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Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact
Ecotoxicology and Environmental Safety 147 (2018) 407–412
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact
MARK
Alessandra Galloa, Raffaele Bonib, Elisabetta Tostia,⁎ a b
Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Napoli, Italy Department of Sciences, University of Basilicata, 75100 Potenza, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Spermatozoon Viability assessment Marine invertebrate Spectrofluorimetry
The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.
1. Introduction Sperm quality is defined as the ability of spermatozoa to successfully fertilize an oocyte, to subsequently allow the development of a normal embryo, and it is influenced by several external factors (Bobe and Labbé, 2010). Assessment of sperm quality in marine species is an important issue due to the increase of ecotoxicology studies looking at the impacts of environmental pollutants on male reproductive health (Gallo and Tosti, 2016). In marine pollution monitoring programs and ecotoxicological studies, sperm cell toxicity tests have been commonly performed and successfully used as a test to monitor and evaluate the adverse effects of environmental contaminants; however, in most cases spermiotoxicity was assessed by evaluating fertilization success and the induction of transmissible damages to the offspring (Gallo et al., 2011; Gallo and Tosti, 2013, 2015; Manzo et al., 2006; Pagano et al., 1996). Sensitive and practical methods are requested for testing different sperm quality parameters to use as endpoint in spermiotoxicity tests.
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Viability is a key determinant of sperm quality whose evaluation is generally achieved using sensitive and specific fluorescent probes in combination with microscopy analysis or flow cytometry (Lewis and Ford, 2012). The dual DNA staining using SYBR-14 and propidium iodide (PI) is among the commonly used stains for assessing sperm viability. The membrane-permanent dye SYBR-14 stains the nucleus of living spermatozoa emitting a bright green fluorescence, while PI only stains DNA in damaged cells that have lost their membrane integrity (Garner and Johnson, 1995). Among marine animals, the dual DNA staining has been successfully used for assessing sperm viability in fishes (Cabrita et al., 2005; Flajšhans et al., 2004; Martínez-Páramo et al., 2013), molluscs (Akcha et al., 2012; Favret and Lynn, 2010; Le Goïc et al., 2013; Paniagua-Chávez et al., 2006; Rolton et al., 2015; Smith et al., 2012; Suquet et al., 2016), crustaceans (Sasson et al., 2012) and echinoderms (Favret and Lynn, 2010). Nevertheless, no attempts have been made to apply this method to the assessment of sperm viability in ascidians.
Correspondence to: Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail address: [email protected] (E. Tosti).
http://dx.doi.org/10.1016/j.ecoenv.2017.07.069 Received 20 April 2017; Received in revised form 24 July 2017; Accepted 28 July 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 147 (2018) 407–412
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The proportion of living and dead spermatozoa is quantified by flow cytometry or fluorescence and confocal microscopy (Favret and Lynn, 2010; Le Goïc et al., 2013; Paniagua-Chávez et al., 2006; Rolton et al., 2015; Sasson et al., 2012; Smith et al., 2012; Suquet et al., 2016). As a key advantage flow cytometry permits to evaluate many parameters on small samples in a very short time providing rapid and accurate results (Graham et al., 1990; Hossain et al., 2011). On the contrary, this technic presents several disadvantages including costs, being flow cytometer an expensive instrument to buy and maintain. Moreover, the computer interfaced flow cytometers are very sophisticated, so skilled and highly trained operators can run it and get any acceptable levels of performance from such an apparatus. Fluorescence and confocal microscopy permit to identify fluorescent signals in individual cells (Ramalho-Santos et al., 2007; Suzuki et al., 1997); however, few cells from the same sample are assessed. These problems can be overcome by using spectrofluorimetry. Compared with flow cytometry, spectrofluorimetry is a high sensitive, specific, simple, easy to operate, and rapid technique that requires a relatively inexpensive instrument (Andrade-Eiroa et al., 2013). However, to the best of our knowledge, no information is available on the use of spectrofluorimetry to assess sperm viability on marine invertebrates. In the present study, we develop a spectrofluorimetric method to assess sperm viability in three different marine invertebrates (the ascidian Ciona intestinalis, the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus). These species were chosen due to their ecological role in marine ecosystems that has led to their use as model for biomonitoring marine pollution. The main objectives of this study were: 1) to determine whether the dual DNA fluorescence staining is a suitable method for sperm viability assessment in C intestinalis, M. galloprovincialis and P. lividus; 2) to develop a simple, accurate, rapid, and sensitive spectrofluorimetric method for the assessment of sperm viability in marine invertebrates.
injected with 0.5 M KCl solution through the peribuccal membrane and then spermatozoa were collected dry. After collection, fresh and dry spermatozoa from all species were held on ice. The concentration and motility of collected spermatozoa were evaluated by a Makler counting chamber and, then, spermatozoa were diluted to the desired concentration in filtered (Millipore 0.22 µm; Milli Q, Medford, MA, USA) natural seawater (FNSW) (38 PSU salinity, pH 8.2 ± 0.1).
