Bovine zygotes produced by in vitro maturation and fertilization (IVM-IVF) were selected according to the time of the first cleavage, and their subsequent developmental ability to the hatched blastocyst-stage and sex were assessed using conventional in vitro culture (IVC) conditions and PCR sexing. Of 669 presumptive zygotes cultured, 124, 227, 88 and 65 cleaving embryos were selected at 22, 26, 30 and 44 h post insemination (hpi), respectively. These embryos were transferred into separate drops of the same medium with cumulus cells and co-cultured for a total of 10 days. Significant correlation was observed between hatching rates and the time of the first cleavage (P< 0.05): 56.5, 40.1, 21.6 and 4.6% of embryos cleaved at 22, 26, 30 and 44 hpi were developed to the hatched blastocyst-stage, respectively. Of 183 hatched blastocysts subjected to PCR for sexing, the sex of 171 embryos was successfully determined and the overall sex ratio was 51.5% (88/171). The sex ratios of early cleaving embryos (22 and 26 hpi) and late cleaving embryos (30 and 44 hpi) were 49.7% (74/149) and 63.6% (14/22), respectively. These ratios were not significantly different from the expected ratio of 1:1 and did not related to the time of the first cleavage. These data suggest that the interval from insemination to the first cleavage of embryos generated in vitro strongly affects their subsequent developmental potential in vitro, but not the sex difference.

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Lectin bindings on the surface of unfertilized mouse ova immediately after ovulation were histochemically examined, and were compared among 30-day-old, 60- to 90-day-old, 180- to 210-day-old and 270-day-old mice. The bindings of PNA, GS-I, DBA, SBA, BPA, MPA, GS-II, WGA and Con A were observed from a weak to an intense degree on the cell surface of ova in mice from different age groups, but those of UEA-I and LPA were not. Although the strength in bindings of PNA, DBA, SBA, GS-II, BPA, WGA and Con A did not differ among the age groups, the strength of GS-I and MPA bindings intensified in 270-day-old mice. From the results, it may be said that the glycoconjugates on the cell surface of mouse ova immediately after ovulation contain galactose, N-acetylgalactosamine, N-acetylglucosamine and mannose, and that the amount of galactose and N-acetylgalactosamine increases as the animals get older.

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The necessity of cell contact during the third cell cycle for the formation of compacted mouse embryos at the 8-cell stage was examined. Early 4-cell embryos (0-2 h post division) were disaggregated into 4 blastomeres, and then reaggregated at the early, mid or late stage of the third cell cycle. The reaggregated embryos were cultured upto the 16-cell stage, and the features of compaction were observed. Embryos reaggregated at the mid or late stage of the third cell cycle, had their compaction retarded by 2 h as compared with control embryos. In embryos reaggregated at the mid or late stage, the establishment of gap junctional communication was also retarded. We concluded that cell contact from the early to the mid stage of the third cell cycle is necessary to regulate the events during compaction, especially for the establishment of the gap junction.

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Using calcium alginate as material for the artificial zona pellucida, the early embryonic development and the implantation of zona-free mouse eggs encapsulated with an artificial zona pellucida were investigated. In the group of two-cell embryos encapsulated with the artificial zona pellucida, 95.0% developed to the blastocyst. On the other hand, 93.1% in the zona intact group and 83.0% in the zona free group developed to the bla-stocyst. The rate of the embryo encapsulated with an artificial zona pellucida developing to the blastocyst was significantly higher than that in the non-capsulated zona-free embryo (P<0.05) and also exceeded that in the zona intact embryo. Twelve normal fetuses were obtained after the transfer of 75 blastocysts encapsulated with the artificial zona pellucida to recipients. These results indicates that calcium alginate used as an artificial zona pellucida is not detrimental to the development of the embryos but plays a protective role in the preimplantation embryo development, and the artificial zona pellucida dissolves timely in the uterus and dose not hinder implantation. If hardness of calcium alginate suitable for humans can be found, clinical application of IVF-ET with zona-free eggs making use of an artificial zona pellucida will become possible.

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Changes in intercellular communication and structural integrity between oocyte and cumulus cells during oocyte maturation were examined in mice injected with pregnant mare serum gonadotropin and human chorionic gonadotropin (hCG). Three and 9 hr after hCG administration, dissolution of the germinal vesicle (GVBD) and progression to the second metaphase occurred in oocytes in both cases. Examination of the expansion of mouse cumulus-oocyte complexes with oocytes having undergone GVBD by electron microscopy revealed the deposition of intercellular materials between cumulus cells and the retraction of the cytoplasmic processes joining the cumulus cells to the oocyte. Cumulus to oocyte coupling decreased progressively with time after hCG administration, as assessed by a progressive reduction in transfer of lucifer yellow from the oocyte to the cumulus cells. Spread of lucifer yellow from the oocyte having undergone GVBD was limited to very few adjacent cumulus cells, but junctional apparatuses were still identified in the area where the processes of cumulus cells contact the oolemma. These results show that coupling occurs between the oocyte having undergone GVBD and its surrounding cumulus cells but that during oocyte maturation, coupling decreases, while materials are deposited in space between cumulus cells.

