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Sublethal effects of phenol on spermatogenesis in sea urchins (Anthocidaris crassispina)
ARTICLE IN PRESS
Environmental Research 93 (2003) 92–98
Sublethal effects of phenol on spermatogenesis in sea urchins (Anthocidaris crassispina) Doris W.T. Au,a, Olga V. Yurchenko,b and Arkadiy A. Reunovb b
a Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, 17 Palchevsky St., Vladivostok 690041, Russia
Received 17 July 2002; received in revised form 6 December 2002; accepted 18 December 2002
Abstract Adult sea urchins, Anthocidaris crassispina, were exposed to 0.1 and 10 mg L1 phenol for 4 weeks. Abnormal sperm development was clearly evident in phenol-treated sea urchins, although no mortality was found throughout the exposure period. Occurrences of multinucleate sperm cells (including spermatocytes to spermatozoa) showed a significant increase from 0.07% in the control to 10.7% and 43.3% in the 0.1- and 10-mg L1 treatments, respectively (Po0.01). Likewise, sperm with electron dense, dark tails increased significantly from 8% in the control to 36.6% and 43.4% in the 0.1- and 10-mg L1 phenol-treated sea urchins, respectively (Po0.01). In addition, disruption of cytoplasmic membranous structures such as loss of mitochondrial cristae and distortion of mitochondrial membranes and nucleus envelope were commonly found in phenol-treated spermatogonia and spermatocytes. Previous studies have clearly demonstrated motility impairment and a concomitant reduction of fertilization capability in sea urchin sperm with dark tails and/or distorted mitochondria. Our current findings therefore suggest that chronic exposure to phenol at 0.1 mg L1 could lower the quality of sperm and reproductive success in sea urchins, which may threaten the survival of these ecologically important species. The observed impairment of spermatogenesis by phenol might also occur in other invertebrate species. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytokinesis defect; Phenol; Sperm tail; Spermatogenesis; Urchin
1. Introduction Phenol is a typical toxicant in municipal and industrial wastes such as oil and paper pulp (Buttino et al., 1991; see review in Mukherjee et al., 1991) and is generally regarded a ubiquitous environmental pollutant. As a result, phenol has been widely employed as a reference toxicant in aquatic toxicity tests (Green et al., 1988; Anderson et al., 1994; Hickey and Martin, 1995). Negative impacts of phenol on reproduction in aquatic animals have been reported for fish (Ghosh, 1983) and invertebrates such as gastropods (Kordylewska, 1980), prawns (Law and Yeo, 1997), and sea urchins (Anderson et al., 1994). While the existing studies have focused mainly on the acute effects of phenol on gamete viability and embryo development, the long-term effects of chronic exposure to phenol leading to reproductive
Corresponding author. Fax: +852-2788-7406. E-mail address: [email protected] (D.W.T. Au).
impairment of aquatic animals are not well known. Information available to date is limited to the influence of phenol on reproduction in females. For instance, reduction of egg production was observed in a copepod (Acartia clausi) after exposure to phenol (0.5 mg L1) for 8 days (Buttino, 1994). Likewise, a gradual decrease of gonadosomatic index was reported in female fish (Cyprinus carpio) after exposure to phenol (8 mg L1) for 45 days (Mukherjee et al., 1991). Exposure of human testis to phenol poses a high risk of testicular cancer (Haughey et al., 1989). However, the chronic effects of phenol on reproduction of male invertebrates are generally not known. Sea urchins play a key role in controlling the structure of subtidal macrophyte communities in temperate and Australian 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). Since reproductive success is a crucial factor in determining species fitness
0013-9351/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0013-9351(02)00094-4
ARTICLE IN PRESS D.W.T. Au et al. / Environmental Research 93 (2003) 92–98
and survival, impairment of reproduction in sea urchins is likely to lead to major ecological impacts on subtidal rocky communities. Our earlier study demonstrated that acute exposure of sea urchin (Anthocidaris crassispina) and mussel (Perna viridis) spermatozoa to phenol could reduce sperm motility, and a parallel ultrastructural study revealed convolution of the plasma membrane, deformation of midpiece, and loss of mitochondrial cristae in phenoltreated sperm (Au et al., 2000). The present study aims to investigate the chronic effects of phenol on spermatogenesis in the adult sea urchin A. crassispina. Findings of this cytological study can provide a better understanding of the long-term impacts of phenol pollution on reproductive processes of male sea urchins. Results of this study will also allow us to evaluate the ecological consequences of chronic phenol pollution on the reproductive success of this ecologically important species.