2.3. Dual DNA fluorescence staining The dual DNA staining was performed using SYBR 14 and PI from a commercially available LIVE/DEAD Sperm Viability Kit (Life technologies, Milan, Italy). Fluorescent staining was carried out as follows: for each species, an aliquot of spermatozoa diluted at 5 × 106 cell/ml were stained by adding SYBR-14 (final concentration 100 nM) and incubated in the dark at 18 ± 1 °C for 15 min according to manufacturer instructions. After SYBR-14 incubation, samples were incubated at 18 ± 1 °C in 12 µM PI dye for 15 min. For microscope examination, samples were immediately analysed; for spectrofluorimetric analysis, samples were washed by adding FNSW, centrifuged at 2000 rpm for 10 min, and then resuspended in FNSW. In order to validate the sperm viability assay, standard curves were generated by using known ratios of live and dead spermatozoa. Spermatozoa collected from each species were separated into two aliquots: one aliquot was maintained at 18 °C to be used as the source of live spermatozoa; the other aliquot was heated to 50 °C for 15 min to kill the spermatozoa as confirmed to be died under microscope observation. Samples were prepared by combining the live and killed aliquots of spermatozoa in percentage ratios of 0:100, 20:80, 40:60, 80:20 and 100:0, then stained and analysed by spectrofluorimetry. This experiment was performed five times in duplicates.
2. Materials and methods 2.1. Ethics statement
2.4. Confocal laser microscopy
The ascidian C. intestinalis, the mollusc M. galloprovincialis, and the sea urchin P. lividus were collected from a location that is protected in any way, according to Italian legislation of the Marina Mercantile (Decreto del Presidente della Repubblica DPR 1639/68, 09/19/1980 confirmed on 01/10/2000). The field studies did not involve endangered or protected species. All animal procedures were in compliance with the guidelines of the European Union (Directive 609/86).
Immediately after incubation, SYBR/PI stained samples were mounted on a microscope slide and observed under a confocal laser scanning microscopy (Zeiss LSM 510) to assess fluorochrome localization within a specific cell compartment.
2.2. Animal and sperm collection
Spectrofluorimetric analysis was performed by using Shimadzu RF5301 spectrofluorometer (Tokyo, Japan) in order to determinate the percentage of live and dead spermatozoa. Spermatozoa were centrifuged, pellets re-suspended in FNSW and transferred to quartz cuvette (10 × 4 mm, high precision mobile, Hellma Analytics, Müllheim, Germany). SYBR 14 and PI fluorescence intensity peaks were measured setting the excitation wavelengths at 488 nm and at 545 nm recording the emission spectra in the range of 500–560 nm and 570–700 nm, respectively. The experiments were performed ten times in duplicates.
2.5. Spectrofluorimetric analysis
Adults of C. intestinalis sp. A were collected from the Gulf of Naples (Italy), transported to Stazione Zoologica Unit of Marine Resources for Research laboratory, and maintained in aquaria with running seawater at 18 °C for at least 7 days until the experiments. After anesthetization of animals on ice, spermatozoa were collected by dissection from the sperm duct. A homogeneous group of adults M. galloprovincialis was collected from the breeding site into the Gulf of Naples, transported dry to Stazione Zoologica Unit of Marine Resources for Research laboratory and maintained in open circuit seawater tanks at 18 °C. After an initial 6-day adaptation phase, mussels were used for the experiments. To collect spermatozoa, mussels were opened with a scalpel, their mantles were slightly cut, and the resulting sperm mass was collected dry with a Pasteur pipette. Sea urchins P. lividus were collected from the Gulf of Naples, transported to Stazione Zoologica Unit of Marine Resources for Research laboratory within 1 h after collection and maintained in tanks with circulating seawater at 18 °C. The animals were acclimated at least for 10 days until use. To induce gamete ejection, sea urchins were
2.6. Statistical analysis Data were reported as the mean ± standard deviation (SD). Pearson correlation test was performed to evaluate the relationship between live and dead sperm population and the one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test was performed to test for significant differences. The significance level was set at p = 0.05.
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Fig. 1. Fluorescence images of Ciona intestinalis (A, B, C), Paracentrotus lividus (D, E, F) and Mytilus galloprovincialis (G, H, I) spermatozoa dual stained with SYBR-14 and propidium iodide (PI). SYBR-14 stained the live spermatozoa with intact plasma membrane (A, D, G) (bright green fluorescence); PI stained dead ones with disrupted plasma membrane (red fluorescence); and dying spermatozoa (yellow/orange fluorescence).
