Arylhydrocarbon receptor (AhR) activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) triggers its downstream signaling pathway to exert adverse effects on vasculature development, which can be initiated by vasculogenesis, followed by angiogenesis, or vascular remodeling, in a variety of animals including avians, piscines and mammals. The placenta, a mammalian organ rich in vasculature, consists of endothelial and trophoblast cells of fetal origin, which proliferate and differentiate under hypoxic condition in the uterine horn. Our studies demonstrated that vascular remodeling occurs prominently in the placenta of the control Holtzman rat strain during the late period of gestation, and induces changes in cell shape and elimination by apoptosis of trophoblasts. As a result, the net volumes of both maternal and fetal blood in the placenta increase to cope with the essential requirements of oxygen and nutrients in the late period of gestation. On the other hand, in utero exposure to TCDD markedly suppressed the development of sinusoids and trophoblast cells and the apoptosis of trophoblast cells with a concomitant increase in the incidence of fetal death under hypoxic condition. A crosstalk between the hypoxia-inducible factor (HIF)-mediated pathway and AhR-mediated pathway is considered to play an important role in this physiological process. No such changes were observed in the Sprague-Dawley rat strain that turned out to have an AhR conformation identical to that of the Holtzman rat strain. In this commentary, we will discuss a possible link of the TCDD toxicities with the AhR signaling pathway and gestation-related diseases.

TCDD (2,3,7,8-tetrachlorodebenzo-p-dioxin) requires the presence of the aryl hydrocarbon receptor (Ahr) gene for its toxic effects, such as reproductive disorders in male offspring of maternally exposed rats and mice. To study the involvement of the Ahr gene in producing the toxic phenotype with respect to testicular development, we administered a relatively high dose of TCDD to mice with three different maternally derived Ahr genotypic traits, and then compared several Ahr-dependent alterations among male reproductive systems on Postnatal Day 14. Reduction in anogenital distance and expression of prostatic epithelial genes in the urogenital complex (UGC) were detected in Ahr(+/+) and Ahr(+/-) mice exposed to TCDD, whereas no difference was observed in Ahr(-/-) mice. In situ hybridization revealed the absence of probasin mRNA expression in the prostate epithelium, despite the obvious development of prostatic lobes in TCDD-exposed mice. In contrast to obvious prostatic dysfunction and induction of cytochrome P450 (CYP) family genes in the UGC by TCDD, no alterations in testicular functions were observed in germ cell/Sertoli cell/interstitial cell marker gene expression or CYP family induction. No histopathological changes were observed among the three genotypes and between control and TCDD-exposed mice. Therefore, mouse external genitalia and prostatic development are much more sensitive to TCDD treatment than testis. Further, the Ahr gene, analyzed in this study, does not significantly contribute to testicular function during perinatal and immature stages, and the developing mouse testis appears to be quite resistant to TCDD exposure.

The maintenance of the placental vasculature is essential for sustaining normal fetal growth. On the basis of our previous observation that fetal death was accompanied by placental hypoxia upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (R. Ishimura et al., 2002a, Toxicol. Appl. Pharmacol. 185, 197-206), we here investigated the effects of TCDD on the placenta, focusing on the development of the labyrinth zone. Holtzman rats were administered a single oral dose of 1.6 mug of TCDD/kg body weight or an equivalent volume of vehicle (control) on gestation day (GD) 15, and placental tissues were analyzed on GD20. Immunohistochemical staining showed that the exposure to TCDD decreased the size of maternal blood sinusoids and caused the constriction of fetal capillaries in the placenta. In contrast, we found that vascular remodeling occurred in the labyrinth zone of normal rat placenta; that is, the vascular development in the normal labyrinth zone during the late gestation (from GD16 to GD20) showed dilated maternal blood sinusoids and fetal capillaries accompanied by a decrease in thickness and the apoptosis of trophoblasts. The present results demonstrate that this remodeling is suppressed by TCDD, which is further supported by the decreased expression level of Tie2 mRNA, the gene which is associated with vascular remodeling, and also by the decrease in the number of apoptotic trophoblasts in TCDD-exposed rats. The present study provided a new finding on the development of the vasculature in the labyrinth zone during the late gestation under normal conditions and showed the inhibition of vascular remodeling in TCDD-exposed rats.

