Signaling Pathway Of C-type Natriuretic Peptide(CNP)on Sperm Capacitation

Part ? The expression of CNP/NPR-B in female rat reproductive tractObjectives: To determinate the expression of CNP and its specific receptor NPR-B in reproductive tract of female rat.Methods: we used immunohistochemical technique to detect the expression and localization of CNP and its specitic receptor NPR-B in the genital tract of female rat?cervix, uterus and oviduct?.Results: Immunohistochemical results showed CNP and its specific receptor NPR-B was expressed at the squamous epithelial cell of cervical mucosa, the endometrial columnar epithelial cells and secretory cells of mucosa epithelium of oviduct;Conclusions: CNP and NPR-B are expressed at the same site of the female rat genital tract, mainly localized at the endometrial columnar epithelial cells and secretory cells of mucosa epithelium of oviduct.Part Effect of CNP on cG?MP content and function of spermObjectives: To investigate the effect of CNP on c GMP content and function of sperm.Methods: The prepared sperm suspensions were incubated in Ham's F?10 medium with the presence of various CNP concentrations?0, 10-7and 10-6 mol/L? for 30 min and the c GMP content was measured by rat c GMP ELISA Kit. We then used CNP, 8-Br-c GMP, a cell membrane-permeable c GMP analog, and KT5823, an inhibitor of c GMP-dependent protein kinases?PKG?, to incubate spermtozoa at 37°C in 5% CO2 incubator. After incubation for 30 minutes, the sperm motility was analysed using computer-assisted sperm analysis system; after incubation for 6h, the sperm acrosome reaction was assessed using the coomassie brilliant blue staining method.Results: 1. Two different concentrations of CNP were both able to increase the amounts of intracellular c GMP?P?0.05?. Moreover, 10-6 mol/L CNP significantly improved the concentration of c GMP?P?0.01?; 2. Compared with the control group, sperm motility and the acrosomal reaction rate were both improved by CNP and 8-Br-c GMP, however, significantly inhibited by the addition of KT5823?P?0.01?.Conclusions: These results suggest that CNP is able to promote sperm motility and the acrosome reaction by increasing the intracellular c GMP content, activating the activation of downstream signaling molecular PKG, and thereby regulating the sperm capacitation.Part Molecular mechanism of CNP/c G?MP on the regulation of sperm capacitationObjectives: To investigate the molecular mechanism of CNP/c GMP on the regulation of sperm capacitation.Methods: We used CNP, 8-Br-c GMP and KT5823 to incubate spermtozoa for 2h at 37°C in 5% CO2 incubator, then, we detected Fluo3-AM fluorescence probe-binding Ca²⁺ in spermotoza by flow cytometry and evaluated sperm protein tyrosine phosphorylation by western blot technique.Results: Compared with the control group, the concentration of intracellular Ca²⁺ was higher at 2h after CNP and 8-Br-c GMP added in sperm suspension, whereas, spermtozoa treated with KT5823 showed a significantly decrease of Ca²⁺. On the other hand, the levels of protein tyrosine phosphorylation in sperm did not changed obviously.Conclusions: CNP/c GMP may activate the downstream signaling molecules PKG, increasing Ca²⁺ content, so as to regulate sperm capacitation.