Fig 1.
Schematic representation of the experimental treatments.
Male C57BL/6 mice were subjected to an intermittent chronic treatment (one day on/one day off for 13 days) with caffeine 5 mg/Kg (CAF), cocaine 10 mg/kg (COC) and their combination (5 mg/kg caffeine + 10 mg/kg cocaine, CAF-COC) in a binge protocol: 3 i.p. injections per day, 1 hour apart. Tissue samples were taken 24 hr after the last binge.
Table 1.
Primer sequences.
Fig 2.
Effect of caffeine, cocaine, and their combination on testicular histology.
Morphometric analysis of interstitial (A) and tubular (B) compartments. DAZL positive cell counts (C) and Dazl mRNA expression (D) in the testis from experimental groups. CAF: caffeine, COC: cocaine: ST: seminiferous tubule, I: interstitium. Values indicate mean ± SEM (N = 6). One way ANOVA- Bonferroni. * p<0.05 different from Vehicle; $ p<0.05 different from COC.
Fig 3.
Effect of caffeine, cocaine, and their combination on protein expression of FSHR, LHR, and proliferation and apoptotic markers.
A) Protein levels of hormone receptors FSHR and LHR and B) proliferation PCNA and pro-apoptotic BAX in the testes of experimental groups. CAF: caffeine; COC: cocaine. Values indicate mean ± SEM (N = 6). One way ANOVA- Bonferroni. * p<0.05 different from Vehicle; $ p<0.05 different from COC.
Fig 4.
Effect of caffeine, cocaine and their combination on testicular lipid peroxidation and antioxidant enzymes mRNA expression.
A) TBARS assay (N = 3–4). B) Antioxidant enzymes Catalase (Cat), superoxide dismutase (Sod1) and glutathione peroxidase (Gpx) mRNA expression (N = 5–6). CAF: caffeine; COC: cocaine. Values indicate mean ± SEM. One way ANOVA- Bonferroni. * p<0.05 different from Vehicle.
Fig 5.
Testicular expression of TH and dopamine receptors.
Testicular expression and cellular localization of the rate-limiting enzyme of catecholamine synthesis TH (A, B) as well as dopamine receptors DRD1 (C) and DRD2 (D) in testicular tissue of naïve mice. ST: seminiferous tubule; LC: Leydig cell; Inset arrows: DRD1 expression in spermatogonia nearest the basal lamina of the seminiferous tubules.
Fig 6.
Testicular TH protein expression after caffeine, cocaine and their combination.
A) TH immunostaining. B) TH protein levels evaluated by western blot. CAF: caffeine; COC: cocaine; I: interstitium; ST: seminiferous tubules; TH: tyrosine hydroxylase. Values indicate mean ± SEM (N = 5–6). One way ANOVA- Bonferroni. * p<0.05 different from Vehicle.
Fig 7.
Testicular mRNA expression of dopamine and adenosine receptors after caffeine, cocaine and their combination.
CAF: caffeine; COC: cocaine. Values indicate mean ± SEM (N = 6). One way ANOVA- Bonferroni. * p<0.05 different from Vehicle; $ p<0.05 different from COC.