A single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioin (TCDD) administered to pregnant Holtzman (HLZ) rats on gestational days 15 (GD15) caused placental dysfunction, resulting in fetal death (Ishimura, R., Ohsako, S., Miyabara, Y., Sakaue, M., Kawakami, T., Aoki, Y., Yonemoto, J., Tohyama, C., 2002a. Increased glycogen content and glucose transporter 3 mRNA level in the placenta of Holtzman rats after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol. 178, 161-171; Ishimura, R., Ohsako, S., Kawakami, T., Sakaue, M., Aoki, Y., Tohyama, C., 2002b. Altered protein profile and possible hypoxia in the placenta of 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed rats. Toxicol. Appl. Pharmacol. 185, 197-206). In order to investigate the mechanism underlying the TCDD-induced fetal death, we compared two outbred strains of rats, namely, the HLZ and the Sprague-Dawley International Genetic Standard rats (SD-IGS), a strain with characteristics resembling those of the HLZ rats. Pregnant HLZ and SD-IGS rats were administered TCDD as a single dose by gavage on GD15, as described within the parentheses (HLZ, 0, 1.6 mug TCDD/kg; SD-IGS, 0, 2, 5, 10 microg TCDD/kg). Whereas a high incidence (14%) of fetal death was observed on GD20 in the HLZ rats, no fetal deaths occurred in the SD-IGS rats, even at the highest dose of TCDD. A histological marker of cellular abnormality at the placental junctional zone, i.e., delay in the disappearance of the glycogen cells and cysts filled with an eosinophilic material (GC-EM), which normally disappear by GD20, was observed in the HLZ rats after exposure to the lowest dose of TCDD (1.6 microg TCDD/kg), but not in the SD-IGS rats even after exposure to the highest dose of TCDD. Furthermore, maternal blood sinusoids in the labyrinth zone were constricted following exposure to TCDD in the HLZ, but not SD-IGS rats. These observations indicate that HLZ rats are more susceptible to the adverse effects of TCDD on fetal growth and placental function, than SD-IGS rats. Direct sequencing analysis of the aryl hydrocarbon receptor (AhR) gene revealed no difference in the primary structure of the receptor between the HLZ and SD-IGS rats. In addition, no significant differences were observed between the two strains of rats in the levels of induction of placental cytochrome P450 1A1, 1B1, AhR, and AhRR mRNAs following administration of serially increasing doses of TCDD (0.0125, 0.05, 0.2, 0.8, and 1.6 microg TCDD/kg), indicating that the activity of TCDD-AhR complex in the placenta is similar between the HLZ and SD-IGS rats. Taken together, the above-described findings indicate that the higher susceptibility of HLZ rats to TCDD-induced placental dysfunction and fetal death may be modulated by other factor(s) in the genetic background of HLZ rats than the AhR.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an extremely toxic, persistent environmental contaminant that disrupts normal development in laboratory animals. In our earlier study, we found that exposure of preimplantation embryos to TCDD markedly induced cytochrome P4501A1 mRNA at the blastocyst stage. In the present study, to determine whether exposure of preimplantation embryos to TCDD affects fetal growth, we exposed preimplantation embryos to TCDD from the 1-cell stage to the blastocyst stage and then transferred them to unexposed recipient mice. On Embryonic Day 14, the fetuses exposed to TCDD during the preimplantation stage weighed less than the fetuses in the unexposed control group. Real-time reverse transcription-polymerase chain reaction analysis revealed that exposure of preimplantation embryos to TCDD tended to decrease the expression levels of the imprinted genes H19 and Igf2 (insulin-like growth factor 2 gene). Use of bisulfite genomic sequencing demonstrated that the methylation level of the 430- base pair H19/Igf2 imprint control region was higher in TCDD- exposed embryos and fetuses than in the controls, and methyltransferase activity was also higher in the TCDD-exposed embryos than in the controls. To our knowledge, the present study is the first to provide evidence that TCDD exposure at the preimplantation stage alters the genomic DNA methylation status of imprinted genes, influences the expression level of imprinted genes, and affects fetal development.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low dose TCDD on placental function, pregnant Holtzman rats were given a single oral dose of 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day 15 (GD15), and changes in the gene expression in the placenta on GD20 were analyzed by two comprehensive methods, representational difference analysis (RDA) and DNA microarray technology. Candidates of TCDD-inducible and -suppressive genes were selected. Quantitative real-time PCR analysis was then performed to verify the induction or suppression levels of the candidate genes. Finally, we identified 81 TCDD-inducible and 21 TCDD-suppressive genes from the placenta of TCDD-treated Holtzman rats on GD20. One of the remarkable profiles of the gene expression was that glucose transporters were strongly up-regulated by the TCDD treatment. Furthermore, many interferon-inducible genes were also up-regulated by the treatment. They included several cytokines such as IP-10 known as a potent angiogenesis inhibitor. In addition, interferon molecules are known to suppress angiogenesis. The above observations suggest that activation of the interferon signaling pathway and the induction of anti-angiogenic factors by TCDD might have a role in causing the inhibition of neovascularization, resulting in the hypoxic state of placenta and increased incidence of fetal death.

