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

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

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

Adrenomedullin Enhances Invasion of Human Extravillous Cytotrophoblast-Derived Cell Lines by Regulation of Urokinase Plasminogen Activator Expression and S-Nitrosylation

Supported by funding from the University of Hong Kong (code 104106)Extravillous cytotrophoblast (EVCT) is responsible for trophoblast invasion, which is an important process during placentation. Dysregulation of the process is associated with a wide range of pregnancy complications. Adrenomedullin (ADM) is a polypeptide expressed most abundantly in first-trimester placentas. We hypothesized that ADM modulated the invasion of human EVCT. Our results showed that ADM enhanced invasion and migration but not proliferation in two EVCT cell lines, JEG-3 and TEV-1. Similar observation can also be obtained in primary EVCTs. JEG-3 and TEV-1 cells expressed ADM receptor components as demonstrated by immunostaining, Western blotting, and RT-PCR. The ADM antagonist ADM22–52 (ADM C-terminal 22-52 amino acid fragment) suppressed ADM-induced invasion and migration, confirming that ADM exerted its biological effects through its classical receptors. The stimulatory effect of ADM on EVCT invasiveness was associated with induction (P < 0.05) of urokinase plasminogen activator (uPA) and nitric oxide synthase (NOS) expression and activity. Silencing of uPA by siRNA transfection abolished the stimulatory effect of ADM, suggesting that uPA is the key mediator for ADM-induced invasion. The involvement of NO in enhancing the invasion and biosynthesis of uPA in EVCT cell lines was confirmed by using pharmacological inhibitors of NOS and NO donors. ADM-mediated NO production also increased protein S-nitrosylation of JEG-3 cells. S-nitrosylation activated uPA in vitro and induced a higher proteinase activity. These findings provide indications that ADM and its downstream NO signaling may play an important role in modulating human EVCT functions.link_to_OA_fulltex

Native human zona pellucida glycoproteins: Purification and binding properties

BACKGROUND: Fertilization starts with the binding of the spermatozoa to the zona pellucida (ZP) of the oocyte. Such binding is a carbohydrate-mediated event and consists of a series of tightly regulated events. Molecular interactions between spermatozoon and ZP in human are not well characterized due to limited availability of oocytes for research. Our current technology cannot generate recombinant human ZP (hZP) glycoproteins with native glycosylation. METHODS AND RESULTS: In this study, hZP glycoproteins, hZP2 (∼120 kDa), hZP3 (∼58 kDa) and hZP4 (∼65 kDa) were purified from ZP (purity >88%) by immunoaffinity columns. The binding sites of the purified native hZP3 and hZP4 were localized to the acrosome region of the capacitated human spermatozoa, and were lost after acrosome reaction. Purified human hZP2 bound to this region only in acrosome-reacted spermatozoa. Differential binding of the three glycoproteins to the post-acrosomal region and the midpiece of the spermatozoa was observed. In addition, hZP3, but not hZP2 and hZP4, induced hyperactivation. The stimulatory activity was dependent partly on N-linked glycosylation of hZP3. CONCLUSIONS: This manuscript describes the biological activities of purified hZP glycoproteins from the native source for the first time. © The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.link_to_OA_fulltex