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Reproductive impairment of sea urchins upon chronic exposure to cadmium. Part I: Effects on gamete quality
Environmental Pollution 111 (2001) 1±9
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Reproductive impairment of sea urchins upon chronic exposure to cadmium. Part I: Eects on gamete quality D.W.T. Au *, C.Y Lee, K.L Chan, R.S.S. Wu Center for Coastal Pollution and Conservation, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong Received 7 July 1999; accepted 18 December 1999
``Capsule'': Gamete quality for male sea urchins was reduced by chronic exposure to cadmium. Abstract Successful reproduction is a determining factor for species survival. Pollution may impair reproductive success of adults through eects on gamete quality. Reproductive impairment of the sea urchin Anthocidaris crassispina upon chronic (4 weeks) exposure to 0.01 and 0.1 mg lÿ1 Cd2+ was investigated. Criteria used for assessing gamete quality included sperm motility, egg morphology, fertilization rate and dynamics of ®rst cleavage. A dose±response relationship was found between Cd2+ levels and changes in sperm motility, and percentage fertilization. Sperm motility, measured by computer-assisted sperm analysis, indicated that percent motile sperm, velocities, and percent sperm with normal trajectory were signi®cantly aected by chronic exposure to 50.1 mg lÿ1 Cd2+. A decline in sperm motility was also accompanied by a decrease in fertilization success of sea urchin sperm. Width/height ratio of sea urchin eggs was not aected by cadmium, but larger egg sizes were found when sea urchins were exposed to 0.1 mg lÿ1 Cd2+. Male sea urchins exposed to Cd2+ produced poorer quality sperm, as indicated by a lower percent fertilization and lower cleavage rate, implying that male sea urchins were more sensitive than females to chronic Cd2+ exposure. Results of the present study provide an explanation of reproductive impairment in marine invertebrates upon chronic exposure to Cd2+. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: CASA; Sperm motility; Fertilization success; Heavy metals; Invertebrate reproduction
1. Introduction Reproductive success is one of the key factors in determining species survival. Pollution may impair reproductive success of adult organisms through decreasing the quality and/or quantity of gametes, which in turn may aect fertilization success, embryo development, larval viability and subsequently species ®tness and survival. Reproductive impairment by a variety of xenobiotics has been well reported in ®sh (Sol et al., 1995; Shatunovskii et al., 1996; Lye et al., 1997) and marine mammals (O'Shea and Brownell, 1994; Jenssen, 1996). The notable eects of tributyltin (TBT) on imposex of gastropods and early development of oysters have been documented (Gibbs and Bryan, 1996; His, 1996). However, the chronic eects of pollution on * Corresponding author. Present address: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong. Fax: +852-2788-7406. E-mail address: [email protected] (D.W.T. Au).
reproductive impairment of marine invertebrates are generally not well known. Sea urchins play a key role in controlling rocky subtidal community structures in coastal waters (Pringle et al., 1982; Fletcher, 1987) and changes in sea urchin populations have led to major alterations in marine community structure (Witman, 1985; Scheibling, 1986). Retarded gonad development was reported in the sea urchin Strongylocentrotus intermedius collected from polluted areas of Peter the Bay (Sea of Japan) (Vaschenko et al., 1993). On the contrary, a ®eld transplantation study showed enhanced gonad development and suppressed fertilization kinetics in Strongylocentrotus purpuratus upon chronic exposure to oil euents (Krause, 1994). Sublethal exposure to heavy metals (e.g. Cu, Zn and Cd) in the laboratory has led to poor embryonic and larval development in the sea urchin Strongylocentrotus intermedius (Khristoforova et al., 1984; Durkina, 1994), Sphaerechinus granularis (Lucu et al., 1991) and Arbacia punctulata (Bowen and Engel, 1996). However, no details were provided on the nature of gamete impairment in the above studies. Also,
0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(00)00035-X
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no attempt has been made to relate changes in gamete quality to subsequent alterations in fertilization capability and larval development. Sperm motility is one of the most important criteria in assessing sperm quality. The computer-assisted sperm analysis system (CASA) provides an objective and quantitative assessment of sperm motility (WHO, 1992). The CASA system, which was ®rst developed to study human sperm, has been employed to study heavy metal eects on the motility of rabbit sperm (Young et al., 1995), and cat®sh sperm (Kime et al., 1996). In an earlier study, we successfully applied the CASA system to study motility of sea urchin and mussel sperm upon direct exposure to Cd2+ and phenol (Au et al., 2000a). Sea urchin eggs are relatively large in size (ca. 100 mm in diameter) and can be examined readily by light microscopy. The CASA image analyzer system in our laboratory is able to provide rapid, quantitative measurements on the area as well as width-to-height ratio of eggs, which allows a quantitative comparison of egg morphology (size and shape) among treatment groups. In this study, these parameters were employed as morphological criteria for assessing egg quality. Quality and viability of eggs and sperm can be appraised based upon their fertilization capability, and the subsequent development of fertilized eggs. Fertilization studies and ®rst cleavage dynamics of sea urchin gametes have been extensively employed for marine pollution monitoring and ecotoxicological studies (Dinnel et al., 1987, 1989; Dinnel, 1995; Kobayashi, 1995; Pagano et al., 1986, 1989; Vaschenko et al., 1995). Kinetics of sea urchin ®rst cleavage has proven to be a sensitive end-point for assessing heavy metal toxicity (Ramachandran et al., 1997). The standard procedure for this kind of assay is to expose sperm in high concentrations of pollutants for a brief period (about 30 min). Eggs are not exposed, although it was common practice in earlier times. In the natural environment, adult sea urchins are normally exposed to lower levels of pollutants for a prolonged period. The quality and subsequent fertilization ability of sperm and eggs produced by adult sea urchins exposed to chronic pollution may be very dierent. However, few studies have been carried out to determine chronic eects of cadmium on sea urchin reproduction. Cadmium is one of the most important toxic metals in industrial discharges, and is included in both US EPA and ECs' priority pollutant lists. Signi®cant levels of cadmium contamination have been reported in Hong Kong's coastal waters (Phillips, 1989; EPD, 1998). Nevertheless, eects of such high levels of cadmium on marine populations in Hong Kong are not known. The short spine sea urchin Anthocidaris crassispina, which is abundant over a wide geographical range in the IndoPaci®c Oceans and East China Seas, is regarded as ecologically important in tropical and subtropical
