1952年以降やツメガエルを用いて行われてきた実験から，オタマジャクシの小腸細胞のような分化した体細胞の核移植によって正常なカエルが得られることが示されている．しかしながら，成体のカエルの体細胞からはオタマジャクシは得られるが，正常なカエルは得られていない．今回Wilmutら[1]は，少なくとも一部の成体の体細胞核は個体への発生能力を維持していることを動物で初めて示した．本稿では，これまでの研究の歩みをふりかえりながら，哺乳動物における体細胞核移植の意義と将来の研究方向を展望してみたい．

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A stereotaxic apparatus was devised for frogs (Rana pipiens pipiens) by adaptation of a commercially available apparatus. An atlas of orienting illustrations emphasizing detailed structure and distribution of forebrain nuclei was prepared from celloidin sections and paraffin sections. Nomenclature of nuclei is discussed and an attempt made to reconcile various interpretations in the published literature.

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日本,韓国およびヨーロッパに分布するトノサマガエル群の進化関係を明らかにするため,5種のカエルと新北沢の1種のカエルを用いて交雑実験を行なった。まず,日本に分布するトノサマガエルRana nigromaculata HALLOWELLと韓国に分布するトノサマガエルとの間で正逆交配を行なったところ,両者間にはほとんど隔離機構がないので,両者は間違いなく同じ種であることがわかった。ヨーロッパのコガタトノサマガエルRana lessonaeCAMERANOとペレジワライガエルRana ridbunda perezi SEOANEは,トノサマガエルから雑種致死によって完全に隔離されているが,ダルマガエルRana brevipoda ITOからは雑種繁殖不能によって完全に隔離されている。韓国のチョウセンプランシーガエルRana plancyi chosenica OKADAはトノサマガエルとダルマガエルの両者から不完全な雑種繁殖不能によって隔離されているが,チョウセンプランシーガエルとトノサマガエルとの隔離の程度はトノサマガエルとダルマガエルとの間よりも弱く,チョウセンプランシーガエルとダルマガエルとの隔離の程度は後者よりも強い。チョウセンプランシーガエルは,ヨーロッパのペレジワライガエルからは完全に,コガタトノサマガエルからは不完全に,共に雑種繁殖不能によって隔離されている。コガタトノサマガエルとペレジワライガエルとは,もし雑種致死による隔離がないときには,雑種繁殖不能によって強く隔離され,時にはこの隔離が完全である。これらの交雑実験の結果から,旧北区のトノサマガエル群の祖先は,アジア系とヨーロッパ系の二つの枝に分かれ,アジア系はダルマガエル,チョウセンプランシーガエルおよびトノサマガエルに分化し,ヨーロッパ系はコガタトノサマガエルとペレジワライガエルになったと考えられる。新北区のRana pipiens SHREBERは,5種の旧北区のカエルから雑種致死によって完全に隔離されている。の雌または雄を用いて交雑を行なうと,受精卵はすべて変圧の初期に死滅する。

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Effects of anthropogenic pollution on aquatic environment have been concerned because some of pollutants have toxicities to aquatic organisms including immunotoxicity. Because immune system is responsible for biological defense and maintenance of good health, toxic effects of chemicals on immune system would decline health condition of organisms. Aquatic organisms which live in polluted areas were reported to be occurred immunotoxic symptoms such as histological changes of lymphoid tissue, changed immune functions and immune cell populations, and changed host resistance to infection pathogens. In addition, aquatic organisms are always thought exposed to pathogen (bacteria, virus, fungi, etc.) via water. Therefore, suppressed immune system by contaminants should increase infection risk. Furthermore, it poses ecological risks such as extinction. In this review, we introduced immunotoxic effects of pollutant contaminants on aquatic organisms including aquatic invertebrates, fish, amphibian and marine mammals, and discussed posed-effects on aquatic population and ecosystem.

