Germ Cell-specific Apoptosis Research Articles

Beneficial effects of Cyclosporine A in combination with Nortriptyline on germ cell-specific apoptosis, oxidative stress and epididymal sperm qualities following testicular ischemia/reperfusion in rats: a comparative study

BackgroundTesticular torsion is a pathological condition which needs emergency surgical intervention. However, after surgical reperfusion, oxidative stress factors cause to germ cell apoptosis. The study was planned to evaluate the efficacy of simultaneous use of Cyclosporine A (CsA) and Nortriptyline (Nort) to repair testicular damages in an experimental torsion/detorsion (T/D) rat model.MethodsMale rats (n = 112) were allocated into 7 groups 16 each in; (Group 1); Control group, (Group 2); T/D group, (Group 3–4); CsA 1 and 5 mg/kg, (Group 5–6); Nort 2 and 10 mg/kg and (Group 7); concurrent group, CsA (1 mg/kg) + Nort (2 mg/kg). Right uni-lateral torsion was inducted by twisting testis 720 degrees in the clockwise direction for 1 h. For short-term and mid-term studies, lipid peroxidation, antioxidant enzyme activities, caspase-3 level, histopathological changes and germ cell apoptosis were evaluated. Moreover, in long-term investigation, semen analysis was performed.ResultsAfter T/D induction, testis abnormalities both functional and structural were appeared. Pre- and post-treatment with CsA and Nort, separately, reduced MDA and caspase-3 levels, normalized antioxidant levels, ameliorate tissue injury and improved sperm criteria.ConclusionThe antioxidant and anti-apoptotic characteristics of CsA and Nort and their protective effects have been shown in our study. Concurrent administration of CsA and Nort in selected low-dose indicated a significant positive effect as compared to the individual drug interventions on the reversal of T/D induced oxidative stress in short-term, apoptosis, and histologic changes in mid-term, as well as semen criteria in the long-term appraisal.

Carvacrol treatment opens Kir6.2 ATP-dependent potassium channels and prevents apoptosis on rat testis following ischemia-reperfusion injury model.

Testicular torsion is a urological problem that causes subfertility and testicular damage in males. Testis torsion and detorsion lead to ischemia–reperfusion (IR) injury in the testis. Testicular IR injury causes the increase of reactive oxygen species (ROS), oxidative stress (OS) and germ cell-specific apoptosis. In this study, we aimed to investigate whether Carvacrol has a protective effect on testicular IR injury and its effects on Kir6.2 channels, which is a member of adenosine triphosphate (ATP)-dependent potassium channels. In the study, 2–4 months old 36 albino Wistar rats were used. For experimental testicular IR model, the left testis was rotated counterclockwise at 720° for two hours, and after two hours following torsion, detorsion was performed. Carvacrol was dissolved in 5% Dimethyl Sulfoxide (DMSO) at a dose of 73 mg/kg and half an hour before detorsion, 0.2 mL was administered intraperitoneally. In testicular tissues, caspase 3 and Kir6.2 immunoexpressions were examined. Serum malondialdehyde (MDA) and testosterone levels were measured. Apoptotic cells and serum MDA levels were significantly decreased and Kir6.2 activation was significantly increased in Carvacrol-administrated IR group. As a result of our study, Carvacrol may activates Kir6.2 channels and inhibits apoptosis and may have a protective effect on testicular IR injury.

Germ cell-specific apoptosis by extracellular clusterin in cryptorchid dog testes

Mammalian testes are maintained at a relatively lesser temperature than the abdominal region so that normal spermatogenesis can occur. Germ cell apoptosis has resulted in heat-damaged testes that occurs as a result of cryptorchidism, but the mechanism is not yet fully understood. To elucidate the cause of germ-cell death by cryptorchidism, cryptorchidism was surgically induced in dog testes and histological and molecular analyses were performed. Histological data indicated that the seminiferous tubules of cryptorchid testes and epididymis contained fewer germ cells. Total RNA sequencing was performed to screen for overexpressed genes in cryptorchid dog testes. Clusterin RNA was in greater abundance (approximately 12.8-fold) in cryptorchid testes than in normal testes. In addition, cleaved caspase-3 and -8 were detected in greater abundance in cryptorchid dog testes. Real time RT-PCR and western blotting analysis indicated there was a greater abundance of clusterin in cryptorchid dog testes. Furthermore, clusterin was detected in extracellular regions of cryptorchid dog testes during the 4 weeks after surgery. Thus, germ-cell specific apoptosis and expression of clusterin genes occur with a resulting presence of this protein in extracellular regions of cryptorchid dog testes. This result will facilitate further study of spermatogenesis and the specific mechanisms by which cryptorchidism results in male infertility.

