Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

The large Japanese field mouse (Apodemus speciosus) is endemic to Japan and may be used as an animal model for studies related to environmental pollution, medical science, and basic biology. However, the large Japanese field mouse has low reproductive ability due to the small number of oocytes ovulated per female. To produce experimental models, we investigated the in vitro developmental potential of interspecies somatic cell nuclear transfer (iSCNT) embryos produced by fusing tail tip cells from the large Japanese field mouse with enucleated oocytes from laboratory mice (Mus musculus domesticus). Only a small number of iSCNT embryos developed to the 4-cell (0-4%) and blastocysts (0-1%) stages under sequential treatment using trichostatin A (TSA) and vitamin C (VC) supplemented with deionized bovine serum albumin (d-BSA). This sequential treatment led to the reduction in H3K9 trimethylation and did not affect H3K4 trimethylation in at least the 2-cell stage of the iSCNT embryos. Moreover, iSCNT embryos that received tail tip cells with exposure treatment to ooplasm from cell fusion to oocyte activation or VC treatment prior to cell fusion did not exhibit significant in vitro development improvement compared to that of each control group. This suggests that large Japanese field mice/laboratory mice iSCNT embryos that received sequential treatment using TSA and VC with d-BSA may have slightly better developmental potential beyond the 4-cell stage. Our results provide insights into the reprogramming barriers impeding the wider implementation of iSCNT technology.

Piezo-actuated intracytoplasmic sperm injection (Piezo-ICSI) is used as an efficient in vitro fertilization method with various animals. With this method, elongated spermatids are collected from testicular tissues and are easier to obtain from animals that unexpectedly die than ejaculate sperm. Additionally, elongated spermatid injection often results in the development of embryos and offspring. To develop assisted reproductive techniques (ARTs) for domestic cats, we examined the effects of oocyte activation on cleavage and embryo development after Piezo-ICSI with motile sperm (experiment 1) and after Piezo-ICSI with either testicular sperm or elongated spermatids (experiment 2). In experiment 1, the proportions of cleaved embryos, morulas, and blastocysts following Piezo-ICSI with ethanol activation were significantly higher (P < 0.05) than in the non-activated groups. However, the proportion of blastocysts and the blastocyst quality did not differ significantly (P > 0.05) between the ethanol-activated and non-activated groups. In experiment 2, the cleavage frequencies of oocytes after Piezo-ICSI of testicular sperm or elongated spermatids and ethanol activation were higher (P < 0.05) than that of oocytes in the non-activated group, but the occurrence of blastocyst formation and quality of blastocysts did not differ between the activated and non-activated groups. In summary, cat embryos can be produced by Piezo-actuated microinjection of elongated spermatids. Ethanol activation increased the frequency of cleavage, but it affected neither the occurrence of blastocyst development nor the quality of blastocysts. These results represent an expansion in the repertoire of ARTs that are potentially applicable to both domestic and endangered species of cats.

The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

Animal cloning has been achieved in many species by transplanting differentiated cell nuclei to unfertilized oocytes. However, the low efficiencies of cloning have remained an unresolved issue. Here we find that the combination of two small molecules, trichostatin A (TSA) and vitamin C (VC), under culture condition with bovine serum albumin deionized by ion-exchange resins, dramatically improves the cloning efficiency in mice and 15% of cloned embryos develop to term by means of somatic cell nuclear transfer (SCNT). The improvement was not observed by adding the non-treated, rather than deionized, bovine serum. RNA-seq analyses of SCNT embryos at the two-cell stage revealed that the treatment with TSA and VC resulted in the upregulated expression of previously identified reprogramming-resistant genes. Moreover, the expression of early-embryo-specific retroelements was upregulated by the TSA and VC treatment. The enhanced gene expression was relevant to the VC-mediated reduction of histone H3 lysine 9 methylation in SCNT embryos. Our study thus shows a simply applicable method to greatly improve mouse cloning efficiency, and furthers our understanding of how somatic nuclei acquire totipotency.

Here, we showed that the Clock gene was important for reproductive performance in mice. We compared outcomes from the four possible mating combinations between wild-type mice (WT) and mice homozygous for the Clock delta-19 mutation (CL). We found that the only significant differences were between the WT male x WT female and CL male x CL female mating groups; these groups differed with regard to elongation of the pregnancy period (19.3 vs. 20.5 days, respectively, P < 0.05) and the number of newborn pups (13.4 +/- 0.8 vs. 8.6 +/- 1.5, respectively, P < 0.05). Because CL dams impregnated by male CLs exhibited normal continuous increases in body weight during the entire gestation period and did not show any signs of spontaneous abortion from mid to late gestation, we reasoned that some embryos were lost before or at the time of implantation. Immediately before implantation (88 hours after fertilization), neither the number of embryos collected from uteri nor the percentage of the embryos that reached the blastocyst stage differed significantly among mating groups. In contrast, immediately after implantation (160 hours after fertilization), the average number of implantation sites was significantly lower for the CL male x CL female mating group than that for the WT male' x WT female mating group (7.0 vs.13.0, P < 0.05); this decrease was accompanied by a significant lowering of the positions of implantation sites in uteri, and this lowering of the implantation sites was more severe when mothers and embryos bore more CL alleles (WT male x WT female > CL male x WT female > WT male x CL female > CL male x CL female), suggesting that the Clock mutation reduced the reproduction performance of the parents by affecting the implantation capacity via such as embryos' ability to implant. (C) 2016 The Author(s). Published by Elsevier Inc.

© 2016, Springer Science+Business Media New York. Purpose: We investigated whether enzymatically fabricated hyaluronan (HA) microcapsules were feasible for use in the cryopreservation of a small number of sperm. Methods: HA microcapsules were fabricated using a system of water-immiscible fluid under laminar flow. Three sperm were injected into a hollow HA microcapsule using a micromanipulator. Capsules containing injected sperm were incubated in a freezing medium composed of sucrose as the cryoprotectant and then placed in a Cryotop® device and plunged into liquid nitrogen. After thawing, the capsule was degraded by hyaluronidase, and the recovery rate of sperm and their motility were investigated. Results: The HA microcapsule measuring 200 μm in diameter and with a 30-μm thick membrane was handled using a conventional intracytoplasmic sperm injection (ICSI) system, and the procedure involved the injection of sperm into the capsule. The HA microcapsules containing sperm were cryopreserved in a Cryotop® device and decomposed by the addition of hyaluronidase. The recovery rate of sperm after cryopreservation and degradation of HA microcapsules was sufficient for use in clinical practice (90 %). Conclusions: Hollow HA microcapsules can be used for the cryopreservation of a small number of sperm without producing adverse effects on sperm quality.

本実験では、死亡個体から採取した筋組織に真空乾燥処理を施し、8〜13 ヶ月間凍結保護剤を用いず- 30℃下にて冷凍保存を行った。保存された組織から修正MIP 法を用いて筋組織由来体細胞核を回収した。得られた体細胞核は、免疫組織学的染色を行い、核内Histone H3 および核膜構成タンパク質Lamin B2 の局在を観察した。さらに、得られた体細胞核の一部を体細胞核移植操作により、前核様構造形成の確認を行った。真空乾燥処理を施した各動物筋組織から体細胞核の回収に成功し、0.64nL 当たりに含まれる体細胞核数は、それぞれ、6.4 個（ マウス）、5.8 個（ケープハイラックス）であった。得られた体細胞核を用いた、免疫組織学的染色では、いずれの組織由来体細胞核にもHistone H3 およびLamin B2 の局在を認めた。体細胞核移植操作による前核様構造の形成率は、マウス74％（43/58））、ケープハイラックス96％（44/46）であった。以上の結果より、真空乾燥処理を施した筋組織由来体細胞核には、核膜構成タンパク質および核内Histone H3 が存在し、前核様構造形成能力を有することから、研究資源としての保存の有効性が期待できると考えられた。In this study, the muscle tissues derived from dead animals were vacuum dried and cryopreserved without cryoprotectant at -30C for 8 to 13 months. The myocyte nuclei were successfully collected from these preserved tissues by modified MIP methods（ Mus musculus : 6.4/0.64nL and Procavia capensis : 5.8/0.64nL）. The normal localization of Histone H3 and Lamin B2 in the collected myocyte nuclei were confirmed by immunohistochemical staining. In addition, somatic cell nuclear transfer was performed using collected myocyte nuclei and the formation of pronuclear-like structure were observed in reconstructed oocytes（ Mus musculus : 74％（43/58） and Procavia capensis : 96％（44/46））. From these results, it was suggested that the combination method of vacuum drying and cryopreservation is the useful method for the preservation of animal muscle tissue for long time as for the genetic resource.

