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[要旨]現在、地球上では多くの野生動物種が絶滅の危機に瀕しており、生物多様性の維持に向けた取り組みが求められている。動物園・水族館などの飼育下で動物を繁殖させる取り組みは「域外保全事業」と呼ばれ、人工繁殖技術はその中心となる技術である。本稿では、多能性幹細胞から生殖細胞を作製することが域外保全の発展に貢献する可能性があると考え、iPS細胞の樹立および生殖細胞の体外作製について、先行研究を体系的にまとめることにより、遺伝的多様性の低下・人工繁殖の難航化・生殖細胞の回収が困難であることへの解決策を提案することを目的とする。[英文抄録] Currently, many animal species on this planet are at risk of extinction, and the conservation of biodiversity is a pressing issue. The conservation program to maintain or recover the population of species outside their natural habitat by artificial reproduction at zoos, aquariums, etc, are called “ex situ conservation”. Establishment of pluripotent stem cells and in vitro production of germ cells have great potential to contribute to the successful ex situ conservation. Here, we introduce the history and current status of the establishment of iPS cells and in vitro production of germ cells in wild animal species, and discuss the future of ex situ conservation., Review

[要旨] 哺乳動物の生殖細胞サイクルでは、受精直後の初期胚と始原生殖細胞(primordial germ cell、PGC)において分化能を獲得するために重要な核のリプログラミングが二度引き起こされる。核のリプログラミングはピストンやDNAのエピジェネティック修飾の変化を伴うクロマチンリモデリングに起因し、直接的な制御因子としてヒストンやDNAを標的としたメチル化やアセチル化酵素が数多く同定されている。また、受精直後の初期胚の雄性ゲノムやPGCのゲノムでは、5一メチルシトシン(5-methylcytosine、5mC)のレベルの低下が能動的に起こることが認められている。現在、能動的にDNAを脱メチル化する酵素は同定されていないものの、能動的DNA脱メチル化機構の解明に向けて新たな局面が最近見られた。例えば、ten-eleven translocation (TET) ファミリータンパク質は5mCを酸化して5-ヒドロキシメチルシトシン(5-hydroxymethylcytosine、5hmC)へ変換する機能を持つことや、activation-induced deaminase(AID)及びapolipoprotein B mRNA editing enzyme catalytic polypeptides (APOBEC) は5mCをチミンへ変換する機能を持つことが明らかになった。これらのタンパク質は、間接的にDNAの脱メチル化を制御することがマウス初期胚及びPGCにおいて明らかにされている。さらに、多能性を持つES細胞では、AIDやTETIはOct4(Pou5f1としても知られている)とNanogにおけるプロモーター領域のDNA脱メチル化に関与することが報告されている。こうした能動的DNA脱メチル化機構の解明は、多能性に関わる遺伝子の発現機構をより明確にし、再生医療の進歩に貢献する。そこで本稿では、マウス初期胚とPGCにおける能動的DNA脱メチル化制御について概説する。 [Abstract] Early embryos and primordial germ cells (PGCs) undergo extensive epigenetic reprogramming in mammals. In these cells, methyltransferases and acetyltransferases have been identified and their activities are involved in the epigenetic reprogramming by mediating the histone modifications and DNA methylation, except for DNA demethylase. Recent reports have indicated mechanisms of active DNA demethylation. One of mechanism are that Ten-eleven translocation 3 (Tet3), which is intensely expressed in oocytes and zygotes, regulates the active DNA demethylation by global conversion of 5-methylcytosine (5mC) to 5-hydroxymethlcytosine (5hmC). In addition, activation-induced deaminase (AID) and apolipoprotein B mRNA editing enzyme catalytic polypeptides (APOBEC) are involved in active DNA demethylation during PGCs development by converting 5mC to thymine. Furthermore, AID is required for induction of Oct4/Pou5f1 and Nanog expression in mouse embryonic stem cells (ESCs), and TET 1 has an important role in mouse ESCs through maintaining the expression of Nanog. Thus, clarifying the mechanisms of active DNA demethylation leads to understanding the reprogramming toward pluripotency and totipotency. Here, we will review mechanisms of active DNA demethylation during preimplantation embryos and PGC development., 本研究は近畿大学生物理工学部戦略的研究No.10-IV-7, 2011の助成を受けた。

マウス胚性幹細胞（mouse embryonic stem cell;mES 細胞）は、各系譜の細胞へと分化可能な分化多能性を有している（1）。この特徴を用いて、様々な分化誘導実験が行なわれてきた。生殖細胞の分化誘導もまた、発生メカニズムの解明に繋がるとして研究の対象となってきた。しかし、受精能を獲得した精子様の細胞は得られたが、同様な卵母細胞様の細胞は得られていない。そこで本実験では、卵巣表面に存在する卵巣上皮細胞（ovarian surface epithelium cells;OSECs）がmES細胞から生殖細胞への分化に関与するかどうか、分子生物学的手法及び免疫化学的手法を用いて未分化マーカー遺伝子であるOct4 遺伝子、生殖細胞関連遺伝子であるMouse vasa homolog（Mvh）、Stella、Fragilis 遺伝子について解析した。その結果、胚様体（Embryoid Body;EB）をOSECs の馴化培養液で接着培養を行った場合、通常の接着培養と比べ、Oct4 遺伝子の発現が維持された。今回の実験系より得られた結果から、OSECs の分泌する液性因子が、mES 細胞から生殖細胞への分化を助長する傾向にあることが示された。

オオミノガ雄幼生期の生殖細胞を材料に,電子顕微鏡を用いて,細胞分裂期におけるミトコンドリアの動態を研究した。細胞分裂期には,ミトコンドリアは極めて長く伸長し,棒状ないし竿状となり,後期ないし終期で両娘細胞を繋げている狭窄部を跨って存在していた。従って,棒状ないし竿状ミトコンドリアは細胞質分裂期に両娘細胞間の縊れ込み部でほぼ均等に二つに分割されることが考えられた。また精子形成過程において,急速なミトコンドリア数の増加は確認出来なかったが,ミトコンドリアの伸長による実質的な容積の増量が行われる事が考えられた。ミトコンドリアの網目構造は確認出来なかった。

マウスでは始原生殖細胞（Primordial germ cell ; PGC）の培養系の研究が進んでおり、in vitro におけるPGC の培養が可能である。そのため、PGC はゲノムインプリンティングの消去、精子・卵子形成の過程の研究材料としてよく用いられている。また、PGC をある環境下で培養することで胚性生殖細胞（Embryonic germ cell ; EG 細胞）を樹立することができる（1）（2）。ウサギではin vitro でのPGC の培養系は整っておらず、EG 細胞も樹立されていないため、ウサギでもin vitro でのPGC 培養系の確立、EG 細胞の樹立によって、より正確な発生メカニズムを解明する手がかりになるかもしれない。本研究ではマウスEG 細胞樹立条件を基にウサギにおけるin vitro でのPGC 培養及びEG 細胞の樹立を試みた。まず、マウスEG 細胞樹立と同じ条件でウサギPGC を培養したところ、ウサギPGC 由来コロニーが得られた。そこで、次に、培養系の改良を目的にウサギPGC 培養にはLIF、bFGF、Forskolin の有効性を検討した。その結果、LIF、bFGF、Forskolin を添加することによってアルカリフォスファターゼ（ALP）活性陽性コロニー数が増加し、ウサギPGC 培養においても有効であったことが示唆された。また、得られたコロニーを免疫化学的手法によって評価したところ、マウスEG 細胞と同様にALP 活性、OCT-4、SSEA-1 の発現が確認された。さらにこれらの細胞の分化能を評価するため胚様体、テラトーマ形成の確認を誘起したところ、マウスEG 細胞と同様に胚様体様構造の形成は見られた。しかし、SCID マウスへの移植を行なった結果、テラトーマの形成は確認されなかった。今回得られた細胞は、未分化細胞マーカーであるALP、OCT-4、SSEA-1 を発現し、胚様体様構造の形成能を有していたがテラトーマの形成能は示さなかったことから、PGC からEG 細胞への過渡期の細胞ではないかと考えられる。

長崎大学学位論文 [学位記番号]博(医歯薬)甲第458号 [学位授与年月日]平成23年9月20日

長崎大学学位論文 [学位記番号]博(医歯薬)甲第458号 [学位授与年月日]平成23年9月20日

本研究により, 1) ブタIVP系によって作出した前核期胚は, SSV法による超低温保存が可能で, 産子への発生能を有することが明らかになった。2) ラット凍結融解精子のIVFを介して産子を作出する場合, 凍結融解精子をIBMX処理し, 細胞内cAMP量が上昇させ, タンパク質のチロシンリン酸化を促進させることで, IVF卵の2PN形成率が改善し, 効率的な産子作出が可能となった。, The objective of the present study was to develop methods for conservation of genetic resources in pigs and rats. In pigs, a high proportion of porcine zygotes were successfully cryopreserved by solid surface vitrification at the pronuclear stage without delipation. Full developmental competence of these zygotes to the piglet stage was preserved. We also show here for the first time successful IVF using cryopreserved rat spermatozoa. These protocols will be useful for the efficient genetic conservation in pig and rats.

