The developmental ability of diploid/tetraploid mosaic mouse embryos produced by electrofusion at the 4-cell stage was examined in vitro and in vivo. Diploid/tetraploid mosaic embryos amounted to 51.3% of embryos treated. They were cultured for 60 h in BMOC-III medium to examine their development, and 77.8% of them developed to blastocysts. This was about the same rate as in unfused diploid embryos. Compaction of the diploid/tetraploid mosaic embryos started at the 6-cell stage, and the mean number of cells in these blastocysts was about three quarters that in control embryos. These embryos which developed to blastocysts were transferred to the uterus of recipients. By 11.5 days of pregnancy, 90.0% of diploid/tetraploid mosaic embryos had been implanted and 25.9% of them had developed into foe-tuses, but in these foetuses, however, there were no tetraploid cells, and they contributed to only some foetal membranes in 3 out of 9 embryos examined. On the 19th day of pregnancy, two recipients were delivered of seven pups altogether. These pups were morphologically normal and grew to be adult mice. Chromosome preparation of the bone marrow cells showed that all of these mice had only diploid chromosome constitutions. These results suggest that tetraploid cells show poor developmental ability after implantation.

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Three methods for subcloning embryonic stem (ES) cells, namely the isolation, precipitation and colony pickup method, were examined as follows. Isolation method: ES cells were trypsinized and classified morphologically as spherical and unspherical cells. Each type of ES cell was isolated with a micropipette and micromanipulator and was cultured separately. Precipitation method: ES cells were trypsinized and incubated for 15-45 min. Both the adhesive and floating cells were cultured separately. Colony pickup method: morphologically undifferentiated ES cell colonies were picked up with a micropipette. The colony was trypsinized and replated on a new feeder layer. Before the subcloning, there were 30-48% of normal diploid sets of 40 chromosomes in ES cells. Both the isolation and precipitation methods have not shown the effect of subcloning on the karyotype. The most effective subcloning method in this study was the colony pickup method. All of three sublines derived by the colony pickup method showed 68-78% of normal karyotypes and simple embryoid and cystic embryoid bodies were formed 3-4 days and 7-8 days after suspension culture, respectively.

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Fertile spermatozoa can be obtained from cauda epididymis after short term preservation at 4°C in paraffin oil. We examined the preservability of fertility under these conditions. Of ICR oocytes, 89% and 69% were fertilized in vitro with ICR and C57BL/6J spermatozoa from cauda epididymis stored in paraffin oil at 4°C for 24 h, respectively. Almost all of these fertilized oocytes developed to the blastocyst stage. The fertility of spermatozoa decreased noticeably with prolonged storage. From the stock stored for 96 h, the percentage of fertilized oocytes from ICR and C57BL/6J mice was only 13% and 1%, respectively. C57BL/6J zygotes which fertilized with either H or F gene disrupted spermatozoa previously stored for 16 h were implanted into individual oviducts. Fifty percent and 41% of these transferred zygotes developed into young, respectively. This study shows that mouse spermatozoa can be simply stored for 24 h within the intact cauda epididymis maintained at 4°C in paraffin oil; this procedure can facilitate the transportation of spermatozoa for this length of time.

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In previous studies we found that pig zona pellucida glycoprotein pZP1 may act as the secondary sperm receptor during penetration into the zona pellucida. In this study, a recombinant pZP1(1-198) protein (r-pZP1) was produced to examine the direct binding of pZP1 to spermatozoa. When boar spermatozoa were co-cultured with pig cumulus oophorus in TC199 medium containing r-pZP1 proteins, r-pZP1 bound to the spermatozoa invaded the cumulus oohorus. The binding site was in the acrosomal region, but no sperm binding of r-pZP1 was observed when they were incubated in the medium without cumulus oophorus. This suggests that the cumulus cells played a role in exposing the ligand molecules for r-pZP1 in the acrosomal region. When boar spermatozoa were incubated with pig cumulus-oocyte complex in the medium containing r-pZP1, r-pZP1 bound to the spermatozoa adhered to the zona pellucida in the postnuclear region and the tail. r-pZP1 also bound to human spermatozoa pretreated with Ca⁺⁺ ionophore A23187 and the binding site migrated from the equatorial region to the tail during incubation. Western blot analysis with crude extracts of boar spermatozoa revealed that three molecules, Mr 55K, Mr 40K and Mr 25K, reacted to r-pZP1.

