No abstract

The uptake of myo-inositol by preimplantation mouse embryos was investigated using [3H]myo-inositol. Uptake increased about 12-fold between one- and two-cell stages and increased again at the blastocyst stage (> 6-fold compared with the two-cell stage). Uptake at the blastocyst stage was time and temperature dependent; it was stimulated by sodium, inhibited by glucose and appeared to take place mainly via a saturable mechanism. Uptake in the presence of 6.25 mmol inositol l-1 was 1424 fmol inositol per blastocyst per h. About 10% of the [3H]inositol taken up by blastocysts during 8 h in culture was incorporated into lipid. Thin layer chromatography of the lipid showed that most of this inositol was incorporated into lipid material co-migrating with phosphatidylinositol with a small proportion co-migrating with phosphatidylinositol 4-phosphate.

Fertilized 1-cell rabbit ova were cultured in the presence of three oxidative phosphorylation inhibitors (cyanide, 2,4-dinitrophenol and oligomycin), two tricarboxylic acid (TCA) cycle inhibitors (malonate and fluoroacetate) and one glycolytic inhibitor (2-deoxyglucose). All three oxidative phosphorylation inhibitors killed ova at the 1-cell stage and the damage caused by each was similar. Malonate was non-toxic at all concentrations whereas some concentrations of fluoroacetate stopped growth at the 1-cell stage. This toxic effect could, in some circumstances, be reversed by the presence of acetate but not of glucose. 2-Deoxyglucose blocked only the transition from morula to blastocyst, and this was prevented by the addition of glucose to the medium; pyruvate, ribose, glycerol, and L-alpha-glycerol phosphate were ineffective. An active oxidative phosphorylation system and tricarboxylic cycle appear to be present and essential in the rabbit embryo from the 1-cell stage, but glycolysis may not be essential until blastocyst formation.

Activation of the phosphatidylinositol (PtdIns) signal transduction system involves stimulation of phospholipase C (PLC) by hormones and other agonists to produce two second messengers, the inositol phosphate, Ins(1,4,5)P3 which releases calcium from intracellular stores, and diacylglycerol which activates protein kinase C (PKC). This study, using activators or inhibitors of PLC and PKC and a calcium ionophore, examined the role of the PtdIns system in mouse embryonic stem (ES) cells. The PLC inhibitor, U-73122, inhibited ES-cell proliferation and also inhibited PLC activation as evidenced by a decrease in inositol phosphate formation in response to fetal calf serum stimulation. The two PKC activators, the diacylglycerol analogue 1,2, dioctanoyl-sn-glycerol (DOG) and the phorbol ester 12-O-tetra-decanoyl phorbol 13-acetate (TPA), increased cell proliferation in a dose-dependent manner, as did the calcium ionophore, ionomycin. However, co-stimulation with either ionomycin and DOG or ionomycin and TPA resulted in a reduced number of cells. The PKC inhibitor, bisindolylmaleimide II (Bis II), significantly decreased the number of ES cells, mainly due to increased apoptosis. The possible feedback effect of PKC on PLC was examined by preincubating ES cells with either the PKC inhibitor Bis II or the activator TPA before stimulation of inositol phosphate production with fetal calf serum; preincubation with Bis II increased inositol phosphate formation whereas preincubation with TPA decreased inositol formation. These results indicate that the PtdIns system is involved in the control of ES-cell proliferation and apoptosis.

The preimplantation period in the rabbit consists of a 3 day cleavage stage during which the number of cells increases with little change in embryo size, followed by a 3-4 day blastocyst stage during which the inner cell mass, the blastocoel and the trophectodermal layer are formed and the embryo grows rapidly in size and protein content. This study used Inositol uptake increased to 0.58 pmol per embryo per h for early blastocysts (day 4) and 23.7 pmol for late blastocysts (day 6). There was a significant linear relationship between inositol uptake and blastocyst diameter and surface area. Efflux of inositol from early morulae was minimal (about 1.25% of embryo content per h), whereas efflux from mid-blastocysts (day 5) was much greater (about 15.6% of embryo content per h). Efflux of inositol from both early morulae and mid-blastocysts was increased by decreasing the osmolality of the incubation medium. Varying the osmolality had no effect on inositol uptake up to 2 h. Inositol uptake was dependent on sodium in cleavagestage embryos but independent of sodium in blastocyst stages. In early morulae, inositol uptake was inhibited by glucose and the sodium-dependent hexose transport inhibitor, phloridzin, but not by the facilitated transport inhibitor, phloretin. Inositol uptake in early morulae was saturable; estimates of 0.227 and 0.288 pmol per morula per h for V max and 0.045 and 0.038 mol l −1 for K m were obtained for sodium-dependent transport in two separate experiments. All of these results are consistent with the hypothesis that transport in cleavage stages occurs via a sodium myo-inositol transporter (SMIT) protein. Uptake in blastocysts was non-saturable. Uptake into blastocysts appeared to take place by a transcellular rather than a paracellular route.