3. Results
3.2. Spectrofluorimetric analysis
3.1. Microscopy analysis
A representative fluorescence emission spectra recorded by spectrofluorimetry on double-stained spermatozoa from the three species tested are reported in Fig. 2. The fluorescence peak value of emission spectra recorded fixing the excitation wavelength at 488 nm and scanned the emission wavelength from 500 nm to 560 nm provide the portion of live spermatozoa (SYBR-14 stained); while the peak value of emission spectrum recorded by scanning the emission wavelengths from 570 nm to 700 nm and fixing the excitation wavelength at 545 nm provide the portion of dead spermatozoa (PI stained). The SYBR-14 and PI-stained sperm populations present in freshly collected spermatozoa were quantified by a spectrofluorimetric analysis. The mean fluorescence intensity value of the SYBR-14 stained spermatozoa was 48.39 ± 1.70 in C. intestinalis, 57.00 ± 2.57 in P.
The microscopic examination of freshly collected spermatozoa stained with the combination of SYBR-14 and PI revealed three populations of sperm staining: live spermatozoa emitting green fluorescence resulting from positive staining by SYBR-14 (Fig. 1A, D, G), dead spermatozoa whose nuclei emitting red fluorescence resulting from positive PI (Fig. 1C, F, I), and dying spermatozoa emitting yellow/orange fluorescence resulting from doubly staining with both dyes (PI/ SYBR) (Fig. 1B, E, H). These three sperm populations were evident for all species.
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Fig. 3. Percentage of live and dead spermatozoa of the three marine invertebrates species quantified by spectrofluorimetric analysis. Mean values of fluorescence emission peak value at 488 nm excitation and 516 nm emission wavelengths provide the portion of live spermatozoa (SYBR-14 stained) while that recorded at 545 nm excitation and 617 nm emission wavelengths provide the portion of dead spermatozoa (PI stained). Data were reported as mean ± SD, n = 10.
In P. lividus, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 82.72 ± 4.4%, 56.14 ± 5.2%, 29.54 ± 3.9% and 9.12 ± 4.7%, respectively. The corresponding groups of dead, PIstained were 8.18 ± 3.4%, 25.16 ± 5.1%, 58.96 ± 4.5%, 74.14 ± 4.9%, 100% respectively. In M. galloprovincialis, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 69.00 ± 5.1%, 59.48 ± 3.8%, 30.59% ± 3.5 and 7.65 ± 4.7%, respectively. The corresponding groups of dead, PI-stained were 8.72 ± 4.3%, 25.58 ± 4.0%, 54.01 ± 3.6%, 70.94 ± 5.3%, 100% respectively. For each species tested, the relationship between the percentage of fresh spermatozoa added to a sample and the percentage of spermatozoa staining as live was statistically significant (R2 = 0.99; P < 0.01). Likewise, the relationship between the percentage of killed spermatozoa added to a sample and the percentage of sperm staining as dead was also significant (R2 = 0.99; P < 0.01). 4. Discussion In the present study, we developed a method using the dual DNA staining with SYBR-14 and PI in combination with spectrofluorimetric analysis for the assessment of sperm viability of three different marine invertebrate species. SYBR-14 and PI double staining was firstly developed on mammalian sperm (Garner and Johnson, 1995), and this is the most commonly used assay to evaluate sperm viability in several species across taxonomic lines (Adams et al., 2003; Donoghue and Donoghue, 1997; Lezcano et al., 2004; McNiven et al., 1992; Salinas-Flores et al., 2005; Segovia et al., 2000; Thomas et al., 1998). Among marine animals, the dual DNA fluorescent staining systems have been used previously to assess sperm viability in different species including molluscs and echinoderms (Favret and Lynn, 2010; Le Goïc et al., 2013; Rolton et al., 2015; Smith et al., 2012; Suquet et al., 2016); however, the present study represents the first report of the assessment of sperm viability with the combination of SYBR-14 and PI in the sea urchin P. lividus and the mollusc M. galloprovincialis. Moreover, the validity of this dual DNA staining for determining live and dead spermatozoa has not been previously demonstrated in ascidian species. In agreement with previous studies, we identified three populations in the spermatozoa of tested species using this staining: live SYBR-14 stained spermatozoa, dead PIstained spermatozoa and dying doubly stained spermatozoa. This population exhibited both green and red fluorescence since dead spermatozoa lose their ability to resist the influx of membrane impermeable dye PI. Consequently, upon cell death, PI rapidly enters and replaces or
Fig. 2. Representative fluorescence emission spectra recorded by spectrofluorimetry on double-stained spermatozoa of Ciona intestinalis (A), Paracentrotus lividus (B) and Mytilus galloprovincialis (C). Sperm were incubated with SYBR-14 for 15 min and then with propidium iodide (PI) for other 15 min, after samples washed emission spectra were recorded by scanning the emission wavelengths from 500 nm to 560 nm for SYBR-14 and from 570 nm to 700 nm for PI and fixing the excitation wavelength at 488 nm and 545 nm, respectively.
