Niklaus A. Bersinger

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.

Evaluation of an In Vitro Dual Perfusion System for the Study of Placental Proteins: Energy Metabolism

The in vitro perfusion of human placenta offers several attractions to the investigator of placental function. Species differences which must be considered when data from animal experiments are discussed are of no concern and there are no ethical or safety questions. Furthermore the isolated in vitro situation allows the controlled modification of various experimental conditions. As in other in vitro techniques — for example, slices or membrane preparations — the tissue function is being studied after isolation from its natural environment, so considerable differences from in vitro behaviour are likely. Maternal and fetal influences which affect placental function by continuously modifying the “perfusate” are excluded, which is advantageous but at the same time present limitations in the interpretation of data.

Effect of Protein Synthesis Inhibitors and Metabolic Blockers on the Production of Placental Proteins by the in vitro Perfused Human Placenta

The capacity of the freshly delivered human term placenta to produce and release placental proteins during in vitro dual perfusion was investigated. The organ was perfused in separate closed circulations and aliquots of medium were taken at regular intervals from both maternal and fetal circuits. The placental proteins human chorionic gonadotrophin (HCG), human placental lactogen (HPL), pregnancy-specific beta 1-glycoprotein (SP1), and pregnancy-associated plasma protein A (PAPP-A) were quantified in these media as well as in the placental tissue before and after the perfusion. It was found that the four above-mentioned proteins were synthesised during the perfusion interval (90 min to 3 h) while pregnancy-associated alpha 2-glycoprotein and prolactin were only washed out. The mean production of HCG, HPL, SP1, and PAPP-A was decreased when either cycloheximide, puromycin, iodoacetic acid, or 2,4-dinitrophenol had been added to the perfusing medium. Amongst these four antimetabolites iodoacetic acid most severely affected both the total release and net synthesis. It is concluded that the above four proteins are synthesised de novo by the perfused placenta in the absence of maternal tissue and that this synthesis is energy-dependent.

De Novo Synthesis of Pregnancy-Specific and Pregnancy-Associated Proteins by the In Vitro Perfused Human Term Placenta

Placental perfusion is one of several in vitro techniques to investigate the production of pregnancy proteins by the trophoblast as well as their control mechanisms. The preparation as described in the chapter by Schneider et al is a closed-circuit system — that is, the released proteins and hormones accumulate in the circulating perfusate. This has the advantage of consuming less medium and yielding smaller volumes of more concentrated solutions when compared with the open perfusion protocol. We are aware that the release kinetics may differ between open and closed circuit protocols. For this reason we have abandoned our first protocol which required a full medium change after two hours of perfusion1 and have followed a new one which required the removal of 5 ml aliquots from both circuits every 30 minutes during the first two hours and every 60 minutes thereafter. The removed aliquots were replaced with fresh medium injected into both circulations.

Production of protein hormones by cultured trophoblast cells isolated from term and early placentae.

PROBLEM: To compare the capacity of de novo hormone synthesis by cultured trophoblast cells isolated from early and term placenta as cytotrophoblast, and to determine the ability of these cells to proliferate in culture.
 METHOD OF STUDY: Cytotrophoblast cells were isolated from term (TP, 38–42 weeks) and early placentae (EP, 8–13 weeks) by enzymatic digestion and subsequent purification on a percoll gradient. The net synthesis of the hormones human placental lactogen (hPL) and human chorionic gonadotropin (hCG) was determined as the release during culture+cell content after culture−cell content before culture. Proliferation was determined using a dedicated colorimetric reagent (CellTiter 96TM).
 RESULTS: Using a percoll gradient we were able to isolate three cell bands with densities of 1.051, 1.058, and 1.063 g/mL, which were predominantly cytotrophoblast cells as shown by immunocytochemical analysis. The cytotrophoblast cells with the highest density (1.063 g/mL) were used because they were found to release the highest amount of hormones and have shown the lowest rate of cell death after 6 days in culture. Both hCG and hPL showed different patterns of release during the first 2–3 days of culture between TP and EP. While the release by EP cytotrophoblast cells continued during 6 days of culture (n=4), the concentrations for TP cytotrophoblast (n=4) reached a plateau between 4 and 6 days. Net de novo synthesis calculated for 3×104 TP trophoblast cells cultured for 6 days (mean±SD, n=4) was 8.65±9.05 mU for hCG and 0.95±0.45 ng for hPL. For EP, it was 395.5±265.5 mU for hCG and 148.8±84.2 ng for hPL. Net synthesis of hCG was>10‐fold (TP) and>70‐fold (EP) higher than the initial cell content. While at term, hPL synthesis was only a fraction of the initial cell content, production by EP cytotrophoblast was 106 times the initial cell content. The extent of cell death after 6 days in culture was significantly ( P<0.02) higher for term (30–40%) than for early trophoblast (10–20%). Using a proliferation detection agent during the first 3 days of culture with first trimester cytotrophoblast cells, we did not find any changes in the proliferative activity.
 CONCLUSIONS: There are differences in the functional activity between trophoblast cells obtained from first and third trimester. The in vitro findings are difficult to reconcile with the different patterns of plasma concentrations of the two hormones observed in vivo during the course of pregnancy.