森 千里

肉眼解剖実習は,医歯科大学生にとって人体の構造および機能を学ぶ上で,重要な基礎科目の一つである。本学において,解剖実習に供される遺体は千葉白菊会会員から提供いただいている。ところで,本学の解剖実習施設は,本学医学生およびコメディカル学生以外には公開していなかった。以前より,実習施設に関しては,会員から「死後自らのご遺体を預ける施設について見学したい」といった要請があったが,その機会を実現するには至らなかった。しかし,平成13年3月に解剖実習施設内の面会室および実習室の改装が終了したことを機に,要請に応えることおよび施設の現状を会員に知っていただくことを目的として,同年10月に開催された千葉白菊会総会時に希望者に対して見学会を実施するに至り,その成果を含めここに報告する。見学会には112名が参加し,見学箇所への移動に支障のない会員を10名程度のグループに分け,面会室,霊安室,遺体保管室および解剖実習室の順に見学を行った。なお,移動の困難な会員については待機場所において映像による見学を行った。後日,見学会に関するアンケート調査を行ったところ,参加いただいた8割の会員から返答をいただき,見学会全体を通して肯定的な回答をいただいた。特に,実際に施設見学を行った会員の回答によって(1)スタッフの対応,(2)見学時間,(3)見学内容,さらには映像による見学を行った会員の回答にみられるとおり,(1)映像の出来映え,(2)映像に関する説明,(3)放映時間については,その肯定的な回答を約6割の会員から得た点からすれば,及第点をクリアーしているといっても過言ではなかろう。

We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays. Newborn male ICR mice were exposed to DES (50 [mug/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis; 11 genes (A1035263, AU080565, AU080361, AU080678, A1131681, AU080631, AA986882, A1037066, AA986537, A1156816, and A1596237) were upregulated and three genes 01131656, A1118968, and All 17606) were down-regulated in DES-treated mouse testes. Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Realtime RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age. Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers. Mol. Reprod. Dev. 63: 17-23, 2002. (C) 2002 Wiley-Liss, Inc.

コメディカル教育機関では「解剖学」は重要な基礎教科であり,多くの機関で解剖実習見学を行っている。本学でも見学を許可してきたが,平成12年「死体解剖資格認定要領」改正に伴う指導講師の解剖資格の有無,医学部の実習時間外の見学による負担増,見学の目的を確認していないといった問題があった。これについて平成12年5月に,11年度の見学実施団体および12年度の見学希望団体に対し,解剖学の必要性,見学希望時間などについてアンケート調査を行い,その回答をもとに新しい見学方法を試み,結果を評価した。新しい実習見学方法は平成12年6月に末学した25団体に対して実施した。見学時間を含めて90分間とし,事前説明を行った後,見学を実施した。見学では,本学教官の指導に加え,スタッフ,見学団体講師,医学部生か補佐を行った。後日,自由記入形式で調査した結果,18団体より回答(回収率68.p%)が得られた。事前説明については「動機付けになった」「献体への理解が深まった」という感想が11団体からあった。事前説明によって見学がより効果的になったと思われる。適当な見学の時間数について質問したところ,「90分」と回答したのが10団体,「2時間」が5団体,他「時間が短い」が2団体であった。従って,時間数はほぼ適当と思われる。内容については「医学生による指導に感謝」と回答したのが8団体,「講師紹介が必要」が3団体であった。諸問題に対する解決策として今回の改訂を実施したが,これに関してはほぼ改善することが出来たと思われる。さらに内容を充実させるためには指摘された点についても配慮していきたいと考えている。

Methamphetamine Induces Apoptosis in Seminiferous Tubules in Male Mice Testis. Yamamoto, Y., Yamamoto, K., Hayase, T., Abirul, H., Shiota, K., and Mori, C. (2002). We investigated whether methamphetamine (MAMP) induces apoptosis in seminiferous tubules in 10-week-old male ICR mice. Methamphetamine was dissolved in saline and injected ip at four doses (1, 5, 10, and 15 mg/kg). TUNEL-positive cells were detected in the seminiferous tubules in animals 24 h after a single treatment with 5, 10, or 15 mg/kg MAMP. The percentage of seminiferous tubules containing more than three TUNEL-positive cells (apoptotic tubules) was considered a reliable indicator for apoptotic changes. After a single treatment with 10 mg/kg MAMP, the percentage of apoptotic tubules increased with time, and it became significant at 24 h, at which time vacuolar changes in spermatogonia. also peaked. Although the percentage of apoptotic tubules increased dose dependently from 5 to 15 mg[kg MAMP, the DNA ladder was detected in the testis of 15 mg/kg MAMP-treated mice. Thus, we have demonstrated that MAMP induces apoptosis in seminiferous tubules in male mice testis. (C) 2002 Elsevier Science (USA).

