ABSTRACT
Patterns of induced protein synthesis and secretion in guinea-pig endometrial epithelial cell cultures in response to oestrone sulphate alone and oestrone sulphate plus progesterone were investigated. Epithelial cells were cultured for 3 days in growth medium, then washed three times in a steroid-free medium. For each experiment, anticytokeratin immunostaining was used to discriminate the epithelial cells from the stromal cells. Only experiments in which the control dishes displayed more than 80% of anticytokeratinimmunostained cells were further processed. After this period oestradiol-17β (20 nmol/l; control), oestradiol-17β (20 nmol/l) plus progesterone (0·5 μmol/l), oestrone sulphate (1 μmol/l) or oestrone sulphate (1 μmol/l) plus progesterone (0·5 μmol/l) were added to the medium for 48 h. An immunocytochemical progesterone receptor assay showed that oestradiol-17β increased the progesterone receptor content of cells, and progesterone added to cultured cells in the presence of oestradiol-17β induced a significant increase in oestrogen sulphotransferase activity assessing the hormone responsiveness of the cultured cells.
In these culture conditions and after 16 h of incubation, oestradiol-17β induced a 1·7-fold increase in [3H]thymidine incorporation into DNA, and [35S]methionine incorporation into cellular proteins was linearly increased up to 8 h. Biochemical changes induced by the different hormone treatments were studied by labelling the proteins with a 6-h pulse of [35S]methionine. The proteins present in the medium and in cells were analysed by two-dimensional polyacrylamide gel electrophoresis, followed by fluorography. Addition of oestrone sulphate alone or with progesterone produced a change in the patterns of cellular and secreted proteins compared with those in cells cultured with either oestradiol-17β or oestradiol-17β plus progesterone. Three cellular proteins (Mr < 14 000, isoelectric point (pI) 5·2 and 5·3; Mr 75 000, pI 4·9) and one secreted protein (Mr 155 000, pI 5·6–5·9) were specifically induced and could serve as markers of oestrone sulphate action.
Journal of Endocrinology (1989) 123, 233–241
Binding of progesterone receptor by nuclear preparations of rabbit and guinea pig uterus.