胚発生の非常に初期に未分化生殖細胞が生じ、個体発生の過程に生殖巣に遊走し到達する。その後、生殖巣の分化に伴って、雌では卵原細胞、雄では精原細胞へと分化する。精巣においては、精原細胞は幹細胞として増殖・分化し、その後精母細胞の分化と減数分裂、減数分裂後の半数体精子細胞特有なクロマチン形成を伴う形態形成が整然と起こる。一連の精細胞分化とそれを支える細胞を含む精巣では、ほとんどすべての遺伝子が発現し、また多くの特異的遺伝子が含まれていることが明らかにされている。さらに、それらの遺伝子の中にレトロトランスポジションによって生じたと考えられているイントロンレス遺伝子が、数多く機能していることが明らかにされた。本稿では、精子形成過程で発現するイントロンレス遺伝子についてまとめ、数多く存在する理由について考察する。

Mechanisms of germ cell formation have been one of intriguing questions in biology. Segregation of the germ line responsible for continuity of the genome through generations from the somatic cells specialized for development of each individual occurs at early stages of development in many multicellular organisms. In mice, presumptive primordial germ cells originate in the proximal region of the pluripotent epiblast abutting on the extraembryonic ectoderm before and at around the onset of gastrulation. Germline specification in mice is thus considered to take place at relatively later stages of embryogenesis among the pluripotent stem cells under the influence from still unidentified environmental factors. Primordial germ cells (PGCs), which are considered to be restricted to the germ line, are first recognized as about 50 cells showing strong alkaline phosphatase (ALPase) activity in the posterior extra-embryonic region of the primitive streak-stage embryos at around 7.25 days post coitum (dpc). They migrate to the gonad anlagen, genital ridge, and proliferate up to about 25000 cells by around 13.5dpc. These PGCs express several surface markers including ALPase and a transcription factor (Oct3/4) which are also expressed in earlier totipotent embryonic stem cells. But they are distinct from totipotent stem cells by their migratory behavior. PGCs are considered to be repressed from totipotency because of their inability to contribute to chimeras when transplanted into early embryos. During investigations of growth factors effective to PGCs in vitro, colonies of cells resembling undiffrentiated pluripotent embryonic stem (ES) cells appeared in the presence of LIF (leukemia inhibitory factor) and bFGF (basic fibroblast growth factor). These cells, named as EG (embryonic germ) cells, showed similarity to ES cells in their morphology, surface markers, and totipotency demonstrated by formation of germline chimeras. Derivation of the totipotent cells from PGCs that are thought to be determined to the germ line, implied dedifferentiation of PGCs and recovery of totipotency. A recent study reported that other combinations of growth factors, LIF and RA (retinoic acid) or FK (forskolin) also supported long term proliferation of PGCs in vitro, but the differentiation potency of these cells was not examined. In this study, I established EG cell lines in the presence of LIF, FK and RA, and performed transplantation of these cells in reaggregates with fetal gonadal somatic cells into the adult mouse testis to test their potency of differentiation. Transplants gave rise to varieties of differentiated somatic tissues, indicating either dedifferentiation of PGCs to pluripotent stem cells, or proliferation of the remaining stem cells within PGCs occured in this combination of growth factors. PGCs after arrival at genital ridges at around 10.5dpc continue to divide mitotically until about 13.5dpc. Then, they take different fates of development according to their surrounding gonadal sex. Germ cells in male gonads are considered to arrest at the G0 stage of cell cycle as prospermatogonia. In female, germ cells enter into meiosis and arrest at prophase of the first meiotic division. Surface markers of PGCs which are common to early totipotent stem cells start to diminish gradually after transition into mitotic arrest or meiosis. These fetal germ cells resume mitosis as spermatogenic stem cells or meiotic progression and oocyte maturation after birth. It has been suggested that sex-differentiation of fetal germ cells is determined by the surrounding gonadal somatic environments, rather than their own chromosomal constitution. However, studies on differentiation of PGCs have been limited to experiments using organ or reaggregate cultures. Thus, examinations of meiotic transition by PGCs and identification of its regulative factors have been impeded, due to the lack of dissociation culture system of PGCs in which meiotic transition could be detected and analyzed in detail. I developed a dissociation culture system in which female PGCs entered into the prophase of first meiotic division. I demonstrated, using this system, that expression of meiotic protein by PGCs occured in the absence of embryonic gonadal somatic cells. This indicates that meiotic transition by PGCs could be determined independently on gonadal environment. I observed a tendency of synchrony of meiotic transition by PGCs in each cell colony formed in vitro. I also demonstrated that LIF-gp130 signal, previously known as an inhibitor of differentiation of ES cells and as a PGCs' survival or growth factor, repressed meiotic transition by PGCs and observed consistent difference of LIF expression between male and female embryonic gonads. In vitro system developed in this study shall provide a novel basis for analysis on mechanisms and factors involved in the development and differentiation of fetal germ cells in mammals.

[要旨]多くの生物種が絶滅の危機に瀕している現在、生物多様性を維持するための取り組みは急務である。その取り組みとして進められているのが生息地における生息数の確保を目的とした「域内保全」と動物園・水族館などにおける飼育下繁殖技術による「域外保全」である。いずれの場合も個体数と遺伝的多様性の双方の維持あるいは回復が求められており、域外保全においては可能な限り多くの個体に由来する生殖細胞を用いた人工繁殖技術の確立が必要である。本稿では、そのような技術の中で、1) 生殖細胞の保存および人工授精、2) 多能生幹細胞の樹立およびそれ由来の生殖細胞の作製、3) 性周期の予測および妊娠判定について述べる。[Abstract] Conservation of biodiversity is an urgent issue on this planet where many species face to extinction now. The conservation programs ongoing in animals are in situ conservation (conservation of ecosystems in the natural habitat to maintain or recover the population of species), and ex situ conservation (maintain or recover the population of species outside their natural habitat by artificial reproduction at zoos, aquariums, etc.). In both conservation programs, maintenance or recovery of population and genetic diversity are required, and the establishment of artificial reproduction using the germ cells from as many individuals as possible for successful ex situ conservation. Here, we introduce and discuss three potential reproductive techniques to achieve successful ex situ conservation, 1) germ cell preservation and artificial insemination, 2) in vitro production of germ cells, and 3) prediction of estrus and detection of pregnancy.

Vasa gene homologs of tiger puffer (Takifugu rubripes, Tp‒vasa) and grass puffer (T. niphobles, Gp‒vasa) were cloned and characterized for use as a molecular marker of germ cells in these species. Analysis of the nucleotide sequence revealed that open reading frames of Tp‒ and GP‒vasa comprise 1,872bp (encoding 623 amino acids) and 1,869bp (encoding 622 amino acids), respectively. These deduced amino acid sequences contained arginine‒glycine or arginine‒glycine‒glycine motifs and eight conserved motifs belonging to the DEAD‒box protein family, and showed highly similarity and identity with vasa homologs of other teleosts. Tissue specific expressions of Tp‒ and Gp‒vasa were detected in testis and ovary. In situ hybridization analysis showed that Tp‒ and Gp‒vasa mRNA expressed in oogonia and oocytes in the ovary, and spermatogonia in the testis, while it was not detected in the primary and secondary spermatocytes, spermatid, spermatozoa or gonadal somatic cells. These findings suggest that Tp‒ and Gp‒vasa should be useful for studies of germ cells specification and differentiation in Takifugu puffers.

本研究課題のもと、マウス胎児期生殖細胞の複数のステージにおいて、ゲノムの3次元構造を明らかにするHi-C解析を行った。今回、マウス胎児期生殖細胞は胎生13.5日目(E13.5)から生後2日目(P2)までのサンプルを用いて行った。その結果、E17.5やE19.5日目においてゲノムの構造がその前後の時期のものと大きく異なることが明らかとなった。具体的には、当該時期においてゲノム上の任意の２つ領域間における染色体内相互作用の頻度が弱まっていた。さらに、E17.5からE19.5の時期において遷移状態にあるクロマチンでは頻繁に染色体相互作用の「繋ぎ替え」が起きていた。この遷移状態を経た染色体はP2の時期になると、初期状態であるE13.5の時期のクロマチン構造に近づく。以上の結果は、E13.5からP2にかけて、クロマチンの三次元構造がリプログラミングを起こし、その後の精子幹細胞への分化に向けたクロマチンレベルの準備を行っていることを示唆している。 Hi-C解析で得られたデータを主成分分析することで、ゲノムのユークロマチンとヘテロクロマチンの領域を知ることが出来る。例えば、ゲノム上の遺伝子砂漠などは主成分分析でヘテロクロマチンと分類される。E13.5の生殖細胞を用いたHi-C解析では、ゲノム領域がユークロマチンとヘテロクロマチンに明確に分類されるのに対して、それをE17.5やE19.5で行うと分類が非常に曖昧なものとなる。この結果は、E17.5やE19.5といった時期の生殖細胞におけるクロマチンではゲノム上の性質が他の時期のクロマチンに比べ非常に異質なものとなっていることを示している。 以上の結果から、本研究課題を通して胎児期の精巣の三次元構造がダイナミックにリプログラミングを起こしていることが明らかとなった。今後、このリプログラミングを引き起こす責任因子を発見するために研究を引き続き行う。 I have performed Hi-C analysis using male embryonic germ cells. From this analysis, I found that the 3D genome architecture at E17.5 as well as E19.5 show a remarkable difference compared with those at flanking stages. The frequency of intra-chromosomal interaction decreases at these intermediate stages. Moreover, I found that the global rewiring of genome interaction frequently occurs during the last stage of male embryonic germ cells. Interestingly, the 3D genome conformation at P2 is more similar to that at E13.5 than E19.5, indicating that reprogramming of chromosome architecture takes place at gonocyte stage. By performing principal component analysis (PCA) with Hi-C dataset, we can differentiate euchromatin region from heterochromatin region. For example, gene desert regions are classified as heterochromatin region with PCA. PCA with E13.5 Hi-C dataset shows every genomic region is clearly assigned as either euchromatin or heterochromatin. In contrast, PCA with E17.5 or E19.5 dataset revealed that the classification of this genome compartment becomes less defined. This finding affirms the former result that 3D genome architecture at E17.5 and E19.5 are in a transitional stage. In sum, under this research project, I found novel 3D genome reprogramming event in gonocyte. In future, I would like to pursue the molecular event that triggers this dynamic chromatin change.

