The in vitro effect of nonylphenol, propranolol, and diethylstilbestrol on quality parameters and oxidative stress in sterlet (Acipenser ruthenus) spermatozoa

Accepted Manuscript The in vitro effect of nonylphenol, propranolol, and diethylstilbestrol on quality parameters and oxidative stress in sterlet (Acipenser ruthenus) spermatozoa

Olena Shaliutina, Ievgeniia Gazo, Anna Shaliutina-Kolešová, Ievgen Lebeda, Marek Rodina PII: DOI: Reference:

S0887-2333(17)30122-4 doi: 10.1016/j.tiv.2017.05.006 TIV 4001

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

26 November 2016 18 April 2017 5 May 2017

Please cite this article as: Olena Shaliutina, Ievgeniia Gazo, Anna Shaliutina-Kolešová, Ievgen Lebeda, Marek Rodina , The in vitro effect of nonylphenol, propranolol, and diethylstilbestrol on quality parameters and oxidative stress in sterlet (Acipenser ruthenus) spermatozoa, Toxicology in Vitro (2017), doi: 10.1016/j.tiv.2017.05.006

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ACCEPTED MANUSCRIPT Revised The in vitro effect of nonylphenol, propranolol, and diethylstilbestrol on quality parameters and oxidative stress in sterlet (Acipenser ruthenus) spermatozoa

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Olena Shaliutina*, Ievgeniia Gazo, Anna Shaliutina-Kolešová, Ievgen Lebeda, Marek

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Rodina

University of South Bohemia in České Budějovice, Faculty of Fisheries and

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Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Research Institute of Fish Culture and Hydrobiology,

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Zátiší 728/II, 389 25 Vodňany, Czech Republic.

*Corresponding author: Tel: +420776079649, E-mail: [email protected] (O. Shaliutina)

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ACCEPTED MANUSCRIPT ABSTRACT The sturgeon is a highly endangered fish mostly due to over-fishing, habitat destruction, and water pollution. Nonylphenol (NP), propranolol (PN), and diethylstilbestrol (DES) are multifunctional xenobiotic compounds used in a variety of commercial and industrial products. The mechanism by which these xenobiotic

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compounds interfere with fish reproduction is not fully elucidated. This study

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assessed the effect of NP, PN, and DES on motility parameters, membrane integrity,

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and oxidative/antioxidant status in sterlet Acispenser ruthenus spermatozoa. Spermatozoa were incubated with several concentrations of target substances for 1

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h. Motility rate and velocity of spermatozoa decreased in the presence of xenobiotics in a dose-dependent manner compared with controls. A significant decrease in

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membrane integrity was recorded with exposure to 5 µM of NP, 25 µM of PN, and 50 µM of DES. After 1 h exposure at higher tested concentrations NP (5-25 µM), PN

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(25-100 µM), and DES (50-200 µM), oxidative stress was apparent, as reflected by

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significantly higher levels of protein and lipid oxidation and significantly greater superoxide dismutase activity. The results demonstrated that NP, PN, and DES can

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induce reactive oxygen species stress in fish spermatozoa, which could impair sperm

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quality and the antioxidant defence system and decrease the percentage of intact sperm cells.

Keywords: Xenobiotics, Lipid oxidation, Spermatozoon motility, Membrane integrity, Sterlet sperm

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ACCEPTED MANUSCRIPT 1. Introduction Most fish populations are exposed to a wide variety of man-made chemicals at concentrations that are not directly toxic. Nevertheless, at sublethal concentrations, exposure can induce harmful effects and potentially reduce populations. In recent years, there has been increasing concern about the potential health effects of

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xenobiotics, since these compounds have been associated with reproductive

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dysfunction in a variety of wildlife (Kime, 1999). Reports have shown that xenobiotics

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can cause disruption of the reproductive endocrine system (Arukwe, 1997) or directly affect gamete development and viability as a result of cytotoxicity (Kime, 1999) or by

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altering the hormonal environment during gamete development (Giesy and Snyder,

Nonylphenol

(NP),

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1998). propranolol

(PN),

and

diethylstilbestrol

(DES)

are

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multifunctional xenobiotic compounds used in a variety of commercial and industrial

