis in which stem cells divide
into mature spermatozoa is controlled by LH and FSH [47,48].
Throughout this cycle, the testes increase in size, which can
be measured by an increase in the gonadosomatic index (GSI;
gonad wt/body wt · 100) and usually is maximal in the breeding
season.
Disruption of testes function can be classified as either
cytotoxicological or endocrine in origin [18]. Cytotoxic effects
are characterized by damage to cellular integrity or alterations
in cell function, whereas endocrine effects involve the disruption of specific cells as a result of alterations in pituitary secretion or gonadal steroidogenesis.
Testicular morphology and spermatogenesis
Compared to control fish, testicular growth and spermatogenesis in murrel were arrested following exposure to inorganic mercury (10 ␮g/L) or a mercurial fungicide (200 ␮g/L)
for six months [25,44,45]. Necrotic areas were visible within
the testes of fish exposed to the mercurial fungicide, suggesting
direct cytotoxic effects [44]; however, no obvious signs of
degeneration were observed after inorganic mercury exposure
[45]. Spermatogenesis also was inhibited and GSI decreased
in male walking catfish after 180 d of exposure to organic and
inorganic mercury at 30 to 50 ␮g/L [49]. Similarly, guppies
(Poecilia reticulata) exposed to 5.6 ␮g/L of methylmercury
for three months [50,51] and medaka (Oryzias latipes) exposed
to 40 ␮g/L of methylmercury for 8 d [52] exhibited signs of
testicular atrophy and arrested spermiation. Wester [50] as well
as Wester and Canton [51] concluded that necrosis of the sperm
was occurring during the process of spermatogenesis, and
those authors proposed a mechanism involving the disruption
of the mitotic spindle. Mercury has been shown to result in
depolymerization of microtubules that form the mitotic spindle
[53,54], which is a possible mechanism underlying mercuryinduced genotoxicity in mammalian cells [55].
K.L. Crump and V.L. Trudeau
Disruption of testicular function also has been observed in
exposures to environmentally relevant levels of methylmercury. Dietary exposure for six months to methylmercury concentrations (0.1–1.0 ␮g/g) similar to those encountered by piscivorous fish in North America resulted in a significant decrease of GSI in juvenile male walleye with mean mercury
body burdens of 0.25 to 2.37 ␮g/g wet weight [56]. The normal
morphology of the testes was significantly altered, with the
appearance of atrophy. Decreased spermatogenesis and atrophied seminiferous tubules were observed in male Nile tilapia
(Oreochromis niloticus) after a seven-month exposure to
methylmercury [57]. Intraperitoneal implants in the male tilapia released methylmercury gradually, with final muscle concentrations ranging from 0.08 to 0.54 ␮g/g wet weight (assuming 80% water content) [57], which are similar to levels
reported for largemouth bass in the northeastern United States
[11]. From these experiments, it can be concluded that both
inorganic and organic mercury exposure via a variety of exposure routes can arrest gonadal development in male fish.
This effect may be cytotoxic in nature, because effects at the
level of the cell include structural alterations in Leydig cells
[25,44,45] and necrotic cell death [50,51]. The inhibition of
spermatogenesis in the murrel was correlated with small and
inactive gonadotrophs of the pituitary [25,45].
The data are inconclusive regarding the impacts of mercury
exposure on testicular growth. In addition to the studies mentioned previously, in several studies no change in GSI occurred. In wild-caught northern pike with muscle mercury content ranging from 0.12 to 0.62 ␮g/g wet weight, no significant
correlations were found between mercury content and GSI or
gonadal sex steroid concentration [58]; however, the small
sample size (n ⫽ 7) and high variability may have masked
any subtle effects. Dietary methylmercury decreased sex steroid levels but had no effect on gonadal development in male
fathead minnows with mean total mercury carcass concentrations of 0.86 to 3.84 ␮g/g wet weight after 250 d of exposure
[59]. Similarly, GSI was not affected in male tilapia exposed
to methylmercury, despite changes in testicular morphology
and spermatogenic arrest [57]. This indicates that histology
may provide a more sensitive indicator than GSI for assessing
the effects of mercury on gonadal development.
Mechanisms underlying testicular atrophy resulting from
mercury exposure are unknown, but they may involve alterations in mitotic activity [50,55]. Alternatively, mercury exposure can reduce both the size and number of gonadotrophs
[46]. In goldfish, human chorionic gonadotropin, which mimics endogenous gonadotropins, acts as a cell survival factor
during the late stage of spermatogenesis [60]. Therefore, methylmercury-induced testicular atrophy may be caused by the
induction of apoptosis through alterations in pituitary-derived
survival factors. Atrophy in developing testes could directly
impact individual reproductive potential.
Testicular hormone production
Gonadotropic regulation of spermatogenesis and spermiogenesis in fish is mediated by androgens secreted by the interstitial cells [61]. Murrel testes undergoing spermiation contain active interstitial cells characterized by large, rounded
nuclei with prominent nucleoli. In contrast, inactive and atrophied interstitial cells were observed in murrels exposed to
inorganic mercury (10 ␮g/L) or a mercurial fungicide (200
␮g/L) for six months [25,44,45,62]. Interstitial cells were inactive and showed signs of degeneration, including atrophy
Mercury-induced reproductive impairment in fish
and pyknosis in the male walking catfish, after mercury exposure [49]. Exposure to methylmercury resulted in inflammation and fibrosis of the interstitium in male guppies [50,51].