[要旨] 1994年、精原幹細胞の精細管移植によりドナー由来の精子を作製できる移植技術が開発された。さらに、2007年には幼若ブタ精巣細胞を免疫不全マウスの皮下へ移植することにより、精子形成の誘導に成功したことが報告された。そこで本研究では、絶滅危惧動物などでの精子形成分化誘導モデルの確立を目的に、ウシ精巣細胞を用いた移植実験を行った。マウス新生子（生後7日以内）、ウシ新生子（生後7 日以内）およびウシ幼若（3～5ヶ月齢）精巣細胞をマトリゲルマトリックスと混合し、免疫不全マウスの皮下に移植することで、精巣様構造の再構築と精子形成の誘導を試みた。移植後、1週～ 44週に組織学的解析を行った。さらに、生殖細胞の生存・増殖・分化について、抗VASA 抗体を用いた免疫組織化学的解析を行った。マウス新生子精巣細胞の移植では、移植後10週以降に再構築精細管および減数分裂に至る精子細胞がみられ、精子形成誘導を可能にする機能的な精細管の構築が示された。一方、新生子ならびに幼若ウシ精巣細胞の移植では、移植後10 週から管構造の再建がみられたものの、免疫組織化学的解析において再構築された精細管様構造の基底膜上に配置される生殖細胞を認めることはできなかった。以上の結果から、異種異所移植法によりウシにおいても精細管様構造が再構築されたことから, 今後移植法の改良によりウシ精子形成の誘導が可能となることが期待される。 [Abstract] Male germ cell transplantation induces donor cell-derived spermatogenesis in the testis of recipient mice. However, this technique is still available only to mice and rats. Recently, xenoectopic transplantation of testicular cells into the subcutis of immunodeficient mice has been developed as a novel technology to rebuild seminiferous tubules and to produce functional sperm. In order to develop an alternative technology for transgenesis and induction of spermatogenesis in domestic animals, we examinedxenoectopic transplantation of bovine testicular cells. Testicular cells from neonatal（within 7 days）and juvenile（3-5 months）bovine and neonatal（within 7 days）mouse testes were prepared at the concentration of 1-5 x 10^7/ml（mouse）or 1 x 10^8/ml（bovine）and then mixed with equal volume of BD Matrigel^ Basement Membrane Matrix（Matrigel）. The testicular cell/Matrigel suspensions were grafted into the subcutis of castrated immunodeficient male mice（BALB/c Slc-nu/nu）. Morphogenesis of the grafts was examined between 1 and 44 weeks after transplantation. To examine the growth and differentiation of donor germ cells, the expression of VASA, which is expressed from gonocytes to early meiotic stages in mammalian male germ cells, was examined by immunohistochemistry. Grafts derived from mouse testicular cells regenerated complete functional seminiferous tubules 20 weeks after transplantation in which haploid spermatids were generated in the lumen. Initiation of reconstruction of seminiferous tubules from bovine testicular cells was also observed 10 weeks after transplantation. However, immunohistochemical analysis demonstrated that bovine germ cells failed to colonize along the basement membrane of reconstructed seminiferous tubules and to proliferate thereafter. Although further investigation for suitable conditions of bovine germ cell colonization and survival in the reconstructed seminiferous tubules is needed, xenoectopic transplantation would make possible to induce bovine spermatogenesis and be usefull for animal sciences, conservation of wild animals and male infertility.

[要旨] 近年、マウスエピブラストより、エピブラスト幹細胞（Epiblast stem cells;EpiS細胞）が樹立された。このEpiS細胞はマウスより樹立されているが、マウス胚性幹細胞（Embryonic stem cells;ES細胞）よりもヒトES 細胞に近い性質を有しており、幹細胞研究の新たなツールとして注目を集めている。このEpiS細胞は、Bone Morphogenetic Protein 4（BMP4）の作用により始原生殖細胞（Primordial germ cells;PGC）の前駆細胞で発現が確認されるBlimp1やStella の発現が増加するが、ヒトES細胞と同様に減数分裂開始前後のPGC様の細胞が誘導可能であるかは不明である。 本実験では、EpiS細胞がBMP4の作用により、PGC様の細胞が誘導可能であるか、移動期で発現するMvh遺伝子や第一減数分裂開始前に発現するStra8遺伝子の発現を確認し検討を行った。 その結果、BMP4 の添加により、EpiS細胞はPGC様の細胞への誘導が促進されることが確認された。 このことから、マウスEpiS細胞は生殖細胞への分化様式がヒトES 細胞と類似している可能性が示された。 [Abstract] Recently, mouse epiblast stem cells（EpiSCs）were established from mouse epiblast. These cell's characters are similar to human embryonic stem cells（ESCs）than mous ESCs. Because of this point, EpiSCs have started to gain attention as a new tool for stem cell research. In this stuy, we examined whether EpiSCs can differentiate into germ cells in a similar manner as human ESCs. Human ESCs candifferentiate to primordial germ cells（PGCs）when bone morphogenetic protein 4（BMP4）is added to culture medium. Similarly, when BMP4 is added EpiSCs can increase Blimp 1 and Stella that are precursor cells of PGCs marker. However, it is not clear whether BMP4 can influence EpiSCs to differentiate into PGCs. As a result, EpiSCs promoted expression of late PGCs marker gene, MVH and Stra8 when BMP4 is added. These results show that EpiSCs are differentiated to germ cells in a manner similar to human ESCs EpiSCs may be available not only for stem cell research, but also in advancing germ cell research with human ESCs.

一側停留精巣を有する36歳の患者で, 対側の陰嚢内精巣に腫瘍が発生した症例を報告した.精巣腫瘍に対しては, 精巣摘除術を施行し, 後腹膜リンパ節転移巣に対して放射線療法を行い, CRを得た.停留精巣は摘除し, 現在, 定期的なテストステロン補充療法を行っている, A 36-year-old man visited the hospital complaining of painless swelling of the right scrotal contents. His left scrotal contents were absent in the scrotum. Right inguinal orchiectomy was performed on May 17, 1993 under the diagnosis of testicular tumor. The pathological diagnosis of the tumor was pure seminoma. Abdominal computed tomography (CT) and magnetic resonance imaging (MRI) revealed the enlargement of para-aortic lymph nodes and there was a soft tissue signal in the left inguinal region which seemed to be the left testis in MRI. His serum testosterone level was low and gonadotropin level was high. The left orchiopexy was performed on June 24, 1993, but the left testis was finally removed because of methicillin-resistant Staphylococcus aureus (MRSA) infections. Radiation therapy was done against the metastatic lesion in the retroperitoneal lymph nodes and complete remission was obtained. The left undescended testis pathologically showed atrophy of germ cells but there was no appearance of atypia of Leydig cells or germ cells.

HZE(高エネルギー重荷電粒子)および宇宙放射線の遺伝的影響を調べるため、ショウジョウバエ(Drosophila melanogaster)の成虫雄と幼虫をスペースシャトル/エンデバー号(STS-47)に搭載し、雄の生殖細胞(精子やその元となる精原細胞など)に起こる伴性劣性致死突然変異と、幼虫の翅原基体細胞に起こる染色体のつなぎかえによる突然変異を調べた。用いた系統は、標準的な野生株(Canton-S)と放射線高感受性株(mei-41)である。各系統で、成虫雄は200匹ずつ、幼虫は約6000匹ずつを搭載し宇宙環境に曝すとともに、ほぼ同数を地上対照群として、宇宙飛行群と同じ環境条件(温度と湿度)で飼育した。宇宙飛行は約8日間であった。帰還した雄のハエは、伴性劣性致死突然変異を調べるため、検出用系統の処女雌に交配し、次々世代で致死遺伝子を保有しているX染色体を検出した。宇宙飛行群の致死遺伝子をもった染色体頻度は、地上対照群の頻度に比べて、野生株では2倍、放射線高感受性株では3倍高かった。幼虫は、帰還時にほとんどが蛹になっており、翌日より徐々に羽化が始まった。羽化した成虫は、順次70%アルコールで貯蔵し、後に翅標本を作成して、染色体突然変異由来の翅毛変異スポットを調べた。野生株では、宇宙飛行群と地上対照群の頻度は、ほぼ同じであった。放射線高感受性株から分離してくるMuller-5個体における頻度は、地上対照群に比べて宇宙飛行群では、1.5倍高かった。しかし、放射線高感受性個体における宇宙飛行群の頻度は、地上対照群に比べて有意に低い頻度を示した。地上対照群に比べて、宇宙飛行群の劣性致死突然変異の高頻度は、生殖細胞において、放射線と微小重力の突然変異誘発作用への相乗効果を示唆している。しかし、この相乗効果は、体細胞の染色体突然変異誘発作用に対しては観察されなかった。, For examining the genetic effects of High Energy Heavy ions (HZE) and cosmic radiation, adult males and larvae of fruit flies (Drosophila melanogaster) were loaded on the Space Shuttle, Endeavor (STS-47), and sex-linked recessive lethal mutation in the male germ cells (spermatozoa and spermatogonia that produce them) and chromosome-recombination-derived mutation on the larva wing basal somatic cells were investigated. The strains used were a standard wild type strain (Canton-S) and a radiation sensitive strain (mei-41). Two-hundred adult males and about 6000 larvae of each strain were loaded and exposed to the cosmic environment. At the same time, almost the same number of adult flies and larvae were bred in the same environmental condition (temperature and humidity) as ground control groups. The flight period was about eight days. The returned male flies were mated with virgin female flies from a detection strain for investigating sex-linked recessive lethal mutation. X chromosomes with lethal genes were detected on the second generation. In the returned flies, the frequency of chromosomes with lethal genes was two times higher in the wild type strain and three times higher in the radiation sensitive strain than that of the ground control groups. Most of the returned larvae were in the pupal stage on the return day, and began to emerge on the next day. The emerged adult flies were stored in 70 percent alcohol solutions, and their wing samples were prepared for investigating the wing mutation spots derived from chromosome mutation. Mutation frequency in the wild type strain was almost the same between the flight and ground groups. The frequency in the Muller-5 individuals separated from the radiation sensitive strain was one and half times higher in the flight group than in the ground control group. However, the frequency in the radiation sensitive strain was significantly lower in the flight group than the ground group. The higher recessive lethal mutation frequency in the flight group suggests the synergistic effect of radiation and minute gravity on mutation induction in germ cells. However, this synergy effect was not observed on the chromosome mutation induction in somatic cells., 資料番号: AA0004115012, レポート番号: NASDA-TMR-940002 V.1

The purpose of this study is to examine the effect of microwaves on germ cells of Drosophila me lanogaster. 1) The materials used were 72-hour old larvae, late third instar larvae, young pupae, and old pupae. The microwaves were applied for five seconds, 10 seconds, and 30 seconds using household electronic range (2.45GHz) with an output power of 200W (Fig. 1). We thus attemped to observe the chromosomal aberrations of meiotic division and to do a somatic protein syntheses analysis of sibling adult flies. 2) In this study, cytological preparations were made by the air-drying method (Tonomura and Shima, 1986) or by the squash method (H. Kayano and A. Kayano, 1987). 3) The primary electrophoresis method was used for the biological analysis protein synthesis by SDG-PAGE on sibling adult bodies and old pupae. We conducted are study by using more than 690 microphotographs of germ cells of 20 females and 20 males taken from each group and exposed to microwaves at various lengths of time to see whether or not cell division had taken place. No abnormal chromosomes were found in larvae exposed to microwaves in the current experiment. However, metaphase polyploidy (?) was found in the first spermatocyte of flies which emerged from young pupa that had been exposed to microwaves for 30 seconds (Fig. 4a). We have also observed chromosomal aberrations in the first spermatocyte and secondary spermatocyte for the 10-second-exposure group of pupae immediately before the emergence (Fig. 4b and c). No chromosomal aberrations were found in the control group (Fig. 5a-c). Electrophoresis using polyacrylamide gel (density; 12 gel) was performed for a biochemical study of protein synthesis in these exposed groups. No abnormalities were found to result from the electrophoresis in male and female flies which had been exposed while they were larvae. However, a loss of protein bands in the molecular-weight region of near 100K dalton was found in flies of the 10-second-exposure group or that had been exposed while they were young pupae or, in old pupae immediately before emergence. This is assumed to be caused by the lack of high-polymer proteins (Fig. 6).