Effect of metformin on germ cell-specific apoptosis, oxidative stress and epididymal sperm quality after testicular torsion/detorsion in rats.

The study was designed to evaluate the effects of metformin on apoptosis and epididymal sperm quality in a rat testicular ischaemia/reperfusion (I/R) injury model. A total of 72 male rats were divided into four groups (n=18 for each group): group 1 (sham-operated group), group 2 (metformin group), group 3 (torsion/detorsion [T/D]+saline) and group 4 (T/D+300mgkg-1 metformin). Testicular torsion was achieved by rotating the right testis 720° in a clockwise direction for 1hr. Tissue malondialdehyde (MDA) level and caspase-3 activity increased and the activities of catalase, superoxide dismutase and glutathione peroxidase decreased in comparison with sham-operated group 4hr after detorsion (p<.001). In six rats of each group 24hr after detorsion, histopathological changes and germ cell apoptosis were significantly deteriorated by measuring mean of seminiferous tubule diameters (MSTD) and TUNEL test. Moreover, 30days after T/D, sperm concentration and motility were examined in six animals per group. Metformin pre-treatment reduced MDA and caspase-3 levels and normalised antioxidant enzyme activities 4hr after detorsion, and germ cell apoptosis was significantly decreased, and the MSTD, as well as sperm functions, was significantly improved. Reduction in oxidative stress and apoptosis may have a major role in cytoprotective effects of metformin.

Protective effects of polydatin on experimental testicular torsion and detorsion injury in rats.

Oxidative stress plays a critical role in the process of testicular torsion and detorsion (T/D). The purpose of the present study was to investigate the protective effect of polydatin (PD) on testicular T/D injury. Rats were randomly divided into three groups, a sham group, a group subjected to 2h torsion followed by 24h detorsion and a group subjected to T/D and injected i.p. with 20mgkg-1 PD 30min before detorsion. Unilateral orchiectomy was performed after 24h of reperfusion. Half the testes were prepared for histological examination by haematoxylin-eosin staining and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) technique. In the remaining tissues, levels of malondialdehyde (MDA), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were determined, as was the expression of several apoptosis-related proteins. Compared with the T/D group, PD pretreatment significantly ameliorated the morphological damage, lowered the Cosentino histological score and increased the mean number of germ cell layers and Johnsen's testicular biopsy score. In addition, PD treatment markedly decreased MDA levels and upregulated CAT, GPx and SOD activity. Furthermore, PD decreased T/D-induced germ cell-specific apoptosis, attenuated the activation of caspase-3, caspase-8, caspase-9 and poly(ADP-ribose) polymerase and increased the Bcl-2/Bax ratio. The findings indicate that PD has a protective effect against testicular T/D injuries, especially at the histological, antioxidative stress and antiapoptotic levels.

Hyperbaric Oxygen Therapy in Rats Attenuates Ischemia-reperfusion Testicular Injury Through Blockade of Oxidative Stress, Suppression of Inflammation, and Reduction of Nitric Oxide Formation

To evaluate the therapeutic utility of hyperbaric oxygen (HBO) therapy on testicular ischemia/reperfusion (I/R) injury and elucidate the underlying molecular mechanism, we tested whether HBO therapy provided rescue of the testes after torsion in rats. Sprague-Dawley rats were randomly divided into 4 groups: control group, control plus HBO therapy, I/R group, and I/R plus HBO therapy. The I/R model was induced by torsion of the right testis. I/R in the testis resulted in disrupted seminiferous tubules, germ cell-specific apoptosis, followed by a marked reduction in testis weight and daily sperm production. HBO therapy preserved seminiferous tubules, suppressed apoptosis, and prevented testicular atrophy in I/R testes. HBO therapy abated oxidative stress in I/R testes, marked by reduced malondialdehyde formation, enhanced activities of superoxide dismutase and heme oxygenase 1 (HO-1), and decreased activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and xanthine oxidase. HBO therapy resulted in a reduction of myeloperoxidase (MPO) activity in I/R testes, a marker of neutrophil recruitment. HBO therapy suppressed inflammation in I/R testes, marked by reduced messenger RNA (mRNA) levels of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β), and CD44. Furthermore, HBO therapy suppressed the activation of nuclear factor kappa B (NFκB), p38, and c-JUN-N-terminal kinase (JNK) signaling pathways in I/R testes. In addition, HBO therapy reduced nitric oxide formation in I/R testes through suppression of inducible nitric oxide synthase and dimethylarginine dimethylaminohydrolase. HBO therapy in rats attenuated I/R-induced testicular injury, possibly through abating oxidative stress, suppressing inflammation, and reducing nitric oxide formation.

