森 千里

To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 It thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment. (C) 2004 Elsevier Inc. All rights reserved.

Objective  The accumulation of dioxins, characterized by its lipophilicity and persistency in human tissue, is a great concern since many of these compounds elicit adverse health effects and developmental toxicity. Although the half-life of dioxins in the human body have been described to be as long as 3-25 years, there are very few drugs or methods that can exclude them from the human body. Thus, it is necessary to establish a new method to reduce them and prevent adverse health effects. Here, a pilot study to reduce the dioxins accumulated in the human body using the cholesterol-lowering drug, colestimide, is reported.<br>  Patients and Methods  Eight male and two female subjects were investigated. All of them were treated with colestimide for six months, and the dioxin level of the blood samples was assessed before and after the treatment. The dioxins in the blood samples were measured by gas-chromatography/mass spectrometry.<br>  Results  Nine out of the ten subjects completed the treatment, and their blood samples were analyzed. The mean dioxin level in the blood samples before the treatment was 44.0±8.22 pg-TEQ/g-fat. Six months later, the mean dioxin level was lowered about 20% on average to 34.7±5.49 pg-TEQ/g-fat.<br>  Conclusion  Previous studies have reported that the blood dioxin level increases with age. In this study, the results suggest that colestimide can decrease the blood dioxin level of humans. A randomized placebo-controlled clinical study including large numbers of subjects are needed to confirm the present result.

Aim: To isolate and transplant germ cells from adult mouse testes for transplantation. Methods: In order to distinguish transplanted cells from endogenous cells of recipients, donor transgenic mice expressing green fluorescent protein (GFP) were used. Germ cells were collected from the donors at 10-12 weeks of age and spermatogonia were concentrated by percoll fractionation and transplanted into recipient seminiferous tubules that had been previously treated with busulfan at 5 weeks of age to remove the endogenous spermatogenic cells. Results: Twenty weeks after the transplantation, a wide spread GFP signal was observed in the recipient seminiferous tubules. The presence of spermatogenesis and spermatozoa was confirmed in sections of 12 out of 14 testes transplanted (86%). However, when germ cells were transplanted without concentration the success rate was zero (0/9). Conclusion: Germ cells from adult mouse testes can be successfully transplanted into recipient seminiferous tubules if the cell population is rich in spermatogonia and the percoll fractionation is useful in obtaining such a cell population.

We examined the immunolocalization of isoforms of muscle proteins, myosin and troponin, in the cremaster muscle of the undescended testis (cryptorchidism). In cryptorchid rats induced by a nonsteroidal androgen antagonist, flutamide, the cremaster muscle contained embryonic myosin and embryonic/cardiac troponin T in both immunofluorescence microscopy and Western blotting using antibodies against myosin and troponin T specific for embryonic, cardiac and fast skeletal muscles. However, in muscles other than the cremaster muscle, i.e., the masseter, pectoral and abdominal muscles, embryonic isoforms of these proteins were undetectable by immunohistochemistry with these antibodies, even in the muscles from cryptorchid rats. Our results showing that high levels of embryonic isoforms of muscle proteins were specifically present in the cremaster muscle of cryptorchid rats induced by flutamide suggest that flutamide treatment of pregnant rats might affect genes controlling the development of the lumbar region of the fetus body resulting in the presence of embryonic protein isoforms in the cremaster muscles which are closely associated with undescended testes.

While the targeted disruption of a gene is a powerful tool for investigating the physiological functions of that gene, disruption of a gene essential for embryogenesis leads to embryonic death. Rescue of the defect(s) causing embryonic death should promote survival, thus permitting further evaluation of the roles that the gene plays later in the developmental process. Disruption of the gene for mouse hepatocyte growth factor/scatter factor (HGF/SF) leads to middle-stage embryonic lethality because of a defect in placental development. Here we report that a single injection of HGF/SF at embryonic day 9.5 (E9.5) into the amniotic cavity of HGF/SF-/- embryos rescued the placental defect and resulted in the survival of the embryos until term. Histological analysis suggested that HGF/SF is also required at the late stage of development for tissue organogenesis. Thus, injection of a secreted factor can be a useful method to rescue the defects causing embryonic lethality. (C) 2000 Wiley-Liss, Inc.