These cytotoxic effects imply potential adverse effects on the
steroidogenic potential of the interstitial cells.
The biosynthesis of androgens and estrogen requires 3␤hydroxy-⌬5-steroid dehydrogenase (3␤HSD); a decrease in
3␤HSD activity has been used to indicate decreased steroidogenic activity [18]. Kirubagaran and Joy [62,63] exposed male
catfish to organic and inorganic mercury at 30 to 40 ␮g/L for
six months and then measured 3␤HSD activity ex vivo in
sections of testes. The activity of 3␤HSD was inhibited completely in testes from fish exposed to methylmercury and, to
a lesser degree, in those from fish exposed to inorganic mercury. The loss of enzyme activity may be a direct action of
mercury on the testes or an indirect one at the hypothalamic–
pituitary level through inhibition of gonadotropin secretion,
because 3␤HSD is influenced by gonadotropin [64,65]. Furthermore, impaired steroidogenesis may have led to the inhibition of gonadal growth and spermatogenesis, as reported
in a similar experiment [49].
In male fish, androgens control gonadal development, secondary sexual characteristics, and sexual behavior [18]. Testosterone and/or 11-ketotestosterone secretion is high during
gonadal recrudescence and declines before spermiation [66].
Plasma testosterone levels significantly decreased in male fathead minnows fed diets containing 0.87 to 3.93 ␮g/g of methylmercury for 250 d [59]. Decreases in plasma 11-ketotestosterone also were observed in male Nile tilapia (O. niloticus)
after a six-month exposure to methylmercury [57] and in largemouth bass after an eight-week exposure to 3.12 to 6.25
␮g/g of dietary methylmercury [67]; no concurrent decrease
in 17␤-estradiol was observed in either exposure. These studies
provide further evidence that mercury alters steroidogenesis
in male fish. Further studies, however, are required to determine whether the effect is direct, at the gonad level, and/or
indirect, via disruption of the hypothalamus or pituitary.
In wild fish, the effects of mercury accumulation on androgens, particularly 11-ketotestosterone, are unclear. Similar
to the results of laboratory studies, both plasma testosterone
and 11-ketotestosterone were negatively and significantly correlated with muscle mercury content (mean value, 0.17 ␮g/g
wet wt) in sexually immature white sturgeon (Acipenser transmonatus) [68]. From these results, a mercury concentration
threshold associated with steroidogenic effects was estimated
to be 0.2 ␮g/g wet weight [69]. This threshold was determined
previously by Beckvar et al. [70] to be protective for juvenile
and adult fish based on a systematic analysis of studies assessing sublethal endpoints (growth, reproduction, development, and behavior) of mercury exposure. No significant alterations in 11-ketotestosterone levels were observed in sexually mature common carp (Cyprinus carpio), brown bullhead
(Ameiurus nebulosus), smallmouth bass (Micropterus dolomieui), or largemouth bass [71] containing mercury concentrations greater than this 0.2 ␮g/g wet weight threshold [69].
Nor were any alterations in testosterone levels correlated with
mercury accumulation in recrudescent northern pike with muscle mercury concentrations ranging from 0.28 to 0.64 ␮g/g
wet weight [58]. Although not statistically significant, testosterone levels in prespawning largemouth bass from a highmercury lake (5.42 ␮g Hg/g wet wt muscle tissue) were more
than 50% lower than those of bass in a control lake (0.30 ␮g
Hg/g wet wt muscle tissue) [72]. Conversely, a positive cor-
Environ. Toxicol. Chem. 28, 2009
899
relation between plasma 11-ketotestosterone concentrations
and mercury residues was observed in the male bass from the
high-mercury lake. To our knowledge, this is the only report
of increased steroidogenesis in male fish associated with methylmercury exposure. Increased steroidogenic enzyme activity
and a concurrent increase in plasma steroid levels were observed in female fish exposed to inorganic mercury. Positive
correlations also were observed between 11-ketotestosterone
and muscle mercury concentrations in female smallmouth bass
(0.21–0.51 ␮g/g wet wt) and common carp (0.06–0.33 ␮g/g
wet wt) [71]. Field surveys are complicated by the presence
of multiple contaminants in addition to methylmercury, which
may impact steroidogenesis. Although correlations exist between mercury concentrations and sex steroid levels, the effect
of additional contaminants cannot be ruled out. This is particularly true for the largemouth bass study showing increased
11-ketotestosterone, because the levels of other contaminants
were not measured in these fish [72]. In addition to the confounding factor of multiple contaminants, the differing results
also may be attributed to species differences and the state of
gonadal development during which the sampling took place.
Cholesterol plays a key role in reproduction in teleosts,
serving as the lipid precursor for gonadal steroids [49]. Lipids
mobilized from the liver gradually increase in the gonads from
the preparatory-to-spawning phase of the reproductive cycle.
Testicular cholesterol levels decreased in male catfish exposed
for 180 d to both organic and inorganic mercury [49], whereas
in freshwater murrel, hepatic cholesterol levels increased after
a 180-d exposure to an organic mercury fungicide (Emisan6; 200 ␮g/L) [73]. Both studies suggest that mercury exposure
resulted in a decreased mobilization of lipids from the liver
to gonads. Impaired lipid metabolism may be responsible, in
part, for the inhibition of spermatogenic and steroidogenic
activities in mercury-treated fish.
