Birgit Gellersen

Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial stromal cells.

Decidualization of human endometrial stromal (ES) cells in vitro is induced by cAMP analogs and ligands that elevate cellular cAMP levels. A marker of this differentiation process is the activation of the decidual PRL (dPRL) promoter. In a primary ES cell culture system we show that relaxin not only acutely but permanently elevates cellular cAMP levels and leads to induction of PRL secretion after 6 days. Northern and Western blot analyses revealed that all regulatory subunit isoforms (RIα, RIβ, RIIα, and RIIβ) and catalytic subunits Cα and Cβ of protein kinase A (PKA) are expressed in ES cells. Transcript levels of PKA subunit isoforms are not altered during decidualization, but in decidualized ES cells, exposed to relaxin for more than 6 days, a significant reduction of RIα protein level occurs, whereas levels of all other forms remain unchanged. Reduction of R subunits might result in a net increase in free C subunit activity. This alteration is not due to a change in the mitotic state of the cells, as...

CCAAT/Enhancer-binding Proteins Are Mediators in the Protein Kinase A-dependent Activation of the Decidual Prolactin Promoter

In the course of decidualization, human endometrial stromal cells (ESC) activate the alternative upstream promoter of the decidual prolactin (dPRL) gene. The dPRL promoter is induced by the protein kinase A pathway in a delayed fashion via the region −332/−270 which contains two overlapping consensus binding sequences, B and D, for CCAAT/enhancer-binding proteins (C/EBP). Here we show that sites B and D both bind C/EBPβ and -δ from ESC nuclear extracts. When decidualization of cultured ESC was induced by treatment with 8-Br-cAMP, complex formation on sites B and D was enhanced. Western blot analysis revealed an elevation of both C/EBPβ isoforms, liver-enriched activator protein and liver-enriched inhibitory protein, with a delayed onset between 8 and 24 h of cAMP treatment, while C/EBPδ expression remained unaffected. Cyclic AMP-mediated activation of dPRL promoter construct dPRL-332/luc3 was abrogated by mutation of sites B and D at −310/−285. An expression vector for liver-enriched activator protein potently induced transcription of dPRL-332/luc3 and further enhanced cAMP-mediated induction, while liver-enriched inhibitory protein expression vector abolished the cAMP response, implying that C/EBPs serve as mediators in the delayed cAMP signal transduction to the dPRL promoter. The ratio between activating and repressing isoforms is likely to dictate the transcriptional output.

Endometrial Stromal Cells of Women with Recurrent Miscarriage Fail to Discriminate between High- and Low-Quality Human Embryos

Background The aetiology of recurrent miscarriage (RM) remains largely unexplained. Women with RM have a shorter time to pregnancy interval than normally fertile women, which may be due to more frequent implantation of non-viable embryos. We hypothesized that human endometrial stromal cells (H-EnSCs) of women with RM discriminate less effectively between high-and low-quality human embryos and migrate more readily towards trophoblast spheroids than H-EnSCs of normally fertile women. Methodology/Principal Findings Monolayers of decidualized H-EnSCs were generated from endometrial biopsies of 6 women with RM and 6 fertile controls. Cell-free migration zones were created and the effect of the presence of a high-quality (day 5 blastocyst, n = 13), a low-quality (day 5 blastocyst with three pronuclei or underdeveloped embryo, n = 12) or AC-1M88 trophoblast cell line spheroid on H-ESC migratory activity was analyzed after 18 hours. In the absence of a spheroid or embryo, migration of H-EnSCs from fertile or RM women was similar. In the presence of a low-quality embryo in the zone, the migration of H-EnSCs of control women was inhibited compared to the basal migration in the absence of an embryo (P<0.05) and compared to the migration in the presence of high-quality embryo (p<0.01). Interestingly, the migratory response H-EnSCs of women with RM did not differ between high- and low-quality embryos. Furthermore, in the presence of a spheroid their migration was enhanced compared to the H-EnSCs of controls (p<0.001). Conclusions H-EnSCs of fertile women discriminate between high- and low-quality embryos whereas H-EnSCs of women with RM fail to do so. H-EnSCs of RM women have a higher migratory response to trophoblast spheroids. Future studies will focus on the mechanisms by which low-quality embryos inhibit the migration of H-EnSCs and how this is deregulated in women with RM.

