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Environmental contaminant-induced spermatozoa anomalies in fish inhabiting Lake Mariut, Alexandria, Egypt.

Abstract

This investigation was designed to evaluate reproductive impairment of tilapia (as a bioindicator organism), inhabiting three sites in Lake Mariut which varied in nature and degree of physicochemical and organic criteria. It is concerned with monitoring bioaccumulation of metal in sperms of Oreochromis niloticus using SEM-X-ray microanalysis and illustrating concomitant ultrastructure sperm abnormalities. Fish caught from polluted sites exhibited a higher proportion of heavy metals, in particular Cu, Zn and Hg, than in reference site. Morphologically, sperms of O. niloticus were anacrosomal aquasperm which is typical of species with external fertilization. It appears that each spermatozoon possesses a short round head, few mitochondria and underdeveloped middle piece. However, sperms of fish caught from the polluted sites showed variable deformations including: changes in head morphology, incomplete chromatin condensation, malformed middle piece, altered axonemal structure, wavy and sometimes ruptured plasma membrane, head-to-head and head-to-tail sperm agglutinations. It is believed that the observed alterations in sperms may result from metal accumulation leading to reproductive impairment. It seems also that the ability of metal exposed fish to produce viable offspring is seriously reduced. Therefore, new recruitment, which is very important to the stability of fish population, will be sharply influenced. Also, alterations in spermatozoa should be included as a model for predicting environmental hazards.

Keywords: Lake Mariut, tilapia, water pollution, reproductive impairment.

Introduction

During the last couple of decades, increases in several pathological disorders in human reproductive system, including cryptochidism (maldescent of the testis), falling sperm counts, decreased semen quality and testicular cancer have been observed (Sharp and Skakkeboek, 1993; Toppari et al., 1995). Abnormalities in male reproductive system of fish, reptiles and mammals have also been observed (Purdom et al., 1994; Guillette et al., 1994; Facemire et al., 1995; Toppari et al., 1995). Recently, it has been suggested that the increasing incidence of male reproductive abnormalities may be a result of environmental pollution by man-made chemicals (Colborn et al., 1993; Sharp and Skakkeboek, 1993; Toppari et al., 1995). Heavy metals, in particular, have been associated with altered steroid levels and inhibited gonadal development in a variety of fish species (Joy and Kirubagaran, 1989; Wester and Canton, 1992). Such reproductive abnormalities not only impair the individual, but may also threaten populations as a whole (Colborn et al., 1993; Toppari et al., 1995).

Since the quality of sperm is a major factor contributing to successful production of fish larvae (Kime et al., 1996), sperm morphology could provide a sensitive and accurate bioindicator of aquatic pollution. Previous studies using copper, methylmercury, mercuric chloride and acid water have used either fertilization rate or a subjective assessment of motility to measure the effects of pollutants on sperm quality (McIntyre, 1973; Duplinsky, 1982; Khan and Weis, 1987; Anderson et al., 1991). Mercury damages sperms and decreases their motility probably by interfering with flagellar function (Mottet and Landolt, 1987). Kime et al. (1996) reported a significant decrease in motility of catfish sperm exposed to sublethal levels of waterborne Cd and Zn. These levels were comparable to reported bioaccumulated levels in the gonads of fish that live in contaminated waters. Furthermore, Gill et al. (2002) described sperm abnormalities in head and tail, e.g. big heads and fuzzy tails, in flounders taken from the lower Tyne estuary in northeast England, a heavily impacted site known to contain endocrine disrupting chemicals.

Stocks of both freshwater and marine fish within Egypt are increasingly threatened by aquatic pollution, but no data are available on the extent by which fertility of individual species has been decreased. Lake Mariut, one of the Nile Delta lakes, receives the strongest human impacts among the Egyptian lakes. It has greatly deteriorated from a productive lake to a heavily polluted and highly eutrophicated basin (Hamza, 1999). The present study was designed to detect metal residues in sperms as well as to assess sperm morphology of fish from three sites in Lake Mariut varied in nature and degree of pollution. Data obtained will be useful in the diagnosis of subtle and obvert effects of heavy metals in polluted habitats.