Sperm morphology and motility
The morphology and motility of sperm are useful in assessing the total impact of mercurials on the male reproductive
system, because they are critical factors for fertilization success in teleosts. Instantaneous (5-s) and short-term (ⱕ24-h)
exposure of sperm are representative of the effect of mercury
on sperm released into a polluted aquatic environment [74].
Short-term (21- to 80-s) exposure of milt to 1.0 ␮g/L of inorganic mercury decreased the motility of African catfish
(Clarius gariepinus) sperm [75]. A short-term (2- to 5-min)
aqueous exposure of mummichog sperm to 50 ␮g/L of methylmercury resulted in decreased motility without any apparent
morphological effects [76,77]. Instantaneous exposure of goldfish sperm to inorganic mercury reduced curvilinear velocity
at 1 ␮g/L, percentage of motile sperm at 10 ␮g/L, and flagella
length at 100 ␮g/L [74]. The decreased motility of sperm and
morphological changes in the flagellum reported in some species may be a result of mercury binding to protein sulfhydryl
groups located on the membranes of the nucleus, midpiece,
and tail. Mercury can react with sulfhydryl groups on microtubules and inhibit microtubule assembly in vitro [53], which
may affect the microtubule sliding mechanism required to generate waves along the flagellum [78]. Mercury also may interfere with the function of sperm mitochondria, resulting in
a decrease in energy production [79].
The success of fertilization events likely are reduced or
inhibited completely by alterations in sperm motility and morphology. Fertilization success was decreased after short-term
900
Environ. Toxicol. Chem. 28, 2009
(ⱕ40-min) exposure of rainbow trout (Oncorhynchus mykiss)
sperm [80,81] and mummichog sperm [76,77] to inorganic
mercury and methylmercury (1–1,000 ␮g/L). It is now known,
however, that fertilization success is dependent on the ratio of
sperm to egg used in each test, because high ratios will mask
toxic effects by providing sufficient spermatozoa to overcome
the spermiotoxic effects [75]. None of the above studies discuss the ratio used and, therefore, may have underestimated
the toxicity of inorganic and organic mercury.
Sperm also can be exposed to mercury in seminal fluids
(i.e., in vivo) of contaminated fish. A 24-h exposure in an
extender buffer has been designed to mimic this type of exposure in vitro. Goldfish sperm, which exhibited no effects
after instantaneous exposure, had decreased flagella length and
motility after preincubation in an extender buffer with 100
␮g/L of inorganic mercury [74]. Attempts have been made to
measure the effects of in vivo sperm exposure on fertilization
success [82]. Thus far, the results have been inconclusive because of the inherent difficulty in assessing the effects on sperm
in isolation from other reproductive impairments.
DISRUPTION OF FEMALE REPRODUCTIVE FUNCTION
Mercury exposure also can affect reproductive endpoints
in female fish. Mercury has been shown to affect ovarian morphology, delay oocyte development, and inhibit steroid hormone synthesis. As with the testes, it is difficult to determine
whether mercury affects the ovaries directly or indirectly, via
alterations at the level of hypothalamus or pituitary, and whether the effects are the cause or the result of changes in steroidogenesis. Unlike in males, female gamete production involves the synthesis and incorporation of a yolk protein, which
may transfer some of the maternal mercury burden to the embryo. The effects of mercury on the female reproductive system discussed in the next section are separated into changes
at the level of ovarian morphology and egg development, steroid hormone and vitellogenin synthesis, fecundity and spawning, and ultimately, egg and embryo survival.
Ovarian morphology and oogenesis
Oogenesis in teleosts involves the maturation from immature or previtellogenic oocytes through the accumulation of
yolk vesicles during vitellogenesis and, finally, the formation
of mature oocytes [83]. This cycle of oogenesis was significantly repressed in the freshwater murrel after exposure to
inorganic mercury or mercurial fungicide (10–20 ␮g/L), with
a concurrent decrease in GSI [25,44,45,84,85]. After 180 d of
exposure, only immature oocytes completely devoid of vitellogenin were present, whereas the control ovaries were fully
developed, with a large number of mature and maturing oocytes [25,44,45,84,85]. Degenerative changes—namely, nucleolar necrosis and atresia (follicular degeneration)—also
were observed in some of the fungicide and inorganic mercury
treatment groups [44,84,85]. Ovarian recrudescence was arrested with some atretic changes and a decrease in GSI of
walking catfish exposed over six months to inorganic and organic mercury (30–40 ␮g/L) [63]. Atretic follicles also were
more common in female Nile tilapia (O. niloticus) after exposure to methylmercury implants; however, no change in GSI
was observed [57]. Common spiny loach (Lepidocephalichthys thermalis) exposed to inorganic mercury for 10 d at
100 ␮g/L had extensive vacuolation in the oocortex, necrosis
of the oolemma, and hypertrophy of follicular cells, leading
to atresia of the oocytes after 20 d [86]. In sexually mature
K.L. Crump and V.L. Trudeau
largemouth bass, the frequency of atretic oocytes was positively correlated with sediment mercury concentrations in a
Tennessee, USA, reservoir system [87]. These degenerative
changes are suggestive of cytotoxic actions on the ovary.