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Nearly 50 multicelluar animal species have ever been flown on the near earth orbit or further. Animals used in space experiments range from Primates to Cnidaria. Widely used laboratory animals on the ground, such as rats and fruit flies, are frequently flown species. Mice, however, seem less popular in space experiments, probably because of their relatively poor resistance to the stressful space environment. To keep rodents onboard the space craft in a health state requires tremendous efforts. Moreover, the use of rodents onboard the International Space Station (ISS) has become extremely difficult, due to the cancellation of the Centrifuge facility. Rearing fish in space, on the other hand, is easier than to keep rodents, since their waste excretions are borne by the water flow and can be effectively removed by filtering. Fish are suitable model for the studies on development, radiation effects and vestibular functions. Among fish, Medaka (Oryzias latipes) will be the most useful species for space experiments, with the inventory of inbred and mutant strains, their genome sequence data, and the established gene knockout technologies. Medaka together with established small laboratory animals, such as C. elegans, are most promising species for the experiments onboard the ISS. Other unique groups of animals in space experiment are those inhabit in the arid area. These animals, such as Mongolian Gerbil (Meriones unguiculatus), generally require few drinking water and produce little amount of waste. This enables flight hardware being simpler. Although the lack of their genome sequence data is a big drawback, they are useful for space experiments, especially for those utilizing recoverable capsule satellites.

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哺乳動物では,一度分化した体細胞からは,再び個体にまで戻すことは不可能かもしれないと考えられていたが,核移植という方法で,未受精卵子細胞質と融合することにより,遺伝子の初期化と正常2倍体核の再構築が可能であることが最近証明されつつある.細胞核と細胞質の相互作用を研究するために用いられてきた技術が,目的遺伝子を有する個体作出のための技術として脚光を浴びつつある.この核移植技術で誕生した,受精卵クローン,体細胞クローンは,動物工場による有用タンパク質や代替臓器の生産,ミルクや牛肉の品質向上と斉一化など多くの分野で変革をもたらす可能性がある.

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Developing organisms are sensitive to estrogenic chemicals. Exposure to estrogens or estrogenic chemicals during critical periods of development induces persistent changes in reproductive as well as nonreproductive organs, including persistent molecular alterations. Chemicals released into the environment potentially disrupt the endocrine system in wild animals and humans, some of which exhibit estrogenic or anti-androgenic activity by binding to estrogen or androgen receptors. Estrogen-responsive genes and critical developmental windows of various animal species, therefore, should be identified to understand the molecular basis of estrogenic activity during embryonic development. To understand molecular mechanisms of estrogenic chemicals in developing organisms, we identified estrogen-responsive genes using cDNA microarrays and a quantitative reverse transcriptase-polymerase chain reaction, and genes related to estrogenindependent vaginal changes in mice induced by estrogens during a critical window. We also analyzed genes in Xenopus laevis embryos related to abnormalities induced by estrogenic chemicals using cDNA microarrays. Estrogen receptors and other steroid hormone receptors were cloned in various species, including the American alligator, crocodile, Florida red-bellied turtle, Silurana tropicalis, roach, Fundulus and mosquito fish. An environmental androgen, trenbolone, was found to induce persistent changes in the gonopodium and ovotestis in mosquito fish. In Daphnia magna, juvenile hormone agonists induce the production of male offspring. Tributyltin (TBT) induces imposex in rockshell through retinoid X receptor (RXR) activation. Microarray technology is a powerful tool for understanding the molecular mechanism of receptor-mediated toxicology in various animal species. Ecotoxicogenomics is a multidisciplinary field concerned with elucidating how the entire genome is involved in the biological response of wild animals exposed to environmental toxicants and stressors. Ecotoxicogenomics combines information from studies of genomic-scale mRNA profiling by microarray analysis (transcriptomics), protein profiling (proteomics), metabolite profiling (metabolonomics) and computational models to understand the roles of gene-environment interaction in the chemical toxicity. To understand the molecular mechanism of chemicals in developing wildlife, the establishment of ecotoxicogenomics is essential in the near future.

