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Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish
Accepted Manuscript Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish Guangfu Liu, Mingjing Ke, Xiaoji Fan, Meng Zhang, Youchao Zhu, Tao Lu, Liwei Sun, Haifeng Qian PII:
S0045-6535(17)31753-8
DOI:
10.1016/j.chemosphere.2017.10.167
Reference:
CHEM 20189
To appear in:
ECSN
Received Date: 22 August 2017 Revised Date:
26 October 2017
Accepted Date: 30 October 2017
Please cite this article as: Liu, G., Ke, M., Fan, X., Zhang, M., Zhu, Y., Lu, T., Sun, L., Qian, H., Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.10.167. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Reproductive and endocrine-disrupting toxicity of Microcystis
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aeruginosa in female zebrafish
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Guangfu Liu 1,2, Mingjing Ke 1, Xiaoji Fan 1, Meng Zhang 1, Youchao Zhu 1, Tao Lu 1,
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Liwei Sun 1,*, Haifeng Qian 1, *
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China
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Department of Food Science and Technology, Zhejiang University of Technology,
Hangzhou 310032, PR China
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College of Environment, Zhejiang University of Technology, Hangzhou 310032, PR
* Corresponding Author: Tel.:+86 571 8832 0742, Fax: +86 571 8832 0599. E-mail address: [email protected] (Sun, L), [email protected] (Qian, H) 1
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Abstract Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa
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can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at
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environmentally relevant levels on the reproductive and endocrine systems of
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zebrafish are still unknown. The present study investigated the reproductive and
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endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by
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short-term exposure (96 h). After exposure, marked histological lesions in the liver or
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gonads, such as nuclear pyknosis and deformation, were observed, and the
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fertilization rate and hatchability of eggs spawned from treated females were both
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significantly lower than they were in females in the control group, suggesting the
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possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M.
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aeruginosa exposure decreased the concentration of 17β-estradiol (E2) and
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testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level
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significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels
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increased. The present findings indicate that M. aeruginosa could modulate endocrine
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function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL)
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axis-related genes, and impair the reproductive capacity of female zebrafish,
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suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in
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Microcystis bloom-contaminated aquatic environments.
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Keywords: Microcystis aeruginosa; Zebrafish; Endocrine-disruption; Reproductive
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toxicity.
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1. Introduction Due to a combination of factors, including eutrophication and warming,
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cyanobacterial blooms frequently occur in freshwater and marine ecosystems and
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have been causing global public health concerns with increasing frequency (Carvalho
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et al., 2013). These blooms have caused serious problems in food webs, drinking
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water safety, water and habitat quality, and the sustainability of freshwater ecosystems
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(Paerl et al., 2011; Paerl and Otten, 2013; Song et al., 2017). Approximately 60,000
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cases of human poisoning resulting from cyanobacterial blooms are reported each
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year, with an overall mortality rate of approximately 1.5% (Ajani et al., 2017; You et
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al., 2017). Cyanobacterial blooms can cause substantial economic losses. For instance,
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the cost of cleanup efforts for Taihu Lake, China, has exceeded US$ 16 billion since
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2007 (Zhang et al., 2016).
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Microcystis aeruginosa is one of the primary species responsible for cyanobacterial blooms. Microcystins (MCs) are the most commonly occurring toxic
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metabolites synthesized by Microcystis, and more than 100 structural variants of MCs
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have been described, of which microcystin-LR (MC-LR), microcystin-RR (MC-RR),
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and microcystin-YR (MC-YR) are the most extensively studied (Lawton et al., 1994;
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Puddick et al., 2014). These cyanotoxins can accumulate in fish tissue (Zimba et al.,
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2001) and cause developmental toxicity (Qi et al., 2016), neurotoxicity (Jonas et al.,
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2015), immune toxicity (Chen et al., 2016a), hepatotoxicity (Chen et al., 2016b),
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cytotoxicity (Dias et al., 2009), and endocrine disruption (Botha et al., 2004; Prieto et
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al., 2006).
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ACCEPTED MANUSCRIPT In addition to the above-mentioned toxins, Microcystis cells and lysates have
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been shown to contain numerous other bioactive substances. For example, Microcystis
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can also produce many peptides classified as aeruginosins, micropeptins, and
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microviridins that have diverse biological functions (Chen et al., 2017). These
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metabolites may also lead to various negative health effects on organisms (Jonas et al.,
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2015). A few studies have demonstrated that crude extracts of cyanobacterial biomass
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or Microcystis lysates have greater effects on organisms than purified cyanotoxins
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(Oberemm et al., 1997; Palíková et al., 2007). Esterhuizen-Londt et al. (2016) found
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that crude cyanobacterial extracts elicited higher oxidative stress in Daphnia pulex
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compared to purified MCs or even MC mixtures, which implies that unidentified
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compounds in crude cyanobacterial extracts may enhance the toxic effects of MCs
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synergistically.
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The zebrafish is an important model organism for biological research (Engeszer et al., 2007). Although several studies have demonstrated the endocrine-disrupting
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activity of single purified MCs in zebrafish, the effects of M. aeruginosa on the
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hypothalamic-pituitary-gonadal-liver (HPGL) axis remain to be elucidated. The
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purpose of the current study was to assess the toxic effects of M. aeruginosa at
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environmental levels on the reproductive and endocrine systems of zebrafish. Changes
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in reproductive capacity, tissue histology, plasma sex steroid hormone level, and
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transcription levels of key genes along the HPGL axis were examined after 96 h of
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exposure. This study will contribute to a better understanding of M. aeruginosa
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reproductive toxicity and relevant endocrine disruption potential in adult fish.
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2. Materials and Methods
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2.1 Algal cultures The freshwater unicellular cyanobacterium M. aeruginosa (FACHB-905) was
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obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan,
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China). The algae were cultivated in an incubator (Laifu Company, China) with a light
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irradiance of 46 µmol photons m-2 s-1 held at a constant temperature of 25 ± 0.5°C and
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a 12h:12h light-dark cycle in 2 L beakers containing 1 L of BG-11 medium (pH = 7.1).
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Algal cell density was monitored at 685 nm by optical density values (UV/Visible
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spectrophotometer, Shimadzu). When the culture reached the middle of the
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exponential growth phase (approximately 3.7×105 cells/mL), the algae were used in
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for subsequent experiments.
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2.2 Zebrafish maintenance, exposure, and sample collection
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Healthy five-month-old adult zebrafish (Danio rerio) from the Institute of
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Hydrobiology of the Chinese Academy of Science were acclimated for two weeks in
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housing tanks containing BG-11 medium (12h:12h light/dark cycle, 25 ± 0.5 °C). The
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fish were fed two times daily with brine shrimp during the acclimation period, which
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stopped 24 h before the start of the toxicity tests.
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Female zebrafish were randomly distributed into the glass tank containing 40 L
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of M. aeruginosa culture (35 fish/tank). The initial concentrations of algae
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corresponded to an optical density (OD) of 0.02, 0.04, and 0.08, hereafter referred to
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as low, medium, and high, respectively. Triplicate tanks were used for each treatment 5
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level. Control tanks with the same amount of BG-11 medium were also established.
