The formation of junctional complexes and the location of actin and cytokeratin were examined in bovine embryos which were being transformed in vitro into blastocysts. In 16-cell embryos and morulae, gap junctions were observed between some pairs of blastomeres, though not very often. In early blastocysts, however, three kinds of junctions, Le., zonula occludens, predesmosomes and gap junctions were found between each pair of trophoblast cells. Gap junctions were seen between a pair of inner-cell-mass cells, and between a trophoblast cell and an inner-cell-mass cell, also. In expanded blastocysts, zonula ocdudens, zonula adherens, desmosomes and gap junctions were found between each pair of trophoblast cells, while predesmosomes and gap junctions were found between each pair of inner-cell-mass cells, and between a trophoblast cell and an inner-cellmass cell. Meanwhile, actin and cytokeratin fluorescences were observed throughout the cytoplasm of embryos at the 16-cell through blastocyst stages, and they were more prevalent in the ectoplasmic zone of trophoblast cells in blastocysts.

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Cynomolgus monkey oocytes were collected from the ovarian follicles of females that died from accidents, diseases of respiratory and digestive organs, or sacrificed for the other experiment. Immature oocytes were collected from 10 females aged 1 to 18 years except 1 female aged 19 days and they were cultured for 24 h in TCM 199 medium supplemented with 10 % FCS and 10 IU/ml PMSG. The 11.9 % oocytes extruded the first polar body and some of them were further used for in vitro fertilization. In two cases, we could observe the presence of male and female pronudei and the second polar body. The ovaries were histlogically different with regard to the number of primordial follicles and the vesicular follicles between sexually immature and mature females. This study suggests that the ovarian oocytes collected from dead animals are useful for the embryological studies in nonhuman primates, and this procedure is applicable to gene banking for individuals died accidentally.

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Ovaries excised from 4-day-old mice were cultured for 20 days in the absence of gonadotrophins. In the ovaries, there were a great number of non-growing oocytes (10-19 μm in diameter) and a group of growing oocytes (20-39 μm). After organ culture, although most oocytes remained in the non-growing pool, a very low percentage of oocytes increased their diameter to 60-75 μm. Those oocytes grown in culture were enclosed with 1-3 layers of granulosa cells. When the oocytes were further cultured for 24 h after isolation from granulosa cells, 1, 9 and 47% of oocytes of 60-64, 65-69 and over 70 μm in diameter underwent germinal vesicle breakdown, respectively. Oocytes smaller than 60 μm failed to resume meiosis. Extrusion of a first polar body was observed in 11% of oocytes larger than 70 μm. Oocyte degeneration, however, was observed frequently during both organ culture and maturation culture.

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Two experiments using pig oocyte-cumulus-granulosa cell complexes dissected from healthy follicles of 4-6 mm in diameter were conducted in this study. In the first experiment, nuclear change of oocytes during maturation culture was examined and the appropriate timing of insemination was determined. In the second experiment, the effects of the time of co-culture with spermatozoa after insemination on fertilization and subsequent early cleavage were examined. Oocytes reached the metaphase II between 30 and 33 h. When oocytes cultured for 30, 36, 42, 48 and 54 h were inseminated by ejaculated spermatozoa, no differences were detected in the proportions of penetrated oocytes in these groups. In contrast, oocytes cultured for 36 and 42 h formed male pronucleus (ei) at significantly higher percentages than the other groups. In oocytes cultured for 36 h and inseminated, sperm penetration and male pronuclear formation were first observed at 9 h after insemination. Oocytes co-cultured with spermatozoa for 9 and 12 h showed higher rates of normal cleavage than oocytes co-cultured for 3 and 6 h. However, the rate of embryos reaching the 4-cell stage or beyond was higher for oocytes co-cultured for 9 h than 12 h. It is concluded from these results that the proportion of normal fertilization of pig follicular oocytes is highest when oocytes are matured for 36 h in vitro and subsequently co-cultured with spermatozoa for 9 h under the culture condition of the present study.

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Rat blastocysts were cryopreserved by vitrification with a simple and rapid method using ethylene glycol (EG)-based solution which was mixed with different concentrations of polyethylene glyco1 (PEG)(5-15%) and sucrose (0.25-1.OM). Effect of time (2-10min) and temperature (on ice or at room temperature) for equilibration on the in vitro development of vitrified-warmed embryos were investigated.Successful vitrification was obtained by the equilibration for 5 min on ice (3-4C). More than eighty percent of the vitrified-warmed embryos developed to the stages of expanded or hatched blastocyst when the embryos were equilibrated on ice for 5 min in the solution containing40%EG, 5%PEG and 0.5M sucrose.The solution containing sucrose (0.5M) was not effective for recovering the vitrified-warmed embryos. About 30% of the vitrified-warmed embryos developed to live fetus after transfer to recipient female rats. The present results indicate that even a simple and rapid method could be successful for the vitrification of rat blastocysts using the solution containing EG, PEG and sucrose.