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products. Detected environmental concentration for NP range from 0.7 ng L-1 to 15 μg L-1 (Petrovic et al., 2002), for PN from 0.59 to1.9 μg L-1 (Owen et al., 2007) and for DES from 0.98 to 51.6 ng g−1 (Lei et al., 2009). During the past decade, concern over

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their widespread usage has increased because of toxicity to both marine and

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freshwater species and their ability to induce estrogenic responses. Estrogenic effects of NP have been observed at 10 – 20 μg L-1 (Carlisle et al., 2009), of PN at 0.5 μg L-1 (Huggett et al., 2002), and of DES at 2 – 10 ng L-1 (Zha et al., 2008). Propranolol is a non-selective beta blocker, blocking the action of epinephrine and norepinephrine on both β1- and β2-adrenergic receptors. Nonylphenol and DES are endocrine disrupting chemicals (EDCs) that can act as xenoestrogens, modulating the endocrine pathways via a receptor-mediated process (Feng et al., 2011; WHO/UNEP, 2013). The presence of estrogen receptors (ERs) and beta-adrenergic 3

ACCEPTED MANUSCRIPT receptors on the spermatozoon membrane has been reported in mammals (Saberwal et al., 2002; Adeoya-Osiquwa et al., 2006), but there is no evidence for these receptors in fish spermatozoa. Therefore, it is necessary to observe direct effects of xenobiotics on fish spermatozoa to gain a better understanding of molecular mechanisms of their toxicity.

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Sperm quality, defined as those traits of sperm that determine its capacity to

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fertilize eggs, is crucial for aquaculture purposes and must be monitored in fish

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farming to predict male reproductive success. Sturgeon spermatozoa are immotile in the testis and acquire the potential for motility after contact with hypoosmotic medium

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(fresh water). Water pollution can damage sperm, affecting their viability (Kime, 1999). Sperm of most fish species can be affected by exposure to a wide variety of

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manmade compounds released into water. Spermatozoon head possess membranes containing polyunsaturated fatty acids, which are highly susceptible to oxidative

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damage (Fabrik et al., 2008). When the production of reactive oxygen species is

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excessive, the gamete's limited defences are rapidly overwhelmed, and oxidative damage induces lipid peroxidation (LPO), with a resulting loss of motility and

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fertilizing potential (Aitken et al. 1998; Shaliutina-Kolesova et al., 2014). Therefore,

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assessment of motility parameters of sperm may be a sensitive and accurate bioindicator of aquatic pollution (Li et al., 2010). The sturgeon is among the world's most valuable wildlife resources. These northern hemisphere fishes can be found in large river systems, lakes, coastal waters, and inland seas throughout Eurasia and North America (Birstein and DeSalle, 1998). Most of the world’s sturgeon populations have undergone significant decline, mainly due to over-fishing, habitat destruction, and pollution (Pikitch et al., 2005). It is logistically difficult and costly to conduct toxicity evaluations on 4

ACCEPTED MANUSCRIPT broodstock (Tashjian et al., 2006), therefore, in this study, we used an in vitro sperm assay with sterlet Acipenser ruthenus as a model to investigate potential adverse effects of the xenobiotics nonylphenol, propranolol, and diethylstilbestrol. The main objectives were to explore effects of short-term (1 h) in vitro exposure to NP, PN, and DES on quality parameters and oxidative stress in spermatozoa of the

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sterlet Acipenser ruthenus by assessing spermatozoon motility, velocity, and

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membrane integrity and analysing oxidative stress indices, including lipid oxidation,

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protein carbonylation, and superoxide dismutase activity.

2. Materials and methods

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All experiments were conducted according to the principles of the Ethics Committee for the Protection of Animals in Research of the University of South Bohemia in

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Ceske Budejovice, Research Institute of Fish Culture and Hydrobiology, Vodnany

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(based on the EU-harmonized Animal Welfare Act of the Czech Republic).