lividus and 86.42 ± 1.65 in M. galloprovincialis. The mean value of the PI stained spermatozoa was 17.33 ± 1.81 in C. intestinalis, 22.66 ± 0.51 in P. lividus and 27.25 ± 1.38 in M. galloprovincialis (Fig. 3). Further to validate the SYBR-14 and PI stain combination, fresh spermatozoa and killed ones by heat treatment were mixed together in known ratios (Fig. 4). In C. intestinalis, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 72.81 ± 3.4%, 50.26 ± 2.7%, 25.38 ± 3.1% and 9.85 ± 4.8%, respectively. The corresponding groups of dead, PI-stained were 14.44 ± 2.9%, 28.24 ± 4.5%, 59.27 ± 3.9%, 76.32 ± 3.4%, 100% respectively. 410
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provides rapid measurements. In addition, this technique offers simple operation as well as availability of a relatively inexpensive instrument. On the contrary, the practical use of flow cytometry has been limited by the need for a skilled operator and expensive instrumentation. Spectrofluorimetric analysis was commonly performed on stained spermatozoa of mammalian species to assess several sperm quality parameters (Aitken et al., 1993; Boni et al., 2017; Crippa et al., 2015; Fang et al., 2016; Krausz et al., 1995); while in marine animals it was applied to evaluate few sperm parameters including intracellular calcium concentration and pH, lipid peroxidation, mitochondrial membrane potential, and DNA fragmentation (Boni et al., 2016; Gallo et al., 2016) and never to assess sperm viability. Herein, the portion of spermatozoa stained with SYBR-14 and PI, representing the live and dead sperm population, were rapidly quantified by spectrofluorimetric analysis. For all species used, the standard deviations did not exceed 5% of the mean values of live spermatozoa, consequently the obtained results could be considered homogenous. When fresh and killed spermatozoa were mixed in known ratios prior to staining, there was a significant relationship between the proportion of live spermatozoa that were present in a sample and the percentage of SYBR-14 stained spermatozoa. Similarly, there was also a significant relationship between the proportion of killed spermatozoa that were added to a sample and the percentage of PI stained spermatozoa. The obtained results confirmed that spectrofluorimetric analysis is an effective, precise and rapid tool for assessing live and dead spermatozoa in the tested species. In summary, in this study, we developed, for the first time, a simple, accurate, rapid, and sensitive method using dual DNA fluorescence staining in combination with spectrofluorimetric analysis for the assessment of sperm viability in test marine invertebrates. Moreover, data reported herein for ascidian, sea urchin, and mollusc spermatozoa suggest that this method may be very useful and promising for a variety of marine invertebrate species. Sperm viability testing is usually used for the assessment of mammalian spermatozoa, (Garner and Johnson, 1995) unlike in marine invertebrates it is confined to a few studies (Akcha et al., 2012; Favret and Lynn, 2010; Lewis and Galloway, 2009). The interest in developing methods to assess sperm viability in marine invertebrates has been motivated by the need to study the impact of environmental pollutants on reproductive success employing bioassays that can be performed rapidly at the least costs and that retain maximum sensitivity. The present study provides a simple and a cost-effective method to investigate spermiotoxicity of pollutants affecting marine environment, so it can be argued that this method could be used in marine pollution monitoring program and ecotoxicological studies.
Author contributions Fig. 4. Percentages of spermatozoa stained with SYBR-14 and PI in different ratios of fresh to killed spermatozoa of three marine invertebrates species. The correlation between the percentages of fresh and killed spermatozoa added to a sample and the percentage of spermatozoa staining as live and as dead, respectively, was significant. Data presented are the mean percentage values ± SD, n = 5.
All authors contributed in designing the study and in writing the manuscript; A.G., R.B. performed the experiments and the statistical analysis of results; E.T. supervised the project.
quenches the fluorescence exhibited by SYBR-14 (Garner and Johnson, 1995). These results confirmed that the dual DNA staining with SYBR14 and PI is effective and reliable means in estimating sperm viability in the test marine invertebrate species. Up to date, after the dual DNA staining, live and dead spermatozoa were determined by fluorescence microscopy or flow cytometry. In the present study, the assessment of dual-stained live and dead spermatozoa was performed by spectrofluorimetry. To our knowledge, no attempt has previously been made to assess sperm viability by spectrofluorimetric analysis. Spectrofluorimetry is a sensitive and simple methodology that
Funding This work was supported by Stazione Zoologica Anton Dohrn Institutional Funds. A.G. has been supported by a Stazione Zoologica Anton Dohrn Post-doc Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing financial interests statement The authors declare no competing financial interests. 411
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Sperm viability assessment in marine invertebrates by fluorescent staining and spectrofluorimetry: A promising tool for assessing marine pollution impact
MARK
Alessandra Galloa, Raffaele Bonib, Elisabetta Tostia,⁎ a b
Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, 80121 Napoli, Italy Department of Sciences, University of Basilicata, 75100 Potenza, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords: Spermatozoon Viability assessment Marine invertebrate Spectrofluorimetry
The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.