Ovaries of 37 human fetuses from 6 to 40 weeks of gestational age were examined with the electron microscope to know the reason why germ cells decrease in number and to know the process how the follicular epithelial cells are formed. In the fetal ovary before 10 weeks of gestational age, there existed mainly primordial germ cells, and between 10 and 14 weeks, there existed mainly oogonia, and after 17 weeks of gestatinal age there existed mainly oocytes. Between 10 and 14 weeks, germ cells increased remarkably in number, but on the other hand extensive necrosis and degeneration of germ cells occurred during this period. After 17 weeks, groups of dividing oocytes were observed in the same stages of meiotic prophase, and intercellular bridges were often present among them. Between 20 and 40 weeks, oocytes grew slowly. In their cytoplasm, stratiform membranous structures were constructed among the large number of mitochondria. Annulate lamellae were often observed in large mature oocytes. At 40 weeks, primordial germ cells were very few, and existed in the ovarian medulla. Most of them were not encapsulated by epithelial cells. On the contrary, oogonia were covered with epithelial cells. Therefore it is suggested that encapsulation of epithelial cells are required for primordial germ cells to differentiate into oogonia. Between the oocytes and granulosa cells, zona pellucida was found, which indicates zona pellucida is already formed in the fetal stage in humans. Surface epithelial cells divided intensively and increased in number, penetrating into the ovarian medulla, and covered the groups of oogonia. After 17 weeks, a number of primary follicles were formed, and follicle cells changed their shape and developped gradually into granulosa cells. From such observation on human ovaries of whole fetal stage, it is suggested that surface epithelial cells are the origin of granulosa cells. As described above, necrosis and degeneration occurred in germ cells after 10 weeks of gestation. These degenerating germ cells concentrated and became smaller, and were often seen in the cytoplasm of pre-granulosa cells. Since electron dense lipid granules and lysosomes were observed in the cytoplasm of these pre-granulosa cells, it was supposed that they were residual bodies as the result of phagocytosis and digestion.

第22回宇宙利用シンポジウム(2006年1月17日-19日, 日本学術会議6階会議室 六本木、東京), The Twenty-second Space Utilization Symposium (January 17-19, 2006: Science Council of Japan, Roppongi, Tokyo, Japan), In the ICE-first experiment, we have analyzed apoptotic activity in germ cells of the nematode C. elegans, following spaceflight. The results indicate that checkpoint and physiological apoptosis in germ cells proceeds normally in the space environment. It suggests that metazoans, including humans, would be able to eliminate cells that have failed to repair DNA lesions introduced by cosmic radiation during spaceflight. We also found that certain genes related to signaling pathway of G-protein coupled receptor protein and to locomotory behavior tended to decrease in the spaceflown samples. In this presentation, we also explain the perspective of space experiments such as stress responses and RNA interference (RNAi) using the C. elegans., 共催: 日本学術会議, Meeting sponsors: The Science Council of Japan, The Institute of Space and Astronautical Science, Japan Aerospace Exploration Agency (ISAS)(JAXA), 資料番号: AA0064113086

A 39-year-old man experiencing cranial nerve symptoms was referred to our neurosurgery department after a brain tumor was detected on computed tomography (CT) scans at a local hospital. Due to convulsive symptoms, the patient was admitted to our hospital for detailed examination. The patient was diagnosed with right testicular tumor, multiple brain metastases, multiple lung metastases and right kidney metastases, and was transferred to our urology department. Since the testicular tumor was staged as IIIC and identified as poor prognosis by the International Germ Cell Consensus classification (IGCCC), Bleomycin Etoposide Cisplatin (BEP) chemotherapy was initiated prior to surgery. A right high orchiectomy was performed after two courses of BEP chemotherapy. Histopathology revealed mixed germ cell tumors (seminoma and/or embryonal carcinoma+teratoma) along with the following results : ly (−) ; Intratubular Malignant Germ Cells (ITMGC) (+, viable) ; tunica albuginea invasion (−) ; spermatic cord invasion (−) ; tumor size (73× 50×45 mm). Two additional courses of BEP chemotherapy and two courses of Paclitaxel Ifomaide Cisplatin(TIP) chemotherapy were performed successively. The CT revealed metastatic lesions shrinking steadily but the metastatic foci still remained. Since tumor markers were not negative, continuous chemotherapy was considered. However, strong side effects were expected, and treatment was discontinued. Since then, the tumors continued to shrink, and the tumor markers became negative. Currently, the patient maintains complete response and is being followed-up.

平成10年度～平成11年度科学研究費補助金(基盤研究(C)(2))研究成果報告書, 研究概要:刷り込み遺伝子の配偶子特異的メチル化機構を探るため、良性卵巣奇形腫(以下、奇形腫)を利用した。最初に45のDNAマーカーで25例の奇形腫の分類を行った。メチル化解析は父性メチル化の代表としてH19、母性メチル化の代表としてSNRPNを選んでサザーン法およびPCR法により行った。また、ヒト内在性レトロウィルス(HERV)K-familyを選び、活性別およびインプリンティングドメイン内外の局在別に解析することを計画した。結果および結論を以下に要約する。1.欧米の報告と異なり、meiosisIIからの奇形腫発生は25例中1例(4％)と稀である。2.奇形腫は遺伝的に異質な(heterogenousu)細胞集団から成る。3.個々の奇形腫におけるH19およびSNRPNのメチル化はきれいな逆相関関係にあり、奇形腫発生の卵形成上におけるステージが進むにつれ、両者のメチル化パターンは卵子のそれに近づく。4.ヒトH19のprimary imprintは卵形成のかなり遅い時期まで完全に消去されない。またSNRPNのprimary imprintも同様に遅くまで確立されない。5.以上より、配偶子特異的primary imprintの形成は漸進的構築過程であること、primary imprintの消去および確立と相反する二つの機構になんらかの共役した過程が存在することが示唆される。6.胎盤、精巣、肺および末血リンパ球で6種類のHERV-Kの発現が認められ、複数の組織で発現していた3つの活性型HERV-Kを単離したが、11p15.5および15q11-q13に局在するHERV-Kはコスミドライブラリーのスクリーニングからもコンピューター検索からも得ることができなかった。, 要約(欧文):In humans, studies of female germ cells are very limited by ethics. The current study investigated the usefulness of benign ovarian teratomas as a substitute for ova in analyses of imprinted genes. Twenty-five human benign ovarian teratomas were typed with 45 microsatellite DNA markers and classified according to their genotypic features. Two oppositely imprinted genes, H19 and SNRPN, were then chosen for analysis of their methylation states in these tumors. These analyses revealed that benign ovarian teratomas consist of a mixture of genetically and epigenetically heterogeneous cell populations. In contrast to previous reports, we could document only one case rising from germ cells by meiosis-II nondisjunction. H19 and SNRPN were methylated in individual teratomas to various degrees, ranging from normal somatic cell to expected ovum levels. The allele with residual methylation of H19 was consistent with that methylated in the patient's blood DNA, thus being of paternal origin. Degrees of H19 hypomethylation and SNRPN hypermethylation increased as the cellular origin of the tumors advanced in oogenesis and were closely correlated in individual teratomas. These results could be best explained by the assumption that the primary imprinting is a progressively organized process and suggest that the establishment of primary imprints on different genes might be mechanistically linked, even when those genes are oppositely imprinted. Transcripts of six genes of human endogenous retrovirus K (HERV-K)-family members were detected by sequencing of RT-PCR clones in the placenta, testis, peripheral blood leukocytes and fetal lung. Three of them, which were expressed at least in the two tissues analysed, were isolated from a BAC library., 未公開：P.7以降(別刷論文のため)

ショウジョウバエ卵巣は，多細胞生物の器官構築過程を研究する良いモデルとなっている。我々が単離した卵巣形態異常の突然変異体のうち，主にchickadee変異体について間接蛍光抗体法と共焦点レーザー顕微鏡で解析した。変異体３令幼虫卵巣では，始原生殖細胞数の減少，２核化が観察された。変異部位は第１イントロン内であった。chickadeeは，収縮環による細胞質分裂の制御を通じて，幼虫期の生殖系列細胞の増殖や分化に関与していると考えられる。, Drosophila ovary serves as a genetically tractable model for the study of stem cell biology, pattern formation, cell migration and cell proliferation. Regulation of cell division is a crucial aspect in the development of multicellular organisms. The embryonic gonad in Drosophila carries about 12 primordial germ cells (PGCs). PGCs proliferate in the female gonad during the subsequent three larval stages until, at the late-third larval stage, the gonad carries over 100 germ cells. Here we show that profilin gene is required for proper mitotic division of PGCs during larval growth. Profilin mutant contains a reduced number of PGCs that are occasionally binucleated. Inverse PCR revealed that the insertion site of our P-element insertion allele is within the intron 1. Three ligands for profilin have been well-characterized in vitro: actin monomers, membrane polyphosphoinositides and poly-L-proline. Profilin may be involved in the formation of cleavage furrows, resulting in proper cytokinesis of PGCs in Drosophila larval ovary

Plasma FSH and LI-I were determined by radioimmunoassay on six normozoospermic men, twelve oligozoospennic (sperm concentration less than 40×10[6]/ml) and thirty-four azoospermic. Klinefeter's syndrome and obstructive azoospermia were excluded from this study. Plasma FSH of the azoospermic group (18.1mIU/ml) was sigificantly higher than that of the normozo-oligozoospermic group (11.1mIU /ml). Azoospermic men having no germ cells on testicular biopsy showed plasma FSH as high as 21.0 mIU/ml. Those having germ cells, however, showed no significant increase of FSH compared with the normozo-oligozoospennic group. There was no correlation between testicular biopsy score count and plasma FSH. Plasma LH of the azoospermic men without germ cells on testicular histology was 27.6 mIU/ml which was significantly higher than that of the normozo-oligozoospermic group (18.2mIU/ml). Plasma LH of the azoospermic men without germ cells showed positive correlation with their plasma FSH. From the above results, it might be suggested that FSH inhibiting factor originating from the germ cells probably gives suppression on production of FSH whereas androgen on release of FSH.

