In the present study, the oxidation of palmitic acid to carbon dioxide and the difference of incorporation rate of the same and oleic acid into embryo lipids were examined. To test the exact role of fatty acids for energy production, the oxidative rates of exogenous palmitic were assessed in M16 medium with and without carbohydrate substrates. The oxidation of palmitic acid in the medium with carbohydrates was lower than that without carbohydrates in the blastocyst stage. Incorporation rates of both palmitic and oleic acids into embryo lipids increased significantly from the 2-cell to the blastocyst stage (P<0.05), whereas the incorporation rates of palmitic acid were significantly higher than those of oleic acid at most of the cell stages except for the blastocyst stage (P<0.05). In the neutral lipid fraction, ³H-palmitic acid was predominately distributed in triacylglycerol species and a little in the other glycerides (P<0.05). However, higher percentages of ¹⁴C-oleic acid were recovered in fatty alcohols, diacylglycerols and monoacylglycerols. In the polar lipid fraction, the percentages of ³H-palmitic acid in choline and ethanolamine phosphatides were significantly higher than those of oleic acid among all cell stages except the 2-cell stage (P<0.05), whereas the percentages of ¹⁴C-oleic acid in inositol and serine phosphatides were significantly higher than those of ³H-palmitic acid during preimplantation development (P<0.05). According to the comparison of incorporations into the embryo lipids and distributions in individual lipid classes between palmitic and oleic acids, it could be inferred that exogenous oleic acid is not a main energy substrate but a major intermediate for synthesis of various embryo lipids.

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The aspiration of excess numbers of follicles was examined as a method for inducing twin pregnancy in cattle. Seven cattle were administered follicle stimulating hormone (FSH) for superovulation. The number of follicles was measured by an ultrasound scanning scope and excess numbers of follicles were punctured and aspirated to decrease the number of ovulations. One or two follicles were left intact in each ovary. Two of six cattle responding to FSH bore twins and one pair of twin calves was born.

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Bovine oocytes were matured and fertilized in a medium supplemented with or without 0.1% linoleic acid-albumin (LAA). The presumptive pronuclear-stage zygotes were frozen-thawed in the medium containing 1.5 M ethylene glycol with or without 0.1 M sucrose. The post-thaw survival of the zygotes was assessed by blastocyst development in vitro. In non-frozen control groups, 66 vs 64% (p=0.757) of the zygotes that were produced in the presence vs absence of LAA cleaved and 25 vs 27% (p=0.883) developed to blastocysts, respectively. After freezing and thawing, similar proportions (53-65%) of the zygotes appeared to be normal regardless of the LAA treatment in the IVM / IVF media (P≥0.775) and sucrose inclusion in the cryoprotective medium (P≥0.177). However, the post-thaw cleavage rate of LAA-treated zygotes (45-54%) was significantly higher (p<0.008) than that of non LAA-treated zygotes (23-29%). The post-thaw development of LAA-treated zygotes into blastocysts (14%) was also higher (p<0.003) than that of non LAA-treated zygotes (2%). Developmental kinetics and cell numbers of the resultant blastocysts were similar between frozen and non-frozen groups. These results suggest that presumptive pronuclear-stage bovine zygotes matured and fertilized in the presence of LAA are relatively tolerant to the process of freezing and thawing, although the post-thaw survival rate needs to be further improved.

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Immature pig oocytes cultured for 36 h in a modified tissue culture medium 199B were freed from cumulus cells and treated for 30 min with fluorescein isothiocyanate-labelled lectins. When examined under fluorescence illumination, Ricinus communis agglutinin (RCA-I) and Lens culinaris agglutinin (LCA) bound to all oocytes, with the strongest fluorescence in either the outer region of the ZP (RCA-I) or throughout the ZP (LCA). However, Bandeiraea simplicifolia lectin-I (BS-I) and Ulex europaeus agglutinin-I (UEA-I) bound with either strong or weak intensity mainly to the outer and inner regions of the ZP, respectively, in ~70-90% of oocytes examined. Bandeiraea simplicifolia lectin-II (BS-II) bound to various regions of the ZP with different intensities in only 60% of oocytes examined. At 2 h after insemination in vitro, significantly fewer spermatozoa were bound to the ZP of lectin-treated oocytes than untrearted control oocytes. Of the lectins, RCA-I and LCA most inhibited sperm binding. At 12 h after insemination, penetration rates were similar in control oocytes and those treated with BS-I or BS-II, but penetration rates were lower in oocytes treated with UEA-I than those in untreated controls, and an almost complete block of sperm penetration was observed in oocytes treated with RCA-I or LCA. The incidence of monospermy was similar in untreated oocytes and those treated with BS-I or UEA-I, but it was higher in oocytes treated with BS-II. These results suggest that β-D-galactose and α-D-mannose residues in the pig ZP may act as primary sperm receptors and/or inducers of the sperm acrosome reaction and β-D-N-acetylglucosamine residues may be involved in the block of polyspermy. The variability in binding of BS-II to the ZP, which correlated with monospermy/polyspermy may reflect differences in the maturation state of individual oocytes. In future, this lectin might provide a means of monitoring maturation in vitro, leading to the development of improved culture systems.

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We attempted to produce a muscle-specific cell marker, which would be useful for examining myogenesis, by introducing pMGN(-4k)LacZ-neo gene into ESD3 cells, and expression of the transgene was analyzed in embryoid bodies. The gene expression analysis of embryoid bodies indicated that transfected cells did not express LacZ gene in an undifferentiated state and that the number of cells with LacZ expression increased progressively. In DMSO treated embryoid bodies, the number of cells expressing LacZ gene tended to increase more rapidly than that of untreated ones. The results suggested that the DMSO may induce mesodermal differentiation of ES cells and that the sublines obtained might be useful as muscle-specific cell markers since they might express lacZ gene at the time of myogenin expression.