C57/BL/6Jマウスを用い、β-ニコチンアミド・モノヌクレオチド(β-NMN)を体外成熟培地に用いることによる体外成熟卵子の細胞質内活性酸素種(ROS)に与える影響および体外成熟卵子の発生能に与える影響について検討した。各濃度のβ-NMNを添加したmTaM体外成熟培地による57/BL/6Jマウス由来GV期卵子を用いた体外成熟成績は、これら各濃度のβ-NMNを添加した体外成熟培地による体外成熟率は1mM区では81%(64/79)、2mM区では84%(70/83)、5mM区では83%(75/90)、10mM区では83%(69/83)で、非添加区における体外成熟後のMII期卵子発生成績74%(135/183)と比べ有意差は認められなかった。ROSの検出における蛍光輝度成熟は74%であり、非添加群と比べ、1mMおよび2mM添加区において蛍光輝度の低下を認めた。これらの結果、mTaM培地にβ-NMN添加は細胞質内のROSの過剰産生を抑制することが認められた。

[要旨] これまで遺伝資源の保存技術の構築のために, 様々な動物種の培養細胞を樹立し遺伝資源の確保と細胞特性の検討を行っている. しかし, 高度な発生工学・生殖工学的技術の習熟には時間を要し, 特に体細胞核移植操作は, 作製した再構築胚の発生率が非常に低率であることから多くの胚を作製しなければならない状況下にある. そこで本実験では, 体細胞核移植（Somatic cell nuclear transfer : SCNT）により作製した初期胚のガラス化保存を実施し, 加温後の正常性を検討した. B6D2F 1マウスへ常法に従い過剰排卵処置後にSCNT を実施した. 続いて発生した胚は簡易ガラス化法によりガラス化保存を行った. 加温後, 形態的に正常と認められた胚は, DAPI 染色および免疫染色（Oct4）による組織学的観察と胚発生を検討した. SCNT により作製し, 生存した卵子を活性化処理後, 2細胞期へ発生した再構築胚は83.2% であり, 一部は胚盤胞期へ発生した（44.8% : 47/105）. これら再構築胚をガラス化保存し一部を加温した結果, 加温後の生存胚は70% 以上であり免疫組織化学的解析により形態的な正常性を認めた. 以上の結果から, 作製されたSCNT胚のガラス化保存による遺伝資源の保存とそれらの胚を用いた移植核の内部構造構築に関する基礎的検討が可能であることが示唆された. [Abstract] Somatic cell nuclear transfer（SCNT） has been used in various research namely investigation into cellular features for establishing of genetic resourses technology from wild animals. However, it requires a high level of developmental engineering and reproduction technology which take time to learn as well as many successfully reconstructed oocytes and embryos. In this experiment, we examined the cryopreservation by simple vitrification of the mouse early embryos produced by SCNT. After warming a part of cloned embryos, morphologically normal embryos were obtained at more than 70%. And they were morphologically normal by the investigation with immunohistochemical analysis, which detected Oct4, puluripotency facter in inner cell mass. In conclusion, these results suggested that the genetic resources preservation were possible by simple vitrification of cloned mouse embryos. Furthermore, fundamental study on the internal structure of the cell nucleus can be made possible using the cloned mouse embryos.近畿大学先端技術総合研究所紀要編集委員会

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly chaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit alpha 4/PSMA7 in the adult mouse testis. ZPAC and alpha 4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of alpha 4 persisted until step 12. We then examined the expression profile of ZPAC and alpha 4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of alpha 4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo-matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%; 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm. (c) 2013 Elsevier Inc. All rights reserved.

Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5 mC) and the accumulation of 5-hydroxymethylcytosine (5 hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5 mC and 5 hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5 mC to 5 hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

During the maternal-to-zygotic transition (MZT), maternal proteins in oocytes are degraded by the ubiquitin-proteasome system (UPS), and new proteins are synthesized from the zygotic genome. However, the specific mechanisms underlying the UPS at the MZT are not well understood. We identified a molecule named zygote-specific proteasome assembly chaperone (ZPAC) that is specifically expressed in mouse gonads, and expression of ZPAC was transiently increased at the mouse MZT. ZPAC formed a complex with Ump1 and associated with precursor forms of 20S proteasomes. Transcription of ZPAC genes was also under the control of an autoregulatory feedback mechanism for the compensation of reduced proteasome activity similar to Ump1 and 20S proteasome subunit gene expression. Knockdown of ZPAC in early embryos caused a significant reduction of proteasome activity and decrease in Ump1 and mature proteasomes, leading to accumulation of proteins that need to be degraded at the MZT and early developmental arrest. Therefore, a unique proteasome assembly pathway mediated by ZPAC is important for progression of the mouse MZT. (C) 2012. Published by The Company of Biologists Ltd.

[要約] 本研究では、卵子の細胞内運搬経路として密接な関係を示す、L- カルニチンを添加した体外成熟培地を用いて、マウス未成熟卵子への体外成熟および体外受精をおこない、その後の初期胚の発生改善に与える影響を検討した。私たちの開発した、修正TaM 培地にL- カルニチン1mM, 2mM, 5mM, 10mM を添加して体外成熟をおこなった結果、82 ～ 90％であり、非添加区（92％：905/987）との有意な差は認めず正常に発生した。次に、体外成熟を経て正常に発生した卵子の体外受精成績は、それぞれ69 ～ 80％であり、非添加区（71％：635/889）との有意な差は認めなかった。続いて、胚盤胞期胚への発生成績は、それぞれ24 ～ 46 ％ であり、2mM添加成熟培地（46 ％：136/293） および5mM添加成熟培地（37％：101/272）では、非添加区（26％：129/499）と比較し初期胚の発生に改善を認めた。さらに、初期胚の発生が改善された、2mM L- カルニチン添加区において、正常な産子への発生を認めた。これらの結果から、体外成熟培地への適切な濃度のL- カルニチン添加は、胚盤胞期胚への発生を有意に改善することが示唆された。 [Abstract] In this study, mouse immature oocytes of ICR used in vitro maturation and in vitro fertilization, since analysised early embryos development. PMSG interperitoneal administration mouse collected immature oocytes removal cumulus oocyte complexes（COCs）. In vitro maturation experimented with modified TaM medium or modified TaM medium supplement with L-Carnitine 1mM, 2mM, 5mM, and 10mM. In vitro maturation oocytes fertilized sperm of ICR. In vitro maturation medium supplement with L-Carnitine of maturation rate was 82 to 90％. There was not significant differential L-Carnitine non supplement. In vitro fertilization rate of L-Carnitine supplement was 67 to 80％.There was not significant differential L-Carnitine non supplement. In vitro early embryos development resulted 26％（129/499）, 27％（86/323）, 46％（136/293）, 37％（101/272） and 24％（71/302） respective. Moreover, Supplement withL-Carnitine 2mM and 5mM of blastcyst rate was significant higher than L-Carnitine 0mM of blastcyst rate （p＜0.05）. In conclusion, in vitro maturation medium supplement with appropriate L-Carnitine suggested that blastcyst rate was improvement.

[要約] 本実験では、麻酔下による外科的処置により卵巣摘出を施すことで、動物個体を処分することなく一部の卵巣組織のみを低侵襲的に回収し、そこから得られる未成熟卵子の発生能を検討した。B6D2F1（BDF1）系およびICR系マウスへPMSGを7.5単位投与し48時間後に麻酔処置をおこなった後、供試雌の卵巣を一部切除した（約2mm角）。回収した卵巣の胞状卵胞より、GV 期の未成熟卵子を回収しmTaM培地を用いて体外成熟および体外受精をおこなったところ、いずれの系統においても成熟培養後M II期卵子への発生を認め、体外受精成績はいずれも65％以上であった。またいずれの系統においても胚盤胞期胚までの発生を認めた。さらに、卵巣切除を施した各系統マウスにおいて、卵巣切片による卵胞観察像により卵子が確認され、自然交配により産子作出が可能であった。以上の結果より、外科的処置による卵巣の低侵襲的回収技術により得られた未成熟卵子由来の体外受精は可能であり、今後、低繁殖能を呈する系統や遺伝子改変マウスあるいは野生小型げっ歯類などの希少な動物個体を処分することなく、遺伝資源の効率的な保存・増殖方法の提示が期待されると示唆された。 [Abstract] This study, the developmental potential of immature oocytes collected low invasive by the surgical manipulation under the anesthesia was examined. Mice were used B6D2F1（BDF1）strain and ICR strain of matured mice. Mice are injected PMSG. After 48 hours, a part of ovaries（about 2mm）were extirpated under the anesthesia. Oocytes were collected from a part of ovary collected under the anesthesia Oocytes are transferred maturation medium（mTaM with 5％FBS）and matured in 5％ CO_2 incubator for 16 hours. In vitro matured oocytes were drilled by laser optimal system（XYClone； Nikko-Hansen Co., Ltd.）. In vitro matured oocytes drilled zona pellucida was performed in vitro fertilization and confirmed embryonic development. In vitro fertilization rate that used in vitro matured oocytes was over 65％ in each mice strain. Embryo development was developed to the blastocyst stage in each mice strain. In addition, oocytes were confirmed in the follicle images of the ovary slice after it had passed between convalescences in each mice strain removed the ovary.