In the course of profiling alterations of gene expression in the male reproductive system induced by anti-androgenic agents, 28 genes expressed in the testis or pituitary of adult rats were examined shortly after subacute administration of the well-known anti-androgen, flutamide (FM). FM (25 mg/kg/day) was orally administered to male rats for six days. On day 8 (D8) after the first dose of FM, intratesticular testosterone (T) levels had dramatically increased, but daily sperm production on D36 was significantly decreased. The mRNA levels of testicular and pituitary genes on D8 were measured by semiquantitative RT-PCR. Among the six testicular steroidogenic enzyme genes, the mRNAs of the P450 side chain cleavage, P450 17 alpha/C(17-20) lyase, and 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) genes significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III slightly decreased. Among the three steroid receptors examined, androgen receptor (AR) and glucocorticoid receptor (GR) mRNAs were significantly down-regulated (29% and 35%, respectively) in the testis, but there was no change in estrogen receptor alpha. There were no clear changes in expression of the gonadotropin receptors and Sertoli cell specific genes, but a slight increase was observed in expression of the lactose dehydrogenase-c mRNA, a germ cell specific gene. Among the three immediate early genes, c-myc mRNA was increased approximately 1.4-fold. In the pituitary, on the other hand, mRNAs for LHbeta and FSHbeta subunits and gonadotropin releasing hormone receptor had increased significantly. These results show that subacute FM administration first affected hypothalamus/pituitary hormone gene expression, then altered gonadotropin secretion, and subsequently induced over-expression of testicular steroidogenic enzyme genes. However, the significant up-regulation of 3betaHSD and down-regulation of AR mRNAs, despite the higher level of intratesticular T, might be explained by an antagonistic action of hydroxyflutamide retained in the testis. The profiles of alterations in gene expression observed will provide important information for the screening of adult male animals for anti-androgenic chemicals.

The effect of vinclozolin (VCZ), used as a fungicide and known to have anti-androgenic effects on spermatogenesis and gene expression in the male rat testis was investigated. In Experiment 1, VCZ (100 mg/kg/day) or flutamide (FM, 25 mg/kg/day) was orally administered to male Holzman rats for six days. 8 days after the last administration (D8), a drastic increase in intratesticular testosterone was detected in FM (4.2-fold over control) but not in VCZ treated animals, whereas on D36 post-administration, both groups showed similar levels. Significant decreases in daily sperm production were seen in both VCZ and FM-treated rats on D36. Semiquantitative RT-PCR analysis with testicular and pituitary mRNAs on D8 revealed that LHbeta and FSHbeta mRNAs were increased in the pituitary by VCZ, as well as by FM. Among the four testicular steroidogenic enzyme genes, cytochrome P450 side chain cleavage (P450scc) and cytochrome P450 17alpha/C(17-20) lyase (P450c17) mRNAs were significantly increased, whereas 17beta-hydroxysteroid dehydrogenase type III (17betaHSD) mRNA was not changed. A significant increase in 3beta-hydroxysteroid dehydrogenase type I (3betaHSD) and a decrease in androgen receptor (AR) mRNA were observed only in FM treated rats. Immunohistochemistry demonstrated intense staining of P450scc in the interstitial cells of VCZ-treated testis on D8. In Experiment 2, hormone levels were measured at 1, 3, 6, 12 and 24 hours after VCZ (100 mg/kg) administration to Sprague-Dawley rats. Serum LH level remained constant for the first 3 hours and started to increase at 6 hrs. In contrast, serum and intratesticular testosterone levels increased 2-fold at 1 hr and maintained the level until 24 hrs. P450c17 mRNA level was 2-fold increased at all periods, whereas no obvious changes were detected in the other steroidogenic enzyme genes. Although not statistically significant, AR mRNA level increased 2-fold, 3 hrs after VCZ administration. These results indicate that VCZ affects the pituitary in a similar manner as FM, but functions differently on testicular gene expression.