1. Introduction Sperm quality is defined as the ability of spermatozoa to successfully fertilize an oocyte, to subsequently allow the development of a normal embryo, and it is influenced by several external factors (Bobe and Labbé, 2010). Assessment of sperm quality in marine species is an important issue due to the increase of ecotoxicology studies looking at the impacts of environmental pollutants on male reproductive health (Gallo and Tosti, 2016). In marine pollution monitoring programs and ecotoxicological studies, sperm cell toxicity tests have been commonly performed and successfully used as a test to monitor and evaluate the adverse effects of environmental contaminants; however, in most cases spermiotoxicity was assessed by evaluating fertilization success and the induction of transmissible damages to the offspring (Gallo et al., 2011; Gallo and Tosti, 2013, 2015; Manzo et al., 2006; Pagano et al., 1996). Sensitive and practical methods are requested for testing different sperm quality parameters to use as endpoint in spermiotoxicity tests.
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Viability is a key determinant of sperm quality whose evaluation is generally achieved using sensitive and specific fluorescent probes in combination with microscopy analysis or flow cytometry (Lewis and Ford, 2012). The dual DNA staining using SYBR-14 and propidium iodide (PI) is among the commonly used stains for assessing sperm viability. The membrane-permanent dye SYBR-14 stains the nucleus of living spermatozoa emitting a bright green fluorescence, while PI only stains DNA in damaged cells that have lost their membrane integrity (Garner and Johnson, 1995). Among marine animals, the dual DNA staining has been successfully used for assessing sperm viability in fishes (Cabrita et al., 2005; Flajšhans et al., 2004; Martínez-Páramo et al., 2013), molluscs (Akcha et al., 2012; Favret and Lynn, 2010; Le Goïc et al., 2013; Paniagua-Chávez et al., 2006; Rolton et al., 2015; Smith et al., 2012; Suquet et al., 2016), crustaceans (Sasson et al., 2012) and echinoderms (Favret and Lynn, 2010). Nevertheless, no attempts have been made to apply this method to the assessment of sperm viability in ascidians.
Correspondence to: Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy. E-mail address: [email protected] (E. Tosti).
http://dx.doi.org/10.1016/j.ecoenv.2017.07.069 Received 20 April 2017; Received in revised form 24 July 2017; Accepted 28 July 2017 0147-6513/ © 2017 Elsevier Inc. All rights reserved.
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The proportion of living and dead spermatozoa is quantified by flow cytometry or fluorescence and confocal microscopy (Favret and Lynn, 2010; Le Goïc et al., 2013; Paniagua-Chávez et al., 2006; Rolton et al., 2015; Sasson et al., 2012; Smith et al., 2012; Suquet et al., 2016). As a key advantage flow cytometry permits to evaluate many parameters on small samples in a very short time providing rapid and accurate results (Graham et al., 1990; Hossain et al., 2011). On the contrary, this technic presents several disadvantages including costs, being flow cytometer an expensive instrument to buy and maintain. Moreover, the computer interfaced flow cytometers are very sophisticated, so skilled and highly trained operators can run it and get any acceptable levels of performance from such an apparatus. Fluorescence and confocal microscopy permit to identify fluorescent signals in individual cells (Ramalho-Santos et al., 2007; Suzuki et al., 1997); however, few cells from the same sample are assessed. These problems can be overcome by using spectrofluorimetry. Compared with flow cytometry, spectrofluorimetry is a high sensitive, specific, simple, easy to operate, and rapid technique that requires a relatively inexpensive instrument (Andrade-Eiroa et al., 2013). However, to the best of our knowledge, no information is available on the use of spectrofluorimetry to assess sperm viability on marine invertebrates. In the present study, we develop a spectrofluorimetric method to assess sperm viability in three different marine invertebrates (the ascidian Ciona intestinalis, the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus). These species were chosen due to their ecological role in marine ecosystems that has led to their use as model for biomonitoring marine pollution. The main objectives of this study were: 1) to determine whether the dual DNA fluorescence staining is a suitable method for sperm viability assessment in C intestinalis, M. galloprovincialis and P. lividus; 2) to develop a simple, accurate, rapid, and sensitive spectrofluorimetric method for the assessment of sperm viability in marine invertebrates.
injected with 0.5 M KCl solution through the peribuccal membrane and then spermatozoa were collected dry. After collection, fresh and dry spermatozoa from all species were held on ice. The concentration and motility of collected spermatozoa were evaluated by a Makler counting chamber and, then, spermatozoa were diluted to the desired concentration in filtered (Millipore 0.22 µm; Milli Q, Medford, MA, USA) natural seawater (FNSW) (38 PSU salinity, pH 8.2 ± 0.1).
