Monoclonal antibodies (Mab) produced from one of the hybridomas (B11C8) which were established by cell fusion of mouse myeloma cells (P3UI) with spleen cells from a mouse immunized with porcine zona pellucida (ZP) were characterized and their inhibitory effect on in vitro fertilization of porcine oocytes by boar spermatozoa was studied. B11C8 Mab was IgG2a and reacted tissue-specifically and species-specifically to porcine ZP. It formed an immune precipitin line against heat solubilized porcine ZP in Ouchterlony's double immunodiffusion test, suggesting that multiple antigen epitopes corresponding to B11C8 Mab were expressed on one antigen molecule. The binding site of the antigen corresponding to the Mab seems to be composed of a peptide-carbohydrate mixed portion. B11C8 Mab, either divalent (IgG) or univalent (Fab) antibody, showed a strong inhibitory effect on the binding of boar spermatozoa to porcine oocytes. This suggests that B11C8 Mab possibly recognized some antigen molecule related to the sperm receptors on porcine ZP.

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As a part of studies to research the possibility of superpregnancy, the early development of fertilized ova which were induced superovulation by the administration of pituitary extract to female rat was observed. Pituitary gland removed from 3-10 months old male rat was homogenized and centrifuged. The supernatant fluid extracted from one pituitary was administered to 3-6 months old female rat at rletestrus. Effects of the pituitary extract injection on ovulation, recovery rate of ova and development of ova in pregnant rat were investigated.
1. In all immature rats treated, the age at vaginal opening was advanced. But the timing of the first ovulation was not affected by administration of pituitary extract.
2. In most of 3-6 months old rats ovulated, the number of ova ovulated was more than 20 and the average of them was 22.8±8.9. On the other hand, in most of rats over 7 months old, the number of ova ovulated was less than 20 and the average of them was 13.3±5.8.
3. Nine of nineteen treated adult female rats were induced superovulation at following estrus and were copulated by fertile male. Mean number of ovulated ova of treated rat was about twofold that of control one on day 1 of pregnancy.
4. Recovery rate of ova on day 3 and 5 of pregnancy in the treated rats were lower (68.9 and 67.0%, respectively) than those of control rats (88.6 and 82.7%, respectively).
5. Large vesicular ovarian follicles were not observed during their early pregnancy.
In conclusion, the pituitary extract acted on ovaries of all treated rats and promoted the growth of their ovarian follicles. Especially, in 3-6 months old rats, administration of the pituitary extract induced superovulation frequently and did not interfere with the early development of fertilized ova in pregnant rats.

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Mammalian chimaeras are used for reserch on embryology and interaction of distinctive cells in genetic disorder. Though chimaeric mice have been produced on a large scale, there are a few reports concerning chimaeric rats. The present study was attempted to produce chimaeric rats by aggregation between Wistar and Hybrid (Wistar x Brown Norway) embryos. The results obtained are summarised as follow.
1. Aggregated embryos cultured in modified medium (Dulbecco's PBS (-) with Whitten's nutrient saline) or Ham's F-12 with 5% fetal calf serum developed to blastocyst in higher proportion compared with modified medium with 10% bovine serum albumin.
2. A total of 185 aggregated embryos was transfered and eighteen of them developed to chimaeric rats. Fifteen of the chimaeric rats grew up to adult.
3. Sex ratio of chimaeric rats was 2: 1.
4. All of the chimaeric rats had reproductive ability.

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This paper describes the effect of EDTA on development of 8-cell golden hamster embryos to the blastocyst stage in vitro. Eight-cell embryos were recovered from mated hamsters that had been superovulated with PMSG. Embryos were cultured for 48 hours in a modified Krebs-Ringer bicarbonate solution (modified KRE solution) containing various concentration of EDTA. At the end of the culture, the numbers of embryos developing to the blastocyst stage were recorded.
The percentage of 8-cell embryos (approx. 70 hours post coitum) developing into blastocysts was improved by adding EDTA to the medium (38.5 to 85.2% with EDTA vs. 16.7% without EDTA). A marked effect of 40-EM EDTA on development were found.

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Present experiments were performed to examine the possibility that the limited development of mouse eggs transferred into rat uterus was due to an immunological rejections. Mature virgin female mice of the ICR strain were used as the donor and recipient. Tree kinds of media were used;(1) KRP buffer;(2) 20% rat serum+KRP buffer; and (3) 20% rat serum was after transfer of mouse eggs into rat uterus especially when the transfer was repeated+KRP buffer. Eggs culture and exposure to 3 kinds of media were transfer to the mouse uterus. As summarized in Table 1, exposure of blastocysts to medium (3) caused a significan (P<0.05) inhibition of the implantation rate. It was suggested from the above results that a species-specific antibody appeared after transfer of mouse eggs into rat uterus especially when the transfer was repeated.

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The rabbit ovary were perfused after ligation of branches of the ovarian artery and cannulation of this artery. perfusion appratus consisted of a pulsatile rotary pump, an oxygenator and a chamber with circulating medium. Tissue culture medium 199 was supplemented with insulin, heparin and antibiotics, and oxgenated by gas mixture of 95% O₂ and 5% CO₂. Follicle rupture, maturation of oocytes and progesterone production of the perfused ovary were observed after administration of hCG or other agents into the medium. Although maturation of oocytes and synthesis of progesterone of the perfused ovary were shown at the same rate as the in situ ovary, follicle rupture occurred earlier. prostaglandin, histamine and norepinephrine did not cause maturation of oocytes nor steroid production but made follicles rupture. No sequence of production of histamine and prostaglandin or interrelationship in their activity could be detected in the ovulatory process.

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タウリン及びエピネフリンを添加した修正タイロード溶液 (mTALP) 中で培養したウサギ射出精子を用いて体外受精土を行った。5時間の培養を行った精子では媒精後速やかに卵子透明帯に結合した。さらに、媒精後1時間以内に囲卵腔中に精子が侵入した。3時間の培養精子でもその割合は低かったが、類似の傾向を示した。しかし、1時間の培養を行った精子では囲卵腔内に侵入したのは媒精後3時間目であった。5時間培養精子による体外受精卵子において、媒精後4-5時問目に雌雄前核土を観察できた、これらの結果はmTAしPが、ウサギ射出精子に比較的短時間で受精能獲得を誘起できることを示した。

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Following attempted in vitro fertilization (IVF), 13 nonfertilized oocytes were examined by scanning electron microscope (SEM), in order to determine the cause of failure. Fifty-eight IVF treatments were performed on 32 infertile couples. Semen analysis and the hamster test were normal in all patients. Each hasband fertilized most of his wife's oocytes and the mean fertilization rate was 93.2±2.6%: of 114 mature oocytes used 101 (88.6%) were fertilized. Nonfertilized oocytes were first examined by light microscope. They were then fixed, and processed for SEM. No oocytes had spermatozoa in the perivi telline space. The outer surface of the zona pellucida displayed a typical porous, net-like appearance in most cases. Oocytes with a smooth surface of the zona were also seen. Many spermatozoa in early stage of penetration were observed. The spermatozoa fixed on the zona had reacted acrosomes. The surface architecture and the sperm-zona interaction observed on the nonfertilized oocytes were not different from those of fertilized ova. It is clear that fertilization failure in human IVF usually oocur at the level of zona pellucida. The subsurface area of the zona seems to be primarily responsible for penetration block.

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