Alicia Quiñonero

The role of the leptin in reproduction.

Purpose of review Since its discovery in 1994, leptin has appeared to be a pleiotrophic hormone, governing energy homeostasis and affecting many tissues in the body. Numerous pieces of evidence have accumulated showing that leptin potentially plays an important role in the control of the reproductive function. Recent findings This review presents the major concepts for the role of leptin in the modulation of reproductive function. As a marker of the nutritional status, leptin affects the hypothalamo–pituitary–gonadal axis, regulating gonadotrophin-releasing hormone and luteinising hormone secretion, and appears to be a permissive factor in the onset of the puberty. This protein and its receptor have been found in the reproductive tissues, indicating that this system could be also implicated in several processes such as embryo development, implantation and pregnancy. Moreover, disorders of the leptin system have been related to some reproductive pathologies such as pre-eclampsia and polycystic ovary syndrome. However, controversy surrounds several aspects of the action of leptin in reproduction that require a deeper investigation of this system. Summary Results to date suggest that this system could be implicated in important reproductive processes such as embryonic development and implantation. Moreover, understanding the role of leptin might be useful for new treatments in reproductive pathologies.

Proteomic analysis of the human receptive versus non-receptive endometrium using differential in-gel electrophoresis and MALDI-MS unveils stathmin 1 and annexin A2 as differentially regulated

BACKGROUND The transcriptome of the endometrium throughout the menstrual cycle has been described in recent years. However, the proteomic of the window of implantation remains unknown. The aim of this study was to compare the proteome of the human endometrium in the pre-receptive phase versus the receptive phase by identifying and quantifying the proteins differentially expressed using differential in-gel electrophoresis (DIGE) and mass spectometry (MS). METHODS Endometrial biopsies were collected at days 2 (pre-receptive) and 7 (receptive) after the urinary luteal hormone surge in the same menstrual cycle from eight fertile women (corresponding to days 16 and 21 of the menstrual cycle). Proteins were extracted and labeled with CyDye DIGE fluorofores and separated using two-dimensional gel electrophoresis. RESULTS Image analysis using the DeCyder software followed by protein identification by matrix-assisted laser desorption/ionization-MS and database searching revealed 32 differentially expressed proteins, although only annexin A2 and stathmin 1 were consistently regulated in the two experiments performed. Validation and localization of annexin A2 and stathmin 1 were performed by western blot and immunohistochemistry. Annexin A2 and stathmin 1 were investigated using an endometrial refractoriness model. The results highlight the key potential of these proteins as possible targets for human endometrial receptivity and interception. CONCLUSION This study shows that the human endometrium has a differential proteomic repertoire during the window of implantation. Consequently, we identified annexin A2 and stathmin 1 as differentially expressed molecules in the receptive endometrium.

CXCL10 and IL-6 induce chemotaxis in human trophoblast cell lines

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.

miRNA Signature and Dicer Requirement during Human Endometrial Stromal Decidualization In Vitro

Decidualization is a morphological and biochemical transformation of endometrial stromal fibroblast into differentiated decidual cells, which is critical for embryo implantation and pregnancy establishment. The complex regulatory networks have been elucidated at both the transcriptome and the proteome levels, however very little is known about the post-transcriptional regulation of this process. miRNAs regulate multiple physiological pathways and their de-regulation is associated with human disorders including gynaecological conditions such as endometriosis and preeclampsia. In this study we profile the miRNAs expression throughout human endometrial stromal (hESCs) decidualization and analyze the requirement of the miRNA biogenesis enzyme Dicer during this process. A total of 26 miRNAs were upregulated and 17 miRNAs downregulated in decidualized hESCs compared to non-decidualized hESCs. Three miRNAs families, miR-181, miR-183 and miR-200, are down-regulated during the decidualization process. Using miRNAs target prediction algorithms we have identified the potential targets and pathways regulated by these miRNAs. The knockdown of Dicer has a minor effect on hESCs during in vitro decidualization. We have analyzed a battery of decidualization markers such as cell morphology, Prolactin, IGFBP-1, MPIF-1 and TIMP-3 secretion as well as HOXA10, COX2, SP1, C/EBPß and FOXO1 expression in decidualized hESCs with decreased Dicer function. We found decreased levels of HOXA10 and altered intracellular organization of actin filaments in Dicer knockdown decidualized hESCs compared to control. Our results provide the miRNA signature of hESC during the decidualization process in vitro. We also provide the first functional characterization of Dicer during human endometrial decidualization although surprisingly we found that Dicer plays a minor role regulating this process suggesting that alternative biogenesis miRNAs pathways must be involved in human endometrial decidualization.