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我が国の現行の中学校理科教科書には，両生類の呼吸方法について，「オタマジャクシはえら呼吸し，カエルは肺呼吸（一部は皮膚呼吸）する」と記述されている。一方，カエル幼生の呼吸機能分担に関する最近の研究によれば，ヒキガエル類を除くカエル類のオタマジャクシ（無尾目の幼生）は，後肢が発達する変態期以前からすでに肺呼吸を始めており，オタマジャクシの肺呼吸は嫌気的水中で生き延びるのに役立っているという。また，系統進化の観点から，オタマジャクシの肺呼吸はセキツイ動物の肺の獲得と進化にかかわる痕跡的な行動とみられている。さらに，ふ化直後のオタマジャクシではエラ呼吸よりも皮膚呼吸の方が重要な役割を果たしていると考えられている。したがって，少なくとも「オタマジャクシはえら呼吸する」という見解は，厳密な意味では，誤りを含む一種のミスコンセプションとみなされる。オタマジャクシの肺呼吸は，すでに1931年にヨーロッパで発見され， 1982 年にはウシガエルの幼生で，肺と皮膚がエラとともに呼吸分担機能をもつことが明らかにされていた。しかしながら，オタマジャクシの呼吸についての不正確な扱いは，我が国の教科書や一部の専門書だけでなく，海外の生物学の専門書にもみられることが報告されている。このようなミスコンセプションが発生するメカニズムには，誤った教育情報の関与も考えられるが，魚類のような水生動物はエラ呼吸し，高等な陸生動物は肺呼吸するという，現生の脊椎動物についての認識から，両生類も水中生活のオタマジャクシはエラで呼吸し，陸上生活期のカエルは肺で呼吸するに違いないという演繹的推論が生まれやすいことも関与しているものと考えられる。このようなミスコンセプションを克服するためには，教育情報の訂正のほかに，オタマジャクシの呼吸生理実験を取り入れた教育プランの活用が必要と考えられる。

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Four experiments examined the motivational manipulation for simultaneous visual discrimination learning in Japanese fire-bellied newts (Cynops pyrrhogaster). A total of ten female newts was trained in a simultaneous visual bright and dark discrimination task using a dry T-maze. Submersion in water was used as reinforcement for a correct response. Experiment 1 showed that the procedure used in a preceding study with red-spotted newts is not effective to train Japanese fire-bellied newts. Experiment 2 showed that the procedure to dehydrate newt’s skin is effective to facilitate instrumental responding of newts, and that prolonged water loss from skin threatens the health of newts. Experiments 3 and 4 suggested that the motivational level for water reinforcement could be controlled by varying the temporal length allowed for access to water during the inter-trial intervals. These results suggest that appropriate water deprivation is important to train Japanese fire-bellied newts in a discrimination task with water reinforcement.

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Over the past two decades, amphibian conservation has been a growing concern with the realization of worldwide amphibian declines and high prevalences of frog deformities. Chemical contaminants have attracted significant attention as a causative agent of these amphibian abnormalities. Japanese amphibians also seem to decrease in number, and deformed frogs have been frequently observed in the Kitakyushu area. Toxicity or teratogenicity tests conducted with Japanese amphibian larvae or Xenopus laevis embryos indicate that the most common herbicide in Japan and some organic compounds detected from the Kitakyushu area have the potential to induce amphibian mortality or malformation. However, these toxicological data have limited relevance to the amphibian abnormalities occurring in the field. In general, laboratory tests conducted by use of wellcontrolled and standardized methods provide only the basic data to evaluate the toxicity of chemicals. Various biological and physical-chemical factors such as geographic, ecological, and physiological diversity in amphibians, susceptibilities that may be variable during larval stages, and environmental fate of chemical contaminants should be in consideration for the risk assessment. Mathematical models may facilitate to assess the effects of chemicals using these complex factors. Amphibian toxicology should therefore make connections with developmental biology, ecology, physiology, mathematical biology, and physical chemistry. Extensive and interdisciplinary investigation would be required to progress toward amphibian conservation focusing on the protection of wild amphibians from chemical contaminants.

