Testicular ischemia reperfusion injury (tIRI) causes oxidative stress-induced DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to establish a direct link between JAK2 activation and the DNA damage response (DDR) signaling pathways and their role in tIRI-induced GCA using AG490, a JAK2 specific inhibitor. Male Sprague Dawley rats (n = 36) were divided into three groups: sham, unilateral tIRI and tIRI + AG490 (40 mg/kg). During tIRI, augmentation in the phosphorylation levels of the JAK2/STAT1/STAT3 was measured by immunohistochemistry. Observed spermatogenic arrest was explained by the presence of considerable levels of DSB, AP sites and 8OHdG and activation of caspase 9, caspase 3 and PARP, which were measured by colorimetric assays and TUNEL. The ATM/Chk2/H2AX and ATR/Chk1 pathways were also activated as judged by their increased phosphorylation using Western blot. These observations were all prevented by AG490 inhibition of JAK2 activity. Our findings demonstrate that JAK2 regulates tIRI-induced GCA, oxidative DNA damage and activation of the ATM/Chk2/H2AX and ATR/Chk1 DDR pathways, but the cell made the apoptosis decision despite DDR efforts.

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.

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress.

The aim of this study is to determine whether the c-Jun N-terminal kinase (JNK) signaling is a regulator of oxidative DNA damage, germ cell apoptosis (GCA), and mitochondrial dysfunction during testicular ischemia reperfusion injury (tIRI) using the JNK inhibitor SP600125. Male Sprague Dawley rats (n = 36) were equally divided into three groups: sham, tIRI only, and tIRI + SP600125 (15 mg/kg). Testicular ischemia was induced for 1 h followed by 4 h of reperfusion prior to animal sacrifice. Spermatogenesis was evaluated by light microscopy, while expression of oxidative stress and GCA-related mRNAs and proteins were evaluated by real-time polymerase chain reaction and colorimetric assays, respectively. Expressions of JNK, p53, and survivin were detected by immunofluorescence (IF) staining. Indicators of mitochondrial dysfunction were examined by western blot analysis and colorimetric assay. In comparison to sham, the tIRI testes showed a significant increase in lipid and protein oxidation products. Oxidative DNA damage was reflected by a significant increase in the number of DNA strand breaks, increased concentration of 8-OHdG, and elevated poly (ADP-ribose) polymerase activity. Spermatogenic damage was associated with the activation of caspase 3 and elevated Bax to Bcl2 ratio. This was also accompanied by a significantly heightened IF expression of the phosphorylated forms of JNK and p53 paralled with the suppression of survivin. Mitochondrial dysfunction was reflected by NAD+ depletion, overexpression of uncoupling protein 2, and increased level of cytochrome c. Such tIRI-induced modulations were all attenuated by SP600125 treatment prior to reperfusion. In conclusion, JNK signaling regulates oxidative DNA damage, GCA, and mitochondrial dysfunction through activation of p53 and suppression of survivin during tIRI.

Testicular ischemia reperfusion injury (tIRI) has been suggested through several studies as the underlying mechanism for testicular torsion and detorsion (TTD) due to the excessive generation of reactive oxygen species (ROS) leading to germ cell apoptosis (GCA). The aim of the study is to explore the mechanism of the apoptosis signal‐regulating kinase 1 (ASK1) signaling pathway during tIRI by using its specific inhibitor, NQDI‐1. We also investigated the effect of NQDI‐1 on the expression of the thioredoxin (Trx) system, a major source of ROS. Male Sprague‐Dawley rats (n=36, 250–300 g) were divided equally into three experimental groups: sham, tIRI and tIRI + NQDI‐1 (10 mg/kg). During tIRI, the testicular artery was occluded for 1 hour followed by 4 hours reperfusion. The NQDI1 was injected intraperitoneally 30 minutes post ischemia. Histological analyses were used to evaluate testicular morphological changes as well as spermatogenesis. Gene expression, protein immunoexpression and enzyme activities were assessed by real‐time PCR, ELISA, IHC and colorimetric assays. Following tIRI, histological examination revealed impairment in spermatogenesis represented by a significant decrease in the Johnsen score. Real‐time PCR confirmed the upregulation of the pro‐apoptosis genes BAD and BAX and the downregulation of anti‐apoptosis gene Bcl2, which were accompanied by activation of the executioner caspase‐3 in the tIRI group. The levels of the antioxidants GSH, SOD, as well as thioredoxin reductase (TrxR) enzyme were also reduced upon tIRI induction. Immunoexpression of ph‐p38 and ph‐ASK1 were increased significantly after tIRI indicating the activation of ASK1 signalling pathway, while a remarkable decrease in Trx was simultaneously observed. The above damaging consequences of tIRI were attenuated after NQDI1 treatment obtaining normalized values similar to sham. Our findings suggest that the redox status of the cell is cooperatively regulated with ASK1 signaling to maintain cellular homeostasis during testicular oxidative stress. Additionally, the interplay between the Trx system and ASK1 suggests their potential role in the pathogenesis of tIRI.Support or Funding InformationThe project was supported by the College of Graduate studies and Research Administration, Kuwait University through grant # YM 09/17 and OMICSRU/RCF project grant # SRUL02/13.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.