This study was conducted to evaluate the ability of Vero cell-conditioned medium for supporting mouse embryo development in vitro.

The mouse late 2-cell embryos were cultured in control media(Ham's F-10 +10%FBS), media with Vero cell monolayer and Vero cell-conditioned media for 4 days and measured the hatching rate and cell number in the blastocyst stage.

The hatching rate in experimental groups was increased significantly compared with embryos in control group(p<0.01). On the other hand, the degree of blastomere fragmentaion exhibited a opposite trend to that of the developmental capacity(p<0.05). And also the cell numbers of expanded blastocysts in experimental groups were increased significantly compared with the control group(p<0.001). There was, however, no difference between experimental groups.

These results indicate that Vero cell-conditioned medium supported the mouse embryo development as a Vero cell monolayer. And the mechanism for enhancement of the development potential of embryos may be releasing the embryotrophic factor during the medium-conditioning period.

Assisted reproductive technology(ART) have contributed significantly to alleviating subfer-tility in the childless couple. In spite of the many advances in the field of ART, the pregnancy and take-home baby rates for in vitro fertilization(IVF) have been very poor. In order to overcome these problems, a variety of coculture systems has been devised. Vero cells, derived from African green monkey kidney, were selected because kidney and genital tract have a common embryonic origin. In addition, these cells are safe for coculture with embryos : they are highly controlled for viruses and other contaminants because they are used for vaccine production. Several investigators showed that cocultureing human embryos with Vero cells in vitro resulted in an improvement of embryo development. However, they did not observe the same results using mouse oocytes and embryos. We thus designed a series of experiments to demonstrate whether or not Vero cells do indeed enhance mouse oocyte maturation and embryo development. In this experiment, Vero cell does not allow the mouse immature oocytes to be enhanced maturation rate in vitro.

To study the 'In-Vitro 2-cell Block' in mouse embryo, we have cocultured ICR one-cell mouse embryos with Vero cell in different medium. In Ham's F-10 the mouse embryos arrested their development prior to 4-cell stage(control 76.7%;coculture 75.0%). In contrast, the coculturing mouse embryos revealed enhanced development(control 0%;coculture 22.8%) in human tubal fluid(HTF) only in late embryonic stages(hatching).

On the other hand, the degree of blastomere fragmentation exhibited a reverse trend to that of the developmental capacity. Embryos from coculture groups(Ham's F-10 & HTF) showed some fragmentation(0% & 4.2%) while 13.3% and 14.3% of the embryos in control groups(Ham's F-10 & HTF) were severely fragmented(P<0.05). Thus the use of coculture systems appears to be dependent on the type of medium used as a support.

The development rate of late 2-cell mouse embryos in Vero cell coculture was no significant differences until blastocyst stage but improved at late developmental stage(control 42.1% ; conculture 70.7%). Thus the Vero cell coculture system was shown to increase the hatching rate of mouse embryos.

The subcellular distribution of glucose transporter in rat hepatocytes was studied in the absence and presence of insulin by measuring glucose-sensitive cytochalasin B binding sites and immunoreactivities to antibodies specific to hepatocyte glucose transporter. Total hepatocyte membranes bound cytochalasin B at a class of glucose-sensitive sites with a K_d of l.6×l0^-6M. and a B_t of 6.8 pmol/mg protein. The glucose-sensitive cytochalasin B binding sites were found in various subcellular membrane fractions with a relative abundance of 47% in a plasma membrane-nuclei-mitochondria-enriched fraction(PM/NM). 29% in a lysosome-enriched fraction(LYSO). 16% in a Golgi-enriched. high density microsomal fraction(HDM) and 8% in the low density microsome fraction(LDM). Relative abundances of two well known plasma membrane markers. 5'-nucleotidase and cell surface carbohydrate label, on the other hand, were found to he 48~50% in PM/NM. 41~43% in LYSO. 6~8% in HDM and l~2% in LDM.

Insulin treatment of intact hepatocytes did not induce any significant changes in the subcellular distributions of the glucose-sensitive. cytochalasin B binding activities, the immunoreactivities to the transporter specific antibodies, or the two cell surface membrane markers. These findings indicate that as much as 15% of the total hepatocyte glucose transporters occur in organelle(s) other than the pasma membrane, most likely representing an intracellular storage pool. which is not decreased by insulin. It is concluded that the rat hepatocyte lacks the insulin-mediated, glucose transporter translocation mechanism, thus would be a valuable experimental system in which one can study the celluar and molecular basis of this deficiency.

Proteoliposomes were prepared by reconstitution of a solubilized band 4.5 protein of human erythrocyte membrane with a mixture of phosphatidylcholine(PC) and phosphatidylethanolamine(PE) of varying ratios. The incorporation of the band 4.5 protein into the reconstituted vesicles was dependent on the stability of the bilayer of lipid added exogeneously when mixed soybean PE and egg PC were used for reconstitution. The protein/phospholipid ratio was maximal at 25% PC and 75%PE, the composition which give maximal bilayer instability. The specific activity of the cytochalasin B binding of the reconstituted band 4.5 protein increased monotonically as PC content increased in egg PC/soybean PE mixture. These coincide with the results of proteoliposomes reconstituted using crude protein extracts containing band 4.5 protein. Band 4.5 protein, however, changed significantly the phase of the lipid in reconstituted vesicles instead of that protein crude extract modified slightly the phase of the lipid. No lipidic particles (LIP, seen by freeze- fracture electron microscopy) were seen in all lipid mixtures studied. From above findings the fact that pre- existing defects in lipid bilayer promote protein incorporation into the bilayer during reconstitution is confirmed and band 4.5 protein somewhat seems to favor the bilayer structure.

Insulin stimulates glucose transport in muscle cell and adipocyte via the rapid redistribution of GLUT4 glucose transporters from intracellular membrane compartments to the cell surface. The mechanism that insulin treggers the translocation of glucose transporters in not known yet whether it is due to the structural differences among glucose transporters or there is cell specific targetting/translocation apparatus insulin-sensitive cells.

This study was planned to examine this question by strdying insulin effect on the glucose transport rate at adipocyte and hepatocyte fused with GLUT1 vesicle, respecitively.

The results showed that treatment of 37nM insulin increased the transport rate of 3-0-methylglucose by 3.8-fold at adipocyte fused with GLUT1 but increased lnly by 1.2-fold at hepatocyte fused with GLUT1.

Therefore, it is suggested that insulin sensitive cell has a cell-specific targetting/translocation machinery which is triggered by insulin-insulin receptor interaction but insulin sensitivity may not dependent on isoform(structural)-specific manner.