Adrenomedullin enhances invasion of human extravillous cytotrophoblast-derived cell lines by regulation of urokinase plasminogen activator expression and s-nitrosylation.

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 ADM(22-52) (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.

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. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

Effects of native human zona pellucida glycoproteins 3 and 4 on acrosome reaction and zona pellucida binding of human spermatozoa.

Acrosome reaction is crucial to the penetration of spermatozoa through the zona pellucida (ZP). Glycosylation of ZP glycoproteins is important in spermatozoa-ZP interaction. Human ZP glycoprotein-3 (ZP3) is believed to initiate acrosome reaction. Recently, human ZP4 was also implicated in inducing acrosome reaction. These studies were based on recombinant human ZP proteins with glycosylation different from their native counterparts. In the present study, the effects of native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding were investigated. Native human ZP3 and ZP4 were immunoaffinity-purified. They induced acrosome reaction and inhibited spermatozoa-ZP binding time- and dose-dependently to different extents. These biological activities of human ZP3 and ZP4 depended partly on their glycosylation, with N-linked glycosylation contributing much more significantly than O-linked glycosylation. Studies with inhibitors showed that both human ZP3- and ZP4-induced acrosome reactions were protein kinase-C, protein tyrosine kinase, T-type Ca2+ channels, and extracellular Ca2+ dependent. G-protein also participated in human ZP3- but not in ZP4-induced acrosome reaction. On the other hand, protein kinase-A and L-type Ca2+ channels took part only in human ZP4-induced acrosome reaction. This manuscript describes for the first time the actions of purified native human ZP3 and ZP4 on acrosome reaction and spermatozoa-ZP binding.

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 (approximately 120 kDa), hZP3 (approximately 58 kDa) and hZP4 (approximately 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.