This research was designed to investigate the effects of luteinizing hormone (LH, 100 ng/ml) on pig oocyte maturation in vitro (Experiment 1), and the relationship between the gap junction and LH (Experiment 2). In Experiment 1, cumulus-enclosed oocytes (CO) and denuded oocytes (DO) were cultured in the presence or absence of LH. After culture for 42-43 hours, nuclear maturation rates which reached the metaphase stage of the second meiotic division (M-II stage) were observed. In Experiment 2, we evaluated whether the gap junction, formed between cumulus cells and oocytes, was associated with the presence of LH in the maturation medium. The addition of LH to the maturation medium significantly enhanced nuclear maturation of both CO and DO (P<0.05) compared with those oocytes which were cultured without LH. The addition of 1-heptanol (5 mM), a blocker of coupling in the gap junction, to the maturation medium supplemented with LH significantly inhibited (P<0.05) the number of CO and DO reaching the M-II stage. These results suggest that the addition of LH to the maturation medium could improve the nuclear maturation of pig oocytes.

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Experiments were conducted to determine the effects of leukemia inhibitory factor (LIF), stem cell factor (SF), and basic fibroblast growth factor (bFGF) on the proliferation of undifferentiated primitive ectodermal cells isolated from bovine inner cell masses (ICM). ICMs of bovine hatched blastocysts produced at 10 days post-insemination (dpi) were immunosurgically isolated. Either one or three ICMs were cultured on mouse embryonic fibroblasts (MEF) in a 100 μl droplet of control medium with or without several combinations of murine recombinant LIF (1,000 units/ml), human recombinant SF (40 ng/ml), and human recombinant bFGF (40 ng/ml) as follows: 1) LIF, 2) SF, 3) bFGF, 4) LIF+SF, 5) LIF+bFGF, 6) SF+bFGF and 7) LIF+SF+bFGF at 37°C under 5% CO₂ in air. Attachment of ICMs to a feeder layer and proliferation of ectodermal cells were observed daily. After 6 to 7 days of culture, the primary ectodermal cells outgrown from ICM clumps were mechanically isolated with a 26G needle and mechanically disaggregated by pipetting through a fine pipette to make small clumps of cells. The primary ectodermal cell clumps were cultured and serially passaged every 6 to 7 days. The attachment of isolated ICMs started 1 day after culture in each treatment, and the rates of attachment ranged from 74 to 94%. The rates of ectodermal cells outgrowing from 1 ICM and 3 ICMs (37 and 56% respectively) were higher in the LIF+SF containing medium than those in the control medium and bFGF containing medium (16 and 17%; 22 and 22% respectively). When primary ectodermal cells that had been isolated from ICMs were cultured in the LIF+SF containing medium, the undifferentiated ectodermal cells were serially passaged (6 passages) during 2 months in culture. The results of this study demonstrate that 1) groups of 3 ICMs gave superior results to single ICM for the formation of ectodermal cell colonies from the ICM clumps and 2) LIF and SF are important factors in supporting the primary outgrowth and serial passage of ectodermal cells, as well as for maintaining an undifferentiated state as determined by a histochemical staining of alkaline phosphatase.

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The distribution of cortical granules (CGs) during in vitro maturation of porcine oocytes was investigated with a confocal laser microscope. The incidence of exocytosis of CGs in oocytes inseminated at various maturation stages was also investigated to determine the dependency of the block of polyspermy on the zona reaction in porcine oocytes matured in vitro. CGs were synthesized but not yet transported beneath the oolemma at 18 h of culture (GVBD stage). CGs transported beneath the oolemma were found at 30 h of culture (at stages metaphase I to anaphase I). From 24 h to 36 h of culture, when a dramatic change in the pattern of distribution of CGs was observed, oocytes were inseminated. Although there was no difference among the groups in the rate of oocytes penetrated, the rate of polyspermy decreased as the culture period of the oocytes was prolonged to more than 30 h. It can be concluded from the results that the oocytes in which cortical granules have been transported beneath the oolemma can react to some degree against excessive penetration of spermatozoa, but polyspermy could not be avoided completely. This may reflect the incomplete amount of CGs synthesized during culture or the incompleteness of exocytosis of CGs in the oocytes matured in vitro.