Systematic Expression Analysis and Antibody Screening Do Not Support the Existence of Naturally Occurring Progesterone Receptor (PR)-C, PR-M, or Other Truncated PR Isoforms

Functional progesterone withdrawal associated with human parturition has been ascribed to various mechanisms modulating the function of the classical progesterone receptors (PRs), B and A, in utero. These include up-regulation of the inhibitory PR-C isoform, described as a 60-kDa protein occurring from translation initiation at codon 595. Our initial attempts to detect PR-C yielded uninterpretable results. To systematically validate antibodies for immunodetection of PR isoforms, we generated expression vectors for PR variants originating from putative start codons AUG-289, -301, -595, -632, and -692 in addition to those for PR-B and PR-A, and for alternative splice variants PR-T, PR-S, and PR-M. All constructs were subjected to in vitro and in vivo translation and immunoblotting with a panel of 13 PR antibodies. Antibodies raised against full-length PR were generally not capable of detecting N-terminally truncated forms, whereas C-terminal antibodies did not or only weakly reacted with PR-B and PR-A but produced prominent nonspecific signals. Thus, immunodetection of N-terminally truncated PR isoforms is prone to artifacts. Proteins of about 64 kDa were expressed from PR-289 and -301, but no corresponding endogenous forms were observed. PR-T, PR-S, and PR-M cDNAs yielded no detectable translation products. No protein was translated from AUG-595 in our PR-C expression vector unless a Kozak sequence was introduced, and the product was not 60 but 38 kDa in size. Thus, the 60-kDa protein called PR-C does not originate from AUG-595 and is not a naturally occurring PR isoform.

The motile and invasive capacity of human endometrial stromal cells: implications for normal and impaired reproductive function

BACKGROUND Mechanisms underlying early reproductive loss in the human are beginning to be elucidated. The migratory and invasive capacity of human endometrial stromal cells (ESCs) is increasingly recognized to contribute to the intense tissue remodelling associated with embryo implantation, trophoblast invasion and endometrial regeneration. In this review, we examine the signals and mechanisms that control ESC migration and invasion and assess how deregulation of these cell functions contributes to common reproductive disorders. METHODS The PubMed database was searched for publications on motility and invasiveness of human ESCs in normal endometrial function and in reproductive disorders including implantation failure, recurrent pregnancy loss (RPL), endometriosis and adenomyosis, covering the period 2000-2012. RESULTS Increasing evidence suggests that implantation failure and RPL involve abnormal migratory responses of decidualizing ESCs to embryo and trophoblast signals. Numerous reports indicate that endometriosis, as well as adenomyosis, is associated with increased basal and stimulated invasiveness of ESCs and their progenitor cells, suggesting a link between a heightened menstrual repair response and the formation of ectopic implants. Migration and invasiveness of ESCs are controlled by a complex array of hormones, growth factors, chemokines and inflammatory mediators, and involve signalling through Rho GTPases, phosphatidylinositol-3-kinase and mitogen-activated protein kinase pathways. CONCLUSIONS Novel concepts are extending our understanding of the key functions of ESCs in effecting tissue repair imposed by cyclic menstruation and parturition. Migration of decidualizing ESCs also serves to support blastocyst implantation and embryo selection through discriminate motile responses directed by embryo quality. Targeting regulatory molecules holds promise for developing new strategies for the treatment of reproductive disorders such as endometriosis and recurrent miscarriage; and harnessing the migratory capacity of progenitor mesenchymal stem cells in the endometrium may offer new opportunities in regenerative medicine.

Honey, we need to talk about the membrane progestin receptors

The recent discovery of three closely related cell surface receptors that bind to progesterone and mediate its actions on various cytoplasmic signalling cascades has been heralded as a major break-through. The reason for this is all too obvious. Progesterone is an essential regulator of all major reproductive events and progestins and antiprogestins are widely used in the treatment of many different gynaecological and obstetrical disorders. The novel membrane progestin receptors (mPRalpha, beta, gamma) reportedly resemble and function as G-protein-coupled receptors and therefore are promising pharmaceutical targets. However, our studies failed to corroborate that mPRs are expressed on the cell surface, that they mediate rapid progesterone signalling events, and even that they are bona fide progestin binding moieties. While the reason for these startling opposing results remains unclear, a critical review of existing data may help to shed some light onto the controversial mPRs. Time has come to talk.