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After 96 h, algal cell density ultimately reached approximately 4.4×105, 7.2×105, and
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10.0×105 cells/mL in the low, medium, and high concentration tanks, respectively. After 96 h exposure, five females were collected randomly from each tank and
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paired with five males from the untreated group. Eggs were immediately collected
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from each tank, and the fertilization rate was calculated. The eggs were then cultured
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in Ringer’s solution at 28 °C, and hatchability was determined after 72 h. The rest of
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the fish in the control and treated groups were anaesthetized in MS 222 (Tricaine,
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Sigma-Aldrich) simultaneously for body weight measurements and blood collection.
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Blood was collected from the caudal vein using a heparinized syringe and maintained
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on ice until centrifugation (1200 g, 15 min). The separated plasma collected from fish
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of each tank was pooled as one sample and stored at -80 °C for later analysis. The
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brain, gonads, and liver from each fish were dissected and weighed, and the
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brain-somatic index (BSI = 100 × [brain weight (g)/body weight (g)]), hepatosomatic
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index (HSI = 100 × [liver weight (g)/body weight (g)]), and gonadosomatic index
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(GSI = 100 × [gonad weight (g)/body weight (g)]) were calculated. The gonads and
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liver were then rinsed with cold phosphate-buffered saline (PBS, pH 7.0) and divided
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into two parts: one part was snap-frozen in liquid nitrogen and preserved at -80 °C for
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gene expression analysis, and the other part was fixed for histological analysis. All
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brain samples were preserved at -80 °C for gene expression analyses.
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2.3 Histological analysis
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For histopathological analysis of liver and brain tissue sections, samples were 6
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ethanol, immersed in xylol, and embedded in paraffin wax. Samples were cut to a
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thickness of 3-5 µm, deparaffinized, and rehydrated by immersion in a sequence of
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xylene, 100% alcohol, 70% alcohol, 50% alcohol, and finally water, stained with
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hematoxylin and eosin (H&E)， and observed under a light microscope.
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2.4 E2, T, and VTG determination
17β-estradiol (E2), testosterone (T), and vitellogenin (VTG) levels in the plasma
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were determined using sex steroid hormones detection kits (Yuanye, Shanghai, China)
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and VTG detection kits (Abebio, Wuhan, China), which were based on the
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enzyme-linked immunosorbent assay (ELISA). The standard curves of these three
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detection kits were obtained to determine the amount of each plasma sample, with a
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satisfactory linearity of R2 > 0.99. E2, T and VTG concentrations were normalized to
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the volume (mL) of the corresponding plasma sample, and the detection limits of the
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E2, T and VTG detection kits were 1.0 pg/mL, 1.0pg/mL and 60 ng/mL, respectively.
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All samples and standards were run in triplicate.
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2.5 Gene expression analyses
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Total RNA was extracted from the brain, gonads, and liver using RNAiso Plus
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(Takara, Dalian, China), and cDNA synthesis and SYBR Green Real-Time PCR were
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performed using commercial kits (Toyobo, Tokyo, Japan) according to protocols we
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have previously described (Chen et al., 2016c). Three biological replicates were used
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for every treatment level. In total, 23 genes related to the HPGL axis, including two
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gonadotropin-releasing hormone genes (gnrh2, gnrh3), four gonadotropin-releasing 7
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hormone beta gene (fshβ), the follicle-stimulating hormone receptor gene (fshr), the
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luteinizing hormone beta gene (lhβ), the luteinizing hormone receptor gene (lhr), two
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hydroxymethylglutaryl CoA reductase genes (hmgra, hmgrb), steroidogenic acute
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regulatory protein genes (star), four cytochrome P450 genes (cyp11a, cyp17,
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cyp19a1a, cyp19a1b), two hydroxysteroid dehydrogenase genes (3βhsd, 17βhsd), two
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estrogen receptor genes (erα, er2β), the androgen receptor gene (ar), and the
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vitellogenin 1 gene (vtg1), were selected. Glyceraldehyde-3-phosphate dehydrogenase
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(gapdh) was used as an internal control gene to normalize the expression profiles. The
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primer sequences for these genes are listed in Table S1.
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2.6 Statistical analyses
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Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL,
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USA). After assessment of normality and homogeneity of variance, a one-way
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analysis of variance (ANOVA) followed by Tukey’s multiple range test was used to
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evaluate differences between the control and exposure groups. Data are presented as
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the mean value ± standard error (SE). Differences between the control and M.
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aeruginosa treatment groups were considered to be statistically significant at p < 0.05.
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3． ．Results
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3.1. Effects of M. aeruginosa exposure on the BSI, HSI and GSI of female
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zebrafish after 96 h
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Microcystis aeruginosa had no effect on the body weight of female zebrafish after
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96 h of exposure (data not shown). Similarly, no effects were observed on the BSI and 8
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and 29.8% in the medium and high groups, respectively, whereas no difference was
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observed in the low group when compared with the control (Fig. 1).
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3.2. Reproductive capacity of female zebrafish after 96 h of M. aeruginosa
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exposure
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The number of eggs spawned by treated females was slightly lower than that by control females, but this difference was not statistically significant. However,
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compared with the control group, the fertilization rate of eggs significantly decreased
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by 9.5% and 10.5% in the medium and high groups, respectively. Additionally,
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hatchability of eggs in the medium and high groups significantly decreased by 8.2%
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and 21.6%, respectively, compared to the control group (Fig. 2).
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3.3. Histological analysis of the liver and ovaries of female zebrafish
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The hepatocytes of control females were densely interconnected, and the cord-like parenchymal structure with central spherical nuclei was clear. The
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cytoplasm of these hepatocytes contained various types of inclusions (Fig. 3A).
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However, M. aeruginosa induced hepatocellular vacuolization in a dose-dependent
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manner, and the spatial community and intercellular spaces were optically empty.
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Some cytoplasmic inclusions were no longer detectable and nuclei appeared pyknotic
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or deformed and parenchyma disorganized indicating partial necrosis of the liver (Fig.
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3B-D).
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Histopathological study of the control group showed normal development of gametogenic populations. Oocyte/vitelline membranes in the vitellogenic stage 9
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by eosin. However, in the ovaries of treated fish, loss of contact between the oocyte
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cell membranes and the follicular cell layer was observed. Irregularity in cell layers
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was seen in the treatment groups (Fig. 3G, H), and atretic oocytes were observed in
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oocytes at mature stages (Fig. 3K, L). The number of early-stage follicles increased in
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the treatment groups compared with the control. In addition, increased oocyte atresia
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and degenerating vitellogenic oocytes were found in treated female fish (Fig. 3E-H).