The testicles of prostatics cancer (10 cases), p rostatic hypertrophy (18 cases), and the control cases of none prostatic disease (above 40 years) were examined histologically and histochemically by the needle biopsies or autopsies. In the testicles of prostatic cancer ard hypertrophy, the decrease or disappearance of germ cells, the thickening of basement membrane of seminiferous tubules, the proliferation of connective tissue of stroma were observed. Above changes of these testicles were nonspecific one and detected commonly in aged testicles. However these changes of testicles were generally more mild than that of the control cases. According to these findings, it was presumed these testicles in prostatic cancer and hypertrophy had a relatively good function. In the cancer testicles of 5 out 10 cases treated with high dosis of estrogen administration, more marked decrease of germ cells, thickening of basement membrane of seminiferous tubules, proliferation of stroma, atrophy and decreasing tendency in number of interstitial cells of Leydig, increase of large lipid granules in the seminiferous tubules and stroma, decrease of alkaline phosphatase reaction in semis iferous tubules were observed .

1. Normal testes of the adult rat and man were ele c tron microscopically observed. Seminoma, teratoma, and embryonal carcinoma were also studied with electron micrographs. 2. As to spermatogenesis, formation of the acrosome in the rat spermatid was demonstrated as well as the structure of the axial fiber bundle which was seen composed of the tubular arrangement of nine axial filaments enveloped with the mitochondrial helix. The mitochondria of the helix were of tubular appearance, being devoid of the mitochondrial cristae but having thick limiting membrane (Fig. 1-4). 3. In general, germ cells were abundant in t he mitochondria and rough surfaced endoplasmic reticulum, but no lipid droplet was found in the cytoplasm. The nuclei were finely granular and of high electron density (Fig. 1, 2, 7, 9, 10). 4. Both Sertoli and Leydig cells were rich in lipid d roplets which are known to be related with steroid production. Lipid droplets in Leydig cell showed two different forms, one was globular and another course granular (Fig. 12). 5. Sertoli cells were small and polygonal in shape, the cytoplasm of which was less dense comparing with germ cells. Cytoplasmic organellae were rather scant. They made network or syncitium with indistinct cytoplasmic border. Leydig cell had vesicular cytoplasm due to vacuoles, some of which contained finely granular substance (Fig. 5, 6, 8, 10). 6. Testicular tumor cells had large nuclei with hypertrophied nucleoli, the nu c leolonema of which were quite prominent. The nucleus of seminoma cell was electron dense with fine granules, whereas that of teratoma was less dense. In embryonal carcinoma the nucleus showed aggregation of course granules arranged in reticular fashion. There were a few interesting findings as to nuclei of the testicular tumor cells. In seminoma, the outer layer of the nuclear membrane was partly detached and protruded into the cytoplasm. In teratoma, "nuclear extrusion" was observed . This phenomenon is, probably, a result of activ e movement of nuclear substance to the cytoplasm. In embryonal carcinoma, osmiophilic substance, RNA from Pars amorpha of the nucleolus, was present close to the nuclear membrane. In cytoplasm of those tumor cells, mitochondria and endoplasmic reticulum were not abundant (Fig. 13-18).

In a mouse, the genital ridge develops around embryonic day (E) 9.5 on the ventral surface of the mesonephros. The genital ridge is identified morphologically around E10.5. At E11.5 to E12.5, the adrenogonadal primordium on the genital ridge separates into the adrenal cortex and the gonad cell populations. However, the precise mechanism of the development is largely unknown. In this study, we report on the production and characterization of a series of monoclonal antibodies (MAbs) against gonad. The antibody, designated as MAb 5B5, yielded the strong signal on germ cells in immunofluorescence staining using the mouse embryo gonad section. Moreover, immunofluorescence analysis using E12.5 mouse various tissue sections revealed that MAb 5B5 also recognizes the cells in neural tube, ventricular zone of cerebral cortex, a part of the epithelial cells, in which tissue-specific stem cells are present abundantly. Furthermore, MAb 5B5 recognized the mouse and human ES and iPS cells. From these findings, it was concluded that MAb 5B5 recognizes stem cells-associated antigen. On the other hand, we found that this antigen expresses cancer cell lines and human tumor tissue. These results suggest that MAb 5B5 may recognize cancer stem cells.

トランスポゾンとその残滓は, ヒトゲノムでは45%もの領域を占めている。トランスポゾンの持つ転移能は個体に有害であり, 特に生殖細胞におけるトランスポゾンの発現抑制は次世代への正確な遺伝情報の伝達を考えた場合に, 生物にとって喫緊の課題である。私は小分子RNAがトランスポゾンの発現を生殖細胞でいかに抑制しているかに関して研究をハエとマウスを用いて行ってきた。ハエにおいては, 特定ゲノム領域に存在するタンパク質群を網羅的に同定する手法を開発してきた。また, マウスにおいては精巣中に含まれる少量の生殖細胞からクロマチン状態をプロファイルし, その様態を明らかにした。 More than 45% of human genome consists of transposons and its remnants. Transpositional events of them are deleterious to their hosts, especially in their gonads. Here, I am engaged in how small RNA would induce transcriptional silencing at transposon sequences, by using both fly and mouse systems. For fly system, I am trying to establish a method to proteomically identify the chromatin components at specific genomic region. For mouse system, I have profiled the chromatin dynamics in the small number of germ cells within the whole testis, and comprehensively described its temporal change in detail, particularly at transposon sequences., 研究種目 : 若手研究(A) 研究期間 : 2014～2016 課題番号 : 26710011 研究分野 : エピジェネティクス

Contents開催･参加・行事報告 ・シンポジウム『カイコ産業の未来』の開催 ・農業生物資源研究所研究成果発表会の開催・“シルク・サミット 2008 in ふくしま”の開催・「まつもと広域工業まつり 2008」への出展・『アグリビジネス創出フェア』への出展報告・2008 年サイエンスキャンプ報告 受賞報告 ・平成 20 年度日本家禽学会優秀論文賞を受賞 “Intense expression of GFP gene in gonads of chicken embryos by transfecting circulating primordial germ cells in vitro and in vivo”

