Fetal Pituitary Gonadotropin as an Initial Target of Dioxin in Its Impairment of Cholesterol Transportation and Steroidogenesis in
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《内分泌学杂志》
Graduate School of Pharmaceutical Sciences (J.M., J.T., K.O., T.I., Y.I., H.Y.), Kyushu University, Fukuoka 812-8582, Japan
Department of Cell Fate Modulation (T.K.), Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan
Division of Active Transport (T.K.), National Institute for Physiological Sciences, Aichi 444-8585, Japan
Abstract
Reproductive and developmental disorders are the most sensitive toxic effects caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is thought to produce many, if not all, of these toxic effects by impairing steroidogenesis and/or steroid action during the prenatal or early postnatal stages. However, the mechanism of the antisex steroid effect of TCDD is not well understood. This study revealed that steroidogenic acute-regulatory protein (StAR), a key transporter of cholesterol for steroidogenesis, in the testes of fetal rats are down-regulated by maternal exposure to TCDD. It was also shown that many mRNAs of steroidogenetic enzymes, including cytochromes P450 11A1, 17, and 11B1 and 3-hydroxysteroid dehydrogenase, are reduced in fetuses of TCDD-treated dams in a testis-specific manner. The same was also observed for the expression of estrogen- receptors and androgen receptors. Whereas StAR expression was not affected by TCDD in cultured fetal testis, the fetal serum content of LH, a pituitary regulator of StAR, was significantly reduced by TCDD. In agreement with this, pituitary expression of LH subunit mRNA in fetuses was reduced by maternal exposure to TCDD, whereas the -subunit remained unchanged. The reduction in LH is suggested to occur by a mechanism different from the reduction in the GnRH level. Direct supply of exogenous gonadotropin to TCDD-exposed fetuses completely abolished the reduction of StAR expression. Taken together, these results demonstrate that TCDD impairs steroidogenesis in the fetus by targeting pituitary gonadotropins.
Introduction
DIOXINS, EXEMPLIFIED BY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are highly persistent environmental pollutants. These substances exert a wide variety of toxic effects in mammals via a mechanism involving the activation of the aryl hydrocarbon receptor (AhR) (1, 2, 3). The AhR is a regulatory factor of gene transcription, and a number of genes are under the control of this receptor (4, 5, 6, 7). Although the AhR plays a crucial role in dioxin toxicity, it is still not known which genes regulated by AhR contribute to dioxin toxicity. Among the possible health injuries after exposure to dioxins, the reproductive disorders are of particular interest because the damage to fetuses and newborn pups appear at doses much lower than those associated with toxic effects in adults (8).
To date, studies using experimental animals have identified multiple forms of reproductive and developmental damage (8). These include teratogenicity, growth retardation of fetuses/infants, and impaired mental and sexual function after development to adults. In the latter case, it has been reported that male offspring born from TCDD-treated dams exhibit a defect in sexual behavior (9, 10). This seems to be closely related to the observations that show the disappearance of male-type behavior in male mice deficient in estrogen synthase [cytochrome P450 19 (CYP19)], estrogen receptors (ER), and androgen receptors (AR) (11, 12, 13, 14). Thus, it is conceivable that TCDD and related compounds exert some forms of reproductive toxic effects by impairing sex steroid synthesis and/or blocking its function. In CYP19-knockout mice, recovery of the defect in sexual behavior needs continuous treatment with exogenous estrogen from the newborn pup stage onward, whereas treatment only at the adult stage fails to improve the impairment. This clearly indicates that estrogen-dependent signaling during the fetal or early neonatal stages is necessary for acquiring some forms of phenotype in adults. The effects of TCDD on reproduction, development, behavior after development, and steroid hormone status have been studied extensively. However, the majority of such research examined the effects of maternal exposure to dioxins on such end points using offspring of pre- or postpubertal age (Refs.9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ; also see references therein), and it is largely unknown how TCDD affects steroidogenesis and steroid action in the fetus. To address this issue, we examined the effect of TCDD on fetal expression of enzymes and transporters involved in steroidogenesis and of steroid hormone receptors and propose a mechanism governing the alteration of these mechanisms.
Materials and Methods
Materials
TCDD was obtained from AccuStandard Inc. (New Haven, CT). [14C]progesterone (2.04 TBeq/mol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Its radiochemical purity was confirmed to be over 99% by thin-layer chromatography (TLC) [Rf = 0.62; solvent used: benzene-acetone = 3:1 (vol/vol)]. An antibody against rat steroidogenic acute-regulatory protein (StAR) was obtained from Abcam Ltd. (Cambridge, UK). The other reagents were of the highest grade commercially available.
Animals and treatments
Female Wistar rats (10–14 wk old) were paired overnight with male Wistar rats. The next morning, sperm in the vaginal smears was checked by microscopy (x100–400) to confirm pregnancy. When the presence of sperm was detected, the day was designated as gestational day (GD)0 of pregnancy. In typical experiments, pregnant rats were treated once with TCDD (1 μg/kg per 2 ml corn oil, orally) at GD15, and their fetuses were removed at GD20 for further experiments. In the dose-response relationship experiment, different doses (0.01, 0.1, and 1 μg/kg per 2 ml corn oil) of TCDD were given to pregnant rats in a schedule described above. In a separate experiment, pregnant rats were treated with TCDD at GD8, followed by removal of fetuses at GD20. When pups born from dams pretreated with TCDD at GD15 were examined, testes were obtained from the pups at a postnatal age of 7 d. The effect of TCDD on StAR expression in the testis was also examined in pubertal rats as a reference experiment. In this case, 44-d-old male Wistar rats were treated once with 1 μg/kg TCDD, and the testes were removed 5 d after the treatment. Tissues of rats were stored at –80 C before mRNA extraction, immunoblotting, and the assay of CYP17 activity.
Direct injection of equine chorionic gonadotropin (eCG) to fetuses was performed as follows. Pregnant rats (GD17) pretreated with TCDD (1 μg/kg) at GD15 were anesthetized with sodium pentobarbital [30 mg/kg per 2 ml PBS, ip]. The abdomen was opened by a 3-cm incision and both uteruses were removed from the body. Surgery was carried out while pouring warm PBS (37 C) on the uteruses to avoid drying of the tissue. Fetuses in the right and left uteruses were treated as control and eCG-treated groups, respectively. eCG in PBS (5 IU per 5 μl) was injected into the back of the fetuses in the uterus using a syringe fitted with a 31G needle. The control fetuses were given PBS. Because fetal sex cannot be judged during this operation, all fetuses were injected with eCG or PBS. Male fetuses were confirmed by detecting the presence of testes when they were dissected. After injection of eCG or PBS into the fetuses, the uterus containing pups was returned to the dam, and 5 ml warm PBS were poured into the abdominal cavity, and the wound was stitched up. The operation described above was completed within 30 min. Treated dams were housed individually, and the fetal testes were removed at GD20.
