Abstract
Tributyltin (TBT) is known to disrupt the development of reproductive organs, thereby reducing fertility. The aim of this study was to evaluate the acute toxicity of TBT on the testicular development and steroid hormone production. Immature (3-week-old) male mice were given a single administration of 25, 50, or 100 mg/kg of TBT by oral gavage. Lumen formation in seminiferous tubule was remarkably delayed, and the number of apoptotic germ cells found inside the tubules was increased in the TBT-exposed animals, whereas no apoptotic signal was observed in interstitial Leydig cells. Reduced serum testosterone concentration and down-regulated expressions of the mRNAs for cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase/C17–20 lyase (P45017α), 3β-hydroxysteroid-dehydrogenase (3β-HSD), and 17β-hydroxysteroid-dehydrogenase (17β-HSD) were also observed after TBT exposure. Altogether, these findings demonstrate that exposure to TBT is associated with induced apoptosis of testicular germ cells and inhibition of steroidogenesis by reduction in the expression of steroidogenic enzymes in interstitial Leydig cells. These adverse effects of TBT would cause serious defects in testicular development and function.
Tributyltin (TBT) has been widely used as an antifouling agent in marine paints, a wood preservative, and a heat stabilizer of polyvinyl chloride (PVC) (Shawky and Emons 1998). In spite of the restrictions established on the use of TBT-based antifouling paints in many countries, TBT deposited in sediments has been a continuous source of exposure for marine biota due to its high stability. Indeed, widespread contamination of TBT is still detectable along the coastal regions of many countries (Inoue et al. 2006; Sudaryanto et al. 2002). TBT is known to induce imposex in some female mollusks, and this sexual abnormality is believed to be caused by problems related to steroidogenesis (Horiguchi 2006; Morcillo and Porte 1999).
Although many studies have focused on marine species, possible exposure to TBT compounds in mammals, including humans, has generated considerable concern within the public and scientific community. In fact, TBT and its metabolites may undergo bioaccumulation through food chains, and due to drinking contaminated water and consumption of seafood from TBT-contaminated waters, mammals can be exposed to significant quantities of TBT (Takahashi et al. 1999; Tsuda et al. 1995). Recently, studies using laboratory animals have also shown a variety of reproductive toxicities associated with TBT compounds. Exposure to TBT chloride during the preimplantation period resulted in early embryo loss and implantation failure in rats (Harazono and Ema 2000; Harazono, Ema, and Ogawa 1996). In the study of two-generation reproductive toxicity in male rats, decreases in body weight and sex organ weights were pronounced, and sperm counts of the testis and caudal epididymis were also decreased in F1 and F2 neonates (Omura et al. 2001). TBT exposure in pubertal male rats also produced spermatogenic disorders characterized by decreasing testicular and epididymal sperm counts, and some motion parameters of sperm in the vas deferens (Yu et al. 2003a, 2003b).
Although the detrimental effects of TBT on the male reproductive system have been reported, exact mechanisms of action are not yet fully understood. Inhibition of aromatization and conjugation of testosterone by TBT has been suggested as a possible cause of imposex in marine animals. The aromatization of testosterone for the synthesis of 17β-estradiol and the conjugation of testosterone for the excretion of testosterone are both common metabolic pathways in mammals. Moreover, a recent study has shown that TBT and triphenyltin inhibit pig testicular 17β-hydroxysteroid dehydrogenase (17β-HSD) activity and suppress 17α-hydroxylase/C17–20 lyase (P45017 α ) transcription and testicular testosterone biosynthesis (Nakajima et al. 2005; Ohno, Nakajima, and Nakajin 2005). Therefore, evaluation of the effect of TBT on mammalian steroidogenic systems may lead us to understand the adverse effects of TBT on testicular development and steroid hormone synthesis.
The Leydig cells of the testis have the capacity to synthesize testosterone from cholesterol (Payne and Youngblood 1995). The biosynthesis of testosterone is dependent on various steroidogenic enzymes. Steroidogenic acute regulatory protein (StAR) is required for the transport of cholesterol to the mitochondrial membrane. Further conversions from cholesterol to testosterone occurring through mitochondria to the smooth endoplasmic reticulum are catalyzed by the cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), P45017 α , and 17β-HSD. Leydig cells also express P450 aromatase, which catalyzes the aromatization of testosterone to estradiol (Payne and Youngblood 1995). Because the differentiation of adult Leydig cells during the immature stage is crucial for testosterone production in the adult stage, it is important to clarify the inhibitory effects of TBT exposure on steroidogenesis during this developmental stage.