Apoptosis is the main trigger for follicle atresia in mammals
[88] and is involved in normal teleost ovarian development
and regression [89]. A significant increase in the number of
apoptotic cells was measured in ovaries of fathead minnows
exposed to dietary methylmercury as juveniles through to sexual maturity (⬃250 d), with resultant mean total mercury carcass concentrations of 0.92 to 3.84 ␮g/g wet weight [90]. Thus,
the induction of apoptosis in steroidogenic gonadal cells is one
possible mechanism by which sex steroid levels are reduced
in fish exposed to methylmercury. Apoptosis may be a result
of oxidative stress [91], which can be induced by methylmercury exposure [21]. Apoptosis occurs spontaneously in
ovarian follicles and is regulated by follicular survival factors,
including FSH, LH, and 17␤-estradiol [92]. Therefore, an alternative mechanism may involve methylmercury-induced alterations in follicular survival factors resulting in apoptosis.
Reduced serum levels of LH were observed in methylmercuryexposed goldfish (Crump and Trudeau, unpublished data), and
decreased serum 17␤-estradiol levels have been correlated with
mercury concentrations in studies of wild-caught fish and laboratory fish [57,59,67,87,93]. Furthermore, in the absence of
degenerative changes, gonadal arrest has been related to endocrine alterations resulting from a decrease in activity of
pituitary gonadotrophs [25,45].
Steroidogenesis
In female fish, 17␤-estradiol is the major reproductive hormone and is produced primarily in the follicle of the developing oocyte [66]. Luteinizing hormone stimulates the production of testosterone, which is then converted into 17␤estradiol via the aromatase enzyme. One of the primary roles
of 17␤-estradiol is to stimulate liver production of the yolk
protein, vitellogenin, which is then incorporated into the maturing oocyte to later serve as nourishment for the developing
fish larva. Similar to testosterone, gonadal 17␤-estradiol secretion and subsequent vitellogenin production both change
with maturation of the oocytes; thus, plasma levels can be used
to monitor the reproductive stage of fish.
In female Nile tilapia (O. niloticus), 17␤-estradiol levels
decreased over the summer months and remained low until
November, when the hormone level surged to prepare for the
new reproductive season [57]. This hormone surge was repressed in fish exposed for seven months to methylmercury
implants (0.3–1.3 ␮g Hg/g wet wt muscle tissue), which may
suggest an interference with the production of stimulatory pituitary hormones or gonadal steroidogenesis. Methylmercury
accumulation also was correlated with decreased 17␤-estradiol
in fathead minnows exposed for 250 d to diets containing 0.87
to 3.93 ␮g/g of methylmercury (mean carcass accumulation,
0.92–3.84 ␮g/g wet wt) [59] and in previtellogenic largemouth
bass after four to eight weeks of exposure to diets containing
3.12 and 6.25 ␮g/g of methylmercury [67]. In addition to 17␤estradiol, 11-ketotestosterone levels were measured in laboratory exposures with female tilapia and largemouth bass;
however, no significant changes were observed. In wild fish,
sex steroid levels have been correlated both negatively and
positively with total mercury concentrations. Similar to the
results of laboratory studies, plasma 17␤-estradiol concentrations in sexually mature female largemouth bass were nega-
Environ. Toxicol. Chem. 28, 2009
Mercury-induced reproductive impairment in fish
tively correlated with total mercury in muscle [87]. A significant negative correlation between plasma 17␤-estradiol and
liver mercury (mean value, 0.14 ␮g/g wet wt) also was detected
in sexually immature white sturgeon collected in the lower
Columbia River, Oregon, USA [68]. No significant correlations
between muscle mercury and 17␤-estradiol were measured in
sexually mature female smallmouth bass and carp from the
Hudson River, New York, USA; however, in the same fish,
11-ketotestosterone was significantly positively correlated
with total muscle mercury (0.09–0.47 ␮g/g wet wt) [71]. Taken
together, these laboratory and field studies suggest that methylmercury exposure, at concentrations currently found in the
environment, can induce alterations in serum sex steroid levels
in female fish.
The synthesis of steroid hormones involves a series of enzymatic reactions that may be sensitive to mercury exposure.
Inorganic mercury specifically binds to oocyte plasma membrane, where it inhibits Na⫹/K⫹–adenosine triphosphatase activity and alters steroidogenesis in murrel [94]. One of the
initial steps in 17␤-estradiol production is the conversion of
pregnenolone to progesterone via 3␤HSD [28]. Both in vitro
and in vivo experiments demonstrated that mercury exposure
results in a decrease in 17␤-estradiol and a buildup of progesterone in the ovary related to an increase in 3␤HSD activity
[94]. Further along the steroidogenic pathway, aromatase catalyzes the conversion of androgens to 17␤-estradiol. Using a
rainbow trout microsome-based assay, methylmercury has
been shown to significantly reduce aromatase activity in both
the ovary (methylmercury concentration that inhibited aromatase activity by 50% [IC50], 0.78 ␮M) and brain (IC50, 11
␮M) [95]. Therefore, alterations in steroidogenic enzyme activity may be one direct mechanism by which methylmercury
exposure reduces serum 17␤-estradiol. Further mechanistic
studies are required to clarify the effects of mercury on the
regulation of steroidogenesis, including the expression and activity of steroidogenic enzymes in vivo. Whether the suppression of 17␤-estradiol is sufficient to induce apoptosis in
ovarian follicular cells is unclear, and whether methylmercury
exposure can alter other follicular survival factors remains to
be determined.
