Studies on the microheterogeneity of chorionic gonadotrophin secreted by the human cytotrophoblast in culture*

Abstract

In the present study, we investigated the biological characteristics of different molecular forms of chorionic gonadotrophin (HCG) secreted by the human cytotrophoblast during its morphological and functional differentiation in culture. Highly purified cytotrophoblasts were prepared from term placentae and cultured for 24 to 96 h in the absence or presence of 8-bromo-3',5'-cAMP. Media were collected at 24 h intervals and the secreted isofonms of HCG were then separated by polyacrylamlde gel isoelectric focusing (pH range 8.0–3.0) and quantified by radloimmunoassay. The secretion of HCG was significantly increased by 8-bromo-cAMP (from 23.5 ± 6.3 ng/ml at 24 h to 1619 ± 835.8 ng/ml at 96 h; controls, 9.3 ± 0.1 ng/ml at 24 h and 26.6 ± 3.5 ng/ml at 96 h, X&macr; ± SD). Analysis of media concentrates from cAMP-stimulated cultures by isoelectric focusing revealed the presence of several distinct peaks of HCG within the pH range of 7.3–4.8; major peaks consistently exhibited isoelectric points (pI) of 7.3–7.0 (peak 1), 5.6–5.4 (peak 2) and 5.1–4.8 (peak 3). The relative HCG content of the most acidic peak (as % of total on gel) progressively increased with time of exposure to the cAMP analogue (from 19.8 ± 1.6% at 24 h to 34.4 ± 4.3% at 96 h, X&macr; ± SEM, P < 0.01). HCG recovered from peak 1 exhibited the highest receptor-binding capacity and in-vitro biological potency. Removal of sialic acid residues from purified HCG (pI of 5.2) by neuraminidase digestion resulted in the production of several peaks of immunoactive HCG with less acidic pIs (6.5, 6.7, 6.9 and 7.1). Only in those concentrates from control media at 72 and 96 h was it possible to identify some peaks of HCG immunoactivity after isoelectric focusing. These components exhibited pIs of 7.8–5.2 and were dissimilarly distributed over the 24 h period analysed. These results demonstrate that the human cytotrophoblast in vitro secretes chorionic gonadotrophin in different molecular forms, thus indicating that some of the previously identified forms of HCG in serum are actually produced by the placenta and then released to the periphery.