Proliferative Human Endometrium Research Articles

Estrogen signaling in the proliferative endometrium: implications in endometriosis.

Even though the physiological role of estrogen in the female reproductive cycle and endometrial proliferative phase is well established, the signaling pathways by which estrogen exerts its action in the endometrial tissue are still little known. In this regard, advancements in cell culture techniques and maintenance of endometrial cells in cultures enabled the discovery of new signaling mechanisms activated by estrogen in the normal endometrium and in endometriosis. This review aims to present the recent findings in the genomic and non-genomic estrogen signaling pathways in the proliferative human endometrium specifically associated with the pathogenesis and development of endometriosis.

Enhanced caveolin-1 expression increases migration, anchorage-independent growth and invasion of endometrial adenocarcinoma cells.

BackgroundCaveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease.MethodsHere, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar.ResultsImmunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4β-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4β-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells.ConclusionOur study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1477-5) contains supplementary material, which is available to authorized users.

Evaluation of Estrogen Treatment in an Immunodeficient Mouse Endometriosis Model

Background/Aims: Endometriosis is known to be an estrogen-dependent disease. However, only a few studies have analyzed the effect of estrogen treatment in mice xenotransplanted with human endometrium. The objective of this study was to adapt a previously developed heterologous murine model to the study of estrogens and test the impact of estrone treatment on endometriosis development. Methods: Human proliferative endometrium was xenotransplanted into the peritoneal cavity of castrated immunodeficient mice. These mice were treated with estrogens by means of subcutaneous estrone-releasing pellets. The effect of estrone on estradiol level, uterine histology and endometriosis development was evaluated after 21 days. Results: Bioactivity of estrone pellets and their metabolization into estradiol were demonstrated. However, there was no impact on endometriosis development (no difference in lesion number, weight, size or fluorescence). This lack of response was not due to absence of estrogen receptor expression, since strong expression was found in all lesions harvested. Surprisingly, castrated nontreated mice presented with lesions showing high proliferative activity, similar to lesions found in treated mice (around 30%). Conclusion: The high proliferation observed in lesions recovered from ovariectomized nontreated mice questions the utility of using estrogens in heterologous murine models.

Mixed origin of neovascularization of human endometrial grafts in immunodeficient mouse models

In vivo mouse models have been developed to study the physiology of normal and pathologic endometrium. Although angiogenesis is known to play an important role in endometrial physiology and pathology, the origin of neovasculature in xenografts remains controversial. The aim of this study was to assess the origin of the neovasculature of endometrial grafts in different mouse models. Human proliferative endometrium (n = 19 women) was grafted s.c. in two immunodeficient mouse strains: nude (n = 8) and severely compromised immunodeficient (SCID; n = 20). Mice were also treated with estradiol, progesterone or levonorgestrel. Fluorescence in-situ hybridization using a centromeric human chromosome X probe, immunohistochemistry (von Willebrand factor and collagen IV) and lectin perfusion were performed to identify the origin of the vessels. More than 90% of vessels within xenografts were of human origin 4 weeks after implantation. Some vessels (9.67 +/- 2.01%) were successively stained by human or mouse specific markers, suggesting the presence of chimeric vessels exhibiting a succession of human and murine portions. No difference in staining was observed between the two strains of mouse or different hormone treatments. Furthermore, erythrocytes were found inside human vessels, confirming their functionality. This article shows that human endometrial grafts retain their own vessels, which connect to the murine vasculature coming from the host tissue and become functional.

FHIT expression in neoplastic, hyperplastic, and normal endometrium

Fragile histidine triad (FHIT), a candidate of tumor suppressor protein, expression was examined on paraffin-embedded specimens in proliferative, secretory, hyperplastic, and neoplastic human endometrium by immunohistochemistry. The results of FHIT immunoreactivity in endometrial carcinomas were compared with prognostic indicators as well as with p53 overexpression. Forty-four cases of endometrial carcinoma, 30 normal functional (15 proliferative, 15 secretory), and 24 hyperplastic endometrium (12 without atypia, 12 with atypia) specimens were studied using polyclonal FHIT antibody. The streptavidin-biotin-peroxidase detection system was used, and the intensity and the distribution of immunoreactivity were evaluated semiquantitatively. There were no significant differences in FHIT expression in the proliferative, secretory, hyperplastic, either with or without atypia, or carcinomatous endometria. No significant difference in FHIT expression of endometrial carcinomas was detected when prognostic parameters or p53 overexpression were considered. Loss or reduced FHIT expression was not found to predict disease-free or cumulative survivals. This study showed that loss or reduction in FHIT protein expression is present in normal functional and hyperplastic endometria as well as in neoplastic endometrium. FHIT protein seems not to be involved directly in endometrial carcinogenesis, but rather, it regulates cell proliferation both in physiologic and in pathologic conditions of endometrium.