[要約] 本研究では、マンモス骨髄組織からの効率的な細胞核の回収方法を開発するために、組織の状態が類似したウシ凍結骨髄組織からの効率的な細胞核の回収方法の開発を試みた。さらに、回収方法の有用性を検討するために、回収したウシ凍結骨髄組織由来細胞核をマウス除核未受精卵子に注入し、回収された核の生物学的特性の有無を検討した。 初めに、凍結保存されたウシ骨髄組織からの効率的な細胞核の回収方法の開発を試みた。ウシ骨髄組織から細胞核を回収する際に、Collagenase 処理を行う事によって、細胞核の回収量が有意に向上した。次に、回収したウシ凍結骨髄組織由来ドナー細胞核をマウス除核未受精卵子へ注入し、再構築卵子の早期染色体凝集および前核様構造物の確認を行った。その結果、早期染色体凝集ならびに前核様構造物の形成は観察されなかったものの、細胞核の注入直後に観察されたPropidium iodide による強い赤色蛍光が、細胞核注入後7 時間ではほぼ消失していた事から、生物学的特性は保持されていると考えられた。以上の結果より、生物学的特性を保持した状態の細胞核を大量に回収できる新しい細胞核の回収方法を開発する事に成功した。 [Abstract] We tried to develop the effective nuclei recovery method from cryopreserved cattle bone marrow tissues for the development of effective nuclei recovery method from mammoth bone marrow tissues. In addition, we studied whether its cell nuclei still kept their biological characteristics by injecting recovered nuclei into mouse enucleated matured oocytes. First, we tried to develop the effective nuclei recovery method from cryopreserved cattle bone marrow tissues. The amount of recovered cell nuclei was significantly increased by using collagenase treatment. Subsequently, we tried to inject recovered cell nuclei into mouse enucleated matured oocytes and checked for premature chromatin condensation and pronuclear-like structure. As a result, there was no oocyte with premature chromatin condensation and pronuclear-like structure, but because the highly-red fluorescence which was observed shortly after injection nearly disappeared after 7 hours, their cell nuclei were shown to keep biological characteristics. Based on these results, our method was shown to be effective in mass recovery of cell nuclei that kept their biological characteristics.

[要約] 本研究では、ICR系統マウスを用いた体外受精ならびに、初期胚の体外培養について検討した。体外受精では、常法により過剰排卵処置を施した成熟雌マウスより卵を回収し、前培養を行うことにより、受精能を獲得させた精子と共に培養し、受精させた。精子前培養培地および受精培地からウシ血清アルブミンを除き、L-カルニチンを添加した修正HTF培地を用いた。その結果、L-カルニチン添加濃度0mMでは48％（162/337）、1mM では12％（35/294）、2mM では0％（0/311）、5mM では0％（0/292）、10mM では0％（0/270）の受精率が得られた。続いて胚培養を行った結果、L-カルニチン添加濃度0mM では78％（127/162）、1mM では57％（20/35）であり、2mM、5mM、10mM では胚の発生には至らなかった。次に、高分子物質のPVA を0.1％添加した培地でも同様の実験を行い、L-カルニチン添加濃度0mM では25％（96/390）、0.1mM では25％（91/362）、0.5mM では30％（77/256）、1mMでは34％（117/347）の受精率が得られた。続いて胚培養を行った結果、L- カルニチン添加濃度0mMでは80％（76/95）、0.1mM では81％（74/91）、0.5mM では95％（70/74）、1mM では73％（85/117）が胚盤胞期胚へ発生した。これらの結果から、L- カルニチン高濃度における受精阻害を呈することが明らかとなった。また、L-カルニチンに高分子物質であるPVA を添加することで、ウシ血清アルブミンの代替物質になる可能性が示唆された。 [Abstract] The present study examined in vitro fertilization that used the ICR mouse and the in vitro culture of the early embryo. Newly ovulated eggs from mature mouse injected PMSG and hCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. Preculture medium of sperm and fertilization medium added the L-carnitine without bovine serum albmin. Percentage of fertilization were 48％（162/337）at L-carnitine 0mM and 12％（35/294） at L-carnitine 1mM. Following the percentage of blastocyst were 78％（127/162） and 57％（20/35）. Next, the same experiment was performed using the medium that added 0. 1% PVA of polymeric mass. Results of the fertilization rate were 24％（95/390） at L-carnitine 0mM and 25％（91/362） at L-carnitine 0.1mM and 30％（77/256）at L-carnitine 0.5mM and 34％（117/347） at L-carnitine 1mM. Following the rate of developed to blastocysts stage were 73-95％. When the high density L-carnitine from these results, it was shown not to retard of fertilization. Moreover, L-carnitine was indicated the possibility for a substitute of bovine serum albumin by adding PVA.

[要約] 私達は、Branched DNA 技術により微量mRNA の検出が可能である、QuantiGene ViewRNA を用いたin situ hybridization技術をマウス単一卵子での実験手法の確立を目的に、組織で検出できる方法を改良することにより卵細胞での評価を行なった。今回用いた卵子の成熟因子に関わるGdf9 およびハウスキーピング因子18S を検討したところ、いずれの因子において、卵子での発現を認めた。本QuantiGene ViewRNAキットを用いたin situ hybridization の手法を確立することが可能となり、今後、未成熟卵子あるいは成熟卵子そして初期胚における単一卵子での遺伝子発現の解析に有効なツールとなることが示された。 [Abstract] We evaluated ooplasm using QuantiGene ViewRNA which was able to detect a small amount of mRNA by the Branched DNA technology with a mouse single ovum. This study aimed to establish experimental method. We investigated Gdf9 which is related to oocyte maturation factor and 18S which is housekeeping factor. Hereby, it was recognized that each factor were expressed in oocytes. It was able to establish the technique of in situ hybridization using this QuantiGene ViewRNA kit. These results suggest that an effective tool for the analysis of the gene expression was mouse immature ovum or the early embryo.

[要約] 本研究では成熟B6D2F1マウスの精巣上体尾部由来精子を用いた冷蔵保存を行い、その後、冷蔵精子の評価および体外受精操作について検討した。精巣上体尾部精子を、各種保存液50μL（R18S3、R18、R18+AFP III 1mg/mL）中で4℃にて冷蔵保存した。加温後のそれらの運動性は、精子運動解析装置（IVOS：ニッコー・ハンセン（株））を用いて、保存期間１～ 5日を評価し、R18+AFP III の保存液では、5日目においても52％の運動性を保持していた。精子細胞膜損傷はLIVE/DEAD sperm viability kit を用いて評価し、R18S3 とR18+AFP IIIは5日目の細胞膜完全性の保持率は共に48％であった。受精能の確認では、常法により採卵し冷蔵精子と受精させた。また、一部の卵子卵丘複合体は透明帯穿孔処理を行い、冷蔵精子と受精させた。透明帯穿孔処理後の卵子との体外受精結果は冷蔵保存後3日目の冷蔵精子において37％（46/126）の受精率であった。冷蔵保存後5日目においても2％（4/162）の受精率を確認できた。 以上の結果より、マウス精子の冷蔵保存は低率ではあるが受精能を保持し、冷蔵保存が可能であることが示された。 [Abstract] The present study was performed to examine the storage of Slc : B6D2F1 mouse epididymal sperm at cold temperature. Afterwards, the evaluation and the in vitro fertilization operation of the refrigeration sperm were examined. The cauda epididymal spermatozoa from adult males were preserved with various solutions（ R18S3, R18, R18+AFP III 1mg/mL） at 4℃ . Sperm motility was assessed by Integrated Visual Optical System（ IVOS）. R18+AFP III maintained the movement of 52％ during 5 days. The integrity of the sperm membrane was determined using Live/Dead sperm viability kit. The percentage of membraneintact spermatozoa that stored in R18S3 and R18+AFP III was 48％ at day 5. Super ovulated eggs were fertilized in vitro with various refrigeration sperm. Moreover a part of eggs was treatment of zona drilled, and it was fertilized with the refrigeration sperm. In vitro fertilized rate of zona drilled oocyte withrefrigeration sperm of 3 days after preserved. The fertilization of the refrigeration sperm of the 5 day of preservation was 2％（ 4/162）. In conclusion were indicate that mouse epididymal spermatozoa stored at 4℃ maintain fertilization ability, although low rate, and can store at cold temperature.

Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti-copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1(G93A) Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.

We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA) Using immunofluorescence staining, we observed that the nuclear Localization of RNAP IT was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi In a transient gene expression assay using a plasmid reporter gene (p beta-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for it least 4 hours after injection We found that the methylation status in the chicken beta-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state Taken together, these results suggest that deposition 01 selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation Ho Never, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryo First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used the se oocytes throughout this study Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the Cl phase of the first cell cycle Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i e, the hsp70 1, MuERV-L, eif-1a and zscan4d genes As a result, we found that onset of expression of the four examined ZGA genes was delayed m both normally developed 2-cell embryos and arrested 1-cell embryos Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos

We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85-100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA. (C) 2010 Elsevier B.V. All rights reserved.

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals. (C) 2010 Elsevier Inc. All rights reserved.

At refrigerated temperatures, mouse embryos can maintain developmental ability for short periods. Previously, we succeeded in transporting vitrified and warmed 2-cell mouse embryos while maintaining developmental ability at refrigerated temperatures for 50 h. Transport of nonfrozen embryos is an easier and more useful means of exchanging genetically engineered mice between laboratories than is transport of cryopreserved embryos. Here we examined the developmental ability of transported 2-cell embryos that were produced through in vitro fertilization using cryopreserved sperm. Results show that 2-cell embryos produced by cryopreserved sperm can develop into blastocysts after cold storage for 24, 48, and 72 h. Transported 2-cell embryos produced by cryopreserved sperm yielded a favorable number of pups in all of the receiving laboratories after transport lasting 48 to 52 h. In summary, cold storage and transport of 2-cell embryos derived from cryopreserved sperm at refrigerated temperatures provides a novel means of transporting genetically engineered mice as an alternative to the transport of cryopreserved embryos and sperm.

本実験では成熟齢C57BL/6J マウスから得られた体外成熟卵子を作出し、レーザー穿孔処理法を用いて各出力条件下（200, 150, 120μsec.）で透明帯穿孔処理を行ない、その後の体外受精および発生能を検討した。C57BL/6J 卵巣より回収した未成熟卵子の体外成熟成績は、91％（1, 517/1, 674）であった。レーザー穿孔処理時間による受精成績は、それぞれ、60％（191/316）, 54％（103/192）, 45％（196/439）であり、対照区（28％：129/463）と比較し有意な差が認められた（P<0.05）。また一部を培養した結果、胚盤胞期への発生率は31％（32/102）, 51％（74/144）, 53％（40/75）であった。２細胞期胚を移植した結果、レーザー出力を低出力にした場合、産子の発生向上が確認された〔6％（5/81）, 13％（10/83）, 21％（12/56）〕。さらにレーザー照射による熱変性を避けるため、卵細胞質を収縮させた透明帯穿孔卵子における受精成績も同様に対照区と比較し有意に向上した（p<0.05）。以上の結果より、C57BL/6J 未成熟卵子の体外成熟およびレーザー穿孔処理の条件を調整することにより、産子への発生を改善することが示唆された。

本研究ではWistar 系ラットの精巣上体尾部由来精子を用いた冷蔵保存について検討した。ラット精巣上体尾部精子を、各種保存液（R18S3、マルベリーIII、それぞれを混合した1：1 稀釈液、2：1 稀釈液）中で4℃にて冷蔵保存した。加温後のそれらの運動性は、運動性指数およびSQA-V よりにて評価した。運動性指数では1：1、2：1 稀釈液で14 日間運動性を保持した。SQA-V ではSMI（精子自動性指数）値を用いて評価し、1：1、2：1 稀釈液で７ 日間自動運動性を確認した。精子頭部における膜損傷の有無をLIVE/DEAD Sperm Viability Kit を用いて評価し、2：1 稀釈液において7 日目の正常膜保持精子率は40％であった。受精能の確認では、常法により過剰排卵処置を施した幼若雌ラットの卵子を透明帯除去後、冷蔵保存精子と共に受精させた。この結果、7 日間保存した精子にて1：1 稀釈液では、20％（17/86）、2：1 稀釈液では30％（36/122）の精子侵入率であった。これらの結果から、Wistar 系ラットの精子冷蔵保存は、低率ではあるが受精能を保持し、冷蔵保存が可能であることが示された。

本研究では、マウスの耐凍剤を用いずに凍結された骨髄組織から回収された細胞を用いて体細胞核移植（somatic cell nuclear transfer; SCNT）を行い、永久凍土中から発見される動物遺物のような、耐凍剤を用いずに非常に長期間、不安定な環境下で凍結保存された生物の細胞をSCNT に用いることが可能か否か検討した。まず、耐凍剤を用いずに凍結保存されたマウスの骨髄細胞の回収と、DNA の断片化状態を確認した。回収された骨髄細胞をHoechst 33342 で染色し観察したところ、核の存在が確認された。コメットアッセイ及びフローサイトメトリーの結果から、耐凍剤を用いずに凍結保存された骨髄細胞では、多くの細胞のDNA が断片化していることが確認された。次に、回収した骨髄細胞を、核ドナー細胞としてSCNT に用いた。その結果、マウス新鮮骨髄細胞および−80℃凍結骨髄細胞を用いた再構築卵子が、胚盤胞期にまで発生した（16.9％、及び1.9％）。以上の結果より、耐凍剤を用いずに凍結保存された骨髄細胞は、凍結保存されることにより、DNA に大きな障害を受けていることが確認されたが、SCNT の核ドナー細胞として利用できる可能性も示された。

Here, we report the recovery of cell nuclei front 14,000-15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues.

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs Could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible, tissues for transplantation.

Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos-the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.

Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24,48 and 72 h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24 h, the rates were markedly decreased to low levels after 48 h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young. (C) 2008 Elsevier Inc. All rights reserved.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.

本研究では、Wistar系ラットを用いた体外受精ならびに、初期胚の体外培養について検討した。体外受精では、常法により過剰排卵処置を施した幼若雌ラットより卵を回収し、前培養を行うことにより、受精能を獲得させた精子と共に培養し、受精させた。その結果、Wistar (HLA) では48％ (61/127)、WistarHannover/Rccでは67％ (62/92) の排卵誘起率が得られた。続いて各種系統において、2,526個、3,238個の卵子がそれぞれ得られ、Wistar (HLA)では一匹あたり41個、WistarHannover/Rccでは一匹あたり52個の卵子が過剰排卵処置により得られた。体外受精においてWistar(HLA)では46％ (1,172/2,526)、 WistarHannover/Rccでは69％ (2,226/3,238) の卵子が受精した。その後、77％ (900/1,172)、88％ (1,954/2,226) の卵子がそれぞれ2細胞期胚に発生した。初期胚の体外培養では、Wistar(HLA) では10％ (40/387)、WitarHannover/Rccでは13％ (122/923)の胚が2細胞期胚から胚盤胞期胚まで発生した。 これらの結果から、今回供試したWistar系幼若ラットは系統により差は生じるものの、過剰排卵処置および体外受精が可能であり、低率ではあるが胚盤胞期胚まで発生することが示された。

体細胞核移植 (Somatic cell nuclear transfer: SCNT)技術は、1997年のWilmutらによる報告後、様々な動物種においてクローン個体の作出を実現してきたが、10余年を経た現在においてもその作出効率は未だに低率である。その原因を解明し作出効率を上げるための研究は活発に行われているが、その根本的な原因は不明なままである。近年、生細胞における分子の動態や、クロマチンの配置などの解析から、核内構造構築が転写活性などの遺伝子発現制御の基盤となることがわかってきた。そして最近、核内のアクチン関連タンパク質 (Arp) がクロマチン構造をする複合体に広く含まれており、Arpファミリータンパク質が細胞核、クロマチンの機能構造やダイナミクスに関与していることが示唆されている。本研究では、受精卵および核移植由来卵子において、クロマチンリモデリング因子SWR1複合体の構成因子であるArp4、Arp6、SWR1の局在を免疫組織化学的染色によって解析した。その結果、受精卵および核移植由来卵で、クロマチン構造に違いが見られた領域でのArp4およびSWR1の発現パターンが異なっていた。このことから、クロマチン配置の相違、あるいは核内構造制御タンパク質の局在パターンが核移植由来卵子での核の不適切なリプログラミングを反映している可能性が示唆された。

In short hairpin RNA (shRNA) transgenic mice, the tissue difference in gene silencing efficiency and oversaturation of microRNA (miRNA) pathway have not been well assessed. We studied these problems in our previously-reported anti-copper/zinc superoxide dismutase (SOD1) shRNA transgenic mice. Although there was a tissue difference (liver and skeletal muscle, >95%; central nervous system and lung, similar to 80%), the target gene silencing was systemic and our anti-SOD1 shRNA transgenic mice recapitulated the SOD1-null mice. Neither endogenous miRNAs nor their target gene levels were altered, indicating the preservation of endogenous miRNA pathways. We think that the shRNA transgenic mice can be utilized for gene analysis. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become Cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they call also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin oil inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.