Exposure to a relatively low dose of 2,3,7,8-tetrachlorodebenzo-p-dioxin (TCDD) during mid-gestation induces a reduction of ventral prostate weight in rat offspring. Recently we reported that a single administration of TCDD (12.5-800 ng/kg body weight) to pregnant Holtzman rats on gestational day (GD) 15 caused a decrease in androgen receptor (AR) mRNA level in the ventral prostate during the prepubertal period, and we proposed that this reduction of AR mRNA is one of the most sensitive adverse endpoints due to perinatal exposure to TCDD (S. Ohsako et al, 2001, Toxicol. Sci. 60, 132-143). In the present study, to investigate the mechanism of a decrease in AR mRNA level, we administered TCDD to rats at other developmental stages and compared possible alterations of the male reproductive system. Pregnant Sprague-Dawley rats were given a single oral dose of 1 mug TCDD/kg body weight on GD 15 or GD 18, or male pups born from untreated dams were subcutaneously given a single dose of 1 jig TCDD/kg body weight on postnatal day 2 (PND 2). Offspring exposed on GD 15, GD 18, and PND 2 were sacrificed on PND 70. TCDD exposure on GD 15 resulted in significant decreases in the urogenital complex and ventral prostate weights and urogenital-glans penis length of male rat offspring, but not on GD 18 and PND 2. Testicular and epididymal weights were also lower than control group only in the TCDD-exposed GD 15 group. Anogenital distance was significantly reduced in the TCDD-exposed GD 15 and GD 18 groups, but not in the TCDD-exposed PND 2 group. Semiquantitative RT-PCR analysis showed that AR mRNA levels were decreased in the TCDD-exposed GD 15 group only, and that the constitutive level of cytochrome P450 1A1 (CYP1A1) mRNA in the ventral prostate was not changed by TCDD in any of the exposed groups. No changes in AR mRNA level were detected in the testis or brain in any of the TCDD-exposed groups. These results suggest the presence of a critical window during development with regard to impairments of male reproductive organs by in utero and lactational exposure to a low dose of TCDD.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during pregnancy causes fetal death in many animal species. In an earlier study we observed alteration of placental glucose kinetics at the same TCDD exposure level that resulted in fetal death (Ishimura et al., Toxicol. Appl. Pharmacol. 178, 161–171, 2002). In the present study, in order to identify the molecules that might explain the alterations of placental function and the mechanism of fetal death, we used two-dimensional gel electrophoresis (2D/E) to detect and identify placental proteins whose amounts changed after exposure to TCDD and we examined the expression properties of these proteins in the placenta during hypoxia by using the uterine artery ligation model. Pregnant Holtzman rats were given a single oral dose of 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestational day (GD) 15 and placental tissue was collected on GD16 and GD20. The 15,000 g supernatant fractions of placental homogenates from the control group and TCDD-exposed group were subjected to the 2D/E analysis, and the protein spots whose amounts had changed after exposure to TCDD were characterized by amino acid sequence analysis. The amounts of heat shock protein 27 (Hsp27) and β-tropomyosin (β-TM) in TCDD-exposed placentas tended to have increased on GD16 and had increased significantly on GD20, and these changes were followed by an approximately twofold increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) on GD20. Next, the uterine-artery ligation model was prepared on GD15, and the hypoxic placentas were collected on GD20. Two-D/E analysis of the 15,000 g supernatant proteins of the placentas revealed an increased level of GAPDH but not of other proteins, including Hsp27 and β-TM. The results of this study showed that the increase in GAPDH level during hypoxia previously observed in endothelial cells occurs in the placenta and indicated that the TCDD-exposed placentas were in a hypoxic state at the end of pregnancy. Finally, the results of this study suggested the possibility that the increased incidence of fetal death after exposure to TCDD was due to the placental hypoxia.

