Human Uterine Fibroblasts Research Articles

Autocrine Prolactin Inhibits Human Uterine Decidualization: A Novel Role for Prolactin1

Human prolactin (PRL) and its receptor (PRLR) are markedly induced during human uterine decidualization, and large amounts of PRL are released by decidual cells as differentiation progresses. However, the role of PRL in decidualization is unknown. In order to determine whether PRL plays an autocrine role in decidualization, human uterine fibroblast cells that were decidualized in vitro with medroxyprogestrerone acetate (1 microM), estradiol (10 nM), and prostaglandin E(2) (1 microM) were exposed to exogenous PRL and/or the pure PRLR antagonist delta1-9-G129R-PRL. As measured by quantitative PCR, cells that were decidualized in the presence of exogenous PRL (0.25-2 microg/ml) expressed significantly lower levels of mRNA for the genes that encode insulin-like growth factor binding protein 1 (IGFBP1), left-right determination factor 2 (LEFTY2), PRL, decorin (DCN), and laminin alpha 1 (LAMA1), all of which are known to be induced during decidualization. These effects were blocked when the cells were exposed simultaneously to PRL and the PRLR antagonist, which confirms the specific inhibitory action of PRL on the expression of decidualization markers. In addition, cells exposed to the PRLR antagonist alone expressed higher levels of the marker gene mRNAs than cells that were decidualized in control media. Taken together, these results strongly suggest that PRL acts via an autocrine mechanism to regulate negatively the extent of differentiation (decidualization) of human uterine cells.

124 A THREE-DIMENSIONAL IN VITRO IMPLANTATION MODEL WITH NONHUMAN PRIMATE EMBRYOS AND EXTRACELLULAR MATRIX UNDER VARIOUS CULTURE CONDITIONS

The need for blastocyst culture and post-implantation embryo research has emerged in the past few years. Our objective is to evaluate a novel in vitro model to study implantation and placenta formation in vitro with rhesus macaque embryos under various culture conditions. A novel nonhuman primate in vitro 3-D system can provide cues for implantation and interaction with the extracellular environment not available in 2-D planar models. Optimization of such a model can be tested with diverse culture environments. We developed and evaluated an in vitro 3-D implantation model utilizing IVF-derived, blastocyst-stage rhesus macaque embryos embedded in 3-D Matrigel droplets cultured with different feeder cells and media. Signs of implantation including enlargement of the embryo mass, invasion and proliferation of trophectoderm cell layers, cystic formation, and cellular outgrowths derived from the embryo were initiated within the first week post-embedding. Trophoblast structures with protrusion and branches growing from the surface of embryo implants were observed. Immunohistochemical staining for chorionic gonadotropin (CG) combined with immunoassays for CG and progesterone indicated differentiation of trophoblastic cell lineages. In addition, we found morphological factors, such as proliferation of embryonic and extraembryonic structures, as well as initiation of protrusions interacting with the extracellular matrix, to predict successful establishment of prolonged embryo development. We further evaluated effects of different types of feeder cells and media combinations, and found that a combination of BRL, Ishikawa cells, and human uterine fibroblasts, provided an optimized culture microenvironment to promote peri-implantation embryo development and hormone secretion including CG and progesterone. In conclusion, we have established a 3-D in vitro system modeling implantation initiation, and demonstrating the capability of the embryo to interact with the extracellular matrix. Further studies will facilitate the methodology of peri-implantation blastocyst culture and accelerate our understanding of nonhuman primate embryo development, with potential for insights into early pregnancy loss and related pathologies. The present study and future directions may be extended to provide retrospective views on blastocyst selection for embryo transfer in assisted reproductive technology. This study was funded by NIH grants RR000167, RR21876, and HD053926.

O-185: Genome-wide expression analysis of human uterine fibroblast cells following in vitro decidualization with IL1β, prostaglandin E2, or butaprost treatment

Prostaglandin E2 (PGE2), butaprost, and IL1β treatments independently stimulate decidualization of human uterine fibroblast cells in vitro. To better understand the molecular mechanisms regulated by these decidualization-inducing molecules, analysis of the expression of over 39,000 unique transcripts in human uterine fibroblast cells (HC11) was performed.

Cytokines Regulate Matrix Metalloproteinases in Human Uterine Endometrial Fibroblast Cells Through a Mechanism That Does Not Involve Increases in Extracellular Matrix Metalloproteinase Inducer

