Inhibition of CatSper and Hv1 Channels and NOX5 Enzyme Affect Progesterone-Induced Increase of Intracellular Calcium Concentration and ROS Generation in Human Sperm

BACKGROUND: Normal sperm function depends on appropriate intracellular calcium (Ca(i)(2+)) and reactive oxygen species (ROS) levels. Calcium activates NADPH oxidase-5 (NOX5) that leads to ROS generation. The calcium channel of sperm (CatSper) is activated by progesterone and intracellular alkalization. Herein, the interactive role of CatSper, Hv1 channels, and NOX5 enzyme on Ca(i)(2+) and ROS generation in human sperm is investigated. METHODS: The present laboratory in vitro study was carried out in the School of Medicine, Shiraz University of Medical Sciences (Shiraz, Iran) during 2016. Normal semen samples (n=15) were washed and diluted to 20×10(6) sperm/mL. The diluted samples were divided into 16 groups containing Ham’s F-10 (the control group), 2 µM NNC (CatSper inhibitor), 1 mM ZnCl2 (Hv1 inhibitor), 1 µM DPI (NOX5 inhibitor), NNC+Zn, NNC+DPI, and NNC+Zn+DPI. The other 8 groups were the same as the above except that they contained 1 µM progesterone. Cell viability and Ca(i)(2+) were analyzed by flou-3 AM probe and PI staining, respectively, using flow cytometric method. ROS generation was assessed by chemiluminescence method. Statistical analysis was performed using the one-way ANOVA followed by Tukey’s test. P values <0.05 were considered statistically significant. RESULTS: Progesterone increased Ca(i)(2+) and ROS generation. The addition of NNC, Zn, or NNC+Zn significantly decreased Ca(i)(2+) in the control and progesterone containing groups. Progesterone-induced ROS generation was decreased significantly in all groups containing NNC, Zn, or DPI and reached to the control level when DPI was added to NNC or Zn. CONCLUSION: There is a functional relationship between CatSper and Hv1 channels in increasing Ca(i)(2+)+. The activity of CatSper and Hv1 channels are required for progesterone-induced ROS generation by NOX5 enzyme.