All experiments were approved by the Institutional Animal Care and Experiment Committee of the Graduate School of Pharmaceutical Sciences of Kyushu University.
Tissue culture of fetal testis
Fetuses were removed from untreated pregnant rats at GD20. A testis was put on a well of a 12-well plate filled with DMEM (1 ml/well) containing 100 U/liter penicillin, 0.01% streptomycin, and 1 mM sodium pyruvate and washed once by changing the medium. After treatment of the tissue with TCDD and/or 8-bromoadenosine-cAMP (8-Br-cAMP), forskolin, and eCG, the testis was cultured at 37 C for 24 h in an atmosphere of 5% CO2 in air. TCDD and forskolin were dissolved in dimethyl sulfoxide at concentrations of 100 pmol/ml medium per 5 μl and 10 μmol/ml medium per 5 μl, respectively. 8-Br-cAMP and eCG were dissolved in PBS at concentrations of 1 μmol/ml medium per 5 μl and 10 IU/ml medium per 5 μl, respectively. The treated tissue was washed twice with PBS and stored at –80 C before use.
Quantitative RT-PCR
Tissue mRNA was determined by RT-PCR according to the method described elsewhere (25). In the preparation of cDNA, 0.1 μg extracted mRNA (liver, testis, adrenal, and pituitary) and 1.0 μg (hypothalamus) was reverse transcribed to cDNA. The primer pairs used for amplification of cDNA are shown in Table 1. The PCR was performed using 1 μl of the reverse transcription reaction mixture in a final volume of 50 μl (25). In the cases of ER (3 μl) and adrenal CYP11B1 (1 μl, 20-fold diluted cDNA solution), the indicated volume of cDNA solution was used for PCR. All PCRs were performed under conditions guaranteeing template quantity-dependent amplification. The conditions used for PCR were as follows: 94 C for 2 min, x-cycles (94 C for 30 sec, y C for 30 sec, and 72 C for 30 sec), and then 72 C for 10 min. The extension cycle and annealing temperature, shown as x and y, respectively, are given in Table 1. Different temperatures for denaturation at the first step of PCR and the initial step in each extension reaction were set as described below: 95 C for StAR, cytochrome P450 1A1 (CYP1A1), CYP11A1, CYP11B1, 3-hydroxysteroid dehydrogenase (HSD), steroidogenic factor-1 (SF-1) and LH receptor (LHR); 96 C for CYP11B2; and 97 C for AR. The extension reaction was carried out for 15 sec (72 C) for GnRH and 60 sec (72 C) for StAR, CYP1A1, CYP11A1, 3-HSD, 17-HSD, SF-1, LHR, AhR, and AhR nuclear translocator (ARNT). The cDNA amplified was electrophoresed in 1.5% agarose gel and stained with ethidium bromide. When necessary, the band image was extracted by scanner and quantified using National Institutes of Health Image software (version 1.62). The amount of quantified mRNA was normalized by -actin mRNA, and, in some figures, the value was expressed as a relative level to the control.
Assay of CYP17 activity
Testes of fetuses (4–11 individuals) in one dam were combined and homogenized in 0.25 M sucrose containing 5 mM potassium phosphate (pH 7.4), 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 20% glycerol (0.05 ml per one testis). [14C]progesterone (7.5 μM, 670 GBeq/mol) was incubated at 37 C with 1 mM nicotinamide adenine dinucleotide phosphate reduced and 30 μg protein testis homogenate in a final volume of 200 μl 0.1 M potassium phosphate (pH 7.4). The mixture was extracted three times with 400 μl ethyl acetate-isooctane (1:1, vol/vol). Pooled organic phase was evaporated to dryness under N2, and the residue was reconstituted in 100 μl methanol containing nonlabeled carries (200 μM progesterone and 17-hydroxyprogesterone). A portion (10 μl) of this solution was subjected to thin-layer chromatography (Silica-gel gel 60 F254; Merck, Darmstadt, Germany; solvent: benzene-acetone 3:1, vol/vol). The area to which the unchanged progesterone (Rf, 0.62) and 17-hydroxyprogesterone (Rf, 0.49) had migrated was visualized under UV light (254 nm), excised, and the radioactivity measured in scintillation cocktail (ACSII; Amersham, Piscataway, NJ).
Other methods
Immunoblotting with antibody against antirat StAR was performed by the method reported previously (25). Briefly, tissue samples were electrophoresed by Laemmli’s method (26), and proteins on the gel were electrically transferred to a polyvinilidene difluoride membrane (27). StAR protein on the membrane was visualized using secondary antibody conjugated with alkaline phosphatase (28). The content of fetal serum LH was determined by ELISA using a commercial kit (Endocrine Technologies, Inc., Newark, CA). Serum of male fetuses in one dam was pooled and diluted twice with diluent supplied with the kit before assay. Statistical differences between two groups were calculated by Student’s t test. Other statistical differences were estimated by ANOVA with a post hoc test (Fisher’s protected least significant difference method).
Results
TCDD-induced change in the expression of fetal proteins involved in steroidogenesis and steroid action
Pregnant Wistar rats were given a single administration of TCDD (1 μg/kg, orally) at GD15, and mRNA expression in fetal tissues was determined at d 20 of gestation. The hepatic expression of CYP1A1 mRNA, a well-known marker for AhR-regulated gene, was markedly increased by TCDD (Fig. 1), and it was evident that TCDD given to dams distributes to fetuses and affects gene expression. StAR and the peripheral benzodiazepine receptor (PBR) play important roles in cholesterol transportation to the mitochondrial inner membrane in which steroidogenetic reactions occur (29). Of these transporters, the fetal expression of testicular StAR mRNA was markedly reduced after TCDD treatment, whereas adrenal StAR remained unchanged (Fig. 2, A and B). A reduction in testicular StAR protein was also confirmed by immunoblotting (Fig. 2C). The 7-d-old offspring born from TCDD-treated dams did not show any reduction in the expression of testicular StAR (Fig. 3A). The absence of TCDD-induced reduction in the expression of testicular StAR was also observed in pubertal rats (49 d old) that were given TCDD at postnatal d 44 (Fig. 3B). Thus, the reduction in the expression of this transporter was specific to the fetus. From these data, we focused our main interest on the StAR alteration and tried to clarify the mechanism in this study.
