mTALP培養液中でcapacitationを誘起したウサギ射出精子を用いて体外受精を行ない、初期受精過程における精子先体の形態変化についてSEMを用いて観察を行なった。5時間の前培養を行なった精子は媒精後15-20分でcumulus cellに接着した。その大部分のものはacroeome intact (AI) であった。Cumulus cell群を通過している精子はほとんどがAIであり、一部形態変化が見られたが先体反応は観察できなかった。完全な先体反応が見られたのは透明帯上の精子のみであった。透明帯上に接着し先体反応を起こした精子は、透明帯を消化して囲卵腔内に侵入した。

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An attempt for obtaining xenogeneic blastocysts (Rat↔Mouse) was undertaken by aggregation method. As a culture medium, Dulbecco's modified eagle medium with 0.3% bovine serum albumin (pH 7.4) was used for the production of xenogeneic chimeric embryos in this experiment.
When the late eight-cell stage rat and mouse embryos were cultured immediately after flushing from the uterus, 88.2%(30/34) of rat embryos and 95.0%(19/20) of mouse embryos developed into expanded blastocyst after 48 hours in culture. When rat and mouse embryos were removed their zona pellucidae with 0.3% pronase before culture, 87.0%(20/23) of rat embryos and 95.7%(22/23) of mouse embryos developed into expanded blastocyst after 48 hours in culture. On the other hand, 87.0%(40/44) of allogenic chimeric rat embryos (Rat↔Rat) and 90.5%(38/42) of allogenic chimeric mouse embryos (Mouse↔Mouse) developed into single, integrated expanded blastocyst after 48 hours in culture respectively. Furthermore, xenogeneic chimeras betwen rat and mouse were produced by aggregating embryos at the late eight-cell stage in this experiment. Out of 41 aggregated embryos from rat and mouse, 34 (82.9%) formed single, integrated expanded blastocyst after 48 hours in culture.
These results showed that Dulbecco's modified eagle medium was available for the production of xenogeneic chimeras between rat and mouse.

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ICRマウスの過排卵卵子と精巣上体精子を用いて体外受精を行い、精子添加後6時間に前核期の卵子を100μMの濃度のEDTA・2Naを含むWhitten培地へ移し、72時間または96時間培養した。得られた桑実胚 (72時間) および胚盤胞 (96時間) を偽妊娠第3日または第4日の受容雌の子宮へ移植した。対照区として、交配後第3日および第4日の妊娠マウスの子宮を灌流して採取した桑実胚および胚盤胞を採取後直ちに偽妊娠マウスに移植した。体外受精由来胚の新生子への発生率は、桑実胚では対照区に比し有意に低かったが、胚盤胞では、偽妊娠第3日および第4日のいずれの移植においても対照区との間に有意差を認めなかった。これらの結果から、'2-cell block'を克服するためにEDTAを添加した培地内で発生した体外受精由来胚は、正常な発生能を有していると考えられる。

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When zona-free hamster egg test is used for assessment of fertilizing ability in human spermatozoa and elucidating the mechanism of fertilization, it needs to know the effect of zonaless, polyspermic, and cross fertilization. In this study, it is shown to observe unusual fertilization and sperm head chromatin decondensation. Multiple flagella sperm was also shown to be capaole of penetrating egg. These observations seems to be very useful for electron microscopic observations.

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The present study was undertaken to examine the effect of storage period of the culture medium, M16 (Table 1), on the development of mouse zygotes to blastocyst stage in vitro, and to live fetuses after transfer to recipients.
Mouse zygotes obtained from the superovulated F1 (C57BL/6×CBA) females 18 hrs after hCG injection were used. The zygotes with two pronuclei were cultured for 4 days in one of the medium either fresh, storaged for 30 days, 60 days, or 90 days in the refrigerator. Blastocysts developed from zygotes cultured in each medium were separately transferred into the uteri of recipients on the third or the 4th day of psedopregnancy. They were killed on day 18 to examine the number of implantation sites and live fetuses.
In fresh medium, 185 of 228 zygotes (81%) were developed to blastocyst stage. The proportion of blastocysts developed from zygotes cultured in preserved media was not significantly different from that obtained in fresh medium (76-85%, in table 2).
The proportions of implantation sites (58 to 65%) and live fetuses (38 to 51%) after transfer of blastocysts developed in preserved media were not also significantly different from those (67% and 46%) obtainded in fresh medium (Table 3).
The present study clearly demonstrated that the chemically defined culture medium, M16, could be preserved at least for 90 days in refrigerator without any detrimental effect on

