The Signaling Pathway And Molecule Mechanism Of Nitric Oxide On Embryo Implantation

Nitric oxide (NO) is produced by a group of enzymes named nitric oxide synthases (NOS), which has three isoforms: endothelial (eNOS), neural (nNOS) and inducible (iNOS). Nitric oxide is a multifunctional messenger molecule and is produced through the oxidation of L-arginine to L-citrulline by the enzyme NO synthase. NO acts as an important intracellular messenger in a variety of systems including reproduction. In mammals, NO is known to be a major paracrine mediator and an important regulator in female reproductive processes, such as ovulation, implantation, pregnancy maintenance, labor and delivery. NO donors may contribute to a reduction of fetal and maternal perinatal morbidity and mortality. NO donors and NOS inhibitors may provide novel, effective, safe and inexpensive drugs to regulate and steer various functions in female reproductive life.In the present study, mouse blastocysts were cultured in the medium containing different treatments according to the experimental design. The implantation capacity of blastocysts was evaluated by calculating the percentage of embryos with attachment or outgrowth after being cultured for 12 h, 24 h and 36 h. MMP-2 mRNA was detected by RT-PCR and MMP-2 protein was detected by gelatin zymography. Inhibition of blastocyst attachment and outgrowth was observed in embryo cultured with NOS inhibitor NG-monomethyl- L-arginine (L-NMMA). Both NO donor S-nitroso-N-acetylpenicillamine (SNAP) and cGMP analogue 8-Br-cGMP blocked this inhibition. The expression and secretion of MMP-2 in the blastocysts were suppressed by L-NMMA and this suppression was also reversed by SNAP or 8-Br-cGMP. These results indicated that NO/cGMP signaling pathway induced embryo implantation by increasing the expression of MMP-2. Based on the above results, the next experiment was designed to elucidate the detailed molecule mechanism of NO in embryo implantation, using JAR cells, a well propagated human first trimester extravillous trophoblast cell line in vitro. The first study was designed to determine the enzyme activity of eNOS in the transfected JAR cell. The results showed that mRNA of eNOS in the transfected JAR cell was significantly increased compared to untreated cells. Immunocytochemical studies revealed a strong green perinuclear immuno -fluorescence in eNOS transfected JAR cell. NOS activity and NO release did not change in transfectants. In this study, three independent methods demonstrated that transfection resulted in over-expression of eNOS in JAR cell, but the enzyme was not active until being treated with calcium ionophore A23187.The effect of endogenous NO on the proliferation, migration and matrix metalloproteinase-2 (MMP-2) production in the JAR cells was investigated by transfected nitric oxide synthase (eNOS) gene. The transfected JAR cells treated with different levels of calcium ionophore A23187 produced different concentrations of endogenous NO. The results indicated that eNOS gene transfer significantly inhibited the proliferation, migration and MMP-2 production in JAR cells when A23187 was at the dosage of 8μM. In contrast, NO at a low concentration produced by the mock cells treated with 8μM A23187 had opposite results in the same model. Furthermore, these effects may be regulated via Akt/protein kinase B (Akt/PKB), a well-known important mediator of cell survival signaling pathways. Exogenous NO donor SNAP (50μM and 500μM ) had the similar effect in this experiment. The current results indicated that different concentrations of NO, regardless of endogenesis or exogenesis, had different effects on the proliferation and migration of the embryos and MMP-2 production, which were regulated by Akt/PKB pathways. These results implied that the dual effects may depend on the NO concentration. Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that interacts with the cytoplasmic domains of integrins and numerous cytoskeleton-associated proteins. ILK has been shown to be involved in the regulation of a number of integrin-mediated pr...