coastal waters (Thompson and Wu, 1981). In this investigation, we examined the eects of chronic exposure to Cd2+ of adult Anthocidaris crassispina, in which gamete quality was assessed by sperm motility, egg morphology, fertilization and dynamics of ®rst cleavage. The relative sensitivity of female and male sea urchins to Cd2+ was compared, based on the performance of their gametes, and an attempt was also made to relate sperm motility to fertilization success. The results of this study allow a better understanding of the chronic eects of cadmium pollution on the reproductive success of sea urchins, and permit an extrapolation to predict population eects on this ecologically important species. 2. Materials and methods In Hong Kong, gonad development of Anthocidaris crassispina is initiated in autumn (November to December); gonads are gravid in spring (March to May), and spawning occurs in summer (June to September) (Chiu, 1988; Au et al., 1998). In May 1998, specimens of Anthocidaris crassispina were collected by SCUBA from subtidal zones of a pristine site (Kat O) in Hong Kong. In the laboratory, sea urchins were acclimated for 48 h in 30 l seawater tanks (®ve sea urchins per tank, 25 C, 30%) with constant aeration. US EPA recommended a criterion of 9.3 mg lÿ1 for cadmium in saltwater, and such elevated levels of Cd2+ have been reported in polluted coastal areas in Hong Kong and China. A preliminary experiment showed that time for 50% mortality (LT50) of 1 mg lÿ1 Cd2+ for Anthocidaris crassispina was 18 days, and since application factors of 10±100 are normally used to extrapolate sublethal eects, sea urchins were exposed to 0.1 and 0.01 mg lÿ1 Cd2+ (prepared from CdCl2) in the present study. Each level of Cd2+ exposure utilized 30 individuals, each held in six separate replicate tanks. Control sea urchins were kept in seawater only. Seawater and Cd2+ solutions were renewed every 2 days and sea urchins were fed with sea lettuce, Ulva lactuca (ca. 4 g wet wt.). No signi®cant dierence in feeding rates (algal wet wt./sea urchin/day) was observed in the dierent treatments or the control (Au et al., 2000b). At the end of the 4-week exposure period, individuals were induced to spawn by injecting 1 ml 0.5 M KCl through the peristomal membrane. The `dry' sperm/eggs collected from each individual were stored separately in micro-tubes, and used for experiments within the same day. 2.1. Sperm motility Sperm motility was measured using the CRISMAS computer-assisted sperm motility analysis system (Image House A/S, Denmark). Sperm movement was
D.W.T. Au et al. / Environmental Pollution 111 (2001) 1±9
recorded using a CCD camera (PULNiX TM-710) attached to an Olympus BH-2 microscope with a phasecontrast objective lens at 2010 magni®cation. The system captured images of sperm heads at a rate of 30 images sÿ1. Software parameters were set according to the manufacturer's instruction (Gade-Nielsen, 1995). Images of cell aggregates or debris were ®ltered from video analysis using the GIPS object feature analysis provided by the CRISMAS system. Motility of sperm collected from each male individual in all treatment groups (four to six males per group) was studied. Sperm were ®rst diluted using ®ltered seawater (density ca. 107 sperm mlÿ1). Sub-samples (50 ml) of sperm were taken at 1-, 30-, 60- and 120-min intervals, and mounted on a concave glass slide [pre-coated with 1% sodium dodecyl sulfate (SDS) to prevent sperm adhesion]. Five microscopic ®elds were examined, and recorded for 5 s each. Percent motile sperm (velocity >10 mm sÿ1) in each ®eld was determined by the CRISMAS. Motile sperm with normal circular trajectory were counted and the percentage calculated. The following motion parameters of individual sperm were also assessed: (1) curvilinear velocity (VCL) (mm sÿ1), the time-average velocity of the sperm head along its actual trajectory; (2) straight line velocity (VSL) (mm sÿ1), the time-average velocity of the sperm head along a straight line between its ®rst detected position and its last position; and (3) average path velocity (VAP) (mm sÿ1), the time-average velocity of a sperm head along its spatial average trajectory. This trajectory was computed by smoothing the actual path according to algorithms of the CRISMAS system. 2.2. Egg morphology
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were mixed with the eggs and sperm from the 0.01 and 0.1 mg lÿ1 Cd2+ treatments (in which a sperm-to-egg ratio of 3000:1 was used) in dierent combinations (Table 1), and this subsequently gave rise to seven experimental subgroups. In each subgroup, three replicates of sperm and egg mixtures were prepared. Procedures for the fertilization study and the dynamics of ®rst cleavage assays for Anthocidaris crassispina followed those of Vaschenko et al. (1999). Parameters measured and sampling intervals are summarized in Table 2. At each sampling interval, a 0.1-ml sub-sample was collected from each replicate subgroup and ®xed in 0.1 ml 8% formaldehyde for subsequent analysis. 2.4. Statistical analysis Sperm motion data were arcsin transformed to achieve equality of variance prior to analysis of variance (ANOVA). The null hypothesis that dierent cadmium treatments do not cause signi®cant changes in sperm motion parameters (%motile sperm, %circular trajectory, VCL, VSL, VAP) was tested. An ANOVA was also used to test for dierences in percent fertilization among experimental subgroups (all data were arcsin transformed prior to ANOVA). Where treatment group means were signi®cantly dierent, pair-wise comparisons between the control and individual treatment groups were carried out using a Dunnett test (Zar, Table 1 The seven experimental subgroups obtained by combining egg and sperm samples from the control and the two Cd2+ treatment groups for fertilization and ®rst cleavage kinetic assays Experimental subgroups
Size and shape of eggs in the control and two treatment groups were assessed and compared by using (1) area and (2) width-to-height ratio. A total of 1000±1200 eggs was analyzed for each treatment group and control, using the CRISMAS image analyzer system (examined at 200 magni®cation). 2.3. Fertilization capability and dynamics of ®rst cleavage `Dry' sperm (ca. 0.5 ml) from all male sea urchins in each group were individually pooled, and the same procedures followed for eggs. In order to test relative sensitivity to Cd2+, sperm and eggs from the control
Abbreviation
1. Both sperm and eggs from the control group
CS+CE
2. Sperm from the control plus eggs from the 0.01 mg lÿ1 Cd2+ group
CS+0.01E
3. Sperm from the 0.01 mg lÿ1 Cd2+ group plus eggs from the control
0.01S+CE
4. Both sperm and eggs from the 0.01 mg lÿ1 Cd2+ group
0.01S+0.01E
5. Sperm from the control plus eggs from the 0.1 mg lÿ1 Cd2+ group
CS+0.1E
6. Sperm from the 0.1 mg lÿ1 Cd2+ group plus eggs from the control
0.1S+CE
7. Both sperm and eggs from the 0.1 mg lÿ1 Cd2+ group
0.1S+0.1E
Table 2 Gamete quality assays: the parameters measured and sampling time selected Assays
Parameter measured
Sampling intervals
1. Fertilization capability 2. Dynamics of ®rst cleavage
%fertilized eggs, as indicated by presence of fertilization membrane %®rst cleavage, as indicated by formation of two-celled embryos (blastomeres)
30 min 52, 54, 56, 58, 60, 64, 68 min
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1996). A w2 distribution test was used to compare differences in frequency distribution of egg parameters between control and treatment groups (area and widthto-height ratio).