In addition to StAR, the expression in the fetal testis of several steroidogenetic enzymes (CYP11A1, CYP17, CYP11B1, and 3-HSD) was reduced by TCDD treatment (Fig. 4A). Changes in these enzymes were also specific for the testis, except for CYP17, which was reduced in the adrenals of both sexes after TCDD treatment (Fig. 4A). In agreement with the change in CYP17 expression, testicular homogenate prepared from fetuses in TCDD-treated dams exhibited a lower ability to use progesterone and produce 17-hydroxylated metabolite (Fig. 4B). As far as the receptors were concerned, a significant decrease in the expression of testicular ER and AR mRNAs was observed (Fig. 4A). Treating dams with TCDD had no effect on the expression of AhR and its partner receptor (ARNT) in the fetal testes and adrenals (Fig. 4A). These results strongly suggest that steroidogenesis and steroid action are impaired in the fetuses in TCDD-treated dams. As can be seen in Fig. 5A, reduced expression of fetal testicular StAR, CYP11A1, and CYP17 was seen only after treating dams with 1 μg/kg TCDD, and lower doses (0.1 and 0.01 μg/kg) had no significant effect on the expression of these mRNAs. Earlier exposure at GD8 to TCDD gave comparable results to treatment at GD15 as far as the expression of StAR, CYP11A1, and CYP17 was concerned (Fig. 5, A and B).
Change in the expression of regulatory factors by TCDD and the effect of TCDD on cultured fetal testis
Expression of the StAR gene is under the control of transcriptional factors involving positive regulators [SF-1, GATA-binding protein 4 (GATA-4)] and a negative regulator [dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1)] (30, 31). Of these factors, SF-1 mRNA was significantly reduced by treating dams with TCDD (Fig. 6). It is, therefore, likely that a reduction in SF-1 contributes to StAR reduction. We then examined the possibility that TCDD could reduce StAR expression by direct action on the fetal testis. To this end, the testes of fetuses removed from untreated dams were cultured, and the effect of TCDD on StAR expression was examined. The results obtained showed that StAR expression in cultured testes is completely unaffected by TCDD treatment (Fig. 7A). Pituitary gonadotropins, such as LH, FSH, and chorionic gonadotropin, play a key role in StAR expression in gonadal tissues. These pituitary factors bind to specific receptors expressed on the plasma membrane of peripheral target tissues and activate protein kinase A-dependent signaling, which further activates transcriptional factors, such as SF-1, GATA-4, and DAX-1 (31, 32, 33). When cultured testis was treated with 8-Br-cAMP, an analog of cAMP, forskolin, and eCG, a significant increase in StAR mRNA was observed as expected (Fig. 7A). This observation showed that the cultured testis is able to respond to gonadotropin to cause gene expression. TCDD failed to suppress induced expression of StAR by 8-Br-cAMP, forskolin, and eCG (Fig. 7A). These data do not support the view that TCDD directly affects the testis to suppress the expression of steroidogenetic mechanisms involving StAR. Because CYP1A1 is normally induced by TCDD (Fig. 7B), TCDD added to cultured testis is assumed to enter the cells.
Effect of TCDD on gonadotropin synthesis in fetal brain and its significance for testicular StAR expression
As described above, pituitary gonadotropins are primary factors regulating the expression of StAR in the peripheral tissues. To identify the mechanism of the TCDD-induced reduction in fetal StAR expression, we then focused on the possibility that dioxin impairs the expression and function of gonadotropin. The expression of the LHR mRNA of the fetal testis was not affected by maternal exposure to TCDD (Fig. 8A). On the other hand, the -subunit mRNA of pituitary LH was significantly reduced in fetal brain by TCDD, whereas the -subunit mRNA did not change (Fig. 8A). In agreement with this, LH in fetal serum was significantly reduced by treating dams with TCDD (Fig. 8B). These data suggest that TCDD down-regulates steroidogenetic proteins in fetal testis by reducing circulating pituitary hormone(s) in fetuses. Hypothalamic GnRH is one of the important factors regulating the synthesis of gonadotropin in the pituitary and its secretion. However, maternal exposure to TCDD produced no effect on the GnRH mRNA in the fetal hypothalamus (Fig. 8C). To confirm the hypothesis that a reduction in circulating gonadotropin is a reason for the TCDD-caused reduction in fetal StAR expression, we tried to combat the StAR reduction in TCDD-exposed fetuses by direct injection of gonadotropin. The results obtained supported the above view, and direct supply of exogenous eCG to male fetuses without removing them from the dam completely restored the expression of testicular StAR to control levels (Fig. 9).
Discussion
We have shown that there is a reduction in the expression of StAR and steroidogenetic enzymes in fetal testis after maternal exposure to TCDD (Fig. 10). Because the reduction in these proteins is specific to the fetal stages, this suggests that such alterations produce reproductive and developmental toxicities by impairing the sex steroid-mediated imprinting of gender-specific phenotypes. These data assume a reduction in the formation of androgen and estrogen in the testis after treatment of the dams with TCDD. However, other workers have reported that testosterone in the testis and serum is not reduced in the fetuses of TCDD-exposed pregnant rats (Han/Wistar, Long-Evans, and Holtzman strains) (34, 35), whereas early reports described a reduction in fetuses (9) and adults (36, 37). The reason for these inconsistencies is unknown. However, because all these studies assayed steroid hormones by immunological techniques using antibodies against target steroids, it is conceivable that the antibody used detects alterations in other steroids to give false-positive or -negative results. Alternatively, there may be great species/strain differences in TCDD-induced down-regulation of StAR and other steroidogenetic proteins. On the other hand, maternal treatment with TCDD is reported to be able to reduce serum -estradiol in fetuses and offspring of prepubertal age (16, 35). The present study shows that the expression of testicular CYP19 remains unchanged in TCDD-exposed fetuses. If androgen and estrogen levels are reduced by TCDD, the supply of precursors of these steroids should be reduced. This same assumption has been proposed by other investigators (36, 37, 38). Thus, although the change in proteins contributing to the early steps of steroidogenesis reported here is thought to be important for the antiestrogenic and -androgenic effects of dioxin, further studies are needed to clarify the alteration in gonadal steroids in the fetus and during the early postnatal stages and the mechanism involved.
