Protective effects of vitamin D on learning and memory deficit induced by scopolamine in male rats: the roles of brain-derived neurotrophic factor and oxidative stress.

The beneficial effects of vitamin D (vit D) on central nervous system disorders have been suggested. In the current research, the protective effects of vit D on learning and memory deficit induced by scopolamine, oxidative stress criteria, brain-derived neurotrophic factor (BDNF), and nitric oxide (NO) in the brain were investigated. Rats were divided into five groups, including (1) Control, (2) Scopolamine (2 mg/kg), (3-5) Scopolamine + Vit D (100, 1000, and 10,000 IU/kg) groups. Vit D administrated for 2 weeks and in the third week scopolamine co-administrated with vit D and behavioral tests, including Morris water maze (MWM) and passive avoidance (PA) tests, were carried out. The cortical and hippocampal tissues were analyzed for BDNF, catalase (CAT), and superoxide dismutase (SOD) activities, thiol content, NO metabolites, and malondialdehyde (MDA) concentration. Scopolamine injection significantly impaired rats' performance on the MWM and PA test. It further enhanced the MDA and nitrite level while decreased thiol content and BDNF levels and SOD and CAT activities in the brain. Administration of both 1000 and 10,000 IU/kg vit D improved cognitive outcome in MWM and PA tests. In addition, vit D elevated thiol content, SOD and CAT activities, and BDNF levels, while reduced nitrite and MDA concentration. Vit D also increased the levels of vit D and calcium in the serum. The results demonstrated that vit D has protective effects on scopolamine-associated learning and memory impairment by improving BDNF levels and attenuating NO and brain tissue oxidative damage.

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 (Cai 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 Cai 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 20×106 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 Cai 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 Cai 2+ and ROS generation. The addition of NNC, Zn, or NNC+Zn significantly decreased Cai 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 Cai 2++. The activity of CatSper and Hv1 channels are required for progesterone-induced ROS generation by NOX5 enzyme.

Effect of CatSper and Hv1 Channel Inhibition on Progesterone Stimulated Human Sperm.

BACKGROUND: Intracellular calcium and proton concentrations are important factors for activating human sperm. Calcium ion (Ca2+) enters sperm through voltage-dependent calcium channel of sperm (CatSper). Proton was extruded from sperm through voltage-gated proton channel (Hv1). In the present study, the selective inhibitors of the CatSper and Hv1 channels, NNC 55-0396 (NNC) and zinc ion, respectively, were used to investigate functions of these channels. METHODS: Normal semen samples (n=24) were washed and diluted to 20×106sperm/ml. The diluted sample was divided into 8 groups, containing Ham's F-10 (the control group), 2 µM NNC, 1 mM ZnCl2 and NNC+Zn. The other 4 groups were the same as above, except that they contained 1 µM progesterone. The computer assisted analysis was done by VT-Sperm 3.1 to determine the percentage of motile sperm and sperm velocity. Acrosomal status was monitored by FITC-PSA and viability assessed by Eosin-Y staining. Statistical comparisons were made using ANOVA followed by Tukey post hoc test. The p<0.05 was considered significant. RESULTS: The percentage of viable and motile sperm, curvilinear velocity and other parameters of motility was reduced in all groups containing NNC, zinc and NNC+ zinc. Progesterone-induced acrosome reaction was abolished by each of these inhibitors. The combination effect of NNC plus zinc on motility and progesterone-induced acrosome reaction was not stronger than NNC by itself. CONCLUSION: CatSper and Hv1 channels play a critical role in human sperm function and viability. It seems that a functional relationship exists between CatSper and Hv1 channels.

Inhibition of the CatSper Channel and NOX5 Enzyme Activity Affects the Functions of the Progesterone-Stimulated Human Sperm.

BACKGROUND: Low levels of reactive oxygen species (ROS) and calcium are necessary for sperm function. NADPH oxidase 5 (NOX5) is a membrane enzyme which produces ROS. This enzyme is dependent on calcium for its activity. We investigated the importance of NOX5 and an important calcium channel (CatSper) on sperm function. METHODS: This laboratory in-vitro study was done in Shiraz, Iran, 2016. Normal semen samples (n=24) were washed and diluted to 20×106 sperm/mL. The diluted samples were divided into 8 groups, containing Ham's F-10 (control group), 2 µM of NNC (CatSper channel inhibitor), 1 µM DPI (NOX5 inhibitor), and NNC+DPI. The other 4 groups were the same as the 1st ones, except that they contained 1 µM of progesterone. Motility assessment was done by VT-Sperm 3.1. Acrosome status was monitored with acrosome-specific FITC-PSA using fluorescent microscopy. Sperm viability was assessed by Eosin Y. Statistical analysis was performed using SPSS 16 software. The comparison between the groups was done using the one-way ANOVA, followed by Tukey. A P<0.05 was considered significant. RESULTS: The percentage of motile sperm, sperm velocity, and viability decreased significantly in the groups containing NNC. DPI reduced sperm progressive motility only in the progesterone-stimulated condition. Progesterone induced acrosome reaction, but this effect was inhibited by NNC and DPI. CONCLUSION: CatSper had a prominent role in the motility, acrosome reaction, and viability of the human sperm. The function of NOX5 was important only in the stimulated sperm. We conclude that CatSper has a more prominent role than NOX5 activity. The functional relation between NOX5 and CatSper is not clear but is very probable.