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The changes of Cx-43 and MAP kinase in cumulus cells that were recovered from porcine cumulus oocyte complexes (COCs) cultured with FSH and LH and/or PI 3-kinase inhibitor, LY294002, were analyzed by immunoblotting. Three migration forms of Cx-43 (43, 45, and 47 kDa) were detected in the cumulus cells of COCs immediately recovered from their follicles. At 12-hr of cultivation with hormones, oocyte nuclear stage was progressed from GV II stage to GV III stage. At this time point, the 43 kDa band almost completely disappeared, although two phosphorylation forms of Cx-43 (45 and 47 kDa) were still detected. However, in the cumulus cells of COCs cultured with LY294002, three prominent bands of Cx-43 were recognizable, as in the cells recovered immediately from the follicles. Although two intense bands of activated MAP kinase (ERK 1, 2) were also seen in the cumulus cells of COCs at 12-hr of cultivation with hormones, the intensity of the activated ERK 2 band was relatively faint in cumulus cells of COCs cultured with LY294002. At the same time point, LY294002 produced a significant decrease in the proportion of oocytes exhibiting GV III stage compared to those observed with hormones. It is concluded that the progression from GV II stage to GV III stage in porcine oocytes may be closely associated with phosphorylation of Cx-43 by virtue of MAP kinase, which was activated through the PI 3-kinase pathway in their cumulus cells.

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Fertilized hamster eggs were recovered from the ampulla of the oviduct at various times after in vivo insemination. To examine the distribution of the egg cortical actin and the position of nuclei from freshly ovulated unfertilized eggs to the 2-cell stage, they were double stained with Rhodamine-phalloidin and Hoechst 33342. In the freshly ovulated hamster eggs arrested at metaphase-II of meiosis, the egg surface above the meiotic spindle was devoid of microvilli and had instead conical projections. This area showed strong fluorescence, indicating that the actin was aggregated. As the sperm head was incorporated and began to decondense in the ooplasm, an incorporation cone was formed at the site of sperm entry. The cone consisted of relatively long, conical projections which closely resembled those of freshly ovulated eggs, and also showed strong fluorescence. This cone lasted while the sperm nucleus was decondensing and diminished at the time when the male pronucleus was formed. The results obtained with regard to the formation of the incorporation cone were comparable with those of mouse and rat eggs. Moreover, as to the elimination of second polar body, it was found that the body underwent two constrictions, resulting in a gourd-like body.

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The authors encountered rabbit fetuses that developed from fertilized eggs implanted in the peritoneal cavity. A laparotomy of a rabbit that showed no signs of parturition even in the late period of pregnancy 32 days after mating revealed two fetuses suspended from the greater curvature of the stomach. Both fetuses were dead, their body weights were 44 g and 37 g, and their body lengths were 8.0 cm and 7.8 cm. No placenta was present. Blood vessels that ran in the greater omentum communicated with the chorio-allantoic membranes of the two fetuses. Since no corpora lutea were observed in the ovaries, and since no injury was noted in the uterus, this case was judged to be primary ectopic pregnancy caused by migration of fertilized eggs in the peritoneal cavity and their implantation in the greater omentum.

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The present study was designed to investigate the immunohistochemical localization and distribution of basic fibroblast growth factor (bFGF) in the goat pituitary gland. We examined various fixatives for tissue preparation, and confirmed that methyl Carnoy-fixed tissues were associated with a better preservation of bFGF. After fixation, paraffin- embedded tissue sections were prepared, and were stained immunohistochemically with mouse monoclonal antibody against bovine bFGF. Approximately 30% of parenchymal cells in the pars distalis of the goat pituitary gland displayed strong immunoreactivity against the bFGF antibody. Endothelial cells occasionally showed positive immunoreactivity. No immunopositive cells were found in either the pars intermedia of the pituitary gland or posterior lobes. Based on analysis of serial sections stained with antibodies against pituitary hormones (luteinizing hormone and adrenocorticotropic hormone), bFGF-positive cells appear to be a subpopulation of gonadotrophs and or corticotrophs. The present findings indicate that immunohistochemical reactivity for bFGF may be partially attributed to differences in fixatives and that bFGF may play critical roles in the goat pituitary gland.

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The process of apoptosis of follicular granulosa cells in porcine ovaries during follicular atresia was investigated by in situ DNA 3'end-labeling at the level of individual cells. Histochemical changes in the follicular basement membrane (BM) were visualized by immunofluorescent staining of BM extracellular matrix (ECM) components, i.e. type IV collagen, laminin and fibronectin. At the first stage, granulosa cells located on the inner surface of the granulosa layer appeared to undergo apoptosis, followed by neighboring granulosa cells. No apoptotic granulosa cells making tight contact with intact BM were observed. Detachment and degeneration of the granulosa cell layer and fragmentation of BM occurred in follicles at the advanced stage of atresia. Finally, intermittent structures of BM and subsequent invasion of macrophages and fibroblasts were observed. Therefore we concluded that granulosa cell apoptosis is an initial symptom of follicular atresia in the porcine ovaries, and the degradation of BM follows granulosa cell apoptosis in the pig. Our results suggest that ECM components of follicular BM act as survival factors on follicular granulosa cells in porcine ovaries.

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