The molecular pathways leading from indifferent mammalian gonad to either testis or ovary are not well understood. A number of genes, including the Y-linked sex determining gene SRY, have been shown to play roles in sex determination or differentiation, but there are clearly many missing elements to be found. We used suppression-subtractive hybridization to construct normalized cDNA libraries enriched for male-specific or female-specific transcripts in mouse fetal gonads. We describe the strategy used to efficiently screen these libraries for candidate sex-determination and gonado-genesis genes. One gene arising from these screens is vanin-1, which encodes a protein implicated in the induction of cell migration into the thymus. We find that vanin-1 is expressed male-specifically in Sertoli cells of the developing testis and may be involved in inducing cell migration from the adjacent mesonephros, a process known to be critical for testis development. This screening approach is likely to be applicable to the isolation and study of genes involved in a variety of developmental systems. (C) 2000 Wiley-Liss, Inc.

Retinoic acid (RA) is mandatory for various biological processes and normal embryonic development but is teratogenic at high concentrations. In rodents, one of the major malformations induced by RA is cleft palate (CP). RA mediates its effects by RA receptors (RARs), but the expression patterns of RARs in the developing palate are still unclear. We investigated the normal expression of RAR alpha, beta, and gamma messenger RNAs (mRNAs) in the fetal mouse secondary palate and the effects of all-trans and 13-cis RAs on the expression of RAR mRNAs by Northern blot analysis. RAR alpha (2.8, 3.8 kb), RAR beta (3.3 kb), and RAR gamma (3.7 kb) mRNAs were detected in the fetal palate on gestational days (GD) 12.5-14.5. The expression of RAR alpha and gamma mRNAs did not show apparent sequential changes, but that of RAR beta mRNA increased at GD 13.5. Treatment of pregnant mice with 100 mg/kg all-trans RA induced CP in 94% of the fetuses and elevated the levels of RAR beta and gamma mRNAs in the fetal palate. The up-regulation of RAR beta mRNA by all-trans RA was more marked than that of RAR gamma mRNA. Treatment with 100 mg/kg 13-cis RA induced CP in only 19% of the fetuses. Although 13-cis RA elevated the RAR beta and gamma mRNA levels in fetal palates, its up-regulation was slower and less marked than that induced by all-trans RA. These findings indicate that the induction of RAR beta mRNA in the fetal palate correlates well with the tissue concentration of all-trans RA after RA treatment, and RAR beta may be one of the most influential candidate molecules for RA-induced teratogenesis.

Spermatogenic cells synthesize a unique 70-kDa heat shock protein (HSP70-2) during prophase of meiosis I, and targeted disruption of the Hsp 70-2 gene has shown that this protein is required for spermatogenic cell differentiation in adult mice, HSP70-2 is associated with synaptonemal complexes formed between paired homologous chromosomes during meiotic prephase, The present study focuses on the nearly synchronous first wave of spermatogenesis in 12- to 28-day old juvenile mice to determine more precisely when HSP70-2 is required and what meiotic processes are affected by its absence. Spermatogenesis in homozygous mutant mice (Hsp70-2(-/-)) proceeded normally until day 15 when increasing numbers of pachytene spermatocytes became apoptotic and differentiation of cells beyond the pachytene stage began to falter, Synaptonemal complexes assembled in Hsp 70-2(-/-) mice and spermatocytes developed through the final pachytene substage, However, synaptonemal complexes failed to desynapse and normal diplotene spermatocytes were not observed, Metaphase spermatocytes were not seen in tissue sections from testes of Hsp70-2(-/-) mice, and expression of mRNAs and antigens characteristic of late pachytene spermatocytes (e.g., cyclin Al) and development of spermatids did not occur. Thus, HSP70-2 is required for synaptonemal complex desynapsis, and its absence severely impairs the transition of spermatogenic cells through the late meiotic stages and results in apoptosis beginning with the first wave of germ cell development in juvenile mice.

We describe a rapid fluorescent in situ hybridization (FISH) procedure with X and Y chromosome-specific DNA probes for sexing human embryos and fetuses using paraffin-embedded tissues. The effects of various fixatives and pretreatment conditions on FISH efficiency were examined in different embryonic and fetal tissues. Fluorescent hybridization signals were identified in tissues which had been fixed in 4% paraformaldehyde (PFA) or 10% formalin but not in Bouin-fixed tissues. The preservation in 4% PFA at 4 degrees C yielded better FISH results than that in 10% formalin at room temperature. Another critical factor for visualization of fluorescent hybridization signals in paraffin sections was the duration of proteinase K digestion. Optimal digestion visualized hybridization signals more clearly probably because it increased the accessibility of DNA probes into nuclear DNA in paraffin sections. We have succeeded in shortening the hybridization time significantly and eliminating an immunocytochemical step for amplifying hybridization signals, and it has become possible to sex human conceptuses in paraffin sections within 5 hr. This FISH technique should be useful to sex fixed human embryos and fetuses retrospectively and to diagnose sex chromosome aneuploidies using fixed tissues.