2. Materials and methods The short spine sea urchin A. crassispina is abundant over a wide geographical range in the IndoPacific Oceans and East China Seas. In Hong Kong, differentiation of spermatogonia of A. crassispina begins in spring (February–March) and spawning occurs during summer (June–September) (Au et al., 1998). During March to April, all stages of male gonadal development (spermatogonia, spermatocytes, spermatids, and spermatozoa) can be found. In the present study, A. crassispina specimens were collected by scuba from subtidal areas of a pristine site (Kat O) in Hong Kong in March of 1999 and acclimated in clean seawater in 30-L tanks (five sea urchins per tank, 251C, 30%), with continuous aeration for 48 h. Sea urchins were randomly exposed to 0.1 and 10 mg L1 phenol–seawater solution (prepared by dilution of a 100-mg L1 stock phenol solution with filtered seawater). Each level of phenol treatment consisted of three replicate tanks, each containing five sea urchins. Control sea urchins were kept in seawater only. Mortality of sea urchins was checked daily. Seawater was renewed every 2 days and sea urchins were fed with the green algae Ulva lactuca. At the end of the 4-week exposure, all sea urchins were scarified. Testes of male urchins were sampled, dissected, and fixed immediately in primary fixative (containing 1% tannic acid and 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 8.55% sucrose, pH 7.5) for 2 h. Fixed tissues were washed (in decreasing concentrations of sucrose–buffer solutions and buffer), postfixed in 2% buffered OsO4 for 2 h, rinsed in buffer and DI water, dehydrated in a graded ethanol series and acetone, infiltrated, and embedded in Spurr’s resin. Male gonads of five sea urchins were studied for each
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treatment, and three tissue blocks were randomly chosen from each gonad for sectioning. Ultrathin sections were stained with 2% alcoholic uranyl acetate and aqueous lead citrate before being examined with a JEOL 100SX or JOEL JEM 100B transmission electron microscope at 80 kV. To determine percentage occurrence of dark tails in the gonad, a total of 1000 sperm tails were counted for each treatment to estimate the number of tails with extraordinary high electron density (the ‘‘dark’’ tail sperm cell). A w2 test was used to test for significant differences in the frequency of ‘‘dark’’ tail sperm cells and ordinary sperm cells in the different treatment and control groups. An average of 4000 sperm cells was counted for each treatment, and percentages of multinucleate spermatogonia, spermatocytes, spermatids, and spermatozoa were calculated. Data on percentage occurrence of multinucleate sperm cells were arcsine-transformed prior to two-way ANOVA to achieve homogeneity of variances. Where significant effects were found, post hoc multiple comparisons between treatment groups were carried out using a Tukey honest significant difference test (a ¼ 0:05).
3. Results The sex ratio of A. crassispina used in these experiments was ca. 45 male:55 female. No mortality was found in both the treatment and the control groups throughout the 4-week exposure period. Gonads of the 10-mg L1 phenol treatment group were apparently regressed while gonad size of the 0.1- mg L1 phenoltreated sea urchins was similar to that of the control. In the 0.1-mg L1 phenol treatment, heterophagosomes of nutritive phagocytes absorbed not only spermatozoa (Fig. 1(1)) but also sperm cells at the earlier stages of development (i.e., spermatogonia and spermatocytes; Figs. 1(2) and (3)). In the 10-mg L1 phenol-treated sea urchin gonads, heterophagosis appeared to be more active, and intact spermatogonia and spermatocytes were rarely observed. Autophagosomes of nutritive phagocytes are specialized for self-destruction after spawning (Fig. 1(4)). Early arising of autophagosomes was another feature observed in sea urchin gonads exposed to both concentrations of phenol. A high occurrence of fragmented clusters of nutritive phagocytes in the 10-mg L1 phenol-treated sea urchin gonads indicated active autophagosis (Fig. 1(5)). Many distorted sperm cells could be found inside these regions (Fig. 1(5)), which were probably destroyed during autophagosis of nutritive phagocytes. The amount of intact sperm cells in testes lumens was apparently reduced in the 10-mg L1 phenol-treated sea urchins as compared to the controls.
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Spermatogenesis in the control urchins was typical of that observed and reported in A. crassispina (Au et al., 1998). Incomplete cytokinesis of sperm cells was rarely seen in the gonads of control urchins (Table 1). However, significant increases in multinucleate sperm cells were observed in phenol-treated gonads (Po0:01), and a dose–response relationship was demonstrated (Table 1). Occurrences of double-nucleate spermatogonia (Fig. 2(6)) and double-nucleate spermatocytes (Fig. 2(7)) were found to be 5.4% and 20.3% in the 0.1- and 10-mg L1 phenol treatments, respectively. These undivided spermatocytes continued to undergo spermiogenesis, marked by simultaneous condensation
1
of nuclei chromatin (Fig. 2(8)). Acrosomes were present in the apical region (Fig. 2(9)) and more than one pair of centrioles were found near the basal region of undivided spermatids (Fig. 2(10)). These abnormal spermatids continued to differentiate into spermatozoa with ‘‘fused’’ heads (Figs. 2(11) and (12)), which are analogous to ‘‘Siamese twins’’. Occurrences of multinucleate spermatids/spermatozoa in the 0.1- (5.3%) and 10mg L1 (23.0%) phenol treatments were significantly higher than that in the control (0.07%) (Po0:01). Another unusual feature observed in the phenoltreated gonads was the presence of ‘‘dark’’ tails in spermatids/spermatozoa. ‘‘Dark’’ tails lost the normal
2
3 4
5 Fig. 1. Gonads exposed to 0.1 mg L1 phenol. (1) Heterophagosomes of nutritive phagocyte containing spermatozoa (arrows). Scale bar, 1 mm. (2) Heterophagosome with absorbed spermatogonia. Scale bar, 1 mm. (3) Heterophagosome containing spermatocyte-like sperm cell. , electron-luscent lysosome. Scale bar, 1 mm. (4) Autophagosome () in nutritive phagocyte specialized for self-destruction. Nu, nucleus. Scale bar, 1 mm. (5) Fragments of nutritive phagocytes aggregate with many sperm cells in the testis lumen. Scale bar, 5 mm.
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9+2 pattern of axoneme and appeared electron dense with amorphous substances (Figs. 2(13) and (14)) (see Au et al., 2001b). Results of the w2 test revealed a significantly higher frequency of ‘‘dark’’ tails in the 0.1and 10-mg L1 phenol treatments (36.6% and 43.4%, respectively) compared with the control (8%) (Po0:01).
Alterations of cytoplasmic membranous structure integrity were observed in spermatogonia and spermatocytes of phenol-treated sea urchins. For example, swelling of nucleus double membrane (Fig. 3(15)), loss of mitochondrial cristae and swollen as well as electronlucent cristae were regularly found in spermatogonia and spermatocytes (Figs. 3(15) and (16)). However, such alterations were not seen in the midpiece of late spermatids and spermatozoa in both of the phenol treatments. Mitochondria in late sperm cells appeared to be more resistant to phenol.
