Glycodelin-A primes zona pellucida-induced acrosome reaction of human spermatozoa via downregulation of extracellular signal regulated kinases and enhancement of zona pellucida-induced calcium influx

Conference Theme: The Intersection Between Genetics, Genomics, and Reproductive BiologypostprintThe 43rd Annual Meeting of the Society for the Study of Reproduction (SSR), Milwaukee, WI., 30 July-3 August 2010. In Meeting Abstracts, 2010, p. 38, abstract no. 17

MicroRNA-34a is a tumor suppressor in choriocarcinoma via regulation of Delta-like1

Background: Choriocarcinoma is a gestational trophoblastic tumor which causes high mortality if left untreated. MicroRNAs (miRNAs) are small non protein-coding RNAs which inhibit target gene expression. The role of miRNAs in choriocarcinoma, however, is not well understood. In this study, we examined the effect of miR-34a in choriocarcinoma.Methods: MiR-34a was either inhibited or ectopically expressed transiently in two choriocarcinoma cell lines (BeWo and JEG-3) respectively. Its actions on cell invasion, proliferation and colony formation at low cell density were examined. The miR-34a putative target Notch ligand Delta-like 1 (DLL1) was identified by adoption of different approaches including: in-silico analysis, functional luciferase assay and western blotting. Real-time quantitative polymerase chain reaction was used to quantify changes in the expression of matrix proteinase in the treated cells. To nullify the effect of miR-34a ectopic expression, we activated Notch signaling through force-expression of the Notch intracellular domain in the miR-34a force-expressed cells. In addition, we studied the importance of DLL1 in BeWo cell invasion through ligand stimulation and antibody inhibition. Furthermore, the induction in tumor formation of miR-34a-inhibited BeWo cells in SCID mice was investigated.Results: Transient miR-34a force-expression significantly suppressed cell proliferation and invasion in BeWo and JEG-3 cells. In silicon miRNA target prediction, luciferase functional assays and Western blotting analysis demonstrated that miR-34a regulated DLL1 expression in both cell lines. Although force-expression of miR-34a suppressed the expression of DLL1 and NOTCH1, the extent of suppression was higher in DLL1 than NOTCH1 in both cell lines. MiR-34a-mediated DLL1 suppression led to reduced matrix metallopeptidase 9 and urokinase-type plasminogen activator expression. The effect of miR-34a on cell invasion was partially nullified by Notch signaling activation. DLL1 ligand stimulated while anti-DLL1 antibody treatment suppressed cell invasion. Mice inoculated with BeWo cells transfected with miR-34a inhibitor had significantly larger xenografts and stronger DLL1 expression than those with cells transfected with the control inhibitor.Conclusions: MiR-34a reduced cell proliferation and invasiveness, at least, partially through its inhibitory effect on DLL1. © 2013 Pang et al.; licensee BioMed Central Ltd.published_or_final_versio

Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.published_or_final_versio

Human chorionic gonadotropin stimulates spheroid attachment on fallopian tube epithelial cells through the mitogen-activated protein kinase pathway and down-regulation of olfactomedin-1

OBJECTIVE: To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro. DESIGN: Experimental study. SETTING: Reproductive biology laboratory. PATIENT(S): Healthy non-pregnant women. INTERVENTION(S): No patient interventions. MAIN OUTCOME MEASURE(S): Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay. RESULT(S): Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression. CONCLUSION(S): Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression.postprin

Study of transforming growth factor alpha for the maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) have great potential for regenerative medicine as they have selfregenerative and pluripotent properties. Feeder cells or their conditioned medium are required for the maintenance of hESC in the undifferentiated state. Feeder cells have been postulated to produce growth factors and extracellular molecules for maintaining hESC in culture. The present study has aimed at identifying these molecules. The gene expression of supportive feeder cells, namely human foreskin fibroblast (hFF-1) and non-supportive human lung fibroblast (WI-38) was analyzed by microarray and 445 genes were found to be differentially expressed. Gene ontology analysis showed that 20.9% and 15.5% of the products of these genes belonged to the extracellular region and regulation of transcription activity, respectively. After validation of selected differentially expressed genes in both human and mouse feeder cells, transforming growth factor a (TGFa) was chosen for functional study. The results demonstrated that knockdown or protein neutralization of TGFa in hFF-1 led to increased expression of early differentiation markers and lower attachment rates of hESC. More importantly, TGFa maintained pluripotent gene expression levels, attachment rates and pluripotency by the in vitro differentiation of H9 under non-supportive conditions. TGFa treatment activated the p44/42MAPK pathway but not the PI3K/Akt pathway. In addition, TGFa treatment increased the expression of pluripotent markers, NANOG and SSEA-3 but had no effects on the proliferation of hESCs. This study of the functional role of TGFa provides insights for the development of clinical grade hESCs for therapeutic applications. © The Author(s) 2012. © Springer-Verlag 2012.published_or_final_versio

Zona pellucida-induced acrosome reaction in human spermatozoa is potentiated by glycodelin-A via down-regulation of extracellular signal-regulated kinases and up-regulation of zona pellucida-induced calcium influx

Background Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction. Methods Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining. Results Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction. Conclusions Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. © 2010 The Author.postprin

SLeX: Potential implications for fertility and contraception

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Adrenomedullin stimulates human trophoblast invasion through the upregulation of nitric oxide and urokinase plasminogen activator activity

Poster Session A - Implantation & pregnancy - placental function: abstract no. 446Conference Theme: Reproduction and the World’s FutureExtravillous cytotrophoblasts (EVCTs) invasion into the uterine endometrium is important to placentation and successful pregnancy. Dysregulation of the process is associated with a wide range of pregnancy complications, like intrauterine growth restriction, choriocarcinoma and preeclampsia. EVCTs produce urokinase plasminogen activator (uPA) that activates plasmin to degrade the extracellular matrix of the endometrium for invasion. Adrenomedullin is a 52-amino acid polypeptide belonging to the calcitonin gene-related peptide family which has diverse physiological functions including vasodilation, differentiation, hormone secretion and cell invasion. Nitric oxide (NO) signaling has been implicated to play a role in the biological functions of adrenomedullin in different cell types. In pregnant women, adrenomedullin is most abundantly expressed in the first-trimester placenta when the EVCTs exhibit the maximal invasive behavior, suggesting a possible role of the peptide in EVCT invasion during early pregnancy. The objective of this study is to investigate the effect of adrenomedullin on human EVCT invasion. JEG3, a choriocarcinoma cell line derived from first trimester human EVCT, was used as the study model. Adrenomedullin treatment significantly enhanced the invasiveness and uPA expression and activity of the JEG3 cells as demonstrated by transwell invasion assay, RT-PCR and uPA activity assay respectively. Immunostaining, western blotting and PCR demonstrated the presence of adrenomedullin receptor components in JEG3 cells. We further demonstrated that the stimulatory effect of adrenomedullin on trophoblast invasion is mediated by NO signaling pathway. Adrenomedullin treatment was shown to increase the NO production and nitric oxide synthase (NOS) expression in JEG3 cells. NOS inhibitors significantly suppressed the stimulatory effect of adrenomedullin on JEG3 invasion, uPA expression and activity. In contrast, NO donors were able to mimic the biological activities of adrenomedullin. The present results suggest that adrenomedullin enhances EVCT invasion by upregulating the uPA expression and activity through a NO-dependent manner.link_to_OA_fulltextThe 44th Annual Meeting of the Society for the Study of Reproduction (SSR 2011), Portland, OR., 31 July-4 August 2011. In Abstracts of the 44th SSR, 2011, p. 106, abstract no. 44