Institute of Oceanology, Chinese Academy of Sciences
Article Information
- XIA Jigang(夏继刚), NIU Cuijuan(牛翠娟)
- Acute toxicity effects of perfluorooctane sulfonate on sperm vitality, kinematics and fertilization success in zebrafish
- Chinese Journal of Oceanology and Limnology, 35(4): 723-728
- http://dx.doi.org/10.1007/s00343-017-6086-5
Article History
- Received Mar. 19, 2016
- accepted in principle Apr. 29, 2016
- accepted for publication Jun. 11, 2016
2 Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering, College of Life Sciences, Beijing Normal University, Beijing 100875, China
Perfluorinated compounds (PFCs) are a class of synthetic chemical compounds that have been used in surfactants for commercial and industrial applications over the past 60 years. This extensive use has resulted in global PFC contamination (Giesy and Kannan, 2001; Kunacheva et al., 2012). As the final degradation product of many commercially used PFCs, perfluorooctane sulfonate (PFOS, C8F17SO3-) is widely detected in aquatic ecosystems (Van Gossum et al., 2009). The concentration of PFOS can be up to 6.57 mg/L in some aquatic environments (Zhang et al., 2013). Currently, PFOS has become the target of public concern and has been declared as a Persistent Organic Pollutant (UNEP, 2009) due to its potential toxicity to the biota, persistence in the environment, and ubiquitous occurrence (Huang et al., 2010; Hagenaars et al., 2014; Pereiro et al., 2014; Xia et al., 2015a, b).
With increasing pollution in the environment, monitoring of PFOS toxicity and understanding of its mechanisms are urgently needed for environmental risk assessment. Toxicological studies have revealed that PFOS has adverse effects on reproductive performance (Ankley et al., 2005; Han and Fang, 2010; López-Doval et al., 2014). A recent research study conducted by López-Doval et al. (2014) revealed that PFOS exposure resulted in morphological change of the hypothalamus, degeneration of gonadotrophic cells, loss and degeneration of the spermatozoids and edema in the testes of adult male rats. In fish, laboratory studies have reported that PFOS affects hormone receptor activity, steroidogenesis and expression of endocrine-related genes both in vitro and in vivo (Du et al., 2013), and these effects lead to up-regulation of the vitellogenin (VTG) levels and VTG mRNA expression in male fish (Liu et al., 2007; Du et al., 2009) and also inhibit the growth of the gonads in female fish (Du et al., 2009). Information is particularly lacking regarding the effects of PFOS-mediated toxicities on fish gamete viability.
Generally, fish spermatozoa mature in the sperm ducts where they are immotile due to the osmolality or ionic content of the seminal plasma (Morisawa and Morisawa, 1994). Mature spermatozoa are usually activated by water after release from the genital papilla into the aquatic environment, and the duration of sperm viability is very short in externally fertilizing oviparous fish (Alavi and Cosson, 2005; Hara et al., 2007). Thus, spermatozoa function and successful fertilization might be greatly affected by environmental contaminants in the water. Therefore, it is important to know whether PFOS may reduce reproductive success through impairments of spermatozoa performance for fish population ecology and resource management. The use of zebrafish (Danio rerio) as an appropriate vertebrate model for investigating various environmental pollutants has increased in popularity in recent years (Hill et al., 2005; Wang et al., 2011). Purpose of the present study was to assess the possible toxicity of PFOS on sperm vitality, kinematics and fertilization success of fish using zebrafish as the experimental model.
2 MATERIAL AND METHOD 2.1 ChemicalsHeptadecafluorooctanesulfonic acid potassium salt (PFOS, purity≥98%), dimethyl sulfoxide (DMSO) and tricaine methanesulfonate (MS-222) were purchased from Sigma-Aldrich (Germany). All other chemicals used in this study were analytical grade. PFOS was initially dissolved in DMSO, and the stock solution (0.5 g/mL) was stored at 4℃ until preparation of the final exposure solutions in water.
2.2 Experimental fish and holding conditionsAdult zebrafish (Danio rerio, AB strain) were maintained according to standard culture protocols (Westerfield, 1995). The male and female fish were housed in separate aquariums for 4 weeks prior to the experiment. The rearing water was dechlorinated and filtered through activated carbon before addition into the aquariums. The water temperature was maintained at 25±1℃, the dissolved oxygen level was kept above 6 mg/L, the pH ranged from 7.0 to 7.8, and the rearing system was maintained under a 14 h L:10 h D light cycle. The fish were fed commercial worms (Tubifex tubifex) at 10:00 am and 17:00 pm every day. After the acclimation period, male and female zebrafish of uniform size (i.e., 0.41±0.003 g, n=120 and 0.49±0.003 g, n=96, respectively) were selected as the experimental fish. All experimental procedures were performed according to the Guidelines on the Humane Treatment of Laboratory Animals established by the Ministry of Science and Technology of the People's Republic of China.