The role of KATP channels on ischemia-reperfusion injury in the rat testis

AimsTo investigate the participation of KATP channels on the ischemia-reperfusion (IR)-induced apoptosis in the rat testis. Main methodsEight-week-old male Sprague–Dawley rats were divided into three groups: control and IR rats without or with cromakalim (300μg/kg intraperitoneally), 30min before the induction of ischemia. The right testicular artery and vein were clamped to induce ischemia in the testis. Sixty minutes after the ischemia, a 24h period of reperfusion followed. Then, expressions of KIR6.1, KIR6.2, caspase-3, PARP, Fas, FasL, and KIR6.1 and KIR6.2 mRNAs were investigated by Western blot analyses and real-time PCR methods, respectively. Furthermore, testicular tissues were processed for histological evaluation and TUNEL staining. Key findingsExpressions of KIR6.1 protein and mRNA were more than 10-fold of those of KIR6.2 protein and mRNA in the testis. IR significantly increased the expressions of KIR6.1 protein and mRNA as well as KIR6.2 mRNA, caspase-3, and TUNEL index in the testis compared to the control. PARP expressions were significantly lower in the IR group than those of the control. Histologically, severe acute germ cell damage was observed in the IR testis. Treatment with cromakalim ameliorated these parameters compared to the non-treated IR group. There were no significant differences on Fas, FasL and protein level of KIR6.2 expressions between any of the groups. SignificanceTreatment with cromakalim has a protective effect against IR-induced testicular damage via activating KATP channels. This is the first study to give evidence for the advantageous effect of cromakalim in the germ cell-specific apoptosis induced by testicular IR.

Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis

BackgroundSpermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’) identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1.MethodsThe testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis.ResultsThe ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter.ConclusionsThe results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.

N79 Coenzyme Q10 treatment reduces lipid peroxidation, inducible and endothelial nitric oxide synthases and germ cell-specific apoptosis in testicular ischemia/reperfusion injury

N79 Coenzyme Q10 treatment reduces lipid peroxidation, inducible and endothelial nitric oxide synthases and germ cell-specific apoptosis in testicular ischemia/reperfusion injury

Coenzyme Q 10 treatment reduces lipid peroxidation, inducible and endothelial nitric oxide synthases, and germ cell–specific apoptosis in a rat model of testicular ischemia/reperfusion injury

In this experimental study, we assessed the preventive effects of coenzyme Q(10) (CoQ(10)) in a rat model of ischemia/reperfusion injury. The results of this study show that CoQ(10) administration before the reperfusion period of testicular torsion provides a significant decrease in testicular lipid peroxidation products and expressions of inducible nitric oxide synthase, endothelial nitric oxide synthase, and germ cell-specific apoptosis.

Caenorhabditis elegans caspase homolog CSP-2 inhibits CED-3 autoactivation and apoptosis in germ cells

In C. elegans, apoptosis in germ cells is mediated by the same core apoptotic machinery that controls apoptosis in somatic cells. These include the CED-3 caspase, the CED-3 activator CED-4, and the cell death inhibitor CED-9. However, germline apoptosis also differs from somatic apoptosis in its regulation. We found that CSP-3, a caspase homolog that blocks CED-3 autoactivation and apoptosis in somatic cells, does not affect apoptosis in germ cells. Interestingly, the second C. elegans caspase homolog CSP-2 shares sequence similarity to both catalytic subunits of the CED-3 caspase, and surprisingly, contains a stretch of sequence that is almost identical to that of CSP-3. Unlike CSP-3 that acts specifically in somatic cells, loss of CSP-2 causes increased apoptosis only in germ cells, suggesting that CSP-2 is a germ cell specific apoptosis inhibitor. Moreover, like CSP-3, CSP-2 associates with the CED-3 zymogen and inhibits its autoactivation, but does not inhibit CED-4-induced CED-3 activation or the activity of the activated CED-3 protease. Thus, two different C. elegans caspase homologs employ the same mechanism to prevent caspase autoactivation and apoptosis in different tissues, suggesting that this could be a generally applicable strategy for regulating caspase activation and apoptosis.

