Progesterone metabolism in human endometrial stromal and gland cells in culture.

Metabolism of progesterone by human endometrium has been described, but the rapidity and extent of progesterone metabolism is incompletely documented in cellular fractions of normal endometrium. Therefore, we evaluated progesterone metabolism in separated stromal and gland cells in culture obtained from normal human endometrium by thin-layer chromatography. We find that in both cell types, the most abundant metabolite is 3beta-hydroxy-5alpha-pregnan-20-one (70%), followed by 5alpha-pregnane-3,20-dione (15%), and 3alpha-hydroxy-5alpha-pregnan-20-one (10%). A small amount is metabolized to 5alpha-pregnane-3alpha/3beta,20alpha-diols and to 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. The metabolism of progesterone in cultured endometrial cells occurs rapidly; 70% of progesterone is metabolised in 8 h, and 90% by 24 h. We conclude that when in vitro experiments are conducted utilizing progesterone treatment, the rapidity and the extent of the metabolism of this steroid should be taken into account.     

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.     

Progesterone in vitro stimulates secretion of a specific protein by ovine epithelial endometrial cells

Cultured ovine epithelial endometrial cells from oestrogen-treated ovariectomized ewes were treated in vitro with combinations of oestradiol-17 beta (E), progesterone (P) and the P-receptor antagonist RU486 (each 10(-6) to 10(-9) M), in the presence of [35S]methionine. Neither DNA content of dishes nor total protein were increased in treatment compared with control dishes. Incorporation of [35S] into secreted protein was lower from cells treated in vitro with P or E + P (10(-9) M) than from those treated with E (10(-9) M, P less than 0.01). Incorporation of [35S] into cellular protein was decreased by P (10(-9) M, P less than 0.025). SDS-PAGE analysis of secreted proteins enabled measurement of levels of a 46K protein which is secreted maximally following E + P administration in vivo. In vitro, P either alone or with E (each 10(-7) M) increased the abundance of the 46K protein in cell secretions by a factor of 1.5 +/- 0.1 (N = 9) or 1.8 +/- 0.3 (N = 10) respectively (P less than 0.01) compared with controls. The administration of E (10(-7) M) or either or both steroids at 10(-9) M, was without effect. RU486 alone (10(-6) to 10(-8) M) was also without effect but in the presence of E + P or P, blocked the increase in the 46K protein, suggesting this effect is mediated via binding of P to its receptor.     

Heat shock induces protein tyrosine phosphorylation in cultured cells

We examined the effect of heat shock on protein tyrosine phosphorylation in cultured animal cells using antiphosphotyrosine antibodies in immunoblotting and immunofluorescence microscopy experiments. Heat shock significantly elevated the level of phosphotyrosine in proteins in most of the cultured cells examined, including fibroblasts, epithelial cells, nerve cells, and muscle cells, but not in Rous sarcoma virus-transformed fibroblasts. The increase in protein tyrosine phosphorylation induced by heat shock occurred in proteins with a wide range of molecular masses and was dependent on the temperature and duration of the heat shock.     

Profound architectural and functional readjustments of the secretory pathway in decidualization of endometrial stromal cells

Certain cell types must expand their exocytic pathway to guarantee efficiency and fidelity of protein secretion. A spectacular case is offered by decidualizing human endometrial stromal cells (EnSCs). In the midluteal phase of the menstrual cycle, progesterone stimulation induces proliferating EnSCs to differentiate into professional secretors releasing proteins essential for efficient blastocyst implantation. Here, we describe the architectural rearrangements of the secretory pathway of a human EnSC line (TERT-immortalized human endometrial stromal cells (T-HESC)). As in primary cells, decidualization entails proliferation arrest and the coordinated expansion of the entire secretory pathway without detectable activation of unfolded protein response (UPR) pathways. Decidualization proceeds also in the absence of ascorbic acid, an essential cofactor for collagen biogenesis, despite also the secretion of some proteins whose folding does not depend on vitamin C is impaired. However, even in these conditions, no overt UPR induction can be detected. Morphometric analyses reveal that the exocytic pathway does not increase relatively to the volume of the cell. Thus, differently from other cell types, abundant production is guaranteed by a coordinated increase of the cell size following arrest of proliferation.     

Progesterone treatment in vitro enhances prostaglandin E and forskolin-promoted cyclic AMP production in human endometrial stromal cells

Confluent human endometrial stromal cell cultures were exposed to steroids for up to 72 h and then stimulated with agonists of adenylate cyclase for 60 min. Neither steroid alone or in combination had significant effect on cyclic AMP production. However, when stromal cell adenylate cyclase was stimulated with a receptor-dependent agonist (prostaglandin E), or with forskolin (which acts at a post-receptor site), progesterone in oestradiol-primed cells markedly enhanced (P less than 0.05) the effect of both agonists. The presence of phenol red, a weak oestrogenic compound, in the standard culture medium was sufficient to allow the progesterone effect to be manifest. Moreover, while oestradiol alone had no significant effect on prostaglandin E or forskolin-stimulated cyclic AMP production, the simultaneous exposure of cells to oestradiol and progesterone was the most effective treatment. Short-term incubation (up to 120 min) with progesterone had no effect on agonist-induced cyclic AMP accumulation, indicating that progesterone elicits its effect by the classic nuclear mechanism of action. It is suggested that the potentiation by progesterone of prostaglandin E-promoted production of cyclic AMP represents an important aspect of the functional role progesterone plays in the preparation of the endometrium for implantation.     