We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.

We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.

本研究では、マウス体細胞核移植（Somatic Cell Nuclear Transfer : SCNT）胚の産子への発生能の検討と作出されたクローンマウスの生育ならびに繁殖能力について検討した。ドナー細胞として卵丘細胞を用いて核移植を行い、得られた2 細胞期の再構築胚を卵管へ移植した。その結果、1 匹のクローン産子を作出することに成功した（生存産子1 匹/ 移植胚数24 個：4.2％）。クローンマウスの生殖能力について検討した結果、2 度の自然分娩で計20 匹の産子を獲得し、正常に子孫を得ることが確認された。また、クローンマウスとその産子それぞれについて経時的に体重測定を行った結果、クローンマウスで従来報告されているような極度の肥満はみられず、またその産子では肥満はみられなかった。さらに、各近交系間のサテライトマーカーを用いた遺伝的背景の解析を行った結果、クローンマウスの遺伝的背景はドナー細胞のB6D2F1 と一致した。これらの結果から、B6D2F1 卵丘細胞由来の体細胞クローンマウスでは、正常な繁殖能力を有し肥満もみられないこと、マイクロサテライトマーカーでの解析においては、遺伝的背景はドナー細胞により決定されることが示された。

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.

体細胞核移植（somatic cell nuclear transfer : SCNT）技術は、1997 年のWakayama らによる報告以来、様々な動物種においてクローン個体の作出を実現してきたが、その作出効率は未だに低率である。マウスにおいては、核移植胚のおよそ半分は着床するが、出生まで至るものはきわめて少ない。着床の成立と維持における胚側の要素として、栄養外胚葉系譜への分化は必須の過程であるが、最近、マウス栄養芽細胞で転写因子、Caudal-related homeobox 2 （Cdx2）が発現し、栄養外胚葉系譜の誘導で重要な役割を果たしていることが報告された。さらに、Cdx2 は、初期胚発生過程でOct3/4 と互いに制御関係にあることが示された。そこで本研究では、着床、胎盤形成の基点となる栄養外胚葉で発現するCdx2 に着目し、Cdx2 とその制御関係にあるOct3/4 について、マウスSCNT胚と顕微授精（ICSI）胚での発現解析を行った。その結果、SCNT胚において、Cdx2 は発現しているものの、ICSI 胚に比べ転写量は約半分と低かった。このことから、Cdx2 の発現の低下が、その後の栄養外胚葉系譜での遺伝子発現に影響し、着床の成立と維持に障害をもたらしている可能性が示唆された。

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

哺乳動物において、その発生の途上において重要ないくつかの遺伝子は、ゲノム刷り込みの影響を受ける。本研究では、父親由来のゲノム刷り込みの個体発生に対する影響を検討する第一段階として、マウス雌雄体細胞、精子、卵子、野生型および雄性発生胚由来ES 細胞におけるH19 遺伝子の5 '上流に存在するDMR におけるCpG のメチル化の状態を解析した。その結果、雄性発生胚由来ES 細胞におけるDMR の5 '上流部分のCpG は、精子と同様に高度にメチル化されていた。しかしながら、DMR の3 ' 下流部分のCpGは、雄性発生胚由来ES 細胞において、既にそのメチル化パターンは、雄性配偶子である精子とは異なることが観察された。

Many autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with copper/zinc superoxide dismutase (SOD1) mutation may be induced by missense point mutations that result in the production of proteins with toxic properties. Reduction in the encoding of proteins from such mutated genes can therefore be expected to improve the disease phenotype. The duplex of 21-nucleotide RNA, known as small interfering RNA (siRNA), has recently emerged as a powerful gene silencing tool. We made transgenic (Tg) mice with modified siRNA, which had multiple mismatch alternations within the sense strand, to prevent the "shutdown phenomenon" of transgenic siRNA. Consequently, the in vivo knockdown effect of siRNA on SOD1 expression did not diminish over four generations. When we crossed these anti-SOD1 siRNA Tg mice with SOD1(G93A) Tg mice, a model for ALS, siRNA prevented the development of disease by inhibiting mutant G93A SOD1 production in the central nervous system. Our findings clearly proved the principle that siRNA-mediated gene silencing can stop the development of familial ALS with SOD1 mutation.

卵丘細胞を注入したマウス再構築胚発生において、再構築卵子活性化処理培地中へのジメチルスルフォキシド（DMSO : dimethyl sulfoxide）の添加が、その後の発生率に及ぼす影響について検討を行った。注入後の卵子は塩化ストロンチウム、サイトカラシンB 及び各濃度のDMSO（0.05,0.1,0.5,1,2,4％）含有のCa2^+ フリーmCZB 培地+0.3％ BSA で活性化処理を行った。活性化処理培地中へDMSO を添加した群において、未添加群と比較したところ、2 細胞期への発生率が改善されている傾向が認められた（2％：57％ ,未添加：36％）。また、DMSO の濃度は2％添加にて、桑実期及び胚盤胞期への発生率が改善される傾向が認められた。DMSO 未添加群と、2％添加群について、活性化処理後24 時間での再構築胚の断片化について検討したところ、2％添加群において、断片化の改善が認められた。以上の結果より、卵丘細胞を用いたマウス再構築胚の発生率及び断片化抑制について、DMSO は有効であることが示唆された。

本研究では、核移植によるクローンマウスの作出を目的として卵丘細胞をドナー細胞に用いた核移植を行い、核移植胚の体外培養における発生能および胚移植後の発生能の検討を行った。胚移植の結果、着床痕が認められ、肥大した胎盤の形成を確認することができた。さらに、マウスのクローン産仔を得ることができた。

現在、生命科学領域の研究に幅広く使われているES 細胞は、その殆どが受精卵より発生した胚に由来している。特に胚の中でも将来の胚体部分（原始外胚葉；エピブラスト）より派生してくるため、ES細胞の最大の特徴でもある分化全能性は胚の個体発生能力によると考えられる。一方、マウスの初期胚はエタノールやストロンチウムの刺激によって単為発生的活性化を受け、雌性ゲノムのみで発生することが知られている。これらの胚は通常、産仔に至ることはないが、原始的な胚体を有するためES 細胞の樹立は可能である。本実験では、単為発生させたBDF1 マウス胚よりES 細胞を樹立し、その分化能力を検討することを目的とした。37 ラインを樹立し、ランダムに選択した7 ライン全てがAP 活性、Oct-4、SSEA-1 抗原を発現していることが確認された。このうち実験に用いた2 ラインはICR マウス胚盤胞へのインジェクションによりキメラマウスに寄与した。また、SCID マウスの大腿部に皮下注射したとき、複雑に発達したテラトーマを形成した。これらのことから、単為発生胚を由来するES 細胞はin vivo で高い分化多能性を有していることが示唆された。さらに、これらのES 細胞はin vitro での分化誘導にも従い、目的に応じた系で神経様細胞、幼若肝細胞、心筋細胞を形成した。以上より、単為発生胚由来ES 細胞は従来の受精胚由来ES細胞に順ずる分化多能性を持っていることが確認された。

本研究では、父親由来のゲノム刷り込みの個体発生に対する影響を検討する第一段階として、マウス体細胞、配偶子、2タイプの胚性幹細胞におけるH19遺伝子の5’上流のCpGのメチル化の状態を決定した。ゲノムDNAはBDF1マウスの尾(雌雄体細胞,mSTとfST)、精子(S)、卵子(O)、野生型および雄性発生胚由来胚性幹細胞(wtESとagES)から単離された。CpGのメチル化の状態は、Naバイサルファイト法によって決定された。H19遺伝子の転写開始部位から－4413から－3946塩基の領域には13個のCpGが存在した。個の領域のメチル化CpGの割合は、mSTで88%(160/182)、fSTで79%(27/130)、Sで93%(230/247)、Oで8%(10/130)、wtESで77%(10/13)そしてagESで89%(314/351)であった。CTCF結合部位のコアモチーフ(CCGCGTGGTGGCAG)ではメチル化CpGの割合は、mSTで93%(26/28)、fSTで80%(16/20)、Sで95%(36/38)、Oで0%(0/20)、wtESで50%(1/2)そしてagESで96%(52/54)であった。agESにおけるCpGは精子と同様に高度にメチル化されていた。しかしながら、agESにおいて、既にCpGのメチル化パタ

Recently, with the acetylation of histone and the modification of chromatin structure, the methylation of cytosine residue within CpG dinucleotides in genomic DNA sequence attracts many researchers' attention as one of major epigenetic regulation systems of gene expression. There are several methods (immunofluorescence with 5-methylcytosine specific antibody and methylationsensitive restriction enzyme-PCR method, etc.) to analyze the methylation of cytosine residue. Bisulfitesequencing method, which is one of methods for analyzing the methylation of cytosine residue in genomic DNA sequence, has advantages of high sensitivity and analyzing the methylation of cytosine residue directly. In this note, the detailed procedure of bisulfite-sequencing method for mouse early Preimplantation embryos is described. © 2005, JAPANESE SOCIETY OF OVA RESEARCH. All rights reserved.