Regarding the antisex steroid effect of dioxin, mechanisms other than the reduced production of steroid hormones have also been proposed. These include: 1) decreased expression of ER, AR, and LHR (22, 39, 40, 41); 2) enhanced inactivation of steroid hormones due to induction of steroid-metabolizing enzymes such as CYP1B1 (42, 43); and 3) an antagonist effect of dioxins on ER-dependent signaling (16, 44, 45). Although the TCDD effect is likely due to a combination of these mechanisms, the present study supports the reduction in AR and ER. LH plays a major role in the testicular production of testosterone as shown by the finding that LHR-deficient mice lose almost all the testosterone in their testes (46). However, the residual testosterone that is produced by an LH-independent mechanism is suggested to be sufficient for some testicular functions, such as spermatogenesis (46). This assumption does not support the view that a reduction in steroid hormone significantly contributes to the antisex steroid effects of TCDD. However, because mice lacking AR and ER exhibit abnormalities in gender-specific phenotypes (13, 14), a marked reduction in the level of sex steroids could cause such disorders. The present study shows that the action of sex steroids in the fetuses of TCDD-treated dams is impaired by several mechanisms including a reduction in steroidogenesis and receptor expression. These alterations show that a reduction in the quantity and function of sex steroids makes a significant contribution. As far as the reproductive and developmental damage produced by dioxins is concerned, much of this is thought to occur by mechanisms involving AhR (47, 48).
It would be of great interest to discover whether AhR contributes to the TCDD-produced change in testicular steroidogenesis in the fetus. Concerning this issue, AhR-deficient mice have been reported to resist a reduction in testicular Cyp11a1 produced by TCDD, whereas TCDD produces a significant reduction in this enzyme in wild-type mice (41). This observation supports the view that AhR plays a role in at least some cases of TCDD-induced impairment of steroidogenesis. As can be seen in Fig. 5, CYP11A1 mRNA seemed to be reduced more markedly after TCDD treatment at GD8 than GD15, although the magnitude of the TCDD-produced reduction in StAR and CYP17 was comparable between the dosing timings. Although the reason for these differences is not known, it is conceivable that the mechanism governing StAR and CYP17 regulation differs from CYP11A1 regulation in which the AhR plays a crucial role. The treatment at GD8 would be assumed to produce a prolonged exposure of fetuses to TCDD because of its persistent nature in the body. It is, therefore, possible to assume that CYP11A1 reduction needs long-term exposure to TCDD.
As shown in Fig. 7A, TCDD failed to down-regulate StAR expression in cultured fetal testis, suggesting a mechanism different from direct action of TCDD on gonadal tissue. In a study examining the effect of toxic coplanar polychlorinated biphenyl (3,3',4,4',5-pentachlorobiphenyl) on steroidogenesis in cultured mouse fetal testis, it has been reported that polychlorinated biphenyl reduces Cyp11a1 but not 3-HSD and Cyp17 (49). Thus, some gonadal mechanisms necessary for steroid synthesis may be damaged by the direct effect of TCDD on the tissue. The present study demonstrates that the initial impairment of pituitary synthesis of gonadotropin(s) by TCDD or other mechanisms, including the storage and release of the hormone, mainly contributes to the reduced expression of steroidogenetic mechanisms in the testis. The effect of dioxins on circulating gonadotropins in adults is much less clear: whereas one group (50) reported a reduction in serum LH in TCDD-treated rats [male Sprague Dawley (SD) strain], other investigators (52) demonstrated either an increase (female SD strain) (51) or no change at all (male SD rats). In fetal rats (Has/Wistar and Long-Evans), the LH content of the pituitary has been reported to be either increased or remain unchanged after maternal exposure to TCDD (34). Considering this observation together with our present data showing that serum LH in fetuses is significantly reduced by TCDD, it may be that TCDD impairs the release of LH from the pituitary as well as affecting its synthesis.
The mechanism whereby TCDD reduces the synthesis of the LH subunit in the pituitary remains unknown. It is well established that GnRH plays an important role in regulating the transcription of LH as well as the -subunit genes (53, 54). As far as the content of GnRH in the fetal hypothalamus is concerned, this was not changed after maternal exposure to TCDD (Fig. 8C). Therefore, TCDD is suggested to act on the following targets: either the regulatory mechanism located downstream of GnRH or a different signaling pathway not involving GnRH. Concerning the first possibility, it has been reported that GnRH produces, after binding to its receptors, activation of protein kinase C and an increase in cellular Ca2+ concentration (55, 56). Because LH and the -subunit are differently regulated by these two factors (57, 58), TCDD may specifically affect either protein kinase C activation or Ca2+ influx in fetal brain to reduce the expression of LH. The secretion pattern of GnRH from the hypothalamus is important for the regulation of LH. Whereas intermittent secretion of GnRH is needed for the expression of LH, a constant pulse of this hormone favorably stimulates the transcription of the -subunit gene (53, 54). It is, therefore, an alternative possibility that TCDD reduces the pulsatile pattern of GnRH secretion, resulting in a reduction in LH expression. Regarding the second possibility described above, the transcription of the LH gene is known to be governed by regulatory factors involving SF-1, early growth response protein-1 (Egr-1) and stimulating protein-1 (SP-1) (59, 60, 61, 62). If GnRH is not a TCDD target, a reduction in the expression or function of the above trans-factors may occur after TCDD treatment. It would be of interest to discover whether AhR contributes to the regulation of LH gene expression. When we searched the xenobiotic-responsive element, a consensus sequence for binding to AhR, by homology matching, we found a core xenobiotic-responsive element (-GCGTG-) at around –580 bp of the rat LH gene upstream. Thus, it may be possible that AhR directly regulates LH gene expression, although this should be clarified in future studies.
In conclusion, this study has provided evidence that maternal exposure to TCDD reduces the synthesis of LH in fetal brain, resulting in a reduction in the expression of testicular StAR. We have also shown a reduction in fetuses of a number of other proteins that are necessary for steroidogenesis and steroid action. The impairment detected is suspected to be one of the reasons for TCDD-induced reproductive and developmental toxicities. The reduction in LH is suggested to be due to reduced synthesis of the LH subunit. The mechanism of this reduction is of much interest, and its clarification would improve our understanding of dioxin toxicity.
Footnotes
This work was supported by a Research Grant 15190101 from the Ministry of Health, Labour, and Welfare in Japan.
The authors have nothing to declare.
First Published Online October 27, 2005
Abbreviations: AhR, Aryl hydrocarbon receptor; AR, androgen receptor; ARNT, AhR nuclear translocator; 8-Br-cAMP, 8-bromoadenosine-cAMP; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1; eCG, equine chorionic gonadotropin; ER, estrogen receptor; GATA-4, GATA-binding protein 4; GD, gestational day; HSD, hydroxysteroid dehydrogenase; LHR, LH receptor; P450 or CYP, cytochrome P450; PBR, peripheral benzodiazepine receptor; SD, Sprague Dawley; SF-1, steroidogenic factor-1; StAR, steroidogenic acute-regulatory protein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Accepted for publication October 18, 2005.