Differences in gene expression in the proliferative human endometrium

To use microdissection and DNA microarray technology to demonstrate differences in gene expression between epithelial and stromal areas in the proliferative human endometrium. Pilot study. University hospital. Patients with normal menstrual cycles and at least one previous intrauterine pregnancy. Uterine endometrial biopsy. Gene expression. From a total of 1,200 genes, 14 were strongly expressed in epithelial areas and 12 were strongly expressed in stromal areas. Among the genes strongly expressed in the stroma, expressions of decorin and discoidin domain receptor were confirmed by real-time polymerase chain reaction. Decorin was localized in the stromal areas by immunohistochemical staining. To confirm the effects of estrogen on gene expression, stromal cells were cultured. When E(2) was added to the culture media, expression of decorin mRNA was increased. The data demonstrated in this study help to understand the physiology of human endometrium. Decorin was strongly expressed in the stromal areas and was regulated by estrogen, and therefore it may be involved in restoration of the endometrium.

The Immune Environment in Human Endometrium during the Window of Implantation

Changes in the immune environment in the endometrium are believed to be important for successful implantation and maintenance of pregnancy. We have previously investigated global gene profiling in human endometrium during the window of implantation by oligonucleotide microarray technology, and analysis of these data underscore the regulation of a group of immune-related genes. The present study was therefore conducted to examine the pattern of expression and regulation of these genes including decay accelerating factor (DAF), indoleamine 2,3 dioxygenase (IDO), interleukin-15 (IL-15), IL-15 receptor alpha subunit (IL-15Ralpha), interferon regulatory factor-1 (IRF-1), lymphotactin (Lpn), natural killer-associated transcript 2 (NKAT2) and NKG5 in secretory and proliferative human endometrium. Endometrial biopsies were obtained from normally cycling women in the late proliferative and mid-secretory phase of the menstrual cycle. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analysis were used to determine the expression and regulation of these genes in secretory and proliferative human endometrium. Cellular localization of NKG5, Lpn and IDO by in situ hybridization in secretory-phase endometrium was also examined. Semi-quantitative RT-PCR and Northern blot results demonstrate that there is a coordinated upregulation of this group of genes during the window of implantation. We demonstrate the upregulation of immune-related genes IL-15Ralpha, Lpn and NKG5 in secretory versus proliferative human endometrium. We also demonstrate a similar upregulation in secretory endometrium of other immune-related genes, viz, DAF, IDO, IL-15, IRF-1 and NKAT2. The functions of these genes include stimulation of proliferation of uterine natural killer (uNK) cells, inhibition of cytolytic activity of uNK cells, inhibition of cell growth of T cells and other pathogens and inhibition of the classical complement pathway. Upregulation of these immune-related genes in the window of implantation suggests their role during the process of implantation and in immune tolerance of the implanting conceptus.

Effects of progestins of human proliferative endometrium: An in vitro model of potential clinical relevance

Endometrium biopsy is a useful indicator of endometrium proliferation and is clinically relevant to diagnose cell proliferation and to evaluate response to progestin treatment and to monitor hormone replacement therapy. The aim of our study was to investigate the in vitro effects of progesterone and synthetic progestins on endometrium explants with a particular focus on estradiol receptor (ER) and progesterone receptor (PR) expression which reflects through cell secretion the hormone treatment efficiency. Most widely used progestagens belonging to three distinctive groups were investigated, i.e, medroxyprogesterone acetate (MPA), norethindrone acetate (NOR) and nomegestrol acetate (TX) which are respectively pregnane, 19-nortestosterone and norpregnane derivatives. We used organ culture from human proliferative endometrium, in which tissue integrity, particularly gland/stroma relationships are preserved. Progestins induce epithelial cell secretion and most effects were observed at the highest concentration tested (10(-7) M) and by TX and MPA on homogeneous and on heterogeneous (including also secretory glands) proliferative endometrium respectively. In these conditions, ER as well as PR expression were decreased on both glandular and stromal cells. In contrast, progesterone at 10(-7) M significantly decreased only PR, in glands and in stroma of homogeneous proliferative endometrium, and just in stroma of heterogeneous endometrium. NOR exhibited less effects. At lower concentrations (10(-8) M, 10(-9) M), significantly less effects were observed by synthetic progestins on proliferative endometrium. The experiments show that the different types of progestins do not exhibit in vitro similar effects. Since progestins variably act on proliferative endometrium, the exposure of endometrium explants to progestins may be a useful tool to predict clinical response to hormone therapy (individual "hormonogram") and to monitor endometrium proliferation.

Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax.

Glandular epithelial cells of the human endometrium initiate apoptosis in the secretory phase of the cycle. To better understand the regulation of apoptosis in this paradigm of endocrine-regulated cell turnover, we studied the expression of the cell death regulatory genes, bax, bcl-2, and bcl-x, in human proliferative and secretory endometria relative to the absence or presence of apoptosis. As assessed by immunohistochemistry, levels of BAX protein were modest in proliferative endometrium and increased dramatically in the secretory phase when apoptosis was most prevalent. Expression of BAX was predominantly localized to epithelial cells of the functionalis layer of the secretory endometrium. In contrast, BCL-2 immunoreactivity was maximal during the proliferative phase and decreased in the secretory phase. Moreover, BCL-2 was topographically concentrated in the basalis layer. Immunoreactive BCL-X protein was observed mostly in glandular epithelial cells of the human endometrium. Compared with proliferative endometrium, secretory endometrium showed stronger BCL-X staining, especially in the functionalis layer. By Western blotting we confirmed that both proliferative and secretory endometrium expressed the long or antiapoptosis form as well as the short or proapoptosis form of the BCL-X protein. Taken together, our results demonstrate a coordinated pattern of expression of bcl-2 gene family members in human endometrium during the menstrual cycle, with a shift toward greater levels of the proapoptosis protein, BAX, occurring in glandular epithelial cells during the secretory phase of the cycle. Therefore, we conclude that cyclic changes in endometrial growth and regression may be precisely regulated by shifts in the ratio or balance of BCL-2 and related proteins in glandular epithelial cells.

Proto-oncogene c-fos and c-jun are expressed in both proliferative and secretory phase human endometrium

Proto-oncogene c-fos and c-jun are expressed in both proliferative and secretory phase human endometrium

Direct effect of gonadotropins on decidualization of human endometrial stromal cells

Decidualization of stromal cells isolated from proliferative human endometrium was achieved by adding to the culture medium human gonadotropins (FSH, FSH + LH, hCG). In addition to changes in the morphology of the stromal cells to the decidual phenotype, decidualization was evident from the expression of prolactin (PRL), demonstrated immunocytochemically, by Western blotting analysis, and by measuring its output into the medium through solid phase enzyme immunoassay. Gonadotropins also induced cAMP formation in the endometrial stromal cells under the same experimental conditions. This finding suggests that the mechanism by which gonadotropins promote decidualization of human endometrial stromal cells in vitro involves the introduction of cAMP, a compound that we have found to elicit the expression of PRL in this system. PRL is likely to be a key intermediate in the process of decidualization since it is by itself capable of inducing differentiation of the endometrial stromal cells to the decidual phenotype. Awareness of direct actions of gonadotropins on the endometrial cells and, in particular, of the decidualizing effects of FSH (Metrodin), FSH + LH (Pergonal) and hCG may contribute to the understanding of physiologic as well as pathophysiologic conditions relevant to endometrial functions and fertility.

Cyclic adenosine 3',5'-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium

In vitro decidualization of stromal cells isolated from human proliferative endometrium and cultured in RPMI-1640 containing 2% charcoal-treated fetal bovine serum and 0.1 U/ml insulin was achieved by adding to the medium cAMP derivatives [(Bu)2cAMP (db-cAMP) and 8-bromo-cAMP] or forskolin. PRL production under these conditions was demonstrated by documenting the synthesis of PRL mRNA (approximately 1.1 kilobase), the output of immunoprecipitable [35S]methionine-labeled PRL migrating as a single 23-kilodalton band during gel electrophoresis, and the time- and concentration-dependent secretion of PRL into the medium, measured by RIA (maximal on days 4-5 using 0.5 mM db-cAMP). Medroxyprogesterone acetate (1 microM) enhanced (1.7- to 2.5-fold) the effect of db-cAMP, 8-bromo-cAMP, or forskolin on PRL production, as evaluated by Western blotting analysis. Further evidence for a participation of db-cAMP in the decidualization process was provided by its ability to induce immunocytochemically detectable heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin, all compounds produced by human decidual cells, but not expressed by stromal cells. The induction of PRL by cAMP may be a key step in the process of differentiation of fibroblast-like stromal cells to the decidual phenotype, as it has been previously reported by this laboratory that, under similar culture conditions, PRL itself is capable of inducing the production of heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin in stromal cells isolated from proliferative endometrium.