2.3 Experimental design and protocolSperm were collected via surgical removal of testes of the fish after application of 0.01% MS-222 anesthesia for 2 min. Adherent tissue was dissected away and the testes were rinsed with Hanks' balanced salt solution (HBSS; NaCl 8.0 g/L, KCl 0.4 g/L, CaCl2·2H2O 0.16 g/L, MgSO4·7H2O 0.2 g/L, NaHCO3 0.35 g/L, Na2HPO4 0.06 g/L, KH2PO4 0.06 g/L, C6H12O6 1.0 g/L, pH 8.0) to remove debris. The testes were then suspended in HBSS and stored at 4℃ according to Jing et al. (2009). The ratio of the testes to HBSS (mass:volume) was 1:100. Sperm were released by gently and repeatedly disrupting the testes with a pipette tip.
Sperm vitality and kinematics were determined with a CASA system (WLJY-9000; Weili New Century Technology Development Co. Ltd.; Beijing; China). To avoid the possible effects of inter-male variation in sperm quality, sperm from the same fish were activated by mixing 1 μL sperm suspension with 9 μL different activation solutions containing a range of PFOS concentrations (0, 0.1, 1 and 10 mg/L). Specifically, the concentrations of PFOS in the treatment groups were 0, 0.09, 0.9 and 9 mg/L. The concentration of DMSO in the activation solutions did not exceed 0.002% (v/v). The viabilities and kinematics of sperm exposed to the different treatments were assessed at 20, 40 60 and 80 s after activation at room temperature (25℃). The percentage of motile sperm, the curvilinear velocity (VCL, i.e., the actual velocity along the trajectory), the straight-line velocity (VSL, i.e., the straight line distance between the start and end points of the track divided by the time required to traverse the track), the angular path velocity (VAP, i.e., the velocity along a derived smoothed path) and the mean angular displacement (MAD) of spermatozoa were calculated for each group from three recordings of at least 50 sperm (30 frames/s). A total of 24 male fish (n=24) were used for each PFOS treatment group.
Eggs (approximately 200) from individual females were collected by gentle abdominal massage, mixed with 100 μL sperm suspension (sperm to egg ratio 3 000:1), and immediately exposed to 50 mL solutions in 9-cm-diameter petri dishes containing a range of PFOS concentrations (0, 0.09, 0.9 and 9 mg/L). After 2 min, the eggs were washed three times and then transferred to uncontaminated water without PFOS. The fertilized eggs at the gastrulation stage were examined using a stereomicroscope, and the fertilization rate was then determined 6 h after exposure to spermatozoa. All of the manipulations were performed at 25℃. A total of 24 male and 24 female fish (n=24) were used for each PFOS treatment group.
2.4 Statistical analysesStatistical analyses were performed using the software program SPSS (version 16.0, SPSS Inc., USA). All values are presented as mean±SEM. The data were firstly examined for normality and homogeneity of variances. When the data did not exhibit homogeneity of variance, a Kruskal-Wallis test was conducted followed by a Dunnett T3 test. The effects of PFOS exposure and exposure time on the percentage of motile sperm and the VCLs, VSLs, VAPs and MADs of spermatozoa were analyzed with two-way analysis of variance (ANOVA). The effects of PFOS on the fertilization rates were detected with one-way ANOVA. ANOVA was followed by Tukey's honest significant difference (HSD) post hoc test (when necessary) to determine the differences among different treatment groups. P < 0.05 was considered statistically significant.
3 RESULT 3.1 Sperm vitalityThe percentage of motile sperm was significantly affected by PFOS (F=14.6, P < 0.001) and exposure time (F=147.5, P < 0.001; Table 1). The percentage of motile sperms showed a negative relationship with PFOS level, and significantly decreased in groups of 0.9 mg/L and higher PFOS level (Fig. 1).
3.2 Sperm kinematicsPFOS exposure profoundly influenced the sperm kinematics. The VCL and MAD of spermatozoa were markedly affected by PFOS (P < 0.05; Table 1). PFOS treatment decreased VCLs after activation for 40 s and 60 s, also MADs after activation for 20 s to 60 s (Fig. 2a, d). However, there were no statistically significant alterations in the VSLs or VAPs in response to PFOS exposure (P>0.05; Table 1; Fig. 2b, c).
3.3 Fertilization ratePFOS treatment imposed significant effects on the fertilization rate (χ2=34.0, P < 0.001; Table 1). Fertilization rate was markedly lowered in treatments of 0.9 mg/L or higher PFOS concentrations (Fig. 3).