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Department of Cell Fate Modulation (T.K.), Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto 860-0811, Japan
Division of Active Transport (T.K.), National Institute for Physiological Sciences, Aichi 444-8585, Japan
Abstract
Reproductive and developmental disorders are the most sensitive toxic effects caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is thought to produce many, if not all, of these toxic effects by impairing steroidogenesis and/or steroid action during the prenatal or early postnatal stages. However, the mechanism of the antisex steroid effect of TCDD is not well understood. This study revealed that steroidogenic acute-regulatory protein (StAR), a key transporter of cholesterol for steroidogenesis, in the testes of fetal rats are down-regulated by maternal exposure to TCDD. It was also shown that many mRNAs of steroidogenetic enzymes, including cytochromes P450 11A1, 17, and 11B1 and 3-hydroxysteroid dehydrogenase, are reduced in fetuses of TCDD-treated dams in a testis-specific manner. The same was also observed for the expression of estrogen- receptors and androgen receptors. Whereas StAR expression was not affected by TCDD in cultured fetal testis, the fetal serum content of LH, a pituitary regulator of StAR, was significantly reduced by TCDD. In agreement with this, pituitary expression of LH subunit mRNA in fetuses was reduced by maternal exposure to TCDD, whereas the -subunit remained unchanged. The reduction in LH is suggested to occur by a mechanism different from the reduction in the GnRH level. Direct supply of exogenous gonadotropin to TCDD-exposed fetuses completely abolished the reduction of StAR expression. Taken together, these results demonstrate that TCDD impairs steroidogenesis in the fetus by targeting pituitary gonadotropins.
Introduction
DIOXINS, EXEMPLIFIED BY 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are highly persistent environmental pollutants. These substances exert a wide variety of toxic effects in mammals via a mechanism involving the activation of the aryl hydrocarbon receptor (AhR) (1, 2, 3). The AhR is a regulatory factor of gene transcription, and a number of genes are under the control of this receptor (4, 5, 6, 7). Although the AhR plays a crucial role in dioxin toxicity, it is still not known which genes regulated by AhR contribute to dioxin toxicity. Among the possible health injuries after exposure to dioxins, the reproductive disorders are of particular interest because the damage to fetuses and newborn pups appear at doses much lower than those associated with toxic effects in adults (8).
To date, studies using experimental animals have identified multiple forms of reproductive and developmental damage (8). These include teratogenicity, growth retardation of fetuses/infants, and impaired mental and sexual function after development to adults. In the latter case, it has been reported that male offspring born from TCDD-treated dams exhibit a defect in sexual behavior (9, 10). This seems to be closely related to the observations that show the disappearance of male-type behavior in male mice deficient in estrogen synthase [cytochrome P450 19 (CYP19)], estrogen receptors (ER), and androgen receptors (AR) (11, 12, 13, 14). Thus, it is conceivable that TCDD and related compounds exert some forms of reproductive toxic effects by impairing sex steroid synthesis and/or blocking its function. In CYP19-knockout mice, recovery of the defect in sexual behavior needs continuous treatment with exogenous estrogen from the newborn pup stage onward, whereas treatment only at the adult stage fails to improve the impairment. This clearly indicates that estrogen-dependent signaling during the fetal or early neonatal stages is necessary for acquiring some forms of phenotype in adults. The effects of TCDD on reproduction, development, behavior after development, and steroid hormone status have been studied extensively. However, the majority of such research examined the effects of maternal exposure to dioxins on such end points using offspring of pre- or postpubertal age (Refs.9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 ; also see references therein), and it is largely unknown how TCDD affects steroidogenesis and steroid action in the fetus. To address this issue, we examined the effect of TCDD on fetal expression of enzymes and transporters involved in steroidogenesis and of steroid hormone receptors and propose a mechanism governing the alteration of these mechanisms.
Materials and Methods
Materials
TCDD was obtained from AccuStandard Inc. (New Haven, CT). [14C]progesterone (2.04 TBeq/mol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Its radiochemical purity was confirmed to be over 99% by thin-layer chromatography (TLC) [Rf = 0.62; solvent used: benzene-acetone = 3:1 (vol/vol)]. An antibody against rat steroidogenic acute-regulatory protein (StAR) was obtained from Abcam Ltd. (Cambridge, UK). The other reagents were of the highest grade commercially available.
Animals and treatments
Female Wistar rats (10–14 wk old) were paired overnight with male Wistar rats. The next morning, sperm in the vaginal smears was checked by microscopy (x100–400) to confirm pregnancy. When the presence of sperm was detected, the day was designated as gestational day (GD)0 of pregnancy. In typical experiments, pregnant rats were treated once with TCDD (1 μg/kg per 2 ml corn oil, orally) at GD15, and their fetuses were removed at GD20 for further experiments. In the dose-response relationship experiment, different doses (0.01, 0.1, and 1 μg/kg per 2 ml corn oil) of TCDD were given to pregnant rats in a schedule described above. In a separate experiment, pregnant rats were treated with TCDD at GD8, followed by removal of fetuses at GD20. When pups born from dams pretreated with TCDD at GD15 were examined, testes were obtained from the pups at a postnatal age of 7 d. The effect of TCDD on StAR expression in the testis was also examined in pubertal rats as a reference experiment. In this case, 44-d-old male Wistar rats were treated once with 1 μg/kg TCDD, and the testes were removed 5 d after the treatment. Tissues of rats were stored at –80 C before mRNA extraction, immunoblotting, and the assay of CYP17 activity.
Direct injection of equine chorionic gonadotropin (eCG) to fetuses was performed as follows. Pregnant rats (GD17) pretreated with TCDD (1 μg/kg) at GD15 were anesthetized with sodium pentobarbital [30 mg/kg per 2 ml PBS, ip]. The abdomen was opened by a 3-cm incision and both uteruses were removed from the body. Surgery was carried out while pouring warm PBS (37 C) on the uteruses to avoid drying of the tissue. Fetuses in the right and left uteruses were treated as control and eCG-treated groups, respectively. eCG in PBS (5 IU per 5 μl) was injected into the back of the fetuses in the uterus using a syringe fitted with a 31G needle. The control fetuses were given PBS. Because fetal sex cannot be judged during this operation, all fetuses were injected with eCG or PBS. Male fetuses were confirmed by detecting the presence of testes when they were dissected. After injection of eCG or PBS into the fetuses, the uterus containing pups was returned to the dam, and 5 ml warm PBS were poured into the abdominal cavity, and the wound was stitched up. The operation described above was completed within 30 min. Treated dams were housed individually, and the fetal testes were removed at GD20.
