マウスの初期分割卵を着床前のマウス子宮に移植したところ、透明帯が完全に剥離した初期分割卵23.6%を得た、このことから着床前の子宮液中にタンパク分解酵素が存在することが示唆された。

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ハムスター卵子の細胞質中ヘマイクロマニピュレイターを用いてウサギ精子を注入して、それ以後に起こる受精変化について観察を行なった。凍結一融解処理、あるいはlysophosphatidyl choline (LC) 感作処理をおこなったウサギ精子頭部はハムスター卵子細胞質中で雄性前核に発生することが確認できた. 各処理群の精子はマイクロインジェクション後、1から4時間において前核形成を示した. 少数の卵子においては形成された雄性前核は卵子の中央部に移動して雌性前核と一見融合する様な像を示した.

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排卵前ウサギ卵胞卵の体外成熟および射出精子による体外受精法について検討した。1) 人工膣で採取した射出精子をBO液中で前培養したところ、前培養3時間の時点で25%、6時間以降で30%程度の精子に先体反応が誘起された。2) HCG注射後9時間に卵胞卵を回収したところ、いずれの卵胞卵も成熟分裂を再開していたが、一部の卵は成熟を完了しておらず、すべての卵が成熟を完了するには4時間の体外培養を必要とした。3) 前培養0および4時間の卵胞卵を2～4時間前培養した射出精子で体外受精し、受精後8時間で固定、染色したところ、前培養0時間の卵胞卵は検査した42個すべてが未受精であったが、前培養4時間の卵胞卵は101個中15個 (14.9%) が受精し、卵胞卵前培養の効果が認められた。4) 交配、HCG注射後24時間に回収した2細胞期胚および1細胞期卵を5種類の培養液で培養し、胚盤胞以降への発育を観察したところ、TCH199+FCS (80.0および60.0%)、M16+RS (92.9および60.0%)、M16+FCS (70.4および62.9%) において良好な発育が認められた。さらに、M16+RSは交配、HCG注射後18時間に回収した1細胞期胚の81.4%を胚盤胞以降に発育させたことから、体外受精卵の発生用培地に適するものと思われた。5) HCG注射後15時間に回収した卵管卵とHCG注射後9時間に回収し、4時間前培養した卵胞卵を体外受精、培養したところ、卵管卵は47個中5個 (10.6%) が2～4細胞期胚に発育し、そのうち1個 (20.0%) が胚盤胞を形成した。これに対して前培養した卵胞卵は113個中7個 (6.2%) が2～4細胞期胚に発育したが、いずれも8～16細胞期胚で発育を停止し、胚盤胞を形成するには至らなかった。

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By microsurgery, bovine spermatozoon was injected into bovine oocytes matured in vitro, to elucidate a role of sperm components such as acrosome, neck, middle piece and tailplaying in fertilization and subsequent development. The injected spermatozoon wasselected from the pretreated sperm as follow: 1) washed sperm 2) preincubated sperm 3) sonicated sperm head 4) freeze-dried sperm head 5) preincubated sperm head. The spermsuccessfully injected were almost decondenced and 10.5-26.4 percent of them developedinto male pronucleus in egg cytoplasm. The results indicate that bovine gametes may beavailable for in vitro fertilization by microsurgery, e. g. microfertilization, in spiteof these pretreatment of sperm, and also this technique shall be useful for elucidationthe mechanism of the development in an interaction between oocytes and spermatozoa.

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The regulative capacity of mouse embryos aggregated differentstages and their developmental potential in the postimplantationhave been examined.
Seventy-eight percent of asynchronously aggregated embryosbetween 4-cell and 8-cell stage developed into integrated blastocysts. However, aggregation between 4-cell and 16-cellstage embryos was not occurred.
Two adult mice have been obtained from asynchronous lyaggregated embryos by embryo transfer to normal pregnant hosts. One showed chimeric features, but the other was apparentlyderived from the one of the pair of asynchronously aggregatedembryos.
These results suggest that early mouse embryos tend to keeptheir own time-chronological development regardless ofaggregation and that younger embryo in asynchronously aggregatedembryos can incorporate into inner cell mass of integrated blastocyst.