Blocking E-selectin inhibits ischaemia–reperfusion-induced neutrophil recruitment to the murine testis

Germ cell-specific apoptosis that occurs after ischaemia-reperfusion (IR) of the murine testis is dependent on neutrophil recruitment to the testis and is dependent upon the cell adhesion molecule E-selectin. In this study, we aimed to inhibit neutrophil recruitment to the IR-induced testis. Mice were subjected to a 2-h period of testicular torsion (ischaemia) followed by detorsion (reperfusion). Shortly after onset of reperfusion, mice received either a function-blocking monoclonal anti-mouse E-selectin antibody (FBmAb) or isotype-matched control antibody. Mice were killed 24 h after reperfusion and cells isolated from the testis were analysed for the presence of neutrophil infiltration by flow cytometry. Administration of FBmAb inhibited neutrophil recruitment to the IR-induced testis dramatically. Therefore, blockage of E-selectin may be a strategy to treat post-ischaemic testis.

Ethyl pyruvate reduces germ cell–specific apoptosis and oxidative stress in rat model of testicular torsion/detorsion

Purpose Testicular torsion/detorsion (T/D) results in enhanced formation of free radical metabolites, which contributes to the pathophysiology of tissue damage. We investigated the protective effects of ethyl pyruvate (EP) against testis tissue damage in an experimental model of testicular torsion. Methods Sprague-Dawley rats were divided into 5 groups. In those animals that underwent T/D, right testes were rotated 720° for 1 hour. Group 1 control rats underwent sham operation. In group 2, the rats underwent T/D. The EP was prepared and injected in the form of Ringer's ethyl pyruvate solution. The rats in group 3, 4, and 5 received 2 doses of 20, 50, and 100 mg/kg EP (30 minutes before and after detorsion), respectively. The right testes of 6 animals from each group were excised 4 hours after detorsion for the measurement of lipid peroxidation, myeloperoxidase (MPO), and antioxidant enzymes activities. Germ cell apoptosis was determined in right testes of 8 animals per group 24 hours after detorsion. The epididymal sperm concentration and motility were evaluated 1 month after treatments. Results Germ cell apoptosis indices were significantly higher in group 2 compared with control group. The level of lipid peroxidation and MPO activity increased, whereas antioxidant enzymes activities decreased after T/D. Sperm count and motility were also reduced 1 month after T/D in group 2 rats. However, EP treatment at doses of 50 and 100 mg/kg significantly decreased the early apoptotic damage and improved long-term sperm count and motility. In the same dosing groups, we observed normalization of oxidant/antioxidant balance and decrement of MPO activity. However, administration of 20 mg/kg of EP conferred no protective effect. Conclusions Administration of Ringer's ethyl pyruvate solution (in appropriate doses) is protective against apoptotic tissue damage following testicular torsion and improves long-term testicular function. The antioxidant and anti-inflammatory properties of EP seem responsible for the protective effects. Our findings suggest this resuscitation solution as a possible substitute for fluid and electrolyte maintenance during surgical detorsion.

Administration of N-Acetylcysteine Reduces Germ Cell-Specific Apoptosis and Oxidative Injury Following Testicular Torsion in Rats

PURPOSE Testicular torsion/detorsion (T/D) RESULTS in enhanced formation of free radical species, which contribute to the pathophysiology of tissue damage. Numerous studies in recent years have shown protective effects of N-acetylcysteine (NAC) on cardiac and renal tissue damage following ischemia/reperfusion. We assessed the effectiveness of systemic administration of NAC – at therapeutic doses – in a rat model one-hour 720° testicular T/D. We chose the dose of NAC that reduced general ischemia/reperfusion syndrome in patients who had undergone abdominal aortic aneurysmectomy. MATERIALS AND METHODS Sprague-Dawley rats were divided into 5 groups, 14 animals in each. Group 1 underwent sham operation as the control group. In group 2, rats underwent T/D and received vehicle injections. Animals in groups 3, 4 and 5 received intraperitoneal injections of 150 mg/kg NAC, 30 minutes before torsion, after torsion and after detorsion, respectively. Markers of oxidative stress as well as germ cell apoptosis indices were determined 4 and 24 hours after detorsion, respectively. RESULTS Apoptosis indices were significantly higher in group 2 compared to control group. Four hours after detorsion, testicular level of lipid peroxidation was significantly increased and antioxidant enzymes activities were significantly decreased in group 2 in comparison with controls. Administration of NAC either 30 minutes before or after torsion (group 3 and 4), significantly improved the germ cell apoptosis indices and oxidant/antioxidant balance. Administration of NAC after detorsion had no significant effects on oxidant/antioxidant balance or germ cell apoptosis. CONCLUSIONS Administration of NAC prior to torsion or detorsion, but not after detorsion, is protective against ischemia/reperfusion tissue damage in the rat model of testicular torsion. Since NAC has an established safety after almost 40 years of clinical use as mucolytic agent and antidote of acetaminophen toxicity, one can suggest this agent as an adjunct therapy for “testicular torsion rescue”. Supplying intracellular glutathione as well as anti-oxidant and anti-inflammatory properties of NAC could be the possible mechanisms of this protective effect.