Endometriosis is the presence of ectopic uterine endometrial tissue in the peritoneal cavity. Peritoneal fluid samples of women with endometriosis show elevated interleukin-1 (IL-1)beta, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta(1) levels, indicating that an altered immune system may play an important role in the pathogenesis of endometriosis. The invasion of ectopic endometrium into peritoneal mesothelium requires matrix metalloproteinases (MMPs) for tissue remodeling. Several MMPs are differentially expressed in human uterine endometrium with menstrual endometrium showing the highest level of expression. MMPs are stimulated by cytokines and also by the protein Extracellular Matrix Metalloproteinase Inducer (EMMPRIN). To determine the role of cytokines in ectopic endometrial invasion, we investigated whether cytokines could regulate MMP production by endometrial fibroblast cells and whether this stimulation occurred through an effect on EMMPRIN expression. Human uterine fibroblasts (HUF) were treated with IL-1beta, TGF-beta(1) and TNF-alpha in a dose dependent and time dependent manner (C, 0.1, 1, 10 ng/mL IL-1beta or TGF-beta(1); C, 2, 10, 50 ng/mL TNF-alpha) for 0, 6, 12, and 24 hr. Cell conditioned medium samples were collected and concentrated at each timepoint for immunoblot analysis. Cellular RNA was collected for real time PCR analysis of MMPs-1, -2, -3 and EMMPRIN mRNA levels. Our results showed that IL-1beta stimulated MMP-1 protein secretion and mRNA levels in a time dependent manner (P < 0.05), MMP-2 mRNA in a time dependent manner and MMP-3 in a time and dose dependent manner. TNF-alpha stimulated MMP-1 and -3 protein secretion in a time dependent manner and stimulated MMP-1, -2 and -3 mRNA levels in a time dependent manner (P < 0.05). Neither IL-1beta nor TNF-alpha treatment affected MMP-2 protein secretion. TGF-beta(1) inhibited MMP-1 and MMP-2 mRNAs at the highest treatment dose after 24 hr but there was no effect on protein secretion. TGF-beta(1) exerted no effect on MMP-3 mRNA or protein secretion (P < 0.05). Neither of the cytokines affected EMMPRIN protein or mRNA levels but the 10 ng/mL TGF-beta(1) treatment did cause a reduction in EMMPRIN mRNA levels. These data show that elevated cytokines may play a role in the establishment of ectopic endometrium in the peritoneal cavity by stimulating MMPs to remodel the mesothelial lining of the peritoneum thus allowing for tissue invasion. The stimulation of MMPs by cytokines occurred without any change in EMMPRIN expression whereas the inhibitory effect of TGF-beta(1) involved a reduction in EMMPRIN mRNA levels.

Extracellular Matrix Metalloproteinase Inducer Regulates Metalloproteinases in Human Uterine Endometrium

Endometrial remodeling occurs during each menstrual cycle in women and also during the establishment of endometriosis. Both processes involve the production of metalloproteinases (MMPs) by uterine endometrial cells. The objective of this study was to determine whether tissue remodeling and endometrial invasion involve activation of MMPs by extracellular matrix metalloproteinase inducer (EMMPRIN). EMMPRIN expression was examined by immunohistochemistry and real-time PCR in ectopic and eutopic endometria. For functional assays, human uterine fibroblasts were treated in the absence or presence of IL-1beta (10 ng/ml) or purified native EMMPRIN (0.5 or 1 microg/ml) for 24 h. Cellular RNA and conditioned medium were assayed by real-time PCR or immunoblotting. EMMPRIN protein localized to epithelial and fibroblast cells of eutopic and ectopic endometria. The pattern of localization was regulated by ovarian hormones. EMMPRIN mRNA levels varied throughout the menstrual cycle in parallel with the cyclic changes in estradiol. EMMPRIN treatment (0.5 microg/ml) of human uterine fibroblast cells stimulated MMP-1 (5.23-fold) and MMP-2 (8.55-fold), but not MMP-3, mRNA levels over levels in control cells (P < 0.05). EMMPRIN treatment (1 microg/ml) stimulated endogenous EMMPRIN (1.6-fold) mRNA levels (P > 0.05). IL-1beta stimulated MMP-1 (5.6-fold), MMP-2 (2.8-fold), and MMP-3 (75-fold) gene expression, but not EMMPRIN, over levels in control cells (P < 0.05). Both EMMPRIN and IL-1beta treatments stimulated MMP-1, -2, and -3, but not EMMPRIN protein secretion, with 0.5 microg/ml producing the greatest response. The ability of EMMPRIN to stimulate MMP secretion by endometrial fibroblasts indicates its potential role in uterine remodeling and the pathogenesis of endometriosis.

Increased Phosphorylation of Myosin Light Chain (MLC) Prevents Human Uterine Fibroblasts (HuF) Decidualization

Increased Phosphorylation of Myosin Light Chain (MLC) Prevents Human Uterine Fibroblasts (HuF) Decidualization

Prostaglandin E 2 and Interleukin-1β Stimulate Interleukin-11 in Human Uterine Fibroblast (HuF) Cells by Distinct Signaling Pathways

Decidualization of stromal cells is an important physiological event for successful implantation of an embryo. Factors such as prostaglandin E2 (PGE2) and IL1β are key regulators of decidualization. Signaling by IL-11 is essential for implantation in the mouse. In this study we examined the regulation of IL-11 secretion by PGE2 and IL-1β in human stromal cells. We also studied the signaling pathways involved in the induction of IL-11 by these factors.