Foetomaternal alloimmune thrombocytopenia (FMAIT) occurs when maternal antibodies of an antigen-negative mother cause destruction of sensitized foetal platelets. In Caucasian populations, 6-12% of human platelet antigen (HPA)-1a-negative women develop anti-HPA-1a, and the incidence of clinically affected cases is estimated to be 10-20% of immunized women. This study was performed in order to elucidate the rate of maternal immunization, incidence of FMAIT and the likely outcome of the condition in Asians. Excluding two or more pregnancies during the period, serum samples from 24630 pregnant women, mainly Japanese, were screened for antibodies against platelet alloantigens by means of mixed passive haemagglutination (MPHA) (Anti-HPA-MPHA, Olympus, Tokyo). Antibodies were detected in 0-91% (223/24630) of the women's samples and the immunization rate was correlated with the number of pregnancies. Antibody specificity included anti-HPA-4b (49), anti-HPA-5a (three), anti-HPA-5b (168), anti-HPA-4b + 5b (one) and anti-Naka (CD36) (two). No alloimmunization was observed within the HPÁ-1, HPA-2, HPA-3 or HPA-6 systems. Among HPA-4b- or HPA-5b-negative women, 24% or 14% estimated, respectively, had antibodies and 26% (10/38) or 10% (12/125) of neonates, respectively, born to these mothers developed thrombocytopenia. Two neonates born to mothers having anti-HPA-4b developed generalized purpura. No cases of intracranial bleeding or death due to FMAIT were recorded. Generalized purpura due to FMAIT occurs in one in 9359 (95% CI: 1 in 77519-1 in 2591) pregnancies solely because of HPA-4b incompatibility.

We developed a novel technique that allowed reversible suppression of glutamatergic neurotransmission in the cerebellar network. We generated two lines of transgenic mice termed Tet and TeNT mice and crossed the two transgenic lines to produce the Tet/TeNT double transgenic mice. In the Tet mice, the tetracycline-controlled reverse activator (rtTA) was expressed selectively in cerebellar granule cells by the promoter function of the GABA(A) receptor alpha6 subunit gene. In the TeNT mice, the fusion gene of tetanus neurotoxin light chain (TeNT) and enhanced green fluorescent protein (EGFP) was designed to be induced by the interaction of doxycycline (DOX)-activated rtTA with the tetracycline-responsive promoter. The Tet/TeNT mice grew normally even after DOX treatment and exhibited a restricted DOX-dependent expression of TeNT in cerebellar granule cells. Along with this expression, TeNT proteolytically cleaved the synaptic vesicle protein VAMP2 (also termed synaptobrevin2) and reduced glutamate release from granule cells. Both cleavage of VAMP2/synaptobrevin2 and reduction of glutamate release were reversed by removal of DOX. Among the four genotypes generated by heterozygous crossing of Tet and TeNT mice, only Tet/TeNT mice showed DOX-dependent reversible motor impairments as analyzed with fixed bar and rota-rod tests. Reversible suppression of glutamatergic neurotransmission thus can be manipulated with spatiotemporal accuracy by DOX treatment and removal. These transgenic mice will serve as an animal model to study the cerebellar function in motor coordination and learning.

We compared the levels of growth hormone (GH) mRNA in the pituitary, plasma GH concentration, and altered phenotype in rats heterozygous and homozygous for an antisense RNA transgene targeted to the rat GH gene, with those in nontransgenic rats. We initially investigated whether the transgene promoter, which is connected to four copies of a thyroid hormone response element (TRE) that increases promoter activity, affected in vivo transgene expression in the pituitary of the transgenic rats. Plasma GH concentration correlated negatively with T-3 injection in surgically thyroidectomized heterozygous transgenic rats. There was a reduction of about similar to 35-40% in GH mRNA levels in the pituitary of homozygous animals compared with those in nontransgenic rats. Plasma GH concentration was significantly similar to 25-32 and similar to 29-41% lower in heterozygous and homozygous transgenic rats, respectively, compared with that in nontransgenic animals. Furthermore, the growth rates in homozygous transgenic rats were reduced by similar to 72-81 and similar to 51-70% compared with those of their heterozygous and nontransgenic littermates, respectively. The results of these studies suggested that the biological effect of GH in vivo is modulated dose-dependently by the antisense RNA transgene. The rat GH gene can therefore be targeted by antisense RNA produced from a transgene, as reflected in the protein and RNA levels. (C) 1995 Wiley-Liss, Inc.

We investigated the onset of paternal gene expression in the early mouse embryo. We obtained transgenic mouse embryos by fertilizing BD (C57BL/6N x DBA) F1 hybrid female oocytes in vitro, with sperm from homozygous transgenic males carrying integrated chicken beta-actin promoter-driven firefly luciferase cDNA. We then examined the RNA and protein synthesis of the luciferase gene in embryos from the 1- to 2-cell stage. RNA transcripts of the luciferase gene were first detected in the 1-cell stage embryos as early as 13 hr postinsemination, just prior to elongation. By photon-count imaging, functional luciferase was identified at the 2-cell stage 23 hr postinsemination. These findings indicate that the paternal endogenous gene is already transcribed in the late 1-cell embryos, although paternally derived protein is not synthesized until the 2-cell stage. Therefore, these results suggest that the embryonic gene is activated as early as the late 1-cell stage. (C) 1994 Wiley-Liss, Inc.

トランスジェニックマウスの作製をより効率化することを目的に, 超急速凍結法で保存した体外受精由来前核期受精卵を用いてトランスジェニックマウスの作製を試みた。体外受精によって得られた前核期受精卵 (C57BL/6N) を超急速凍結し, 融解後の卵子に遺伝子 (βact-Luc) を顕微注入した。また, 対照区には新鮮な体外受精卵を用いた。注入後, それぞれ70.8% (131/185) , 71.9% (159/221) の卵子が生存し, それらを偽妊娠第1日目のレシピエント雌卵管内に移植した結果, 凍結区で13.6% (17/125) , 対照区で14.1% (21/149) の産子を得た。更にサザンプロット解析により, いずれの区においても2匹に導入遺伝子の組み込みを認め (凍結区, 2/17: 12%; 対照区, 2/21: 10%) , また, 各トランスジェニックマウスの脳組織についてルシフェラーゼ活性を調べた結果, 全てのマウスで遺伝子の発現を確認した。以上の結果より, 超急速凍結法で凍結保存した体外受精由来前核期受精卵よりトランスジェニックマウスの作製が可能であることが示された。

当研究室で作製したトランスジェニックラット (Anti-rGH・Tg) の系統保存を目的に, 既にマウスにおいて確立されている超急速凍結法を用いて前核期受精卵の凍結保存を行った。導入遺伝子をホモ接合に持つトランスジェニックラットの精巣上体尾部精子を用いた体外受精によって得られた前核期受精卵を凍結し, その一部97個を融解した結果, 形態的に正常な卵子の割合は41% (40/97) であった。更に正常卵子を移植した結果, 10% (4/40) が新生児へ発生した。また, 得られた産子すべてに導入遺伝子の伝達が認められ, 矮小化を呈する表現型が観察された。また, その兄妹交配により導入遺伝子をホモ接合に持つトランスジェニックラットを作製することができた。以上の結果より, 超急速凍結法を用いた受精卵の凍結保存が, トランスジェニックラットの系統保存に利用できる可能性が示された。

ラット胚性幹細胞(embryonic stem cell;ES細胞)の樹立を目的として,ラット初期胚内部細胞塊(ICM)由来細胞の増殖に関する検討を行なった.マウスES細胞樹立に使用されるBuffalo ratliver細胞上清(BRL-CM)および白血病抑制因子(Leukemia inhibiting factor; LIF)の培養液への添加はICMの増殖に対して無効であったが,BRL-CMを酸処理したのち分子量5000の限外ろ過膜で分けた低分子分画(CM/La)とLIFを加えた場合のみ極めて高い頻度でICMの増殖が観察され,ほぼすべての接着胚でICMがドーム状に増殖した.さらにその一部を継代することにより,小型球形細胞からなる不定形のコロニーを形成して増殖する細胞株2株を樹立した.このICM由来細胞株はCM/Laを除去すると線維芽様細胞,神経様細胞,心筋様細胞へと分化し,また,浮遊培養により胚様体を形成したことから,多分化能を有しているものと考えられた.また,CM/LaはラットICMおよびラットICM由来細胞に対して増殖促進および分化抑制活性を持つことが示唆された.BRL-CMに由来するCM/La分画は従来困難であったラット初期胚由来細胞株の樹立•維持に有効であると考えられた.