Cyclic adenosine 3',5'-monophosphate induces prolactin expression in stromal cells isolated from human proliferative endometrium.

In vitro decidualization of stromal cells isolated from human proliferative endometrium and cultured in RPMI-1640 containing 2% charcoal-treated fetal bovine serum and 0.1 U/ml insulin was achieved by adding to the medium cAMP derivatives [(Bu)2cAMP (db-cAMP) and 8-bromo-cAMP] or forskolin. PRL production under these conditions was demonstrated by documenting the synthesis of PRL mRNA (approximately 1.1 kilobase), the output of immunoprecipitable [35S]methionine-labeled PRL migrating as a single 23-kilodalton band during gel electrophoresis, and the time- and concentration-dependent secretion of PRL into the medium, measured by RIA (maximal on days 4-5 using 0.5 mM db-cAMP). Medroxyprogesterone acetate (1 microM) enhanced (1.7- to 2.5-fold) the effect of db-cAMP, 8-bromo-cAMP, or forskolin on PRL production, as evaluated by Western blotting analysis. Further evidence for a participation of db-cAMP in the decidualization process was provided by its ability to induce immunocytochemically detectable heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin, all compounds produced by human decidual cells, but not expressed by stromal cells. The induction of PRL by cAMP may be a key step in the process of differentiation of fibroblast-like stromal cells to the decidual phenotype, as it has been previously reported by this laboratory that, under similar culture conditions, PRL itself is capable of inducing the production of heat shock protein-27, insulin-like growth factor-binding protein-1, desmin, and laminin in stromal cells isolated from proliferative endometrium.

Activation of phospholipase A2 and phospholipase C by endothelin-1 in human endometrium.

Human endometrium contains specific binding sites for iodinated endothelin (ET)-1, ET-2 and ET-3, and ET-1 stimulates prostaglandin (PG) F2 alpha synthesis from explants of proliferative endometrium in short-term culture. This study has investigated the cellular responses of normal proliferative endometrium to ET-1. Radioimmunoassay was used to measure PG release and Dowex anion-exchange column chromatography was utilized to assess the accumulation of inositol phosphates. Endothelin-1 induced a significant increase in PGF2 alpha release (basal median: 1465 pg/mg per 60 min (range: 541-3935 pg/mg per 60 min); ET-1-stimulated: 1813 pg/mg per 60 min (1021-5714 pg/mg per 60 min); P < 0.04 using Wilcoxon signed rank test). The effect of ET-1 was attenuated in the presence of the phospholipase A2 inhibitor quinacrine. Endothelin-1 induced a rapid, transient and concentration-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), measured by the accumulation of tritiated inositol phosphates. Following a 1-min stimulation with ET-1 (100 nmol/l), [3H]inositol mono-, bis- and trisphosphate fractions increased from median values of 490.0 d.p.m./mg dry wt (range: 348.0-807.0 d.p.m./mg dry wt), 120.0 d.p.m./mg dry wt (93.6-144.1 d.p.m./mg dry wt) and 67.0 d.p.m./mg dry wt (54.2-85.0 d.p.m./mg dry wt) to 939.0 d.p.m./mg dry wt (635.9-1596.0 d.p.m./mg dry wt; P < 0.03), 145.0 d.p.m./mg dry wt (127.0-293.9 d.p.m./mg dry wt; P < 0.05) and 146.0 d.p.m./mg dry wt (77.5-187.0 d.p.m./mg dry wt; P < 0.03) respectively. These results suggest that ET-1 activates the phospholipase A2 and PtdIns(4,5)P2-specific phospholipase C in human proliferative endometrium, resulting in the generation of PGF2 alpha and second messengers respectively which are pivotal to endometrial function.