Our recent study clearly shows that fetuses are exposed to multiple chemicals including endocrine disruptors in Japan. Although the embryo and fetus stages are the most sensitive period to chemicals in humans' life cycle, the health effects of the chemicals such as endocrine disruptors to them are largely unknown. The conventional risk assessment method cannot assess the risk to fetuses precisely. Now we need a new risk assessment, in which the target is fetuses and not the adults, in addition to the conventional risk assessment. At the same time, we also need a new strategy to practically eliminate the risk for the future generations. To make the strategy effective, we suggest a new approach to reduce the risk and avoid the possible adverse health effects, using primary, secondary and tertiary preventions as they are used in public health. We also suggest a new concept of "pre-primary prevention" to reduce the risk for fetuses. Furthermore, to make this method even more practical, we suggest a new risk communication method. In this paper, we present a framework of risk avoidance of multiple chemical exposure to fetuses.

近年,環境中に放出された化学物質の多くにホルモン様あるいは抗ホルモン様作用があることが見出され,化学物質の内分泌撹乱作用による生態系への悪影響が,社会問題となっている。その理由は,内分泌撹乱物質の曝露が野生動物の生殖腺や生殖腺付属器官の発生や機能分化に異常をきたし,ホルモン代謝に悪影響を及ぼしていることが判明してきたためである。諸外国より発表された論文では,ヒトにおける「精子数の減少」や「男性生殖器の発生異常の増加」などの生殖異変の原因として,内分泌撹乱物質をあげている。さらに,ヒトの疫学調査から内分泌撹乱物質の悪影響として,精巣ガン,乳ガン,子宮内膜症,女性の思春期の早期化,免疫系・神経系への影響,さらに次世代への影響が懸念されている。ただし,内分泌撹乱物質のヒトに対する影響は,現時点では明白でない。よって,本稿では,「内分泌撹乱物質とその健康影響」と題し,内分泌撹乱物質の定義や種類および生体作用の特徴,作用メカニズム,検出法等について概説し,内分泌撹乱物質のヒトヘの影響の可能性について整理する。特に,ヒトヘの影響に関しては不明な点も多いため,当方が行っている調査研究結果の一部も紹介しながら「男性生殖能の異変」に関する報告を中心に概説する。

Some chemicals are known to induce limb malformations in mice. The occurrence of limb abnormality induced by chemical reagents is due to changes in the programmed cell death (PCD), 5-Bromodeoxyuridine (BrdU) is known as a potent teratogen and has been reported to induce polydactyly and other limb malformations in rodents (DiPaolo, Science 145, 501-503, 1964). Here, we undertook the morphological and genetic analyses of fetuses with limb malformations in BrdU-treated mice, in order to investigate an alteration of gene expression that resembles that of mutant mice with similar limb malformations. The fetuses of the BrdU-treated mice exhibited preaxial polydactyly and preaxial triphalangism of the hindlimb at a high incidence. Our observations showed that the PCD in the preaxial necrotic zone was found to be delayed or absent on day 11 of pregnancy. Histological analyses of these fetuses showed that the preaxial apical ectodermal ridge (AER) of the hindlimb was hyperplastic and consisted of several irregular layers. In observation of the whole-mount in situ hybridization, we detected the anterior-extended overexpression of Hoxd-11 and Hoxd-13 genes in the mesenchyme cells and the overexpression of Fgf4 and Fgf8 genes in the anterior region of the AER of hindlimbs of BrdU-treated fetuses. Our study shows that the injection of BrdU changed the PCD and gene expression during limb development and induced time-specific limb malformations during fetal development. This examination of the changes of the PCD and gene expression will be useful markers for the investigation of toxicities and teratologieties of other chemicals now present in the world environment. (C) 2000 Academic Press.

Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3-phosphate dehydrogenase-s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homologue of glyceraldehyde 3-phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72-amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is proline-rich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PGR) to identify exon-intron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exon-intron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of similar to 56 000, although the calculated molecular weight is 44 501. The conserved nature of the mouse, rat. and human enzymes suggests that they serve similar roles in these and other mammalian species.

To investigate the incorporation of oligonucleotides (ODNs) into the tissues of cultured fetal mouse palates and their effects on in vitro palatogenesis, we cultured day-12.5 fetal mouse palates in a chemically defined serumless medium supplemented with either antisense or sense ODNs to epidermal growth factor receptor (EGF-r). The EGF-r ODNs were found to be incorporated into the palatal tissue and remained detectable for at least 72 hr. Immunohistochemical and immunoblot analyses revealed that the treatment with 5μM EGF-r antisense ODN suppressed the production of EGF-r protein. No pathological change was observed in the explanted palates when they were treated with 5 μM EGF-r antisense or sense ODNs, but the treatment with 10 or 20 μM ODN caused pyknotic changes in the palatal epithelium, probably due to the ODN toxicity. The present results indicate that under optimal conditions, antisense ODNs to EGF-r can be incorporated into fetal organs cultured in vitro and specifically inhibit the production of EGF-r protein. Since the suppression of the production of EGF-r protein did not prevent the palate fusion, EGF and/or EGF-r alone may not play a critical role in palatogenesis, as suggested by previous studies. The antisense ODN technique could be of potential use for analyzing the roles of specific molecules in normal and abnormal morphogenesis.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase, However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Retroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd, In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a "promoter-trap" fashion, One such promoter ("promoter B" at the Mintb locus) was found in a CPG island, associated with an upstream enhancer ("enhancer B"), Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit ("R" for reverse), which apparently interfered with transcription from promoter B, Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U), Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.