The oxidation and incorportion of [U-¹⁴C] glucose were examined in preimplantation rat embryos. An increasing capacity for incorporation and oxidation of glucose after the 1-cell stage was observed and the oxidative turnover of this substrate at the blastocyst stage was ten times more than that at the 1-cell stage. To evaluate how glucose is utilized for the synthesis of embryo lipids, 2-cell embryos and blastocysts were cultured for 5 h in medium containing [U-¹⁴C] glucose, and total lipids extracted from the embryo were separated into various neutral lipids and phospholipids by thin layer chromatography and radioactivities of these lipid fractions were measured. Most of the radioactivity was recovered in triacylglycerols in both stages, and radioactivities were also found in other neutral lipids and phospholipids. These results indicate that [U-¹⁴C] glucose was certainly utilized for oxidation and the synthesis of various lipids in embyros at the preimplamtation stage.

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The physiologic roles of the tubal endothelium and tubal secretions are as yet poorly understood. In this study, we examined the tubal surfactant-like materials (SF-phospholipids) found in the tubal endothelium and lumen in order to elucidate their roles in the maintenance and development of the zygote. Evaluation of tubal infertility based on the presence or absence of SF-phospholipids in human tubal endothelium and tubal secretions was carried out in order to determine the degree to which tubal function was intact. By using fluorescing phosphin E, we found fluorescence in both the endothelium and the tubal secretions throughout the entire length of the tubes. In electron microscopic observations of the tubal endothelium, we were able to identify high electron density, lamellar inclusion bodies, either scattered or clustered in the areas close to the tubal lumen, in both ciliated and secretory endothelial cells. These lamellar granules in the tubal secretions appear to be secreted by a merocrine process and are observed in the secretions as a dispersion. Tubes from the proliferatory phase and the secretory phase were evaluated with the following results. The constituents of the tubal secretions were found to be phospholipids and proteins. The phospholipids were most abundant at or around the time of ovulation. This material caused capacitation of sperm by inducing an acrosomal reaction in the hamster sperm penetration test.

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In this study we investigated the effects of gravity over 1 G on the distribution of cortical granules (CGs) in mouse oocytes. We had found eggs incubated in a centrifuge culture medium showing a high rate of polyspermy, but there had been no difference between 2 G and 3 G loads in the incidence of polyspermy. We focused on cortical granules (CGs) which are known to act as a protection against polyspermy. The CG-free area of mouse oocytes subjected to increased G, when compared to that of control oocytes, was found to be significantly larger. Therefore, it can be assumed that the increased G caused an abnormal CG distribution which resulted in a high incidence of polyspermy in oocytes subjected to increased G. Further, as the centrifugation force was increased, the CGs free area of oocytes did not tend to expand. Observation was facilitated by using FITC-LCA fluorescent dye and TEM.

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The present study was carried out to determine the time-dependent changes in the cortical granule (CG) distribution and the glycoprotein compositions of zona pellucida (ZP) after egg activation by electrostimulation in porcine oocytes matured in vitro. CG exocytosis was observed by staining with fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) and laser confocal microscopy, and ZP modification was analyzed by using enhanced chemiluminescent (ECL) detection of the biotinylated ZP derived from a single oocyte. In the oocytes matured in vitro, CGs staining with FITC-PNA had formed a continuous monolayer underlying the oolemma, and three major bands (ZP1, ZP2 and ZP3) were observed in a biotinylated ZP subjected to SDS-PAGE followed by ECL detection. Electrostimulation to induce artificial activation caused a decline in the fluorescent intensity of the CGs with a concomitant decrease in the amounts of ZP1 and ZP2 bands, but CG exocytosis according to oocyte activation occurred slowly, and the incidence of oocytes with complete CG exocytosis was first observed 1.0 h after electrostimulation (9%). Similarly, the complete ZP modification, i.e. a maximum decrease in amounts of ZP1 and ZP2 bands, required more than 3.5 h after artificial activation, and the ZP dissolution time caused by 0.1% protease action increased apparently as incubation time was prolonged after electrostimulation. Results of these experiments suggest that the slower ZP modification due to the delayed CG exocytosis in porcine oocytes may reflect the remarkable increase in polyspermy frequency in in-vitro fertilization.

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The reverse transcription-polymerase chain reaction (RT-PCR) method is widely used for studying mRNA expression in cells and tissues. In this study, we examined whether the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene could be used as an endogenous control for gene expression in single bovine oocytes and early embryos. First, sequencing of partial bovine G3PDH cDNA from ovarian tissue was performed. The sequence analysis of bovine G3PDH cDNA after subcloning indicated a high homology with human, mouse and rat G3PDH cDNA. Next, we examined whether G3PDH could be detected in single bovine oocytes and early embryos by using the RT-PCR method. Signals for G3PDH mRNA were detected in single immature and mature oocytes and single embryos at the one-cell to blastocyst stages. Thus, G3PDH is suitable as an endogenous control for examining mRNA expression even with single bovine oocytes or early embryos by using the RT-PCR method.