Materials and Methods

Study Area

Lake Mariut, located southwest of Alexandria, is the heavily polluted lake in Egypt and its water is pumped to the sea. It is now divided artificially into four basins, namely the lake proper, the fish farm, the southeast (S.E.) and the southwest (S.W.) basins. The locations of the sampling sites in Lake Mariut are shown in Figure 1. Information about the health state of the lake is available (Saleh et al., 1983; Adham et al., 1997, 2001; Massoud, 2003). The lake proper (sampling location II) represents the main basin of the lake. It receives most of its water bulk from the polluted El-Kalaa Drain, which carries agricultural wastewater as well as city sewage collected from Semouha Drain and the eastern sewage treatment plant. Other sources of pollution include industrial waste effluents discharged at the northeastern corner, Gheit El-Enab Drain that receives sewage from Karmouz, and El-Kabbari Outfall that discharges raw sewage at the northwestern side. According to Saleh et al. (1983) and Adham et al. (1997), the SW basin (sampling location III) is contaminated with higher amounts of heavy metals, particularly lead and mercury, in addition to mineral oils discharged by the cooling pipes of El-Nasr Petroleum Company. The SE basin (sampling location I) receiving runoff water from the relatively unpolluted Noubariia canal and El-Umoum Drain (Adham et al., 1997, 2001) was originally selected as a reference site.

[FIGURE 1 OMITTED]

Sampling Strategy

Random fish sample of sexually maturing Oreochromis niloticus were collected in close meshed nets from the three sampling sites. Sampling was conducted every 4-6 weeks from December (2002) through February (2003) and from June through August (2003). Fish were sampled during the same period and at the same location each year. After capture, fish were transferred into large vessels filled up with aerated lake water. Ten to 20 male fish were randomly selected (from each site at each sampling time), dissected, and the testes were excised.

Bioaccumulation of Metals

Levels of accumulated elements in spermatozoa were detected in un-coated specimens using Jeol scanning electron microscope -5300 equipped with a Link-Isis energy dispersive X-ray microanalyzer. A stationary spot (X500) was analyzed at random for 110 sec. Due to heterogeneous distribution of trace metals, at least three points on each specimen were measured to check for internal variability of trace metal composition.

Preparation of Specimens for Electron Microscopy

Small pieces of testis were fixed immediately in 4% formalin and 1% glutaraldehyde ([sub.4][F.sub.1]G) fixative mixture in 0.1M phosphate buffer (pH 7.4) for 24 hrs. at 4[degrees]C. Specimens were then post fixed in 2% [O.sub.s][O.sub.4] in the same buffer for 2 hrs. at 4[degrees]C. Samples were washed in the buffer and dehydrated at 4[degrees]C through a graded series of ethanol. For scanning electron microscopy, dehydrated specimens were dried by the critical point method, mounted on an Al-stub and coated with gold in a sputter-coating. Observations of sperm morphology were performed by a JSEM-5300 operated at 20 Kv. For transmission electron microscopy, dehydrated specimens of testes were embedded in Epon-araldite resin mixture. Semithin sections (1 [micro]m thick) stained with Toluidine blue were examined by LM to identify suitable area for ultrastructural evaluation. Ultrathin sections (60 nm thick) were double stained with uranyl acetate for 1/2 hr. and lead citrate for 20-30 min. and were examined in JTEM (Reynolds, 1963).

Results

Metal Accumulation Patterns

X-ray spectra of sperms revealed the presence of 14 elements including Na, Al, P, S, Cd, K, Ca, Fe, Ni, Cu, Zn, Pb, Hg and As. Cu, Zn, Hg and Al were the toxic metals showing considerable high levels and large variations among the different sites. Trends in proportions of Na, P, S, K and Ca were inconsistent. Lower levels of Cd, Fe, Ni, Pb and As were occasionally recorded.