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The purposes of this study were to determine the optimal conditions for inducing parthenogenetic activation by strontium and to evaluate the developmental ability of the activated oocytes in mice. MII oocytes collected from B6CBF1 and CD-1 mice were cultured for 2-60 min in medium containing 1.7 mM strontium. The proportion of oocytes activated increased in a time-dependent manner, with an apparent difference between the B6CBF1 and CD-1 strains in sensitivity to strontium. In oocytes derived from B6CBF1 mice more than 90% of oocytes were activated by the treatment with strontium for 30 min, whereas more than 80% of the oocytes from CD-1 mice were activated by the treatment for 5 min. The majority of oocytes formed the second polar body and a single pronucleus in both cases. Of the parthenogenetically activated 1-cell embryos with the haploid genome, around 39% developed to the blastocyst stage in the B6CBF1, and in contrast the developmental ability was low (11%) in the CD-1 strain. The ability to develop to blastocysts was significantly improved in diploid parthenogenetic embryos of both the strains; 93% and 58% were developed to the blastocyst stage, respectively. After transfer of the diploid parthenogenetic embryos to recipient mice, 11% of the embryos developed to day 10 of gestation. The record of intracytoplasmic Ca²⁺ concentrations showed that the exposure of oocytes to medium containing 1.7 mM strontium induced repetitive intracellular Ca²⁺ transients. These data clearly showed that strontium is a potent stimulus for inducing parthenogenetic activation and supporting in vitro and in vivo development in mouse oocytes.

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The present study was conducted to examine the effect of repetitive electrical pulses applied to recipient ooplasts and donor blastomeres on the development of nuclear transferred bovine embryos. A series of repetitive electrical pulses at 30 min. intervals improved the rates of activated oocytes and the pronuclear formation in oocytes matured in vitro. After fusion with a donor nucleus, more than 20% of the reconstituted embryos developed to the blastocyst stage in vitro. Additional electrical pulses before fusion to the donor blastomeres and recipient ooplasts significantly enhanced the percentage of reconstituted embryos developing to blastocysts (38%). These results suggest that a series of repetitive electrical pulses can clearly induce suitable oocyte activation and enhance the development of nuclear transferred bovine embryos.

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The purpose of the present study was to evaluate the therapeutic effect of intrauterine chitin suspension on endometritis in cows. In experiment 1, 50 ml chitin suspension (60 and 80 mg/ml in saline), definitely induced estrus. Therefore, 50 ml of the 60 mg/ml suspension was injected into the uteri of Japanese Black cows (3-5 years old, average weight 530 kg) with a normal estrus cycle or endometritis in further experiments. In experiment 2, 21 parous Japanese Black cows with a normal estrus cycle received chitin on day 8-12 of estrus (the day on which estrus occurred was designated day 0). Nineteen of the 21 (90.5%) came into estrus 5-8 days after administration, and 3 control cows receiving saline solution came into estrus 13 days after administration (21 days after the preceding estrus). Progesterone levels in 3 randomly selected cows were reduced from 6.3, 6.3 and 2.6 ng/ml on the day of chitin administration to 0.3, 0.2 and 0.2 ng/ml, respectively, 6 days after administration. In addition, many leucocytes were detected in uterine mucosal biopsy specimens 2 to 3 days after chitin administration. In experiment 3, estrus was induced in 5 cows with endometritis 6-8 days after chitin administration and the endometritis was improved with the disappearance of pus. Normal embryos were obtained in 2 of 4 cows (8/12 ova; 75%) that received superovulatory treatment during the estrus cycle induced by chitin. These findings suggest that the intrauterine injection of chitin can induce estrus and subsequent ovulatory response by its therapeutic effect on endometritis in cattle.

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Pig early antral follicles (0.5-1.0 mm in diameter) containing oocytes approximately 100 μm in diameter were collected from ovaries. The intact early antral follicles, oocyte-cumulus-granulosa cell complexes (OCGs), and oocyte-cumulus complexes (OCs) dissected from the follicles were embedded in collagen gels, cultured in Waymouth's medium containing 5% fetal calf serum and 0.05 mg/ml sodium pyruvate for 8 days, and the oocyte morphology was examined. During the culture, follicular cells grew out of the complexes and spread into the collagen gels. All of the recovered oocytes had been detached from cumulus cells in the complexes after the culture. Although no significant increase in diameter was observed, 7% of the oocytes showed normal morphology in the cultured follicles, as did 53% in the OCGs and 47% in the OCs. Before culturing, oocytes from early antral follicles had decondensed filamentous chromatin or thicker stringy chromatin distributed throughout the germinal vesicle (GV0 stage). In the cultured follicles, OCGs and OCs, 0% (0/42), 16% (7/43) and 12% (5/43) of the oocytes, respectively, were at the GVI-MI (the first metaphase) stage, and 2% (1/42), 21% (9/43) and 10% (4/43) of the oocytes, respectively, formed female pronuclei. These results suggest that 37% (16/43) and 21% (9/43) of oocytes in OCGs and OCs, respectively, survived the 8-day culture and advanced their meiotic stage.