(Fig. 3). A dose±response relationship was demonstrated between percent fertilization of sperm from Cd2+treated sea urchins and Cd2+ concentration (Fig. 3).
3. Results
A higher percent ®rst cleavage was demonstrated in the control (subgroup CS+CE) at all time intervals as compared to that of the other subgroups using sperm or/and egg from sea urchins exposed to Cd2+ (Fig. 4). At 0.01 mg lÿ1 Cd2+, percent ®rst cleavages after 60 min incubation for the subgroups 0.01S+CE and 0.01S+0.01E was distinctly lower than that of subgroup CS+0.01E. In the 0.1 mg lÿ1 Cd2+ treatment, percent cleavage among the subgroups CS+0.1E, 0.1S+CE and 0.1S+0.1E were generally not dierent from each other during the period of study.
Mortality of sea urchins in the 0.01 and 0.1 mg lÿ1 Cd2+ treatment groups was <5% within 4 weeks and similar to the seawater control. The sex ratio of Anthocidaris crassispina used in this experiment was found to be 45 male:55 female. 3.1. Sperm motility Motility of `control' sperm, as measured by %motile sperm, %motile sperm with circular trajectory, VCL, VSL and VAP, exhibited a decreasing trend with time (Fig. 1). A dose±response relationship was generally demonstrated between sperm motion parameters and Cd2+ concentration. No signi®cant decrease in sperm motility was observed in the 0.01 mg lÿ1 Cd2+ treatment within 60 min as compared to the control (Fig. 1a±e). After 120 min, however, both percent motile sperm (Fig. 1a), and percent motile sperm with normal circular trajectory (Fig. 1b) were signi®cantly lower in the 0.01 mg lÿ1 Cd2+ treatment (P<0.01). For the 0.1 mg lÿ1 Cd2+ treatment, two motion parameters, percent motile sperm (Fig. 1a) and percent motile sperm with circular trajectory (Fig. 1b), were lower than the control at all time intervals (P<0.01) (Fig. 1a, b). Dierences in initial VCL, VSL and VAP between the control and 0.1 mg lÿ1 Cd2+ treatment sperm were signi®cant (P<0.01) except after 60 min in seawater (Fig. 1c±e). 3.2. Egg morphology Results of a w2 test for independence showed that areas of eggs were aected by cadmium (P<0.05), and that sea urchins exposed to 0.1 mg lÿ1 Cd2+ produced a higher percentage of eggs with larger areas (>10103 mm2) (Fig. 2). Shapes of sea urchin eggs in terms of width-to-height ratio (range: 0.7±1.2) were, however, not aected by Cd2+ treatment (Fig. 2). 3.3. Fertilization capability Upon exposure to 0.01 mg lÿ1 Cd2+, percent fertilization using sperm and/or eggs from the Cd2+-treated sea urchins (subgroups CS+0.01E, 0.01S+CE, 0.01E+0.01S) was similar to that of the control (CS+CE) with >85% fertilization after 30 min incubation (Fig. 3). At 0.1 mg lÿ1 Cd2+ treatment, a signi®cant reduction in percent fertilization was observed in the subgroups 0.1S+CE and 0.1S+0.1E (P<0.05)
3.4. Dynamics of ®rst cleavage
4. Discussion Results of the present investigation demonstrated severe gamete impairment in Anthocidaris crassispina after chronic exposure to 0.01±0.1 mg lÿ1 Cd2+. Sperm motility was apparently impaired when sea urchins were exposed to 0.1 mg lÿ1 Cd2+ for 4 weeks, and the decrease in sperm motility indicated a dose±response relationship to cadmium levels. A reduction in percent motile sperm, change in sperm movement pattern and a decline in initial velocities (VCL, VSL, VAP) of motile sperm were apparent when sea urchins were exposed to 0.1 mg lÿ1 Cd2+. Such alterations may indicate that Cd2+ has caused structural impairments to sperm during development (Au et al., 2000b). Movement of sperm is supported by propagation of bending waves along sperm tails, and sperm tail bending is the result of sliding between pairs of doublet microtubules (A, B) in the axoneme. Active sliding (which is ATP dependent) occurs all along the axoneme so that the resulting bends are propagated without damping (Takahashi and Kamimura, 1983; Lodish and Darnell, 1995). Alterations in microtubules/axoneme of sperm tail and ATP supply for microtubule sliding can result in a direct impact on sperm movement. Reduction in sperm motility has been related to inherited defects associated with sperm tails (Ryder et al., 1990; Hellander et al., 1991) and midpiece mitochondria (Earnshaw et al., 1986). Cadmium has been shown to inhibit microtubule sliding in bovine sperm axoneme (Kanous et al., 1993). In this study, we suggest that accumulation of Cd2+on axoneme of Anthocidaris crassispina sperm may reduce swimming velocity as well as changing movement patterns, thereby altering sperm movement. In this connection, a parallel study has been conducted to relate sperm ultrastructural changes in motility and chronic exposure to Cd2+ (Au et al., 2000b).
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Fig. 1. Sperm motion parameters of Anthocidaris crassispina after being exposed to dierent levels of cadmium for 4 weeks. Motility of sperm was measured between 1 min and up to 120 min after being discharged into seawater. Data are expressed by meanS.E.M. Values signi®cantly diering from the control within the same time intervals are indicated by asterisks (*P<0.05; **P<0.01).
A `circular' trajectory was common among `control' Anthocidaris crassispina sperm in this study. An earlier study, using a stroboscopic darkground illumination technique, also reported a circular or helical path of progression for normal sea urchin sperm (Gary, 1955).
In the present study, percent motile sperm with circular trajectory gradually declined with time in normal sperm of Anthocidaris crassispina. This may re¯ect aging/ incompetence of sperm after being discharged into seawater. Investigations on the mechanics of sperm
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Fig. 2. Percent distribution of egg morphological parameters: area and width-to-height ratio in Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks.
trajectory, and physical factors leading to trajectory alterations, are not the focus of this study. Nevertheless, erratic sperm trajectories (e.g. spiky, see-saw patterns) were consistently and frequently observed in the Cd2+treated samples. The relationship between alterations in sperm trajectory and subsequent changes in sperm quality (e.g. fertilization success) is not well understood and warrants further investigation. With regard to egg quality, the shape of Anthocidaris crassispina eggs in terms of width/height ratio was not aected by both levels of Cd2+. Female sea urchins exposed to 0.1 mg lÿ1 Cd2+ produced eggs relatively larger in area, but degenerative changes were not observable. Adverse eects might be detected only after prolonged exposure (>4 weeks), as degenerative changes were found in growing oocytes of the sea urchin Strongylocentrotus intermedius after >10 weeks of exposure to 0.05±0.1 mg lÿ1 Cd2+ (Gnezdilova et al., 1987). Fertilization capability of Anthocidaris crassispina eggs was not aected by chronic exposure to Cd2+ (CS+0.01E, CS+0.1E). This oers further support to the study of Khristoforova et al. (1984) who reported normal fertilization capacity for Strongylocentrotus intermedius exposed to 0.1 mg lÿ1 Cd2+ for 20 days. In contrast, in this study a dose±response relationship was observed in percent fertilization when male sea urchins were exposed to the same level of Cd2+. In the 0.1 mg
Fig. 3. Percent fertilization of gametes from Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks. Values signi®cantly diering from the control are indicated by asterisks (*P<0.05; **P<0.01).
