Impact of TBT on the vitellogenesis and sex hormones in freshwater prawn Macrobrachium rosenbergii (De Man, 1879)

Freshwater ecosystem is under increasing threat due to rapid expanding population and the subsequent modernization process resulted in invisible exploitation of natural resources leading to pollution. Rivers are very vulnerable towards pollution, since the industrial, domestic and farm effluents are directly released into them. During the past few decades, rising trends of population explosion, development of modern technology, industrialization and dramatic increase in the production and consumption of large variety of new synthetic chemicals were reported. These kinds of modernization are the reason behind the enormous release of pollutants into aquatic environment [1]. Accumulation of industrial effluents and agricultural runoff in water bodies has become a major concern [2]. TBT is an ubiquitous persistent xenobiotic that can be found in freshwater, estuarine and marine ecosystem [3].

Organotin compounds, particularly TBT, have been reported to be strong endocrine disrupting compound (EDC). TBT is highly toxic to many aquatic organisms and is still detected in aquatic environments though it had banned in antifouling paints because of its usage as biocides in a variety of consumer and industrial products. The level of TBT in the aquatic environment is still cause of great concern [4]. Effects of TBT have been investigated in several aquatic organisms, including algae [5] and crustaceans [6]. Champ [7] reported that TBT inhibits growth, reproduction [8, 9] and sexual differentiation in fishes [10]. In our previous study, we documented the inhibition of organogenesis as well as embryonic development in M. rosenbergii due to TBT toxicity [11].

Histological analysis appears to be a very sensitive parameter and is crucial in determining cellular changes that may occur in target organs [12]. Besides, biochemical parameters are the best indicators of stress situations caused by xenobiotics [1]. Vitellogenesis is a biomarker of reproductive disruption by xenobiotics [13]. As evidence, a wide range of agrochemicals, industrial and municipal contaminations can decrease gonadal development and steroid levels [14]. Sex hormones derived from the gonads play crucial roles in sexual differentiation, maturation and behavior in vertebrates [15]. It is well known that 17β-estradiol once secreted into the circulation, stimulates the hepatic production of vitellogenin, necessary for oocyte maturation [16]. Some studies have additionally assessed the impact of xenobiotics on endogenous steroid levels, which may in turn be an indication of altered steroid synthesis and metabolism [17].

Extensive literatures dealing with the adverse impact of TBT in molluscs but in contrast only a few articles have addressed the effects of TBT on crustaceans. With reference to TBT toxicity, not much information is available on freshwater organisms especially on the commercially important species M. rosenbergii[11]. Therefore, the present study was conducted to examine the impact of TBT on vitellogenesis and sex hormones in freshwater female prawn, Macrobrachium rosenbergii.

In the present study the impact of TBT on vitellogenesis in the adult freshwater female prawn M. rosenbergii was studied with the analysis of survival rate, growth as a measure of body weight, GSI, HSI, histological, immunofluorescence, biochemical changes and quantification of vitellogenin, vitellin content and female specific hormones in different reproductive tissues of both control and TBT treated prawns.

Survival rate and bodyweight

The maximum survival rate of 98.0 ± 1.05% was obtained in the control group. However, in the TBT treated groups, survival rate was decreased compared to control group. At 10 ng/L, 100 ng/Land 1000 ng/L, the survival rate was decreased to 95.0±1.03%, 90.0±2.01% and 84.0±1.04% respectively after 90 days of exposure (Figure 1).

The body weight of control prawns was recorded as 25.0 ±1.75 g at the end of the experiment. TBT treatment found to decrease the body weight of prawns compared to control. At10ng/L of TBT treatment, the body weight of prawns was recorded as 23.0 ± 1.21 g. Besides, the body weights remarkably reduced to 17.0±0.72 g and 16.3 ±0.43 g at 100 ng/l and 1000 ng/l TBT, respectively at the end of experiment (Figure 1). Statistical analysis indicated that the changes in survival rate and body weight of TBT treated groups differed significantly to that of control group (P < 0.05).

Assessment of reproductive activity

TBT had significantly reduced the GSI and HSI values in treated prawns. In control, the GSI and HSI values were recorded as 7.8 ±0.39% and 2.1 ±0.33% respectively. Whereas, the GSI and HSI values were steadily declined as the concentration of TBT increases. At higher concentration of 1000 ng/l TBT treated group, the GSI and HSI values were decreased drastically to 0.2±0.07% and 0.5 ±0.05% respectively after 90 days exposure (Figure 2).

