Impact of Environmental Contaminants on the Testes of Oreochromis niloticus with Special Reference to Ultrastructure of Spermatozoa in Lake Manzala (Egypt)

Copyright: © 2012 Shalaby FM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Impact of Environmental Contaminants on the Testes of Oreochromis niloticus with Special Reference to Ultrastructure of Spermatozoa in Lake Manzala (Egypt)


Introduction
The importance of Nile tilapia, Oreochromis niloticus in aqua cultures is due to its spreading in most countries of the world [1]. It breads in captivity and wide variety of water conditions [2]. Lake Manzalah (LM) is one of the most important lakes in Egypt that provides more than 70% of the total fishery of the country [3] and considered as a highly polluted with toxic heavy metals and pesticides due to progressive increase of industrial and agricultural drainage, as well as sewage out fall from the surrounding governorates [4]. The mean values of some heavy metals (Cd, Pb, Fe, Cr and Mn) recorded in LM were higher than the world permissible levels [5] and according to the Egyptian laws [6]. Such heavy metals are not lethal to fish, but concentrated in their flesh and create a hazard to the consumer [7,8]. Cd, Pb, Zn, and Cr are considered the main causes of pollution in aquatic ecosystem [9]. The serious effect of these metals is derived from their persistence, toxicity and bioaccumulation, and consequently exert dangerous problem to man [10]. Experimental studies on heavy metals proved that they could impair the respiratory functions of the gills; consequently death in fish is probably caused by tissue hypoxia [11]. Metals affect not only the fish morphology, but also all biological activities [12]. Among the heavy metals; Cd and Pb are the serious heavy metals that produce histopathological alterations including liver damage [13], respiratory dysfunctions [14], testicular and ovarian alterations [15][16][17]. These pathological alterations were primarily attributed to damage of cell membranes which allows higher uptake of Cd and Pb and thus the injury extends to more critical targets [18]. Many studies focused on bioaccumulation of heavy metals in fish and biological studies. Thus we aimed in this work to study the testicular histopathological alterations of Oreochromis niloticus using light microscope and the mature sperm using TEM.

Samples collection
Samples of water and 48 specimen of Nile tilapia, Oreochromis niloticus about 14-20 cm in length and 120-200 g in weight were collected from two areas; Demitta branch of RN at Mansoura city as a control site and LM as a polluted area during the spawning season from March-June and transported to the laboratory in tanks provided with oxygen pump. The fish were weighed, dissected and the testes were removed carefully wiped with filter paper and weighed. The gonadosomatic index (GSI) was then calculated for each fish according to the following formula:-GSI=Gonad weight (g)/total body weight (g) ×100 [19].

Water analysis
According to the measurement of Saeed and Shaker and Elewa [20,21] (Tables 1,2) . The samples of water from the two studied areas were analyzed by using a flame atomic absorption spectrophotometer (Model 2380, Perkin-Elmer) at the chemistry department, Faculty of Sciences, Mansoura University, Egypt. *PL: permissible limits according to USEPA (1986)

Light microscopy
The testes were cut into small pieces and fixed in Bouin's solution, dehydrated in ascending grades of ethyl alcohol, cleared in xylene and mounted in molten parablast at 58-62°C. Specimens were sectioned 5 µm by microtome, stained with Hematoxylin & Eosin [22] and examined using light microscope.

Electron microscopy
Testicular tissues prepared for TEM were cut into very small pieces and immediately fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for two hours. After rinsing in 0.1 M cacodylate buffer, samples were post fixed in a buffered solution of 1% osmium tetroxide at 4°C for 1.5 hour. This was followed by dehydration in ascending grades of ethyl alcohol and embedded in epoxy-resin [23]. Ultrathin sections were obtained with a diamond knife on a LKB microtome and mounted on formvar-coated grids, stained with uranyl acetate and lead citrate for 30 mins and examined using a Joel 1200 EXII at the Electron Microscope unit in Alexandria University, Egypt. Then these sections were examined and photographed with an Olympus CX41 digital camera.