4 DISCUSSIONThe ecological risk associated with PFOS has been the subject of growing international concern (Dorts et al., 2011; Pereiro et al., 2014; Xia et al., 2015a). This study is the first to investigate the acute toxicity effects of PFOS on fish sperm in vitro. We clearly demonstrated that PFOS exposure resulted in dysfunction of the spermatozoa as manifested by reduced percentages of motile sperm, decreased VCLs and MADs and lowered fertilization rate. The effects of PFOS on sperm vitality, kinematics and fertilization success in zebrafish were dose-dependent. The lowest observed effect concentration (LOEC) of PFOS in terms of the above parameters was 0.9 mg/L, indicating that the sperm quality of zebrafish may serve as considerable ecologically relevant biomarkers in PFOS pollution accidents.
The fish spermatozoon is differentiated into a head, midpiece, and flagellum (Lahnsteiner and Patzner, 2008). Plasma membrane is the first target that receives the signals that trigger intracellular signaling cascades mediated from the aquatic environment. The axoneme is activated by cAMP-or Ca2+-dependent phosphorylation/dephosphorylation of specific proteins (Inaba, 2007; Hatef et al., 2013), and the mitochondria located in the midpiece produce the ATP required for spermatozoon motility (Ingermann, 2008). In the current study, the decreased performance of sperm due to PFOS exposure might have resulted from impairments at a number of loci, including structural damage to the plasma membrane and functional dysfunction of the energy-producing mitochondria. Previous studies have indeed documented that PFOS increase the permeability and fluidity of cell membranes (Hu et al., 2003), alter the activities of mitochondrial citrate synthase (CS) and cytochrome c oxidase (CCO), impair the ubiquitin-proteasome system and energy metabolism in the gills of the European bullhead Cottus gobio (Dorts et al., 2011), and decrease gill Na+-K+ ATPase activity in Sebastes schlegeli (Jeon et al., 2010). Furthermore, PFOS can elicit oxidative stress and damage DNA in spermatozoa, which subsequently impairs the function of the spermatozoa (Zhang et al., 2009; Hagedorn et al., 2012; Pereiro et al., 2014).
In general, the trajectories of spermatozoa are smooth curves due to the symmetrical waveform of the flagellum (Alavi et al., 2009). VCL, VSL and VAP are useful parameters for assessing sperm performance (Hatef et al., 2013). Interestingly, we found that PFOS exposure reduced the VCL but did not influence the VSL or VAP. The above phenomenon might be partly attributable to alterations in the flagellar waveform, which would in turn influence the MAD (Alavi et al., 2011). The model of the spermatozoa velocity response to PFOS in this study is somewhat consistent with the the results of a study of African catfish (Clarias gariepinus) spermatozoa following instant exposure to a combination of zinc and cadmium (Kime et al., 1996; Hatef et al., 2013). Decreases in spermatozoa velocity parameters that are caused by contaminants provide clues that indicate that knowledge about alterations in intracellular signaling cascades is essential for understanding disruptions in the physiological function and molecular structure of the axoneme (Hatef et al., 2013).
Fertilization success is the most integrative estimator of sperm quality and represents the culmination of nearly all other reproductive endpoints (Bobe and Labbé, 2010). The decrease in fertilization success is indicative of direct toxic effects of PFOS on fish reproductive fitness. Previous studies have reported decreases in the fertilization abilities of killifish (Fundulus heteroclitus) and African catfish spermatozoa following instant exposure to 0.01 mg/L methyl mercury and 1 mg/L mercury, respectively (Khan and Weis, 1987; Rurangwa et al., 1998). Moreover, Rosety et al. (2003) demonstrated that exposure to the anionic surfactant linear alkylbenzene sulphonate (LAS) significantly decreased the quality of gilthead (Sparus aurata L.) sperm, which may at least partially explain the impairment of fertilization success. In the present study, the fertilization rate was reduced by 10.4% and 19.8% after being exposed to 0.9 and 9 mg/L PFOS, respectively. In addition to the deterioration in sperm vitality and kinematics, the adverse effects of PFOS on fertilization success may also be partly attributable to the underperformance of the eggs during fertilization (Lahnsteiner et al., 2005). Regardless, PFOS exhibited considerable toxic effects that hindered the process of sperm-egg contact.
In summary, the adverse effects of PFOS in terms of both the physiological performance of zebrafish spermatozoa (e.g., decreased vitality and VCL) and the potential ecological consequences (e.g., reduced fertilization success) were revealed. Our findings indicate that waterborne PFOS exposure for even very short periods between ejaculation and fertilization will elicit potent ecotoxicological effects on the reproductive fitness of fish.
5 ACKNOWLEDGEMENTWe are grateful to Prof. FU Shijian for his valuable comments in preparing the manuscript.
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