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.     

Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro.

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.     

Influenza virus ns1 protein induces apoptosis in cultured cells

The importance of influenza viruses as worldwide pathogens in humans, domestic animals, and poultry is well recognized. Discerning how influenza viruses interact with the host at a cellular level is crucial for a better understanding of viral pathogenesis. Influenza viruses induce apoptosis through mechanisms involving the interplay of cellular and viral factors that may depend on the cell type. However, it is unclear which viral genes induce apoptosis. In these studies, we show that the expression of the nonstructural (NS) gene of influenza A virus is sufficient to induce apoptosis in MDCK and HeLa cells. Further studies showed that the multimerization domain of the NS1 protein but not the effector domain is required for apoptosis. However, this mutation is not sufficient to inhibit apoptosis using whole virus.     

Decrease of estrogen receptors induced by 17 beta-estradiol and progesterone in cultured rabbit endometrial cells

A whole cell technique to measure estradiol receptors in cultured rabbit endometrial cells is described. The estradiol receptors measured appear to be composed of at least two components: one component has high affinity but low capacity, while the other component has low affinity but high capacity. Using the assay, the effects of estradiol and progesterone pretreatment were examined on the estradiol receptor levels. It was found that both of the hormones decreased the number of estrogen receptors in the cultured cells. The finding that estradiol decreased its own receptors was unexpected and its possible relevance is discussed.     

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

Summary Proteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.Supported by grant Ki 154/6-7 from the Deutsche Forschungsgemeinschaft     

Mouse endometrial stromal cells produce basement-membrane components

During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The decidualization of stromal cells is characterized by the accumulation of pericellular basement-membrane material. Decidualized stromal cells of pregnancy differ in this respect from stromal cells obtained from nonpregnant uteri, which are not associated with any significant amounts of basement-membrane components. Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells synthesize laminin, entactin, fibronectin, type-IV collagen, and heparan sulfate proteoglycan. Stromal cells isolated from nonpregnant uteri and explanted in vitro produced the same basement-membrane components. Apparently, these cells were stimulated by the procedure used during isolation and explanation to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components.     

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.     

Isolation of a storage and secretory organelle containing Von Willebrand protein from cultured human endothelial cells

Von Willebrand protein was synthesized and secreted by human endothelial cells in culture. Ca²⁺ ionophore A23187 and phorbol myristate acetate stimulated the release of Von Willebrand protein from the cultured cells. Stimulated release was accompanied by the disappearance of rod-like structures from the cultured endothelial cells immunostained for Von Willebrand protein, suggesting the existence of a storage organelle for Von Willebrand protein in these cells (Loesberg, C., Gonsalves, M.D., Zandbergen, J., Willems, C., Van Aken, W.G., Stel, H.V., Van Mourik, J.A. and De Groot, P.G. (1983) Biochim. Biophys. Acta 763, 160–168). Cultured human endothelial cells were fractionated on a density gradient of colloidal silica. Von Willebrand protein was found in two organelle populations: a buoyant one sedimenting with a variety of cell organelle marker enzymes, including those of the Golgi apparatus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum and plasma membrane fragments (peak density of this fraction: 1.08 g·ml^?1), and a dense one with a peak density of 1.12 g·ml^?1. The dense organelles containing Von Willebrand protein were apparently free of other organelles. Stimulating Von Willebrand protein release with phorbol myristate acetate or Ca²⁺ ionophore A23187 resulted in a decrease or even complete disappearance of Von Willebrand protein from the high-density organelle fraction, implying a role of this organelle in the stimulus-induced release of Von Willebrand protein. The Von Willebrand protein content of the buoyant fraction was lowered to some extent or did not change upon incubation of the cells with ionophore A23187 and phorbol myristate acetate. Restoration of Von Willebrand protein content of the dense organelle fraction after stimulation occurred within 2 days; this was accompanied by recurrence of immunostaining of rod-shaped structures in cells and an increase in cellular Von Willebrand protein. The excretion of restored Von Willebrand protein could be stimulated again.     

Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep)

Exogenous progesterone given early in the ovine estrous cycle results in precocious luteolysis. It has been suggested that under this circumstance regression of the corpus luteum is caused by an advancement in the timing of uterine secretion of prostaglandin F2 alpha. Uterine tissues were obtained from ewes following administration of progesterone (or injection vehicle), fixed in paraformaldehyde, and embedded in paraffin. Tissue sections were hybridized using an 35S-labeled cRNA probe specific for cyclooxygenase mRNA. There was approximately an eight-fold increase in the level of hybridization signal, localized mainly to uterine glands, consequential to treatment with progesterone. Thus, progesterone appears to control expression of the endometrial gene and(or) the stability of the message encoding for a rate-limiting enzyme involved in metabolism of arachidonic acid to its luteolytic product, thereby resolving the length of the nonpregnant cycle.