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To preserve genetic resources efficiently in rats, sperm cryopreservation is essential. This study aimed to confirm the ability of cryopreserved and transported rat spermatozoa to fertilize through intrauterine insemination. The Komeda miniature rat Ishikawa is a mutant caused by the autosomal recessive mutation mri. The epididymal sperm was frozen with egg yolk medium and banked at the National BioResource Project (NBRP), Kyoto University. The sperm was transported to Azabu University, then thawed at 37℃. The thawed semen was inseminated into the uterine horns of recipients; its motility was around 10%. Seven of 15 inseminated female rats became pregnant and 13 live pups were born. The results indicate that rat spermatozoa cryopreserved at NBRP are capable of restoring genetic resources through intrauterine insemination. We also confirmed the usefulness of assisted reproductive technologies for the rat including sperm cryopreservation and intrauterine insemination.

ビオチン欠乏時および回復後のラット海馬歯状回のニューロジェネシスについて免疫組織化学的に検証した。Wistar系雄ラットを用いた。離乳直後からビオチン欠乏飼料を与え,ペアフィーディング(以下PF)を行い,ビオチン欠乏群(BD群)には生理食塩水を,対照群(BS群)にはビオチン(200μg/kg)をそれぞれ毎日腹腔内投与した。半数(BDI群・BS I群)は5週間後に剖検し,残りの半数(BD II群・BS II群)はBD群ヘビオチンを投与し4週間回復させた後に剖検し,脳を摘出した。脳は海馬の同一部位を厚さ20μmの凍結切片を個体ごとに6枚作製し,神経細胞検マーカーである抗Neuronal Nuclei (NeuN)抗体と抗BrdU抗体を用いた二重免疫染色を施し,BrdU陽性細胞数およびBrdU/NeuN陽性細胞数を計測した。BD I群・BS I群において,BrdU陽性細胞数とBrdU/NeuN陽性細胞数のどちらも有意に減少していた(P, We previously confirmed that learning ability of a biotin-deficient (BD) rat decreased and LTP of its hippocampus was suppressed. In this study, the neurogenesis of hippocampal dentate gyruses of rats in BD conditions were investigated immunohistochemically. Wistar strain rats, 3-week-old, were fed BD diet. BD group was subjected to daily i.p. injection of saline for 5 weeks. Control (BS) group received same diet with daily injection of biotin (200 μg/kg, i.p.). The rats of the BD and BS groups were administered 5-Bromo-2'-deoxyuridine (BrdU, 50 mg/kg, i.p.) every 5 days, at a total of 4 times before autopsy. After exsanguination under anesthesia, the brains were removed, fixed with 4 % para-formaldehyde, sectioned 20 μm thick, and stained immunohistochemically with anti-neuronal nuclei (NeuN) and anti-BrdU antibodies. The numbers of BrdU positive cells of BD and BS were 39.1 ± 1.0 and 53.0 ± 0.8, respectively, and the numbers of BrdU/NeuN positive cells were 26.8 ± 0.7 and 39.3 ± 0.6, respectively. Both numbers of BrdU and BrdU/NeuN positive cells of the BD group showed significantly small values (p < 0.001). The neurogenesis of the hippocampus was suppressed in the BD condition. This suggests that biotin has an important role in the development and maintenance of brain functions.

