Stimulation of rat placental lactogen-II (rPL-II) secretion by cultured trophoblasts by insulin: development of a rat placental cell culture system and effects of peptide hormones on rPL-II secretion in vitro

摘要

The purpose of this study was to develop a primary culture system using serum-free medium for rat placental trophoblast cells and to investigate the factors that control rat placental lactogen-II (rPL-II) secretion in vitro. The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase. Dissociated cells were fractionated into five segments by centrifugation through a 40% Percoll density gradient and incubated on rat tail collagen bed in medium SFM-101 for up to 7 days. Fraction B at the Percoll gradient density of 1.05 g ml?1 was enriched with rPL-II-producing cells and the time course of rPL-II secretion was characterized by a rapid increase in the first 2 days, remaining at high values (mean: 14–16 ng μg?1 DNA) for the following 2–3 days and decreasing thereafter. The rPL-II-producing cells from faction B identified by immunocytochemical examination accounted for approximately 69% of total cultured cells and consisted of a few giant cells and polygonal cells. Growth factors (bovine insulin, 0.1–20 μg ml?1; recombinant human insulin-like growth factor (IGF)-I, IGF-II, 0.1–1.0 μg ml?1; murine epidermal growth factor (EGF), 0.001–10 μg ml?1), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1–10 μg ml?1) and hypothalamic hormones (human growth hormone-releasing hormone (GHRH), corticotrophin-releasing hormone (CRH), LHRH, 0.1–10 μg ml?1) were individually added to the culture medium to investigate the putative factors that directly control rPL-II secretion by the trophoblast cells. Insulin and GHRH stimulated rPL-II secretion in a dose-dependent manner and their effective doses were found to be 0.1 μg insulin ml?1 and 1 μg GHRH ml?1. IGFs, EGF, rat growth hormone, rat prolactin, CRH and LHRH did not affect rPL-II secretion for 2–3 days of incubation. These results indicate that this in vitro culture system is suitable for elucidating the regulation of rPL-II secretion and that rat growth hormone and rat prolactin did not directly inhibit rPL-II secretion. They also suggest that insulin may play a role in regulating rPL-II secretion in vivo.