All experiments were approved by the Institutional Animal Care and Experiment Committee of the Graduate School of Pharmaceutical Sciences of Kyushu University.
Tissue culture of fetal testis
Fetuses were removed from untreated pregnant rats at GD20. A testis was put on a well of a 12-well plate filled with DMEM (1 ml/well) containing 100 U/liter penicillin, 0.01% streptomycin, and 1 mM sodium pyruvate and washed once by changing the medium. After treatment of the tissue with TCDD and/or 8-bromoadenosine-cAMP (8-Br-cAMP), forskolin, and eCG, the testis was cultured at 37 C for 24 h in an atmosphere of 5% CO2 in air. TCDD and forskolin were dissolved in dimethyl sulfoxide at concentrations of 100 pmol/ml medium per 5 μl and 10 μmol/ml medium per 5 μl, respectively. 8-Br-cAMP and eCG were dissolved in PBS at concentrations of 1 μmol/ml medium per 5 μl and 10 IU/ml medium per 5 μl, respectively. The treated tissue was washed twice with PBS and stored at –80 C before use.
Quantitative RT-PCR
Tissue mRNA was determined by RT-PCR according to the method described elsewhere (25). In the preparation of cDNA, 0.1 μg extracted mRNA (liver, testis, adrenal, and pituitary) and 1.0 μg (hypothalamus) was reverse transcribed to cDNA. The primer pairs used for amplification of cDNA are shown in Table 1. The PCR was performed using 1 μl of the reverse transcription reaction mixture in a final volume of 50 μl (25). In the cases of ER (3 μl) and adrenal CYP11B1 (1 μl, 20-fold diluted cDNA solution), the indicated volume of cDNA solution was used for PCR. All PCRs were performed under conditions guaranteeing template quantity-dependent amplification. The conditions used for PCR were as follows: 94 C for 2 min, x-cycles (94 C for 30 sec, y C for 30 sec, and 72 C for 30 sec), and then 72 C for 10 min. The extension cycle and annealing temperature, shown as x and y, respectively, are given in Table 1. Different temperatures for denaturation at the first step of PCR and the initial step in each extension reaction were set as described below: 95 C for StAR, cytochrome P450 1A1 (CYP1A1), CYP11A1, CYP11B1, 3-hydroxysteroid dehydrogenase (HSD), steroidogenic factor-1 (SF-1) and LH receptor (LHR); 96 C for CYP11B2; and 97 C for AR. The extension reaction was carried out for 15 sec (72 C) for GnRH and 60 sec (72 C) for StAR, CYP1A1, CYP11A1, 3-HSD, 17-HSD, SF-1, LHR, AhR, and AhR nuclear translocator (ARNT). The cDNA amplified was electrophoresed in 1.5% agarose gel and stained with ethidium bromide. When necessary, the band image was extracted by scanner and quantified using National Institutes of Health Image software (version 1.62). The amount of quantified mRNA was normalized by -actin mRNA, and, in some figures, the value was expressed as a relative level to the control.
Assay of CYP17 activity
Testes of fetuses (4–11 individuals) in one dam were combined and homogenized in 0.25 M sucrose containing 5 mM potassium phosphate (pH 7.4), 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 20% glycerol (0.05 ml per one testis). [14C]progesterone (7.5 μM, 670 GBeq/mol) was incubated at 37 C with 1 mM nicotinamide adenine dinucleotide phosphate reduced and 30 μg protein testis homogenate in a final volume of 200 μl 0.1 M potassium phosphate (pH 7.4). The mixture was extracted three times with 400 μl ethyl acetate-isooctane (1:1, vol/vol). Pooled organic phase was evaporated to dryness under N2, and the residue was reconstituted in 100 μl methanol containing nonlabeled carries (200 μM progesterone and 17-hydroxyprogesterone). A portion (10 μl) of this solution was subjected to thin-layer chromatography (Silica-gel gel 60 F254; Merck, Darmstadt, Germany; solvent: benzene-acetone 3:1, vol/vol). The area to which the unchanged progesterone (Rf, 0.62) and 17-hydroxyprogesterone (Rf, 0.49) had migrated was visualized under UV light (254 nm), excised, and the radioactivity measured in scintillation cocktail (ACSII; Amersham, Piscataway, NJ).
Other methods
Immunoblotting with antibody against antirat StAR was performed by the method reported previously (25). Briefly, tissue samples were electrophoresed by Laemmli’s method (26), and proteins on the gel were electrically transferred to a polyvinilidene difluoride membrane (27). StAR protein on the membrane was visualized using secondary antibody conjugated with alkaline phosphatase (28). The content of fetal serum LH was determined by ELISA using a commercial kit (Endocrine Technologies, Inc., Newark, CA). Serum of male fetuses in one dam was pooled and diluted twice with diluent supplied with the kit before assay. Statistical differences between two groups were calculated by Student’s t test. Other statistical differences were estimated by ANOVA with a post hoc test (Fisher’s protected least significant difference method).
Results
TCDD-induced change in the expression of fetal proteins involved in steroidogenesis and steroid action
Pregnant Wistar rats were given a single administration of TCDD (1 μg/kg, orally) at GD15, and mRNA expression in fetal tissues was determined at d 20 of gestation. The hepatic expression of CYP1A1 mRNA, a well-known marker for AhR-regulated gene, was markedly increased by TCDD (Fig. 1), and it was evident that TCDD given to dams distributes to fetuses and affects gene expression. StAR and the peripheral benzodiazepine receptor (PBR) play important roles in cholesterol transportation to the mitochondrial inner membrane in which steroidogenetic reactions occur (29). Of these transporters, the fetal expression of testicular StAR mRNA was markedly reduced after TCDD treatment, whereas adrenal StAR remained unchanged (Fig. 2, A and B). A reduction in testicular StAR protein was also confirmed by immunoblotting (Fig. 2C). The 7-d-old offspring born from TCDD-treated dams did not show any reduction in the expression of testicular StAR (Fig. 3A). The absence of TCDD-induced reduction in the expression of testicular StAR was also observed in pubertal rats (49 d old) that were given TCDD at postnatal d 44 (Fig. 3B). Thus, the reduction in the expression of this transporter was specific to the fetus. From these data, we focused our main interest on the StAR alteration and tried to clarify the mechanism in this study.
