Histopathological Effects of Mercury on Male Gonad and Sperm of Tropical Fish Gymnotus carapo in vitro

Hg is toxic metal mostly due to adverse effects on structure and function of tissues and organs in humans and animals. Male reproductive systems of fish species are also sensitive to Hg action. However, the histological alterations in tropical fish testis are less known and little information is available concerning the underlying mechanisms of metal pathogenesis in reproductive functions. Further investigations dealing with Hg direct effects on tissue and organs of tropical species are a need. The present study investigated HgCl2 toxic effects in testes and sperms of tropical fish Gymnotus carapo. The histopathology, germ cell structure and number were analysed to elucidate the pathological process during exposure to increasing metal concentrations (1 μM 30 μM). Fishes exposed to 20μM and 30μM reached testicular Hg concentrations of 5.1 μg.g and 5.2 μg.g, respectively. No significant alterations in gonadosomatic index (GSI) occurred between control, and Hg exposed fishes. Untreated fishes showed characteristic organization of testicular tissue, with germ epithelium organized in cysts where spermatogenesis occurs. Germ cells and spermatozoa are seen an inner the cysts. HgCl2 induced severe damages characterized by complete disorganization of seminiferous lobules, proliferation of interstitial tissue, congestion of blood vessels, reduction of germ cells and sperm aggregation. Exposed fishes showed a decrease in the sperm's number. Initial reduction of a sperm's number (36,8%) was observed after 20 μM/24h treatment and subsequent decrease (48,7%) was obse rved after 20μM/96h. Hg (20μM) also altered sperm morphology in 24h and 96h where sperm head abnormalities were present. In conclusion, the present study showed HgCl2 progressive damages in testicular tissue, sperm count and morphology of tropical species Gymnotus carapo. The effects in testicular tissues were observed since low Hg concentrations. These results are important to establish a direct correlation between the mercury accumulation and severity of lesions.


Introduction
Mercury (Hg) is a toxic environment pollutant that induces several adverse effects in many tissues and organs of humans and animals.Male reproductive system is also sensitive to Hg (Boujbiha et al.,200).In fishes, Hg can inhibit gametogenesis, induce testicular atrophy, and impair individual reproduction.Despite some of Hginduced damages are known, there is still less information about the metal accumulation on tissue and its relationship with ultrastructure disorganization of fish testis, especially for tropical species.
Sperms are also useful in assessing the Hg impacts on male reproductive system.In rodents, acute contamination decreased reproductive quality of gametes, where sperm morphology, count, motility and viability were affected.Further investigations in such parameters are needed for different fish species since they may dramatically decrease sperm performance in aquatic environment affecting fertilization success and alter fish populations.
Gymnotus carapo (tuvira) is a tropical freshwater In the present study, the HgCl 2 toxic effects in testes and sperms of a teleost Gymnotus carapo were observed, elucidating the pathological process during in vitro exposure to increasing concentrations (1 µM -30 µM).

Fish contamination
Gymnotus carapo specimens (n=116) used in the present study were all male in same sexual maturity stage, obtained from Cima Lake, northern of Rio de Janeiro state (21º 46' S e 41º 31' W).Heavy metal's distribution in sediments and biota of Cima Lake has already been described and characterized as area with low levels of metal pollution (Ferreira et al., 2003).
For each HgCl 2 contamination, 6 male adult fish were selected, four exposed to HgCl 2 concentrations (5 µM/10 µ M/ 20 µ M/ 30µM) and two kept as the control group.The fishes were exposed at different exposure times (24 h/48 h/72 h/96 h) and control fishes were always dissected in 24h and 96h of exposure.In order to increase the sampling for chemical and histological analysis the procedure was repeated consecutive times for each time/concentration tested.
Contamination was achieved by intraperitoneal injection of HgCl 2 solution while the control group was injected with phosphate buffer solution.To avoid differences in treatment, all fish used for this study were of similar size (length: 32 ±1 cm/ weight: 125.8 ±17.8g).After each exposure time, the specimens were measured, weighed and dissected to obtain testis for further analysis.

Hg chemical determination in the testes
For mercury detection, testis samples from control and contaminated fishes followed strong acid digestion according to methodology described by Bastos et al. (1998).All the Hg determinations were performed by spectroscopy of atomic emission method using the equipment ICP-AES (Varian, Liberty II models) with cold vapor accessory (VGA-77).The method limit detection was calculated according to Skoog and Leary (1992) as being of 0.23 µg.g -1 .