lÿ1 Cd2+ treatment (Fig. 3), percent fertilization was signi®cantly reduced in the subgroup 0.01S+CE when sperm from Cd2+-treated sea urchins were combined with control eggs. Although percent fertilization was not aected at a lower concentration (i.e. 0.01 mg lÿ1 Cd2+), the percent cleavage of fertilized eggs in the subgroups 0.01S+CE and 0.01S+0.01E was noticeably lower when compared to CS+0.01E and CS+CE (Fig. 4). Similarly, larval development was found to be more profoundly aected when sperm of the sea urchin Arbacia punctulata exposed to 0.1 mg lÿ1 Cd2+ for 2 weeks were used for fertilization (Bowen and Engel, 1996). Based on the results of earlier studies and the present investigation, it is clear that male sea urchins (and sperm) are relatively more sensitive than female (and eggs) to chronic Cd2+ pollution, which is important to support the current practice of exposing sperm alone in sea urchin fertilization test. It is noteworthy that the US EPA recommended a criterion of 9.3 mg lÿ1 for cadmium in saltwater (US EPA, 1994). Based on the results of this study, such a level may only aord marginal protection to sea urchin reproduction.
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motility. Discrepancies in the ®ndings among various studies may be related to the dierences in methods used in measuring sperm motility, and/or dierent motility parameters used for correlation. Experiments are currently being undertaken in our laboratory relating sperm motion parameters to fertilization success of sea urchins. 5. Conclusion
Fig. 4. Dynamics of ®rst cleavage of gametes from Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks.
4.1. Fertilization success and sperm motility Swimming velocity of sperm is an important factor in¯uencing successful fertilization in free spawning invertebrates (Chia and Barker, 1996). A concomitant increase in fertilization capacity was found with an increase in sperm motility in the sea urchin Strongylocentrotus intermedius (Berdyshev et al., 1995). Studies of fertilization biology in ®sh have shown that fertilization capacity improved with prolonged and intensi®ed sperm motility, e.g. in northern pike (Esox lucius) (Rieniets and Millard, 1987) and rainbow trout (Oncorhynchus mykiss) (Scheerer and Thorgaard, 1989; Chechun et al., 1994; Lahnsteiner et al., 1997). However, such a relationship was not found in Atlantic cod (Gadus morhua) (Trippel et al., 1991), salmon (Salmo salar) (Aas et al., 1991) and thawed sperm of northern pike (Babiak et al., 1995). When Anthocidaris crassispina was exposed to 0.1 mg lÿ1 Cd2+, sperm motion parameters (e.g. abundance of motile sperm, sperm trajectory, swimming velocity) were adversely aected, and a concomitant reduction in percent fertilization was also observed in the subgroup 0.1S+CE using sperm from Cd2+-treated sea urchins for fertilization. The present ®ndings support the proposition that a decrease in fertilization capacity of Anthocidaris crassispina sperm may be related to sperm
Chronic exposure of the adult sea urchin Anthocidaris crassispina to 0.01±0.1 mg lÿ1 Cd2+ for 4 weeks induced apparent impairment in sperm quality. A dose±response relationship was generally demonstrated between sperm motility, percent fertilization and Cd2+ levels. Sperm motility, in terms of percent motile sperm, swimming velocities and patterns of sperm movement, were signi®cantly aected after sea urchins were exposed to 50.1 mg lÿ1 cadmium. A decline in sea urchin sperm motility was also accompanied by a concomitant decrease in fertilization success. No degenerative changes in egg morphology and reduction in fertilization capability of eggs were observed in sea urchins after cadmium treatments, although the size of eggs became larger. In general, male sea urchins were more sensitive to chronic Cd2+ pollution than females, as demonstrated by the poor sperm performance in the fertilization and ®rst cleavage dynamics studies. Acknowledgements We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (7000989) to support this study. We would like to thank Michael W.L. Chiang and the technical sta in the Department of Biology and Chemistry for their technical assistance. References Aas, G.H., Refstie, T., Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture 95, 125±132. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 1998. Four lines of spermatid development and dimorphic spermatozoa in the sea urchin Anthocidaris crassispina (Echinodermata, Echinoida). Zoomorphology 118, 159±168. Au, D.W.T., Chiang, M.W.L., Wu, R.S.S., 2000a. Eects of cadmium and phenol on motility and ultrastructure of sea urchin and green mussel sperm. Archives of Environmental Contamination and Toxicology 38, 455±463. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 2000b. Reproductive impairment of the sea urchin upon chronic exposure to cadmium. II. Eects on sperm development. Environmental Pollution 111, 11±20. Babiak, I., Glogowski, J., Luczynski, M.J., Kucharczyk, D., Luczynski, M., 1995. Cryopreservation of the milt of the northern pile. Journal of Fish Biololgy 46, 819±828.
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D.W.T. Au et al. / Environmental Pollution 111 (2001) 1±9 ¯at®sh. In: Proceedings of the International Symposium on North Paci®c Flat®sh, pp. 547±560. Alaska Sea Grant Collection Program, Fairbanks, Alaska. Takahashi, K., Kamimura, S., 1983. Dynamic aspects of microtubule sliding in sperm ¯agella. Journal of Submicroscope Cytology 15, 1±3. Thompson, G.B., Wu, R.S.S., 1981. Toxicity testing of oil dispersants in Hong Kong. Marine Pollution Bulletin 12, 233±238. Trippel, E., Perley, P., Neilson, J., 1991. Spermatocrit and sperm swimming speed do not correlate with fertilization success in Atlantic cod (Gadus morhua). Special Publication of the European Aquaculture Society No. 15. US EPA, 1994. Water Quality Standards Handbook, 2nd Edition (Report No. EPA-823-B-94-0059). US EPA, Washington DC. Vaschenko, M.A., Zhadan, P.M., Karaseva, E.M., Lukyanova, O.N., 1993. Disturbance of the reproductive function of the sea urchin Strongylocentrotus intermedius in polluted areas of Peter the Bay, Sea of Japan. Russian Journal of Marine Biology 19, 35±40. Vaschenko, M.A., Zhadan, P.M., Malakhov, V.V., Medvedeva, L.A., 1995. Toxic eect of mercury chloride on sexual cells and embryos
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of the sea urchin Strongylocentrotus intermedius. Biological Morya Marine Biology 21, 333±340. Vaschenko, M.A., Zhang, Z.P., Lam, P.K.S., Wu, R.S.S., 1999. Toxic eects of cadmium (CdCl2) on fertilizing capability of spermatozao, dynamics of the ®rst cleavage and pluten formation in the sea urchin Anthocidaris crassispina (Agassiz). Marine Pollution Bulletin 38, 1097±1104. Witman, J.D., 1985. Refuges, biological disturbance, and rocky subtidal community structure in New England. Ecological Monographs 55, 421±445. WHO, 1992. World Health Organisation Laboratory Manual for Examination of Human Semen and Sperm±Crevical Mucus Interaction. Cambridge University Press, New York, pp. 87±91. Young, R.J., Bodt, B.A., Heitkamp, D.H., 1995. Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility. Molecular Reproduction and Development 41, 239±248. Zar, J.H., 1996. Biostatistical Analysis, 3rd Edition. Prentice-Hall, New York.
