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

The purpose of this study was to develop a primary culture system usin g serum-free medium for rat placental trophoblast cells and to investi gate the factors that control rat placental lactogen-II (rPL-II) secre tion in vitro. The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase. Dissociat ed cells were fractionated into five segments by centrifugation throug h a 40% Percoll density gradient and incubated on rat tail collagen be d in medium SFM-101 for up to 7 days. Fraction B at the Percoll gradie nt density of 1.05 g ml(-1) was enriched with rPL-II-producing cells a nd the time course of rPL-II secretion was characterized by a rapid in crease in the first 2 days, remaining at high values (mean: 14-16 ng m u g(-1) DNA) for the following 2-3 days and decreasing thereafter. The rPL-II-producing cells from faction B identified by immunocytochemica l 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 mu g ml(-1); recombinant human insulin-like g rowth factor (IGF)-I, IGF-II, 0.1-1.0 mu g ml(-1); murine epidermal gr owth factor (EGF), 0.001-10 mu g ml(-1)), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1-10 mu g ml(-1)) and hypothalamic ho rmoIles (human growth hormone-releasing hormone (GHRH), corticotrophin -releasing hormone (CRH), LHRH, 0.1-10 mu g ml(-1)) were individually added to the culture medium to investigate the putative factors that d irectly 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 mu g insulin ml(-1) and 1 mu g G HRH 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 res ults indicate that this in vitro culture system is suitable for elucid ating the regulation of rPL-II secretion and that rat growth hormone a nd 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.