In samples collected from the less polluted location I during the breeding period, a representative X-ray spectrum of sperms showed smaller peaks of Al, Fe, Cu and Zn. In location II, Hg was detected in considerable proportion as well as Al. However, Ni, Fe, Cu, Zn and Pb were present in small proportions. On the other hand, Al, Hg, Cu and Zn attained their highest levels in sperms of fish from location III. Traces of Cd, Fe, Ni and Pb were also detected (fig.2). In the non-breeding period, sperms contained higher levels of Cu and Zn in the polluted groups. Levels of Hg residues were greatest in Location II, less so in III and the least in I. It is of considerable interest that Cd was not detected in all locations and levels of Al were uniform across locations. Furthermore, sampling sites contained traces of Fe, Ni and Pb in location I, traces of Ni, As and Pb in location II, and traces of Fe, Ni and As in location III (fig.3). Close inspection of the energy dispersive X-ray analyses showed that during the non-spawning season (Dec-Feb), the sperms had much more pronounced accumulation of heavy metals as compared to the spawning season (Jun-Aug).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Normal Morphology of Tilapian Sperm from Low Polluted Waters (Location I):

Our electron micrographs revealed that mature spermatozoa of fish collected from location I were more or less normal in the two sampling periods with little aberrations. They possessed a spherical head (2 [micro]m in diameter), a small eccentric midpiece (about 1.5 [micro]m in length), and a rather short flagellum (about 20.3 [micro]m in length) (figs.4a & b). The nuclear material comprising the head portion consisted characteristically of many electron-dense chromatin globules, which formed a mass by close adhesion leaving irregular interspaces, among them, enclosed completely by the nuclear membrane. The uneven contour of the head portion was evident in scanning electron microscopic aspects of the spermatozoon. No acrosomal structure was detected in the head. At the base of the midpiece, generally 8 to 10 mitochondria (0.5[micro]m average diameter) appeared to be closely aggregated without fusion with each other. Caudal to the mitochondrial mass, a thin sheath of cytoplasm (about 1.2[micro]m in length) extended to surround the proximal portion of flagellum. The axoneme of flagellum (9+2 microtubules) and its plasma membrane was extended symmetrically into two ridges that were clearly observed in TEM (fig.4c). It is of considerable interest that in spite of the large number of sperms in the lumina of seminiferous lobules and in sperm ducts, head-to-head and head-to-tail agglutinations among sperms of fish caught from the reference site during spawning period (fig.4d) were rarely observed. Spermatogenesis was very rapid therefore sperms were present in the testes in non-spawning season, i.e. prior to the usual time of spawning.

Ultrastructural Changes of Sperms of Tilapia Fish Caught from Moderately Polluted Waters (Location II):

As an expected result, the sperms of fish collected during spawning period revealed variable degrees of deformation, including changes in head morphology. Also, the midpiece appeared highly deteriorated and its mitochondrial sheath showed definite signs of degeneration as it became separated from the axial filament with a wide and clear space. The mitochondria showed signs of disintegration and disorganization (fig.5a). Also, various degrees of degeneration of axoneme were noted. Loss of some of the peripheral and/or central microtubules was commonly observed, in addition to normal axonemal configurations. Higher incidences of sperms with neck vacuoles were evident in most animals (fig.5b). Sperm agglutinations were occasionally observed (fig.5c). The sperms were mainly bound to each other by their heads, although occasionally the head was bound to the tail of other spermatozoa.

[FIGURE 4 OMITTED]

Morphologically changed spermatozoa were frequently observed in males collected during non-spawning period. The head of the sperm was often swollen, enlarged and had abnormal patches of chromatin material (fig.6a). Sperms with abnormally forked nuclei and degenerating mitochondria in the middle piece were also noticed in these specimens (fig.6b). Furthermore, altered axonemal structure was frequently observed. Microtubules of the axial filament displayed abnormal arrangements such as 7+1, 9+0, etc. (fig.6a). In addition, SEM preparations of many samples revealed that there were spermatozoa, which seemed to be agglutinated, with some portions of their heads bound mutually (fig.6c). Generally, the agglutinated spermatozoa were different morphologically from normal spermatozoa of reference fish.

Ultrastructural Changes of Sperms of Tilapia Fish Caught from Highly Polluted Waters (Location III):

A remarkable increase in sperm abnormalities in fish collected during the active reproductive period was detected in location III as compared to the reference site. Among the commonest head anomalies was the deformed head shape (fig.7a) and abnormal chromatin condensation. Malformed sperms with double heads were noticed in few specimens (fig.7b). On the other hand, the most obvious alterations observed were the mitochondrial changes, in number, arrangement and internal structure of cristae. These mitochondrial abnormalities were mostly accompanied by defects in the structure of axoneme, in the form of missing peripheral microtubules or, more commonly, missing central microtubules. Complete degeneration of tail was also observed. Thus, in SEM preparations, sperms with biflagellate tail were observed (fig.7c). This abnormality occurred occasionally. Moreover, deep sutures in plasma membranes of sperm heads were frequently noted (fig.7d). Sperm agglutinations were observed as well.

