Abbie L. Esterman

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.

The effect of hypoxia on term trophoblast: Hormone synthesis and release

Isolated trophoblast in culture remained viable when exposed to severe hypoxia (Po2 12-14 mmHg) for at least 72 h as indicated by trypan blue exclusion and the synthesis and secretion of metabolically labelled proteins. However, release of hCG, hPL, progesterone and estradiol was reduced to < 10 per cent when compared to trophoblast in normoxia (Po2 120-130 mmHg). hCG mRNA was also reduced demonstrating interruption of synthesis at transcription. Acute exposure to hypoxia (2 h) suppressed progesterone release but not hCG, whereas inhibitors of oxidative phosphorylation suppressed hCG release but not progesterone. hCG release increases progressively during culture in normoxia, peaking at 72 h. Exposure of trophoblast to hypoxia for 48 h after 24, 48 and 72 h in normoxia interrupted this progression but did not suppress hCG release. Progesterone release, in contrast, was reduced by hypoxia. Exogenous dibutyryl cAMP increased hCG and progesterone release by normoxic trophoblast but not by hypoxic cells. Trophoblast returned to normoxia after 24 h in hypoxia increased hCG and progesterone release, suggesting early recovery. Conservation of oxygen and ATP by reducing hormone synthesis may contribute to survival of trophoblast in hypoxia.

Synthesis and secretion of apolipoprotein E by human placenta and choriocarcinoma cell lines

Abstract The synthesis and secretion of apolipoproteins (apos) by cells from a human choriocarcinoma cell line, JAR, were examined by [35S]-methionine labeling followed by immunoprecipitation and SDS/PAGE. Apo E, but not aposA-I, A -IV, or B, was synthesized and secreted. Apo E was also synthesized by fragments of chorionic villi from human placenta and by another choriocarcinoma line, BeWo. Pulse-chase experiments with JAR cells revealed that apo E was initially synthesized as a 33 kDa protein followed by a 34 KDa protein, probably the result of glycosylation. The latter was secreted into the medium where it was detected coincident with a 21 22 kDa doublet, possibly proteolyticfragments ofapo E. Approximately 50 per cent of the apo E in the medium was complexed with lipid as indicated by ultracentrifugation at a density of 1.21 g/ml. The amount of apo E produced by JAR was not affected by preincubation with dibutyryl cAMP and theophylline, or by the cholesterol content of the cells. Following perfusion ofan isolated lobule ofhuman placenta with [14C]-amino acids, [14C]-apo E was detected by immunoprecipitation of the maternal and fetal perfusates with 88 per cent in the maternal perfusate. These studies suggest that apo E, which promotes receptor-mediated lipoprotein uptake, is secreted by the trophoblast to facilitate uptake of maternal lipoproteins.

Two Mechanisms for IgG Uptake in Cultured Human Trophoblast: Evidence for a Novel High Affinity Fc Receptor

ABSTRACT: The mechanism of IgG transport by the placental trophoblast was examined by studying IgG uptake by purified trophoblast maintained in culture. This model retains the ability to bind and endocytose human IgG from human serum. Comparison of the relative IgG uptake by the trophoblast among the four subclasses of both human and mouse IgG indicates that the trophoblast IgG receptor has different affinities from those described for the three known human Fcγ receptors, FcRγI, FcRγII, and FcRγIII. These results suggest the presence of a novel trophoblast Fcγ receptor. Although FcγRIII has been reported to be present on trophoblasts, immunocytochemical studies failed to detect binding to the cell surface of antibody-specific for FcγRIII, 3G8 MAb. In addition, blocking studies with MAb 3G8 did not interfere with IgG uptake. Scatchard analysis of human IgG uptake revealed a biphasic curve consistent with two distinct mechanisms for the transport of IgG by the trophoblast. The first is a higher affinity system (Ka = 1.7 × 107 M-1 1.7 × 104 binding sites/cell) which exhibits IgG subclass and species specificity, and the second is a low affinity system (Ka = 6.9 × 103 M-1, 7.5 × 107 binding sites/cell).

Synthesis of α 1 -Antichymotrypsin and α 1 -Antitrypsin by Human Trophoblast

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.

Synthesis of alpha 1-antichymotrypsin and alpha 1-antitrypsin by human trophoblast.

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.

Zellweger Syndrome Amniocytes: Morphological Appearance and a Simple Sedimentation Method for Prenatal Diagnosis

ABSTRACT: Zellweger syndrome is the prototype of a growing group of genetic diseases caused by an absence or deficiency of peroxisomes. The defect causes the enzyme catalase to remain in the cytosol instead of being packaged into peroxisomes. This mislocalization can be easily detected by sedimentation analysis. Amniocytes were homogenized and then centrifuged to pellet organelles. Catalase was found to sediment with the peroxisomes in the homogenates of normal cells, but to remain in the supernatant with Zellweger syndrome amniocyte homogenates. This striking difference is unambiguous and reproducible, and provides a simple method for prenatal diagnosis. Moreover, it allows one to differentiate diseases in which peroxisomes are deficient from other peroxisomal diseases in which the organelle is intact, but one enzyme is defective. Electron microscopic observations support the biochemical determinations. Normal amniocytes contain small peroxisomes in which a weak cytochemical reaction for catalase may be demonstrated. Zellweger amniocytes appear to lack these organelles, although some cells have rare structures that might be residual or abnormal peroxisomes.

Uptake of human immunodeficiency virus envelope protein gp120 by human trophoblast in culture

OBJECTIVE Our purpose was to determine whether human trophoblast has a cell surface CD4 antigen that will bind to gp120, the envelope protein of human immunodeficiency virus. STUDY DESIGN Uptake of iodine 125-labeled gp120 by trophoblast in culture was measured. Particular attention was paid to technical details that may have caused the contradictory results reported by previous investigators: the source of the recombinant gp120, the method of radioiodination, and the isolation procedure of trophoblast to ensure elimination of contaminating cells, particularly macrophages. RESULTS Uptake of transferrin-free iodine 125-labeled gp120 to trophoblast was unaffected by adding a 200 molar excess of gp120, by preincubating gp120 with soluble CD4 to block the CD4 binding sites on gp120 and by preincubation of trophoblast with a blocking antibody to CD4 (OKT4a). In contrast, uptake of gp120 by CD4-positive H9 human lymphocytes was reduced 79% by a 200 molar excess of gp120 and > 50% by a CD4-blocking antibody. CONCLUSIONS Uptake of gp120 to trophoblast is by a high capacity, CD4-independent mechanism that is probably nonspecific and may be related to the mechanism for binding other circulating glycoproteins in maternal blood.

Synthesis of α1-antichymotrypsin and α1-antitrypsin by human trophoblast

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.

RESPONSE OF HUMAN TROPHOBLAST TO HYPOXIA: MORPHOLOGY, GLUCOSE TRANSPORT AND METABOLISM, ENDOCRINE SYNTHESIS. † 1230

RESPONSE OF HUMAN TROPHOBLAST TO HYPOXIA: MORPHOLOGY, GLUCOSE TRANSPORT AND METABOLISM, ENDOCRINE SYNTHESIS. † 1230