Therefore, the present study was designed to investigate the detrimental effects of TBT on growth and steroid hormone production of the testis after exposure to TBT. The changes in the expressions of the steroidogenic enzymes at the mRNA and protein levels were investigated in order to clarify whether changes in testosterone production coincided with the altered Leydig cell function.
MATERIALS AND METHODS
Animals and Treatments
Male ICR mice (21 days old; average body weight, 12.5 ± 0.5 g) were purchased from Samtako BioKorea and utilized in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Mice were housed in a room with controlled lighting (12-h light:12-h dark) and temperature (20° C to 22° C), and given food and tap water ad libitum. The experimental animals were given a single administration of 0.1 ml corn oil alone (vehicle-exposed control; CV) or corn oil containing tributyltin acetate (25, 50, or 100 mg/kg body weight [BW]) by oral gavage. These animals were sacrificed three days after TBT exposure. In the preliminary experiments, no differences were found in the testicular growth and steroid hormone levels between unexposed and corn oil–exposed animals (Kim et al. 2007).
Measurement of Body and Organ Weights
Body and organ weights were measured three days after TBT exposure. Longitudinal and horizontal lengths of testes were measured by a caliper. The volume of the testis was calculated using the formula: (width2 × length × π/6). The gonad index (GI) was calculated using the formula: (testis weight (g)/BW (kg) × 100).
Radioimmunoassay for Determination of Serum Testosterone and Estradiol Concentration
Measurements of testosterone and estradiol were carried out by conventional radioimmunoassay as described previously (Lee et al. 2000; Kim et al. 2004). The lower detection limits of testosterone and estradiol were 10 and 20 pg, respectively. The inter- and intra-assay coefficients of variation for testosterone and estradiol were all below 10%.
Hematoxylin-Eosin Staining
The testes were fixed in Bouin’s solution, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. Sections (4 μm thick) were mounted on glass slides and stained with Harris’ hematoxylin and eosin (Sigma Chemical, St Louis, MO).
In Situ TUNEL Staining for Detection of Apoptosis
Labeling of DNA fragmentation in each testicular section was performed using TUNEL (deoxynucleotidyl transferase-mediated dUTP nick end labeling) method according to the protocol of the In Situ Apoptosis Detection Kit (Apoptaq; Oncor, Gaithersburg, MD). TUNEL-positive cells were detected by diaminobenzidine (DAB) substrate development, counterstained with Mayer’s hematoxylin (Sigma Chemical) and mounted with Canada balsam (Sigma Chemical). Total seminiferous tubules, TUNEL-positive tubules, and TUNEL-positive cells of three sections per animal were determined from three animals per dose level. The “apoptotic index” was calculated by multiplying the percentage of tubules containing apoptotic germ cells by the number of apoptotic germ cells per tubule after exposure to TBT (Yu et al. 2001).
Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) Analysis of Testicular Gene Expressions
Total RNA from the whole testis was isolated using the TRIzol (Invitrogen, Carlsbad, Canada) method. Complementary DNA was synthesized from 1 μg of the total RNA using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s protocol. The cDNAs obtained were used as a template for subsequent PCR amplification using 1 U of Taq DNA polymerase (Roche). Mouse-specific primers for the genes of interest were: 5′-GTG GGC CGC TCT AGG CAC CAA-3′ and 5′-CTC TTT GAT GTC ACG CAC GAT TTC-3′ for β-actin; 5′-AGT GGC AGT CGT GGG GAC AGT-3′ and 5′-TAA TAC TGG TGA TAG GCC ACC-3′ for P450 scc ; 5′-CAG ACC ATC CTA GAT GT-3′ and 5′-AGG AAG CTC ACA GTT TCC A-3′ for 3β-HSD; 5′-CCC ATC TAT TCT CTT CGC CTG GGT A-3′ and 5′-GCC CCA AAG ATG TCT CCC ACC GTG-3′ for P450 17 α ; 5′-ATT TTA CCA GAG AAG ACA TCT-3′ and 5′-GGG GTC AGC ACC TGA ATA ATG-3′ for 17β-HSD; and 5′-CCT GAC ACC ATG TCG GTC ACT-3′ and 5′-GGG CTT AGG GAA GTA CTC GAG-3′ for Aromatase. Analysis of the respective bands was performed using an image analyzer (Viber Louvmat, Marne-la-Vallée, France). The intensity of each mRNA band was normalized to the intensity of the corresponding β-actin band.