当研究室で作製したトランスジェニックマウスの系統保存を量的に, 既に確立されている超急速凍結法を用いて2種類のトランスジェニックマウス (ラット成長ホルモン・アンチセンス遺伝子が導入されたトランスジェニックマウスおよびホタル・ルシフェラーゼ遺伝子が導入されたマウス) 由来の初期胚の凍結保存を行った。導入遺伝子をヘミ接合に持つ雄マウス精子を用いた体外受精によって得られた2細胞期胚の凍結融解後の生存率は, 双方とも70%以上と高率であり, さらに移植後の新生児への発生率は前者で53%, 後者で16%であった。また, 産児の尾部組織より抽出したDNAをサザンプロッティング解析で調べたところ, いずれも約40%の個体に外来遺伝子の伝達が認められた。以上のことから, 超急速凍結法によるトランスジェニックマウス由来初期胚の凍結が可能であることが示された。

β-アクチン/ホタル・ルシフェラーゼ融合遺伝子を導入したトランスジェニック雄マウス (1匹) の精巣上体尾部から得られた精子を凍結保存後, その1/4を融解し, その生存性について検討した。凍結融解した精子を用いて体外受精を行なった結果, 検査卵65個中36個が受精 (55.4%) し, 2細胞期へ発生した。さらにこれら胚を偽妊娠第一日の受容雌の卵管に移植したところ, 23匹 (63.9%, 23/36) の新生児が得られた。また, 得られた新生児の一部 (15匹) について, サザンプロット法にてDNAの解析を行なった結果, すべての新生児に導入遺伝子が伝達されていることが確認された。また, 得られた新生児すべてにおいてルシフェラーゼ遺伝子の発現が確認された。

The appearance of Hanganutziu and Deicher (HD) antibody in the sera of patients suffering from various diseases, including malignancies of some organs and liver disorders, was investigated by enzyme-linked immunosorbent assay using N-glycolylneuraminyl-lactosylceramide (HD3) and 4-O-acetyl-HD3 as the antigenic molecules. More than 25% of sera from patients suffering from malignancies, cholelithiasis and liver cirrhosis had HD antibody, whereas none of 41 sera from healthy persons had HD antibody. The percentage of HD antibody-positive patients was similar in stages I, II and III of gastric cancer and recurrence cases. Antibody titers of the positive patients in each stage were also not different from those in each other stage. These results indicated that HD antigenic expression on cancerous tissue is not dependent on the cancerous malignancy. The HD antibody level was elevated after surgical removal of cancerous tissues in 5 of 6 patients examined, indicating that tumor growth absorbed the serum antibody. Serum antibody against 4-O-acetyl-HD3 was detected independently of HD3 antibody in some cases however, in most cases, correlation between the two antibody titers was observed.

Thrombopoiesis, as well as antibody production, is one of the major events in which interleukin-6 (IL-6) has been reported to be involved. Polyclonal antimurine IL-6 receptor antibody was prepared to examine the effect of the antibody on these events in IL-6-treated mice. Administration of the anti-mIL-6R antibody inhibited the IL-6-induced increase in the number of platelets. Enhancement of the serum level of DNP-specific antibody by intraperitoneal injection of IL-6 was inhibited completely with simultaneous administration of the anti-mIL-6R antibody. The level of DNP-specific antibody was decreased, even below the basal value, by the higher dose of anti-mIL-6R antibody, indicating its effect also on endogenous IL-6. This work provides evidence that anti-IL-6R antibody inhibits IL-6 function in vivo, and provides an animal model of the therapeutic use of anti-IL-6R antibody for IL-6-related disease. © 1991.

マウス胚性幹細胞の分化過程における細胞核染色体テリトリーと遺伝子座の動態について解析した。

マウス胚性幹細胞におけるABCトランスポーター・Bcrp1 mRNAアイソフォームAの転写調節領域の解析

マウス胚性幹細胞の分化過程における細胞核染色体テリトリーと遺伝子座の動態について3D-FISH法を用いて解析した。

マウス体細胞核移植由来卵子におけるクロマチンリモデリング複合体、SWR1複合体の構成因子の発現について解析した。

マウスＥＳ細胞の分化誘導過程におけるArpファミリータンパク質とクロマチンリモデリング因子複合体の動態について解析した。

若齢ウシ精巣細胞を用いたヌードマウス皮下への異種異所移植による精子形成誘導について試みた。

マウス体細胞核移植由来卵子におけるクロマチンリモデリング複合体、SWR1複合体の構成因子の発現について解析した。

マウスES細胞の未分化維持機構においてABCトランスポーターBcrp1が与える影響について解析した。

FGF4がマウス体細胞核移植胚の発生に与える効果について検討した。

若齢ウシ精巣細胞を用いたヌードマウス皮下への異種異所移植による精子誘導について試みた。

Fibroblast growth factor4 (FGF4)がマウス体細胞核移植胚の胚発生に与える効果について解析した。

若齢ウシ雄性生殖細胞のヌードマウス皮下への異種異所移植による再構成精細管の構築について報告した。

トリコスタチンＡ処理によるマウス体細胞核移植胚の着床初期における胚発生に関わる遺伝子発現について解析した。

若齢ウシ雄性生殖細胞の同定ならびにヌードマウス皮下移植による精子形成誘導について試みた。

マウスＥＳ細胞分化誘導過程におけるにABCトランスポーター Bcrp1 アイソフォームの発現様式およびRNAiによるアイソフォームAノックダウンES細胞樹立について試みた。

幹細胞マーカーおよび生殖細胞マーカーを用いて若齢ウシ精巣ならびに初代培養したウシ精巣細胞におけるゴノサイトおよび精原幹細胞を同定した。

マウスＥＳ細胞の分化誘導過程におけるにABCトランスポーター Bcrp1 アイソフォームの発現様式について解析した。

トリコスタチンA処理によるマウス体細胞核移植胚の着床初期における転写因子の発現について解析した。

マウスＥＳ細胞の分化誘導過程におけるにABCトランスポーター Bcrp1 アイソフォームの発現様式について解析した。

脂肪酸不飽和化酵素FAD2を過剰発現するトランスジェニックマウスの表現型に対する遺伝子相互作用の解明を目的として、マイクロアレイを用いた網羅的遺伝子発現解析を行なった。

酵母two-hybrid systemにより、rhophilin-2と相互作用する因子を探索し、候補タンパク質については共免疫沈降法により相互作用の有無を確認し、同定した。

ALSの原因のひとつであるSOD1遺伝子変異に対し、RNAiによる変異ﾀﾝﾊﾟｸ質の発現抑制を試みた。本研究では、ES細胞への遺伝子導入によるSOD1ﾉｯｸﾀﾞｳﾝﾏｳｽの作製に成功し、個体におけるSOD1ﾀﾝﾊﾟｸ質の抑制状態について解析した。

マウス初期胚に対する時計遺伝子群の関与について検討することを目的に、本研究では発現プロファイルについて調べた。

マウス体外受精卵に導入した発現ベクターの発現に対するエピジェネテック修飾の関与について検討した。

トランスジェニックマウスにおける導入遺伝子の染色体上挿入位置を、Inverse PCRを用いて同定した。

マウス初期胚でその発現が変わる低分子量Gタンパク質伝達に関与するrhophilin-2に着目して、two-hybrid法によって相互作用する因子を探索した。

マウス初期胚における時間依存的細胞分裂制御の可能性について検討した。

本研究では、siRNAによる遺伝子治療の有用性の検証を目的として、ALSの原因遺伝子のひとつであるSOD1についてsiRNA過剰発現によるノックダウンマウスの作製を受精卵注入法およびES細胞への遺伝子導入法により試みた。その結果、ES細胞への遺伝子導入法でのみ、標的ﾀﾝﾊﾟｸ質の抑制効果のみられるノックダウンマウスが作製された。