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Distributional changes in lysosome-like bodies (LBs) in mouse, rat, rabbit and bovine embryos during the course of blastocyst formation were studied with acridine orange (AO) staining. AO-stained LBs were distributed throughout the cytoplasm of all round blastomeres in untransformed 8- and 16-cell mouse embryos, 32-cell rabbit embryos and 16-cell bovine embryos, whereas they were observed from the nucleus to the apical cytoplasm of all round blastomeres in 8-cell rat embryos. In morulae of these animals, LBs were present in the entire cytoplasm of inner round blastomeres, and were localized around the nucleus in outer flattened (mouse) and cuboidal (rabbit and cow) blastomeres or the apical cytoplasm in outer flattened (rat) blastomeres.In blastocysts, LBs were present in the entire cytoplasm of inner-cell-mass cells and around the nucleus of trophoblast cells. Such distribution of LBs in mouse embryos was also observed in those developed in vitro. From these results, it was suggested that there is a close relationship between distributional changes in LBs and the transformation and differentiation of blastomeres in mouse, rabbit and bovine embryos.

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The effect of the nuclear stage during in vitro maturation (IVM) on the fertilizing ability of frozen-thawed bovine oocytes was investigated. Oocytes with compact cumulus cells were cultured for 0, 6, 12 and 24 h, and were subjected to 2-step freezing in the presence of 10% ethylene glycol and 0.1 M sucrose. After thawing, the oocytes were additionally cultured to give 24 h for a total IVM period, and were inseminated with 3.5 to 5.0 × 10⁶ cells/ml frozen-thawed spermatozoa for 20 h in TALP solution containing 20 μg/ml heparin. In vitro fertilization rates of oocytes frozen-thawed at 0, 6, 12 and 24 h of IVM were 40, 47, 49 and 54%, respectively, all of which were significantly lower than that of nonfrozen control oocytes (77%). A higher frequency of polyspermic fertilization was observed in the oocytes frozen-thawed at 24 h of IVM (53 vs. 18% in nonfrozen control oocytes). Lowering the sperm concentration during insemination from 5.0 × 10⁶ cells/ml to 1.0 × 10⁵ cells/ml did not decrease the rate of polyspermic fertilization in the frozen-thawed oocytes.

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To examine the sensitivity of mammalian oocytes and embryos to osmotic shrinkage, which can occur during the process of removal of cryoprotectant from cryopreserved cells, the effect of shrinkage on the survival of fresh and vitrified mouse oocytes at metaphase II and embryos at 1-cell to blastocyst was examined. Oocytes and embryos were suspended in PBS media containing various concentrations of sucrose for 30 min at 25°C. They were then returned to isotonic PBS, and the survival was assessed in vitro. Fresh oocytes and embryos were almost entirely insensitive to shrinkage in a solution with 0.75 M sucrose, but in solutions with 1.0-1.5 M sucrose, oocytes and 8-cell embryos were more sensitive to the hypertonic stresses, whereas 2-cell embryos and expanded blastocysts were less sensitive. Vitrified embryos were more sensitive to hypertonic stresses than were fresh ones, but the sensitivity was reduced when the embryos had been cultured for a short period before subjecting them to the hypertonic stress.

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We examined the ultrastructural features of secretion by the murine oviductal epithelia. In ampullae, mucus material accumulated in the apical parts of the secretory cells in proestrus, estrus and metestrus. In estrus and metestrus, numerous secretory granules of various sizes were seen. Fusion of secretory granules and the plasma membrane, i.e. exocytosis, was observed in metestrus. In diestrus, however, the secretory cells were atrophied. Secretory products from the ampullar epithelium may support fertilization and embryonic development in early stages. At the isthmuses, largely-expanded rough endoplasmic reticulum was seen in metestrus. Secretory granules were observed in proestrus and estrus, and these were released by exocytosis especially in proestrus. The material contained in these granules may play an important role in sperm capacitation. The present results indicate that the secretory cells of ampullae and isthmuses are regulated differentially by the estrous cycle dependent factors in mice.