Morphological variation of reproductive tissues

Morphological alterations have occurred in both hepatopancreas and ovary of TBT exposed prawns. Control prawns showed fully mature ovary representing late vitellogenic stage. At 10 ng/L TBT, the ovarian development proceeded up to early vitellogenic stage. In the intermediate concentration of 100 ng/L TBT, ovarian development was seen up to pre vitellogenic stage and thereafter the development was arrested. However, at higher concentration of 1000 ng/l TBT, the ovarian development was completely ceased and remained at spent stage. Moreover, hepatopancreas structure also distorted in all TBT treated groups. Gross morphology of both ovary and hepatopancreas also decreased apparently in TBT treated prawns compared to control (Figures 3A-D).

Cellular level changes in different reproductive tissues

Hepatopancreas of control prawn showed normal architecture of hepatopancreatic tubule, lumen and basement membrane thickness. However, on exposure to TBT (10 ng/l), the hepatopancreas exhibited swelling of hepatopancreatic tubule and abnormal lumen. Hepatopancreas of prawn treated with 100 ng/l TBT showed decrease in basement membrane thickness and disruption of hepatopancreatic tubules. At 1000 ng/l TBT, hepatopancreatic tubule size had reduced unusually and was seen with abnormal lumen. Besides disassociation of epithelial cells from the hepatopancreatic tubules was also observed (Figures 4A-D).

On the other hand, control prawn showed normal development of ovary with vitellogenic oocytes containing distinct ooplasm filled with yolk globules. The oocytes were enveloped by a row of characteristic follicle cells with prominent nucleus and nucleolus. At 10 ng/L TBT, the ovary showed previtellogenic oocytes and reduction in the yolk globules as well as disruption of follicle cells. At 100 ng/L TBT, ovary showed marked variation in the cellular architecture of oocytes such as fusion of developing oocytes and disassociation of follicle cells. At 1000 ng/L TBT, the ovary showed immature oocytes and reduction in size of yolk material (Figures 5A-D).

Identification of vitellin in ovary

The results of immunofluorescence study with specific antivitellin (primary antibody) and FITC conjugation (secondary antibody) clearly indicated the high amount of vitellin content as a measure of increased immunostaining with anti vitellin and FITC in control. Besides moderate to less immunostaining in the ovary of TBT (10 ng/L) treated prawn indicated less vitellin content. Interestingly, at 100 ng/L of TBT, ovary showed fusion and reduction of vitellin content as evident with immunostaining. At higher concentration of TBT (1000 ng/l), very low intensity of vitellin content was observed in the ovary (Figures 6A-D).

Quantification of vitellogenin and vitellin content

The results clearly indicated that vitellogenin and vitellin content decreased significantly from the exposure of TBT, compared to control. In control prawn, the vitellogenin content in hepatopancreas and hemolymph was recorded as 0.7±0.15 μg/g and 2.5±0.36 μg/mL respectively. Interestingly, at higher concentration of TBT (1000 ng/l), vitellogenin content reduced drastically in both hepatopancreas (0.3±0.14 μg/g) and hemolymph (0.50±0.32 μg/ml) after 90 days of exposure. On the other hand, vitellin content in the treated prawns remarkably decreased from 18.7±4.12 μg/g at 10 ng/l to 0.2±0.11 μg/g at 1000 ng/l TBT (Figure 7). The variation of vitellogenin and vitellin content in TBT treated groups differd significantly from that of control group (P < 0.05).

Quantification of sex hormones

17β-estradiol level in different reproductive tissues

In TBT treated prawns, 17β-estradiol level decreased in all reproductive tissues compared to control (Figure 8). In control prawn, 17β-estradiol levels in ovary, hemolymph and hepatopancreas was recorded as 60.5±2.50 pg/g, 58.0±2.10 pg/ml and 30.5±2.40 pg/g respectively. On exposure to TBT (10 ng/l), 17β-estradiol level reduced to 33.0±2.10 pg/g in ovary, 32.0±1.80 pg/ml in hemolymph and 20.6±1.70 pg/g in hepatopancreas. At 100 ng/L TBT, 17β-estradiol level in ovary, hemolymph and hepatopancreas amounted to 22.0±1.60 pg/g, 23.0±1.30 pg/ml and 17.0±1.50 pg/g, respectively. However, at higher concentration of TBT (1000 ng/l), 17β-estradiol level decreased significantly in ovary (16.0±1.30 pg/g), hemolymph (11.0±0.80 pg/ml) and hepatopancreas (8.0±0.60 pg/g). The variation of 17β-estradiol level in control and treated groups at higher concentration was significant (P<0.05).

