The effect of PTH on the epidermal growth factor (EGF) receptor was analyzed during the in vitro differentiation of human cytotrophoblasts. The cytotrophoblasts were isolated by a trypsin-DNase method from first trimester and term placentas and purified on a Percoll gradient. In culture, these cells aggregated and fused together to form a syncytium. This in vitro differentiation was associated with a 2-fold increase in 125I-EGF binding after 48 h of culture. The addition of 0.1 microM PTH (PTH-treated cells) to the culture medium induced a significant 2- to 3-fold increase (P less than 0.005) in EGF binding. The effect was dose related with a maximum obtained at a 1 nM concentration. Scatchard analyses revealed that PTH-treated cells possess a 2-fold higher number of high affinity sites as compared to control cells from early placenta (0.71 +/- 0.06 pmol/mg protein and 0.34 +/- 0.04 pmol/mg protein, respectively) and from term placenta (1.24 +/- 0.10 pmol/mg protein and 0.61 +/- 0.07 pmol/mg protein, respectively). The apparent Kd values for high affinity sites (0.15 nM) and for low affinity sites (4 nM) were not altered either by the gestational age of the cells or by PTH treatment. With respect to the EGF-dependent phosphorylation in membranes of trophoblast cells in culture, it was found that the phosphorylation of two major proteins of 175 kilodaltons and 35 kilodaltons, is greatly increased in PTH-treated cell membranes in the presence of EGF. This PTH-induced effect on EGF receptors was associated with an augmented functional response of trophoblastic cells to EGF. PTH increased the EGF-stimulated secretion of hCG. These results demonstrate that PTH increases the number of biologically active EGF receptors during the in vitro differentiation of human trophoblast cells. This PTH-induced effect suggests a role for this hormone in the regulation of the growth and the endocrine functions of these cells.

In this study, we have cultured in vitro purified trophoblastic cells from first-trimester and term human placenta. These cells were obtained by specific enzymatic digestion and centrifugation through a Percoll gradient. Using 2 specific monoclonal antibodies, the pituitary 22-kD growth hormone (GH) and the placental GH variant were assayed in the culture medium by radioimmunoassay. After 48 h of culture, only the placental GH variant was measured in the medium corresponding to first-trimester placenta (3.4 ng/24 h/10⁵ cells). Surprisingly, an immunoactivity pattern of pituitary GH type was found in 3 out of 5 media conditioned with term placenta cells, while GH immunoactivity was very low, around the detection level, in the 2 others. These secretions are not modified with the time in culture and the state of differentiation of the cells from cytotrophoblast to syncytiotrophoblast. Neither in early nor in term placenta does the addition of GH-releasing factor (10^-6M in the culture medium) stimulate the secretion of pituitary 22-kD GH or placental GH variant.

The binding and the total uptake of [3H]asialoorosomucoid was studied in normal rats, streptozotocindiabetic rats and insuliii-treated diabetic rats.1. The binding of [3H]asialoorosomucoi'd to normal rat hepatocytes gave a curvilinear plot when analysed by the Scatchard method, suggesting either two classes of independent receptor sites with different apparent association constants or a negative cooperativity in binding. The data, plotted according to the method of De Meyts and Roth showed a decrease of the affinity constant with respect to site occupancy.2. The Scatchard plot obtained for the [3H]asialoorosomucoid binding to streptozotocin-diabetic rat hepatocytes was curvilinear. According to the independent site model, the receptor number of each class was decreased, without any change of the apparent association constants. According to the model of De Meyts and Roth, no modification of the interaction factor and apparent affinity constants was observed, whilst the total number of receptor sites was decreased by half in streptozotocin-diabetic rats as compared with normal rats.3. Study of the total uptake of [3H]asialoorosomucoId by hepatocytes from each group of rats showed a decrease by half of the endocytosis in streptozotocin-diabetic rats as compared with normal rats.4. Insulin treatment of the streptozotocin-diabetic rats restored both parameters studied to normal level, whilst the withdrawal of insulin treatment decreased their level to that of streptozotocin-diabetic rats.These results are related to possible modifications of the hepatic binding protein, receptor for the plasma asialoglycoproteins, in the diabetic state.

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