[FIGURE 5 OMITTED]

Deformed sperms were frequently observed in specimens collected during non-spawning season. Morphologic assessment of sperms revealed a high incidence of abnormal head shape, degenerated middle piece, undulating plasma membrane and deformed axoneme structure (figs.8a &b). These changes were usually accompanied by nuclear distortion and abnormal chromatin condensation.

[FIGURE 6 OMITTED]

[FIGURE 7 OMITTED]

[FIGURE 8 OMITTED]

Discussion

The fisheries economy of Egypt depends principally on the Egyptian lakes. One of these lakes, namely Lake Mariut, has suffered much from intensive pollution in recent years, although at one time it was a highly productive lake. The untreated industrial and sewage wastes increased the heavy metals in the water and sediments of Lake Mariut (El-Rayis and Saad, 1990). When fish are exposed to elevated levels of metals in aquatic ecosystems, they tend to accumulate these metals up directly from their environment through the food web (Adham et al., 1999). It is assumed that most metals are taken up in the ionic form (Zamuda and Sunda, 1982). Transports of metals in the fish occur through the blood where ions are usually bound to proteins (High et al., 1997). The metals are thus brought into contact with the organs and tissues of the fish and consequently accumulated to a different extent in different organs/tissues (Ravera, 2001). It is of considerable interest that all the metals taken up are not accumulated because fish have the ability to regulate their body metal concentration to a certain extent (Heath, 1987). The amount of a metal bioaccumulated is influenced by various factors, leading to differences between different individuals, species, seasons and sites (Kotze et al., 1999).

In the present study, X-ray spectra of sperms of O. niloticus from mildly and heavily polluted sites of Lake Mariut demonstrated higher proportions of heavy metals, in particular Zn, Cu and Hg comparable to the less polluted reference site. Despite of the occasional occurrence of minor levels of Hg in location I of the lake, it is considered the least polluted site. A number of field studies on fish indicate the presence of heavy metals in their gonads (Maletin et al., 1991; Chen and Chen, 2001). It is of considerable interest that the present results represent the first report concerning the incorporation of toxic metals, especially Cu, Zn and Hg in sperms of O. niloticus from Lake Mariut. The accumulation of Cu and Zn in the reproductive organs of some fish species has been reported by Windom et al. (1973), who found that the reproductive organs of such fish species accumulated elevated levels of Zn in comparison with the other organs. Shakweer and Abbas (1996) similarly demonstrated higher concentrations of Zn in the gonads of O. niloticus than that of the liver collected from Lake Edku, Egypt. They also showed that liver accumulated high concentrations of Cu when compared with concentrations in the flesh or gonads. Wong et al. (1999) confirmed that the order of Cu concentration in both fingerlings and subadults of the silver sea bream after long term exposure to Cu were: intestine > liver > gonad > gills > skin > muscle.

Copper and zinc are two metals occurring in elevated levels in the water of Lake Mariut. This is of concern since they are tentatively classified as highly toxic metals by Hellawell (1986) and are bioaccumulated in aquatic organisms (Adham et al., 1999). Carino and Cruz (1990) suggested that higher concentrations of zinc impair reproductive success and survival potential of Tilapia nilotica in zinc-contaminated ecosystems. On the other hand, very little data have been published on the concentrations of toxic heavy metals present in sperm cells. There are several hypotheses that suggest how reduced male fertility may result from incorporation of heavy metals into sperm chromatin (Casswell et al., 1987). One hypothesis suggests that these metals, which bind tightly to free thiols, replace or compete with the zinc, forming more stable metal-SH bonds that ultimately prevent proper decondensation of sperm chromatin following fertilization (Johansson and Pellicciari, 1968). An alternate hypothesis is that the presence of tightly bound metal may prevent normal disulfide bond formation within and among protamines during the final stages of sperm maturation. The disruption of this process, or the sequestration of free thiols that may be required for protamine removal from DNA after fertilization, could lead to dominant lethal mutations (Shelby et al., 1986).

