Glyphosate-induced mitochondrial reactive oxygen species overproduction activates parkin-dependent mitophagy to inhibit testosterone synthesis in mouse leydig cells

Glyphosate (GLY), one of the most extensively used herbicides in the world, has been shown to inhibit testosterone synthesis in male animals. Mitochondria are crucial organelles for testosterone synthesis and its dysfunction has been demonstrated to induce the inhibition of testosterone biosynthesis. However, whether low-dose GLY exposure targets mitochondria to inhibit testosterone synthesis and its underlying mechanism remains unclear. Here, an in vitro model of 10 μM GLY-exposed mouse Leydig (TM3) cells was established to elucidate this issue. Data firstly showed that mitochondrial malfunction, mainly manifested by ultrastructure damage, disturbance of mitochondrial dynamics and mitochondrial reactive oxygen species (mtROS) overproduction, was responsible for GLY-decreased protein levels of steroidogenic enzymes, which leads to the inhibition of testosterone synthesis. Enhancement of autophagic flux and activation of mitophagy were shown in GLY-treated TM3 cells, and further studies have revealed that GLY-activated mitophagy is parkin-dependent. Notably, GLY-inhibited testosterone production was significantly improved by parkin knockdown. Finally, data showed that treatment with mitochondria-targeted antioxidant Mito-TEMPO (M-T) markedly reversed GLY-induced mitochondrial network fragmentation, activation of parkin-dependent mitophagy and consultant testosterone reduction. Overall, these findings demonstrate that GLY induces mtROS overproduction to activate parkin-dependent mitophagy, which contributes to the inhibition of testosterone synthesis. This study provides a potential mechanistic explanation for how GLY inhibits testosterone synthesis in mouse Leydig cells. Schematic diagram of the proposed mechanism by which accumulated mtROS promotes parkin-dependent mitophagy to inhibit testosterone synthesis in GLY-exposed mouse Leydig cells. [Display omitted] •Glyphosate or mitochondrial reactive oxygen species is abbreviated as GLY or mtROS.•Low dose GLY exposure inhibits testosterone synthesis in mouse Leydig cells.•GLY-induced mtROS overproduction results in mitochondrial fragmentation.•Parkin-dependent mitophagy facilitates GLY-inhibited testosterone synthesis.•Excessive mtROS production contributes to GLY-activated parkin-dependent mitophagy.