2.1. Fish handling and sperm collection

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The breeding and culture of sterlet Acipenser ruthenus was carried out at the Genetic Fisheries Center, Faculty of Fisheries and Protection of Waters, University of South Bohemia in České Budějovice. Six males age six years, 0.5–2 kg, were used. Prior to experimentation, fish were held in hatchery tanks with water temperature 15 °C. Spermiation was induced by intramuscular injection of carp pituitary powder dissolved in 0.9% (w/v) NaCl solution at 4 mg per kg of body weight. After 24 h, semen was obtained from the urogenital tract using a 5–7 mm plastic catheter

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ACCEPTED MANUSCRIPT connected to a 20 mL plastic syringe. Care was taken to avoid contamination by urine, mucus, faeces, or water. Syringes (one per male) were placed on ice and immediately transported to the laboratory for analyses. Spermatozoon concentrations were examined microscopically (Olympus BX 41) at magnification 20x and estimated using a Burker cell haemocytometer. Mean spermatozoon concentration was 1.26 ±

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0.7 × 109 ml-1.

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2.2. Sperm dilution and exposure

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Diethylstilbestrol (DES) [4,4'-(3E)-hex-3-ene-3,4-diyldiphenol; (E)-11,12-Diethyl4,13-stilbenediol; empirical formula: C18H20O2; MW: 268.35; (≥99% (HPLC); SigmaUSA)]

and

propranolol

(PN)

[(RS)-1-(1-methylethylamino)-3-(1-

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Aldrich,

naphthyloxy)propan-2-ol; empirical formula: C16H21NO2; MW: 259.34; (≥99% (TLC)

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Sigma-Aldrich, USA)] were first dissolved in dimethyl sulphoxide (DMSO) at 200 mM

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for DES and 100 mM for PN and further diluted with DMSO to obtain stock solutions of 10, 25, and 50 mM for PN and 10, 50, 100 mM for DES. Nonylphenol (NP) [4-(2,4-

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dimethylheptan-3-yl)phenol; empirical formula: C15H24O; MW: 220.35; PESTANAL®, analytical standard; Sigma-Aldrich, USA] was dissolved in ethanol at 100 mM and

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diluted with ethanol to obtain stock solutions of 1, 5, 10, and 25 mM. For getting the final concentration in µM, stock solutions were diluted with an immobilization medium (IM) (20 mM Tris, 30 mM NaCl, 2 mM KCl, pH 8.5) at a dilution ratio of 1:1000 to obtain the final concentration of solvents 0.1% (v/v) in incubation medium. Stock solutions were prepared daily. The individual sperm samples from six males were centrifuged at 300 x g and 4 °C for 30 min to remove seminal plasma and subsequently diluted with IM to obtain spermatozoon densities of 5 × 108 cells mL-1. The sperm sub-samples were separately exposed for 1 h to final concentrations of 6

ACCEPTED MANUSCRIPT NP (1, 5, 10, and 25 µM); PN (10, 25, 50, and 100 µM) and DES (10, 50, 100, and 200 µM) at 4 ºC. A control group for NP was exposed to IM with 0.1% ethanol, and the control group for PN and DES was exposed to IM with 0.1% of DMSO.

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2.3. Spermatozoon motility and velocity recording

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The spermatozoa motility parameters were measured as described in our previous works (Linhartova et al., 2013; Gazo et al., 2013). In brief, curvilinear

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velocity (VCL, µm s-1) and percent of motile spermatozoa (motility, %) were evaluated

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at 10, 30, and 60 s post-activation under dark-field microscopy (Olympus BX 50, Tokyo, Japan) at ×20 objective. For motility activation sperm was diluted 1:5000 in

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activation medium (10 mM Tris, 10 mM NaCl, 1 mM CaCl2, pH 8.5). Recordings were made using a video recorder (Sony SVHS, SVO-9500 MDP, Japan). The movements

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of the spermatozoa heads were analyzed using Olympus MicroImage software

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(Version 4.0.1. for Windows with a special macro by Olympus C & S). Spermatozoa head positions on five successive frames are assigned different colors: frame 1 red,

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frames 2–4 green, and frame 5 blue. Those that moved were visible in three colors, while non-moving spermatozoa were white. The percent of motile spermatozoa was

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calculated from the number of white and red cells. 20 to 40 spermatozoa were counted for each frame. Spermatozoa motility activation and measurement was performed in triplicate for each sample.