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3.4. Changes in plasma hormones
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Compared with the control, plasma E2 levels were significantly decreased in the low, medium, and high groups by 18.6%, 20.1%, and 25.6%, respectively, following
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96 h of M. aeruginosa exposure (Fig. 4A). Plasma T concentrations significantly
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decreased (-18.8%) in the high group, and no change was found in the low and
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medium groups (Fig, 4B). The E2/T ratio only significantly decreased in the high
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group (-26.7%) compared with that of the control (Fig. 4C). Plasma VTG levels in
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treatment groups increased in dose-dependent manner compared with that of the
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control group, and in the medium and high groups, this increase was significant
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(68.6% and 84.7% greater than the control group, respectively) (Fig. 4D).
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3.5. Transcriptional changes in genes of the HPGL axis
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In the brain, transcript levels of gnrhr1, gnrhr2, and gnrhr4 in the treated groups
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all increased significantly to more than 1.3 fold higher than the levels in the control
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after 96 h of exposure, whereas expression of gnrh2, gnrh3, and gnrhr3 did not
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change. Significant upregulation of fshβ and lhβ was observed. In the low, medium, 10
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respectively; and lhβ expression increased to 1.5 fold, 1.8 fold, and 2.1 fold higher
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than that in the control, respectively. Significant downregulation of cyp19a1b and erα
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was observed in the treated groups. In the three treated groups, cyp19a1b expression
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decreased to 65.7%, 83.8%, and 42.8% of the levels of control, respectively, and erα
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expression decreased to 71.1%, 53.6%, and 22.4% of the levels in the control.
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However, expression of ar and er2β did not change (Fig. 5).
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the levels in the control in the low, medium, and high groups, respectively, and
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expression of cyp17 decreased to nearly half that of that in the control in the three
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treated groups. However, expression of cyp11a did not change. The expression of
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hmgra was decreased to 55%-75% of that in the control in the three treated groups
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after 96 h of M. aeruginosa exposure, whereas transcript levels of hmgrb did not
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change. The expression of 17βhsd in the low, medium, and high groups decreased to
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58.8%, 68.6%, and 30.3% that of the control, respectively, whereas no change was
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found in the transcription of 3βhsd. The estrogen receptor α, follicle-stimulating
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hormone receptor, and luteinizing hormone receptor genes in the treated groups
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exhibited different degrees of upregulation. In the three treated groups, expression of
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erα increased by 20.8%, 80.3%, and 115.0% compared to the control, respectively;
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that of fshr increased by 20%-40% compared to the control, and that of lhr increased
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by 26%-47% compared to the control. There were no changes in the transcript levels
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of star (Fig. 6).
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In the liver of female zebrafish, transcript levels of vtg1 exhibited a dose-dependent decrease following 96 h of treatment with M. aeruginosa and were
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71.1%, 67.6%, and 22.4% of the control levels in the low, medium, and high groups,
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respectively. The transcription levels of erα decreased to 56.8%, 35.1%, and 42.8% of
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the control in the three treatment groups, respectively (Fig. 7).
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4. Discussion
The concentrations of M. aeruginosa used in this study were over 100,000
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cells/mL, which is similar to the environmental cyanobacterial density during a bloom
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(Saker and Eaglesham, 1999). We measured the concentration of MC-LR, one of the
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most toxic and prevalent congeners among the variants of cyanotoxins, in the algal
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cells and the water at the end of the exposure period. In the low, medium, and high
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groups, the MC-LR concentration was 275.24 ± 8.04 (mean ±SE), 425.54 ± 18.08,
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and 571.95 ± 15.71 µg/L, respectively, in algal cells, whereas it was 24.53 ± 1.97,
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41.85 ± 0.79, and 55.57 ± 5.34 µg/L, respectively in the water (unpublished data).
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Similar concentrations have commonly been encountered in cyanobacterial bloom
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events, indicating that this work has environmental significance (Kardinaal et al.,
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2007; Watson et al., 2017).
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The results from both field and laboratory studies have shown that stress induced
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by toxic substances can impair the liver of fish, and the liver is the primary target
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organ for MCs (Chen et al., 2016b). In the present study, M. aeruginosa exposure
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caused a dose-dependent increase in hepatic histopathological lesions characterized by 12
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necrotic cells. Similarly, severe hepatic injury, including dilatation of sinusoid and
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hyperemia, with blood stasis and inflammatory cells, were observed in adult zebrafish
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after exposure to 200 µg/L MC-LR for 96 h (Wei et al. 2017). Hepatocytes also
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exhibit different degrees of damage after being injected with crude extracts of
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cyanobacteria (Paulino et al., 2017).
Several biomarkers, such as GSI, fecundity rate, fertilization rate, and
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hatchability of eggs, in conjunction with more specific biomarkers, are widely used to
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examine possible adverse effects of environmental pollutants, including
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endocrine-disrupting chemicals, on reproduction (Xu et al., 2008; Naderi et al., 2014).
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Reproductive dysfunction in adult zebrafish caused by M. aeruginosa was observed in
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the present study, which manifested as decreased GSI, reduced fertilization rate, and
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inhibited hatchability. Moreover, a declining trend in the number of spawn was also
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observed, although the difference was not statistically significant. The decrease in GSI
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could be ascribed to pathomorphological changes in the ovaries (Wu et al., 2014), and
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histological lesions, such as vacuolation or irregularity in cell layers, were observed in
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gonadal tissue in the present study. The gonadosomatic tissue damaged could decrease
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the reproductive performance of fish. It has been reported that the reproductive
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performance of medaka fish, including the fecundity and the hatchability, was
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adversely affected by 5 µg/L MC-LR and Microcystis extract (5 µg/L of equivalent
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MC-LR) after 28 days of treatment (Qiao et al., 2016). Wu et al. (2015) also found
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that exposure to less than 40 µg/L MC-LR can lead to subfertility in female mice. Our
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results were in accordance with these previous studies, suggesting that M. aeruginosa
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inhibited the reproductive capacity of female zebrafish and may have
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transgenerational effects. Sex steroid hormone measurements are useful for monitoring reproductive
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systems, as these hormonal changes play crucial roles in the reproduction of fish and
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correspond to alterations in steroidogenic gene transcription (Devlin and Nagahama,
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2002; Ji et al., 2013b). Exposure to M. aeruginosa significantly altered plasma sex
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hormone levels and the expression of genes involved in the steroidogenesis pathway.
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Changes in sex steroid hormone concentrations may result in subsequent reproductive
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dysfunction, including decreased fecundity rate, fertilization rate, and hatch success,
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as observed in this study. The activity and expression of steroidogenic genes control
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steroid hormone synthesis and metabolism (Hogg et al., 2013). For instance, in
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zebrafish gonads, cyp17 catalyzes the conversion of 17-hydroxyprogesterone to
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androstenedione, which is catalyzed into T by 17βhsd (Fernandes et al., 2011).