Vitellogenesis
Estradiol stimulates the liver to synthesize the phospholipoglycoprotein yolk precursor vitellogenin in oviparous vertebrates [96]. Altered expression of vitellogenin mRNA and
its protein is one of the most commonly used biomarkers of
aquatic contaminant exposure [97]. In males, vitellogenin normally is not expressed, but it can be induced by exposure to
estrogenic chemicals. In control female catfish, serum vitellogenin levels doubled after 17␤-estradiol injection [98]. After
injection of inorganic mercury, however, serum vitellogenin
levels decreased and did not respond to subsequent 17␤-estradiol injection. Vitellogenin synthesis also was inhibited in
catfish after a 90- to 180-d exposure to inorganic mercury (50
␮g/L) and organic mercury (40 ␮g/L), as indicated by a decrease in 32P-labeled phosphoprotein content in the liver and
ovaries [99]. In largemouth bass exposed to dietary methylmercury (3.12 and 6.25 ␮g/g) for 56 d, females had significantly reduced levels of vitellogenin, whereas levels increased
in males fed the high methylmercury diet [67]. Female largemouth bass with reduced vitellogenin levels also had significantly reduced levels of 17␤-estradiol. A survey of freshwater
species from various sites along the Hudson River provided
901
equivocal data; vitellogenin levels were negatively correlated
with muscle mercury concentrations in female smallmouth
bass (0.21–0.51 ␮g/g wet wt) and male common carp (0.09–
0.41 ␮g/g wet wt) but were not related to muscle mercury
concentrations in either sex of largemouth bass (0.12–0.61
␮g/g wet wt) or brown bullhead (0.055–0.52 ␮g/g wet wt)
[71]. This inhibition of vitellogenin synthesis on exposure to
inorganic or organic mercury may be a result of impaired
steroidogenesis or alterations in estrogen signaling pathways.
Recent studies have demonstrated alterations in vitellogenin
mRNA levels in livers from fish exposed to dietary methylmercury in laboratory-based and in situ exposures. Similar to
the reduction in vitellogenin protein observed in catfish and
bass, vitellogenin mRNA was down-regulated by two- to fourfold in female fathead minnows exposed for 600 d to dietary
methylmercury (3.93 ␮g/g) [100]. Conversely, some male fathead minnows had elevated vitellogenin levels, but the results
were highly variable and not statistically significant. Cutthroat
trout from a high-mercury lake (0.054 ␮g Hg/g wet wt wholefish tissues) had vitellogenin mRNA levels fivefold lower than
those of fish sampled from a low-mercury lake (0.016 ␮g
Hg/g wet wt whole-fish tissues) based on cDNA microarray
analysis of liver RNA [30]. Unfortunately, the study with trout
did not consider gene expression in males and females separately, thus making specific conclusions regarding the impact
of methylmercury exposure on vitellogenin expression difficult. On the whole, significant evidence suggests that methylmercury alters vitellogenesis in females, with reductions
measured in mRNA, protein, and lipid levels after exposure.
Increased vitellogenin expression in males may be caused by
an estrogen-like activity of methylmercury, because it has been
shown to bind to and activate estrogen receptors [101].
Lipids
During the gonadal cycle, a reciprocal relationship exists
between hepatic and gonadal lipids, the balance of which may
be altered by pollutants, such as mercury. In the peak of reproductive activity, hepatic lipid levels decrease, with a concomitant increase in gonadal levels. Phospholipids are an important component in the structure of proliferating and developing germ cells as reserve food in the form of yolk or as
energy sources. Cholesterol, on the other hand, is the precursor
for steroid hormones. Lipid content increased in the liver (and
muscle) with a concurrent decrease in the ovaries of bronze
featherback (Notopterus notopterus) exposed for 30 d to inorganic mercury (44–88 ␮g/L) [102]. Lipid levels (total lipids,
phospholipids, and cholesterol) also increased in the liver of
walking catfish, with a simultaneous decrease in all forms of
lipids in the ovaries after a 180-d exposure to inorganic mercury (50 ␮g/L) and methylmercury (40 ␮g/L) [99]. Murrel
exposed to 200 ␮g/L of inorganic mercury, however, had a
significant reduction of lipid and cholesterol in the liver and
ovary [103]. In summary, these results suggest a mercuryinduced change in lipid retention and/or inhibition of seasonal
mobilization of lipids from liver to ovary during ovarian recrudescence. Disturbance of the lipid balance by mercury may
result in other disruptions, including impairment of vitellogenesis (implicated in arrest of oogenesis) and steroidogenesis.