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Reproductive impairment of sea urchins upon chronic exposure to cadmium. Part I: Eects on gamete quality D.W.T. Au *, C.Y Lee, K.L Chan, R.S.S. Wu Center for Coastal Pollution and Conservation, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong Received 7 July 1999; accepted 18 December 1999
``Capsule'': Gamete quality for male sea urchins was reduced by chronic exposure to cadmium. Abstract Successful reproduction is a determining factor for species survival. Pollution may impair reproductive success of adults through eects on gamete quality. Reproductive impairment of the sea urchin Anthocidaris crassispina upon chronic (4 weeks) exposure to 0.01 and 0.1 mg lÿ1 Cd2+ was investigated. Criteria used for assessing gamete quality included sperm motility, egg morphology, fertilization rate and dynamics of ®rst cleavage. A dose±response relationship was found between Cd2+ levels and changes in sperm motility, and percentage fertilization. Sperm motility, measured by computer-assisted sperm analysis, indicated that percent motile sperm, velocities, and percent sperm with normal trajectory were signi®cantly aected by chronic exposure to 50.1 mg lÿ1 Cd2+. A decline in sperm motility was also accompanied by a decrease in fertilization success of sea urchin sperm. Width/height ratio of sea urchin eggs was not aected by cadmium, but larger egg sizes were found when sea urchins were exposed to 0.1 mg lÿ1 Cd2+. Male sea urchins exposed to Cd2+ produced poorer quality sperm, as indicated by a lower percent fertilization and lower cleavage rate, implying that male sea urchins were more sensitive than females to chronic Cd2+ exposure. Results of the present study provide an explanation of reproductive impairment in marine invertebrates upon chronic exposure to Cd2+. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: CASA; Sperm motility; Fertilization success; Heavy metals; Invertebrate reproduction
1. Introduction Reproductive success is one of the key factors in determining species survival. Pollution may impair reproductive success of adult organisms through decreasing the quality and/or quantity of gametes, which in turn may aect fertilization success, embryo development, larval viability and subsequently species ®tness and survival. Reproductive impairment by a variety of xenobiotics has been well reported in ®sh (Sol et al., 1995; Shatunovskii et al., 1996; Lye et al., 1997) and marine mammals (O'Shea and Brownell, 1994; Jenssen, 1996). The notable eects of tributyltin (TBT) on imposex of gastropods and early development of oysters have been documented (Gibbs and Bryan, 1996; His, 1996). However, the chronic eects of pollution on * Corresponding author. Present address: Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong. Fax: +852-2788-7406. E-mail address: [email protected] (D.W.T. Au).
reproductive impairment of marine invertebrates are generally not well known. Sea urchins play a key role in controlling rocky subtidal community structures in coastal waters (Pringle et al., 1982; Fletcher, 1987) and changes in sea urchin populations have led to major alterations in marine community structure (Witman, 1985; Scheibling, 1986). Retarded gonad development was reported in the sea urchin Strongylocentrotus intermedius collected from polluted areas of Peter the Bay (Sea of Japan) (Vaschenko et al., 1993). On the contrary, a ®eld transplantation study showed enhanced gonad development and suppressed fertilization kinetics in Strongylocentrotus purpuratus upon chronic exposure to oil euents (Krause, 1994). Sublethal exposure to heavy metals (e.g. Cu, Zn and Cd) in the laboratory has led to poor embryonic and larval development in the sea urchin Strongylocentrotus intermedius (Khristoforova et al., 1984; Durkina, 1994), Sphaerechinus granularis (Lucu et al., 1991) and Arbacia punctulata (Bowen and Engel, 1996). However, no details were provided on the nature of gamete impairment in the above studies. Also,
0269-7491/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0269-7491(00)00035-X
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no attempt has been made to relate changes in gamete quality to subsequent alterations in fertilization capability and larval development. Sperm motility is one of the most important criteria in assessing sperm quality. The computer-assisted sperm analysis system (CASA) provides an objective and quantitative assessment of sperm motility (WHO, 1992). The CASA system, which was ®rst developed to study human sperm, has been employed to study heavy metal eects on the motility of rabbit sperm (Young et al., 1995), and cat®sh sperm (Kime et al., 1996). In an earlier study, we successfully applied the CASA system to study motility of sea urchin and mussel sperm upon direct exposure to Cd2+ and phenol (Au et al., 2000a). Sea urchin eggs are relatively large in size (ca. 100 mm in diameter) and can be examined readily by light microscopy. The CASA image analyzer system in our laboratory is able to provide rapid, quantitative measurements on the area as well as width-to-height ratio of eggs, which allows a quantitative comparison of egg morphology (size and shape) among treatment groups. In this study, these parameters were employed as morphological criteria for assessing egg quality. Quality and viability of eggs and sperm can be appraised based upon their fertilization capability, and the subsequent development of fertilized eggs. Fertilization studies and ®rst cleavage dynamics of sea urchin gametes have been extensively employed for marine pollution monitoring and ecotoxicological studies (Dinnel et al., 1987, 1989; Dinnel, 1995; Kobayashi, 1995; Pagano et al., 1986, 1989; Vaschenko et al., 1995). Kinetics of sea urchin ®rst cleavage has proven to be a sensitive end-point for assessing heavy metal toxicity (Ramachandran et al., 1997). The standard procedure for this kind of assay is to expose sperm in high concentrations of pollutants for a brief period (about 30 min). Eggs are not exposed, although it was common practice in earlier times. In the natural environment, adult sea urchins are normally exposed to lower levels of pollutants for a prolonged period. The quality and subsequent fertilization ability of sperm and eggs produced by adult sea urchins exposed to chronic pollution may be very dierent. However, few studies have been carried out to determine chronic eects of cadmium on sea urchin reproduction. Cadmium is one of the most important toxic metals in industrial discharges, and is included in both US EPA and ECs' priority pollutant lists. Signi®cant levels of cadmium contamination have been reported in Hong Kong's coastal waters (Phillips, 1989; EPD, 1998). Nevertheless, eects of such high levels of cadmium on marine populations in Hong Kong are not known. The short spine sea urchin Anthocidaris crassispina, which is abundant over a wide geographical range in the IndoPaci®c Oceans and East China Seas, is regarded as ecologically important in tropical and subtropical