2.4. Spermatozoon membrane integrity assessment To assess membrane integrity, sperm samples were diluted with PBS to a volume of 2 mL and concentration of 10 000 cells/ml. Diluted samples were stained 7

ACCEPTED MANUSCRIPT with 5 µL of 5X SYBR Green (10,000X, S9430, Sigma, Singapore) for 5 min and then with 5 µl of 4.8 mM propidium iodide, which penetrates non-viable spermatozoa when the plasma membrane is disrupted, for 30 min. A minimum of 2000 cells were analyzed by CUBE 8 (Partec, Germany) cytometer with flow speed 0.2 µl s-1. The data were processed by CyView 1.3 (Partec, Germany). For each sample, numbers

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of cells with high and low concentration of propidium iodide were compared to

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calculate percentage of cells with intact vs. damaged membranes.

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2.5. Lipid peroxidation and antioxidant enzyme activity

Sperm samples were centrifuged at 5000 x g at 4 °C for 10 min. The supernatant

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was collected and discarded. The spermatozoon pellet was suspended in 50 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA and 0.1 mM PMSF to

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obtain a spermatozoon concentration of 5 x 108 cells mL-1 and homogenized in an ice

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bath using a Sonopuls HD 2070 ultrasonicator (Bandelin Electronic, Berlin, Germany). A portion of the homogenate was used for measuring thiobarbituric acid

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reactive substances (TBARS) and carbonyl derivatives of proteins (CP), and the remaining was centrifuged at 12 000 x g for 30 min at 4 °C to obtain the post-

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mitochondrial supernatant for the antioxidant enzyme activity assay. The TBARS was measured as described by Lushchak et al. (2005) as the product of lipid peroxidation (LPO). Its concentration was calculated by absorption at 535 nm wavelength in the spectrophotometer using a molar extinction coefficient of 156 mM cm-1. The bicinchoninic acid kit for protein determination (Sigma-Aldrich, USA) was used to measure the protein concentration in samples. The TBARS content was expressed as nmol per mg of protein. The CP level was detected by 8

ACCEPTED MANUSCRIPT reaction with 2,4-dinitrophenylhydrazine (DNPH) according to the method described by Lenz et al. (1989). The quantity of CP was measured spectrophotometrically at 370 nm using a molar extinction coefficient of 22 mM cm -1, expressed as nmol mg-1 of protein. Oxidative stress indices were obtained in triplicate for each sample from each individual.

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Total superoxide dismutase (SOD) activity was determined by the pyrogallol

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autoxidation method described by Marklund and Marklund (1974) and was assessed

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spectrophotometrically at 420 nm. Antioxidant activity data were obtained in triplicate

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for each sample from each individual.

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2.6. Data presentation and statistical analysis

All measurements were conducted in triplicate. Normality and the homogeneity of

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variance of all data were first tested with the Kolmogorov test and the Bartlett test,

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respectively. Statistical comparison was made by analysis of variance (ANOVA) followed by Tukey’s HSD test for each analyzed parameter. Values were expressed

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as means ± SD (n = 6). All analyses were performed at a significance level of 0.05

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using STATISTICA 9.0 software for Windows.

3. Results

3.1. Spermatozoon motility and velocity The effects of NP, PN, and DES on spermatozoon motility and VCL were assessed 10, 30, and 60 s post-activation (Figs. 1-3). The percentage of motile cells in the control, expressed as spermatozoon motility, was approximately 85% at 10 s 9

ACCEPTED MANUSCRIPT post-activation. In the presence of xenobiotics, motility and velocity decreased in a dose-dependent manner compared with the control. At 10, 30, and 60 s postactivation, no significant (p > 0.05) differences were found between control and samples exposed to NP at concentration of 1 µM, whereas significant decline of spermatozoon motility was observed at 10–25 µM (Fig. 1A). A significant reduction in

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motility rate was observed 30 and 60 s post-activation in the group exposed to 25 µM

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NP, compared to other NP concentrations (Fig. 1A). Similarly, at 10, 30, and 60 s

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post-activation spermatozoa exposed to 10–25 µM of NP showed significantly lower (ANOVA; p < 0.05) VCL than did the control (Fig. 1B).

