Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

Voltage dependent calcium channel (VDCC), one of the most important regulator of Ca 2+ concentration in neuron, play an essential role in the central processing of nociceptive information. The present study investigated the antinociceptive effects of L, T or N type VDCC blockers on the formalin-induced orofacial inflammatory pain. Experiments were carried out on adult male Sprague-Dawley rats weighing 220-280 g. Anesthetized rats were individually fixed on a stereotaxic frame and a polyethylene (PE) tube was implanted for intracisternal injection. After 72 hours, 5% formalin (50 μL) was applied subcutaneously to the vibrissa pad and nociceptive scratching behavior was recorded for nine successive 5 min intervals. VDCC blockers were administered intracisternally 20 minutes prior to subcutaneous injection of formalin into the orofacial area. The intracisternal administration of 350 or 700 μg of verapamil, a blocker of L type VDCC, significantly decreased the number of scratches and duration in the behavioral responses produced by formalin injection. Intracisternal administration of 75 or 150 μg of mibefradil, a T type VDCC blocker, or 11 or 22 μg of cilnidipine, a N type VDCC blocker, also produced significant suppression of the number of scratches and duration of scratching in the first and second phase. Neither intracisternal administration of all VDCC blockers nor vehicle did not affect in motor dysfunction. The present results suggest that central VDCCs play an important role in orofacial nociceptive transmission and a targeted inhibition of the VDCCs is a potentially important treatment approach for inflammatory pain originating in the orofacial area.