Immunohistochemistry
Immunostaining was performed using a commercial immunohistochemistry kit (Innogenex, San Ramon, CA) according to the suggested protocol of the manufacturer. Antigen retrieval was performed by heating the sections in 10 mM citric acid solution (pH 6.0). Primary antibody for P450scc was obtained from Chemicon International, CA. A final coloring reaction was performed using liquid DAB. The slides were then counterstained with Mayer’s hematoxylin.
Statistical Analysis
Results shown indicate the averages of three independent experiments using five animals per different dose level. The values are expressed as means ± standard error of the means (SEM). For the determination of statistical significance, results were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni’s test procedure for multiple comparisons with a vehicle-exposed control. p values less than .05 were judged to be statistically significant.
RESULTS
Body and Organ Weights
Body and organ weights of immature male mice (24 days old) following exposure to TBT are shown in Table 1. A significant reduction in the mean body weight was observed in the animals exposed to 50 and 100 mg/kg of TBT compared to those in vehicle-exposed animals. Absolute testicular weight was reduced dose-dependently, although statistical significance was only observed in the groups exposed to 100 mg/kg of TBT (Table 1). The gonad index and testis volumes were not significantly reduced in the groups of TBT-exposed animals (Table 2). The weights of the liver and other accessory organs such as the seminal vesicle, epididymis, prostate, and vas deferens were also reduced dose-dependently after exposure to TBT. However, statistically significant reduction was only found in seminal vesicle weight in the mice exposed to 100 mg/kg of TBT.
Serum Testosterone and Estradiol Concentrations
To evaluate the effect of TBT on steroid hormone production, we first attempted to measure circulating testosterone and estradiol levels after TBT exposure. The serum testosterone concentration in the animals exposed to 100 mg/kg of TBT at the immature stage was significantly reduced to almost 26% of the value observed in the vehicle-exposed animals (Figure 1A ). The serum estradiol concentration in the animals exposed to 100 mg/kg of TBT was also reduced to 65% of the value observed in the vehicle-exposed animals (Figure 1B ). However, this change on estradiol concentration was not statistically significant due to relatively high interindividual variation.
Histological Structure of Testis
Compared to that of the vehicle-exposed controls (Figure 2A ) lumen formation of seminiferous tubules was delayed (Figure 2C and D ), and the overall number of germ cells in the tubules was reduced in the testis of mice exposed to 100 mg/kg of TBT (Figure 2B ). In addition, the number of cells with pyknotic nuclei (Figure 2D , inset) and multinucleated bodies (Figure 2C , inset) were increased in the tubules of TBT-exposed animals.
The in situ TUNEL method was performed to investigate whether the increased cell death is accompanied by apoptosis. The apoptotic index was calculated by multiplying the percentage of tubules containing apoptotic germ cells by the number of apoptotic germ cells per tubule after exposure to TBT. The apoptotic index was markedly increased in animals exposed to TBT in an apparent dose-response manner (Table 3 and Figure 2, lower panels). The primary spermatocyte was found to be the predominant cell type undergoing apoptosis. Apoptotic signals were not observed in interstitial Leydig cells.
Transcriptional Expressions of Steroidogenesis-Related Genes
Because data from our in situ TUNEL analysis suggested that the decreased serum testosterone concentration did not result from a reduction in the number of Leydig cells due to apoptosis, we next evaluated the expression levels of several steroidogenesis-related genes that are mainly expressed in interstitial Leydig cells and are involved in steroid hormone synthesis. The mRNA expressions of P450scc and P45017 α were markedly decreased in the testis of mice exposed to 50 and 100 mg/kg of TBT (Figure 3A to C). In the case of 3 β -HSD and 17β-HSD, animals exposed to 100 mg/kg of TBT showed significant reductions of gene expression in the testis compared to the vehicle-exposed control (Figure 3A, D , and E), but no significant changes were observed in the mRNA expression of aromatase (Figure 3A and F ).