ブタ体細胞核移植胚の発生能は低く,産子作出効率は極めて低い。しかし,体細胞核移植を介したトランスジェニックブタの作製法は,DNA顕微注入法よりも有利な点が多い。本研究はブタ体細胞核移植胚の発生能の改善を目的とし,単為発生胚と体細胞核移植胚を集合させ,その集合胚の発生能を検討した。実験1: ブタ初期胚の集合胚作製条件を検討するために,発生ステージが異なる単為発生胚どうしの集合胚の集合率および胚盤胞への発生率を調べた。単為発生胚は,48h体外成熟培養後の卵母細胞に1kV/cm,64μsec,0.5sec間隔で三回印可による活性化誘起処理を施し,サイトカラシンBで倍数体処理をおこなって作出した。活性化処理後48あるいは72hに05%pronaseにより透明帯を除去し,発生ステージの異なる単為発生胚を3通りの組み合わせ(48h+48h区,48h+72h区,72h+72h区)で集合させ,その集合率,胚盤胞への発生率および胚盤胞の細胞数を調べた。活性化処理48h後に透明帯除去し,集合させずに培養したものを対照区とした。透明帯除去単為発生胚の集合率および胚盤胞への発生率は,48h+48h区で58.3%(60/103)および50.5%(52/103),48h+72h区で28.5%(43/151)および22.5%(34/151),72h+72h区で38.6%(71/184)および28.8%(53/184)となり,48h+48h区が他の区に比べ有意に(P, Somatic cell nuclear transfer cloned (SCNT) embryos have still lower developmental competence in the pig. However, transgenesis through somatic cell cloning in pigs has more advantage than conventional DNA micro-injection method. The aim of the present study was to improve the competence of SCNT porcine embryos, we examined that embryonic development in vitro of aggregated embryos between SCNT and parthenogenetic embryos. In Experiment I, rates of successful aggregation and development to the blastocyst stage were assessed. In vitro matured porcine oocytes were electro-stimulated (1kV/cm, 64μsec, three times, interval of 0.5sec) and cultured with 5μg cytochalasin B for 2 hours (h) to obtain diploid parthenogenetic embryos. At 48 or 72h, the zona pellucidae of activated oocytes were removed with 0.5% pronase, the zona-free embryos were treated with aggregation (48h+48h, 48h+72h, 72h+72h), and cultured in vitro. The zona-free embryos at 48h post electro-activation were also cultured as control. The successful aggregation rates and the blastocyst formation rates were 58.6% (60/103) and 50.5% (52/103) in the 48h+48h treatment, 28.5% (43/151) and 22.5% (34/151) in the 48h+72h tratment, and 38.6% (71/184) and 28.8% (53/184) in the 72h+72h treatment, respectively. The rate of the 48h+48h treatment was significantly (P

雌豚では,発情期に外陰部の腫脹や充血などの特徴的な変化が見られる。この変化はEstrogenの生理的作用である。このとき生殖器官組織の経時的変化も認められ,その変化に関係する生理的に多様な機能を有する肥満細胞(TB陽性細胞)分布の動態に注目をした。17-βestradiol(E_2略)を用いて発情期の状態を再現し,その時の生殖器に見られる組織学的変化と肥満細胞の関連性を検討した。供試動物は,生後月齢のランドーレス,大ヨークシャー雑種の雌に。E_2投与した後,1・2・4・6・8・10日目に各々卵巣,子宮角,子宮頚管,膣部,外陰部とその周囲の皮膚を採取した。生殖器官の組織学的変化と肥満細胞の分布について観察し,検査はHE法とTB(異調性染色)法を用て組織中の肥満細胞を算定した。結果は,副生殖器官と外陰部周囲の皮膚部における組織変化が,E2投与で粘膜及び表皮細胞の増殖,粘膜固有層の拡張と水腫,好中球・リンパ球の浸潤等が2日目から4日目に顕著に認められた。卵巣は,4日目から6日目に卵胞の発育が見られた。採材した器官すべてに組織中に4日目から6日目に肥満細胞の分布が増加した。更に,特徴的に卵巣髄質,外陰部と,その周囲皮膚の真皮層に肥満細胞は多数認められた。これらの事から未成熟雌豚にE_2投与すると生殖器組織が経時的変化し,特に粘膜組織の増殖と水腫性,血管拡張性ならびに白血球浸潤変化が著明に認められた。この変化と合致するように肥満細胞の増数的変化が見られた。以上のことから肥満細胞の変化は,E_2と極めて密接な関係性があることが示唆された。, Specific changes, such as swelling and erythema of the external genitalia, take place in sows at the time of estrus. These changes are caused by estrogen. At the same time, progressive histological changes are also observed in the reproductive organs. In this study we focused on the changes in the distribution of mast cells (toluidine blue [TB] positive cells), which have various physiological functions and are related to the changes described above. We used 17-β estradiol (E_2) to induce estrus in sows, and then examined the relationship between histological changes in the reproductive organs and mast cells. We administered E_2 to one-month- and sixmonth-old Landrace x Large White sows, and sampled tissue from the ovary, uterine horn, cervical canal, vagina, external genitalia and skin around the external genitalia on days 1, 2, 4, 6, 8 and 10 following administration. Histological changes in the reproductive organs and the distribution of mast cells were examined by means of the HE method and TB (metachromatic staining) method. The number of mast cells in tissue and around arteries/veins was counted. With regard to histological changes in the accessory reproductive organs and skin surrounding the external genitalia, thickening of the mucosa, proliferation of epidermal cells, expansion of lamina propria mucosae, edema, and infiltration of neutrophils and lymphocytes were pronounced between day 2 and day 4. In the ovary, significant growth in the follicles was seen between day 4 and day 6. In all organ tissues, the distribution of mast cells increased between day 4 and day 6. In addition, a large number of mast cells were observed, particularly in the medulla of the ovary, the external genitalia and the dermis surrounding the external genitalia. As such, when E_2 was administered to immature sows, the reproductive organs showed progressive histological changes, and in particular, thickening of the mucosal tissue, edema, dilation of blood vessels, and infiltration of white blood cells were significant. The number of mast cells increased in response to these changes. These findings suggest that changes in the number of mast cells are closely related to E_2.