Heavy metals in water samples
The water analysis of RN at Mansoura city showed slight pollution in comparison with that of LM. This study showed that the mean values of heavy metals especially cadmium (Cd) and lead (Pb) were higher than those of RN, whereas these heavy metals were at the permissible levels during spawning season (Table 3).

Gonadosomatic index
The mean values of male gonado-somatic index (GSI) were increased gradually from March -June and showed that the mean percentage of GSI was ranged between 0.34 and 1.17 for RN and between 0.14 and 0.91 for LM (Table 3). These results indicated that the minimum value of mean percentage of GSI in both RN and LM was observed in March, while maximum value was in June (Table 3).

Histological examination
Histological examination of the testes of O. niloticus from both RN and LM in the studied period showed that each testis is lobular in shape and formed of many seminiferous tubules. Each one contains the germ cells that arranged in clusters and occurs in several places along the length of each tubule. During the spawning period, all spermatogenic stages were observed. Depending upon the morphology and size of the nucleus these stages could be described.
In march: In the specimens of RN, the seminiferous tubules showed intensive clusters of spermatogenic cells of large spherical spermatogonia containing large rounded central nuclei with distinct nucleoli. The primary spermatocytes possessed a darkly stained nucleus, whereas that of secondary spermatocytes was smaller than the primary spermatocytes with clump chromatin material. The smaller deeply stained spermatids and reduced number of sperms were also observed ( Figure 1). The test is collected from specimen of LM showed several histological alternations such as degenerative changes, vacuolation in some parts of seminiferous tubules and the number of different spermatogenic cells were fewer than those of RN at the same time of collection ( Figure 2).

In april:
The seminiferous tubules of specimens of RN showed obvious packed great number of spermatogenic cells especially spermatids ( Figure 3). However, in comparison with those of LM there were a dispersion, degeneration and decrease in number of the spermatogenic cells of the seminiferous tubules, as well as, vacuolization in interstitial tissue (Figure 4-6).
In may and june: The seminiferous tubules of specimens from RN were packed with sperm masses but the tail portions were rarely visible     by light microscopic. While the specimens from LM showed sever decreases in the total number of sperm cells with the presence of some empty seminiferous tubules (Figures 7-13).

Ultrastructure observations
The TEM examination of specimens collected from RN showed late stages of spermatids and mature spermatozoa, with densely chromatin nuclei and extensive mitochondria ( Figure 14). However, the specimens collected from LM reveals different early and late stages of spermatids and spermatozoa. The early stage of spermatids possesses spherical small round nuclei with electron dense & prominent chromatin patches interconnected by the cytoplasmic bridge ( Figure  15). The middle stages of spermatids are characterized by electron dense nuclei with prominent batches of heterochromatin, appear the implantation fossa, both centrioles at the basal pole of the nucleus & the distal centriole starts to form the flagellum. Some mitochondria are located near the centrioles and aggregated irregularly on both sides of the mid piece around the flagellum (Figure 15). In late stage of spermatids, the nucleus becomes indented and a nuclear fossa is formed, the diplosome flagellar axis become perpendicular to the base of the nucleus and the proximal & distal centrioles are connected with electron dense filaments and located within the nuclear fossa, (Figure  16,17). Moreover, specimens of LM showed that, the cytoplasm of spermatid contains many vacuoles and few numbers of large sized mitochondria comparing to those from RN ( Figure 16 and Figure 17).
The mature spermatozoon of RN is simple elongated cell composed of a head, short mid piece and a tail or flagellum. The head has no acrosome and consists of nucleus that has very dense homogenous chromatin. The head becomes completely surrounded by the cytoplasm, with dispersed large population of mitochondria ( Figure 18). The neck region shows well developed proximal and distal centrioles in the implantation fossa (Figure 18,19). The proximal centriole, showed a