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Spermatozoa after treatment with PN showed a significantly lower motility rate (p < 0.05) at concentrations of 50-100 µM compared to the control at all tested time-

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points after activation (Fig. 2A). Similarly, a significant dose-dependent reduction of VCL (p < 0.05) was observed in spermatozoa treated with 25-100 µM PN (Fig. 2B).

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For example, at 10 s post-activation, VCL reached 180 µm s-1 in the control group

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compared to 122 µm s-1 in spermatozoa exposed to 100 µM PN (Fig. 2B). The minimum concentration of DES that affected sturgeon spermatozoon motility

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at 10 s post-activation was 100 µM; however, at 30 s post-activation, motility was

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decreased with 10 µM (Fig. 3A). On the other hand, VCL was significantly reduced with 10 µM at all tested time-points (Fig. 3B). A motility rate of 48% and VCL of 124.61 ± 5.6 µm s-1 were observed in sperm samples exposed to 200 µM of DES at 10 s post-activation (Figs 3A and 3B).

3.2. Membrane integrity

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ACCEPTED MANUSCRIPT Indices of spermatozoon membrane integrity in control groups and groups exposed to NP, PN, and DES are shown in Fig. 4. There was no significant difference (p > 0.05) in membrane integrity of spermatozoa in the control groups with that exposed to the lowest concentrations of NP, PN, and DES. The percentage of intact spermatozoa after exposure to concentrations of NP over 5 µM was

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significantly lower than in the control (p < 0.05). Samples exposed to the highest

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concentration (100 µM) of PN showed 53.01 ± 5.91% intact cells (Fig. 4). A

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significantly lower percentage of intact spermatozoa after exposure to DES at concentrations of 50 µM was observed compared to control (p < 0.05; Fig. 4). The

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highest DES concentration (200 µM) was associated with 43.3 ± 4.41% intact

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spermatozoa.

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3.3. Indices of oxidative stress and antioxidant response

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To assess the occurrence of oxidative imbalance induced by NP, PN, or DES, LPO levels, as indicated by TBARS level, along with CP levels were measured

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(Table 1). The NP control group (ethanol) TBARS level was 0.23 ± 0.02 nmol mg-1 of protein and, for PN and DES controls (DMSO) was 0.25 ± 0.03 nmol mg-1 of protein.

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The CP level in the NP control was 12.96 ± 1.45 nmol mg-1 of protein and 12.47 ± 2.02 nmol mg-1 of protein in PN and DES controls. There was no significant difference (p > 0.05) in TBARS level of spermatozoa in the control groups and those treated with the lowest concentrations of the xenobiotics. TBARS levels observed at 5 µM NP, 25 µM PN, and 50 µM DES were significantly higher (p < 0.05) than controls, correlating with decrease in motility parameters. The samples exposed to 25 µM NP, 50 µM PN, and 50 µM DES showed approximately equal TBARS levels (0.42 ± 0.03 nmol mg-1 of protein; 0.43 ± 0.04 nmol mg-1 of protein, and 0.43 ± 0.02 nmol 11

ACCEPTED MANUSCRIPT mg-1 of protein, respectively), which may indicate difference in toxicity of these compounds. Compared to the controls, CP was significantly higher with exposure to NP at 5 µM, PN at 10 µM, and DES at 50 µM (Table 1). The highest detected CP levels were 27.30 ± 5.76 nmol mg-1 of protein in spermatozoa exposed to the 25 µM

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concentration of NP, 32.11 ± 5.00 nmol mg-1 of protein in spermatozoa exposed to

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100 µM of PN, and 34.88 ±3.90 nmol mg-1 of protein in sperm exposed to 200 µM of

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DES. These data correspond to the lowest motility rate and the lowest membrane integrity.