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Additionally, aromatase (cyp19) enzymes are known to catalyze the final conversion
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of androgen to estrogen (Trant et al., 2001), and in zebrafish, T is secreted from the
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follicle cell and finally converted to E2 by aromatase (Clelland and Peng, 2009). In
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the present study, M. aeruginosa exposure inhibited the expression of most
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steroidogenic genes (cyp19a1a, cyp19a1b, 17βhsd, cyp17, and hmgra), corresponding
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to the changes of E2 and T levels in the plasma. For example, plasma T concentrations
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significantly decreased in the high group, but no change was found in the low and
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medium groups. Those changes were consistent with the concentration-dependent
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in the ovary may inhibit the conversion of T to E2 such that the ratio of E2/T is
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reduced. This reduced ratio suggests that exposure to M. aeruginosa disrupts the
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balance of sex hormones in fish and results in adverse effects on oogenesis and
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reproduction (Orlando et al., 2004). Similar to our observations, Qiao et al. (2013)
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found that E2 levels were significantly decreased after exposure to 20 µg/L MC-LR,
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whereas no change in T was observed. When zebrafish were exposed to 50 µg/L
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MC-LR, both E2 and T levels were significantly decreased (Zhao et al. 2015).
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In teleosts, the regulation of reproduction is primarily coordinated via interactions between genes along the HPGL axis in addition to genes involved in the
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steroidogenesis pathway (Hilscherova et al., 2004; Villeneuve et al., 2008). Studies in
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female zebrafish have revealed that FSH is primarily involved in vitellogenesis,
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whereas maturation of oocytes is primarily controlled by LH (Sarkar et al. 2014). In
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the present study, the transcription of fsh and lh genes in zebrafish after M. aeruginosa
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treatment increased significantly, suggesting possible promotion of oogenesis and
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maturation. However, these effects were not observed in the histological and
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reproductive examination. It is well known that endocrine-disrupting chemicals alter
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normal patterns of gene expression, by either steroid hormone receptor-mediated
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pathways or compensatory effects (Villeneuve et al., 2007). Insufficient E2 content
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could initiate a feedback mechanism that could stimulate the pituitary to respond,
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leading to increased secretion of gonadotropins (Hou et al., 2016). Therefore, the
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upregulation of fshβ and lhβ observed in this study indirectly indicates the existence
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ACCEPTED MANUSCRIPT of a feedback mechanism in the HPGL-axis. Other studies have noted that exposure to
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prochloraz decreases the plasma concentration of E2 and causes compensatory
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upregulation of genes of the HPGL in small fish (Ankley et al., 2009; Liu et al., 2010),
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strongly supporting our observations.
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Changes in VTG levels in female fish have been identified as a sensitive
endpoint for assessing the levels endocrine-disrupting chemicals (Dang et al., 2011).
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VTG is a yolk precursor protein that is synthesized by the liver in response to
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estradiol stimulation and is essential for vitellogenesis, oocyte maturation, and yolk
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biosynthesis (Aravindakshan et al., 2004; Yuan et al., 2013). Seven known vtg genes
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are located differently in the zebrafish genome, and vtg1 is the most highly expressed
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(Tong et al., 2004; Wang et al., 2005). As the expression of vtg genes in the livers of
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female fish is modulated by E2 (Bemanian et al. 2004), the observed downregulation
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of the hepatic vtg1 gene in the present study might have resulted from decreased E2
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levels in the plasma. Liver lesions caused by M. aeruginosa in the present study may
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also have been responsible for the downregulation of vtg1 mRNA expression.
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However, plasma VTG levels significantly increased in a dose-dependent manner in
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this study, which may be due to the impairment of the ovaries making it difficult for
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plasma VTG to incorporate into the oocytes, resulting in accumulation in the blood.
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Similarly, Qiao et al., (2013) also reported that MC-LR exposure decreased hepatic
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vtg1 gene expression and increased VTG levels in female zebrafish with MC-LR
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exposure.
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5. Conclusions In summary, the present study demonstrated that short-term (96 h) exposure to M. aeruginosa could impair reproductive capacity and disrupt the endocrine system in
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female zebrafish, damaging the liver and gonadal tissue, altering plasma sex hormone
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levels, and changing the transcription of HPGL axis-related genes. Moreover, the
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decreased fertilization rate and hatchability of eggs spawned from treated females
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demonstrated the possibility of transgenerational effects of M. aeruginosa exposure.
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Future work is necessary to study the reproductive toxicology of M. aeruginosa in a
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systematic manner after long-term exposure to environmentally relevant
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concentrations of M. aeruginosa.
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We gratefully acknowledge the National Natural Science Foundation of China (Nos.21377118 and 21577128) for supporting this research.
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Supplementary data
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Table S1. Sequences of primers for the target genes
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Acknowledgments
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Figure captions
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Fig. 1. Brain index (BSI), hepatosomatic index (HSI), and gonadosomatic index (GSI)
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of female zebrafish exposed to M. aeruginosa for 96 h.
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Fig. 2. Effects of M. aeruginosa on the reproductive capacity of female zebrafish.
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Fig. 3. Histological changes in the liver (A-D) and ovary (E-L) of zebrafish exposed
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to M. aeruginosa for 96 h. Microphotographs show liver and ovary sections (5 µm)
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stained with hematoxylin and eosin (H&E). The original magnification was ×400
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(A-D), ×100 (E-H), ×200 (I-L).
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(A-D): H&E-stained liver section. A: Control group. B: Low group. C: Medium group. D: High group. Black asterisk: hyperemia. White asterisk: dilatation of
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sinusoid. Black arrow: pyknotic or deformed nucleus. White arrow: vacuolation.
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White ellipse: parenchyma disorganization.
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(E-L): H&E-stained ovary section. E, I: Control group. F, J: Low group. G, K: Medium group. H, L: High group. White arrow: the loss of contacts (ellipses) between
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the oocyte cell membranes and the follicular cell layer. Black arrow: the vacuolation
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of the gonadosomatic tissue. White triangle: Irregularity in cell layers. Black asterisk:
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oocyte atresia. White asterisk: degenerating vitellogenic oocytes.
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Fig. 4. Effects of M. aeruginosa on plasma levels of (A) 17β-estradiol (E2), (B)
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testosterone (T), (C) the E2/T ratio, and (D) vitellogenin (VTG) after 96 h of M.
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aeruginosa exposure.
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Fig. 5. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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Fig. 6. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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genes in the ovary of adult female zebrafish after exposure to M. aeruginosa.
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Fig. 7. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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genes in the liver of adult female zebrafish after exposure to M. aeruginosa.
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M. aeruginosa caused histological lesions in liver and gonad of female zebrafish.
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M. aeruginosa impaired reproductive capacity and caused transgenerational
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effects. M. aeruginosa exposure altered the level of plasma hormones in female zebrafish.