The spermatozoa of some marine animals have distinctly high zinc content. Martin et al. (1973) reported that the spermatozoa of Octopus dofleini martini contained 18,509,500 [micro]g zinc/g dry weights. This result agrees to a good extent with the present study, which shows high proportions of zinc in sperms of tilapia from the reference site. On the other hand, the enhanced levels of Zn in polluted locations may highlight the beneficial role of zinc in providing protection against lead and mercury-induced testicular toxicity (Afonne et al., 2000, 2002).

Although Pb levels in Lake Mariut were higher than permissible (Shakweer and Abbas, 1997), decreased proportions of Pb were observed in sperms of fish from all locations selected. This agrees with the results of Marchlewicz (1994), who observed no deposits of lead either in germinal cells or Sertoli cells of mature male rats after long-term exposure to lead acetate. Furthermore, the results of X-ray microanalysis indicated high levels of toxic Hg in sperms of fish inhabiting locations II and III. Ernst and Lauritsen (1991) reported that Hg compounds decreased the number of motile spermatozoa. In general, heavy metals, especially Hg, is well known to reduce sperm motility in fish (Kime et al., 1996; Rurangwa et al., 1998). Mercury is known to bind strongly to sulfhydryl groups and it becomes more toxic during embryogenesis (Nriagu, 1979). The major processes affected might include cellular differentiation, proliferation, and inhibition of mitotic cell division (Friberg and Vortal, 1972) to produce chromosomal aberrations, polyploidy and somatic mutation with gonadal accumulation, all of which substantially impair development.

Our findings of a complete lack of Cd in sperms of fish from the polluted locations are consistent with the results of Bench et al. (1999) who reported that mice subjected to long-term intraperitoneal Cd exposure did not incorporate Cd into their sperm chromatin. In addition, Betka and Callard (1999) showed that intracellular 109Cd in the testis of the shark Squalus acanthias was stage dependent resulting in a 3-to-5 fold gradient: germinal zone > premeiotic > meiotic > postmeiotic stages. Cd in semen plasma has also been associated with sperm motility (Xu et al., 1993).

Spermatozoa of the tilapia examined showed spherical head without acrosome. The middle piece elongated into a rather long cytoplasmic sheath surrounding the proximal part of the flagellum. In the middle piece, 8-10 mitochondria accumulated in its proximal region without any fusion. Baccetti et al. (1984), reported that the flagellum was rather short in comparison with that of the cyprinid fishes and two lateral ridges formed by the plasma membrane extended from the flagellum. The occurrence of similar ridges in spermatozoon tails has been demonstrated in various teleost fishes such as rainbow trout Salmo gairdneri (Billard, 1983). These are considered to correspond to the undulating membrane occurring in spermatozoa of various animal species.

Fish spermatozoa exhibit a great variety of shapes and structures, reflecting, to a large degree, reproductive patterns. In teleosts, the only common aspect is the absence of an acrosome, which has been related to the presence of a micropyle in the egg (Riehl, 1997). Energy required by the spermatozoon is supplied by mitochondria, which consume the endogenous substrate in the middle piece (Baccetti and Afzelius, 1976). Internal fertilization can therefore be related to a long middle piece and external fertilization to a short one (Mattei, 1991). The spermatozoon of O. niloticus is of the type 1 anacrosomal "aquasperm" as defined by Lou and Takahashi (1989), which is typical of species with external fertilization, i.e., it has a short head, few mitochondria, and barely differentiated middle piece.

