The Thioredoxin System is Regulated by the ASK-1/JNK/p38/Survivin Pathway During Germ Cell Apoptosis

Abstract

The aim is to explore the mechanism of the apoptosis signal-regulating kinase-1 (ASK-1) signaling pathway and the involvement of the thioredoxin (Trx) system during testicular ischemia reperfusion injury (tIRI) by using ASK-1 specific inhibitor, NQDI-1. Male Sprague-Dawley rats (n = 36, 250-300 g) were equally divided into 3 groups: sham, tIRI, and tIRI + NQDI-1 (10 mg/kg, i.p, pre-reperfusion). For tIRI induction, the testicular cord and artery were occluded for 1 h followed by 4 h of reperfusion. Histological analyses, protein immunoexpression, biochemical assays, and real-time PCR were used to evaluate spermatogenesis, ASK-1/Trx axis expression, enzyme activities, and relative mRNA expression, respectively. During tIRI, ipsilateral testes underwent oxidative stress indicated by low levels of superoxide dismutase (SOD) and Glutathione (GSH), increased oxidative damage to lipids and DNA, and spermatogenic damage. This was associated with induced mRNA expression of pro-apoptosis genes, downregulation of antiapoptosis genes, increased caspase 3 activity and activation of the ASK-1/JNK/p38/survivin apoptosis pathway. In parallel, the expression of Trx, Trx reductase were significantly reduced, while the expression of Trx interacting protein (TXNIP) and the NADP⁺/ nicotinamide Adenine Dinucleotide phosphate (NADPH) ratio were increased. These modulations were attenuated by NQDI-1 treatment. In conclusion, the Trx system is regulated by the ASK-1/Trx/TXNIP axis to maintain cellular redox homeostasis and is linked to tIRI-induced germ cell apoptosis via the ASK-1/JNK/p38/survivin apoptosis pathway.

Keywords: ASK-1; JNK; NQDI-1; germ cell apoptosis; oxidative DNA damage; survivin; testicular ischemia reperfusion injury; thioredoxin.

Figure 1

NQDI-1 protects against damage to testicular histology and spermatogenesis. hematoxylin and eosin (H&E)stained-slides were visualized under light microscopy revealing destruction in the Seminiferous Tubule (ST) structure accompanied by alterations in the germ cell layer arrangement after tIRI induction. In contrast, sham and NQDI-1 treated groups showed normal ST histology and proper cellular stages of spermatogenesis. Contralateral testes showed no significant difference between the three animal groups. NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. Images were taken at 10x and 40x magnification with a scale bar of 50 μm.

Figure 2

NQDI-1 inhibits testicular oxidative stress. Colorimetric assays were used to analyze (a) SOD activity; (b) GSH levels and (c) MDA concentration, while Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) fluorescent staining was used to assess (d) DNA strand breaks. During tIRI, ipsilateral testes exhibited lower levels of the antioxidants SOD and GSH but high levels of MDA and DNA strand breaks in comparison to sham. NQDI-1 treatment attenuated testicular OS. Contralateral testes showed no significant difference between the three animal groups (p-value > 0.05). NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. Data are presented as mean ± SD (n = 6/group). * tIRI compared to sham and # NQDI-1 compared to tIRI. I = Ipsilateral and C = Contralateral.

Figure 3

NQDI-1 prevents DNA strand breaks. Oxidative DNA strand breaks were assessed by counting TUNEL positive nuclei from 55 STs in testicular tissue sections/animal group. The STs of tIRI-subjected testes showed an increased number of fluorescently labelled free DNA 3′ ends in comparison to sham and NQDI-1-treated rats (p-value < 0.0001). Contralateral testes showed no significant difference between the three animal groups (p-value > 0.05). NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. Fluorescently stained tissue sections were visualized using the LSM700 confocal microscope. Images were taken at 10× and 40× magnification with a scale bar of 50 μm.

Figure 4

NQDI-1 attenuates Caspase 3 Activity. Utilizing a biochemical assay, tIRI-induced Caspase 3 activity was normalized by NQDI-1 treatment and similar to sham levels (p-value < 0.0001). Contralateral testes showed no significant difference between the three animal groups (p-value > 0.05). NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. Data are presented as mean ± SD (n = 6/group). * tIRI compared to sham and # NQDI-1 compared to tIRI. I = Ipsilateral and C = Contralateral.

Figure 5

Immunofluorescence (IF) detection of the ph-ASK-1/ph-JNK/ph-p38/survivin apoptosis signaling pathway. The immuno-expression of the pro-apoptosis kinases ph-ASK-1/ph-JNK/ph-p38 was increased in spermatocytes in the ipsilateral testes-subjected to tIRI than in sham and after NQDI-1 treatment (p-value < 0.0001). The immune-expression of the IAP survivin was localized to spermatids and spermatozoa, however, it was significantly decreased in the tIRI-I group compared to sham and after NQDI-1 treatment (p-value < 0.0001). Contralateral testes showed no significant difference between the three animal groups (p-value > 0.05). NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. DAPI staining (blue) was carried out for all three animal groups. Fluorescently stained tissue sections (4 μm) were visualized using the LSM700 confocal microscope. Images were taken at 40× magnification with a scale bar of 50 μm.

Figure 6

NQDI-1 regulates the expression of the Trx system. In the ipsilateral testes of tIRI-subjected rats, elevated levels of (a) NADP⁺/NADH ratio and (b) TXNIP were measured by a colorimetric assay and ELISA, respectively, however, Low (c) TrxR activity was measured using a kinetic assay in comparison to the sham group. Treatment with NQDI-1 reverted the levels of NADP⁺/NADH ratio, TXNIP and TrxR to sham levels. Contralateral testes showed no significant difference between the three animal groups (p-value > 0.05). NQDI-1 (10 mg/kg) was i.p. injected 30 min post ischemia. Data are presented as mean ± SD (n = 6/group). * tIRI compared to sham and # NQDI-1 compared to tIRI. I = Ipsilateral and C = Contralateral.

Figure 7

NQDI-1 modulates the expression of the ASK-1/Trx axis. The immunoexpression of the ASK-1, ph-ASK-1, and Trx were evaluated by IHC staining under light microscopy. Both Trx and ASK-1 showed reduced immunoreactivity in the tIRI-subjected testes in comparison with sham NQDI-1 treated groups. As for ph-ASK-1, it showed strong immunoreactivity in the tIRI-subjected testes in comparison with sham NQDI-1 treated groups. Contralateral testes showed no significant difference between the three animal groups. NQDI-1 (10 mg/kg) was injected i.p. 30 min post ischemia. Images were taken at 10× and 40× magnification with a scale bar of 50 μm.