生物の生活環境中には遺伝子レベルでの障害あるいは染色体異常を誘発させる化学物質が多数存在している。 それらの中，特に遺伝的障害を発現する化学変異原物質を検出するために数多くの検出系が考案されている。マウスを用いての実験においては生殖細胞の染色体異常の検出系として優性致死試験や遺伝性転座試験が多く用いられている。しかし，これらの検出系は実験の規模が大きく簡便とはいえない。一般に個体の発生は生殖細胞が合体した1細胞期胚から始まると言える。従って，両親のうちのいずれかの生殖細胞において誘発された障害，あるいはこの障害をもつものを親とする次世代における染色体レベルの遺伝的障害は，さかのぼって1細胞期胚における染色体異常の有無を検索することにより，事前に発見できる可能性があると考えられる。さいわいマウスの1細胞期胚の染色体は特異的で，精子由来と卵子由来の染色体が識別できるので雌雄いずれからの異常かが判別可能である。しかし，1細胞期胚の染色体異常に関する報告は少なくさらに1細胞期胚の染色体異常と他の遺伝的障害との関連性についても明らかにされていない。このことは1細胞期胚の染色体標本作製技術にも問題があると考えられる。 今回の研究は，まず第1にマウス1細胞期胚の染色体標本作製法の改良・開発，第2に，この改良・開発した方法を利用することによって，1細胞期胚の染色体分析結果が環境変異原により発現される優性致死作用や次世代の転座ヘテロ個体の誘発頻度を事前評価できる簡便な検出系になりうるかどうかを検討することを目的とした。そのため既知の化学変異原物質を雄マウスに投与することにより，受精前の配偶子あるいは 受精後の1細胞期胚及び障害を持つ雄による次世代のF_1個体に亘っての障害を明らかにするため種々の実験を実施した。 実験材料と方法 9週齢の雄マウス(Slc-BDF_1)を用い，体重1kgあたり，以下の各変異原物質のそれぞれの量を単独で1回，腹腔内に投与した。 Methyl methanesulfonate (MMS, 100mg) Cyclophosphamide (CPA, 240mg) Trimethylphosphate (TMP, 300mg) Nitrogen mustard-N-oxide hydrochloride (HN_2-O, 100mg) iso-propyl methanesulfonate (i-PMS, 200mg) Procarbazine hydrochroride (PC, 800mg) Mitomycin C (MC, 5mg) Fosfestrol (DSDP, 300mg) なお，X線照射(500 rad)雄マウスについても比較のための実験を行った。 これらの処置をされた雄マウスを経時的に成熟正常雌マウスと交配させ，著者の考案した一連の術式を用いて雌マウスより1細胞期胚を採取し，精子由来の染色体を検査した。 他方，交配後1細胞期胚を採取せず，妊娠を持続せしめた雌マウスについても経時的に着床や胚死の状況を追求すると共に分娩にまで至ったものについては新生仔中のF_1雄個体について遺伝的障害因子保有の有無を知るため，性成熟後に正常成熟雌マウスと交配させ，着床や胚死等の有無を含め，受胎状況を観察し，変異原物質を処置された初代雄の精子による次世代雄への遺伝的障害の有無についても追求した。 また，同様処置した雄マウスの精巣の組織学的所見および精子の形態異常についても検討した。 この他，雄生殖細胞のDNAレベルの障害とその修復機能を観察するため，アイソトープ(thymidine-methyl-^3H)を精巣に投与後，経時的に精子を回収し，オートラジオグラフを作製して不定期DNA合成の発現の有無についても適宜検査した。 また，精子による遺伝的障害以外に精液を介して薬物の雌体内への機械的な搬入による障害等も考慮し，精液中の薬物検出も一部実施した。 実験結果 本研究で改良・開発したマウス1細胞期胚の染色体標本作製法は従来の方法と異なり，標本作製が簡便で，しかも低数性の染色体の発現頻度が低く染色体分析の可能な標本作製についての成功率が高かった。この方法を用いて，精子細胞および精子をMMS，CPA，TMP，HN_2-O，i-PMSおよびX線で処置し，その精子による1細胞期胚の染色体分析を行った。その結果，精子由来の染色体において主に染色体型の異常が観察された。しかし，PCおよびMCで精子細胞および精子を処置した場合は，主に染色分体型の異常であった。一方，精原細胞および精母細胞をPC，MC，i-PMSおよびX線で処置した場合は不受精卵が高頻度に観察された。 変異原物質を雄生殖細胞に処置した場合の各雄生殖細胞に対する優性致死誘発率は，化学変異原物質の種類により異なり，精原細胞では高いが，精母細胞，精子細胞および精子の順に優性致死率が減少するタイプ(PC，MCおよびX線)，精子細胞ないし精子に対しては優性致死誘発作用が高いタイプ(MMS，CPA，TMPおよびHN_2-O)，全ての生殖細胞に対して優性致死誘発作用が高いタイプ(i-PMS)に分類された。また精原細胞および精母細胞の障害に起因する優性致死現象は着床前の胚の損失によるものであったが，精子細胞および精子に対する化学物質の影響による優性致死現象は主に着床前の胚の損失と着床後の胚死によるものであった。これらの実験結果はこれまでの報告とほぼ一致した。 精子細胞や精子に対してMMS, i-PMSおよびX線を処置した雄を用いて交配させ，排卵後72時間目の胚を回収して分析した結果，卵割遅延および卵割停止が観察され，それらの障害の程度は変異原物質の種類により異なった。 精子をHN_2-Oで処置した後に交配受精させ，出産させた次世代F_1雄の妊孕性試験の結果においては，着床後の胚の死亡を主に引き起こす半不妊および着床胚が全く観察されない不妊のF_1雄個体が高頻度に発現した。これらのF_1雄の半不妊および不妊個体の精巣組織学的検索では，不妊個体において精母細胞での精子形成阻害が観察され，一方，半不妊個体では正常な精子形成が認められた。また，これらの半不妊個体の精母細胞の染色体分析では，非相同染色体間の相互転座を示す鎖状および環状の4価染色体が観察され，相互転座ヘテロ個体であることが確認された。 他方，MMS，MCおよびi-PMSを投与された処置当代における雄について精巣の精子形成および精子の形態に対する影響について組織学的に検索した結果，精母細胞，精子細胞および精子の顕著な細胞死および精子形成阻害は観察されなかった。しかし，MCおよびi-PMSを投与した場合は，精原細胞のDNA合成阻害による一時的あるいは永久的な分裂阻害が観察され，これらの精巣は萎縮が観察された。MCを投与した場合の精子の形態異常は主に精母細胞および精原細胞処置のものに観察された。一方，精子変態を過ぎた段階での精子をMC，MMS，i-PMSおよびX線で処置した場合，精子の形態異常は観察されなかった。 生殖細胞のDNA傷害の修復を示唆する不定期DNA合成はMMS，TMPおよびPCを精母細胞および精子細胞に対して処置した場合誘発されたが，MC処置の場合は誘発されなかった。一方，精子に対してMMSおよびMCを処置した場合は誘発されなかった。 考察 以上の実験結果から，精原細胞および精母細胞処置による優性致死試験で観察された着床前の卵の損失は精原細胞の分裂阻害あるいは障害をうけた大部分の精母細胞が発生して精子変態過程において精子形態異常を引き起こし，受精能に支障を生じ，その結果，不受精卵が生じたものと推察された。一方，精子細胞や精子処置により観察された着床前後の胚の損失は，1細胞期胚の精子由来の染色体異常に起因し，染色体異常を有した1細胞期胚が発生し卵割遅延あるいは卵割停止を生じた結果胚死亡を引き起こしたものと推察された。 不定期DNA合成がDNA障害の結果発現したDNAの修復現象と仮に考えると，精子では不定期DNA合成が発現されず，修復能が欠損しているものと推察された。また，精子形成過程に対する優性致死の発現時期と不定期DNA合成の発現時期との相関性が認められないことより，この2つの現象は発現機構を異にするものと思考された。 化学変異原物質を精子細胞および精子に処置し，それを用いて受精した1細胞期胚の染色体分析で主に染色体型の異常を誘発する化学変異原物質は次代のF_1雄に高頻度に半不妊および不任を示す転座ヘテロ個体を誘発し，一方，染色分体型の異常を発現する化学変異原物質の処置では転座ヘテロ個体の誘発頻度が低い。このように，1細胞期胚で観察された染色体異常の種類および発現頻度とF_1を対象とした場合の転座ヘテロ個体の発現頻度とは相関関係が認められた。 体細胞の染色体に対する放射線と化学物質の作用の違いは，放射線は細胞周期と無関係に染色体異常を発現するものに対し，化学変異原物質はどの細胞周期に処置しても染色体異常を発現するには処置した細胞がDNA合成期を経過する必要があり，そこに発現される染色体異常は主に染色分体型であると言われてきた。しかし，本研究においては化学変異原物質を細胞周期のG_1期にあたる精子細胞および精子に処置し，受精後1回目のDNA合成期を経過した1細胞期胚の染色体分析を行った結果，体細胞の場合とはかなり異なり，主に染色体型の異常が発現された。従って，変異原物質による次世代への遺伝的障害の事前評価には生殖細胞を用いた検出系の必要性を指摘したい。 なお，化学物質を雄個体に処置した場合，その化学物質が射出精液を介して雌個体に持ち込まれ，母体に作用し，二次的に卵の発生あるいは着床に影響を及ぼす場合がありうる結果がえられた。そのため化学物質による優性致死試験の結果の評価においては，遺伝的影響以外に化学物質の射出精液を介した雌マウスへの持ち込みによる影響をも併せて考慮する必要性が指摘される。 以上の実験結果より以下の結論が得られた。 1) 本研究において改良・開発されたマウス1細胞期胚の染色体標本作製法は，簡便であるとともに，信頼度の高い方法である。 2) 1細一期胚の染色体分析は，化学物質に暴露された配偶子の受精能に対する影響も判定できる。 3) 優性致死試験で胎仔の認められない場合の原因について，これが不受精によるのか，染色体異常によるのかの判定には，1細胞期胚の染色体の分析が有効である。 4) 顕著なDNA障害を有した精子でも受精は可能であり，この場合，1細胞期胚において染色体異常が観察される。 5) 体細胞の場合と異なり，雄生殖細胞を化学物質で処置した場合は，1細胞期胚の染色体分析で，おもに染色体型の異常が発現され異常の発見が容易である。 6) 染色体異常を有した大部分の1細胞期胚は，初期胚および胎仔において発生遅延や発生停止を引き起こし，致死経過をとる。 7) 化学変異原物質で雄生殖細胞を処置した後，1細胞期胚の染色体分析で主に染色体型の異常を発現させる化学変異原物質は，次世代のF_1において転座ヘテロ個体を高頻度に誘発する。 従って1細胞期胚の染色体分析は優性致死および次世代の転座ヘテロ個体の発現頻度を事前評価でき，環境変異原物質を対象とした簡便な遺伝毒性の検出系として充分利用できるものと考えられる。, A large number of chemical mutagens that may exert potentially hazardous effects on DNA of animal life exist in our environments. A large variety of test methods have been introduced for the detection of these substances that engender genetic risks. However, a single test method does not permit detection of all genetic damages, because of the multifariousness and multiformity of damages. Genetic damages can be categorized into two main types; gene mutation and chromosomal aberration. Mammalian mutagenesity tests for the detection of chromosomal aberrations induced in germ cells by various agents are usually achieved by dominant lethality test and heritable translocation test. However, these tests are costly and time-consuming. Furthermore, they require a very large number of animals. Since life begins with one-cell embryo, chromosomal aberrations induced in male or female germ cells or transmissible chromosomal aberrations actually transmitted to a next generation may be preestimated by cytological analysis of one-cell embryo. At the first cleavage metaphase of mice, paternal and maternal chromosome sets can be readily distinguished by differential chromosome condensation. Therefore, it is possible to discriminate the origin of chromosomal aberrations at the first cleavage metaphase. The purpose of this study is to investigate various genetic disorders induced in male gamete, one-cell embryo, early embryogenesis before and after implantation and F_1 offspring by treatment of male mice with known chemical mutagens. Important aspects of this experiment are to determine whether chromosomal aberrations detected in one-cell embryo correlate with various genetic damages induced by treatment with chemical mutagens and, furthermore, to evaluate the usefulness of cytological analysis of one-cell embryo as a simple test method that permits preestimation of dominant lethal effects and heritable translocations due to environmental mutagens.Materials and Methods Slc-BDF_1 (C57BL/6 X DBA/2) mice, 9 weeks old at the commencement of the experiment, were used. In all experiments, male mice were treated by intraperitoneal injectin of a single dose of chemicals. The chemicals used were methyl methanesulfonate (MMS, 100mg/kg), cyclophosphamide (CPA, 240mg/kg), trimethylphosphate (TMP, 300mg/kg), nitrogen mustard-N-oxide hydrochloride (HN2-O, 100mg/kg), isopropyl methanesulfonate (iPMS 200mg/kg), procarbazine hydrochloride (PC, 800mg/kg), mitomycin C (MC, 5mg/kg) and fosfestrol (DSDP, 300mg/kg). X-ray irradiation (500 rad) was also used for comparison with the chemical mutagens. After treatment, each male was mated with a normal female. One-cell embryos of the first cleavage metaphase were then collected from the ampulla, and chromosome specimens prepared by a specially devised technique were analyzed for chromosomal aberrations in the paternal chromosome sets. Also, embryos were flushed from the oviduct and uterus on day 4 of pregnancy to examine the conditions of embryonic development. Furthermore, in the dominant lethality test, the numbers of live and dead embryos were counted in females dissected on day 13 of pregnancy. A fertility test of F_1 male offspring was also performed to investigate whether chemically induced damages in paternal germ cells were transmitted to the F_1 generation as heritable tanslocation carriers. The testes and sperm from the vasa deferentia were removed at various times from animals treated with chemicals. The testis sections were observed to evaluate spermatogenesis and to determine stages at which arrest of spermatogenesis occurred. Sperm shape abnormalities were also examined. Unscheduled DNA synthesis in male germ cells, which reflects a repair of chemically damaged DNA, was also investigated using thimidine-methyl-^3 H. Detection in females of ejaculated chemicals that had been injected intraperitoneally into males was also attempted, because it was considered possible that the transfer of injected chemicals by ejaculation to females may exert an embryotoxic effect apart from genetic damages.Results and Discussion The new method of preparing chromosome specimens employed in the present experiment was a reliable and simple technique enabling karyological examination of embryos from one-cell to blastocyst of mice. Excellent spreading of chromosomes can be obtained with a low frequency of hypoploid mtaphase. Mainly a chromosome type of aberrations was detected in the paternal chromosome sets at the first cleavage metaphase after treatment of spermatozoa and spermatids with MMS, CPA, TMP, HN2-O, iPMS or X-ray. However, chromosomal aberrations detected after their treatmen with PC or MC consisted mainly of a chromatid type of aberrations. On the other hand, treatment of spermatogonia and spermatocytes with PC, MC, iPMS or X-ray resulted in an increased frequency of unfertilized eggs in analysis of one-cell embryos. The dominant lethality by treatment of male mice with the mutagens showed differential spermatogenic responses depending on mutagens. The dominant lethality patterns specific to germ cell stages were divided into three types. In type 1 (PC, MC or X-ray), the dominant lethality induction decreased with spermatogenesis from spermatogonia to mature sperm. In type 2 (MMS, CPA, TMP or HN2-O), the induction of dominant lethality was frequent in spermatozoa and late spermatids, and infrequent or not observed in spermatocytes and spermatogonia. In type 3 (iPMS), the dominant lethality was induced frequently in all the spermatogenic stages. The majority of dominant lethality induced in spermatozoa and spematids resulted from the pre- and post-implantation losses of embryos, while those induced in spermatocytes and spermatogonia resulted from the preimplantation loss. The embryonic development was considerably retarded in emryos of day 4 of pregnancy as a result of MMS, iPMS or X-ray treatment of male postmeiotic germ cells. This developmetal retardation differed by the kind of mutagens. A heritable translocation test was performed in F_1 male mice collected from mothers that had been mated with males treated with HN2-O. The test showed a high frequency of sterile and semi-sterile F_1 male offspring. Histological testicular analysis of the sterile and simi-sterile F_1 males revealed arrest of spermatogenesis at the stage of spermatocyte in the majority of sterile F_1 males, but normal spermatogenesis in semi-sterile F_1 males. Cytological analysis of these sterile and semi-sterile F_1 males at diakineisis-metaphase I of spermatocytes revealed the presence of chain- or ring-shaped four configuration in most of them, confirming them to be translocation carriers. Histological analysis of the seminiferous tubules after treatment of the testes with MMS, MC or iPMS demonstrated no disturbance of the development of spermatocytes, spermatids and spermatozoa by these mutagens. However, by treatment with MC or iPMS, spermatogonia underwent temporary or permanent inhibition of division and necrosis. The testes with inhibited spermatogonial division were small. Sperm abnormalities increased in the stage of spermatogonia, spermatocyte and spermatid after treatment with MC. However, mutagenic treatment with MC, MMS, iPMS or X-ray produced no measurable increase of sperm abnormality in spermatozoa after spermiogenesis. Unscheduled DNA synthesis (UDS) which reflects a repair of chemically damaged DNA was demonstrated in spermatocytes and spermatids treated with MMS, TMP or PC but not in those treated with MC. UDS after treatment with MMS or MC was not detected in spermatozoa which represent the most mature germ cell stage. The results of the present experiment indicated that the frequency of preimplantation loss of embryos in the dominant lethality test after premeiotic cell treatment with the mutagens was closely correlated with that of unfertilized eggs. The occurrence of unfertilized eggs seems to have principally resulted from oligospermia or azospermia due to inhibition of spermatogonial division or fertilization failure which had been caused by sperm abnormalities induced during the spermatogenesis of spermatids that developed from damaged spermatocytes. therefore, the preimplantation loss of embryos in the dominant lethality test by premeiotic cell treatment may be attributed to unfertilized eggs. the frequency pre- and post-implantation loss of embryos was closely correlated to that of one-cell embryos with chromosomal aberrations. It seems probable that one-cell embryos with chromosomal aberrations observed in the paternal chromosome sets reflected the cleavage failure in the very early stage of embryogeneis and thus be described as pre- and post-implantation losses. If unscheduled DNA synthesis should be interpreted as a repai of damaged DNA, any damaged DNA in spermatozoa of the most mature cell stage seemed unrepairable. There was no correlation between the frequency patterns of dominant lethality and unscheduled DNA synthesis in the male germ cell stage. These phenomena, dominant lethality and unscheduled DNA synthesis, may be considered to represent different levels of DNA responses when exposed to mutagenic agents. Chemical mutagens that induce a chromosome type of aberrations in paternal chromosome sets at the first cleavage metaphase by treatment of postmeiotic male germ cells tend to producd high rates of heritable translocations in F_1 offspring. On the other hand, chemical mutagens that induce a chromatid type of aberrations cause a low incidence of heritable translocations. It is postulated that the incidence of heritable translocations induced in F_1 offspring may be estimated on the basis of the type of chromosomal aberrations that occur at the first cleavage metaphase. Chromosomal aberrations produced in somatic cells by chemical agents and ionizing radiation are known to differ in that ionizing radiation provokes chromosomal aberrations regardless of treatment cell cycles, while chemical agents at any cell cycle provoke chromosomal aberrations to appear after first cell division. Chemical agents also induce mainly a chromatid type of aberrations. In this study, however, chemical treatment of postmeitic male germ cells produced chromosomal aberrations that differed from those seen in somatic cells and the chromosomal aberraions induced were mainly a chromosome type. This indicates the necessity of genetic assay using mammalian germ cells for preestimation of various human genetic defects by chemical substances. The chemical agents injected intraperitoneally into male mice was transferred into females by ejaculation during mating and caused retardation of tubal embryonic development or inhibition of intrauterine implantation of embryos. These phenomena indicate that the chemicals transferred by ejaculation into females do not exert a direct effect on embryos but disturb the physiological integrity of the endometrium which is required for proper implantation. The effects of ejaculation-mediated transfer of chemicals ought also to be taken into consideration in estimation of dominant lethality test results after injection into male mice of chemicals such as steroid hormones.Conclusion Cytogenic analysis at the first cleavage metaphase allows preestimation of dominant lethality and heritable translocation carriers produced among F_1 offspring. It is, furthermore, a simple assay technique for determination of heritable germ line effects of chemical mutagens.