Since details of spermiogenesis may vary considerably even amongst closely related teleost species (Grier, 1981), the morphology of spermatozoa has also become of interest from a taxonomic point of view in recent years (Jamieson and Leung, 1991). In particular head morphology, nuclear invagination, and number of mitochondria differ considerably among different teleosts (Lahnsteiner and Patzner, 1996). The differences in the shape of spermatozoa, the number of mitochondria, the arrangement of centrioles, as well as the occurrence of lateral flagellar ribbons among teleost species may have consequences in the swimming behavior with respect to sperm velocity, swimming types, and the head detachment (Lahnsteiner and Patzner, 1995). In the present investigation, ultrastructural diagnosis identified high incidences of various sperm morphological abnormalities in the polluted groups as compared to those examined in the reference site. TEM and SEM were used to make a detailed qualitative analysis and intracellular detailed features of the structure of spermatozoa of fish collected from locations II and III. In fact, sperm morphology assays provide a quantitative method for locating genetic damage in male germ cell lines as reported previously by Soares et al. (1979). Incidences of structurally abnormal sperm shapes are reported to be genetically controlled by numerous autosomal and sex-linked genes (Krazanowska, 1976). Thus, the obvious relative decrease in the density of chromatin condensation seen in sperms of fish from polluted locations might be associated with chromosomal abnormalities, which was reported to decrease fertility potential (Abramsson, et al. 1982). In accordance, Acharya et al. (2003) reported chromosomal aberrations, increased sperm head abnormalities and decreased sperm count profile in lead-treated mice.

In some affected specimens, the mitochondrial sheath in the midpiece became separated from axial filament and showed signs of disintegration and disorganization. Similarly, Au et al. (2000) indicated that exposure to toxicants changed the size and shape of the midpiece of spermatozoa of mussel and sea urchin, and this might affect the balance of spermatozoa in their swimming. They also reported disorganization of mitochondrial membranes and cristae and disrupted ATP supply for sperm movement. Moreover, the present study showed that mitochondrial abnormalities were accompanied by flagellar degeneration in the polluted samples. Axonemal alterations included missing of some of the peripheral and more commonly the central microtubules. Similarly, Au et al. (2001) reported cytological distortion of sperm tails and mitochondrial cristae deformation in sperm cells of sea urchin chronically exposed to cadmium. Similarly, biflagellate sperms commonly observed in fish from location III were also detected with lack of dynein arms in humans, by Baccetti et al. (1993).

In most aquatic species with external fertilization, spermatozoa are immotile in the testis and become motile at release into the external medium (Perchec et al., 1995). At the initiation of sperm motility, ATP synthesized in the midpiece mitochondria, is hydrolyzed by dynein ATPase, and its contents decrease coupled to the movement, as described in various animals, e.g. trout (Christen et al., 1987) and carp (Perchec et al., 1995). On the other hand, Hancock and de Krester (1992) found that a reduction of axonemal components caused lowered motility. This was correlated with chromosomal abnormalities. Among those genetically determined congenital defects in cilia and flagella is the 'immotile cilia syndrome'. It is caused by the lack of dynein arms that is normally link microtubular doublets (Afzelius et al., 1975). They also suggested a genetic inability of these individuals to synthesize the protein 'dynein'.

The present study demonstrated occasional occurrence of sperm agglutinations in the polluted groups. The spermatozoa were agglutinated in a head-to-head manner, which invariably makes them fail to fertilize the egg as reported by Mochida and Takahashi 1993. It seems likely that the sperm agglutination results from an influence of autologous IgM, which comes to penetrate the efferent duct as autoimmune responses in the testis progress. Lou and Takahashi (1991) showed that in the Nile tilapia, autoantigens existed locally on the head of spermatozoa as determined by immunocytochemical method using anti-sperm autoantibody purified from the serum of immunized fish.

The micromorphological changes in spermatozoa presented in the current study should be included as a model for predicting environmental hazards. In addition, such investigations might provide a more insight into how the toxic metals can alter the natural biology of feral fish and their ability to produce new offspring. This is an alarm bell to the governmental authorities indicating that the fish populations in Mariut lagoon need more protection.

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Nabila E. Abdelmeguid *, Abdel-Moneim M. Kheirallah, Cecil A. Matta and Ashraf M. Abdel-Moneim

Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt

* Correspondence to: Nabila E. Abdelmeguid E-mail address: dr_nabila_elsayed2000@Yahoo.com
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Author:Abdelmeguid, Nabila E.; Kheirallah, Abdel-Moneim M.; Matta, Cecil A.; Abdel-Moneim, Ashraf M.
Publication:International Journal of Applied Environmental Sciences
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Geographic Code:7EGYP
Date:Jun 1, 2007
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