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Biologically active materials were characterized chemically from various kinds of amphibian skin, and from the venom of wasps or spiders. The assay system employed for the biological activity on smooth muscle, were vaso-constriction and-delation, smooth muscle contraction and relaxation. Cytotrophic action including haemolysis, granulocyte degranulation, platelet aggregation, chemotaxis of leucocyte, and modulation of neural synaptic transmission were adopted for screening of the other materials. Many kinds of active peptide honologous to mammaliam peptide hormone, for example thyrotropin-releasing hormone (TRH), neurotensin, cholecystokinin (CCK), angiotensin, were found in high concentration from the methanol extract of the frog skin. A new cytotrophic peptide family was found both in frog skin and wasp venom. These peptides were rich in hydrophobic amino acids and basic ones. A peptide named mastoparan acts directly on the GTP binding protein as the hormone mimetic action and degranulates histamine granules of the mast cells. Another peptide revealing chemotactic activity for neutrophils acts directly on the FMLP receptor of the cells. These types of cytotrophic peptides modulate a cell signalling system via GTP-binding protein in such cells. Finally, over 15 kinds of neurotoxin were found from the spider venom which inhibit synaptic transmission of glutamate, both for lobster neuromuscular junction and mammaliam hippocampal neurons. A specific binding protein for this type of spider toxin was isolated from the bovin brain. The protein is a family of calreticulin previously isolated as a calcium regulating protein from sarcoplasmic reticulum in skeletal muscle.

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The basic embryonic plan of mammals is inherently feminine. The diversion of this plan is carried out by the Y chromosome which directs the embryonic indifferent gonad to organize a testis instead of an ovary. It has been assumed that the Y chromosome contains a determinant that is essential for the development of a testis (testis-determining gene). On the basis of ubiquitous expression and of widespread phylogenetic conservatism, it has been proposed that serologically-defined H-Y antigen is the product of the testis-determining gene. The proposal has been tested on a considerable variety of exceptional individuals whose gonadal sex does not agree with their chromosomal sex. Any individual who posseses testes in spite of the apparent absence of the Y chromosome should express H-Y antigen on his cells. A series of extensive tests performed on such extraordinary individuals yielded no exception from the above expectation. Gonadal cells dissociated from the neonate of mouse and rat reorganize histotypic aggregates in the Moscona-type aggregation experiment. Testicular cells, lysostripped of H-Y antigen, reorganized ovarian follicular aggregates whereas ovarian cells in the presence of H-Y antigen partially reorganized semiferous tubules-like aggregates. Organ culture for 5 days of bovine XX embryonic indifferent gonads in the presence of the H-Y antigen, excreted from Daudi human male Burkitt lymphoma cells, induced very precocious testicular organogenesis. The above clearly support the proposed testis-organizing function of H-Y antigen.

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This study reviewed recent studies of road-kill, barrier effect and mitigation techniques on wild animals. It also surveyed road-crossing structures used in Japan. By comparing Japanese and international case studies, we addressed the possibility of studies on road-kill, barrier effect and mitigation technique development in Japan. The road-studies in Japan were mostly studies of the road-kill of Sika deer, Cervus nippon yesoensis, and Raccoon dog, Nyctereutes procyonoides. There were fewer Japanese studies on roadkill and barrier effect examining species, taxa, and landscapes than international studies. Most of the road-crossing structures were underpasses, box-culverts, and pipe-culverts targeting large and mid-sized mammals. There were fewer eco-bridges targeting arboreal mammals and amphibian tunnels targeting herptiles in Japan. In the future, it will be necessary to analyze the factors influencing road-kill and barrier effect of various species and taxa in order to develop mitigation techniques targeting arboreal mammals and herptiles, and to develop quantitative methods for monitoring wild mammals utilizing road-crossing structures as movement corridors.

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