2.3. Dual DNA fluorescence staining The dual DNA staining was performed using SYBR 14 and PI from a commercially available LIVE/DEAD Sperm Viability Kit (Life technologies, Milan, Italy). Fluorescent staining was carried out as follows: for each species, an aliquot of spermatozoa diluted at 5 × 106 cell/ml were stained by adding SYBR-14 (final concentration 100 nM) and incubated in the dark at 18 ± 1 °C for 15 min according to manufacturer instructions. After SYBR-14 incubation, samples were incubated at 18 ± 1 °C in 12 µM PI dye for 15 min. For microscope examination, samples were immediately analysed; for spectrofluorimetric analysis, samples were washed by adding FNSW, centrifuged at 2000 rpm for 10 min, and then resuspended in FNSW. In order to validate the sperm viability assay, standard curves were generated by using known ratios of live and dead spermatozoa. Spermatozoa collected from each species were separated into two aliquots: one aliquot was maintained at 18 °C to be used as the source of live spermatozoa; the other aliquot was heated to 50 °C for 15 min to kill the spermatozoa as confirmed to be died under microscope observation. Samples were prepared by combining the live and killed aliquots of spermatozoa in percentage ratios of 0:100, 20:80, 40:60, 80:20 and 100:0, then stained and analysed by spectrofluorimetry. This experiment was performed five times in duplicates.
2. Materials and methods 2.1. Ethics statement
2.4. Confocal laser microscopy
The ascidian C. intestinalis, the mollusc M. galloprovincialis, and the sea urchin P. lividus were collected from a location that is protected in any way, according to Italian legislation of the Marina Mercantile (Decreto del Presidente della Repubblica DPR 1639/68, 09/19/1980 confirmed on 01/10/2000). The field studies did not involve endangered or protected species. All animal procedures were in compliance with the guidelines of the European Union (Directive 609/86).
Immediately after incubation, SYBR/PI stained samples were mounted on a microscope slide and observed under a confocal laser scanning microscopy (Zeiss LSM 510) to assess fluorochrome localization within a specific cell compartment.
2.2. Animal and sperm collection
Spectrofluorimetric analysis was performed by using Shimadzu RF5301 spectrofluorometer (Tokyo, Japan) in order to determinate the percentage of live and dead spermatozoa. Spermatozoa were centrifuged, pellets re-suspended in FNSW and transferred to quartz cuvette (10 × 4 mm, high precision mobile, Hellma Analytics, Müllheim, Germany). SYBR 14 and PI fluorescence intensity peaks were measured setting the excitation wavelengths at 488 nm and at 545 nm recording the emission spectra in the range of 500–560 nm and 570–700 nm, respectively. The experiments were performed ten times in duplicates.
2.5. Spectrofluorimetric analysis
Adults of C. intestinalis sp. A were collected from the Gulf of Naples (Italy), transported to Stazione Zoologica Unit of Marine Resources for Research laboratory, and maintained in aquaria with running seawater at 18 °C for at least 7 days until the experiments. After anesthetization of animals on ice, spermatozoa were collected by dissection from the sperm duct. A homogeneous group of adults M. galloprovincialis was collected from the breeding site into the Gulf of Naples, transported dry to Stazione Zoologica Unit of Marine Resources for Research laboratory and maintained in open circuit seawater tanks at 18 °C. After an initial 6-day adaptation phase, mussels were used for the experiments. To collect spermatozoa, mussels were opened with a scalpel, their mantles were slightly cut, and the resulting sperm mass was collected dry with a Pasteur pipette. Sea urchins P. lividus were collected from the Gulf of Naples, transported to Stazione Zoologica Unit of Marine Resources for Research laboratory within 1 h after collection and maintained in tanks with circulating seawater at 18 °C. The animals were acclimated at least for 10 days until use. To induce gamete ejection, sea urchins were
2.6. Statistical analysis Data were reported as the mean ± standard deviation (SD). Pearson correlation test was performed to evaluate the relationship between live and dead sperm population and the one-way analysis of variance (ANOVA) followed by Fisher's least significant difference (LSD) test was performed to test for significant differences. The significance level was set at p = 0.05.
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Fig. 1. Fluorescence images of Ciona intestinalis (A, B, C), Paracentrotus lividus (D, E, F) and Mytilus galloprovincialis (G, H, I) spermatozoa dual stained with SYBR-14 and propidium iodide (PI). SYBR-14 stained the live spermatozoa with intact plasma membrane (A, D, G) (bright green fluorescence); PI stained dead ones with disrupted plasma membrane (red fluorescence); and dying spermatozoa (yellow/orange fluorescence).