Analysis of testis morphology
Samples of control and Hg exposed testis were fixed in 10% neutrally buffered formalin for 24 hours.The samples were then dehydrated in a progressive series of alcohol, cleared in xylene, embedded in paraffin.The samples sectioned (5 µm) and stained with hematoxylin and eosin (H&E) for examination by light microscopy.Samples of the testis (approximately 1 mm 3 ) were also fixed in formaldehyde 4%, glutaraldehyde 2.5%, cacodylate buffer 0.1 M, sucrose 5%, calcium chloride 5 mM and post-fixed (1:1) in osmium teroxide 1% and potassium ferricyanide 0.8%, dehydrated with acetone, embedded in Epon®.Semithin slices (0.4µm) were obtained using an ultramicrotome Reichercuts Leica.Slices stained with toluidine blue (1%) were observed with light microscopy.

Sperm Sampling
Following the damages observed in testis, Hg effects in sperm were evaluated in 20 µM concentration after 24h and 72h.Testis from control (n=4), Hg exposed to 20 µM/24h (n=2) and 20 µ M/72h (n=2) were minced with anatomic scissors in 2 mL of cacodylate buffer 0.1M (pH 7.2) for 5 minutes at room temperature.After dilution, the sperm's number was counted in hemocytometer under light microscopy using phase contrast at x400 magnification.

Statistic analysis
All the values are expressed as mean±SD.Significant differences were determined with Graph-prism v.4 Software (GraphPad Software, Inc. CA, USA).Two-way analysis of variance followed by Bonferroni test was performed for Hg concentration's data and one-way analysis of variance followed by Tukey test was used for sperm data.Differences were considered significant when p < 0.05.

Results and Discussion
External investigation was performed in each fish before and after the execution of the experiments.Control and Hg treated fishes showed to be healthy according to conditions of their gills, eyes and scales.Internal organs, as liver, kidney and especially the testis did no present macroscopic anatomic alteration.
As described in figure 1, testicular tissue in untreated fishes showed characteristic organization of cysts arrangement where spermatogenesis occurs (Fig 1a).Inner the cysts, germ cells (Fig 1b, c) in different stages of differentiation are distributed as: primary (SPGI) and secondary (SPGII) spermatogonia, primary (SPCI) and secondary (SPCI) spermatocytes and spermatids (SPD).Hg chemical analysis revealed that treated fishes with 20µM and 30µM reached highest testicular concentrations of 5.1 µg.g -1 and 5.2 µg.g -1 , respectively (Table 1).Hg concentrations in control and treated with 5µm, 10 µm were bellow the detection limits of the method (Table 3).These results are in agreement with severe damages observed by histopathological analysis and indicate that even low Hg doses can induce morphological alterations in testis.
The present study also enhances the knowledge about progressive accumulation of HgCl 2 and adverse effects in testicular tissue structure.In important point, this study showed that histology damages started at concentrations not detectable by the limit of accumulation method (< detection limit).These results are important to establish a direct correlation between the mercury accumulation and the severity of tissue lesions (Boujbiha et al. 2009).
Hg induced severe damages in testis arrangement, affecting the germ cells that are involved in spermatogenesis process.Therefore, investigations in sperms were also performed for overall evaluation of Hg effects in male gonad.

Conclusion
The present study showed HgCl 2 progressive damages in testicular pathology, sperm count and morphology of tropical fish species Gymnotus carapo.These results are important to establish a direct correlation between the Hg accumulation and the severity of lesions since the testis analysis was performed since Hg concentrations bellow limit of method detection until higher doses that induced severe damages.Moreover, this work enhances the data about Hg toxicological effect in fishes from tropical regions.
These cells undergo a number of cell divisions until sperm formation inner the cysts (arrowheads) (Fig 1b, c).Between the cysts interstitial tissue (it) is present being composed by Leydig cells, blood/lymphatic vessels and connective tissue (Fig 1b).Hg induced changes in treated fishes for all concentrations administrated and the effects become more severe with increase of dose/time.Hg treatment induced complete disorganization in cysts arrangement (Fig 2) with congestion of blood vessels (Fig 2b) and proliferation of interstitial tissue (Fig 2b).Severe damages were observed in higher concentrations (20 µM and 30 µM) as reduction of germ cells (Fig 2d), marked variations of cyst size (Fig 2c), interstitial and lobular disintegration (Fig 2d), sperm aggregation (Fig 2c).
nd: not detected by equipment since concentrations bellow limit of method detection.The letters a, b indicate the indicate groups at means that are significant different at 5% of significance level.

Fig. 3 .
Fig. 3. Effect of mercury on sperm count after 20 µM treatment for different exposure times (24h and 96h).The letters a, b and c indicate that are significant different at 5% of significance level.