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S0045-6535(17)31753-8
DOI:
10.1016/j.chemosphere.2017.10.167
Reference:
CHEM 20189
To appear in:
ECSN
Received Date: 22 August 2017 Revised Date:
26 October 2017
Accepted Date: 30 October 2017
Please cite this article as: Liu, G., Ke, M., Fan, X., Zhang, M., Zhu, Y., Lu, T., Sun, L., Qian, H., Reproductive and endocrine-disrupting toxicity of Microcystis aeruginosa in female zebrafish, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.10.167. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Reproductive and endocrine-disrupting toxicity of Microcystis
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aeruginosa in female zebrafish
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Guangfu Liu 1,2, Mingjing Ke 1, Xiaoji Fan 1, Meng Zhang 1, Youchao Zhu 1, Tao Lu 1,
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Liwei Sun 1,*, Haifeng Qian 1, *
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China
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Department of Food Science and Technology, Zhejiang University of Technology,
Hangzhou 310032, PR China
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College of Environment, Zhejiang University of Technology, Hangzhou 310032, PR
* Corresponding Author: Tel.:+86 571 8832 0742, Fax: +86 571 8832 0599. E-mail address: [email protected] (Sun, L), [email protected] (Qian, H) 1
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Abstract Microcystis aeruginosa, a primary species in cyanobacterial blooms, is ubiquitously distributed in water. Microcystins (MCs) purified from M. aeruginosa
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can exert reproductive toxicity in fish. However, the effects of M. aeruginosa at
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environmentally relevant levels on the reproductive and endocrine systems of
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zebrafish are still unknown. The present study investigated the reproductive and
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endocrine-disrupting toxicity of M. aeruginosa on female zebrafish (Danio rerio) by
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short-term exposure (96 h). After exposure, marked histological lesions in the liver or
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gonads, such as nuclear pyknosis and deformation, were observed, and the
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fertilization rate and hatchability of eggs spawned from treated females were both
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significantly lower than they were in females in the control group, suggesting the
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possibility of transgenerational effects of M. aeruginosa exposure. Moreover, M.
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aeruginosa exposure decreased the concentration of 17β-estradiol (E2) and
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testosterone (T) in female zebrafish. Interestingly, the vtg1 transcriptional level
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significantly decreased in the liver, whereas plasma vitellogenin (VTG) protein levels
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increased. The present findings indicate that M. aeruginosa could modulate endocrine
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function by disrupting transcription of hypothalamic-pituitary-gonadal-liver (HPGL)
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axis-related genes, and impair the reproductive capacity of female zebrafish,
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suggesting that M. aeruginosa causes potential adverse effects on fish reproduction in
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Microcystis bloom-contaminated aquatic environments.
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Keywords: Microcystis aeruginosa; Zebrafish; Endocrine-disruption; Reproductive
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toxicity.
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1. Introduction Due to a combination of factors, including eutrophication and warming,
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cyanobacterial blooms frequently occur in freshwater and marine ecosystems and
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have been causing global public health concerns with increasing frequency (Carvalho
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et al., 2013). These blooms have caused serious problems in food webs, drinking
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water safety, water and habitat quality, and the sustainability of freshwater ecosystems
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(Paerl et al., 2011; Paerl and Otten, 2013; Song et al., 2017). Approximately 60,000
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cases of human poisoning resulting from cyanobacterial blooms are reported each
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year, with an overall mortality rate of approximately 1.5% (Ajani et al., 2017; You et
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al., 2017). Cyanobacterial blooms can cause substantial economic losses. For instance,
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the cost of cleanup efforts for Taihu Lake, China, has exceeded US$ 16 billion since
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2007 (Zhang et al., 2016).
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Microcystis aeruginosa is one of the primary species responsible for cyanobacterial blooms. Microcystins (MCs) are the most commonly occurring toxic
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metabolites synthesized by Microcystis, and more than 100 structural variants of MCs
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have been described, of which microcystin-LR (MC-LR), microcystin-RR (MC-RR),
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and microcystin-YR (MC-YR) are the most extensively studied (Lawton et al., 1994;
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Puddick et al., 2014). These cyanotoxins can accumulate in fish tissue (Zimba et al.,
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2001) and cause developmental toxicity (Qi et al., 2016), neurotoxicity (Jonas et al.,
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2015), immune toxicity (Chen et al., 2016a), hepatotoxicity (Chen et al., 2016b),
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cytotoxicity (Dias et al., 2009), and endocrine disruption (Botha et al., 2004; Prieto et
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al., 2006).
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been shown to contain numerous other bioactive substances. For example, Microcystis
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can also produce many peptides classified as aeruginosins, micropeptins, and
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microviridins that have diverse biological functions (Chen et al., 2017). These
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metabolites may also lead to various negative health effects on organisms (Jonas et al.,
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2015). A few studies have demonstrated that crude extracts of cyanobacterial biomass
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or Microcystis lysates have greater effects on organisms than purified cyanotoxins
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(Oberemm et al., 1997; Palíková et al., 2007). Esterhuizen-Londt et al. (2016) found
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that crude cyanobacterial extracts elicited higher oxidative stress in Daphnia pulex
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compared to purified MCs or even MC mixtures, which implies that unidentified
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compounds in crude cyanobacterial extracts may enhance the toxic effects of MCs
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synergistically.
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The zebrafish is an important model organism for biological research (Engeszer et al., 2007). Although several studies have demonstrated the endocrine-disrupting
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activity of single purified MCs in zebrafish, the effects of M. aeruginosa on the
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hypothalamic-pituitary-gonadal-liver (HPGL) axis remain to be elucidated. The
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purpose of the current study was to assess the toxic effects of M. aeruginosa at
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environmental levels on the reproductive and endocrine systems of zebrafish. Changes
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in reproductive capacity, tissue histology, plasma sex steroid hormone level, and
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transcription levels of key genes along the HPGL axis were examined after 96 h of
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exposure. This study will contribute to a better understanding of M. aeruginosa
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reproductive toxicity and relevant endocrine disruption potential in adult fish.
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2. Materials and Methods
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2.1 Algal cultures The freshwater unicellular cyanobacterium M. aeruginosa (FACHB-905) was
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obtained from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan,
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China). The algae were cultivated in an incubator (Laifu Company, China) with a light
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irradiance of 46 µmol photons m-2 s-1 held at a constant temperature of 25 ± 0.5°C and
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a 12h:12h light-dark cycle in 2 L beakers containing 1 L of BG-11 medium (pH = 7.1).
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Algal cell density was monitored at 685 nm by optical density values (UV/Visible
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spectrophotometer, Shimadzu). When the culture reached the middle of the
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exponential growth phase (approximately 3.7×105 cells/mL), the algae were used in
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for subsequent experiments.
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2.2 Zebrafish maintenance, exposure, and sample collection
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Healthy five-month-old adult zebrafish (Danio rerio) from the Institute of
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Hydrobiology of the Chinese Academy of Science were acclimated for two weeks in
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housing tanks containing BG-11 medium (12h:12h light/dark cycle, 25 ± 0.5 °C). The
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fish were fed two times daily with brine shrimp during the acclimation period, which
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stopped 24 h before the start of the toxicity tests.
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Female zebrafish were randomly distributed into the glass tank containing 40 L
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of M. aeruginosa culture (35 fish/tank). The initial concentrations of algae
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corresponded to an optical density (OD) of 0.02, 0.04, and 0.08, hereafter referred to
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as low, medium, and high, respectively. Triplicate tanks were used for each treatment 5
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level. Control tanks with the same amount of BG-11 medium were also established.