Genomic imprinting refers to the parental-origin-specific gene expression of a subset of autosomal genes in mammals. Disruption of imprinting causes embryonic or postnatal lethality, growth retardation, abnormal behavior and many human diseases. It has been proposed that DNA methylation marks the imprinted genes differently during male and female gametogenesis. The epigenetic differences (imprints) between the two gametes lead to parental-origin-specific gene expression in the offspring. These imprints are maintained during development but erased in the fetal germ cells, and then re-established during gametogenesis in a sex-specific manner. Mice deficient for Dnmt1, which is a maintenance methyltransferase, show embryonic lethality, genome-wide demethylation and disruption of genomic imprinting. This indicates Dnmt1 is essential for the maintenance of imprints postfertilization. On the other hand, the primary germline imprints are thought to be established during gametogenesis through the action of a de novo DNA methyltransferase(s). However, targeted disruption of the de novo DNA methyltransferases (Dnmt3a and Dnmt3b) in mice results in embryonic or early postnatal lethality. Therefore, the role of DNA methylation in the establishment of the germline imprints cannot be addressed in these mice. To circumvent this problem, I took advantage of the Cre-loxP system to inactivate the two de novo DNA methyltransferases in a germline-specific manner. The floxed Dnmt3a and Dnmt3b alleles were disrupted in both male and female germlines by introducing Cre recombinase driven by the endogenous TNAP (tissue non-specific alkaline phosphatase) promoter. These germline-specific Dnmt3a and Dnmt3b knockout mice should be viable and expected to grow up to adulthood because the deletion should occur only in germ cells. Analysis of the embryos and gametes from these germline-specific Dnmt3a and Dnmt3b knockout mice will determine the function of each enzyme in the establishment of genomic imprints. The conditional Dnmt3a knockout mice that I generated were indeed viable and grew up to adulthood, though somatic tissues had various degrees (56%-80%) of the deletion. Recombination efficiency was determined in fetal germ cells, oocyte and sperm, which suggested that the Dnmt3a alleles was mostly inactivated before the onset of methylation imprints. Offsprings from the conditional Dnmt3a knockout females crossed with wild-type males died around embryonic day 9.5-10.5 (E9.5-10.5). All embryos that I examined had recombined allele only, suggesting that the recombination efficiency was 100%. Embryos appeared grossly normal at E9.5, but they showed growth retardation, defects in neural tube closure and lack of branchial arches by E10.5. At E11.5, only resorptions were seen. The phenotype was similar to that of the embryos conceived by Dnmt3L knockout females, which are defective in establishing the maternal methylation imprints during oogenesis. Indeed, the maternal methylation imprints and the allele-specific expression of several imprinted genes that I examined were lost in the embryos conceived by the conditional Dnmt3a knockout mice. These results indicate that Dnmt3a is a key enzyme that establishes maternal methylation imprints during oogenesis. Since Dnmt3L does not have any detectable DNA methyltransferase activity, it is conceivable that Dnmt3a and Dnmt3L cooperate in the process of maternal methylation imprinting. The conditional Dnmt3a knockout males were also viable but showed impaired spermatogenesis, again resembling the Dnmt3L knockout males. At postnatal day 11 (P11), the testes from these conditional Dnmt3a knockout males appeared normal, but, at 11 weeks of age, the size and weight of the testes were significantly reduced. Virtually no spermatids or spermatozoa were observed in the seminiferous tubules of these testes. These results showed that Dnmt3a, as well as Dnmt3L, is required for spermatogenesis. Both the conditional Dnmt3b knockout males and females were viable and grew up to adulthood, in contrast to the Dnmt3b-null mice, which die in late gestation. No deletion was observed in most somatic tissues examined, except for a small proportion of skeletal muscle cells. Most fetal germ cells had recombined Dnmt3b allele, suggesting that the floxed Dnmt3b allele was mostly deleted before the onset of methylation imprints. A total of 88 pups were born from the conditional Dnmt3b knockout females and 87 pups had the recombined allele, suggesting a high rate of recombination by Cre. These pups grew up to adulthood and were fertile. No abnormalities were observed in the embryos. The methylation levels of the imprinted genes and minor satellite DNA, which is the target sequence of Dnmt3b, were also normal. These results suggest that Dnmt3b is not required for oogenesis or the establishment of maternal methylation imprints. The pups and embryos derived from the conditional Dnmt3b knockout males were also normal and showed 100% recombination efficiency (102/102). No abnormal phenotype, imprinting defect, or change in methylation level was observed. Also, histological sections of the testes from these conditional Dnmt3b knockout males showed normal spermatogenesis. These results suggest that Dnmt3b is not required for spermatogenesis or the establishment of paternal methylation imprints. I also examined whether the Dnmt3b allele recombined by Cre was functionally null. Embryos homozygous for the recombined Dnmt3b allele were embryonic lethal and the phenotype (growth retardation, rostral neural defects and demethylation of centromeric minor satellite repeats) was very similar to that of Dnmt3b-null embryos. These observations confirmed that the floxed Dnmt3b allele was successfully inactivated by the Cre recombinase. This work suggests that Dnmt3a, but not Dnmt3b, is responsible for the establishment of the maternal imprints. The results from my work also suggest that the primary imprints established in gametogenesis is DNA methylation. Dnmt3a is also required for spermatogenesis, but whether Dnmt3a is responsible for the establishment of paternal methylation imprints is yet to be investigated.