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The process of early development in diploid parthenogenetic mouse embryos from the 2-cell to the blastocyst stages was observed by time-lapse videomicrography, and was compared with that in fertilized embryos. Parthenogenetic 2-cell embryos cleaved and developed to 8-cell embryos after 23.0 hrs of culture. Transformation of blastomeres occurred at the 8-cell stage: namely, outer blastomeres were flattened. The embryos compacted at the morula stage, and then developed to blastocysts after 54.0 hrs of culture. A slit in the zona pellucida was formed 42.7 hrs after blastocyst formation, and trophectoderm cells protruded out of the zona pellucida through the slit. Protrusion of trophectoderm cells from the zona pellucida could arise from either side, polar trophectoderm or mural trophectoderm. After 7.0 hrs, the blastocysts completely escaped from the zona pellucida in either state, expansion or contraction. Fertilized 2-cell embryos showed morphological changes similar to those of parthenogenetic embryos and developed to blastocysts after 51.9 hrs of culture. Fertilized blastocysts took a significantly shorter time, 31.1 hrs, to start hatching, but required a significantly longer time, 23.8 hrs, to complete hatching, compared with parthenogenetic blastocysts. Hatching patterns of fertilized blastocysts were consistent with those of parthenogenetic blastocysts, except that hatching began with protrusion of trophectoderm cells from small holes in zonae pellucidae of fertilized blastocysts. From these results, it was confirmed that the developmental ability of early diploid parthenogenetic embryos prepared by the treatment with ethanol and cytochalasin B is comparable to that in fertilized embryos.

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The necessity of the germinal vesicle (GV) for the activation of MPF during porcine oocyte maturation was investigated. Porcine follicular oocytes were enucleated without damaging the oolemma, and the enucleated oocytes without culture or cultured for 30 h were examined for their protein synthesis and MPF activities. The protein synthesis pattern in enucleated oocytes changed between before and after 30 h of culture, and this change was exactly the same as that of intact oocytes between before and after GV breakdown. The MPF activity increased about 7 and 5 times after 30 h of culture in cumulus enclosed and denuded oocytes, respectively. The activation of MPF was also observed in the enucleated oocytes after 30 h of culture and the MPF activity was the same level as that of denuded oocytes. These results indicate that the presence of GV is not required for normal protein synthesis during porcine oocyte maturation and that the generally suggested nucleus-cytoplasmic interactions, cyclin B movement into GV and the mixing of karyoplasm with cytoplasm, are not required for the MPF activation.

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Bovine cumulus-enclosed oocytes were matured in vitro (IVM) in PVA-HEPES-TCM199 supplemented with 0, 0.5, 5, 50 or 500 μM cysteamine for 24 h and fertilized in vitro (IVF). After removal of cumulus cells, IVM-IVF oocytes were cultured in vitro for 8 days. Cysteamine had no effect on oocyte maturation or fertilization rates. In contrast, 5 μM cysteamine in the maturation medium enhanced subsequent in-vitro development of IVM-IVF oocytes to the blastocyst stage. Glutathione (GSH) content of oocytes cultured in medium with 0 or 0.5 μM cysteamine was significantly lower than that of oocytes before culture. When oocytes were cultured in medium with 5, 50 or 500 μM cysteamine, GSH content of oocytes remained constant (5 μM) or was significantly increased (50 or 500 μM) compared with that of oocytes before culture. These results indicate that the addition of cysteamine to a serum-free maturation medium enhances the efficiency of in vitro production of bovine embryos by maintaining GSH content of IVM oocytes.

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The aim of this study was to investigate basically, using the gametes of golden hamster, whether acridine orange (AO) fluorescent dye was efficient for detecting the sperm-egg fusion in vitro, and to evaluate the usefulness of this dye by comparison with other staining methods such as toluidine blue and Giemsa. Before insemination with zona-free hamster mature oocytes (metapase-II), the nuclei of acrosome reacted spermatozoa collected from the cauda epididymis emitted bright green AO fluorescence. Chronologically sperm nuclei changed AO fluorescence from green to red before it began to decondense within the ooplasm. Condensed nuclei attached to the oolemma of GV oocytes and pronuclear stage eggs, which were thought to be fused, were stained red 1 hour after insemination. On the other hand, although the spermatozoa incubated with zona-free, metaphase-II oocytes under calcium-magnesium free conditions had condense shaped nuclei after 1 hour insemination, the AO stained nuclei were completely green. The AO staining indicates the thiol-disulfide status of sperm nuclei, namely, green and red fluorescences mean S-S rich and S-S poor, respectively. Therefore, since nucleoproteins of the sperm nucleus should be reduced after being mixed with the ooplasma during the sperm-egg fusion event, the condense shaped and red nuclei are considered fused with oolemma before nuclear decondensation occurred. The AO fluorescence dye was proven to be effective compared with other staining methods reported previously, and AO staining will be useful for a precise and efficient means of detecting and investigating sperm-egg fusion events in mammals.

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Ovaries from 4-day-old mice were organ-cultured and the effects of progesterone, androstenedione and estradiol-17β on early follicular development were investigated. Both androstenedione (40-1,000 ng/ml) and estradiol-17β (8-1,000 ng/ml) promoted proliferation of granulosa cells in developing follicles, while progesterone showed no remarkable effect. Cyproterone acetate (4,000 ng/ml), an androgen receptor antagonist, partially inhibited the granulosa cell proliferation induced by androstenedione (40 ng/ml). Dihydrotestosterone (8-1,000 ng/ml), which is not converted to estrogens, also induced proliferation of granulosa cells. These results suggest that androgens directly promote proliferation of granulosa cells in early developing mouse follicles in vitro.

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The purpose of the present study was to try to improve the western blotting method in order to reduce the number of sampling ova for protein detection. We established a small-scale western blotting system, named "micro-western blotting", in which the width of each lane was 1 mm and 2 μl sample was applied to each lane in SDS-PAGE. In this method, only 4 porcine ova were required to detect and analyze the phosphorylation states of p34^cdc2, a catalytic subunit of maturation promoting factor, and two major components of the MAP kinase cascade, ERK and MEK. The number of ova required for protein detection in this method was about one tenth of that required for the normal-scale method and the resolution qualities were comparable with the normal-scale method. The present system might be useful for analyzing biological molecules in small and rare materials such as mammalian oocytes and embryos.

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