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After 96 h, algal cell density ultimately reached approximately 4.4×105, 7.2×105, and
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10.0×105 cells/mL in the low, medium, and high concentration tanks, respectively. After 96 h exposure, five females were collected randomly from each tank and
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paired with five males from the untreated group. Eggs were immediately collected
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from each tank, and the fertilization rate was calculated. The eggs were then cultured
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in Ringer’s solution at 28 °C, and hatchability was determined after 72 h. The rest of
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the fish in the control and treated groups were anaesthetized in MS 222 (Tricaine,
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Sigma-Aldrich) simultaneously for body weight measurements and blood collection.
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Blood was collected from the caudal vein using a heparinized syringe and maintained
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on ice until centrifugation (1200 g, 15 min). The separated plasma collected from fish
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of each tank was pooled as one sample and stored at -80 °C for later analysis. The
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brain, gonads, and liver from each fish were dissected and weighed, and the
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brain-somatic index (BSI = 100 × [brain weight (g)/body weight (g)]), hepatosomatic
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index (HSI = 100 × [liver weight (g)/body weight (g)]), and gonadosomatic index
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(GSI = 100 × [gonad weight (g)/body weight (g)]) were calculated. The gonads and
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liver were then rinsed with cold phosphate-buffered saline (PBS, pH 7.0) and divided
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into two parts: one part was snap-frozen in liquid nitrogen and preserved at -80 °C for
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gene expression analysis, and the other part was fixed for histological analysis. All
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brain samples were preserved at -80 °C for gene expression analyses.
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2.3 Histological analysis
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For histopathological analysis of liver and brain tissue sections, samples were 6
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ethanol, immersed in xylol, and embedded in paraffin wax. Samples were cut to a
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thickness of 3-5 µm, deparaffinized, and rehydrated by immersion in a sequence of
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xylene, 100% alcohol, 70% alcohol, 50% alcohol, and finally water, stained with
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hematoxylin and eosin (H&E)， and observed under a light microscope.
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2.4 E2, T, and VTG determination
17β-estradiol (E2), testosterone (T), and vitellogenin (VTG) levels in the plasma
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were determined using sex steroid hormones detection kits (Yuanye, Shanghai, China)
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and VTG detection kits (Abebio, Wuhan, China), which were based on the
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enzyme-linked immunosorbent assay (ELISA). The standard curves of these three
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detection kits were obtained to determine the amount of each plasma sample, with a
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satisfactory linearity of R2 > 0.99. E2, T and VTG concentrations were normalized to
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the volume (mL) of the corresponding plasma sample, and the detection limits of the
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E2, T and VTG detection kits were 1.0 pg/mL, 1.0pg/mL and 60 ng/mL, respectively.
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All samples and standards were run in triplicate.
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2.5 Gene expression analyses
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Total RNA was extracted from the brain, gonads, and liver using RNAiso Plus
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(Takara, Dalian, China), and cDNA synthesis and SYBR Green Real-Time PCR were
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performed using commercial kits (Toyobo, Tokyo, Japan) according to protocols we
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have previously described (Chen et al., 2016c). Three biological replicates were used
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for every treatment level. In total, 23 genes related to the HPGL axis, including two
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gonadotropin-releasing hormone genes (gnrh2, gnrh3), four gonadotropin-releasing 7
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hormone beta gene (fshβ), the follicle-stimulating hormone receptor gene (fshr), the
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luteinizing hormone beta gene (lhβ), the luteinizing hormone receptor gene (lhr), two
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hydroxymethylglutaryl CoA reductase genes (hmgra, hmgrb), steroidogenic acute
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regulatory protein genes (star), four cytochrome P450 genes (cyp11a, cyp17,
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cyp19a1a, cyp19a1b), two hydroxysteroid dehydrogenase genes (3βhsd, 17βhsd), two
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estrogen receptor genes (erα, er2β), the androgen receptor gene (ar), and the
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vitellogenin 1 gene (vtg1), were selected. Glyceraldehyde-3-phosphate dehydrogenase
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(gapdh) was used as an internal control gene to normalize the expression profiles. The
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primer sequences for these genes are listed in Table S1.
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2.6 Statistical analyses
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Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL,
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USA). After assessment of normality and homogeneity of variance, a one-way
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analysis of variance (ANOVA) followed by Tukey’s multiple range test was used to
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evaluate differences between the control and exposure groups. Data are presented as
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the mean value ± standard error (SE). Differences between the control and M.
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aeruginosa treatment groups were considered to be statistically significant at p < 0.05.
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3． ．Results
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3.1. Effects of M. aeruginosa exposure on the BSI, HSI and GSI of female
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zebrafish after 96 h
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Microcystis aeruginosa had no effect on the body weight of female zebrafish after
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96 h of exposure (data not shown). Similarly, no effects were observed on the BSI and 8
ACCEPTED MANUSCRIPT HSI in treated zebrafish (Fig. 1). However, the GSI significantly decreased by 25.8%
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and 29.8% in the medium and high groups, respectively, whereas no difference was
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observed in the low group when compared with the control (Fig. 1).
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3.2. Reproductive capacity of female zebrafish after 96 h of M. aeruginosa
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exposure
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The number of eggs spawned by treated females was slightly lower than that by control females, but this difference was not statistically significant. However,
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compared with the control group, the fertilization rate of eggs significantly decreased
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by 9.5% and 10.5% in the medium and high groups, respectively. Additionally,
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hatchability of eggs in the medium and high groups significantly decreased by 8.2%
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and 21.6%, respectively, compared to the control group (Fig. 2).
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3.3. Histological analysis of the liver and ovaries of female zebrafish
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The hepatocytes of control females were densely interconnected, and the cord-like parenchymal structure with central spherical nuclei was clear. The
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cytoplasm of these hepatocytes contained various types of inclusions (Fig. 3A).
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However, M. aeruginosa induced hepatocellular vacuolization in a dose-dependent
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manner, and the spatial community and intercellular spaces were optically empty.
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Some cytoplasmic inclusions were no longer detectable and nuclei appeared pyknotic
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or deformed and parenchyma disorganized indicating partial necrosis of the liver (Fig.
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3B-D).
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Histopathological study of the control group showed normal development of gametogenic populations. Oocyte/vitelline membranes in the vitellogenic stage 9
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by eosin. However, in the ovaries of treated fish, loss of contact between the oocyte
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cell membranes and the follicular cell layer was observed. Irregularity in cell layers
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was seen in the treatment groups (Fig. 3G, H), and atretic oocytes were observed in
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oocytes at mature stages (Fig. 3K, L). The number of early-stage follicles increased in
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the treatment groups compared with the control. In addition, increased oocyte atresia
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and degenerating vitellogenic oocytes were found in treated female fish (Fig. 3E-H).