3. Results
3.2. Spectrofluorimetric analysis
3.1. Microscopy analysis
A representative fluorescence emission spectra recorded by spectrofluorimetry on double-stained spermatozoa from the three species tested are reported in Fig. 2. The fluorescence peak value of emission spectra recorded fixing the excitation wavelength at 488 nm and scanned the emission wavelength from 500 nm to 560 nm provide the portion of live spermatozoa (SYBR-14 stained); while the peak value of emission spectrum recorded by scanning the emission wavelengths from 570 nm to 700 nm and fixing the excitation wavelength at 545 nm provide the portion of dead spermatozoa (PI stained). The SYBR-14 and PI-stained sperm populations present in freshly collected spermatozoa were quantified by a spectrofluorimetric analysis. The mean fluorescence intensity value of the SYBR-14 stained spermatozoa was 48.39 ± 1.70 in C. intestinalis, 57.00 ± 2.57 in P.
The microscopic examination of freshly collected spermatozoa stained with the combination of SYBR-14 and PI revealed three populations of sperm staining: live spermatozoa emitting green fluorescence resulting from positive staining by SYBR-14 (Fig. 1A, D, G), dead spermatozoa whose nuclei emitting red fluorescence resulting from positive PI (Fig. 1C, F, I), and dying spermatozoa emitting yellow/orange fluorescence resulting from doubly staining with both dyes (PI/ SYBR) (Fig. 1B, E, H). These three sperm populations were evident for all species.
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Fig. 3. Percentage of live and dead spermatozoa of the three marine invertebrates species quantified by spectrofluorimetric analysis. Mean values of fluorescence emission peak value at 488 nm excitation and 516 nm emission wavelengths provide the portion of live spermatozoa (SYBR-14 stained) while that recorded at 545 nm excitation and 617 nm emission wavelengths provide the portion of dead spermatozoa (PI stained). Data were reported as mean ± SD, n = 10.
In P. lividus, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 82.72 ± 4.4%, 56.14 ± 5.2%, 29.54 ± 3.9% and 9.12 ± 4.7%, respectively. The corresponding groups of dead, PIstained were 8.18 ± 3.4%, 25.16 ± 5.1%, 58.96 ± 4.5%, 74.14 ± 4.9%, 100% respectively. In M. galloprovincialis, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 69.00 ± 5.1%, 59.48 ± 3.8%, 30.59% ± 3.5 and 7.65 ± 4.7%, respectively. The corresponding groups of dead, PI-stained were 8.72 ± 4.3%, 25.58 ± 4.0%, 54.01 ± 3.6%, 70.94 ± 5.3%, 100% respectively. For each species tested, the relationship between the percentage of fresh spermatozoa added to a sample and the percentage of spermatozoa staining as live was statistically significant (R2 = 0.99; P < 0.01). Likewise, the relationship between the percentage of killed spermatozoa added to a sample and the percentage of sperm staining as dead was also significant (R2 = 0.99; P < 0.01). 4. Discussion In the present study, we developed a method using the dual DNA staining with SYBR-14 and PI in combination with spectrofluorimetric analysis for the assessment of sperm viability of three different marine invertebrate species. SYBR-14 and PI double staining was firstly developed on mammalian sperm (Garner and Johnson, 1995), and this is the most commonly used assay to evaluate sperm viability in several species across taxonomic lines (Adams et al., 2003; Donoghue and Donoghue, 1997; Lezcano et al., 2004; McNiven et al., 1992; Salinas-Flores et al., 2005; Segovia et al., 2000; Thomas et al., 1998). Among marine animals, the dual DNA fluorescent staining systems have been used previously to assess sperm viability in different species including molluscs and echinoderms (Favret and Lynn, 2010; Le Goïc et al., 2013; Rolton et al., 2015; Smith et al., 2012; Suquet et al., 2016); however, the present study represents the first report of the assessment of sperm viability with the combination of SYBR-14 and PI in the sea urchin P. lividus and the mollusc M. galloprovincialis. Moreover, the validity of this dual DNA staining for determining live and dead spermatozoa has not been previously demonstrated in ascidian species. In agreement with previous studies, we identified three populations in the spermatozoa of tested species using this staining: live SYBR-14 stained spermatozoa, dead PIstained spermatozoa and dying doubly stained spermatozoa. This population exhibited both green and red fluorescence since dead spermatozoa lose their ability to resist the influx of membrane impermeable dye PI. Consequently, upon cell death, PI rapidly enters and replaces or
Fig. 2. Representative fluorescence emission spectra recorded by spectrofluorimetry on double-stained spermatozoa of Ciona intestinalis (A), Paracentrotus lividus (B) and Mytilus galloprovincialis (C). Sperm were incubated with SYBR-14 for 15 min and then with propidium iodide (PI) for other 15 min, after samples washed emission spectra were recorded by scanning the emission wavelengths from 500 nm to 560 nm for SYBR-14 and from 570 nm to 700 nm for PI and fixing the excitation wavelength at 488 nm and 545 nm, respectively.