本研究では、モデル生物の1つである線虫C.elegansを用いて、微小重力や宇宙放射線をはじめとする複合的な宇宙環境ストレスが生物に及ぼす影響について調べることを目的としている。(1)我々はこれまでに、国際宇宙線虫実験ICE-first(2004年4月フライト)に参画し、宇宙で成長した線虫においても地上の対照区と同様に、減数分裂チェックポイント制御下のアポトーシスならびに卵母細胞成熟に伴う発生制御下のアポトーシスがともに正常に行われること、(2)筋肉関連の幾つかの遺伝子群ならびにタンパク質が発現レベルで顕著に低下することを見出してきた。(3)また、過重力による影響は受精直後の卵核の減数第1・第2分裂に最も影響を及ぼすことを証明してきた。(4)さらに、様々な環境ストレス(放射線など)の生物影響について、各種突然変異体やRNA干渉法(RNA interference: RNAi)による特異的な遺伝子発現の抑制体とDNAマイクロアレイを用いることにより、ゲノムワイドな分子モニターができることを明らかにしてきた。これらの結果から、複合的な宇宙環境ストレスが個体レベルに及ぼす分子影響を解析する上で、モデル生物の線虫が大変有効であることが示唆された。今後は、第1に宇宙環境におけるRNA干渉機構(RNAi)の効果を検証し、RNAiによりDNA損傷のチェックポイント制御を抑制した場合における細胞内での諸変化について調べる。さらに、アクチンや細胞骨格の再構築を介し、重力感受にも関わる可能性が想定されるRho-guanine nucleotide exchange factor(RhoGEF)の一変異体であるunc-73と野生株N2との遺伝子およびタンパク質発現の変化を比較し、シグナル伝達を含めたタンパク質のリン酸化に対する宇宙環境の影響について明らかにしたい。また、宇宙環境下で長期間、連続的な世代交代を通して生じる適応と変異・進化の方向性について明らかにすることを次なる研究課題として位置づけている。, It is important for human life in space to study the effects of environmental factors during spaceflight on a number of physiological phenomena. In the ICE-first experiment (International Caenorhabditis elegans Experiment-1, Mission DELTA, April 2004), we found that (1) both pachytene-checkpoint apoptosis and physiological apoptosis in germ cells occurred normally under spaceflight conditions and (2) certain genes related to signaling pathway of G-protein coupled receptor protein and to locomotory behavior tended to decrease in the spaceflown C. elegans. (3) In the hypergravity condition, we determined that the most susceptible aspect in the life cycle of this organism is the oocyte meiotic division for exclusion of polar bodies and the anterior-posterior polarization shortly after fertilization. (4) We also found several genes specifically responding to environmental stresses such as radiation by transcriptome analyses of entire genome using DNA microarray in several mutants and RNA interference (RNAi) animals. Since these phenomena and the identified genes are highly conserved in mammalian systems, the model experiments using this organism are useful to study the effects of space environment on human life. We are now attempting to study the RNAi effect, signal transduction, adaptation and evolution responding to space environment in future space experiments using the nematode C. elegans., 資料番号: AA0049199001, レポート番号: JAXA-RR-05-033

症例1:42歳男.5年5ヵ月の不妊を主訴として受診し, 無精子症を指摘された.全身所見で異常はなく, 精液検査所見も無精子症であった.染色体検査では末梢リンパ球培養G band法で細胞20個において検査を行ったところ46, XXと女性の核型であった.また2番染色体短腕p11, 長腕q13を切断点とする腕間逆位が認められた.以上の所見よりXX male症例と診断した.さらにPCR法によるsex-determining region Y(SRY)は陽性, deleted in azoospermia(DAZ)は陰性であった.患者は挙児は希望せず, 現在も外来で経過観察中である.症例2:29歳男.1年3ヵ月の不妊を主訴として受診した.症例1と同様に無精子症を指摘され, 全身所見で異常はなく, 精液検査所見も無精子症であった.染色体検査でも症例1と同様に末梢リンパ球培養G band法で, 細胞20個において検査を行ったところ46, XXと女性の核型であり, 以上の所見より, XX male症例と診断した.また, PCR法による測定を行い, SRYは陽性, DAZは陰性であった.挙児を希望し, 非配偶者間人工授精により正常男児がえられた, We report two cases of XX male with chief complaints of infertility. Physical examination of both patients aged 42 and 29 demonstrated normal male habitus except for small testes. Semen analyses demonstrated no spermatozoa. Endocrinological examinations showed hypergonadotrophic hypogonadism. Vesiculograms demonstrated normal seminal tracts. Histological examination of their testes did not reveal germ cells; one case lacked seminiferous tubules and there was hyalinization in the seminiferous tubule in another case. Chromosomal analyses of peripheral blood demonstrated 46, XX. The sex-determining region Y gene was positive and DAZ (deleted in azoospermia) gene was negative in both cases.

From the images of domestic animals photographed with digital cameras and the images of fertilization of gametes and early development of zygotes in various mammals taken by a CCD color camera connected to an inverted microscope and a time-lapse video cassette recorder, dynamic visual aids have been created and developed. These dynamic visual aids that have been independently created were utilized in actual classes and their educational effectiveness was clarified through questionnaire surveys. With respect to the query upon whether characteristics were comprehended regarding the mammalian germ cells (sperm, eggs and embryos) as resources and the biotechnological new techniques in animal production, 66 or 80% of the students replied in the affirmative far exceeding the number of students who replied in the negative. Furthermore regarding the query as to whether seeing the slides of eggs and embryos in various mammals and their video images was instrumental in their respective understanding of them, over 84% of the students answered in the positive. From the above, the use of independently developed slides and video images as teaching materials was judged as being undoubtedly effective in the comprehension of the content of lectures. From the results of questionnaire survey, it was concluded that the educational effectiveness of courses which positively employed such visual aids was undoubtedly large.

無精子症または乏精子症と診断された男性99名(21～55歳)の精漿を検体に, ゲル電気泳動による乳酸脱水素酵素(LDH)アイソザイムキットを用い, 不妊症患者の精漿中のLDH-C4を定量した.対照群の健常者10名の血清中総LDHは99.5±5.6IU/l, 不妊症患者99名の精漿中の総LDHは2487±1384IU/lと健常者に比べ, 約25倍の値を示した.無精子症7例中4例の精漿中にLDH-C4が検出された.精漿LDH-C4は不妊症患者の精巣の精子形成能と密接な関係を持っており, 男子不妊症の診断, 治療において従来のセミノグラムを補う有用な指標に成り得ると考えられる, Lactate dehydrogenase C4 (LDH-C4) is the specific isozyme of LDH produced by germ cells. We measured LDH-C4 activity in seminal plasma from infertile men with oligozoospermia or azoospermia using gel electrophoresis. Total LDH activity in seminal plasma from infertile patients (n = 99) was 2, 487 +/- 1, 384 IU/l (mean +/- SD). LDH-C4 isozyme activity was detected in 63 out of 75 seminal plasma samples from infertile patients with a mean of 383 +/- 356 IU/l (13.8 +/- 8.6% of total LDH). Sperm count was positively correlated with LDH-C4 (r = 0.298; P < 0.05), but not with any other LDH isozymes or with total LDH. Seminal LDH-C4 was significantly lower in patients with varicoceles (253 +/- 223 IU/l) than without varicoceles (474 +/- 262 IU/l). Six azoospermia patients were treated with hCG and hMG. Three out of four patients whose seminal plasma revealed LDH-C4 activity responded to the treatment, whereas none of the other two patients without seminal LDH-C4 activity did. These results indicate the clinical usefulness of seminal LDH-C4 as a potential marker for seminal epithelium activity in the diagnosis and treatment of male infertility.

Chimeric mice containing a human chromosome were produced by introducing a human chromosome into mouse embryonic stem cells via microcell-mediated chromosome transfer. In present study, the dead foetuses of the chimeric mice containing a human chromosome 2-derived fragment (hChr.2f) or a human chromosome 21 (hChr.21) were studied anatomically in order to examine the effects of introduction of a human chromosome to mouse. Polydactyly were obsered in almost of the chimeric foetuses containing hChr.2f, but were not detected in the chimeric foetuses containing hChr.21. Aplasia of the sternum and delayed ossification or deletion of the cervical vertebrae centrum were universally observed in both the chimericfoetuses. Histological abnormalities in heart and liver were frequently detected in both the chimeric foetuses. Interestingly, the number of germ cells decreased remarkably in all of the chimeric foetuses containing hChr.21.