Discussion
Heavy metals, especially Cd and Pb are considered the most serious pollutants; and if present in high concentrations could have a negative effect not only on the river system, but also on fish population [24]. The values of heavy metals concentrations at RN were fewer than that for LM and differ from month to another. The heavy metals uptake by fish depends upon exposure time, concentration, temperature and diet. Among heavy metals Cd and Pb promote an early oxidative stress and lead to the development of serious pathological conditions because of their long retention in several tissues [18,25,26]. The dangerous effect     Spermatozoa of specimens of LM showed the same main features of those of RN, however, the nucleus was more or less irregular in shape with irregularly dense chromatin clumps of electron-dense material and numerous electron-lucent areas ( Figure 19). The two lateral fins of sperms of LM were shorter, swelling and degraded than those of RN. There are also degenerative changes in the plasmalema of the lateral fins and the microtubule construction ( Figure 21).
The gonado-somatic index is generally used to determine the maturity stage of fish and subsequently degree of gonadal development. The increase of gonadosomatic index is related with the percentages of ripe females and males towards the spawning season [28,29]. In the present investigation, the data obtained showed that the values of male gonado-somatic index of Oreochromis niloticus were higher at RN than that in LM giving an idea about the exert effect of high level of pollution in LM. Such effect was reflected by the decrease of sperm account in the ripe testes during the spawning season as a result of high heavy metal concentration [30].
Many studies showed that the spermatid and sperm increase in number toward the end of spawning season [31,32]. The specimens of LM showed decrease in spermatogenesis, disruption of spermatogenic cells and reduction in the spermatids and spermatozoa. This implies the heavy metals bad effects that can disrupt and impair reproduction in       [17,33]. It was noticed that increasing histopathological alterations is associated with increasing the concentration of Cd and Pb pollution that causing severe testicular atrophy with arrested spermatogenesis, necrotic spermatogenic cells, and vacuolization in the interstitial tissue [34]. Also, animals that exposed to high doses of Cd and Pb exhibits severe testicular atrophy [35,36] as well as biochemical processes [37,38]. The marked inhibition in the process of spermatogenesis due to Cd and Pb pollution simulates with that described by Mousa and Mousa [24] who found a decline in the gonadal activity as reflected by decreasing of sperm amount in the ripe testis. The high levels of heavy metals may interfere with the release of hormones and disturb the feedback mechanisms of gonadal cycle) [39].
The effects of pollution on spermiogenesis of O. niloticus were reflected on the modifications in nucleus, flagellum, spermatids, distribution and organization of the cytoplasmic organelles and implantation fossa of spermatids [40]. Ultrastructural study of mature sperm of O. niloticus inhabited RN showed the normal structure of spermatozoa that consists of head with no acrosomes, an ovoid nucleus, short middle piece and bifin (lateral fins) flagellum with a classical axoneme pattern (9+2 arrangement), these results coincide with many authors [41][42][43][44]. The absence of acrosomes in sperms is compensated by presence of micropyle in the oocytes that allows the sperm penetration and has a role in the prevention of polyspermy [45]. On the other hand, the ultrastructural morphology of sperm of O. niloticus collected from LM displays some alternations including nuclear distortion changes in head morphology, incomplete chromatin condensation, and shortage of bilateral fins of the flagellum, wavy and ruptured plasma membrane. As well as, the presence of some vacuoles and the relative decreases in the density of chromatin condensation might be associated with chromosomal abnormalities, which is associated with decrease fertility potential [46,47]. Moreover, LM fish's spermatozoa revealed the presence of few numbers and large sized mitochondria in front of the proximal centriole toward the lateral aspects of the nucleus. The enlarged size of the mitochondria appeared to be a considerable hypertrophy and a sign of acclimatization of fish in these samples rather than RN [48,49]. The bilateral fins of the spermatozoa that formed by the plasma membrane extended from the flagellum is very important for perfect motility and exhibits higher fertilization capacity [40]. The present results indicated that the samples of RN exhibited more differentiation and development of the flagellum than that of LM samples. The deformities change of the sperm's flagellum of O. niloticus of LM may lead to decreasing the capacity of sperm motility and reducing its fertilization capacity, and consequently lead to failure of reproduction [50,51]. Most probably these alterations in sperms may result from environmental hazards increasing the levels of Cd and Pb. The changes in sperms structure as a result of environmental hazards