Table 1 Occurrences (%) of multinucleate sperm cells in gonads of sea urchins exposed to phenol for 4 weeks Cell types
Control
0.1 mg L1 phenol
10 mg L1 phenol
Spermatogonia Spermatocytes Spermatids Spermatozoa
0 0 0.05 0.02
0.8 4.6 3.2 2.1
1.9 18.4 12.2 10.8
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4. Discussion No mortality was observed in A. crassipina after exposure to the two levels of phenol treatment for 4
Significantly different from the control (Po0:01).
N1 N1 N2
N2
6
N1
9
10
7
8
11
12
N2
13
14
Fig. 2. Gonads exposed to 0.1 mg L1 phenol. (6) Double-nucleate spermatogonium. Scale bar, 1 mm. (7) Early spermatid with double nuclei, N1 and N2. Scale bar, 1 mm. (8) Late spermatid with double nuclei, N1 and N2. Scale bar, 1 mm. (9) Two acrosomes (arrowheads) in a double-nucleate (N1 and N2) spermatozoon. Scale bar, 0.5 mm. (10) Centrioles (arrowheads) in the middle part of an undivided spermatid. Scale bar, 0.5 mm. (11) Doublenucleate spermatozoon with two tails (arrow heads). Scale bar, 1 mm. (12) Double-nucleate spermatozoa. Scale bar, 1 mm. (13) Dark tail (l.s.). Scale bar, 0.5 mm. (14) Dark tails (arrowheads) (x.s.). Scale bar, 0.5 mm.
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15
16
Fig. 3. Gonads exposed to 0.1 mg L1 phenol. (15) Spermatocyte with dilated nucleus membrane (arrowheads) and electron-luscent vacuoles. Mitochondria were swollen with membrane disruption (arrows). Scale bar, 1 mm. (16) Spermatogonium. Swelling and loss of mitochondrial cristae. Scale bar, 0.5 mm.
weeks. Gonads of sea urchins in the 10-mg L1 phenol treatment appeared to be regressed in size as compared to those of the controls. In conjunction, a reduction of sperm cells was generally observed in the lumen of testis. Severe loss of sperm cells and regression of testis in A. crassispina exposed to 10 mg L1 phenol could be explained by active heterophagocytosis and autophagocytosis in nutritive phagocytes. Under normal circumstances, spermatozoon is the major cell type for absorption by heterophagosomes, and the formation of autophagosomes is a natural process to reduce the number of nutritive phagocytes in gonads of A. crassispina after spawning (D.W.T. Au et al., unpublished data). However, under the influence of phenol, heterophagosomes digested all kinds of sperm cells, and autophagocytosis of nutritive phagocytes prior to spawning destroyed sperm cells in the vicinity. Subsequently, the amount of spermatozoa in the phenoltreated sea urchins was reduced. Gonad regression has been reported in the ovaries of fish (C. carpio) exposed to 8 mg L1 phenol for 45 days (Mukherjee et al., 1991). Similarly, reduction of egg production was reported in a copepod (A. clausi) after exposure to 0.5 mg L1 phenol for 8 days (Buttino, 1994). Our results demonstrate regression of testis size in sea urchins exposed to phenol for 4 weeks and offer further evidence that chronic exposure to phenol may affect gamete production in aquatic animals. With regard to the quality of sperm cells, our ultrastructural findings revealed abnormal sperm development in A. crassipina after a 4-week exposure to 0.1 mg L1 phenol. Significant sperm impairments were exemplified by the formation of multinucleate sperm cells, high occurrences of sperm cells with ‘‘dark’’ tails,
and disruption of cytoplasmic membranous structures (e.g., mitochondria, nucleus membrane). Under the influence of phenol at levels X0.1 mg L1, incomplete cytokinesis occurred during mitotic and meiotic divisions of sperm cells, resulting in the formation of undivided spermatogonia/spermatocyte/ spermatids/spermatozoa in the gonads of A. crassipina. In Drosophila, cytokinesis defects occurred during spermatogenesis, also leading to the formation of undivided sperm cells with multinuclear, ‘‘fused’’ heads (Carmena et al., 1998). Phenol, which is highly soluble in lipids, can readily penetrate the cell membrane (Kordylewska, 1980). However, the mechanism involved in the inhibition of cell division by phenol is not known. Cytokinesis inhibitors such as stypoldione and pseudopterolide are known to induce the formation of microtubule spiral asters, inhibit microtubule polymerization, and covalently bind with sulfhydryl groups of protein responsible for the formation and function of the contractile ring (O’Brien et al., 1989; Grace et al., 1992). Coincidently, our study showed that the 9+2 pattern of microtubules in the axoneme of sperm tails was affected by phenol, resulting in significantly higher percentages of ‘‘dark tail’’ sperm in the phenol-treated groups. Although it is clear that disturbance of the microtubule assembly may lead to cytokinesis defects and loss of tail integrity in sperm cells, whether phenol may affect microtubules in sperm cells requires further verification. In addition to cytokinesis and sperm tail defects, disruptions of cytoplasmic membrane structures (e.g., mitochondrial membrane and cristae, nuclear membrane) were commonly found in spermatogonia and spermatocytes exposed to 0.1 mg L1 phenol. Although