Testosterone level in ovary

The level of testosterone gradually increased in the ovary of TBT treated groups (Figure 9). The testosterone level in the ovary of control prawns was recorded as 11.3±1.60 pg/g. The testosterone level showed a marginal increase of 12.1±0.70 pg/g and 12.9±0.50 pg/g at 10 ng/L and 100 ng/L respectively. At higher concentration of TBT (1000 ng/l), testosterone level increased to 13.2±0.30 pg/g in the ovary. The testosterone levels significantly increased in treated groups compared to control (P<0.05).

Our results clearly demonstrated that TBT had considerably reduced the survival rate as well as growth in terms of body weight in all TBT treated prawns compared to control. Similarly, Lignot et al. [18] reported that TBT had significantly reduced the survival rate in Penaeus japonicus. The weight of prawn decreased to 65.2-fold at higher concentration in TBT treated group. The developmental rate slowdown in all TBT treated groups and even lower concentrations had effect on growth in M. rosenbergii. Likewise, Laughlin et al. [19] also described the effect of TBT on growth in mud crab, Rhithropanopeus harrisii. Lobster larvae (Homarus americanus) exhibited decreased growth and increased mortality at 1 μg/L [20]. Similarly, in our previous study also reported that TBT had substantially reduced the growth rate in M. rosenbergii[11].

The present study clearly demonstrated that TBT had impaired the ovarian development as evidenced by the level of GSI, HSI indices and oocyte diameter in M. rosenbergii. Likewise, Zhang et al. [21] suggested that TBT can affect the GSI and HSI in female Sebastiscus marmoratus. The results revealed the dose dependent toxicity of TBT on the reproductive performance of M. rosenbergii. The higher concentration of TBT (1000 ng/l) had entirely arrested the ovarian development and led to spent stage in M. rosenbergii. Similar observation has been reported in freshwater prawn M. rosenbergii when exposed to cadmium [22]. Rodriguez et al. [23] also documented the impairment of ovarian development after exposure to cadmium in fiddler crab, Uca pugilator.

In crustaceans, the hepatopancreas are considered to function as a storage organ of organic nutrients in the form of protein, lipid and carbohydrate, which are mobilized during the reproductive cycle, in order to meet the specific requirements of maturing gonads [24]. After exposure to TBT, the hepatopancreas exhibited rumpling of basement membrane, abnormal lumen, disorganization of epithelial cells, disruption in the hepatopancreatic tubule and occurrence of more number of vacuoles. These cellular and structural damages in hepatopancreas of M. rosenbergii significantly affect the absorption, secretion, digestive functions and the production of precursor material for major yolk protein; vitellogenin synthesis. Likewise, Sreeram and Menon [25] reported that the function of hepatopancreas of Metapenaeus dobsoni affected by the exposure of petroleum hydrocarbons (PHC). Klobucar et al. [26] also found swelling and damage of cell membranes in digestive gland of snail Planorbis corneus exposed to polychlorinated biphenyls (PCB).

From the present study, it was obvious that, TBT had substantially decreased and retarded the sexual maturation in M. rosenbergii. It also affected the ovarian development, as indicated by the reduction in oocyte diameter, yolk globules, fusion of developing oocytes and disruption of follicle cells. Sex steroid hormones are synthesized by follicle cells in the ovaries of female fish [27]. These follicle cells are sensitive to environmental stressors. It has been demonstrated that some natural and xenobiotic chemicals induce apoptosis in follicle cells [28]. The results of the current study clearly revealed that the follicle cells had remarkably affected and this may cause impairment in the steroid synthesis in experimental prawns. In the ovary, 17β-estradiol is catalyzed by the steroid synthesizing enzymes, in particular the P450 aromatase. In teleosts, change in the activity and expression of P450 aromatase have been shown to drive changes in the ovarian production of 17β-estradiol during the reproductive cycle [29].

