The effect of hypoxia on human trophoblast in culture: morphology, glucose transport and metabolism.

The response to hypoxia of trophoblast isolated from term placenta and maintained in culture was studied. Trophoblast exposed to normoxic (PO2 120-130 mmHg) or hypoxic (PO2 12-14 mmHg) conditions were examined by electron microscopy. After 48 h, the cytoplasm of the hypoxic cells was more electron-dense with increased numbers of mitochondria, lysosomes and vacuoles. Compared to normoxic cells, the surface microvilli of the hypoxic cells were sparse, short and unevenly distributed. [3H]thymidine incorporation by both hypoxic and normoxic trophoblast fell rapidly and equivalently after 2 days in culture. The percentage of cells with the proliferation-associated nuclear antigen, Ki 67, also decreased, but remained higher in hypoxic cells suggesting that hypoxia retarded completion of the cell cycle (normoxia, 10.80 +/- 2.51 s.e.; hypoxia, 19.87 +/- 2.73, P < 0.01). Glucose consumption was elevated in hypoxia (3.73 +/- 1.07 s.e. mumol/10(6) cells/24 h) as compared to normoxia (1.46 +/- 0.83, P = 0.01). Although lactate production was consistently higher in hypoxia, the difference was not statistically significant (hypoxia 5.38 +/- 1.54 mumol/10(6) cells/24 h versus normoxia, 1.52 +/- 0.29, P = 0.07). After 48 h, uptake of [3H]2-deoxglucose ([3H]2DG) by hypoxic cells was reduced to 12 per cent +/- 4.3 s.e. of that in normoxic cells; return to normoxia resulted in recovery within 10 min. Lineweaver-Burk plots of [3H]2DG uptake indicated high affinity (KM 2.2 +/- 0.4 x 10(-4) M) and low affinity transporters (KM 4.5 +/- 1.6 x 10(-3) M). Northern blot analysis identified mRNA for GLUT1 and GLUT3. In hypoxia, steady-state GLUT1 and GLUT3 mRNA were approximately three- and 10-fold higher than in normoxia respectively. Inhibitors of oxidative metabolism of glucose increased the uptake of [3H]2DG within 2 h, whereas hypoxia reduced uptake. Hence, trophoblast in culture survives in extreme hypoxia, but manifests striking changes in morphology and in glucose metabolism and transport. Completion of cell cycle appears to be retarded.