In Vitro Studies of Cultured Rat Trophoblast Cells

Abstract

This study was designed to explore the feasibility of using rat trophoblast cells as an animal model for studies of placental growth and regulation. The structural and functional similarities between the giant cells of the rat placenta, and the syncytio-trophoblasts of the human placenta warrant such an investigation. In this study, trophoblast cells were cultured in vitro. Particular attention was paid to the cytodifferentiation of cytotrophoblasts into giant trophoblast cells, and the time at which they appeared after they were first seeded in culture. Trophoblast cells derived from the isolated basal zone of the chorio-allantoic placenta from pregnant rats sacrificed on days 12, 16, and 20 of gestation were used for experimentation. These cells were enzymatically dispersed and cultured in vitro as primary monolayers. The morphology of these cells was studied using histochemical methods under light, phase contrast, and fluorescent microscopy. Human chorionic gonadotropin (hCG) as a self regulatory/tropic agent, and diethylstilbestrol (DES) as a maternal estrogen were added to placental cell cultures to investigate their effects on steroidogenesis. Radioimmunoassay (RIA) was employed to analyze the culture media for the determination of progesterone hormone secretion by the trophoblast cells. The basal secretion of P by the placental cells was highest in day 16 trophoblast with an average of 3.0ng P/18h. Day 12 trophoblast produced the second highest amount with 0.8ng P/18h, and a marked reduction was seen in day 20 trophoblast cultures with only 0.3ng P/18h. DES treatment had no significant effect on day 16 and 20 trophoblast. However, day 12 trophoblast showed a biphasic response with an intense stimulatory effect (26.6ng P/18h) at the 10-10M; with higher concentrations causing an inhibitory effect (0.36ng P/18h) at 10-5M. HCG stimulated both day 12 (2.4ng P/18h) and day 16 (6.7ng P/18h) trophoblast with peak P production at the lowest concentration of 10mIU. However, hCG had no significant effect at any dosage level on day 20 trophoblast; while increased concentrations did not further stimulate day 12 and day 16 trophoblast to secrete P above the levels attained at 10-10 M. Morphological observations of the isolated basal zone cells revealed six distinct cell types: 1) mononucleated polygonal cells, 2) binucleated cells, 3) large multinucleated cells, 4) multinucleated giant cells, 5) mononucleated giant cells, and 6) fibroblasts. Within each of the 3 placental cell cultures we have observed that the formation of giant trophoblast cells occurs through several stages of differentiation of the mononuclear cytotrophoblast cells by means of cell and nuclear fusion. Therefore, isolated and cultured trophoblast cells are destined to become giant cells, and this differentiation can occur in the absence of maternal and fetal cues.