Propofol reduces nitric oxide‐induced apoptosis in testicular ischemia–reperfusion injury by downregulating the expression of inducible nitric oxide synthase

The aim of the present study was to investigate the underlying mechanisms in the preventive effects of intravenous anesthetics on testicular ischemia-reperfusion injury. Forty male Wistar Albino rats were randomly assigned to four groups of 10 rats each. Anesthesia was induced and maintained with thiopental in groups 1 and 2 and with propofol in groups 3 and 4. Groups 2 and 4 received left testicular ischemia (torsion) for 1 h and reperfusion (detorsion) for 24 h. Groups 1 and 3 (control groups) had no testicular torsion and detorsion. At 24 h of reperfusion, animals were killed and ipsilateral testes were removed for determination of tissue nitric oxide (NO) levels and immunohistochemical evaluation of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and apoptosis protease-activating factor 1 (APAF-1). Between groups 1 and 3, there were no differences in tissue NO levels and eNOS, iNOS, and APAF-1 expressions. iNOS and APAF-1 expressions were markedly increased in group 2, but these parameters were at the mild to moderate level in group 4 at 24 h of reperfusion. Also, elevated expression of iNOS was accompanied by a high NO production in group 2 compared with group 4. Although eNOS expressions were increased in both the groups (groups 2 and 4), there were no significant differences between these groups. Propofol as an anesthetic agent may attenuate germ cell-specific apoptosis and decrease NO biosynthases through downregulation of iNOS expression in an animal model of testicular torsion and detorsion.

Blocking Both E-Selectin and P-Selectin Inhibits Neutrophil Recruitment into the Murine Testis after Ischemia-Reperfusion-Induced Injury

Ischemia-reperfusion (IR) injury of the testis results in germ cell specific apoptosis, a process in which neutrophil recruitment to the testes plays a critical role. Adhesion molecules, in particular E- and P-selectins, play a critical role in this recruitment. The present study sought to characterize the inhibitory effect of function-blocking monoclonal anti-mouse E- and P-selectin antibodies on the migration of neutrophils into the IR-induced testis of the mouse. Mice were subjected to a 2 hr period of testicular torsion (ischemia) followed by detorsion (reperfusion). Ten minutes after the onset of reperfusion mice received either a mixture of 200 μg function-blocking monoclonal E-selectin and P-selectin antibody (FBMAb group; 100 μg; each) intravenously or 200 μg of isotype-matched control-antibody (IMCAb group). Separate groups of mice underwent shamoperation (SO group) or received 500 ng of TNFα (IF group) to induce inflammation. Mice were sacrificed 24 h after reperfusion and testicular interstitial cells were isolated and analyzed for the presence of neutrophils by means of flow cytometry. The mixture of function-blocking monoclonal E- and P-selectin antibody (FBMAb) decreased neutrophil recruitment to the IR-induced testis significantly (FBMAb group as compared to the IMCAb group 20.2 ± 2.8 vs. 51.9 ± 4.0 % Gr-1+CD11b+ of total leukocytes; p = 0.0002). Therefore, blocking both E- and P-selectin may be therapeutically beneficial to protect postischemic testis.

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress, BAX, and caspase 2

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function.