In addition to StAR, the expression in the fetal testis of several steroidogenetic enzymes (CYP11A1, CYP17, CYP11B1, and 3-HSD) was reduced by TCDD treatment (Fig. 4A). Changes in these enzymes were also specific for the testis, except for CYP17, which was reduced in the adrenals of both sexes after TCDD treatment (Fig. 4A). In agreement with the change in CYP17 expression, testicular homogenate prepared from fetuses in TCDD-treated dams exhibited a lower ability to use progesterone and produce 17-hydroxylated metabolite (Fig. 4B). As far as the receptors were concerned, a significant decrease in the expression of testicular ER and AR mRNAs was observed (Fig. 4A). Treating dams with TCDD had no effect on the expression of AhR and its partner receptor (ARNT) in the fetal testes and adrenals (Fig. 4A). These results strongly suggest that steroidogenesis and steroid action are impaired in the fetuses in TCDD-treated dams. As can be seen in Fig. 5A, reduced expression of fetal testicular StAR, CYP11A1, and CYP17 was seen only after treating dams with 1 μg/kg TCDD, and lower doses (0.1 and 0.01 μg/kg) had no significant effect on the expression of these mRNAs. Earlier exposure at GD8 to TCDD gave comparable results to treatment at GD15 as far as the expression of StAR, CYP11A1, and CYP17 was concerned (Fig. 5, A and B).
Change in the expression of regulatory factors by TCDD and the effect of TCDD on cultured fetal testis
Expression of the StAR gene is under the control of transcriptional factors involving positive regulators [SF-1, GATA-binding protein 4 (GATA-4)] and a negative regulator [dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1)] (30, 31). Of these factors, SF-1 mRNA was significantly reduced by treating dams with TCDD (Fig. 6). It is, therefore, likely that a reduction in SF-1 contributes to StAR reduction. We then examined the possibility that TCDD could reduce StAR expression by direct action on the fetal testis. To this end, the testes of fetuses removed from untreated dams were cultured, and the effect of TCDD on StAR expression was examined. The results obtained showed that StAR expression in cultured testes is completely unaffected by TCDD treatment (Fig. 7A). Pituitary gonadotropins, such as LH, FSH, and chorionic gonadotropin, play a key role in StAR expression in gonadal tissues. These pituitary factors bind to specific receptors expressed on the plasma membrane of peripheral target tissues and activate protein kinase A-dependent signaling, which further activates transcriptional factors, such as SF-1, GATA-4, and DAX-1 (31, 32, 33). When cultured testis was treated with 8-Br-cAMP, an analog of cAMP, forskolin, and eCG, a significant increase in StAR mRNA was observed as expected (Fig. 7A). This observation showed that the cultured testis is able to respond to gonadotropin to cause gene expression. TCDD failed to suppress induced expression of StAR by 8-Br-cAMP, forskolin, and eCG (Fig. 7A). These data do not support the view that TCDD directly affects the testis to suppress the expression of steroidogenetic mechanisms involving StAR. Because CYP1A1 is normally induced by TCDD (Fig. 7B), TCDD added to cultured testis is assumed to enter the cells.
Effect of TCDD on gonadotropin synthesis in fetal brain and its significance for testicular StAR expression
As described above, pituitary gonadotropins are primary factors regulating the expression of StAR in the peripheral tissues. To identify the mechanism of the TCDD-induced reduction in fetal StAR expression, we then focused on the possibility that dioxin impairs the expression and function of gonadotropin. The expression of the LHR mRNA of the fetal testis was not affected by maternal exposure to TCDD (Fig. 8A). On the other hand, the -subunit mRNA of pituitary LH was significantly reduced in fetal brain by TCDD, whereas the -subunit mRNA did not change (Fig. 8A). In agreement with this, LH in fetal serum was significantly reduced by treating dams with TCDD (Fig. 8B). These data suggest that TCDD down-regulates steroidogenetic proteins in fetal testis by reducing circulating pituitary hormone(s) in fetuses. Hypothalamic GnRH is one of the important factors regulating the synthesis of gonadotropin in the pituitary and its secretion. However, maternal exposure to TCDD produced no effect on the GnRH mRNA in the fetal hypothalamus (Fig. 8C). To confirm the hypothesis that a reduction in circulating gonadotropin is a reason for the TCDD-caused reduction in fetal StAR expression, we tried to combat the StAR reduction in TCDD-exposed fetuses by direct injection of gonadotropin. The results obtained supported the above view, and direct supply of exogenous eCG to male fetuses without removing them from the dam completely restored the expression of testicular StAR to control levels (Fig. 9).
Discussion
We have shown that there is a reduction in the expression of StAR and steroidogenetic enzymes in fetal testis after maternal exposure to TCDD (Fig. 10). Because the reduction in these proteins is specific to the fetal stages, this suggests that such alterations produce reproductive and developmental toxicities by impairing the sex steroid-mediated imprinting of gender-specific phenotypes. These data assume a reduction in the formation of androgen and estrogen in the testis after treatment of the dams with TCDD. However, other workers have reported that testosterone in the testis and serum is not reduced in the fetuses of TCDD-exposed pregnant rats (Han/Wistar, Long-Evans, and Holtzman strains) (34, 35), whereas early reports described a reduction in fetuses (9) and adults (36, 37). The reason for these inconsistencies is unknown. However, because all these studies assayed steroid hormones by immunological techniques using antibodies against target steroids, it is conceivable that the antibody used detects alterations in other steroids to give false-positive or -negative results. Alternatively, there may be great species/strain differences in TCDD-induced down-regulation of StAR and other steroidogenetic proteins. On the other hand, maternal treatment with TCDD is reported to be able to reduce serum -estradiol in fetuses and offspring of prepubertal age (16, 35). The present study shows that the expression of testicular CYP19 remains unchanged in TCDD-exposed fetuses. If androgen and estrogen levels are reduced by TCDD, the supply of precursors of these steroids should be reduced. This same assumption has been proposed by other investigators (36, 37, 38). Thus, although the change in proteins contributing to the early steps of steroidogenesis reported here is thought to be important for the antiestrogenic and -androgenic effects of dioxin, further studies are needed to clarify the alteration in gonadal steroids in the fetus and during the early postnatal stages and the mechanism involved.