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such alterations were not observable in the midpieces of spermatids/spermatozoa in the 10-mg L1 phenol group, damage may still occur at higher phenol levels and/or after longer periods of exposure. Acute exposure of the spermatozoa of A. crassipina to 500 mg L1 phenol for 30 min was able to induce distortion of mitochondrial integrity (e.g., irregular shape, loss of cristae, and an electron-lucent matrix) and convolution of the plasma membrane (Au et al., 2001b). Likewise, an ultrastructural study of the sperm of the bivalve Spisula solidissima revealed a pronounced swelling of the midpiece and an irregular appearance of sperm plasma membrane after exposure to gossypol, a phenolic compound (Burgos et al., 1997). Moreover, exposing gastrula of a gastropod (Limnaea stagnalis) to 1 mg L1 phenol for 1–4 h led to ultrastructural changes in cell shape, swollen nuclei, and destruction of mitochondrial membrane/cristae and cell membranes, and cell limits became less visible (Kordylewska, 1980). Clearly, destruction of membranous structures is a characteristic cytological damage induced by phenol exposure. From the viewpoint of reproduction and survival fitness, the most important concern is whether the quality of sperm (e.g., motility, fertilization capability) is affected after chronic exposure of sea urchins to phenol. Our earlier studies demonstrated impairment of motility in sea urchin sperm with ‘‘dark’’ tails and/or distorted mitochondria (Au et al., 2000, 2001a, b). Notably, a significant correlation has been successfully established between sea urchin sperm motility and sea urchin fertilization success (Au et al., 2002). Our cytological data revealed a significant increase of ‘‘dark tail’’ sperm in gonads exposed to X0.1 mg L1 phenol. The present results therefore indicate impairment of sperm motility and subsequent reduction of fertilization capability in the phenol-treated sperm. Importantly, the deleterious effects of chronic phenol pollution on reproduction of this ecological important species can be expected at levels as low as 0.1 mg L1 and possibly lower. USEPA (1987) reported that chronic toxicity of phenol to freshwater aquatic life generally occurs at 2.56 mg L1, while acute toxicity in saltwater occurs at 5.8 mg L1. In Malaysia DOE-MU, (1986) recommended an interim standard of phenol of not exceeding 9.9 mg L1 at any time, to protect aquatic life. Our data show that reproductive impairment can occur in male sea urchins when exposed to as low as 0.1 mg L1 phenol. Thus, the criteria for phenol recommended by USEPA and DOE-MU may not offer protection to reproduction of sea urchins and possibly also reproduction of other invertebrates. Although the Australian water quality criteria (AWRC, 1984) recommended a criterion of 0.1 mg L1 phenol for protection of aquatic life, our findings suggest that the water quality standard for phenol in seawater should be 0.1 mg L1 or less.
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Acknowledgments This work was partially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (No. 873001) and grants from the President of the Russian Federation (RFBR No. 00-15-99354) and the RFBR-primorye (No. 01-0496930), Russia. We thank M.W.L. Chiang, Debbie Cheng, and staff in the Electron Microscope Unit of the University of Hong Kong for technical assistance, and Mr. D. Fomin for his kind help in the TEM Unit of the Institute of Marine Biology RAS FEB.
References Anderson, S.L., Hose, J.E., Knezovich, J.P., 1994. Genotoxic and developmental effects in sea urchins are sensitive indications of effects of genotoxic chemicals. Environ. Toxicol. Chem. 13, 1033–1041. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 1998. Four lines of speratid 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., 2000. Effects of cadmium and phenol on motility and ultrastructure of sea urchin and mussel spermatozoa. Arch. Environ. Contam. Toxicol. 38, 455–463. Au, D.W.T., Lee, C.Y., Chan, .K.L., Wu, R.S.S., 2001a. Reproductive impariment of sea urchins upon chronic exposure to cadmium. Part I: effects on gamete quality. Environ. Pollut. 111, 1–9. Au, D.W.T., Reunov, A.A., Wu, R.S.S., 2001b. Reproductive impairment of sea urchin upon chronic exposure to cadmium. Part II: effects on sperm development. Environ. Pollut. 111, 11–20. Au, D.W.T., Chiang, M.W.L., Tang, J.Y.M., Yuen, B.B.H., Wang, Y.L., Wu, R.S.S., 2002. Impairment of sea urchin sperm quality by UV–B radiation: predicting fertilization success from sperm motility. Mar. Pollut. Bull. 44, 583–589. Australian Water Research Council (AWRC). 1984. In: Nicholson, B.C. (Ed.), Australian Water Quality Criteria for Organic Compounds. Australian Government Publishing Service, Canberra. Burgos, M.H., Ito, S., Segal, S.J., Tran, P.T., 1997. Effect of gossypol on the ultrastructure of spisula sperm. Biol. Bull. 193, 228–229. Buttino, I., 1994. The effect of low concentrations of phenol and ammonia on egg production rates, fecal pellet production and egg viability of the calanoid copepod Acartia clausi. Mar. Biol. 119, 629–634. Buttino, I., Filippi, M., Cardellicchio, N., 1991. Tossicitia dei composti fenoloco nell’ambiente marino. Acqua. Aria. 9, 853–861. Carmena, M., Riparbelli, M.G., Minestrini, G., Tavares, A.M., Adams, R., Gallaini, G., Glover, D.M., 1998. Drosophila polo kinase is required for cytokinesis. J. Cell Biol. 3, 659–671. Fletcher, W.J., 1987. Interactions among subtidal Australian sea urchins, gastropods and algae: effects of experimental removals. Ecol. Monogr. 57, 89–109. Ghosh, T.K., 1983. Effect of phenol pollution on aquatic life. Environ. Ecol. 1, 1–3. Grace, K.J.S., Medina, M., Jacobs, R.S., Wilson, L., 1992. Selective inhibition of cytokinesis in sea urchin embryos by the marine natural product pseudopterolide. Mol. Pharmacol. 41, 631–638. Green, D.W.J., Williams, K.A., Hughes, D.R.L., Shaik, G.A.R., Pascoe, D.R.L., 1988. Toxicity of phenol to Asellus aquaticus (L.) effects of temperature and episodic exposure. Water Res. 22, 225–231.