The effect of progesterone and human interferon α-2 on the release of PGF 2α and PGE from epithelial cells of human proliferative endometrium

Progesterone and interferon-like trophoblastic proteins modulate prostaglandin (PG) synthesis from endometrium in early ovine and bovine pregnancy. Enriched epithelial cells were prepared from human endometrium removed in the proliferative phase of the menstrual cycle (n=8). Progesterone at a concentration of 1 μM suppressed PGE release from the cells during the first 24 hours in culture. After 48 hours in culture progesterone at a dose of 100 nM and 1 μM suppressed both the release of PGF 2α and PGE from the cells and this suppression was maintained for a further two days. Addition of exogenous 30 μM arachidonic acid (AA) abolished this effect of progesterone on both PGF 2α and PGE release. Interferon α-2 did not suppress the basal release of PGF 2α nor PGE. In the presence of progesterone, interferon α-2 attenuated the progesterone mediated suppression of PGF 2α but not PGE release from endometrial cells. These findings suggest that progesterone suppresses the basal release of PGs from human endometrium, but unlike the sheep, interferon α-2 does not exert this action on human endometrium.

In vitro decidualization of human endometrial stromal cells

Stromal cells isolated from proliferative human endometrium undergo morphologic and biochemical changes when exposed to a mixture of ovarian hormones, acquiring characteristics of decidual cells. In addition to the previously reported progestin-induced secretion of prolactin (PRL) by explants of human proliferative endometrium, and of PRL and laminin by stromal cells in culture, “ in vitro” induction of several other decidual cell products was demonstrated in the present study, using cultures of stromal cells isolated from proliferative endometrium. Incubation of stromal cells with a mixture of estradiol, medroxyprogesterone acetate and relaxin, at a concentration reported to yield maximal stimulation of PRL production, resulted in changes from elongated to rounder cells, approx. 90% of which showed immunostaining for PRL under these conditions. Immunocytochemical procedures were carried out on cytospins of decidual cells isolated from decidual tissue adherent to fetal membranes collected at delivery (positive controls), and on stromal cells cultured in Lab-Tek chamber-slides, in the absence (negative controls) or in the presence of added hormones. Antibodies to 24K (a heat-shock protein also named HRP27), desmin (present in intermediate filaments), p29 (a protein associated with the estrogen receptor), and PP12 (an insulin growth factor-1 binding protein), did not react with stromal cells isolated from proliferative endometrium but showed immunostaining of the rounder cells obtained after hormonal treatment when tested with the peroxidase-labeled second antibody complex. In another series of similar experiments, in which the same decidualization end-points were emploted, changes in 24K, desmin and PP12 expression were obtained by adding to the insulin-containing medium PRL instead of the hormonal mixture, a finding suggesting sequential steps during the decidualization process.

Insulin-Like Growth Factor Binding Proteins in Human Endometrium: Steroid-Dependent Messenger Ribonucleic Acid Expression and Protein Synthesis*

The insulin-like growth factor (IGF) autocrine/paracrine system is believed to play a role in endometrial differentiation and trophoblast growth. The IGFs bind with high affinity to a family of binding proteins (IGFBPs) that regulate the actions of the IGFs at their target cells. IGFBP-1, one of three well-characterized IGFBPs in humans, has been shown by numerous investigators to be present in secretory but not proliferative endometrium; however, no information is available with regard to two other human IGFBPs, IGFBP-2 and IGFBP-3, in normal human endometrium. We have examined expression of the messenger RNAs (mRNAs) encoding IGFBP-2 and IGFBP-3 in human proliferative endometrium [under the influence of estradiol (E2)] compared to secretory endometrium (under the influence of E2 and progesterone). Using a complementary DNA probe specific for IGFBP-2, by Northern analysis, expression of a 1.4 kilobase mRNA was found to be differentially expressed in secretory compared to proliferative phase endometrium. Using a complementary DNA probe encoding IGFBP-3, a 2.4 kilobase mRNA was found in endometrium and was also found to be differentially expressed in secretory compared to proliferative phase endometrium. Endometrial stromal cells were established in culture and revealed constitutive synthesis and secretion of IGFBP-2 and IGFBP-3 into the conditioned medium (CM), as detected by Western ligand blot analysis of the CM. Identification of the IGFBPs in the CM was made using IGFBP-2 and IGFBP-3 specific antiserum. In the presence of E2 and progesterone, there was an enhancement in the amount of IGFBP-3 and a marked enhancement of IGFBP-2 in the conditioned media, suggesting sex steroid-dependence of IGFBP-2 and, to a lesser extent, IGFBP-3 protein synthesis. Western ligand blot analysis of IGFBPs in serum throughout the menstrual cycle revealed no hormonal dependence in the serum IGFBPs, suggesting local action of the IGFBPs in endometrium. These data thus show that mRNAs encoding IGFBP-2 and IGFBP-3 are expressed in human endometrium, that their expression is differentially regulated in secretory compared to proliferative phase endometrium (different steroidogenic environments), and that the synthesis of both IGFBP-2 and IGFBP-3 proteins is regulated by steroid hormones.