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ICR雌マウスに、PMSGとhCGを投与して過排卵を誘起し、同系の雄と交配後、受精卵を回収して第1分割期の染色体標本を作製し、Cバンド染色を施した。染色体の分析率は96.6%であり、3倍体の出現率0.5%、4倍体0.2%、1倍体0.9%、高2倍体0.9%、構造的異常0.5%であった。また、Cバンド染色標本においてのY染色体の識別による性判別率は93.8%と高いものであった。1次性比 (雄率) は45.2%であり、理論値の50%から有意な偏りを示さなかった。

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This study was undertaken to discover any influences of hyperestrogenism on implantation of mouse embryos in vivo and in vitro.
I. In. Vivo Implantation Experiment: Mouse oocytes were collected from superovulated Crj-CD1 (ICR) females. In vitro fertilization was performed according to the method of Toyoda et.al. Pronuclear stage eggs were transferred to m-WM medium added 100 μM EDTA six hours after insemination. Blastocysts developed after ninety-six hours of culture, were transferred into uterine horn of recipients on Day4 of pseudopregnancy which mated with BDF1 sterilized male mice by a modified McLaren's technique. Each amounts (0, 0.1, 1, 10, 100μg/mouse) of estradiol-17β were injected subcutaneously to recipient mice during Day1 to Day5 (Group I) or on Day4 and 5 (Group II) of pseudopregnancy. On the other hand, 1 or 5mg of progesterone was injected simultaneously with _E2 injection to determine the effect of luteal support on implantation. The recipients were killed on Day 9 of pseudopregnancy by cervical dislocation. The proportion of implantation sites and live embryos were examined in bilateral uterine horn.
II An Vitro Implantation Experiment: Uteri were removed from mice at D₄ of pseudopregnancy. The monolayer cells were made by the digestion of uterine epithelium with a trypsin-EDTA-DNase solution according to the technique of Salomon et. al. The uterine cells were seeded with a concentration of 6×10⁶ cells/dish in a 35mm culture dish (Falcon 3001) containing BME+AA medium (Spindle, A. I., et. al.), and were allowed to settle and attach to the dish for 24 hours. Five blastocysts fertilized in vitro were transferred directly onto the monolayer cells. Hormonal treatment was performed from the day 1 of co-culture with addition of E₂ or E₂ & P in the medium. Attachment, TbOG and two layered ICM were scored by counting under a dissecting microscope.
Results; In Group I, 1μg or more of E₂ treatment had of on implantation in vivo. In Group II, 10μg or more of E₂ had of on implantation. However, the injection of 1 or 5 mg of progesterone with E₂ overcame the influences. Addition of E₂ or E₂ and P had no affect on attachment, TbOG and two-layered ICM up to a concentration of 10ng/ml E₂. 10² ng/ml or more of E₂ had influence on the number of two-layered ICM, and 10³ng/ml or more of E₂ had affect on TbOG. However, progesterone had no influence on these phenomena.

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We have studied the method of cryopreservation of mouse embryos.
First of all we examined the effect of propanediol (PROH) as a cryoprotectant on survival of frozen-thawed embryos. The highest survival rate was obtained when we used Embryo Transfer Freezing Medium (ETFM) included 1.5M PROH as a cryoprotectant.
Then we studied the effect of addition of sucrose or trehalose to freezing media (1.5M PROH, 1.5M Glycerol, 1.5M dimethylsulphoxide (DMSO)) on survival of mouse embryos. The survival of embryos frozen in the medium containing 1.5M PROH + 0.075M trehalose was considerably increased compared with others.
These results suggest that the medium containing 1.5M PROH + 0.075 M trehalose may be useful as the freezing medium of human embryos.

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Zona drilled hamster and human oocytes were examined by scanning electron microscope. In the hamster mechanical drilling by micropipette created a beautiful hole mimicking sperm penetration hole left on the zona. Chemical drilling with acidic solusion (PH 2.2) made a round, straight hole with the size corresponding to the micropipette used. In the human oocytes, mechanical drilling formed a round, somewhat irregular, micropipette sized hole. Neither the zona surface, nor the microvilli were damadged. On the contrary, drilling with ascidic solusion resulted in the formation of a large, V-shaped hole, reflecting the different solubility properties of human zona pellucida. The microvilli on the plasma membrane were apparently affected. These results indicate that a simple drilling by micropipette is enough to create a hole sufficient for sperm passage, and that acidic solusion is not recommended for human zona drilling because of potential damadge to the vitellus.

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The number of spontaneous ovulations and number of ovulations induced by gonadotropic hormone were investigated in 12 strains of mice (nine inbred strains, one hybrid, two closed colonies). The number of spontaneous ovulations were in the 9.1-12.8 range in the inbred strains and 10.3-15.4 in the hybrid and closed colonies.The response to induction of ovulation differed among the strains, and the rateof increase in ovulations with respect to spontaneous ovulations was in the 1.7-4.8 fold range.

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凍結保存マウス卵子における受精能の低下の原因を知る目的で、卵丘除去卵子の受精能に及ぼすDMSOの影響について検討した。その結果、1.5MのDMSOを室温で添加すると、30秒以内に卵子の透明帯を変化させ、精子の侵入が不可能になることが知られた。一方、牛胎児血清 (FCS) の分子量約30, 000以上の画分に、この変化を防ぐ効果があることが明らかになり、20%FCSを含む1.5MDMSO溶液内で凍結保存された未受精卵の体外受精から正常な胎子が得られることが確認された。

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