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Haughey, B.P., Graham, S., Brasure, J., Zielezny, M., Sufrin, G., Burnett, W.S., 1989. The epidemiology of testicular cancer in upstate New York. Am. J. Epidemiol. 130, 25–36. Hickey, C.W., Martin, M.L., 1995. Relative sensitivity of five benthic invertebrate species to reference toxicants and resin-acid contaminated sediments. Environ. Toxicol. Chem. 14, 1401–1409. Kordylewska, A., 1980. Effect of phenol on the cell structure on the embryos of Limnaea stagnalis. L. (gastropoda, pulmonata). Light and electron microscopic study. Acta Biol. Cracov. Ser. Zool. 22, 89–98. Law, A.T., Yeo, M.E., 1997. Toxicity of phenol on Macrobrachium rosenbergii (de Man) eggs, larvae, and post-larvae. Bull. Environ. Contam. Toxicol. 58, 469–474. Malaysia DOE-MU, 1986. In: Goh, S.H., Yap, S.Y., Lim, R. P., (Eds.), Water Quality Criteria and Standards for Maylaysia, Vol. 4, Criteria and Standards for Organic Constituents. Malaysia/IPT, University of Malaya, Malaysia, DOE. Mukherjee, D., Guha, D., Kumar, V., Chakrabarty, S., 1991. Impairment of steroidogenesis and reproduction in sexually mature
Cyprinus carpio by phenol and sulfide under laboratory conditions. Aquat. Toxicol. 21, 29–40. O’Brien, E.T., Asai, D.J., Jacobs, R.S., Wilson, L., 1989. Selective inhibition of cytokinesis in sea urchin embryos by low concentrations of stypoldione, a marine natural product that reacts with sulfhydryl groups. Mol. Pharmacol. 35, 635–642. Pringle, J.D., Sharp, G.J., Caddy, G.J., 1982. Interaction in help bed ecosystems in the Northwest Atlantic: review of a workshop. Fish Aquat. Sci. (Canadian Special Publication). 59, 108–115. Scheibling, R., 1986. Increased macroalgal abundance following mass mortalities of sea urchin (Strongylocentrotus droebachiensis) along the Atlantic coast of Nova Scotia. Oceologia 68, 186–198. USEPA, 1987.Quality criteria for water (440/5-86-001). US Environment Protection Agency, EPA, 1986. Witman, J.D., 1985. Refuges, biological disturbance, and rocky subtidal community structure in New England. Ecol. Monogr. 55, 421–445.
Environmental Research 93 (2003) 92–98
Sublethal effects of phenol on spermatogenesis in sea urchins (Anthocidaris crassispina) Doris W.T. Au,a, Olga V. Yurchenko,b and Arkadiy A. Reunovb b
a Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, 17 Palchevsky St., Vladivostok 690041, Russia
Received 17 July 2002; received in revised form 6 December 2002; accepted 18 December 2002
Abstract Adult sea urchins, Anthocidaris crassispina, were exposed to 0.1 and 10 mg L1 phenol for 4 weeks. Abnormal sperm development was clearly evident in phenol-treated sea urchins, although no mortality was found throughout the exposure period. Occurrences of multinucleate sperm cells (including spermatocytes to spermatozoa) showed a significant increase from 0.07% in the control to 10.7% and 43.3% in the 0.1- and 10-mg L1 treatments, respectively (Po0.01). Likewise, sperm with electron dense, dark tails increased significantly from 8% in the control to 36.6% and 43.4% in the 0.1- and 10-mg L1 phenol-treated sea urchins, respectively (Po0.01). In addition, disruption of cytoplasmic membranous structures such as loss of mitochondrial cristae and distortion of mitochondrial membranes and nucleus envelope were commonly found in phenol-treated spermatogonia and spermatocytes. Previous studies have clearly demonstrated motility impairment and a concomitant reduction of fertilization capability in sea urchin sperm with dark tails and/or distorted mitochondria. Our current findings therefore suggest that chronic exposure to phenol at 0.1 mg L1 could lower the quality of sperm and reproductive success in sea urchins, which may threaten the survival of these ecologically important species. The observed impairment of spermatogenesis by phenol might also occur in other invertebrate species. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Cytokinesis defect; Phenol; Sperm tail; Spermatogenesis; Urchin
1. Introduction Phenol is a typical toxicant in municipal and industrial wastes such as oil and paper pulp (Buttino et al., 1991; see review in Mukherjee et al., 1991) and is generally regarded a ubiquitous environmental pollutant. As a result, phenol has been widely employed as a reference toxicant in aquatic toxicity tests (Green et al., 1988; Anderson et al., 1994; Hickey and Martin, 1995). Negative impacts of phenol on reproduction in aquatic animals have been reported for fish (Ghosh, 1983) and invertebrates such as gastropods (Kordylewska, 1980), prawns (Law and Yeo, 1997), and sea urchins (Anderson et al., 1994). While the existing studies have focused mainly on the acute effects of phenol on gamete viability and embryo development, the long-term effects of chronic exposure to phenol leading to reproductive
Corresponding author. Fax: +852-2788-7406. E-mail address: [email protected] (D.W.T. Au).