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low-dose TCDD on placental function in this study, pregnant Holtzman rats were given a single oral dose of 800 or 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day (GD) 15 and the results were observed on GD16 and GD20. The number of fetal deaths increased in the animals exposed to TCDD. Although fetal and placental weight did not differ significantly between the control group and the TCDD groups, histological differences from the control rats were clearly observed in the junctional zone (JZ) of the placentas of the TCDD-exposed rats. In the control placenta, glycogen cells occupied the majority of the JZ on GD16, but then decreased in number and almost disappeared by GD20, whereas on GD20 the placenta of the TCDD-exposed rats exhibited a larger area occupied by the glycogen cells and cysts filled with eosinophilic material surrounded by glycogen cells in the JZ than that of the control group. Glycogen assay revealed that the glycogen content of the placentas from the TCDD-exposed rats was higher than in the control rats. Semiquantitative RT–PCR analysis was performed to assess the expression of glucose transporter 1 (GLUT1) and GLUT3, the two major placental glucose transporter isoforms. On GD20 the level of expression of GLUT1 mRNA in the placentas was not different between the control and TCDD groups, whereas the level of expression of GLUT3 mRNA approximately doubled in both the 800 and 1600 ng/kg TCDD groups. GLUT3 mRNA expression was restricted to the labyrinth zone of placenta, where zone-specific expression of mRNA arylhydrocarbon receptor and induction of cytochrome P450 1A1 mRNA by TCDD were observed, and none was detected in the JZ. These results, including the increase of glycogen content and GLUT3 mRNA level in TCDD-exposed placentas, provide the first evidence of alteration of glucose kinetics in the placenta by TCDD.

Bisphenol‐A Affects Spermatogenesis in the Adult Rat Even at a Low Dose: MotoharuSakaue,et al.Environmental Health Sciences Division, National Institute for Environmental Studies—Bisphenol‐A (BPA), a xenobiotic estrogenic compound widely used as a plastics monomer, has been suspected to have a so‐called low dose effect on the reproductive system when administered transplacentally. In the present study, we investigated possible low‐dose effects of BPA on spermatogenesis in adult rats. Male rats (13 weeks old; W13) were administrated a daily oral dose of BPA, ranging from 2 ng to 200 mg/kg, for 6 days and examined for testicular weight (TW) and daily sperm production (DSP) at W14 and W18. A BPA dose as low as 20 jug/kg tended to decrease TW and significantly reduced both DSP and the efficiency of spermatogenesis (DSP per gram testis) at W18, showing that BPA suppressed a normal increase in DSP and TW from W13 to W18. A single administration of 20figBPA/kg to W13 rats affected the intensity or mobility of several protein spots in the testicular cytosol fraction as shown by two‐dimensional gel electrophoresis analysis. The present study showed that BPA at a low dose affects spermatogenesis in the adult rat.

We previously analysed the plasma membrane proteins of rat placenta and prepared a database of 150 plasma membrane proteins, expressed in a stage-specific manner, utilizing two-dimensional gel electrophoresis (2D/E) [Mol. Cell. Endocrinol. 115(1995)149]. In this study, we focused on the proteins, tentatively named psL-I (MW 36.2 kDa, pI 5.3) and psL-II (35.9 kDa, 5.3), which were expressed mainly in late pregnancy. Close to psL-I and psL-II on 2D/E gels, we also recognized more abundant proteins [psC-I (36.2 kDa, 5.4) and psC-II (35.9 kDa, 5.4), respectively] arranged side by side with the same MW but different pI. Expression of psL-I and psL-II was detected only in junctional zone of placenta, whereas psC-I and psC-II were expressed in both labyrinth and junctional zones. In addition, psL-I and psL-II began to increase on day 16 of pregnancy and peaked at term, whereas expression of psC-I and psC-II was relatively constant. The analysis of these four proteins (psL-I, psL-II, psC-I and psC-II) by preparative 2D/E, peptide mapping, amino acid sequence and mass spectrometry (MALDI-TOF-MS) revealed that psC-I was a G protein β1 subunit, and psC-II was a β2 subunit, and showed that psL-I and psL-II were molecular modified forms of psC-I and psC-II, respectively. Expression of these G protein β subunits (psL-I, psL-II, psC-I and psC-II) was also observed in rat choriocarcinoma cells, Rcho-1 cells. Expression of psC-I and psC-II was much higher than those of psL-I and psL-II, and their level was relatively constant regardless of the stage of differentiation in vitro. Interestingly, expression of psL-I and psL-II gradually increased in association with the differentiation. Since the expression of β1 and β2 subunit proteins and their mRNAs was constant during the process of differentiation in Rcho-1 cells, the expression of these lower pI forms of G protein subunits (psL-I and psL-II) was thought to be post-translationally regulated. In conclusion, there are modified forms of G protein β1 and β2 subunits, in the placenta and Rcho-1 cells, which are expressed in a pregnancy-stage or differentiation stage specific manner.