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The SOD activity, an indicator of antioxidant activity in fish spermatozoa, was

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significantly higher in all treatment groups compared to the controls (P < 0.05) (Table 1). Total SOD was 7.04 ± 1.04 mU mg -1 of protein in spermatozoa exposed to 25 µM

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of NP compared to 1.12 ± 0.37 mU mg-1 of protein in the ethanol control. It was 8.09

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± 1.07 mU mg-1 at 100 µM of PN, and 8.71 ± 1.05 mU mg-1 at 200 µM of DES,

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4. Discussion

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compared to 1.80 ± 0.30 mU mg-1 of protein in the DMSO control.

Environmentally relevant concentrations of chemical compounds have been shown to have adverse effects on reproduction in various species including fish (Hulak et al., 2013; Linhartova et al., 2013), quail (Razia et al., 2006), and rat (McClusky et al., 2007). In fish, successful fertilization and subsequent embryo development require adequate spermatozoon motility, as well as acrosomal and chromatin integrity (Bobe and Labbe, 2009). The current study showed that DES, PN, and NP reduced sturgeon spermatozoon motility parameters, membrane 12

ACCEPTED MANUSCRIPT integrity, and the antioxidant defence system with in vitro exposure at relatively high toxicological concentrations. In sturgeon, spermatozoon motility is often used for assessment of sperm quality (Cosson et al., 2000). This is a highly sensitive and reliable endpoint for toxicology studies, since it can be rapidly assessed and analysed. Unlike in vivo studies, sperm

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in vitro assays do not incur ethical issues. Data from the present study demonstrated

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that NP, PN, and DES, significantly decreased sterlet spermatozoon motility rate and

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velocity in a dose-dependent manner. These results are in agreement with studies on common carp Cyprinus carpio (Nicotra and Senatori, 1992) and Japanese medaka

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Oryzias latipes (Hara et al., 2007) that showed lower spermatozoon motility with

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exposure to DES and NP.

These compounds potentially affect spermatozoon motility activation and

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movement in several ways: (1) through binding to membrane and/or nuclear

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receptors thereby influencing intracellular signalling and metabolic processes; (2) by inducing excessive production of reactive oxygen species (ROS), leading to development of oxidative stress. In vitro studies on rat spermatozoa indicated

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significant reduction in acrosome integrity and motility after exposure to 1 and 250 µg

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mL-1 NP, concentrations close to nonylphenol IC50 for ER binding (Uguz et al., 2015; Blair et al., 2000). Studies with DES (Tayama et al., 2006) have shown reproductive toxin to be associated with abnormal sperm morphology and lower sperm count in mouse at concentration 1 µg kg-1 body weight. However, DES and NP mechanism of action may differ in mammalian and fish spermatozoa, since mammalian spermatozoa possess membrane estrogen receptors (ER) (Saberwal et al., 2002). In fish, Thomas and Doughty (2004) observed that non-estrogenic, as well as estrogenic organic compounds, could interfere with rapid non-genomic progestin 13

ACCEPTED MANUSCRIPT action to up-regulate motility in Atlantic croaker

Micropogonias undulatus

spermatozoa. However, they suggest that ERs are unlikely to be involved in spermatozoa response to xenoestrogens, since the high concentrations of estradiol17β used in the study are inconsistent with an ER-mediated mechanism (Thomas and Doughty, 2004). Results of the current study also present indirect evidence that

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fish spermatozoa likely lack membrane ERs, since decreased motility, observed only

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at relatively high concentrations of DES and NP, was correlated with decreased

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membrane integrity and elevated levels of lipid and protein oxidation. This indicate that investigated multifunctional xenobiotic compounds have toxic effect on sperm at

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relatively high concentrations; however, no effect on sperm motility related to receptor binding was observed at lower, pharmacological, concentrations. We

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suggest that mitochondrial damage associated with chemical exposure can result in the formation of reactive oxygen species and could be one of the major causes of

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reduced sperm motility. However, it is still unclear to what extent NP and DES

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influence the energy performance of mitochondria and its effect on fertilization. It is known that xenobiotics, able to block β-adrenergic receptors, interfere with

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sperm motility (Ahmadi et al., 2014). Adrenergic monoamines possibly modulate spermatozoon motility by both a calcium-dependent and a cyclic nucleotide-