全身で蛍光タンパク質を発現するトランスジェニックラットの作製を目的とし,蛍光タンパク質遺伝子(EGFP,d2EGFP,DsRed)を前核期胚の前核へ注入(MI)し,注入胚をレシピエントヘ移植した。前核期胚のドナーとしては4-6週齢,交配用には12-36週齢,レシピエントとしては12-18週齢のWistar系ラットを用いた。eCGおよびhCG投与による過剰排卵処置した雌と成熟した雄を自然交配させ,hCG投与29時間後に卵管灌流により前核期胚を採取した。MIに用いた遺伝子コンストラクトは以下のように作製した。蛍光タンパク質遺伝子としては, EGFP,d2EGFP,DsRedを購入した。それぞれの蛍光タンパク質遺伝子の上流にCMV-IEプロモーター,下流にSV40ターミネーターを含んでいる。この様なプラスミドを構築し,導入遺伝子として増殖させた。これらをプロモーター上流の1箇所の制限酵素サイトで切断し,5mg/mlに調整してMIに用いた。前核期胚の雄性前核に蛍光タンパク質遺伝子(EGFP,d2EGFP,DsRed)をMIし,その16時間後に形態的に正常な1-2細胞期胚を偽妊娠誘起したレシピエントの卵管に移植した。えられた産子は離乳後,尾部組織よりゲノムDNAを抽出しPCR法により注入遺伝子の存在を調べた。DsRedを652個の胚にMIして11匹のレシピエントに卵管移植した結果,7匹が妊娠し,20匹の産子をえた。この産子のうち2匹がDsRed導入ラットであった。EGFPを241個の胚にMI後,5匹のレシピエントに移植した結果,2匹が妊娠し,9匹の産子がえられた。このうち2匹がEGFP導入ラットであった。また, d2EGFPを281個の胚にMIし,2匹のレシピエントに移植した結果,2匹が妊娠して7匹の産子がえられたが,H2EGFPが導入された産子はえられなかった。本研究により,全身で蛍光タンパク質遺伝子(EGFP,DsRed)の発現が期待されるトランスジェニックラットを作製することができた。これらのトランスジェニックラットは,様々な研究分野でのマーカーとして有望で,広範な分野での応用が期待される。, The objective of the present study was to generate transgenic rats carrying and expressing the fluorescent protein gene all over their body. Rat pronucleus stage embryos were microinjected with the transgenes (EGFP, d2EGFP, DsRed), then transferred into recipient female rats. Females aged at 4-6 weeks as embryo donors, at 12-18 weeks as embryo recipients, and males aged at 12-36 weeks for mating, were used in the present study. All the used rats were Wistar strain. Embryo donors were superovulated by injection of eCG and hCG, and naturally mated with matured males. Pronucleus stage embryos were collected from the donors at 29 hrs after the hCG injection. The gene constructs as transgenes were made as fellows. The fluorescent protein gene (EGFP, d2EGFP, DsRed), which were included the promoter of the CMV-IE and the terminator of the SV40, were purchased commercially. The DNA (5μg/ml) solution was prepared for microinjection. Pronucleus stage embryos were microinjected with the DNA solution. Morphological normal embryos selected among the embryos cultured for 16 hrs after microinjection were then transferred into recipient females that were mated with vasectomized male rats to induce pseudo-pregnancy. The extracted DNA from the tails of weaned pups was tested for the presence of the transgene by the PCR method. The 241, 28land 652 embryos injected with the EGFP, d2EGFP and DsRed, were transferred to the 5, 2 and 11 recipients, 2, 2 and 7 females of the recipients gave birth to 9, 7 and 20 new born pups. The 2, 0 and 2 pups of them were confirmed as transgenic founders. These results indicate that it can be generated that transgenic rats were expected expressing the fluorescent protein gene all over their body.