Effects on ovulation and spawning
Mercury exposure can alter fecundity (number of eggs produced) and spawning of female fish. Zebrafish produced fewer
eggs following exposure (19–25 d) to 1.0 ␮g/L of a mercurial
902
Environ. Toxicol. Chem. 28, 2009
fungicide [104]. A multigenerational study with brook trout
(Salvelinus fontinalis) exposed to aqueous methylmercury observed no spawning in second-generation trout exposed to 0.93
␮g/L or more methylmercury for 108 weeks [105]. Similarly,
no spawning occurred in fathead minnows (whole-body concentrations, 4.47 ␮g Hg/g wet wt) after 41 weeks of inorganic
mercury exposure at and above 1.02 ␮g/L [106]. Fertilization
success was reduced in offspring (whole-body mercury concentrations, 1.1–1.2 ␮g/g wet wt) of mummichog fed methylmercury-contaminated diets [107]. Recently, two studies
were performed with fathead minnows exposed to dietary
methylmercury as juvenile fish through sexual maturation to
determine the effects on reproduction [59,82]. In these studies,
an overall decrease in spawning success was observed as a
consequence of reduced gonadal growth correlated to a decrease in 17␤-estradiol levels, decreased fecundity, and increased number of days to spawning in fish, with carcass mercury concentrations ranging from 0.57 to 3.68 ␮g/g wet
weight. Days to spawning appears to be influenced by the
female, because contaminated females that mated with control
males took 3 d longer to spawn, on average, than control
females that mated with contaminated males [82]. In male
fathead minnows with carcass mercury concentrations of 0.71
to 4.23 ␮g/g wet weight, spawning behavior and spawning
success were reduced after dietary methylmercury exposure
as juveniles through to sexual maturity [108]. Because androgens are critical in regulating reproductive behavior, methylmercury-induced impairment of reproduction may be caused
by alterations in sex steroids that, in turn, result in altered
behavior. A female-specific delay in spawning can be especially detrimental if sperm release is not similarly delayed.
Delayed spawning can have significant effects on the offspring,
because it may disrupt the timing of feeding relative to seasonal
food resources. If young fish are unable to find food as an
indirect effect of mercury exposure, their growth may be affected, leading to increased susceptibility to predation and reduced survival.
Eggs and embryos
Mercury in eggs originates primarily from maternal transfer
and is predominantly (⬎90%) in the methylated form [109–
111]. Originally, it was believed that maternal transfer occurred by mobilization and transfer of stored mercury to the
gonads during oogenesis [112]. The actual amount transferred,
however, is only 0.2 to 2.3% of the maternal burden [110–
112]. In contrast, organochlorines are transferred to the eggs
at 5.5 to 25.5% of the whole-body burden [112]. The diet of
the maternal adult during oogenesis, rather than the body burden, is now accepted as the principal source of methylmercury
in eggs [113]. Therefore, the exposure of embryos to maternally transferred methylmercury depends on the levels of mercury in the prey of the adult during oogenesis, which can vary
intra- and interannually.
Mercury exposure may diminish the reproductive potential
of maternal fish by reducing the hatching success of embryos.
Hatching success of zebrafish embryos in unpolluted water
was decreased after maternal exposure (19–25 d) to 0.2
␮g/L of a mercurial fungicide [104]. Maternal exposure also
may alter the sensitivity of eggs to mercury in the environment.
Mummichog eggs from a polluted site and a control site were
exposed to methylmercury (0.5–5.0 mg/L) for 20 min before
fertilization with untreated sperm [114]. Eggs from the polluted creek demonstrated a higher tolerance to methylmercury.
K.L. Crump and V.L. Trudeau
Scanning-electron microscopy revealed that the control eggs
exposed to 1.0 mg/L of methylmercury for 20 min had an
increased incidence of spontaneous micropyle blockage, resulting in reduced fertilization success [115]. These studies
indicate that pre-exposure of eggs to mercury in maternal fish
can decrease hatching success in the laboratory, whereas multigenerational exposure in wild populations may convey increased resistance to mercury pollution.
In addition to maternal transfer, mercury bioaccumulation
in eggs and embryos can occur via bioconcentration of mercury from the water. On spawning, eggs are released into the
aquatic environment, where they may be exposed to additional
methylmercury. Mercury was initially bound primarily to the
chorion of Chinook salmon (Oncorhynchus tshawytscha)
eggs, and over a 5-d exposure period, mercury partitioned into
the yolk mass [116]. Bioconcentration factors averaging 2,025
were measured after a 16-d exposure of medaka embryos to
inorganic mercury (10–30 ␮g/L) [117]. Fathead minnow embryos quickly accumulated methylmercury to a final bioconcentration factor of 70 after only 96 h [118]. In these embryos,
protein synthesis was initially stimulated, but this was followed
by a significant reduction after longer exposure to 25 ␮g/L of
methylmercury. Hatching success was decreased in embryos
exposed to 15 ␮g/L, with no hatching occurring at higher
concentrations. Short-term (1- to 5-d) exposure of mummichog
embryos to higher concentrations of inorganic mercury (40
␮g/L) and methylmercury (30 ␮g/L) decreased survival,
whereas a subchronic (32-d) exposure to 10 ␮g/L decreased
hatching success [119,120]. Embryos of the mummichog exposed to 5 ␮g/L of methylmercury until 3 d posthatch demonstrated no change in their morphology yet exhibited impairment in the ability of larvae to capture prey [121]. Similarly, Atlantic croaker (Micropogonias undulatus) larvae exposed to maternally derived methylmercury (0.29–4.6 ng/g
spawned eggs) had impaired survival skills as measured by
the responsiveness to a vibratory startle stimulus [122]. Taken
together, these studies clearly show that at ␮g/L concentrations, aqueous mercury can quickly accumulate in eggs and
have negative impacts on hatching success, embryonic development, and larval behavior. These effects are significant, but
considering that mercury concentrations in natural habitats are
in the ng/L range, their relevance to exposure in the environment may be limited.