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The present paper describes the in vitro development of single blastomeres isolated from 2-cell eggs in mice.
Two-cell eggs were recovered from CF 1f1 females which were injected with 5 IU of PMS followed 48 hours later by 5 IU of HCG and were mated with fertile CF#1 males. Zona pellucidae were removed by incubation in Hanks medium containing 0.2 pronase for 5 to 8 minutes, and the zona free eggs were washed two times with Hanks medium. Blastomeres were separated mechanically with a glass pipet in Ca⁺⁺and Mg⁺⁺free Hanks medium containing 0.02% EDTA. Separated blastomeres were incubated in drops of culture medium (Whitingham 1971) coverd with paraffin oil at 37°C in an atmosphere of 5% CO₂., 95% air for 72 hours.
Of the 576 single blastomeres isolated from 288 2-cell eggs, 416 (72.2%) developed to blastocyst in culture for 72 hours. This rate was similar to the rate of development into blastocyst in zona free 2-cell eggs, although lower than that of normal 2-cell eggs. The proportion of monozygotic pair embryos developed to blastocyst was 61.5%, in this culture.

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It is well known that in mammalian oocytes meiotic division is resumed by LH surge. Oocytes and preovulatory follicles from immature rats injected with PMS or PMS-hCG were used for studies of the changes in energy metabolism during oocyte maturation. The activity of phosphofructokinase, a rate-limiting enzyme in glycolysis, and ATP content in one oocyte were measured using an ultramicroassay called the enzymatic cycling method. They increased markedly in parallel with meiotic maturation after h CG injection. The activity of phosphofructokinase increased after PMS injection dissociated from meiosis, which may be mediated by production of estrogen within the follicles. Oocyte maturation should be considered meiotic and cytoplasmic separately. It is proposed that activated energy metabolism, that is, glucose metabolism, is so good an indicator for cytoplasmic maturation as germinal vesicle breakdown and polar body emission for meiotic maturation.

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Many kinds of culture medium are used for human IVF-ET at various institutions around the world, but the reason for their use is not always clear.
We examined such culture mediums for human IVF as Hoppe & Pitts, mEarles, Ham's F-10, T6 and for animal IVF as mBWW, mKRB, TALP-2 for their applicability to fertilization and to embryo culture by the hamster test and cultural experiment of mouse 2-cell stage embryo, respectively. The mBWW showed the most favorable results in both experiments. It seems important for the mBWW to be reconsidered as a culture medium for human IVF.

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Loss of the zona pellucida in the blastocyst is an essential process at the initiation of implantation, but its mechanism has not been completely clarified. It is generally known that loss of the zona pellucida is retarded during delayed implantation. In this experiment, we compared and investigated how the varied methods of delayed implantation effected the time when loss of the zona pellucida occurred.
Mature virgin female mice of ICR strain, 6-9 weeks old and 25-35 g in body weight, were kept in a light controlled room, supplied with water and a standard laboratory diet ad libitum. The mice at proestrus were caged overnight with males, and were checked for copulation plug the morning of the following day, which was set as the first day of pregnancy.
The results obtained were:(1) Loss of the zona pellucida began as early as the afternoon of the 4th day and was over by 2 o'clock of the 5th day;(2) the loss got significantly delayed in the groups of tubal ligation, recerpin, atropine sulfate, indomethacine, overiectomy (OVX), and OVX + progesterone, the most retarded among those groups being the one that got OVX;(3) Uterine environment and ovarian hormones did not seem directly participate in loss of the zona pellucida, judged from the fact that such loss occurred even in the groups of tubal ligation and OVX;(4) loss of the zona pellucida was prompted by the application of a higher concentration of progesterone and / or of estrone treatment. It may be said that the gestagens and estrogens are indirectly responsible for the ratio of zona-free mouse blastocysts.

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A large number of normal ova or embryos are needed in basic researches on the ovum including ET, IVF and embryo engineering. For those purposes, ova are obtained by perfusing the extirpated oviduct or uterus. The authors succeeded in collecting embryos through the vagina from the uterus in situ of a rabbit with superovulation treatment, using their original 2-way balloon catheter for the boy's bladder.
A total of 555 ova were obtained from 17 subject rabbits; cleaved ova included 391 morula and 46 early blastocyst. The best yield from a single rabbit was 65 ova including 63 cleaved ova.
This is a very efficient method for embryo collection from the rabbit as it enables us to obtain many ova from the uterus in situ and even in repetition from the same animal.

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