coastal waters (Thompson and Wu, 1981). In this investigation, we examined the eects of chronic exposure to Cd2+ of adult Anthocidaris crassispina, in which gamete quality was assessed by sperm motility, egg morphology, fertilization and dynamics of ®rst cleavage. The relative sensitivity of female and male sea urchins to Cd2+ was compared, based on the performance of their gametes, and an attempt was also made to relate sperm motility to fertilization success. The results of this study allow a better understanding of the chronic eects of cadmium pollution on the reproductive success of sea urchins, and permit an extrapolation to predict population eects on this ecologically important species. 2. Materials and methods In Hong Kong, gonad development of Anthocidaris crassispina is initiated in autumn (November to December); gonads are gravid in spring (March to May), and spawning occurs in summer (June to September) (Chiu, 1988; Au et al., 1998). In May 1998, specimens of Anthocidaris crassispina were collected by SCUBA from subtidal zones of a pristine site (Kat O) in Hong Kong. In the laboratory, sea urchins were acclimated for 48 h in 30 l seawater tanks (®ve sea urchins per tank, 25 C, 30%) with constant aeration. US EPA recommended a criterion of 9.3 mg lÿ1 for cadmium in saltwater, and such elevated levels of Cd2+ have been reported in polluted coastal areas in Hong Kong and China. A preliminary experiment showed that time for 50% mortality (LT50) of 1 mg lÿ1 Cd2+ for Anthocidaris crassispina was 18 days, and since application factors of 10±100 are normally used to extrapolate sublethal eects, sea urchins were exposed to 0.1 and 0.01 mg lÿ1 Cd2+ (prepared from CdCl2) in the present study. Each level of Cd2+ exposure utilized 30 individuals, each held in six separate replicate tanks. Control sea urchins were kept in seawater only. Seawater and Cd2+ solutions were renewed every 2 days and sea urchins were fed with sea lettuce, Ulva lactuca (ca. 4 g wet wt.). No signi®cant dierence in feeding rates (algal wet wt./sea urchin/day) was observed in the dierent treatments or the control (Au et al., 2000b). At the end of the 4-week exposure period, individuals were induced to spawn by injecting 1 ml 0.5 M KCl through the peristomal membrane. The `dry' sperm/eggs collected from each individual were stored separately in micro-tubes, and used for experiments within the same day. 2.1. Sperm motility Sperm motility was measured using the CRISMAS computer-assisted sperm motility analysis system (Image House A/S, Denmark). Sperm movement was
D.W.T. Au et al. / Environmental Pollution 111 (2001) 1±9
recorded using a CCD camera (PULNiX TM-710) attached to an Olympus BH-2 microscope with a phasecontrast objective lens at 2010 magni®cation. The system captured images of sperm heads at a rate of 30 images sÿ1. Software parameters were set according to the manufacturer's instruction (Gade-Nielsen, 1995). Images of cell aggregates or debris were ®ltered from video analysis using the GIPS object feature analysis provided by the CRISMAS system. Motility of sperm collected from each male individual in all treatment groups (four to six males per group) was studied. Sperm were ®rst diluted using ®ltered seawater (density ca. 107 sperm mlÿ1). Sub-samples (50 ml) of sperm were taken at 1-, 30-, 60- and 120-min intervals, and mounted on a concave glass slide [pre-coated with 1% sodium dodecyl sulfate (SDS) to prevent sperm adhesion]. Five microscopic ®elds were examined, and recorded for 5 s each. Percent motile sperm (velocity >10 mm sÿ1) in each ®eld was determined by the CRISMAS. Motile sperm with normal circular trajectory were counted and the percentage calculated. The following motion parameters of individual sperm were also assessed: (1) curvilinear velocity (VCL) (mm sÿ1), the time-average velocity of the sperm head along its actual trajectory; (2) straight line velocity (VSL) (mm sÿ1), the time-average velocity of the sperm head along a straight line between its ®rst detected position and its last position; and (3) average path velocity (VAP) (mm sÿ1), the time-average velocity of a sperm head along its spatial average trajectory. This trajectory was computed by smoothing the actual path according to algorithms of the CRISMAS system. 2.2. Egg morphology
3
were mixed with the eggs and sperm from the 0.01 and 0.1 mg lÿ1 Cd2+ treatments (in which a sperm-to-egg ratio of 3000:1 was used) in dierent combinations (Table 1), and this subsequently gave rise to seven experimental subgroups. In each subgroup, three replicates of sperm and egg mixtures were prepared. Procedures for the fertilization study and the dynamics of ®rst cleavage assays for Anthocidaris crassispina followed those of Vaschenko et al. (1999). Parameters measured and sampling intervals are summarized in Table 2. At each sampling interval, a 0.1-ml sub-sample was collected from each replicate subgroup and ®xed in 0.1 ml 8% formaldehyde for subsequent analysis. 2.4. Statistical analysis Sperm motion data were arcsin transformed to achieve equality of variance prior to analysis of variance (ANOVA). The null hypothesis that dierent cadmium treatments do not cause signi®cant changes in sperm motion parameters (%motile sperm, %circular trajectory, VCL, VSL, VAP) was tested. An ANOVA was also used to test for dierences in percent fertilization among experimental subgroups (all data were arcsin transformed prior to ANOVA). Where treatment group means were signi®cantly dierent, pair-wise comparisons between the control and individual treatment groups were carried out using a Dunnett test (Zar, Table 1 The seven experimental subgroups obtained by combining egg and sperm samples from the control and the two Cd2+ treatment groups for fertilization and ®rst cleavage kinetic assays Experimental subgroups
Size and shape of eggs in the control and two treatment groups were assessed and compared by using (1) area and (2) width-to-height ratio. A total of 1000±1200 eggs was analyzed for each treatment group and control, using the CRISMAS image analyzer system (examined at 200 magni®cation). 2.3. Fertilization capability and dynamics of ®rst cleavage `Dry' sperm (ca. 0.5 ml) from all male sea urchins in each group were individually pooled, and the same procedures followed for eggs. In order to test relative sensitivity to Cd2+, sperm and eggs from the control
Abbreviation
1. Both sperm and eggs from the control group
CS+CE
2. Sperm from the control plus eggs from the 0.01 mg lÿ1 Cd2+ group
CS+0.01E
3. Sperm from the 0.01 mg lÿ1 Cd2+ group plus eggs from the control
0.01S+CE
4. Both sperm and eggs from the 0.01 mg lÿ1 Cd2+ group
0.01S+0.01E
5. Sperm from the control plus eggs from the 0.1 mg lÿ1 Cd2+ group
CS+0.1E
6. Sperm from the 0.1 mg lÿ1 Cd2+ group plus eggs from the control
0.1S+CE
7. Both sperm and eggs from the 0.1 mg lÿ1 Cd2+ group
0.1S+0.1E
Table 2 Gamete quality assays: the parameters measured and sampling time selected Assays
Parameter measured
Sampling intervals
1. Fertilization capability 2. Dynamics of ®rst cleavage
%fertilized eggs, as indicated by presence of fertilization membrane %®rst cleavage, as indicated by formation of two-celled embryos (blastomeres)
30 min 52, 54, 56, 58, 60, 64, 68 min
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1996). A w2 distribution test was used to compare differences in frequency distribution of egg parameters between control and treatment groups (area and widthto-height ratio).