哺乳類において単為発生は,致死である。ブタ体外成熟卵母細胞由来のブタ雌性単為発生胚移植により21日齢の胎子を作出し,それらの発育とインスリン様増殖因子2遺伝子(IGF-II)の発現について調べた。ブタ体外成熟卵母細胞に電気剌激による人為的活性化およびサイトカラシンBを添加したNCSU-37にて培養し,2倍体のブタ雌性単為発生胚を作出した。次いでこれらを発情同期化させたレシピエントに移植した。卵子の活性化の21日後にブタ雌性単為発生胎子を採取した。比較対照として,両性由来のゲノムを有する胎生21日齢人工授精由来胎子を用いた。えられた胎子の一部を組織標本に用い,残りの胎子から個体ごとにtotal RNAを抽出し,Real Time RT-PCRを用いてIGF-IIの発現量を調べた。IGF-IIの発現量は,個体ごとのβ-actinに対する相対値として表した。結果,16個体のブタ雌性単為発生由来および19個体の人工授精由来の心拍を確認した生存胎子をえた。組織標本から,ブタ雌性単為発生胎子は受精由来の胎子に比べ発育が遅延していることがわかった。ブタ雌性単為発生胎子におけるIGF-IIの発現量は,人工授精由来胎子における発現量の6%であった。以上から,マウスやヒツジと同様にブタ胎生21日齢においてもIGF-IIは父性発現であり,単為発生胎子の発育遅延に関連していることが示唆された。, We examined development and expression of the insulin-like growth factor II gene (IGF-II) in porcine parthenotes on day-21 post artificial activation derived from in vitro matured (IVM) oocytes. IVM oocytes were stimulated by electric pulses, and were subsequently cultured contained cytochalasin B to obtain diploid oocytes. The oocytes were then transferred to recipients. Parthenotes were collected from the recipients at 21 days post electro-stimulation. As control, bi-parental fetuses through artificial insemination were also recovered on day-21 of gestation. Fetuses were fixed for histological preparations. From the other of the fetuses the m-RNA expressing of IGF-II by real-time RT-PCR. Total 16 of parthenotes and 19 of bi-parental fetuses were obtained and examined. Developmental delay was observed histologically in the parthenotes compared to bi-parental fetuses. The relative expression of IGF-II was detected as 6 % in the parthenotes of bi-parental fetuses. The results suggested that IGF-II was imprinted and paternally expressed in pigs as well as mice and sheep, and that the expression of IGF-II could be associated with the developmental delay of porcine parthenotes.

本研究は,ビオチン欠乏がラット海馬へ及ぼす影響について,Timm染色法を用いて海馬組織を組織計測するとともに,DNAマイクロアレイ法を用いて海馬組織における遺伝子発現を検証した。組織学的観察では,歯状回,CA1,CA3の各エリアにおいてBD群の方がBS群より神経細胞が小さい傾向を示し,ビオチン欠乏により海馬神経細胞の代謝活性が低下していることが示唆された。また,Hilus,Lucidum領域のシナプス密度は,背側海馬(頭側)では両群の間に差は認められなかったが,腹側海馬(尾側)ではBD群の方がBS群より有意に増加しており,ヒト側頭葉てんかんに見られる所見に類似することが示唆された。さらに海馬組織の遺伝子発現については,BD群では細胞間や細胞内情報伝達に関わる複数の遺伝子(アセチルコリン作動性受容体,AMPA型受容体,神経軸策伸張に関わる関連遺伝子)が抑制されており,ビオチンが遺伝子発現にも重要な働きを示すものと推察された。ビオチンは脳機能の維持,特に記憶・学習に関与している可能性がある。, Biotin participates in gluconeogenesis, amino acid metabolism, as well as fatty acid synthesis. In former studies, we observed that long-term potentiation in the biotin-deficient rat hippocampus was inhibited and the rats' maze-learning ability deteriorated. This study examined how biotin deficiency effects the rat hippocampus using Timm's staining and DNA microarray. Wistar rats were used in this study. They were fed biotin deficient diet. Experimental (BD) group was given a vehicle and control (BS) group was given biotin (200μg/ml/kg, i.p.) by daily. Four weeks later, the rats were euthanized and their hippocampuses were removed. Six hippocampuses from each group were embedded in paraffin and were stained with HE and Timm's method to measure the neuronal cell number and synapse density of the hippocampuses. The remaining two hippocampuses were used to examine gene expression using analysis of DNA microarray. In histological observation, the cells in the dentate gyros, CA1, and CA3 regions of the BD group were smaller than the BS group. The synapse density of ventral hippocampuses (caudal) of the BD group was significantly higher than the BS group. Also, expression of multiple intercellular signaling related genes, such as acetylcholinergic receptors, AMPA-type receptors, and nerve axon elongation related genes, was inhibited in the BD group. It is likely that biotin participates in maintaining brain functioning, especially in memory and learning functions.

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.