The above hypothesis was consistent to the present study as the inhibition of gonadal maturation associated with increased levels of testosterone and decreased levels of 17β-estradiol in the ovary of prawns exposed to TBT, which should be accompanied with inhibition of P450 aromatase activity. Thus the changes of sex hormone levels would finally influence the ovarian development of prawn. In recent years, there is an upsurge in the interest of toxicity on the endocrine disruption in crustaceans which ultimately affect the process of gametogenesis. Accordingly, Zhang et al. [21] reported that environmentally realistic concentrations of TBT had an adverse effect on ovarian development in cuvier S. marmuratus. Here, ovary showed several spherical structures formed by the fragmentation of nucleus lying randomly in the chromatin network, yolk vesicles at the periphery, vacuolization in the nucleus, stromal hemorrhage and damage of germinal epithelium. Histo-anatomical abnormalities in ovaries may be caused by xenobiotics [30].

The alteration in basic biochemical metabolism ultimately posing a detrimental effect on growth and reproductive activity of M. rosenbergii exposed to TBT. Major biochemical constituents such as protein and lipid content also decreased in all the treated groups. The depletion of protein content suggests the increase in proteolysis and possible utilization of the product of their degradation for metabolic purpose. Products of proteolysis may be mobilized into trichloroacidic acid cycle through amino acid metabolism to cope up with the excess demand of energy during toxic stress. The fall in protein level during pollutant exposure may be due to increased catabolism and decreased anabolism of proteins. Similarly, depletion of protein content in different tissues of Macrobrachium kistnensis exposed to TBTCL was reported by Kharat et al. [1]. The decrease in protein and lipid content was reported in freshwater prawn M. kistnensis exposed to organotin pesticides [31]. The total lipid content also decreased because of the utilization of fatty acid deposits instead of glucose for energy purpose under TBT toxicity. Accordingly, West et al. [32] reported the decrease of total lipid content in hepatopancreas with increased activity of lipase, the enzyme is responsible for the breakdown of the lipid into free fatty acids and glycerol. Since lipids form a rich energy reserve whose caloric value is reported to be twice that of an equivalent weight of carbohydrate or proteins, the mobilization of lipid reserve testifies the imposition of high energy demands. Therefore, the amount of total lipid found to be lower in tissues of prawns exposed to TBT.

In the present study, a noticeable reduction of glycogen content in the hepatopancreas resulted the elevation of glucose level in hemolymph exposed to TBT. The decrease in glycogen content may be due to enhanced breakdown of glycogen to glucose through glycogenolysis under toxic stress of TBT in M. rosenbergii. Depletion in glycogen level might be due to its rapid utilization to meet the energy demands under toxic stress and to supply energy demand in the form of glucose which undergoes breakdown to produce energy rich ATP. Similarly, depletion of glycogen content in hepatopancreas of M. kistnensis exposed to TBTCL was reported by Kharat et al. [1]. Holwerda and Herwig [33] also reported that the dibutyltinchlorine exposed clam Anodonta anatine, showed notable decrease in carbohydrate content. In crustaceans, elevation of hemolymph glucose was also observed In vivo when they were subjected to pollutants [34]. Chin-Chyuan Chang et al. [35] found that the depletion of glycogen stores should be accompanied by an increase in glucose content after exposure of M. rosenbergii to trichlorfon. The glucose content increased with an increase of trichlorfon concentrations based on the dose-dependent manner.

Our results clearly demonstrated the impact of TBT on the vitellogenesis by reducing the vitellogenin (Vg) and vitellin (Vt) content in M. rosenbergii. Low concentration of Vg and Vt is an indication of malfunction of reproductive activity resulted in inhibition of reproductive performance in M. rosenbergii. Similarly, Vijayavel et al. [36] also found that the reduction in vitellogenin content influenced by naphthalene stress. Vitellogenin content can also act as a biomarker to study the abnormality in vitellogenesis. Low level of vitellogenin content is an indication of malfunction of reproductive endocrine system and inhibited the ovarian development [37].

Overall, the results clearly demonstrated the increased testosterone level and decreased 17β-estradiol levels in the ovary of prawns exposed to TBT, which should be associated with an inhibition with P450 aromatase activity. Thus the changes of sex hormone levels will influence the ovarian development of prawn. Zhang et al. [21] reported similar changes in ovarian development as well as sex hormonal changes in S. marmoratus.

Thus, TBT had reduced the vitellogenin and vitellin content by retarding vitellogenesis. On the other hand, imbalance of sex hormones such as decrease in 17β-estradiol and increase in testosterone has led to endocrine imbalance and ultimately that would have impaired the oogenesis in the freshwater female prawn M. rosenbergii.