Immunohistochemistry of P450scc in Testis
A decreased expression of P450scc after TBT exposure was further evaluated at the protein level using immunostaining. Intense P450scc immunoreactivity was primarily observed in interstitial Leydig cells (Figure 4A ). Positive immunostaining of P450scc was also observed near the nuclei of the secondary spermatocytes and in the cytoplasm of Sertoli cells. In contrast, exposure to 100 mg/kg of TBT caused a decrease in the overall expression level of P450scc in interstitial Leydig cells compared to exposure to vehicle alone (Figure 4).
DISCUSSION
Although previous studies have demonstrated that acute and chronic exposure to various environmental chemicals in male fetuses, neonates, or adult animals have adverse effects on testicular development, relatively little has been reported on the exact mechanism for the harmful effects of TBT on testicular development and testosterone production, especially in mammals. In the present study, we have focused on the effect of acute exposure to TBT on testosterone production in mouse testis by determining the changes in the testicular expressions of various steroidogenic enzymes.
An important finding of our study was the marked decrease in testosterone secretion after exposure to TBT. Acute exposure to TBT is unlikely to have caused any significant effects on the viability of Leydig cells because data from in situ TUNEL analysis showed no significant increase in apoptosis of Leydig cells. Therefore, the suppression in testosterone production was not the result of a decrease in the number of viable Leydig cells at the doses of TBT administered in the present study. The production of testosterone in Leydig cells is dependent on a series of steroidogenic enzymes. Therefore, the reduction in testosterone level may result from altered expressions of the steroidogenic enzymes due to exposure to TBT. The transcriptional expressions of P450scc, P450 17 α , 3β-HSD, and 17β-HSD were all markedly decreased in the animals exposed to TBT during the immature stage. This suggested that reduced testosterone production was due to decreased expression of these enzymes in Leydig cells, as the steroidogenic enzymes are rate-limiting enzymes in testosterone production in the testis. These findings are in concordance with a previous report that described that TBT chloride suppresses the P450 17 α transcription under gonadotropin stimulation and inhibits testosterone production in cultured pig Leydig cells (Nakajima et al. 2005). Data from the present study provide evidence that TBT exposure is also associated with a decrease in the translational expression of P450scc in interstitial Leydig cells. Therefore, our observations suggest that the inhibited expressions of the steroidogenic enzymes in Leydig cells upon exposure to TBT resulted in the decreased serum testosterone concentration.
The reduction of testosterone production by TBT exposure may also occur through the alteration of the activities of these steroidogenic enzymes. In fact, many in vitro studies have shown direct inhibitory effects of TBT and other related organotins on the activities of human 5α-reductases in the brain and prostate (Doering et al. 2002), rat testicular 3β-HSD (McVey and Cooke 2003), human placental 17β-HSD, adrenal 3β-HSD, and testicular 17β-HSD (Lo et al. 2003), and pig testicular 17β-HSD (Ohno, Nakajima, and Nakajin 2005). Results from these in vitro studies suggest that the reduced production of testosterone following exposure to TBT may be caused not only by reduced expressions of the steroidogenic enzymes but also by inhibited activities of these enzymes in the present study.
Although the present study shows an association between TBT exposure and reduced expressions of steroidogenic enzymes involved in testosterone production, the exact mechanism underlying the inhibitory effect of TBT on the transcription of these enzymes remains unclear. Steroidogenesis is dependent on pituitary hormones. In Leydig cells, hormonal stimulation triggers the activation of an intracellular signaling pathway that typically involves cycle adenosine monophosphate (cAMP) production, activation of protein kinanse A (PKA), and phosphorylation of target transcriptional factors (Payne and Youngblood 2005). SF-1 and Dax-1 are known to positively regulate the expressions of multiple cytochrome P450 steroid hydroxylases (Peter and Dubuis 2000). GATA-4 and -6 are also suggested as novel downstream effectors of hormonal signaling in steroidogenic tissues, and are known to control the transcriptions of steroidogenic genes (Tremblay and Viger 2003). Further investigations are needed to determine whether the pituitary hormonal pathways and their underlying signaling molecules are involved in the TBT-induced alteration in steroid biosynthesis of Leydig cells.