Expression of tumor necrosis factor‐α stimulated gene‐6 mRNA in cultured human uterine cervical fibroblasts

Uterine cervical ripening process is an active biochemical process similar in part to inflammatory reaction. In this process, hyaluronan plays important roles including facilitation of tissue hydration, release of matrix metalloproteinases and migration of inflammatory cells. The activities of hyaluronan are mediated by the hyaluronan binding proteins, hyaladherins. In the present study, we investigated the mRNA expression of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a member of the hyaladherin family, in cultured human uterine fibroblasts and uterine cervical tissues. We developed one-step RT-PCR method for the quantification of TSG-6 mRNA and quantified the expression of TSG-6 mRNA in cultured human uterine fibroblasts, treated with or not proinflammatory cytokines, and TSG-6 mRNA in uterine cervical tissues. We clarified that [1] TSG-6 mRNA was expressed constitutively in cultured human uterine cervical fibroblasts, [2] expression of TSG-6 mRNA was upregulated in a dose dependent manner by proinflammatory cytokines, such as IL-1beta and TNF-alpha, which were key mediators in the cervical ripening process, [3] expression of TSG-6 mRNA in uterine cervices during parturition was significantly (P < 0.05) higher than that in a non-pregnant state. Our results suggest that TSG-6 might participate in the cervical ripening process.

Internalization of urinary trypsin inhibitor in human uterine fibroblasts.

We have characterized the molecular species and internalization of urinary trypsin inhibitor (UTI) in human uterine fibroblasts. Link protein (LP) has previously been identified as one of the cell-associated UTI binding proteins. The truncated forms of UTI were readily detectable in the cells after incubating the cells with purified UTI. Immunoblotting analysis with a panel of domain-specific antibodies revealed that the UTI species lacked the amino-terminal domain of UTI, but contained the carboxyl-terminal domain. We have examined whether LP is involved in the UTI internalization in the cells. Internalization of 125I-labelled UTI was blocked by the intact UTI, but not by the carboxyl-terminal domain of UTI. Treatment with a polyclonal antibody to the UTI binding domain of LP partially inhibited UTI binding to the cells, but did not significantly prevent UTI internalization. In addition, preincubation of the cells with hyaluronidase reduced the UTI binding to the cells, but had no effect on the rate with which UTI was internalized. These data allow us to conclude that there are at least two different mechanisms for internalization of UTI. The major one is via unknown UTI receptors in a Ca2+, Mg2+-sensitive manner and another is via LP.

Effects of Estradiol and Testosterone on the Synthesis, Expression and Degradation of Androgen Receptor in Human Uterine Endometrial Fibroblasts.

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17beta-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p < 0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p < 0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts. Copyright 1995 S. Karger AG, Basel

Effect of 5-bromodeoxyuridine incorporation on survival of cultured mammalian cells

The analog 5-bromodeoxyuridine replaced 93–100% of DNA thymine in human-uterine fibroblasts and in mouse firboblasts when amethopterin was used to inhibit the synthesis de novo of thymidylate. The incorporation of the analog was followed by unbalanced growth and finally cell death. Even when extensive incorporation had occurred, some cells were able to resume normal growth in analog-free medium. This involved a drastic selection revealed by the appearance of isolated clones after 2.5–4 weeks. It is suggested that the few surviving cells are those which have escaped a lethal mutation. The possible occurrence of resistance to the analog or to amethopterin is excluded. Prolonged growth of amethopterin-resistant Sarcoma-180 cells in the analog-containing medium failed to interfere with the capacity of these cells to produce large amounts of folic acid reductase.

Vitamin requirements of uterine fibroblasts, strain U12-79; their replacement by related compounds

Choline, folic acid, meso-inositol, nicotinamide, pantothenic acid, pyridoxal, riboflavine, and thiamine were found to be required for the serial propagation of human uterine fibroblasts, strain U12-79, in a defined medium supplemented with 5% ( v v ) dialyzed horse serum. The concentration of vitamin which permits one-half optimal proliferation as determined by propagating the cells serially in media containing graded concentrations of each compound ranged from 2.5 to 50 × 10 −5 m M. Six amino acids which are not required for the growth of U12-79 replace pyridoxal under the conditions employed. Of the 16 cofactor derivatives and related compounds tested for their ability to replace the corresponding vitamin, all were active except p-aminobenzoic acid. Flavine adenine dinucleotide and leucovorin promoted a more rapid rate of growth, whereas the other compounds were either equally or less effective than the corresponding vitamin with regard to rate of growth or concentration required.

Synthesis of Cholesterol by a Strain of Human Uterine Fibroblasts Propagated in vitro.

SummaryStrain U12–79 of human uterine fibroblasts propagated in vitro for almost 5 years was grown in a medium containing acetate-2-C14. C14-cholesterol was isolated from both the cells and the medium.