Summary. Normal guinea-pig endometrial cells, grown in primary culture, were made quiescent by serum depletion. Quiescent cells cultured in the control medium (containing 1% fetal calf serum treated with dextran-coated charcoal, DCC-FCS) showed a steady and weak rate of [³H]thymidine incorporation, but the addition of 15% fetal calf serum (FCS) or 10% DCC-FCS to the control medium induced a significant increase of DNA synthesis, demonstrating the responsiveness of the quiescent cells to stimulation. A lower but significant increase in [³H]thymidine incorporation was elicited by epidermal growth factor (EGF, 100 ng/ml) or insulin (10 μg/ml) added to the basal medium.

Oestradiol-17β added to the control medium at concentrations ranging from 10⁻¹⁰ to 10⁻⁵ mol/l not only failed to increase but even inhibited [³H]thymidine incorporation at the highest concentrations tested. An additive effect was noticed when quiescent cells were incubated with oestradiol-17β (10⁻⁹ mol/l) in the presence of 10% DCC-FCS, but no synergistic effect occurred when 2 × 10⁻⁹ mol oestradiol-17β/l was combined with either EGF (100 ng/ml) or insulin (10 μg/ml). Oestradiol-17β appears unable alone to stimulate DNA synthesis in normal endometrial cells, but requires factor(s) present in fetal calf serum.

Keywords: serum; oestradiol-17β; DNA; endometrium; cell culture; guinea-pig

Summary. Ovariectomized guinea-pigs were treated with oestradiol-17β (E₂), oestrone sulphate (E₁S) and progesterone (P) and the in-vitro incorporation of ³⁵SO₄ was studied in uterine fragments. The net uptake of ³⁵SO₄ into tissue was only increased by oestradiol-17β plus progesterone. The incorporation of ³⁵SO₄ in the tissue-associated proteins was increased after treatment with E₂ and E₁S compared with untreated controls (3·1- and 2·5-fold, respectively). For secreted proteins, all hormone treatments induced an increase in protein sulphation, the highest increase occurring when progesterone was administered after oestrogens. Tyrosine ³⁵SO₄ was identified in protein extracts from tissues and media and values were greater after hormone treatments. The biggest increase in tyrosine ³⁵SO₄ was observed in secreted proteins in the E₁S + P treatment group.

The patterns of ³⁵S-sulphate-labelled proteins were examined by SDS-polyacrylamide gel electrophoresis. In tissue extracts, the most striking differences related to the hormone treatments were observed in the M _r 94 000–190 000 region. A sulphated protein band of M _r 102 000 was specifically found in the E₂+ P group and a band of M _r 125 000 only in the E₁S+ P group. The M _r 125 000 band was also found in tissue proteins from the E₁S+ P-treated animals after the incorporation of ³⁵SO₄ in vivo. This protein band may be a marker of the action of oestrone sulphate plus progesterone. For secreted proteins, those with a molecular weight > 100 000 were more abundant in the oestrogen plus progesterone-treated groups than in the oestrogen-treated groups. The content of tyrosine sulphate in each protein band ranged from 8 to 25% of the total radioactivity. No protein sulphated exclusively on the tyrosine residues was found.

These studies provide the first description of the effects of steroid hormones on sulphated proteins in the guinea-pig uterus and suggest that oestrone sulphate is a potent biologically active hormone in the uterus.

Keywords: guinea-pig; protein; sulphation; uterus; hormones

Endometrial glandular epithelial cells were subcultured on matrix-coated filters in bicameral chambers in a serum-free chemically defined medium. The cells were untreated or treated with 50 nmol progesterone l⁻¹ or 10 nmol oestradiol l⁻¹ or 10 nmol oestradiol l⁻¹ plus 50 nmol progesterone l⁻¹ and the proteins secreted into the basal or apical compartment were analysed after [³⁵S]methionine labelling. Compared with the untreated cells, oestradiol treatment did not affect the electrophoretic profiles of proteins secreted by the glandular epithelial cells in either compartment. Progesterone treatment induced a decrease in the labelling of 88 and 53 kDa proteins secreted in the apical and basal compartments and an increase in the labelling of a 28 kDa protein. Moreover, progesterone specifically induced the apical secretion of a 137 kDa protein. Interaction between the epithelial and stromal cells was also investigated. When stromal cells were cultured in the basal compartment under the epithelial monolayer, the progesterone effect on the apical secretion of the 137 kDa protein and basal secretion of the 88 and 28 kDa proteins were altered, whereas this progesterone effect was not altered when the epithelial cells were cultured alone in media conditioned with stromal cells. Interactions between epithelial and stromal cells modified the effect of progesterone on protein secretion by the epithelial cells.