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Golden hamster embryos were recovered from aged multiparous dams (15 to 16 months old), and young nulliparous females (3 to 4 months old) after mating with fertile males. The numbers of embryos and fetuses were counted at 1-cell, morula or blastocyst, and 8-day stages, respectively. The average number of normal eggs per dam was without significant difference, although slightly larger in young nulliparous females (young group) than in senescent multiparous females (aged group). However, the average number of anomalous eggs per dam increased significantly in the aged group. The average number of the embryos at morula or blastocyst stage per dam was significantly large in the young group. The average numbers of deciduae and fetuses, which were recovered from females at 8 days after mating, decreased significantly in the aged group. The frequency of appearance of mitotic metaphases in first-cleavage embryos decreased significantly in the aged group. These results indicate that the aging of dams causes delay of fertilization, or asynchronous development of embryos and degeneration of the resultant embryos through the embryo development. In senescent golden hamster females, large numbers of unfertilized eggs, "shell eggs" without cytoplasm, which were previously ovulated and then degenerated, were recovered. They were accumulated in oviducts or uterotubal junctions.

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The fatty acid content of bovine serum albumin fraction-V (BSA-V) was analysed and the effect of fatty acids bounded with BSA was evaluated. Gas-liquid chromatography was employed to analyse the fatty acids contents and culture experiments were conducted to evaluate the effect of fatty acids on 4-cell mouse and 8-cell rat embryo development. Linoleic acid was identified to be the major fatty acid (54.55%) bound with BSA, followed by oleic(25.8%), stearic (12.27%) and linolenic(7.36%) acids. BSA binding fatty acids had considerable effect on both mouse and rat embryo development in general but they provided only the rat embryo with energy. The combined effect of different kind of fatty acids (chemically defined lipid concentrate) was more effective in both mouse and rat embryo development.

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This study was designed to examine whether hyaluronic acid improved development of the electro-activated porcine diploid eggs. In-vitro matured oocytes were subjected to a single square pulse of direct current for 100 μsec at 1,500 V/cm for activation. The activated eggs were treated with 5 μg/ml cytochalasin B for 4 h. Eggs with only the first polar body that were judged as diploids were further cultured either in the modified KRB solution (mKRB) or Whitten's medium, each with or without addition of 0.5 mg/ml hyaluronic acid. The frequency of eggs developed to blastocysts was significantly higher in Whitten's medium (12% at 144 h and 14% at 168 h after electro-activation) than in mKRB (6% both at 144 h and 168 h) (P<0.05). The frequency of development to blastocysts increased to 13% in mKRB and to 28% in Whitten's medium by addition of hyaluronic acid when measured at 168 h. At this time, frequencies of abnormal eggs were lower in both media in the presence of hyaluronic acid. Expanded blastocysts were observed at 168 h but only when the medium included hyaluronic acid. There were no significant differences in the mean number of cells per blastocyst among the four media, and the number of cells per blastocyst ranged from 21 to 61 at 168 h.

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In vitro fertilized 8-16-cell stage embryos, co-cultured with cumulus cells for 117 hours, were divided into two groups. Embryos in one group (control group) were further co-cultured with cumulus cells or cultured in TCM199 supplemented with 10 μM β-mercaptoethanol (β-ME). Embryos in the other group were used as donor nuclei for nuclear transfer. The nuclear transferred eggs were co-cultured with cumulus cells to the 8-16-cell stage. They were divided into two; half of them were further co-cultured with cumulus cells and the others were cultured in TCM199 supplemented with β-ME to the bla-stocyst stage as control group. The proportions of embryos which developed to blastocysts in β-ME supplemented medium in both groups were not significantly different from those obtained in the co-culture system. However, the proportions of nuclear transferred eggs which developed to blastocysts in both groups were significantly different from those obtained in in vitro fertilized eggs.

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The present study was conducted to clarify changes of intermediate filaments, cytokeratin, vimentin and desmin, in primary, subcultured and cryopreserved bovine oviduct epithelial cells by subculture or cryopreservation and to assess their sustaining ability for the embryo development. Any morphological change of cells isolated from bovine oviducts and cultured in vitro was not observed up to the fifth generation, including cryopreserved cells. Result of indirect immunofluorescent staining study strongly suggested that the primary cells were epithelial cells because immunofluorescence to anti-cytokeratin antibody was detected but not observed anti-vimentin and anti-desmin antibody. From second generation, however, the reaction of vimentin was become to be detected, suggesting that the nature of the epithelial cells changed. Sustaining ability of epithelial cells was assessed by co-culture of bovine parthenogenetic embryos with the oviduct epithelial cells. The results showed that sustaining ability of primary and subcultured-cryopreserved bovine oviductal cells was equivalent in which 29.8% and 28.3% of the parthenogenones in each cases developed to blastocysts. These results suggested that the expression of vimentin in culturing bovine oviduct epithelial cells and their sustaining ability for bovine parthenogenetic embryos had no relation.

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Three expanded blastocysts were collected from the Japanese Black cow on the 7 day after insemination, and cryopreserved in LN₂. One of the frozen-thawed embryos contained two cell masses in the zona pellucida. The embryo was transfered to the recipient cow, and twin calves (females: birth weight, 13.0 kg, 14.0 kg) were born on the 261 day after transfer.

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