In vitro induction of prolactin production and aromatase activity by gonadal steroids exclusively in the stroma of separated proliferative human endometrium

Prolactin production by decidualized human endometrium has been demonstrated in organ explant cultures and presumed to be of stromal origin by immunocytochemistry and the presence of simultaneous stromal histologic changes. To test the hypothesis that endometrial prolactin production is exclusively of stromal origin, late proliferative human endometrium was separated into glands and stroma and cultured for 3 days. Confluent cultures were incubated with progesterone, 50 ng/ml; estradiol, 5 ng/ml; or both progesterone and estradiol for 4, 8, and 15 days. Prolactin concentration was measured at 2-day intervals by specific radioimmunoassay throughout a 15-day culture in all samples. Aromatase activity at 15 days was assayed by the production of tritiated water from 1 beta 3H-androstenedione. Possible contamination of gland cultures by stromal cells was controlled by the substitution of D-valine in the culture media. No prolactin was detected in the D-valine-treated gland cultures. Stromal cell cultures demonstrated increasing prolactin with continuous incubation with progesterone (15-day control: 11.28 +/- 0.91 ng/100 micrograms deoxyribonucleic acid versus 15-day progesterone: 245.8 +/- 4.24 ng/100 micrograms). Prolactin levels increased after progesterone was removed at day 4 and 8, reaching peak production 4 days later, followed by a steady decline. Estradiol induced a sustained increase in prolactin production even after withdrawal of steroids. Aromatase activity in gland cells exposed to steroids exhibited no change with time. Steroid-treated stromal cell cultures showed an increase in aromatase activity in all 15-day (controls: 21.6 +/- 0.7 pmol substrate converted per mg of deoxyribonucleic acid per 24 hours versus progesterone: 43.4 +/- 4.2 versus estradiol: 34.2 +/- 3.5 versus progesterone and estradiol: 47.5 +/- 2.4) but not in 4- or 8-day incubations. These studies support these conclusions: (1) nongestational endometrial prolactin is of stromal, not glandular origin; (2) stromal prolactin production is induced and maintained by progesterone: (3) stromal prolactin production is induced by estradiol but does not require continued exposure for maintenance; (4) aromatase activity is increased by long-term exposure to progesterone and estradiol in stroma but not in glands.

The effect of cortisol on the synthesis of prostaglandins (PGF 2α, PGE 2) by human endometrial fibroblasts in vitro with and without addition of estradiol-17β or progesterone

Cortisol is known as a potent inhibitor of phospholipase A 2 activity in several tissues. In fibroblast monolayer cell cultures from proliferative human endometrium cortisol alone does not affect the basal PGF 2α or PGE 2 synthesis. After stimulation of PGF 2α production by 10 −7 mol/l estradiol-17β increasing concentrations of cortisol up to 10 −5 mol/l dosedependently reduce the PGF 2α production. Also the progesterone (10 −4 mol/l ) stimulated increase of PGF 2α and PGE 2 synthesis is inhibited by cortisol (10 −7 mol/l).

Effects of progesterone and oestradiol-17 beta on 17 beta-ol-dehydrogenase activity in stromal cells of human endometrium under in-vitro conditions.

A 17 beta-ol-dehydrogenase activity could be demonstrated in fibroblast monolayer cell cultures of proliferative human endometrium. After 24 h incubation 100 nCi [6,7-3H]oestradiol-17 beta was completely oxidized to oestrone. Progesterone was not able to enhance the metabolizing velocity. In contrast, progesterone incubation revealed a decreasing oxidation rate with increasing molarity. Histological changes after transformation of the endometrium are discussed to explain in-vivo results showing an increased 17 beta-ol-dehydrogenase activity in the secretory phase of the cycle.