impairment of aquatic animals are not well known. Information available to date is limited to the influence of phenol on reproduction in females. For instance, reduction of egg production was observed in a copepod (Acartia clausi) after exposure to phenol (0.5 mg L1) for 8 days (Buttino, 1994). Likewise, a gradual decrease of gonadosomatic index was reported in female fish (Cyprinus carpio) after exposure to phenol (8 mg L1) for 45 days (Mukherjee et al., 1991). Exposure of human testis to phenol poses a high risk of testicular cancer (Haughey et al., 1989). However, the chronic effects of phenol on reproduction of male invertebrates are generally not known. Sea urchins play a key role in controlling the structure of subtidal macrophyte communities in temperate and Australian 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). Since reproductive success is a crucial factor in determining species fitness
0013-9351/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0013-9351(02)00094-4
ARTICLE IN PRESS D.W.T. Au et al. / Environmental Research 93 (2003) 92–98
and survival, impairment of reproduction in sea urchins is likely to lead to major ecological impacts on subtidal rocky communities. Our earlier study demonstrated that acute exposure of sea urchin (Anthocidaris crassispina) and mussel (Perna viridis) spermatozoa to phenol could reduce sperm motility, and a parallel ultrastructural study revealed convolution of the plasma membrane, deformation of midpiece, and loss of mitochondrial cristae in phenoltreated sperm (Au et al., 2000). The present study aims to investigate the chronic effects of phenol on spermatogenesis in the adult sea urchin A. crassispina. Findings of this cytological study can provide a better understanding of the long-term impacts of phenol pollution on reproductive processes of male sea urchins. Results of this study will also allow us to evaluate the ecological consequences of chronic phenol pollution on the reproductive success of this ecologically important species.
2. Materials and methods The short spine sea urchin A. crassispina is abundant over a wide geographical range in the IndoPacific Oceans and East China Seas. In Hong Kong, differentiation of spermatogonia of A. crassispina begins in spring (February–March) and spawning occurs during summer (June–September) (Au et al., 1998). During March to April, all stages of male gonadal development (spermatogonia, spermatocytes, spermatids, and spermatozoa) can be found. In the present study, A. crassispina specimens were collected by scuba from subtidal areas of a pristine site (Kat O) in Hong Kong in March of 1999 and acclimated in clean seawater in 30-L tanks (five sea urchins per tank, 251C, 30%), with continuous aeration for 48 h. Sea urchins were randomly exposed to 0.1 and 10 mg L1 phenol–seawater solution (prepared by dilution of a 100-mg L1 stock phenol solution with filtered seawater). Each level of phenol treatment consisted of three replicate tanks, each containing five sea urchins. Control sea urchins were kept in seawater only. Mortality of sea urchins was checked daily. Seawater was renewed every 2 days and sea urchins were fed with the green algae Ulva lactuca. At the end of the 4-week exposure, all sea urchins were scarified. Testes of male urchins were sampled, dissected, and fixed immediately in primary fixative (containing 1% tannic acid and 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 8.55% sucrose, pH 7.5) for 2 h. Fixed tissues were washed (in decreasing concentrations of sucrose–buffer solutions and buffer), postfixed in 2% buffered OsO4 for 2 h, rinsed in buffer and DI water, dehydrated in a graded ethanol series and acetone, infiltrated, and embedded in Spurr’s resin. Male gonads of five sea urchins were studied for each
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treatment, and three tissue blocks were randomly chosen from each gonad for sectioning. Ultrathin sections were stained with 2% alcoholic uranyl acetate and aqueous lead citrate before being examined with a JEOL 100SX or JOEL JEM 100B transmission electron microscope at 80 kV. To determine percentage occurrence of dark tails in the gonad, a total of 1000 sperm tails were counted for each treatment to estimate the number of tails with extraordinary high electron density (the ‘‘dark’’ tail sperm cell). A w2 test was used to test for significant differences in the frequency of ‘‘dark’’ tail sperm cells and ordinary sperm cells in the different treatment and control groups. An average of 4000 sperm cells was counted for each treatment, and percentages of multinucleate spermatogonia, spermatocytes, spermatids, and spermatozoa were calculated. Data on percentage occurrence of multinucleate sperm cells were arcsine-transformed prior to two-way ANOVA to achieve homogeneity of variances. Where significant effects were found, post hoc multiple comparisons between treatment groups were carried out using a Tukey honest significant difference test (a ¼ 0:05).
3. Results The sex ratio of A. crassispina used in these experiments was ca. 45 male:55 female. No mortality was found in both the treatment and the control groups throughout the 4-week exposure period. Gonads of the 10-mg L1 phenol treatment group were apparently regressed while gonad size of the 0.1- mg L1 phenoltreated sea urchins was similar to that of the control. In the 0.1-mg L1 phenol treatment, heterophagosomes of nutritive phagocytes absorbed not only spermatozoa (Fig. 1(1)) but also sperm cells at the earlier stages of development (i.e., spermatogonia and spermatocytes; Figs. 1(2) and (3)). In the 10-mg L1 phenol-treated sea urchin gonads, heterophagosis appeared to be more active, and intact spermatogonia and spermatocytes were rarely observed. Autophagosomes of nutritive phagocytes are specialized for self-destruction after spawning (Fig. 1(4)). Early arising of autophagosomes was another feature observed in sea urchin gonads exposed to both concentrations of phenol. A high occurrence of fragmented clusters of nutritive phagocytes in the 10-mg L1 phenol-treated sea urchin gonads indicated active autophagosis (Fig. 1(5)). Many distorted sperm cells could be found inside these regions (Fig. 1(5)), which were probably destroyed during autophagosis of nutritive phagocytes. The amount of intact sperm cells in testes lumens was apparently reduced in the 10-mg L1 phenol-treated sea urchins as compared to the controls.