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dependent mechanism (Ahmadi et al., 2014). Effects of β‐ adrenergic antagonists on carp spermatozoa indicate that PN affects fish spermatozoon motility and causes vesicle formation in sperm (Nicotra and Senatori, 1992). However, the exact mechanism of PN action should be further studied, since the presence of adrenergic receptors has not been confirmed in fish spermatozoa. Our results indicate that PN effects on sterlet spermatozoon motility were accompanied by decreased percentage of intact cells and increased indices of oxidative stress at high, cytotoxic 14

ACCEPTED MANUSCRIPT concentrations. Similar with other tested xenobiotics, no receptor-mediated effect on sperm motility was observed at lower concentrations. In this study, DES, PN, and NP-induced alterations in spermatozoon membrane integrity appeared to be a sensitive indicator of exposure. Significant loss of membrane integrity of sterlet spermatozoa was observed at NP exposures of 5 µM,

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25 µM of PN, and 50 µM of DES, indicated that these compounds affect different

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segments of membrane function.

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Lipid metabolism in spermatozoa is important for cell structure and for energy production. Spermatozoon membranes of both mammals (Lenzi et al., 1996) and fish

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(Pustowka et al., 2000) contain high levels of polyunsaturated fatty acids (PUFA).

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The LPO cascade is initiated when reactive oxygen species (ROS) attack PUFAs in the spermatozoon cell membrane (Storey, 1997). As a consequence of LPO, the

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plasma membrane loses the fluidity and integrity. In addition to affecting membrane

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fluidity, ROS-induced peroxidation can also reduce the antioxidant defences of the spermatozoa and increase peroxidation of membrane phospholipids (Thomas et al., 1998). In our work, we found that the TBARS level, as a bio-indicator of oxidative

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damage, increased with addition of NP, PN, or DES in a dose-related manner. The

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high production of ROS may be associated with mitochondrial stress and decreased spermatozoon motility, defective acrosome reaction, and loss of fertility (Aitken et al., 1993).

The formation of carbonyl groups is used as an index of oxidative modification of proteins (Levine et al., 1994). An increase in carbonyl content and protein oxidation may occur as the consequence of attack by free radicals (DuTeaux et al., 2004). Protein carbonyls (CP) are useful biomarkers of ROS-mediated protein oxidation (Levine et al., 1990). Previous studies have shown that protein oxidation and 15

ACCEPTED MANUSCRIPT accumulation of lipid hydroperoxides in the plasma membrane can profoundly affect fertilization ability of spermatozoa (DuTeaux et al., 2004). In our experiment, CP level in sterlet spermatozoa significantly increased, compared to the control, at the concentrations of 5 µM NP, 10 µM PN, and DES 50 µM, evidence that CP is more sensitive than LPO as an indicator of oxidative stress in sterlet spermatozoa. In

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addition, based on these results, it is likely that oxidative stress induced by xenobiotic

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compounds resulted in impaired motility and velocity via enzyme inactivation.

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It is widely accepted that ROS and LPO-induced damage in living sperm can be reduced or even eliminated by the action of enzymatic and non-enzymatic

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antioxidants (Partyka et al., 2012). Superoxide dismutase is considered the first line of defence against the effects of oxyradicals in the cell through catalysing the

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dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen (Shaliutina-Kolesova et al., 2014). In the present study, the highest SOD activity was

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observed at 100 µM of PN and 200 µM of DES. However, significantly higher SOD

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activity compared to controls was observed in all treatment groups. This is likely to be an adaptive response to toxic stress and serves to neutralize the impact of increased

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ROS generation.

5. Conclusions

Our data suggested that nonylphenol, propranolol, and diethylstilbestrol as multifunctional xenobiotic compounds have detrimental effects on sterlet sperm characteristics partially through the incensement of lipid peroxidation and free radical formation. Further studies are needed to clarify the mechanism of xenobiotics effect on fish spermatozoa and possible consequences for offspring. Focus should also be 16

ACCEPTED MANUSCRIPT on possible synergistic interactions of xenobiotics, through which even low, environmentally relevant, concentrations could affect the reproductive processes. These preliminary results will be applicable to future studies of mechanisms of

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xenobiotics action and induction of oxidative stress in sterlet spermatozoa.