Regarding the antisex steroid effect of dioxin, mechanisms other than the reduced production of steroid hormones have also been proposed. These include: 1) decreased expression of ER, AR, and LHR (22, 39, 40, 41); 2) enhanced inactivation of steroid hormones due to induction of steroid-metabolizing enzymes such as CYP1B1 (42, 43); and 3) an antagonist effect of dioxins on ER-dependent signaling (16, 44, 45). Although the TCDD effect is likely due to a combination of these mechanisms, the present study supports the reduction in AR and ER. LH plays a major role in the testicular production of testosterone as shown by the finding that LHR-deficient mice lose almost all the testosterone in their testes (46). However, the residual testosterone that is produced by an LH-independent mechanism is suggested to be sufficient for some testicular functions, such as spermatogenesis (46). This assumption does not support the view that a reduction in steroid hormone significantly contributes to the antisex steroid effects of TCDD. However, because mice lacking AR and ER exhibit abnormalities in gender-specific phenotypes (13, 14), a marked reduction in the level of sex steroids could cause such disorders. The present study shows that the action of sex steroids in the fetuses of TCDD-treated dams is impaired by several mechanisms including a reduction in steroidogenesis and receptor expression. These alterations show that a reduction in the quantity and function of sex steroids makes a significant contribution. As far as the reproductive and developmental damage produced by dioxins is concerned, much of this is thought to occur by mechanisms involving AhR (47, 48).
It would be of great interest to discover whether AhR contributes to the TCDD-produced change in testicular steroidogenesis in the fetus. Concerning this issue, AhR-deficient mice have been reported to resist a reduction in testicular Cyp11a1 produced by TCDD, whereas TCDD produces a significant reduction in this enzyme in wild-type mice (41). This observation supports the view that AhR plays a role in at least some cases of TCDD-induced impairment of steroidogenesis. As can be seen in Fig. 5, CYP11A1 mRNA seemed to be reduced more markedly after TCDD treatment at GD8 than GD15, although the magnitude of the TCDD-produced reduction in StAR and CYP17 was comparable between the dosing timings. Although the reason for these differences is not known, it is conceivable that the mechanism governing StAR and CYP17 regulation differs from CYP11A1 regulation in which the AhR plays a crucial role. The treatment at GD8 would be assumed to produce a prolonged exposure of fetuses to TCDD because of its persistent nature in the body. It is, therefore, possible to assume that CYP11A1 reduction needs long-term exposure to TCDD.
As shown in Fig. 7A, TCDD failed to down-regulate StAR expression in cultured fetal testis, suggesting a mechanism different from direct action of TCDD on gonadal tissue. In a study examining the effect of toxic coplanar polychlorinated biphenyl (3,3',4,4',5-pentachlorobiphenyl) on steroidogenesis in cultured mouse fetal testis, it has been reported that polychlorinated biphenyl reduces Cyp11a1 but not 3-HSD and Cyp17 (49). Thus, some gonadal mechanisms necessary for steroid synthesis may be damaged by the direct effect of TCDD on the tissue. The present study demonstrates that the initial impairment of pituitary synthesis of gonadotropin(s) by TCDD or other mechanisms, including the storage and release of the hormone, mainly contributes to the reduced expression of steroidogenetic mechanisms in the testis. The effect of dioxins on circulating gonadotropins in adults is much less clear: whereas one group (50) reported a reduction in serum LH in TCDD-treated rats [male Sprague Dawley (SD) strain], other investigators (52) demonstrated either an increase (female SD strain) (51) or no change at all (male SD rats). In fetal rats (Has/Wistar and Long-Evans), the LH content of the pituitary has been reported to be either increased or remain unchanged after maternal exposure to TCDD (34). Considering this observation together with our present data showing that serum LH in fetuses is significantly reduced by TCDD, it may be that TCDD impairs the release of LH from the pituitary as well as affecting its synthesis.
The mechanism whereby TCDD reduces the synthesis of the LH subunit in the pituitary remains unknown. It is well established that GnRH plays an important role in regulating the transcription of LH as well as the -subunit genes (53, 54). As far as the content of GnRH in the fetal hypothalamus is concerned, this was not changed after maternal exposure to TCDD (Fig. 8C). Therefore, TCDD is suggested to act on the following targets: either the regulatory mechanism located downstream of GnRH or a different signaling pathway not involving GnRH. Concerning the first possibility, it has been reported that GnRH produces, after binding to its receptors, activation of protein kinase C and an increase in cellular Ca2+ concentration (55, 56). Because LH and the -subunit are differently regulated by these two factors (57, 58), TCDD may specifically affect either protein kinase C activation or Ca2+ influx in fetal brain to reduce the expression of LH. The secretion pattern of GnRH from the hypothalamus is important for the regulation of LH. Whereas intermittent secretion of GnRH is needed for the expression of LH, a constant pulse of this hormone favorably stimulates the transcription of the -subunit gene (53, 54). It is, therefore, an alternative possibility that TCDD reduces the pulsatile pattern of GnRH secretion, resulting in a reduction in LH expression. Regarding the second possibility described above, the transcription of the LH gene is known to be governed by regulatory factors involving SF-1, early growth response protein-1 (Egr-1) and stimulating protein-1 (SP-1) (59, 60, 61, 62). If GnRH is not a TCDD target, a reduction in the expression or function of the above trans-factors may occur after TCDD treatment. It would be of interest to discover whether AhR contributes to the regulation of LH gene expression. When we searched the xenobiotic-responsive element, a consensus sequence for binding to AhR, by homology matching, we found a core xenobiotic-responsive element (-GCGTG-) at around –580 bp of the rat LH gene upstream. Thus, it may be possible that AhR directly regulates LH gene expression, although this should be clarified in future studies.
In conclusion, this study has provided evidence that maternal exposure to TCDD reduces the synthesis of LH in fetal brain, resulting in a reduction in the expression of testicular StAR. We have also shown a reduction in fetuses of a number of other proteins that are necessary for steroidogenesis and steroid action. The impairment detected is suspected to be one of the reasons for TCDD-induced reproductive and developmental toxicities. The reduction in LH is suggested to be due to reduced synthesis of the LH subunit. The mechanism of this reduction is of much interest, and its clarification would improve our understanding of dioxin toxicity.
Footnotes
This work was supported by a Research Grant 15190101 from the Ministry of Health, Labour, and Welfare in Japan.
The authors have nothing to declare.
First Published Online October 27, 2005
Abbreviations: AhR, Aryl hydrocarbon receptor; AR, androgen receptor; ARNT, AhR nuclear translocator; 8-Br-cAMP, 8-bromoadenosine-cAMP; DAX-1, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1; eCG, equine chorionic gonadotropin; ER, estrogen receptor; GATA-4, GATA-binding protein 4; GD, gestational day; HSD, hydroxysteroid dehydrogenase; LHR, LH receptor; P450 or CYP, cytochrome P450; PBR, peripheral benzodiazepine receptor; SD, Sprague Dawley; SF-1, steroidogenic factor-1; StAR, steroidogenic acute-regulatory protein; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Accepted for publication October 18, 2005.
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