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A square case containing ova and culture medium was vibrated with a voice coil motor to concentrate the ova at the center of the case. Only those ova which conform to the memorized shape are then selected with a picture processing device and are positioned one by one at the point of a holding pipette. A system with the functions mentioned was experimentally constructed. This system can automatically position any ovum in a certain position, thereby shortening the time of the process to search out an ovum and position it at the point of the holding pipette.

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The formation of junctional complexes and the location of actin and cytokeratin were morphologically examined in parthenogenetic mouse embryos during the course of blastocyst formation. Although there was no junctional complex in untransformed 8- and 16-cell embryos, zonula occludens, predesmosomes and gap junctions appeared between each pair of flattened blastomeres of transformed 8- and 16-cell embryos. In these transformed embryos, gap junctions were observed between every two round blastomeres as well as between a flattened blastomere and a round one. Desmosomes also appeared between some pairs of blastomeres in morulae. In blastocysts, zonula occludens, zonula adherens, desmosomes, predesmosomes and gap junctions existed between each pair of trophoblast cells, and predesmosomes and gap junctions existed between every two inner-cell-mass cells and between a trophoblast cell and an inner-cell-mass cell. The kinds and locations of junctional complexes observed in parthenogenetic embryos and the developmental stages of their appearance did not differ from those of fertilized embryos. The location of actin and cytokeratin in parthenogenetic embryos was also similar to that in fertilized embryos, whereas in parthenogenetic embryos at the morula and blastocyst stages, there existed a small number of blastomeres or cells devoid of these proteins. From these results, it was inferred that the cell-binding in parthenogenetic embryos is comparable to that of fertilized embryos.

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This study was carried out to examine whether EGF, IGF-I and insulin could directly modify the meiotic maturation of bovine denuded oocytes in vitro, and to see whether the stimulatory effect of EGF on bovine oocyte meiotic maturation is mediated through the tyrosine kinase pathway. In the first three experiments, cumulus-oocyte complexes (COCs) were removed from their cumulus cells and the denuded oocytes were cultured in vitro with various concentrations of EGF, IGF-I and insulin. Nuclear maturation was examined after 24 h of culture. EGF, IGF-I as well as insulin effectively increased the proportion of oocytes developed to the metaphase II stage. The higher rates of MII oocytes were achieved with 50 ng/ml EGF (69.4%), 100 ng/ml IGF-I (66.1%) or 5 μg/ml insulin (63.1%) compared to about 48 to 54% in the controls. In experiment 4, whereas EGF significantly (p<0.05) increased the proportion of MII oocytes for denuded oocytes derived from the collected COCs, it had no stimulatory effect on MII formation of oocytes found cumulus-free or denuded at the time of aspiration. In experiment 5, with erbstatin, a specific tyrosine kinase inhibitor, the stimulatory effect of EGF on meiotic maturation of denuded oocytes was abolished. These results indicate that EGF, IGF-I and insulin could directly stimulate meiotic maturation of bovine denuded oocytes removed from the surrounding cumulus cells after oocyte collection. Moreover, the results also suggest that the stimulatory effect of EGF on bovine denuded oocyte meiotic maturation is mediated by the tyrosine kinase signal transduction pathway.

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Both ³H-palmitic and ¹⁴C-oleic acids were simultaneously incorporated into mouse embryo lipids from the 1-cell to the blastocyst stage. Rates of incorporation of the two fatty acids into the embryo lipids increased and differed from each other significantly after 8-cell stage (P<0.05). That of palmitic acid into the polar lipid fraction was significantly higher than that of oleic acid at all corresponding cell stages, but that of palmitic acid into the neutral lipids was significantly higher than that of oleic acid at the morula and blastocyst stages (P<0.05). In the neutral lipid fraction, the radioactivity of ³H-palmitic acid distributed in triacylglycerols, monoakyldiglycerols and diacylglycerols were significantly higher than that of ¹⁴C-oleic acid at most corresponding cell stages with the exception of high monoacyldiglycerol content in ¹⁴C-oleic acid at the 2-cell stage (P<0.05). But that of ³H-palmitic acid in fatty alcohols and monoacylglycerols was significantly lower than that of ¹⁴C-oleic acid at the most corresponding cell stages (P<0.05). In the polar lipid fraction, the amounts of ³H-palmitic acid distributed in choline phosphatides, ethanolamine phosphatides and sphingomyelins were significantly greater at most corresponding cell stages than that of ¹⁴C-oleic acid (P<0.05) but the radioactivity of ¹⁴C-oleic acid recovered in inositol or serine phosphatides, lysophosphatidylcholines, monoglycosylglycerides was significantly higher than that of ³H-palmitic acid at almost every corresponding cell stage (P<0.05). These results demonstrated that the patterns of incorporation and distribution of double fatty acids in various lipid species were significantly different from those of the same single fatty acids in our previous report.

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