(Fig. 3). A dose±response relationship was demonstrated between percent fertilization of sperm from Cd2+treated sea urchins and Cd2+ concentration (Fig. 3).
3. Results
A higher percent ®rst cleavage was demonstrated in the control (subgroup CS+CE) at all time intervals as compared to that of the other subgroups using sperm or/and egg from sea urchins exposed to Cd2+ (Fig. 4). At 0.01 mg lÿ1 Cd2+, percent ®rst cleavages after 60 min incubation for the subgroups 0.01S+CE and 0.01S+0.01E was distinctly lower than that of subgroup CS+0.01E. In the 0.1 mg lÿ1 Cd2+ treatment, percent cleavage among the subgroups CS+0.1E, 0.1S+CE and 0.1S+0.1E were generally not dierent from each other during the period of study.
Mortality of sea urchins in the 0.01 and 0.1 mg lÿ1 Cd2+ treatment groups was <5% within 4 weeks and similar to the seawater control. The sex ratio of Anthocidaris crassispina used in this experiment was found to be 45 male:55 female. 3.1. Sperm motility Motility of `control' sperm, as measured by %motile sperm, %motile sperm with circular trajectory, VCL, VSL and VAP, exhibited a decreasing trend with time (Fig. 1). A dose±response relationship was generally demonstrated between sperm motion parameters and Cd2+ concentration. No signi®cant decrease in sperm motility was observed in the 0.01 mg lÿ1 Cd2+ treatment within 60 min as compared to the control (Fig. 1a±e). After 120 min, however, both percent motile sperm (Fig. 1a), and percent motile sperm with normal circular trajectory (Fig. 1b) were signi®cantly lower in the 0.01 mg lÿ1 Cd2+ treatment (P<0.01). For the 0.1 mg lÿ1 Cd2+ treatment, two motion parameters, percent motile sperm (Fig. 1a) and percent motile sperm with circular trajectory (Fig. 1b), were lower than the control at all time intervals (P<0.01) (Fig. 1a, b). Dierences in initial VCL, VSL and VAP between the control and 0.1 mg lÿ1 Cd2+ treatment sperm were signi®cant (P<0.01) except after 60 min in seawater (Fig. 1c±e). 3.2. Egg morphology Results of a w2 test for independence showed that areas of eggs were aected by cadmium (P<0.05), and that sea urchins exposed to 0.1 mg lÿ1 Cd2+ produced a higher percentage of eggs with larger areas (>10103 mm2) (Fig. 2). Shapes of sea urchin eggs in terms of width-to-height ratio (range: 0.7±1.2) were, however, not aected by Cd2+ treatment (Fig. 2). 3.3. Fertilization capability Upon exposure to 0.01 mg lÿ1 Cd2+, percent fertilization using sperm and/or eggs from the Cd2+-treated sea urchins (subgroups CS+0.01E, 0.01S+CE, 0.01E+0.01S) was similar to that of the control (CS+CE) with >85% fertilization after 30 min incubation (Fig. 3). At 0.1 mg lÿ1 Cd2+ treatment, a signi®cant reduction in percent fertilization was observed in the subgroups 0.1S+CE and 0.1S+0.1E (P<0.05)
3.4. Dynamics of ®rst cleavage
4. Discussion Results of the present investigation demonstrated severe gamete impairment in Anthocidaris crassispina after chronic exposure to 0.01±0.1 mg lÿ1 Cd2+. Sperm motility was apparently impaired when sea urchins were exposed to 0.1 mg lÿ1 Cd2+ for 4 weeks, and the decrease in sperm motility indicated a dose±response relationship to cadmium levels. A reduction in percent motile sperm, change in sperm movement pattern and a decline in initial velocities (VCL, VSL, VAP) of motile sperm were apparent when sea urchins were exposed to 0.1 mg lÿ1 Cd2+. Such alterations may indicate that Cd2+ has caused structural impairments to sperm during development (Au et al., 2000b). Movement of sperm is supported by propagation of bending waves along sperm tails, and sperm tail bending is the result of sliding between pairs of doublet microtubules (A, B) in the axoneme. Active sliding (which is ATP dependent) occurs all along the axoneme so that the resulting bends are propagated without damping (Takahashi and Kamimura, 1983; Lodish and Darnell, 1995). Alterations in microtubules/axoneme of sperm tail and ATP supply for microtubule sliding can result in a direct impact on sperm movement. Reduction in sperm motility has been related to inherited defects associated with sperm tails (Ryder et al., 1990; Hellander et al., 1991) and midpiece mitochondria (Earnshaw et al., 1986). Cadmium has been shown to inhibit microtubule sliding in bovine sperm axoneme (Kanous et al., 1993). In this study, we suggest that accumulation of Cd2+on axoneme of Anthocidaris crassispina sperm may reduce swimming velocity as well as changing movement patterns, thereby altering sperm movement. In this connection, a parallel study has been conducted to relate sperm ultrastructural changes in motility and chronic exposure to Cd2+ (Au et al., 2000b).
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Fig. 1. Sperm motion parameters of Anthocidaris crassispina after being exposed to dierent levels of cadmium for 4 weeks. Motility of sperm was measured between 1 min and up to 120 min after being discharged into seawater. Data are expressed by meanS.E.M. Values signi®cantly diering from the control within the same time intervals are indicated by asterisks (*P<0.05; **P<0.01).
A `circular' trajectory was common among `control' Anthocidaris crassispina sperm in this study. An earlier study, using a stroboscopic darkground illumination technique, also reported a circular or helical path of progression for normal sea urchin sperm (Gary, 1955).
