Downregulation of vdac2 inhibits spermatogenesis via JNK and P53 signalling in mice exposed to cadmium.

Previous studies have reported the reproductive toxicity of cadmium (Cd); however, the effect of Cd on spermatogenesis and the underlying mechanism remain to be elucidated. In this study, mouse Leydig TM3 cells were treated with CdCl2 (0, 5, 10 and 50 µM) for 24 h to evaluate cytotoxicity, and C57BL/6 mice were treated intragastrically with 0.4 mL CdCl2 (0, 0.01, 0.05 and 0.1 g/L) for 2 months to investigate changes in spermatogenesis. The results showed that Cd aggravated apoptosis and proliferation in a dose-dependent manner, concomitant with deteriorated spermatogenesis and testosterone synthesis. For mechanism exploration, RNA-seq was used to profile alterations in gene expression in response to Cd, and the results indicated focus on P53/JNK signalling pathways and membrane proteins. We found that P53/JNK signalling pathways were activated upon Cd treatment, with the Cd-triggered downregulation of the vdac2 gene. P53/JNK pathway blockade ameliorated the Cd-induced inhibition of steroidogenic acute regulatory protein (STAR) expression and testosterone synthesis. Additionally, vdac2 knockdown in TM3 cells contributed to the phosphorylation of JNK/P53 and reduced the testosterone content. Vdac2 overexpression rescued the aforementioned Cd-induced events. Collectively, our study identified an innovative biomarker of Cd exposure in mice. The results demonstrated that vdac2 downregulation inhibits spermatogenesis via the JNK/P53 cascade. This finding may contribute to our understanding of the regulatory mechanism of Cd reproductive toxicity and provide a candidate list for sperm abnormality factors and pathways.

Glycogen synthase kinase 3 inhibition slows mitochondrial adenine nucleotide transport and regulates voltage-dependent anion channel phosphorylation.

Inhibition of glycogen synthase kinase (GSK)-3 reduces ischemia/reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors before ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and were treated with uncoupler or cyanide or were made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be attributable to inhibition of ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC) and/or adenine nucleotide transporter (ANT) or to inhibition of the F(1)F(0)-ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not caused by opening of the mitochondrial permeability transition pore. Because GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed Western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. Liquid chromatographic-mass spectrometric analysis revealed 1 of these proteins to be VDAC2. Taken together, we found that GSK-mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and that VDAC may be an important regulatory site in ischemia/reperfusion injury.