Modeling Human Endometrial Decidualization from the Interaction between Proteome and Secretome

CONTEXT Decidualization of the human endometrium, which involves morphological and biochemical modifications of the endometrial stromal cells (ESCs), is a prerequisite for adequate trophoblast invasion and placenta formation. OBJECTIVE This study aims to investigate the proteome and secretome of in vitro decidualized ESCs. These data were combined with published genomic information and integrated to model the human decidualization interactome. DESIGN Prospective experimental case-control study. SETTING A private research foundation. PATIENTS Sixteen healthy volunteer ovum donors. INTERVENTION Endometrial samples were obtained, and ESCs were isolated and decidualized in vitro. MAIN OUTCOME MEASURES Two-dimensional difference in-gel electrophoresis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry, Western blot, human protein cytokine array, ELISA, and bioinformatics analysis were performed. RESULTS The proteomic analysis revealed 60 differentially expressed proteins (36 over- and 24 underexpressed) in decidualized versus control ESCs, including known decidualization markers (cathepsin B) and new biomarkers (transglutaminase 2, peroxiredoxin 4, and the ACTB protein). In the secretomic analysis, a total of 13 secreted proteins (11 up- and 2 down-regulated) were identified, including well-recognized markers (IGF binding protein-1 and prolactin) and novel ones (myeloid progenitor inhibitory factor-1 and platelet endothelial cell adhesion molecule-1). These proteome/secretome profiles have been integrated into a decidualization interactome model. CONCLUSIONS Proteomic and secretomic have been used as hypothesis-free approaches together with complex bioinformatics to model the human decidual interactome for the first time. We confirm previous knowledge, describe new molecules, and we have built up a model for human in vitro decidualization as invaluable tool for the diagnosis, therapy, and interpretation of biological phenomena.

Human Endometrial CD98 Is Essential for Blastocyst Adhesion

Background Understanding the molecular basis of embryonic implantation is of great clinical and biological relevance. Little is currently known about the adhesion receptors that determine endometrial receptivity for embryonic implantation in humans. Methods and Principal Findings Using two human endometrial cell lines characterized by low and high receptivity, we identified the membrane receptor CD98 as a novel molecule selectively and significantly associated with the receptive phenotype. In human endometrial samples, CD98 was the only molecule studied whose expression was restricted to the implantation window in human endometrial tissue. CD98 expression was restricted to the apical surface and included in tetraspanin-enriched microdomains of primary endometrial epithelial cells, as demonstrated by the biochemical association between CD98 and tetraspanin CD9. CD98 expression was induced in vitro by treatment of primary endometrial epithelial cells with human chorionic gonadotropin, 17-β-estradiol, LIF or EGF. Endometrial overexpression of CD98 or tetraspanin CD9 greatly enhanced mouse blastocyst adhesion, while their siRNA-mediated depletion reduced the blastocyst adhesion rate. Conclusions These results indicate that CD98, a component of tetraspanin-enriched microdomains, appears to be an important determinant of human endometrial receptivity during the implantation window.

Inhibition of Histone Deacetylase Activity in Human Endometrial Stromal Cells Promotes Extracellular Matrix Remodelling and Limits Embryo Invasion