Environmentally relevant concentrations of waterborne
methylmercury were used to examine the effects on the embryonic and larval stages of walleye [109]. Hatching success
significantly declined with increasing waterborne methylmercury (0.1–7.8 ng/L), whereas the methylmercury concentration
in the eggs did not have any significant effect. In the same
way, parental exposure to dietary methylmercury had no significant effect on development, hatching success, or larval survival in fathead minnows [82] and mummichog [107]. The
effect of waterborne methylmercury likely results from bioconcentration during development, because the larvae contained from 2- to 21-fold higher methylmercury concentrations
compared with those of the unfertilized eggs. Therefore, independent of maternal transfer, methylmercury in aqueous environments at current concentrations has the potential to negatively impact embryonic development of wild-fish populations.
CONCLUSION
The present review has collated data from both laboratory
and field studies supporting the hypothesis that mercury in the
4
↓ Testosterone and 11-ketotestosterone synthesis
c
b
a
4
5
↓ Gonadosomatic index
↓ 17␤-Estradiol synthesis
Juvenile to spawning,
recrudescent to
spawning, spawning, sexually mature,
postspawning to
recrudescent
Recrudescent to spawning, spawning
Juvenile to spawning,
immature, recrudescent to spawning, spawning
Juvenile to spawning,
immature, previtellogenic, postspawning
to recrudescent, sexually mature
Juvenile, postspawning
to recrudescent
Juvenile
Recrudescent to spawning
Juvenile, immature,
recrudescent to
spawning
Juvenile to spawning,
immature, previtellogenic, postspawning to recrudescent
Subchronic, chronic
Subchronic, chronic
Diet, intraperitoneal,
environment
Water, diet, environment
Water
Subchronic, chronic
Subchronic, chronic
Subchronic
Water, diet, intraperito- Short term, sub chronneal, environment
ic, chronic
Diet, intraperitoneal,
environment
Water, diet, environment
Water, diet, intraperito- Subchronic, chronic
neal
Water
Short term, subchronic
Water
Subchronic
Subchronic
Subchronic
Short term, subchronic
Subchronic
Length of exposureb
Organic
Inorganic, organic
Inorganic, organic
Inorganic, organic
Organic
Organic
Organic
Inorganic, organic
Organic
Inorganic, organic
Inorganic, organic
Inorganic, organic
Inorganic, organic
Hg species
[21,33]
␮g/L, ␮g/g
␮g/L, ng/g, environmental
␮g/L, ␮g/g, ng/g,
environmental
␮g/L
␮g/L, ␮g/g, ng/g,
environmental
␮g/g, ng/g, environmental
[57,59,67,68,87]
[44,45,59,63,68,82,
84,85]
[25,44,45,63,84,85]
[44,57,84–87,90]
[57,59,67,68]
[44,49,56,68]
[50–52]
[25,44,45,49]
␮g/L
␮g/L
␮g/L, ng/g, environmental
[50,51,56]
␮g/L, ng/g
[25,44–46]
[33,37]
␮g/L
␮g/L
[32,33]
References
␮g/L
Hg concentration
5-HT ⫽ serotonin; DA ⫽ dopamine; MAO ⫽ monoamine oxidase.
Short term ⫽ less than one to two weeks; subchronic ⫽ ⬍10% of the life span of the organism; chronic ⫽ a significant fraction of the organism’s lifetime (⬎200 d).
Equivocal data.
2
↓ Oogenesis
6
4
↓ Gonadosomatic indexc
Ovaries
Histopathological changes
2
2
3
2
Water, diet
Water
Water
Route of exposure
Recrudescent to spawn- Water
ing
Juvenile, recrudescent
to spawning
2
2
Larval to juvenile,
recrudescent
Larval, recrudescent
Life stage
2
No. of
species
Sperm necrosis
↓ Spermatogenesis
Testes
Histopathological changes
Pituitary
Reduced and/or inactive
gonadotrophs
Brain
↓ Stimulatory neurotransmitter
(5-HT)
↑ Inhibitory neurotransmitter
(DA)
↓ Degradation enzyme (MAO)
Endpointa
Table 1. Summary of consensus effects of mercury on the reproductive axis in wild and laboratory-exposed fish
Mercury-induced reproductive impairment in fish
Environ. Toxicol. Chem. 28, 2009
903
904
Environ. Toxicol. Chem. 28, 2009
aquatic environment negatively impacts the reproductive
health of fish. The evidence presented suggests that the inhibitory effects of mercurials on reproduction may occur at multiple sites within the reproductive axis. A variety of endpoints
have been measured, but the majority of studies have focused
on the level of the gonads, measuring alterations in GSI, gametogenesis, steroidogenesis, and vitellogenesis. Some notable variations in response to mercury exposure may be attributed to one or more of the following factors: Species, life stage
(larval, juvenile, or adult), state of gonadal development (recrudescent, spawning, or regressed), route of exposure, length
of exposure, mercurial species, and mercury concentration. For
example, spawning success was reduced in fathead minnows
exposed as juveniles but not as sexually mature adults [82],
suggesting that some life or gonadal development stages may
be more sensitive to methylmercury exposure. Species differences likely are important, because negative correlations were
significant between vitellogenin levels and mercury content in
smallmouth bass and carp but not in bullhead or largemouth
bass collected from the same locations and with comparable
mercury burdens [71]. The heterogeneity of species sensitivity
to mercury also was demonstrated by Birge et al. [123], who
studied lethal concentrations of inorganic mercury in embryo–
larval stages of a variety of freshwater species. Nevertheless,
across all studies, there appears to be a trend for reduced
gonadal development, impaired gametogenesis, and decreased
sex steroid levels. A consensus list of the effects of mercury
exposure on the reproductive axis in fish was developed
through extensive surveying of the literature. The effects observed in at least two different fish species and exposure regimes are listed in Table 1. These alterations are associated
with altered vitellogenesis and reduced spawning success, suggesting that methylmercury-induced reproductive impairment
may have negative impacts on wild-fish populations.