lividus and 86.42 ± 1.65 in M. galloprovincialis. The mean value of the PI stained spermatozoa was 17.33 ± 1.81 in C. intestinalis, 22.66 ± 0.51 in P. lividus and 27.25 ± 1.38 in M. galloprovincialis (Fig. 3). Further to validate the SYBR-14 and PI stain combination, fresh spermatozoa and killed ones by heat treatment were mixed together in known ratios (Fig. 4). In C. intestinalis, the percentages of fresh spermatozoa staining with SYBR-14 as live in ratio groups of 100:0, 75:25, 50:50, 25:75 and 0:100 live:killed were 100%, 72.81 ± 3.4%, 50.26 ± 2.7%, 25.38 ± 3.1% and 9.85 ± 4.8%, respectively. The corresponding groups of dead, PI-stained were 14.44 ± 2.9%, 28.24 ± 4.5%, 59.27 ± 3.9%, 76.32 ± 3.4%, 100% respectively. 410
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provides rapid measurements. In addition, this technique offers simple operation as well as availability of a relatively inexpensive instrument. On the contrary, the practical use of flow cytometry has been limited by the need for a skilled operator and expensive instrumentation. Spectrofluorimetric analysis was commonly performed on stained spermatozoa of mammalian species to assess several sperm quality parameters (Aitken et al., 1993; Boni et al., 2017; Crippa et al., 2015; Fang et al., 2016; Krausz et al., 1995); while in marine animals it was applied to evaluate few sperm parameters including intracellular calcium concentration and pH, lipid peroxidation, mitochondrial membrane potential, and DNA fragmentation (Boni et al., 2016; Gallo et al., 2016) and never to assess sperm viability. Herein, the portion of spermatozoa stained with SYBR-14 and PI, representing the live and dead sperm population, were rapidly quantified by spectrofluorimetric analysis. For all species used, the standard deviations did not exceed 5% of the mean values of live spermatozoa, consequently the obtained results could be considered homogenous. When fresh and killed spermatozoa were mixed in known ratios prior to staining, there was a significant relationship between the proportion of live spermatozoa that were present in a sample and the percentage of SYBR-14 stained spermatozoa. Similarly, there was also a significant relationship between the proportion of killed spermatozoa that were added to a sample and the percentage of PI stained spermatozoa. The obtained results confirmed that spectrofluorimetric analysis is an effective, precise and rapid tool for assessing live and dead spermatozoa in the tested species. In summary, in this study, we developed, for the first time, a simple, accurate, rapid, and sensitive method using dual DNA fluorescence staining in combination with spectrofluorimetric analysis for the assessment of sperm viability in test marine invertebrates. Moreover, data reported herein for ascidian, sea urchin, and mollusc spermatozoa suggest that this method may be very useful and promising for a variety of marine invertebrate species. Sperm viability testing is usually used for the assessment of mammalian spermatozoa, (Garner and Johnson, 1995) unlike in marine invertebrates it is confined to a few studies (Akcha et al., 2012; Favret and Lynn, 2010; Lewis and Galloway, 2009). The interest in developing methods to assess sperm viability in marine invertebrates has been motivated by the need to study the impact of environmental pollutants on reproductive success employing bioassays that can be performed rapidly at the least costs and that retain maximum sensitivity. The present study provides a simple and a cost-effective method to investigate spermiotoxicity of pollutants affecting marine environment, so it can be argued that this method could be used in marine pollution monitoring program and ecotoxicological studies.
Author contributions Fig. 4. Percentages of spermatozoa stained with SYBR-14 and PI in different ratios of fresh to killed spermatozoa of three marine invertebrates species. The correlation between the percentages of fresh and killed spermatozoa added to a sample and the percentage of spermatozoa staining as live and as dead, respectively, was significant. Data presented are the mean percentage values ± SD, n = 5.
All authors contributed in designing the study and in writing the manuscript; A.G., R.B. performed the experiments and the statistical analysis of results; E.T. supervised the project.
quenches the fluorescence exhibited by SYBR-14 (Garner and Johnson, 1995). These results confirmed that the dual DNA staining with SYBR14 and PI is effective and reliable means in estimating sperm viability in the test marine invertebrate species. Up to date, after the dual DNA staining, live and dead spermatozoa were determined by fluorescence microscopy or flow cytometry. In the present study, the assessment of dual-stained live and dead spermatozoa was performed by spectrofluorimetry. To our knowledge, no attempt has previously been made to assess sperm viability by spectrofluorimetric analysis. Spectrofluorimetry is a sensitive and simple methodology that
Funding This work was supported by Stazione Zoologica Anton Dohrn Institutional Funds. A.G. has been supported by a Stazione Zoologica Anton Dohrn Post-doc Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing financial interests statement The authors declare no competing financial interests. 411
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