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Spermatogenesis in the control urchins was typical of that observed and reported in A. crassispina (Au et al., 1998). Incomplete cytokinesis of sperm cells was rarely seen in the gonads of control urchins (Table 1). However, significant increases in multinucleate sperm cells were observed in phenol-treated gonads (Po0:01), and a dose–response relationship was demonstrated (Table 1). Occurrences of double-nucleate spermatogonia (Fig. 2(6)) and double-nucleate spermatocytes (Fig. 2(7)) were found to be 5.4% and 20.3% in the 0.1- and 10-mg L1 phenol treatments, respectively. These undivided spermatocytes continued to undergo spermiogenesis, marked by simultaneous condensation
1
of nuclei chromatin (Fig. 2(8)). Acrosomes were present in the apical region (Fig. 2(9)) and more than one pair of centrioles were found near the basal region of undivided spermatids (Fig. 2(10)). These abnormal spermatids continued to differentiate into spermatozoa with ‘‘fused’’ heads (Figs. 2(11) and (12)), which are analogous to ‘‘Siamese twins’’. Occurrences of multinucleate spermatids/spermatozoa in the 0.1- (5.3%) and 10mg L1 (23.0%) phenol treatments were significantly higher than that in the control (0.07%) (Po0:01). Another unusual feature observed in the phenoltreated gonads was the presence of ‘‘dark’’ tails in spermatids/spermatozoa. ‘‘Dark’’ tails lost the normal
2
3 4
5 Fig. 1. Gonads exposed to 0.1 mg L1 phenol. (1) Heterophagosomes of nutritive phagocyte containing spermatozoa (arrows). Scale bar, 1 mm. (2) Heterophagosome with absorbed spermatogonia. Scale bar, 1 mm. (3) Heterophagosome containing spermatocyte-like sperm cell. , electron-luscent lysosome. Scale bar, 1 mm. (4) Autophagosome () in nutritive phagocyte specialized for self-destruction. Nu, nucleus. Scale bar, 1 mm. (5) Fragments of nutritive phagocytes aggregate with many sperm cells in the testis lumen. Scale bar, 5 mm.
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9+2 pattern of axoneme and appeared electron dense with amorphous substances (Figs. 2(13) and (14)) (see Au et al., 2001b). Results of the w2 test revealed a significantly higher frequency of ‘‘dark’’ tails in the 0.1and 10-mg L1 phenol treatments (36.6% and 43.4%, respectively) compared with the control (8%) (Po0:01).
Alterations of cytoplasmic membranous structure integrity were observed in spermatogonia and spermatocytes of phenol-treated sea urchins. For example, swelling of nucleus double membrane (Fig. 3(15)), loss of mitochondrial cristae and swollen as well as electronlucent cristae were regularly found in spermatogonia and spermatocytes (Figs. 3(15) and (16)). However, such alterations were not seen in the midpiece of late spermatids and spermatozoa in both of the phenol treatments. Mitochondria in late sperm cells appeared to be more resistant to phenol.
Table 1 Occurrences (%) of multinucleate sperm cells in gonads of sea urchins exposed to phenol for 4 weeks Cell types
Control
0.1 mg L1 phenol
10 mg L1 phenol
Spermatogonia Spermatocytes Spermatids Spermatozoa
0 0 0.05 0.02
0.8 4.6 3.2 2.1
1.9 18.4 12.2 10.8
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4. Discussion No mortality was observed in A. crassipina after exposure to the two levels of phenol treatment for 4
Significantly different from the control (Po0:01).
N1 N1 N2
N2
6
N1
9
10
7
8
11
12
N2
13
14
Fig. 2. Gonads exposed to 0.1 mg L1 phenol. (6) Double-nucleate spermatogonium. Scale bar, 1 mm. (7) Early spermatid with double nuclei, N1 and N2. Scale bar, 1 mm. (8) Late spermatid with double nuclei, N1 and N2. Scale bar, 1 mm. (9) Two acrosomes (arrowheads) in a double-nucleate (N1 and N2) spermatozoon. Scale bar, 0.5 mm. (10) Centrioles (arrowheads) in the middle part of an undivided spermatid. Scale bar, 0.5 mm. (11) Doublenucleate spermatozoon with two tails (arrow heads). Scale bar, 1 mm. (12) Double-nucleate spermatozoa. Scale bar, 1 mm. (13) Dark tail (l.s.). Scale bar, 0.5 mm. (14) Dark tails (arrowheads) (x.s.). Scale bar, 0.5 mm.
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15
16
Fig. 3. Gonads exposed to 0.1 mg L1 phenol. (15) Spermatocyte with dilated nucleus membrane (arrowheads) and electron-luscent vacuoles. Mitochondria were swollen with membrane disruption (arrows). Scale bar, 1 mm. (16) Spermatogonium. Swelling and loss of mitochondrial cristae. Scale bar, 0.5 mm.