Two populations of Leydig cells arise in the course of testicular development. In mice, the “fetal” population arises soon after the differentiation of testis, at about 12.5 days post coitum. “Adult” Leydig cells arise after birth, their capacities to produce steroid hormones steadily increase during postnatal development, and the expressions of steroidogenic enzymes and related transcriptional factors increase until they peak around puberty (O’Shaughnessy, Willerton, and Baker 2002). The secretion of testosterone by this “adult” population is critical for initiation and maintenance of spermatogenesis, and for the expression of male secondary sex characteristics. In the present study, 21-day-old mice were exposed to TBT and its acute effect on steroidogenesis and testosterone production was monitored 3 days after exposure. In this stage, the testosterone is produced from the “adult” Leydig cell population in the testis, and any alteration of steroidogenesis by exposure to TBT during this period may adversely affect the normal development of the testis and spermatogenesis.
A significant reduction in mean body weight was observed in animals exposed to 100 mg/kg of TBT compared to those in the vehicle-exposed animals. Various estrogenic and antiestrogenic compounds have been shown to alter the hypothalamic-pituitary-gonad feedback, and the secretion and regulation of growth hormones are related to this axis (Pons and Torres-Aleman 1993). Therefore, changes in the levels of insulin growth factor (IGF)-1 and IGF-binding protein 3 may provide additional evidence for understanding the mechanism of reduced body weight following TBT exposure.
Although the absolute testicular weight is reduced by exposure to TBT, the calculated testis volume and gonad index were not significantly changed because of the concomitant reduction in mean body weight after acute exposure to TBT. However, an alteration of the histological structure of testis was observed. Delayed lumen formation in seminiferous tubules and reduction in the overall number of germ cells in the tubules were seen in the animals exposed to TBT compared to those in the vehicle-exposed animals. Several multinucleated bodies and cells with pyknotic nuclei in the tubules were also observed, and data from in situ TUNEL analysis indicated that those testicular germ cells underwent apoptosis upon exposure to TBT. The significant increases in apoptotic germ cells were observed in the animals exposed to ≥ 25 mg/kg of TBT, although the expressions of steroidogenic enzymes and serum testosterone levels were not altered in these animals. These results suggest that induced apoptosis in testicular germ cells may not be caused by altered testosterone levels, and that testicular germ cells may actually be more sensitive to the cytotoxic effects of TBT. The cytotoxic effect of TBT has been reported previously by others in different cell types such as rat thymocytes (Umebayashi et al. 2004) and hepatocytes (Jurkiewicz et al. 2004). Jurkiewicz et al. (2004) showed the involvement of mitochondrial and death receptor pathways in TBT-induced apoptosis in rat hepatocytes. These pathways are also known to play important roles in both spontaneous and heat- or chemical-induced apoptosis of testicular germ cells (Lee et al. 1997; Sinha Hikim et al. 2003). Studies using different environmental toxicants such as 2-bromopropane and phthalates showed that expressions of Fas and FasL, involved in the death receptor pathway, were induced in the testis (Yu et al. 2001; Park, Habeebu, and Klaassen 2002). Additionally, previous studies (Kim et al. 2004) showed decreased mRNA expression of an anti-apoptotic factor, bcl-xL, in the testis following prepubertal exposure to 4-tert-octylphenol. Further studies are needed to evaluate the possible direct effect of TBT on apoptosis of testicular germ cells and on the expressions of apoptosis-related molecules.
These results indicate that acute exposure to TBT is associated with decreased transcriptional expressions of the steroidogenic enzymes in differentiating Leydig cells of the immature mouse testis, and results in decreased testosterone synthesis. Because the development and differentiation of Leydig cells during this period of exposure are essential later during adult life, the alteration of steroidogenesis by exposure to TBT may adversely affect the normal development of the testis and spermatogenesis.
Footnotes
Figures and Tables
This work was supported by a grant from Korean Research Foundation, funded by the Government of the Republic of Korea (Minister of Education and Human Resources Development, MOEHRD), KRF 2003-C00051 to Dr. Y.-D. Yoon.