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Effects of leukemia inhibitory factor (LIF) on the developmental ability of mouse 8-cell embryos in vitro and in vivo were examined.(1) Eight-cell embryos were cultured with M16+LIF medium in vitro to examine the development to blastocysts and the attachment to culture dish.(2) Blastocysts were transferred to recipient mice following the injection of LIF into the uterine lumen to examine the number of live youngs on Day 17. The results obtained were as follows.(1) When embryos were cultured in a medium supplemented with LIF, the proportion of embryos developed to hatched blastocysts was increased. Blastocysts cultured in the presence of LIF and 10% FCS attached to the surface of the culture dish at a significantly higher frequency than control blastocysts (42vs71-90%).(2) The proportion of embryos that developed into live young was significantly increased, when the LIF was directly injected into the uterine lumen, compared with the control group (15 vs 35%).

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Development of chimeric mouse embryos cultured in a Brinster's medium and a Dulbecco's modified Eagle medium (DMEM) was examined in an attempt to produce chimeric mouse using 8-16-cell stage aggregated embryos. Growth of chimeric mouse embryos cultured in a Brinster's medium or DMEM in vitro was slower than that of normal embryos in vivo. Both Brinster's Medium and DMEM were slightly modified by addition of glucose (0.2% concentration). When modified Brinster's medium and modified DMEM were used to culture aggregated chimeric embryos, growth of aggregated chimeric embryos were faster than aggregated chimeric embryos cultured in several basic media that containing 0.1% glucose. Furthermore, modified Brinster's medium and modified DMEM were incubated with ovaries for 2 hour in advance. When modified and conditioned Brinster's medium and modified and conditioned DMEM were used to culture aggregated chimeric embryos, growth of aggregated chimeric embryos were faster than aggregated chimeric embryos cultured in several modified and not conditioned media that containing 0.2% glucose. Transfer of aggregated chimeric embryos cultured in modified and conditioned Brinster's medium to recipients resulted in live pups.

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Mouse eggs, freed from cumulus cells and zona pellucida, were inseminated at various sperm concentrations in vitro. The fertilization rates of these eggs were in direct proportion to the logarithm of sperm concentration. Sperm concentration for 50% fertilization rate in the zona-free eggs was about 1/25 of that for cumulus-free eggs. Frequency of the number of penetrating sperm in zona-free eggs closely fitted Poisson distribution at low sperm concentration (≤0.9 calls/μl) suggesting that the fertilization was dependent on the random collision of an egg and a fertile spermatozoon. At higher sperm concentrations, however, incidence of monospermic fertilization was significantly higher than that expected from Poisson distribution, showing the vitelline block in these zona-free eggs. When zona-free eggs were coincubated with sperm for restricted periods, sperm attachment started in almost all eggs with 30 sec and eggs with binding sperm reached 87%at 3 min. But these binding sperm were not able to penetrate into cytoplasm immediately and sperm exposure time for more than 30 min was requred to obtain high (>90%) fertilization rate. Increase in total sperm number in the fertilization medium resulted in a significant increase in the fertilization rate at a fix sperm concentration (10 sperms/μl). These result indicate that in vitro frtilization of zona-free mouse eggs is dependent on sperm: egg ratio in the fertilization medium.

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Production of chimeras between embryonic stem (ES) cells and zone-free embryos was examined in mice under coculture conditions. A3-1 ES cells were cocultured at concentrations of 0.5×10⁶ or 1.0×10⁶ cells/ml with zona-free embryos of 8-cell or morula stage in Dulbecco's modified Eagle's medium containing 5% fetal calf serum (FCS) and 23mM lactate (DMEM) or Whitten's medium containing 10% FCS (WM). Three hours later the cocultured embryos were transferred into WM and cultured again for 22-24 hr. The embryos developed to blastocyst stage were transferred into foster mother, and 17 days later the pups including chimeras were obtained. When morula stage embryos were cocultured with ES cells at a concentration of 0.5×10⁶ cells/ml in DMEM, the highest rate of production of chimeric mice (5.4%) was obtained. These our results indicate that the coculture method is useful for the production of chimeras between A3-1 ES cells and zona-free morula embryos.

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