Assessment of doxorubicin-induced mouse testicular damage by the novel second-harmonic generation microscopy

Microtubules, maintaining a non-linear structure, are suitable for direct observation in living mammalian by second-harmonic imaging microscopy (SHIM) (a new kind of confocal microscopies). Testes constituted by vast seminiferous microtubules (SM), serve as good candidates for visualization by SHIM. This study employs the SHIM and Western-blot (WB) to assess the cellular-molecular levels of doxorubicin (Dox)-induced mouse testicular damage. The SHIM examination was able to clearly identify the integrity of normal architecture of the living mouse testis, namely, the anatomical features of SM, smooth muscle wall of SM, manchette microtubules, exoplasmic microtubules in Sertoli cells and interstitial connective tissue, as well as the destructive feature of SM in Dox-treated mice (n = 6 per group). By day 21 after Dox-treatment, the testicular weight and testicular length were significantly progressively decreased as Dox dosage was stepwise increased, i.e., 0/5/10/15/20 mg/kg/body-weight (BW) (all p<0.0001). The cross-section area of SM was significantly lower in Dox-treated (15 mg/kg-BW) mice than that in controls (p<0.001). The protein expression of vimentin was significantly progressively increased whereas the protein expression of β-tubulin/androgen-receptor was significantly progressively decreased in stepwise increased Dox dosage (all p<0.001). The protein expressions of inflammatory (MMP-9/IL-1β/TNF-α/iNOX), oxidative-stress (NOX-1/NOX-2/NOX-4/oxidized protein), apoptotic (mitochondrial-Bax/cleaved-caspase-3/PARP), fibrotic (Smad3/TGF-ß) mitochondrial/DNA-damaged (cytosolic cytochrome-C/γ-H2AX/ATM/KU70), and cell apoptotic/death (PTEN/p53) biomarkers were significantly higher in Dox-treated (15 mg/kg-BW) group than those in controls (all p<0.001). In conlusion, the dose-dependent Dox-caused mouse testicular damage can be not only detected by WB in molecular level but also clearly identified by SHIM in living mice.