Doxorubicin and 4-Hydroperoxycyclophosphamide Alter Mitochondrial Dynamics and miR-34 Expression in Spermatogonia and Spermatocytes In Vitro.

Doxorubicin (DOX) and cyclophosphamide (CP) are widely used chemotherapeutic agents with known gonadotoxic effects. Since miRNAs play a key role in the transcriptional regulation of spermatogenesis, they are suggested as biomarkers for male infertility. This study aimed to investigate changes in vitro the expression of proteins related to mitochondrial dynamics, apoptotic cell profiles and cell cycle distribution after treating mouse spermatogonia and spermatocytes with DOX and 4-hydroperoxycyclophosphamide (4-HC). Additionally, we also aimed to evaluate the correlation between mitochondrial function-related miR-34 family expression and mitochondrial dynamics in both cells. Mitochondrial fusion, fission, mitophagy and apoptosis markers were analysed by immunocytochemistry and qPCR. miR-34 family expressions were assessed by qPCR. Apoptosis profiles and cell cycle distributions were determined using flow cytometry. We found that DOX and 4-HC increased the percentage of cells in S and G2/M phases and apoptosis levels. Mitochondrial fusion and mitophagy gene expressions decreased, whereas fission gene expression increased. Moreover, miR-34a expression rose, whereas miR-34b and miR-34c expressions significantly decreased after treatment. These miR-34 family expression changes correlated with mitochondrial dynamics-related gene expressions, suggesting a potential role of the miR-34 family in mitochondrial dysfunction.