雄性のゲノムインプリンティングが個体発生の中でどのような役割をしているのかを検討するために、二倍体雄性発生胚由来の胚性幹細胞を樹立し、そのゲノムにおいてインプリンティングの状態が雄性のインプリンティングが維持されているのか否かを検討した。

本実験では、マウス1細胞期胚におけるより詳細な遺伝子発現時期を検討することを目的に、レポーター遺伝子として改良型ホタルルシフェラーゼ遺伝子luc＋を前核に顕微注入した胚及びluc＋mRNAを細胞質に注入した胚において遺伝子の転写及び翻訳時期を調べた。

本研究の目的は、初期胚発生における母性タンパク質分解へのN末端則経路のかかわりを明らかにすることで、全能性獲得メカニズムの解明に迫ることである。 受精直後の胚は卵細胞質に蓄積された母性タンパク質を利用して発生を進めるが、それと同時に母性タンパク質を分解することも必要であり、これは全能性獲得の過程のひとつであると考えられている。N末端則とは、タンパク質の寿命がアミノ末端(N末端)残基に依存するという法則で、真核生物で広く保存されている。特にN末端残基がアルギニンであるペプチドは急速に分解されることが知られており、その反応はアルギニル化とよばれる翻訳後修飾により促進される。このメカニズムは、短時間に劇的な変化が必要な初期胚発生や全能性獲得の特性と合致すると思われ、N末端則経路と全能性獲得のかかわりを解明することは学術的に大きな意義をもつ。 本研究で実施することは、1) N末端則経路を介して分解される母性タンパク質の同定、2) アルギニル化が起こらない胚の解析、3) 加齢卵子の質の向上の試みの3点であり、2021年度はN末端則経路を介して分解されるタンパク質の候補として特にアルギニル化されるタンパク質の同定、生殖細胞特異的にアルギニル化が起こらないマウス（ATE1-CKOマウス）の生殖能力の検定を試みた。未受精卵および初期胚においてアルギニル化されることが予想されるタンパク質の検出に成功し、これらの中には未受精卵と初期胚で共通に検出された母性効果因子も含まれており、全能性獲得メカニズムへのアルギニル化の関与が示唆される。

これまで、マウス体細胞核移植技術の産児作出効率の向上によって、希少動物や絶滅危惧種の保全、絶滅動物の復活への横断的研究が可能となった。また分子遺伝学的解析の更新がさらなる技術の更新が可能となりつつある。当該研究課題においても、マウス体細胞核移植胚の発生率を劇的に改善し「産子発生の向上」ならびに「初期化抵抗性遺伝子群の同定」とその作用機序を明らかにした(Azauma et al.,2018)。 本申請課題においては、異種間核移植(iSCNT)において、これらの方法を用いた効率的な異種間クローン胚の作出を目的に検討を進めている。これまでに、アカネズミドナー細胞を用いて作製した融合卵子は、93%(93/100)の卵子が早期染色体凝集を形成した。活性化処理後、78%(78/93)の卵子が前核構造の形成を認め、83%以上が2細胞期胚へ発生することをみとめている。本年度は発生した異種間体細胞クローン胚の発生メカニズム免疫細胞染色しにより、H3K9me3の動態を確認した。これらの結果、iSCNTへ供試するアカネズミ由来細胞の培養下へ添加するVC処理条件(optimal condition)を検討することによって、H3K9me3を低下させることが可能であった。しかしながら、H3K9me3を低下させることだけではiSCNT胚の発生は改善されなことを認めた。さらに、H3K4me3レベルには、変化を認めなかった。これらのことから、今回調べたヒストン修飾は、ドナー細胞種および動物種が変化することによって異なるメチル化レベルを示すことが確認できた。これらの成果は、Journal of Reproduction Developmentへ投稿をおこなった。

卵巣内に存在する未成熟な卵子の利用は、今後生殖補助医療分野への展開へ繋がる技術として考えられている。申請者は未成熟卵子を用いた体外成熟操作の改善と体外受精方法を検討し、近交系マウスを用いた産児作出に成功した。また成熟操作培地の開発を進め、卵子初期の遺伝子発現がアミノ酸構成要素により影響を与えることを認めた。さらに小型げっ歯類を供試して体細胞の樹立を検討し、得られた体細胞ストックは、体細胞核移植により再構築卵子を作成し活性化処理後、クローン胚の作出に成功した。

高効率・安定的な遺伝子改変マウスの開発をめざし、非B型DNA人工配列によるRNAポリメラーゼII型ならびにIII型依存性の転写活性の賦活化作用について検討した。その結果、非B型DNA人工配列モデルとしたT20配列について、(1)ES細胞におけるPolII型転写活性の賦活化作用、(2)ES細胞の分化過程における作用の安定性とクロマチン構造の動態、(3)T20配列導入マウスの作製に関する新たな知見が得られた。

pp14-33　新聞・雑誌

　新聞・雑誌　白浜町のテーマパーク「アドベンチャーワールド」は、近畿大学生物理工学部（紀の川市）などとの共同研究で、人工授精によるキングペンギンの繁殖に成功したと発表した。同園では昨年に続き2羽目、国内では2018年の鴨川シーワールド（千葉県鴨川市）を含め3羽目となる。

　インターネットメディア　アドベンチャーワールド（和歌⼭県⽩浜町）は近畿⼤学⽣物理⼯学部（和歌⼭県紀の川市）および先端技術総合研究所（和歌⼭県海南市）との共同研究において、昨年に続き２年連続でキングペンギンの⼈⼯授精に成功しました。２０２１年２⽉６⽇に誕⽣した⾚ちゃんはDNA鑑定の結果、精⼦を提供したオスの遺伝⼦を持つことがわかりました。今回の成功は国内３例⽬（３⽻⽬）となります。現在⾚ちゃんは⺟親と育ての⽗親とともにペンギン王国で元気に過ごしています。

　テレビ・ラジオ番組　マンモス復活プロジェクトについて、研究を進めていく中で間近に感じた、マンモスのスゴさを分かりやすく紹介した。

　テレビ・ラジオ番組　9月22日迄開催されたマンモス展について、マンモス復活プロジェクトの研究成果を解説した。

　テレビ・ラジオ番組　マンモス展 その「生命」は蘇るのか開催にあたり、研究成果について解説した。

　新聞・雑誌　アドベンチャーワールドと近畿大学がキングペンギンの人工授精に成功した。キングペンギンの人工授精は鴨川シーワールドに続いて国内2例目の誕生である。

　新聞・雑誌　アドベンチャーワールド（和歌山県白浜町）は近畿大学生物理工学部（和歌山県紀の川市）および近畿大学先端技術総合研究所（和歌山県海南市）との共同研究において、国内で2例目となるキングペンギンの人工授精に成功した。

　インターネットメディア　アドベンチャーワールド（和歌⼭県⽩浜町）は近畿⼤学⽣物理⼯学部（和歌⼭県紀の川市）および先端技術総合研究所（和歌⼭県海南市）との共同研究において、国内で２例⽬となるキングペンギンの⼈⼯授精に成功しました。２０２０年１⽉３１⽇に誕⽣した⾚ちゃんのDNA鑑定の結果、⽗親は精⼦を提供したオスであることがわかり ました。現在⾚ちゃんは⺟親と育ての⽗親とともにペンギン王国で過ごしています。

　インターネットメディア　近畿大学は、アドベンチャーワールド(所在地：和歌山県白浜町)におけるキングペンギンの人工授精に成功し、研究成果や開発技術を活かした今後の取組などを発表した。アドベンチャーワールドは、開園当初から各種ペンギンの飼育を行っており、1990年に自然界で暮らすペンギンコロニーの再現を目指すプロジェクトを立上げ、人工孵化や育雛による繁殖の実績をあげている。同大学は、2017年3月に同施設の運営会社である(株)アワーズと産学連携協定を締結し、展示・希少動物の繁殖のための共同研究の一環として、精子等の遺伝資源の保存技術開発を進めている。今回、人工的に精液を採取する技術は確立されているものの、国内では人工授精が1例しか報告されていない「キングペンギン」について、同大学との共同研究に基づき、人工授精が行われ、2020年1月31日にメスの雛が誕生した(孵化日数：56日)。精子の運動性を維持する凍結保存方法の開発まで完了しており、今後は凍結保存した精子による人工授精を試みるという。

　インターネットメディア　アドベンチャーワールドと近畿大学生物理工学部は、バンドウイルカおよびキングペンギンの精子凍結保存に必要な保存液の開発と精子の運動性評価に関する研究をスタートさせました。技術を確立させ、当園では初となるバンドウイルカと、まだ世界でも事例の無いキングペンギンの人工授精の確立を目指します。

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　新聞・雑誌