Invasion of the trophoblast into the maternal decidua is regulated by both the trophoectoderm and the endometrial stroma, and entails the action of tissue remodeling enzymes. Trophoblast invasion requires the action of metalloproteinases (MMPs) to degrade extracellular matrix (ECM) proteins and in turn, decidual cells express tissue inhibitors of MMPs (TIMPs). The balance between these promoting and restraining factors is a key event for the successful outcome of pregnancy. Gene expression is post-transcriptionally regulated by histone deacetylases (HDACs) that unpacks condensed chromatin activating gene expression. In this study we analyze the effect of histone acetylation on the expression of tissue remodeling enzymes and activity of human endometrial stromal cells (hESCs) related to trophoblast invasion control. Treatment of hESCs with the HDAC inhibitor trichostatin A (TSA) increased the expression of TIMP-1 and TIMP-3 while decreased MMP-2, MMP-9 and uPA and have an inhibitory effect on trophoblast invasion. Moreover, histone acetylation is detected at the promoters of TIMP-1 and TIMP-3 genes in TSA-treated. In addition, in an in vitro decidualized hESCs model, the increase of TIMP-1 and TIMP-3 expression is associated with histone acetylation at the promoters of these genes. Our results demonstrate that histone acetylation disrupt the balance of ECM modulators provoking a restrain of trophoblast invasion. These findings are important as an epigenetic mechanism that can be used to control trophoblast invasion.

Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation

Annexin A2 (ANXA2) is present in vivo in the mid‐ and late‐secretory endometria and is mainly localized in the luminal epithelium. Our aim was to evaluate its function in regulating the human implantation process. With an in vitro adhesion model, constructed to evaluate how the mouse embryo and JEG‐3 spheroids attach to human endometrial epithelial cells, we demonstrated that ANXA2 inhibition significantly diminishes embryo adhesiveness. ANXA2 is also implicated in endometrial epithelial cell migration and trophoblast outgrowth. ANXA2 was seen to be linked to the RhoA/ROCK pathway and to regulate cell adhesion. We noted that ANXA2 inhibition significantly reduces active RhoA, although RhoA inactivation does not alter the ANXA2 levels. RhoA inactivation and ROCK inhibition also moderate embryo adhesiveness to endometrial epithelial cells. We corroborated that the induction of constitutively active RhoA partially reverses the effects of ANXA2 inhibition on endometrial adhesiveness. These molecules colocalize on the plasma membrane of endometrial epithelial cells, and a large proportion of ANXA2 and RhoA are colocalized in the F‐actin networks. The functional effects of ANXA2 inhibition and RhoA/ROCK inactivation are associated with significant alterations in F‐actin organization and its depolymerization. ANXA2 may act upstream of the RhoA/ROCK pathway by regulating F‐actin remodeling and is a key factor in human endometrial adhesiveness.—Garrido‐Gómez, T., Dominguez, F., Quiñonero, A., Estella, C., Vilella, F., Pellicer, A., Simon, C. Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F‐actin regulation. FASEB J. 26, 3715–3727 (2012). www.fasebj.org

Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

Defective decidualization during and after severe preeclampsia reveals a possible maternal contribution to the etiology

Significance We provide evidence of a decidualization defect in the endometrium of women with severe preeclampsia (PE) that was detected at the time of delivery and persisted years after the affected pregnancy. We went on to link this defect to impaired cytotrophoblast invasion. The transcriptional signature of the defect could enable its detection before (or after) conception, which would aid the development of therapies focused on improving decidualization and perhaps preventing severe PE. In preeclampsia (PE), cytotrophoblast (CTB) invasion of the uterus and spiral arteries is often shallow. Thus, the placenta’s role has been a focus. In this study, we tested the hypothesis that decidual defects are an important determinant of the placental phenotype. We isolated human endometrial stromal cells from nonpregnant donors with a previous pregnancy that was complicated by severe PE (sPE). Compared with control cells, they failed to decidualize in vitro as demonstrated by morphological criteria and the analysis of stage-specific antigens (i.e., IGFBP1, PRL). These results were bolstered by global transcriptional profiling data that showed they were transcriptionally inert. Additionally, we used laser microdissection to isolate the decidua from tissue sections of the maternal–fetal interface in sPE. Global transcriptional profiling revealed defects in gene expression. Also, decidual cells from patients with sPE, which dedifferentiated in vitro, failed to redecidualize in culture. Conditioned medium from these cells failed to support CTB invasion. To mimic aspects of the uterine environment in normal pregnancy, we added PRL and IGFBP1, which enhanced invasion. These data suggested that failed decidualization is an important contributor to down-regulated CTB invasion in sPE. Future studies will be aimed at determining whether this discovery has translational potential with regard to assessing a woman’s risk of developing this pregnancy complication.