Some evidence suggests that fish and other wildlife species
may adapt to mercury in their environment. Mercury exposure
can affect reproduction by reducing gamete quantity and/or
quality, and surviving offspring could be more resistant to
mercury toxicity. This natural selection scenario could result
in populations made up of individuals who have greater resistance to the effects of mercury. Studies with mummichog
from both a clean reference site and a mercury-contaminated
site have provided evidence for this occurring in wild-fish
populations [76]. Gametes and embryos of contaminated mummichogs exhibited tolerance to methylmercury and, as a result,
were relatively unaffected by concentrations that had severe
effects on gametes and embryos from the reference site. The
effects in mummichog are stage-specific, with contaminated
adults exhibiting reduced growth and condition [124]. Receptor-binding studies in wild mink have shown correlations between neurotransmitter receptor densities and mercury concentrations in the brain, which those authors suggest may represent adaptive mechanisms to maintain normal neurotransmission [39]. Adaptation and/or natural selection for mercury
resistance may occur to some extent in wild populations; however, the long-term effects and ecological significance remain
unknown.
Despite the large number of studies indicating the harmful
effects of mercurials on reproduction, there remains a paucity
of studies examining the specific mechanism and site of action.
Methylmercury is both an endocrine-disrupting chemical and
a neurotoxicant [18,37,82,125]. In mammalian models, research has focused primarily on the underlying mechanisms
K.L. Crump and V.L. Trudeau
of methylmercury-induced neurotoxicity while research into
potential endocrine-disrupting effects has been limited to developmental alterations in the fetus. Conversely, the majority
of fish studies have followed the classical endocrine-disruptor
approach of measuring reproductive impairment through fecundity, spawning success, and alterations in the hypothalamic–pituitary–gonadal axis. Studies in fish have identified impacts of methylmercury on reproduction. To understand the
underlying mechanisms of these effects, however, it is critical
to look at alterations in neuroendocrine control, because the
central nervous system is the primary target for methylmercury
action [126].
Several physiological and cellular mechanisms have been
proposed in the present review. Reproductive impairment may
be caused by alterations in any of the factors involved in
regulating the hypothalamic–pituitary–gonadal axis. First,
changes in neurotransmitter levels or other parameters of neurotransmission could affect the release of GnRH and/or LH
from the hypothalamus and pituitary. Second, inhibition of
GnRH or LH synthesis and release would cause a cascade
effect, resulting in decreased gonadal hormone production and
subsequent decreases in vitellogenesis, gonadal growth, and
GSI. Third, alterations in gonadal steroidogenesis would affect
gamete production and pituitary hormone synthesis and release
via positive-feedback mechanisms. Last, altered lipid balance
may lead to disruption of normal processes, including steroidogenesis and vitellogenesis. Cellular mechanisms also
have been proposed that may result in general toxicity and/or
specific impairment of endocrine processes. The cytoskeleton
is a target for methylmercury, and disruption of cytoskeletal
components could impair the normal mitotic division that occurs during gamete development. Apoptosis also has been proposed as a mechanism by which methylmercury impairs the
steroidogenic capacity of gonads. The reproductive axis is an
integrated system; however, many of the studies reviewed here
examined only a single parameter of the axis. Furthermore,
data implicating the direct involvement of any of these mechanisms in reproductive impairment are still lacking. These
studies provide evidence of reproductive inhibition, but they
do not clarify whether the primary site of action is the gonad
itself or whether it is the caused by inhibition of pituitary
secretion or altered neurotransmission in the hypothalamus or
other brain regions. Additionally, changes at the level of the
transcriptome or proteome associated with reproductive dysfunction remain to be determined.
In summary, there appears to be sufficient evidence from
laboratory studies to link exposure to mercury with reproductive impairment in many fish species. It is now necessary to
determine the primary site of action and the mechanisms involved in endocrine disruption at environmentally relevant exposures to methylmercury. Linking reproductive impairment
with an ecologically relevant impact on real fish populations
is a further challenge. Fish are a large food source for both
humans and wildlife, and the effects of mercury exposure on
fish, as described in the present review, may similarly occur
in these piscivorous vertebrates. Therefore, mercury pollution
is a concern for both fish populations and their consumers,
because mercury can act both indirectly, to decrease the food
supply, and directly, by concentrating in the food chain itself
to affect top predators, such as humans.
Acknowledgement—The authors would like to acknowledge Doug
Crump, Vicki Marlatt, and Colin Cameron for constructive and critical
Environ. Toxicol. Chem. 28, 2009
Mercury-induced reproductive impairment in fish
assessment of an earlier version of the manuscript. Preparation of this
review was supported in part by the NSERC-PGS (K.L. Crump) and
NSERC-DG (V.L. Trudeau) programs.
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