In Vivo Detection of Apoptotic Cells in the Testis Using Fluorescence Labeled Annexin V in a Mouse Model of Testicular Torsion

It is well established that experimental testicular torsion induces germ cell specific apoptosis. Annexin V (BD Pharmingentrade mark) binds phosphatidylserine that becomes exposed on the cell membrane in apoptotic cells. In vivo detection of apoptotic cells with fluorescently labeled annexin V is an emerging technique that we evaluated for detecting apoptotic germ cells in a mouse model of testicular torsion. Annexin V labeled with an indocyanine fluorophore (bisfunctional succinimidyl ester of cyanine 5.5) (Amersham, Little Chalfont, United Kingdom) was injected intravenously in mice 18 hours after the repair of unilateral 720-degree testicular torsion for 2 hours. Serial fluorescence images were obtained 21, 24, 28 and 42 hours after torsion repair. Relative fluorophore localization was visualized in vivo using an optical small animal imaging system mounted with a filter in near infrared light. Average fluorescence intensity in torsed and sham testes was quantified in images of testes in situ exposed through an abdominal incision and in ex vivo testes. A significant increase in fluorescence intensity was found in images of torsed vs sham operated testes. This was seen in ex vivo, exposed and in vivo testes (215%, 250% and 161%, respectively, p <0.05). Bisfunctional succinimidyl ester of cyanine 5.5 conjugated to dehydrogenase, a protein with a size similar to that of annexin V, was used to assess for capillary leakage. It was also more localized to the torsed testis relative to its contralateral sham control whether exposed or ex vivo (174% and 176%, respectively). To our knowledge this study demonstrates for the first time the possibility of in vivo near infrared fluorescence imaging of apoptotic germ cells after testicular torsion in mice. It shows important confounding factors that must be considered as this new imaging technique is developed for detecting apoptotic cells in vivo in testes or in any other organ.

Pre-Ischemic Administration of Nitric Oxide Synthase Inhibitors Reduced Germ Cell Apoptosis after Spermatic Vessel Ligation in the Rat Testis

Introduction: The Fowler-Stephens maneuver,a modeofspermatic vessel ligation, is a method of choice in the management of high testes. The aim of the present study is to investigate the effects of pre-ischemic administration of N^G-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, in preventing testicular ischemic damage and germ cell-specific apoptosis in rats. Materials and Methods: 30 min before ligation of the spermatic vessels, L-NAME was administered intraperitoneally to a group of rats and saline was given to another group of rats (controls). Biochemical assessments of NO and germ cell-specific apoptosis were performed. Results: Testicular NO levels in the L-NAME group showed significant decreases in the ipsilateral (p = 0.004) and contralateral (p = 0.015) testes in relation to those of the control group. The apoptotic indices were found in 2.3% of the L-NAME group and 3.1% of the control group. Conclusion: Pre-ischemic administrations of the NOS inhibitor, L-NAME, effectively decreased NO production and to some degree caused a reduction in germ cell apoptosis in the rat testes after spermatic vessel ligation. Further studies are mandatory to confirm our preliminary results and to address the potential introduction of NOS inhibitors into clinical practice.

The effect of spermatic vessel ligation on testicular nitric oxide levels and germ cell-specific apoptosis in rat testis

Management of high testis may vary but the most popular method in surgical treatment is the Fowler-Stephens maneuver. The aim of the present study was to investigate the effects of spermatic vessel ligation on testicular nitric oxide (NO) levels, expression of inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) and germ cell-specific apoptosis in both ipsilateral and contralateral testes in rats. Twenty-eight animals were randomly allocated into four groups (n=7 each). The spermatic vessels were ligated as a simulation of the Fowler-Stephens maneuver. The groups of animals were sacrificed at 2 h (group 1), 4 h (group 2) and 24 h (group 3) after ligation, respectively. Sham-operated animals served as controls (group 4). Biochemical assessment of testicular NO levels was performed by the Griess method. iNOS and eNOS expression and apoptosis were studied in ipsilateral and contralateral testes. Testicular NO levels at 24 h after the simulated Fowler-Stephens maneuver were found to be significantly increased in both ipsilateral and contralateral testes when compared with the sham-operated group. eNOS expression was clearly increased in ipsilateral testes, whereas moderate expression was detected in the contralateral seminiferous tubules at 24 h after ligation. Mild focal iNOS immunostaining was also observed in seminiferous tubules of the ipsilateral testis at 24 h after the simulated Fowler-Stephens maneuver. Apoptosis was dramatically increased in ipsilateral testes; however, it was only detected in single cells in the contralateral side at 24 h after ligation. In conclusion, the simulated Fowler-Stephens maneuver induces testicular nitric oxide synthesis and germ cell-specific apoptosis in the ipsilateral testis. These results suggest that high levels of NO induce apoptosis and may impair spermatogenesis thus explaining the unsuccessful outcome of the Fowler-Stephens maneuver.