Cloning of Guinea Pig Cyclooxygenase-2 and 15-Hydroxyprostaglandin Dehydrogenase Complementary Deoxyribonucleic Acids: Steroid-Modulated Gene Expression Correlates to Prostaglandin F2α Secretion in Cultured Endometrial Cells1

Prostaglandin F2α (PGF2α) secretion is lowest at midcycle and highest on day 15 at luteolysis in the cycling guinea pig uterus and is inversely related to serum progesterone levels. An increase in 17-β estradiol (E2) occurs only towards the end of the cycle. To investigate the effect of steroids on the control of uterine PGF2α metabolism at the level of gene expression we established a primary cell culture model of day 15 cycling guinea pig endometrial cells. We cloned guinea pig cDNAs for cyclooxygenase 2 (COX-2), 15-hydroxyprostaglandin dehydrogenase (PGDH) that converts PGF2α to biologically inactive 13,14-dihydro-15-keto PGF2α (PGFM) and a fragment of cyclooxygenase-1 (COX-1). They were found to bear 87% and 90% homology at the amino acid level to their human counterparts for COX-2 and PGDH, respectively, retaining all functional sites. Purified epithelial and stromal cell subcultures were primed with medium containing either E2 or medroxyprogesterone acetate (MPA) for 24 h. They were then treated for...

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Expansion of human trophoblastic spheroids is promoted by decidualized endometrial stromal cells and enhanced by heparin-binding epidermal growth factor-like growth factor and interleukin-1β

Successful pregnancy in humans depends on deep invasion of the maternal decidua by extravillous trophoblast cells (EVTs), a process regulated by autocrine and paracrine signals in the decidual-trophoblast microenvironment. Here we examined whether trophoblast invasion is affected by decidual differentiation of endometrial stromal cells (ESC) and modulated locally by cytokines and growth factors. Trophoblast spheroids were generated from the EVT-derived cell line AC-1M88 and placed onto monolayers of either undifferentiated or decidualized ESC, or directly onto tissue culture surface. Co-cultures were treated with epidermal growth factor (EGF), hepatocyte growth factor, heparin-binding EGF-like growth factor (HB-EGF), interleukin-1β (IL-1β) and leukaemia inhibitory factor (LIF). Expansion of spheroids over 2-3 days was significantly enhanced by a monolayer of undifferentiated ESC compared with tissue culture surface and further increased if ESC had been decidualized. HB-EGF and IL-1β, alone or in combination with LIF, stimulated spheroid expansion but only on undifferentiated ESC. CEACAM1, an adhesion molecule implicated in trophoblast invasion, was up-regulated in AC-1M88 cells by conditioned medium from decidualized ESC, and by HB-EGF, IL-1β and LIF in combination. Treatment of ESC with HB-EGF or IL-1β increased the level of the tetraspanin CD82, a metastasis suppressor found in decidual cells at the implantation site. We suggest that decidualized ESC support trophoblast invasion by paracrine signals that may include HB-EGF, IL-1β and LIF.

Something new about early pregnancy: decidual biosensoring and natural embryo selection

It may have involved only 23 good-quality human embryos, and the findings were published as a technical report in Nature Medicine, but the study of Vanneste and colleagues deserves a closer look1. Using a new array-based approach that allows combined screening of genome-wide copy number and loss of heterozygosity in single blastomeres, these investigators examined the incidence of gross chromosomal errors in cleavage-stage embryos from young fertile women. The incidence was staggering. In fact, only two out of the 23 cleavage-stage in-vitro fertilization (IVF) embryos were found to have a normal karyotype in all blastomeres, approximately half of the embryos had no normal cells at all and the remainder were mosaic for large-scale structural chromosomal imbalances, caused predominantly by mitotic non-disjunction1. Whether or not spontaneously conceived pre-implantation human embryos display similar frequency and complexity of gross chromosomal errors remains untested, although there are few, if any, reasons to doubt it. If confirmed, the data imply that the number of ‘healthy’ live births far outstrips the number of ‘normal’ human embryos at the time of implantation. While the study of Vanneste and coworkers sheds new light on some key issues in reproductive medicine, it simultaneously raises some difficult new questions. For example, the mitotic error rate in cleavage-stage embryos was found to be much higher than the meiotic aneuploidy rate. Consequently, the genome of one or two blastomeres is unlikely to be representative of the whole embryo, which explains the failure of preimplantation genetic screening to improve IVF outcome2. The remarkable prevalence of gross chromosomal instability in human embryos, irrespective of maternal age, is likely to be a major factor in the low monthly conception rates, averaging approximately 20%, which compares unfavorably with many other species. However, if a transient period of chromosomal instability in human embryonic development is the norm, rather than the exception, what could be the possible biological Jan Brosens Birgit Gellersen