In the present study, percent motile sperm with circular trajectory gradually declined with time in normal sperm of Anthocidaris crassispina. This may re¯ect aging/ incompetence of sperm after being discharged into seawater. Investigations on the mechanics of sperm
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Fig. 2. Percent distribution of egg morphological parameters: area and width-to-height ratio in Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks.
trajectory, and physical factors leading to trajectory alterations, are not the focus of this study. Nevertheless, erratic sperm trajectories (e.g. spiky, see-saw patterns) were consistently and frequently observed in the Cd2+treated samples. The relationship between alterations in sperm trajectory and subsequent changes in sperm quality (e.g. fertilization success) is not well understood and warrants further investigation. With regard to egg quality, the shape of Anthocidaris crassispina eggs in terms of width/height ratio was not aected by both levels of Cd2+. Female sea urchins exposed to 0.1 mg lÿ1 Cd2+ produced eggs relatively larger in area, but degenerative changes were not observable. Adverse eects might be detected only after prolonged exposure (>4 weeks), as degenerative changes were found in growing oocytes of the sea urchin Strongylocentrotus intermedius after >10 weeks of exposure to 0.05±0.1 mg lÿ1 Cd2+ (Gnezdilova et al., 1987). Fertilization capability of Anthocidaris crassispina eggs was not aected by chronic exposure to Cd2+ (CS+0.01E, CS+0.1E). This oers further support to the study of Khristoforova et al. (1984) who reported normal fertilization capacity for Strongylocentrotus intermedius exposed to 0.1 mg lÿ1 Cd2+ for 20 days. In contrast, in this study a dose±response relationship was observed in percent fertilization when male sea urchins were exposed to the same level of Cd2+. In the 0.1 mg
Fig. 3. Percent fertilization of gametes from Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks. Values signi®cantly diering from the control are indicated by asterisks (*P<0.05; **P<0.01).
lÿ1 Cd2+ treatment (Fig. 3), percent fertilization was signi®cantly reduced in the subgroup 0.01S+CE when sperm from Cd2+-treated sea urchins were combined with control eggs. Although percent fertilization was not aected at a lower concentration (i.e. 0.01 mg lÿ1 Cd2+), the percent cleavage of fertilized eggs in the subgroups 0.01S+CE and 0.01S+0.01E was noticeably lower when compared to CS+0.01E and CS+CE (Fig. 4). Similarly, larval development was found to be more profoundly aected when sperm of the sea urchin Arbacia punctulata exposed to 0.1 mg lÿ1 Cd2+ for 2 weeks were used for fertilization (Bowen and Engel, 1996). Based on the results of earlier studies and the present investigation, it is clear that male sea urchins (and sperm) are relatively more sensitive than female (and eggs) to chronic Cd2+ pollution, which is important to support the current practice of exposing sperm alone in sea urchin fertilization test. It is noteworthy that the US EPA recommended a criterion of 9.3 mg lÿ1 for cadmium in saltwater (US EPA, 1994). Based on the results of this study, such a level may only aord marginal protection to sea urchin reproduction.
D.W.T. Au et al. / Environmental Pollution 111 (2001) 1±9
7
motility. Discrepancies in the ®ndings among various studies may be related to the dierences in methods used in measuring sperm motility, and/or dierent motility parameters used for correlation. Experiments are currently being undertaken in our laboratory relating sperm motion parameters to fertilization success of sea urchins. 5. Conclusion
Fig. 4. Dynamics of ®rst cleavage of gametes from Anthocidaris crassispina after exposure to dierent levels of cadmium for 4 weeks.
4.1. Fertilization success and sperm motility Swimming velocity of sperm is an important factor in¯uencing successful fertilization in free spawning invertebrates (Chia and Barker, 1996). A concomitant increase in fertilization capacity was found with an increase in sperm motility in the sea urchin Strongylocentrotus intermedius (Berdyshev et al., 1995). Studies of fertilization biology in ®sh have shown that fertilization capacity improved with prolonged and intensi®ed sperm motility, e.g. in northern pike (Esox lucius) (Rieniets and Millard, 1987) and rainbow trout (Oncorhynchus mykiss) (Scheerer and Thorgaard, 1989; Chechun et al., 1994; Lahnsteiner et al., 1997). However, such a relationship was not found in Atlantic cod (Gadus morhua) (Trippel et al., 1991), salmon (Salmo salar) (Aas et al., 1991) and thawed sperm of northern pike (Babiak et al., 1995). When Anthocidaris crassispina was exposed to 0.1 mg lÿ1 Cd2+, sperm motion parameters (e.g. abundance of motile sperm, sperm trajectory, swimming velocity) were adversely aected, and a concomitant reduction in percent fertilization was also observed in the subgroup 0.1S+CE using sperm from Cd2+-treated sea urchins for fertilization. The present ®ndings support the proposition that a decrease in fertilization capacity of Anthocidaris crassispina sperm may be related to sperm
Chronic exposure of the adult sea urchin Anthocidaris crassispina to 0.01±0.1 mg lÿ1 Cd2+ for 4 weeks induced apparent impairment in sperm quality. A dose±response relationship was generally demonstrated between sperm motility, percent fertilization and Cd2+ levels. Sperm motility, in terms of percent motile sperm, swimming velocities and patterns of sperm movement, were signi®cantly aected after sea urchins were exposed to 50.1 mg lÿ1 cadmium. A decline in sea urchin sperm motility was also accompanied by a concomitant decrease in fertilization success. No degenerative changes in egg morphology and reduction in fertilization capability of eggs were observed in sea urchins after cadmium treatments, although the size of eggs became larger. In general, male sea urchins were more sensitive to chronic Cd2+ pollution than females, as demonstrated by the poor sperm performance in the fertilization and ®rst cleavage dynamics studies. Acknowledgements We are grateful to the Research Committee, City University of Hong Kong, for the award of a Strategic Grant (7000989) to support this study. We would like to thank Michael W.L. Chiang and the technical sta in the Department of Biology and Chemistry for their technical assistance. References Aas, G.H., Refstie, T., Gjerde, B., 1991. Evaluation of milt quality of Atlantic salmon. Aquaculture 95, 125±132. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 1998. Four lines of spermatid development and dimorphic spermatozoa in the sea urchin Anthocidaris crassispina (Echinodermata, Echinoida). Zoomorphology 118, 159±168. Au, D.W.T., Chiang, M.W.L., Wu, R.S.S., 2000a. Eects of cadmium and phenol on motility and ultrastructure of sea urchin and green mussel sperm. Archives of Environmental Contamination and Toxicology 38, 455±463. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 2000b. Reproductive impairment of the sea urchin upon chronic exposure to cadmium. II. Eects on sperm development. Environmental Pollution 111, 11±20. Babiak, I., Glogowski, J., Luczynski, M.J., Kucharczyk, D., Luczynski, M., 1995. Cryopreservation of the milt of the northern pile. Journal of Fish Biololgy 46, 819±828.
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