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ACCEPTED MANUSCRIPT Acknowledgements The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects CENAKVA (No. CZ.1.05/2.1.00/01.0024), CENAKVA II (No. LO1205 under the NPU I program) and by the Czech Science Foundation (15-03044S). The Lucidus Consultancy, UK is gratefully acknowledged

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for English correction and suggestions.

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Adeoya-Osiquwa, S.A., Gibbons, R., Fraser, L.R., 2006. Identification of functional alpha2- and beta-adrenergic receptors in mammalian spermatozoa. Hum.

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Ahmadi, R., Hajighorbani, M., MasaeeManesh, M.B., 2014. In vitro effects of propranolol on human sperm motility. International Conference on Earth, environment and life sciences (EELS-2014) 23-24, Dubai (UAE).

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Aitken, R.J., Harkiss, D., Buckingham, D.W., 1993. Analysis of lipid peroxidation

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ACCEPTED MANUSCRIPT Uguz, C., Varisli, O., Agca, C., Evans, T., Agca, Y., 2015. In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa. Andrologia 47(8), 910-919. WHO (World Health Organization)/UNEP (United Nations Environment Programme), 2013. The State-of-the-Science of Endocrine Disrupting Chemicals – 2012,

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rarus). Ecotoxicol. Environ. Safety 71, 390–399.

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ACCEPTED MANUSCRIPT Figure legends Fig. 1. In vitro effect of nonylphenol (NP) on spermatozoon motility (A) and velocity (B) in Acipenser ruthenus (number of males, n = 6). Data are presented as mean±SD. Different letters denote significant difference between treatments (ANOVA, p ˂ 0.05).

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Fig. 4. Membrane integrity of Acipenser ruthenus spermatozoa after in vitro exposure

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ACCEPTED MANUSCRIPT Table 1 Influence of nonylphenol (NP), propranolol (PN), and diethylstilbestrol (DES) on TBARS, CP, and SOD level in Acipenser ruthenus spermatozoa. Data represent mean±SD (number of males, n = 6). Different letters indicate significant differences

SOD

(µM)

(nmol mg-1 of protein)

(nmol mg-1 of protein)

control

0.23 ± 0.02a

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12.96 ± 1.45ab

1.12 ± 0.37a

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0.26 ± 0.04 ab

16.81 ± 3.4b

3.29 ± 0.29b

5

0.30 ± 0.04 b

20.98 ± 4.18c

4.53 ± 0.83c

10

0.37 ± 0.03c

24.80 ± 3.79c

5.70 ± 1.11cd

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0.42 ± 0.03c

27.30 ± 5.76c

7.04 ± 1.04d

control

0.25 ± 0.03a

12.47 ± 2.02a

1.80 ± 0.30a

10

0.29 ± 0.05ab

17.29 ± 2.67b

2.69 ± 0.21b

0.35 ± 0.03b

25.64 ± 4.45c

5.10 ± 0.71c

0.43 ± 0.04c

29.28 ± 4.86c

7.55 ± 0.61d

0.52 ± 0.05c

32.11 ± 5.00c

8.09 ± 1.07d

control

0.25 ± 0.04a

12.47 ± 4.02a

1.80 ± 0.30a

10

0.31 ± 0.04a

17.81 ± 3.91b

2.90 ± 0.59b

50

0.43 ± 0.02b

22.98 ± 3.98bc

4.82 ± 0.82c

100

0.47 ± 0.03b

26.90 ± 3.12c

5.33 ± 0.92c

200

0.55 ± 0.04c

34.88 ± 3.90d

8.71 ± 1.05d

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ACCEPTED MANUSCRIPT Highlights

o The effect of NP, PN, and DES on sperm of sterlet was investigated o NP, PN, and DES induced a loss of sperm motility at relatively high concentrations

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o The highest concentrations of NP, PN, and DES led to oxidative stress in fish sperm

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o Fish sperm could be used for monitoring pollutants in aquatic environment

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