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The effects of activators of protein kinase C (PKC) on the development of mouse late morulae were examined by morphological techniques. Late morulae were cultured with low levels of the PKC activators phorbol 12-myristate 13-acetate (PMA; 0.01-1 nM) and 1-oleoyl 2-acetyl-sn-glecerol (OAG; 0.1-10 μM) for 24 h, their blastulation ratios were determined and morphological changes in intracellular distribution of actin filaments were examined. PMA treatment induced the decompaction of morulae and delayed blastocoel formation. OAG treatment induced delayed embryonic development. Both PMA and OAG treatment induced reorganization of actin filaments in late morulae. It is assumed that PKC activation induces reversible arrest of embryonic morphogenesis in mouse late morulae.

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Immature and in vitro matured bovine oocytes were cryopreserved in a solution containing propanediol as a cryoprotectant to compare their developmental ability following the treatment. After cryopreservation, the oocytes were inseminated with frozen-thawed bovine spermatozoa and cultured in vitro for a given period of time to determine the developmental capacity of the embryos. The average rates for cleavage, which were measured 2 days after in vitro fertilization (IVF), were 71.2% for freshly collected immature oocytes, 3.4% for frozen-thawed immature oocytes and 6.8% for cryopreserved in vitro matured oocytes, respectively, though none of the cryopreserved oocytes developed to the blastocyst stage. Following cryogenic storage, the oocytes were processed for transmission electron micro-scopy (TEM). Freezing and thawing of the immature oocytes induced remarkable ultrastructural changes in oolemma, microvilli, mitochondria and vesicles in oo-plasm and cumulus cells surrounding the oocytes, whereas the integrity of cell organelles was relatively better preserved in in vitro matured oocytes. The present results suggest that the cryogenic storage of bovine oocytes, both immature and in vitro matured, induced various kinds of ultrastructural damage which was associated with the low developmental capacity of post freeze-thaw oocytes. On the other hand, in vitro matured oocytes were considered to maintain their structural integrity better than immature oocytes following cryopreservation with propanediol.

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Four hundred fifty-one oocytes were recovered from 115 Holstein cows with ovarian cyst(s) and 159 oocytes were recovered from 36 noncystic normal Holstein cows, respectively. The oocytes recovered from cystic cows were classified as healthy or degenerative by cumulus investment and by nucleus configuration, as well. The relative growth relating follicle diameter to the oocyte diameter in each classification was examined by using regression models. The best fitting models in each group were the hyperbolic regressions with R² values of 0.994 to 0.999. The growth rates of the oocyte calculated from the differentiated equations of the hyperbolic equations were found to be an asymptotic depression into zero less than 0.5 μm since the follicle grew more than 6.0 mm in diameter. The linear regressions for the growth of the follicle more than 6.01 mm in diameter in relation to that of the oocyte in each abovementioned group all indicated that the true slopes (β)=0 lay somewhere in the 95% confidence intervals of these regressions by the statistical analyses. These results corroborated that the oocytes did not grow but the follicles grew more than 6.0 mm in diameter in both normal and cystic cows. There was no difference between the growth patterns in cystic cows from which healthy and degenerative oocytes were recovered. This was regardless of the judgment by either cumulus investment or nucleus configuration.

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In the present study, preimplantation development of chimeric mouse embryos cultured in Dulbecco's Modified Eagle Medium (DMEM) was examined by using 4 to 16-cell stage aggregated embryos. Three types of embryos, embryos with intact zona, zona-free embryos and aggregated chimeric embryos (BALB/c⇔C57BL/6J), were cultured for 60 h in vitro. Aggregated embryos, 4-cell⇔4-cell, 8-cell⇔8-cell and 16-cell⇔16cell developed into an integrated blastocyst at percentages of 82.2, 89.1 and 84.7, respectively. Development of chimeric embryos into morula was delayed about 12 h more than that of intact embryos, but high rates of developmental of chimeric embryos from morula into blastocyst were obtained. These results suggested that DMEM was suitable for the in vitro culture of chimeric mouse embryos.

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