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3.4. Changes in plasma hormones
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Compared with the control, plasma E2 levels were significantly decreased in the low, medium, and high groups by 18.6%, 20.1%, and 25.6%, respectively, following
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96 h of M. aeruginosa exposure (Fig. 4A). Plasma T concentrations significantly
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decreased (-18.8%) in the high group, and no change was found in the low and
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medium groups (Fig, 4B). The E2/T ratio only significantly decreased in the high
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group (-26.7%) compared with that of the control (Fig. 4C). Plasma VTG levels in
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treatment groups increased in dose-dependent manner compared with that of the
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control group, and in the medium and high groups, this increase was significant
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(68.6% and 84.7% greater than the control group, respectively) (Fig. 4D).
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3.5. Transcriptional changes in genes of the HPGL axis
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In the brain, transcript levels of gnrhr1, gnrhr2, and gnrhr4 in the treated groups
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all increased significantly to more than 1.3 fold higher than the levels in the control
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after 96 h of exposure, whereas expression of gnrh2, gnrh3, and gnrhr3 did not
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change. Significant upregulation of fshβ and lhβ was observed. In the low, medium, 10
ACCEPTED MANUSCRIPT and high groups, fshβ was upregulated by 52.0%, 81.3%, and 71.5% of the control,
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respectively; and lhβ expression increased to 1.5 fold, 1.8 fold, and 2.1 fold higher
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than that in the control, respectively. Significant downregulation of cyp19a1b and erα
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was observed in the treated groups. In the three treated groups, cyp19a1b expression
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decreased to 65.7%, 83.8%, and 42.8% of the levels of control, respectively, and erα
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expression decreased to 71.1%, 53.6%, and 22.4% of the levels in the control.
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However, expression of ar and er2β did not change (Fig. 5).
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In the ovary, expression of cyp19a1a decreased to 52.0%, 38.1%, and 20.6% of
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the levels in the control in the low, medium, and high groups, respectively, and
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expression of cyp17 decreased to nearly half that of that in the control in the three
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treated groups. However, expression of cyp11a did not change. The expression of
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hmgra was decreased to 55%-75% of that in the control in the three treated groups
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after 96 h of M. aeruginosa exposure, whereas transcript levels of hmgrb did not
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change. The expression of 17βhsd in the low, medium, and high groups decreased to
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58.8%, 68.6%, and 30.3% that of the control, respectively, whereas no change was
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found in the transcription of 3βhsd. The estrogen receptor α, follicle-stimulating
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hormone receptor, and luteinizing hormone receptor genes in the treated groups
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exhibited different degrees of upregulation. In the three treated groups, expression of
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erα increased by 20.8%, 80.3%, and 115.0% compared to the control, respectively;
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that of fshr increased by 20%-40% compared to the control, and that of lhr increased
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by 26%-47% compared to the control. There were no changes in the transcript levels
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of star (Fig. 6).
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In the liver of female zebrafish, transcript levels of vtg1 exhibited a dose-dependent decrease following 96 h of treatment with M. aeruginosa and were
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71.1%, 67.6%, and 22.4% of the control levels in the low, medium, and high groups,
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respectively. The transcription levels of erα decreased to 56.8%, 35.1%, and 42.8% of
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the control in the three treatment groups, respectively (Fig. 7).
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4. Discussion
The concentrations of M. aeruginosa used in this study were over 100,000
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cells/mL, which is similar to the environmental cyanobacterial density during a bloom
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(Saker and Eaglesham, 1999). We measured the concentration of MC-LR, one of the
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most toxic and prevalent congeners among the variants of cyanotoxins, in the algal
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cells and the water at the end of the exposure period. In the low, medium, and high
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groups, the MC-LR concentration was 275.24 ± 8.04 (mean ±SE), 425.54 ± 18.08,
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and 571.95 ± 15.71 µg/L, respectively, in algal cells, whereas it was 24.53 ± 1.97,
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41.85 ± 0.79, and 55.57 ± 5.34 µg/L, respectively in the water (unpublished data).
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Similar concentrations have commonly been encountered in cyanobacterial bloom
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events, indicating that this work has environmental significance (Kardinaal et al.,
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2007; Watson et al., 2017).
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The results from both field and laboratory studies have shown that stress induced
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by toxic substances can impair the liver of fish, and the liver is the primary target
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organ for MCs (Chen et al., 2016b). In the present study, M. aeruginosa exposure
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caused a dose-dependent increase in hepatic histopathological lesions characterized by 12
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necrotic cells. Similarly, severe hepatic injury, including dilatation of sinusoid and
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hyperemia, with blood stasis and inflammatory cells, were observed in adult zebrafish
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after exposure to 200 µg/L MC-LR for 96 h (Wei et al. 2017). Hepatocytes also
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exhibit different degrees of damage after being injected with crude extracts of
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cyanobacteria (Paulino et al., 2017).
Several biomarkers, such as GSI, fecundity rate, fertilization rate, and
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hatchability of eggs, in conjunction with more specific biomarkers, are widely used to
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examine possible adverse effects of environmental pollutants, including
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endocrine-disrupting chemicals, on reproduction (Xu et al., 2008; Naderi et al., 2014).
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Reproductive dysfunction in adult zebrafish caused by M. aeruginosa was observed in
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the present study, which manifested as decreased GSI, reduced fertilization rate, and
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inhibited hatchability. Moreover, a declining trend in the number of spawn was also
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observed, although the difference was not statistically significant. The decrease in GSI
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could be ascribed to pathomorphological changes in the ovaries (Wu et al., 2014), and
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histological lesions, such as vacuolation or irregularity in cell layers, were observed in
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gonadal tissue in the present study. The gonadosomatic tissue damaged could decrease
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the reproductive performance of fish. It has been reported that the reproductive
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performance of medaka fish, including the fecundity and the hatchability, was
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adversely affected by 5 µg/L MC-LR and Microcystis extract (5 µg/L of equivalent
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MC-LR) after 28 days of treatment (Qiao et al., 2016). Wu et al. (2015) also found
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that exposure to less than 40 µg/L MC-LR can lead to subfertility in female mice. Our
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results were in accordance with these previous studies, suggesting that M. aeruginosa
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inhibited the reproductive capacity of female zebrafish and may have
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transgenerational effects. Sex steroid hormone measurements are useful for monitoring reproductive
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systems, as these hormonal changes play crucial roles in the reproduction of fish and
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correspond to alterations in steroidogenic gene transcription (Devlin and Nagahama,
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2002; Ji et al., 2013b). Exposure to M. aeruginosa significantly altered plasma sex
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hormone levels and the expression of genes involved in the steroidogenesis pathway.
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Changes in sex steroid hormone concentrations may result in subsequent reproductive
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dysfunction, including decreased fecundity rate, fertilization rate, and hatch success,
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as observed in this study. The activity and expression of steroidogenic genes control
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steroid hormone synthesis and metabolism (Hogg et al., 2013). For instance, in
292
zebrafish gonads, cyp17 catalyzes the conversion of 17-hydroxyprogesterone to
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androstenedione, which is catalyzed into T by 17βhsd (Fernandes et al., 2011).