weeks. Gonads of sea urchins in the 10-mg L1 phenol treatment appeared to be regressed in size as compared to those of the controls. In conjunction, a reduction of sperm cells was generally observed in the lumen of testis. Severe loss of sperm cells and regression of testis in A. crassispina exposed to 10 mg L1 phenol could be explained by active heterophagocytosis and autophagocytosis in nutritive phagocytes. Under normal circumstances, spermatozoon is the major cell type for absorption by heterophagosomes, and the formation of autophagosomes is a natural process to reduce the number of nutritive phagocytes in gonads of A. crassispina after spawning (D.W.T. Au et al., unpublished data). However, under the influence of phenol, heterophagosomes digested all kinds of sperm cells, and autophagocytosis of nutritive phagocytes prior to spawning destroyed sperm cells in the vicinity. Subsequently, the amount of spermatozoa in the phenoltreated sea urchins was reduced. Gonad regression has been reported in the ovaries of fish (C. carpio) exposed to 8 mg L1 phenol for 45 days (Mukherjee et al., 1991). Similarly, reduction of egg production was reported in a copepod (A. clausi) after exposure to 0.5 mg L1 phenol for 8 days (Buttino, 1994). Our results demonstrate regression of testis size in sea urchins exposed to phenol for 4 weeks and offer further evidence that chronic exposure to phenol may affect gamete production in aquatic animals. With regard to the quality of sperm cells, our ultrastructural findings revealed abnormal sperm development in A. crassipina after a 4-week exposure to 0.1 mg L1 phenol. Significant sperm impairments were exemplified by the formation of multinucleate sperm cells, high occurrences of sperm cells with ‘‘dark’’ tails,
and disruption of cytoplasmic membranous structures (e.g., mitochondria, nucleus membrane). Under the influence of phenol at levels X0.1 mg L1, incomplete cytokinesis occurred during mitotic and meiotic divisions of sperm cells, resulting in the formation of undivided spermatogonia/spermatocyte/ spermatids/spermatozoa in the gonads of A. crassipina. In Drosophila, cytokinesis defects occurred during spermatogenesis, also leading to the formation of undivided sperm cells with multinuclear, ‘‘fused’’ heads (Carmena et al., 1998). Phenol, which is highly soluble in lipids, can readily penetrate the cell membrane (Kordylewska, 1980). However, the mechanism involved in the inhibition of cell division by phenol is not known. Cytokinesis inhibitors such as stypoldione and pseudopterolide are known to induce the formation of microtubule spiral asters, inhibit microtubule polymerization, and covalently bind with sulfhydryl groups of protein responsible for the formation and function of the contractile ring (O’Brien et al., 1989; Grace et al., 1992). Coincidently, our study showed that the 9+2 pattern of microtubules in the axoneme of sperm tails was affected by phenol, resulting in significantly higher percentages of ‘‘dark tail’’ sperm in the phenol-treated groups. Although it is clear that disturbance of the microtubule assembly may lead to cytokinesis defects and loss of tail integrity in sperm cells, whether phenol may affect microtubules in sperm cells requires further verification. In addition to cytokinesis and sperm tail defects, disruptions of cytoplasmic membrane structures (e.g., mitochondrial membrane and cristae, nuclear membrane) were commonly found in spermatogonia and spermatocytes exposed to 0.1 mg L1 phenol. Although
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such alterations were not observable in the midpieces of spermatids/spermatozoa in the 10-mg L1 phenol group, damage may still occur at higher phenol levels and/or after longer periods of exposure. Acute exposure of the spermatozoa of A. crassipina to 500 mg L1 phenol for 30 min was able to induce distortion of mitochondrial integrity (e.g., irregular shape, loss of cristae, and an electron-lucent matrix) and convolution of the plasma membrane (Au et al., 2001b). Likewise, an ultrastructural study of the sperm of the bivalve Spisula solidissima revealed a pronounced swelling of the midpiece and an irregular appearance of sperm plasma membrane after exposure to gossypol, a phenolic compound (Burgos et al., 1997). Moreover, exposing gastrula of a gastropod (Limnaea stagnalis) to 1 mg L1 phenol for 1–4 h led to ultrastructural changes in cell shape, swollen nuclei, and destruction of mitochondrial membrane/cristae and cell membranes, and cell limits became less visible (Kordylewska, 1980). Clearly, destruction of membranous structures is a characteristic cytological damage induced by phenol exposure. From the viewpoint of reproduction and survival fitness, the most important concern is whether the quality of sperm (e.g., motility, fertilization capability) is affected after chronic exposure of sea urchins to phenol. Our earlier studies demonstrated impairment of motility in sea urchin sperm with ‘‘dark’’ tails and/or distorted mitochondria (Au et al., 2000, 2001a, b). Notably, a significant correlation has been successfully established between sea urchin sperm motility and sea urchin fertilization success (Au et al., 2002). Our cytological data revealed a significant increase of ‘‘dark tail’’ sperm in gonads exposed to X0.1 mg L1 phenol. The present results therefore indicate impairment of sperm motility and subsequent reduction of fertilization capability in the phenol-treated sperm. Importantly, the deleterious effects of chronic phenol pollution on reproduction of this ecological important species can be expected at levels as low as 0.1 mg L1 and possibly lower. USEPA (1987) reported that chronic toxicity of phenol to freshwater aquatic life generally occurs at 2.56 mg L1, while acute toxicity in saltwater occurs at 5.8 mg L1. In Malaysia DOE-MU, (1986) recommended an interim standard of phenol of not exceeding 9.9 mg L1 at any time, to protect aquatic life. Our data show that reproductive impairment can occur in male sea urchins when exposed to as low as 0.1 mg L1 phenol. Thus, the criteria for phenol recommended by USEPA and DOE-MU may not offer protection to reproduction of sea urchins and possibly also reproduction of other invertebrates. Although the Australian water quality criteria (AWRC, 1984) recommended a criterion of 0.1 mg L1 phenol for protection of aquatic life, our findings suggest that the water quality standard for phenol in seawater should be 0.1 mg L1 or less.
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Acknowledgments This work was partially supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (No. 873001) and grants from the President of the Russian Federation (RFBR No. 00-15-99354) and the RFBR-primorye (No. 01-0496930), Russia. We thank M.W.L. Chiang, Debbie Cheng, and staff in the Electron Microscope Unit of the University of Hong Kong for technical assistance, and Mr. D. Fomin for his kind help in the TEM Unit of the Institute of Marine Biology RAS FEB.
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