Seiichiroh Ohsako

Distinct response to dioxin in an arylhydrocarbon receptor (AHR)-humanized mouse

There are large inter- and intraspecies differences in susceptibility to dioxin-induced toxicities. A critical question in risk assessment of dioxin and related compounds is whether humans are sensitive or resistant to their toxicities. The diverse responses of mammals to dioxin are strongly influenced by functional polymorphisms of the arylhydrocarbon receptor (AHR). To characterize responses mediated by the human AHR (hAHR), we generated a mouse possessing hAHR instead of mouse AHR. Responses of these mice to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene were compared with the responses of naturally sensitive (C57BL/6J) and resistant (DBA/2) mice. Mice homozygous for hAHR exhibited weaker induction of AHR target genes such as cyp1a1 and cyp1a2 than did C57BL/6J (Ahrb-1/b-1) mice. DBA/2 (Ahrd/d) mice were less responsive to induction of cyp genes than C57BL/6J mice. hAHR and DBA/2 AHR exhibit similar ligand-binding affinities and homozygous hAHR and Ahrd/d mice displayed comparable induction of AHR target genes by 3-methylcholanthrene. However, when TCDD was administered, a greatly diminished response was observed in homozygous hAHR mice compared with Ahrd/d mice, indicating that hAHR expressed in mice is functionally less responsive to TCDD than DBA/2 AHR. After maternal exposure to TCDD, homozygous hAHR fetuses developed embryonic hydronephrosis, but not cleft palate, whereas fetuses possessing Ahrb-1 or Ahrd developed both anomalies. These results suggest that hAHR may define the specificity of the responses to various AHR ligands. Thus, the hAHR knock-in mouse is a humanized model mouse that may better predict the biological effects of bioaccumulative environmental toxicants like TCDD in humans.

Bisphenol-A Affects Spermatogenesis in the Adult Rat Even at a Low Dose.

Bisphenol‐A Affects Spermatogenesis in the Adult Rat Even at a Low Dose: Motoharu Sakaue,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 20 fig BPA/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.

Exposure of Mouse Preimplantation Embryos to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Alters the Methylation Status of Imprinted Genes H19 and Igf2

Abstract 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.

Reproliferation and relocation of mouse male germ cells (gonocytes) during prespermatogenesis

In the prespermatogenesis period, male germ cells (gonocytes) begin to reproliferate and move to the basement membrane of the seminiferous tubule. Although these two events—reproliferation and relocation—are important for establishment of spermatogenesis, they have not been greatly analyzed both in a mechanical and in an endocrine or paracrine aspect. In this study, the relationship between reproliferation and relocation of gonocytes was examined, using the thymidine analog bromodeoxyuridine (BrdU) labeling method and transmission electron microscopy (TEM). BrdU was injected into the fetuses [day 13.5 post coitus (dpc) to 18.5 dpc] and pups [day 0.5 post partum (dpp) to 6.5 dpp] of C57BL/6J mice. Two hours later, BrdU positive gonocytes were examined immunohistochemically and these data were analyzed. TEM and LM observation was carried out as well. Gonocytes began to relocate on the basement membrane from 18.5 dpc (1.4%) while BrdU‐labeled gonocytes were first detected on 1.5 dpp (13.6%). Relocated BrdU‐negative gonocytes were recognized from 18.5 dpc (1.4%), and relocated BrdU‐labeled gonocytes were recognized from 1.5 dpp (8.4%). On the other hand, non‐relocated BrdU‐labeled gonocytes were detected from 1.5 dpp (5.2%). Gonocyte relocation began 2 days earlier than reproliferation during the late fetal period. After birth, the two events occured at random. These results indicate that the reproliferation of the gonocyte does not correlate with relocation. The two events may be regulated by different mechanisms. Anat Rec 258:210–220, 2000.

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.

Testicular cytochrome P450scc and LHR as possible targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the mouse.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult animals has been reported to perturb the regulation of steroidogenesis in the testis, possibly by arylhydrocarbon receptor (AhR). To clarify how AhR is involved in the testicular steroidogenesis, we carried out comparative experiments using wild-type and AhR-null male mice that were intraperitoneally administered TCDD. The TCDD administration to wild-type mice showed significant reduction of P450scc and LHR in the testis, whereas the levels in the AhR-null mouse testis were unchanged. To compare anti-androgenic properties on hypothalamo-pituitary-gonadal (HPG) axis, estradiol-3-benzoate (EB), a synthetic estrogen agonist, was administered to mice, the expression of the LHalpha/FSHalpha, LHbeta, FSHbeta and GnRHR genes was severely impaired in the pituitary gland, in contrast to no observed effects in the TCDD-treated mice. In addition, the expression of the LHR gene was increased in the testis of the EB-treated mice. These observations suggest that the target of TCDD is different from that of EB on HPG axis and that TCDD treatment suppresses the P450scc and LHR genes in the testis in an AhR-dependent manner.

Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: A possible estrogenic effect of di(n-butyl) phthalate

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Effects of flutamide in the rat testis on the expression of occludin, an integral member of the tight junctions

In an effort to uncover the gonadal impairment by the antiandrogen flutamide (FM) in males, the effect of subacute administration of FM on the expression of tight junction (TJ) genes that build the blood-testis barrier (BTB) was investigated in adult rat testis. At 13 weeks old of age male rats were given vehicle (corn oil) or FM (25 mg/kg per day, in corn oil) orally for 6 days. At 8 days (D8) after the first dose, testicular expression of the occludin, claudin-1, and -11 was analyzed by semiquantitative RT-PCR. The testicular weight of the FM-treated rats on D8 was a little but significantly higher than in the control group. On D8 the expression of occludin in the FM-treated animals was significantly decreased but claudin-1 and -11 were not altered significantly. Because FM administration inhibits germ cell differentiation, it is likely that the down-regulated occludin expression in FM-rat testes may be attributed to the alteration in the paracrine interaction between Sertoli cells and germ cells in testis. It also emphasized that FM might have differentially affected the transcription of TJ genes in Sertoli cells building the BTB. These findings provide a rationale for a number of observations on the gonadal impairment by FM in males and suggest that FM is potentially harmful to spermatogenesis by alteration of the BTB.

Di(n-butyl) phthalate induces vimentin filaments disruption in rat sertoli cells: a possible relation with spermatogenic cell apoptosis.

With 3 figures

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on preimplantation mouse embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.