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Additionally, aromatase (cyp19) enzymes are known to catalyze the final conversion
295
of androgen to estrogen (Trant et al., 2001), and in zebrafish, T is secreted from the
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follicle cell and finally converted to E2 by aromatase (Clelland and Peng, 2009). In
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the present study, M. aeruginosa exposure inhibited the expression of most
298
steroidogenic genes (cyp19a1a, cyp19a1b, 17βhsd, cyp17, and hmgra), corresponding
299
to the changes of E2 and T levels in the plasma. For example, plasma T concentrations
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significantly decreased in the high group, but no change was found in the low and
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medium groups. Those changes were consistent with the concentration-dependent
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in the ovary may inhibit the conversion of T to E2 such that the ratio of E2/T is
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reduced. This reduced ratio suggests that exposure to M. aeruginosa disrupts the
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balance of sex hormones in fish and results in adverse effects on oogenesis and
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reproduction (Orlando et al., 2004). Similar to our observations, Qiao et al. (2013)
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found that E2 levels were significantly decreased after exposure to 20 µg/L MC-LR,
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whereas no change in T was observed. When zebrafish were exposed to 50 µg/L
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MC-LR, both E2 and T levels were significantly decreased (Zhao et al. 2015).
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In teleosts, the regulation of reproduction is primarily coordinated via interactions between genes along the HPGL axis in addition to genes involved in the
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steroidogenesis pathway (Hilscherova et al., 2004; Villeneuve et al., 2008). Studies in
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female zebrafish have revealed that FSH is primarily involved in vitellogenesis,
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whereas maturation of oocytes is primarily controlled by LH (Sarkar et al. 2014). In
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the present study, the transcription of fsh and lh genes in zebrafish after M. aeruginosa
316
treatment increased significantly, suggesting possible promotion of oogenesis and
317
maturation. However, these effects were not observed in the histological and
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reproductive examination. It is well known that endocrine-disrupting chemicals alter
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normal patterns of gene expression, by either steroid hormone receptor-mediated
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pathways or compensatory effects (Villeneuve et al., 2007). Insufficient E2 content
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could initiate a feedback mechanism that could stimulate the pituitary to respond,
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leading to increased secretion of gonadotropins (Hou et al., 2016). Therefore, the
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upregulation of fshβ and lhβ observed in this study indirectly indicates the existence
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prochloraz decreases the plasma concentration of E2 and causes compensatory
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upregulation of genes of the HPGL in small fish (Ankley et al., 2009; Liu et al., 2010),
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strongly supporting our observations.
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Changes in VTG levels in female fish have been identified as a sensitive
endpoint for assessing the levels endocrine-disrupting chemicals (Dang et al., 2011).
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VTG is a yolk precursor protein that is synthesized by the liver in response to
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estradiol stimulation and is essential for vitellogenesis, oocyte maturation, and yolk
332
biosynthesis (Aravindakshan et al., 2004; Yuan et al., 2013). Seven known vtg genes
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are located differently in the zebrafish genome, and vtg1 is the most highly expressed
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(Tong et al., 2004; Wang et al., 2005). As the expression of vtg genes in the livers of
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female fish is modulated by E2 (Bemanian et al. 2004), the observed downregulation
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of the hepatic vtg1 gene in the present study might have resulted from decreased E2
337
levels in the plasma. Liver lesions caused by M. aeruginosa in the present study may
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also have been responsible for the downregulation of vtg1 mRNA expression.
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However, plasma VTG levels significantly increased in a dose-dependent manner in
340
this study, which may be due to the impairment of the ovaries making it difficult for
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plasma VTG to incorporate into the oocytes, resulting in accumulation in the blood.
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Similarly, Qiao et al., (2013) also reported that MC-LR exposure decreased hepatic
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vtg1 gene expression and increased VTG levels in female zebrafish with MC-LR
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exposure.
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5. Conclusions In summary, the present study demonstrated that short-term (96 h) exposure to M. aeruginosa could impair reproductive capacity and disrupt the endocrine system in
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female zebrafish, damaging the liver and gonadal tissue, altering plasma sex hormone
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levels, and changing the transcription of HPGL axis-related genes. Moreover, the
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decreased fertilization rate and hatchability of eggs spawned from treated females
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demonstrated the possibility of transgenerational effects of M. aeruginosa exposure.
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Future work is necessary to study the reproductive toxicology of M. aeruginosa in a
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systematic manner after long-term exposure to environmentally relevant
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concentrations of M. aeruginosa.
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We gratefully acknowledge the National Natural Science Foundation of China (Nos.21377118 and 21577128) for supporting this research.
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Supplementary data
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Table S1. Sequences of primers for the target genes
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Acknowledgments
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Figure captions
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Fig. 1. Brain index (BSI), hepatosomatic index (HSI), and gonadosomatic index (GSI)
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of female zebrafish exposed to M. aeruginosa for 96 h.
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Fig. 2. Effects of M. aeruginosa on the reproductive capacity of female zebrafish.
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Fig. 3. Histological changes in the liver (A-D) and ovary (E-L) of zebrafish exposed
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to M. aeruginosa for 96 h. Microphotographs show liver and ovary sections (5 µm)
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stained with hematoxylin and eosin (H&E). The original magnification was ×400
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(A-D), ×100 (E-H), ×200 (I-L).
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(A-D): H&E-stained liver section. A: Control group. B: Low group. C: Medium group. D: High group. Black asterisk: hyperemia. White asterisk: dilatation of
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sinusoid. Black arrow: pyknotic or deformed nucleus. White arrow: vacuolation.
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White ellipse: parenchyma disorganization.
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(E-L): H&E-stained ovary section. E, I: Control group. F, J: Low group. G, K: Medium group. H, L: High group. White arrow: the loss of contacts (ellipses) between
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the oocyte cell membranes and the follicular cell layer. Black arrow: the vacuolation
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of the gonadosomatic tissue. White triangle: Irregularity in cell layers. Black asterisk:
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oocyte atresia. White asterisk: degenerating vitellogenic oocytes.
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Fig. 4. Effects of M. aeruginosa on plasma levels of (A) 17β-estradiol (E2), (B)
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testosterone (T), (C) the E2/T ratio, and (D) vitellogenin (VTG) after 96 h of M.
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aeruginosa exposure.
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Fig. 5. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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Fig. 6. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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genes in the ovary of adult female zebrafish after exposure to M. aeruginosa.
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Fig. 7. Relative transcript levels of hypothalamic-pituitary-gonadal-liver (HPGL) axis
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genes in the liver of adult female zebrafish after exposure to M. aeruginosa.
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M. aeruginosa caused histological lesions in liver and gonad of female zebrafish.
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M. aeruginosa impaired reproductive capacity and caused transgenerational
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effects. M. aeruginosa exposure altered the level of plasma hormones in female zebrafish.
M. aeruginosa changed the transcriptions of genes involved in endocrine
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M. aeruginosa can disrupt reproductive and endocrine system in fish.
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system.