Unilateral Testicular Ischemia Research Articles

Protective Effects of Topical Application of Nitrite on Testicular Ischemia-Reperfusion Injury in Rats.

Testicular torsion is a urologic emergency induced by torsion of the spermatic cord, interrupting blood circulation to the testis. Therapeutic options for testicular torsion, except surgical restoration of testis, are rarely applied in clinical practice. This study, therefore, investigated whether topical application of nitrite (NO2−) is beneficial in tissue damage due to testicular ischemia-reperfusion (I/R) injury in rats. Pubertal Sprague-Dawley rats were assigned to seven groups: group A, sham-operated control group; group B, I/R with no treatment; groups C, D, and E, I/R followed by treatment with three different doses of nitrite; group F, I/R followed by administration of nitrite and a NO scavenger, C-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt); and group G, I/R followed by administration of nitrate (NO3−). Unilateral testicular ischemia was maintained for 5 h, followed by reperfusion for 24 h. Nitrite and nitrate were topically administered before reperfusion. Compared to group A, germ cell apoptosis, oxidative stress, antioxidant enzymatic function, and lipid peroxidation were significantly increased, along with abnormal morphology and impaired spermatogenesis in group B (P < 0.05). In contrast, testicular damage was generally attenuated in the nitrite treatment groups due to a reduction in superoxide and peroxynitrite levels and the inhibition of caspase-3-dependent apoptosis (P < 0.05 vs. group B). These therapeutic effects of nitrite-derived NO were suppressed after injection of C-PTIO, which showed in group F. Taken together, our results demonstrate that topical application of nitrite may be one of the therapeutic strategies for testicular ischemia-reperfusion injury.

A novel pathway for the induction of DNA damage in human spermatozoa involving extracellular cell-free DNA

DNA damage is a common feature of human spermatozoa associated with an impaired capacity to fertilize the oocyte and an increased mutational load in the offspring. However, the etiology of this damage remains poorly defined. In this study we demonstrate that a major pathway for the induction of DNA damage in mammalian spermatozoa is triggered by exposure to exogenous cell free DNA (cfDNA). Exposure of human and mouse spermatozoa to cfDNA (calf thymus, mouse liver and salmon testes) in vitro induced a dose-dependent increase in sperm DNA damage that could be effectively suppressed by the concomitant presence of DNase. The induction of such damage was not accompanied by any concomitant change in sperm motility or vitality and was not directly associated with the induction of oxidative stress. In vivo the injection of exogenous DNA again precipitated an increase in sperm DNA fragmentation that could be reversed by the prior administration of DNase. Similarly, the induction of a transient unilateral testicular ischemia induced an increase in DNA fragmentation that was evident within 24 h and sustained for at least 14 days via mechanisms that could be completely suppressed by the prior administration of DNase. We conclude that exogenous cfDNA activates a defensive response in human spermatozoa associated with the nuclease-mediated induction of DNA fragmentation, possibly involving the participation of TLR9 and CD4. These novel insights have significant implications for our understanding of DNA fragmentation in the male germ line and open up new pathways for the remediation of this condition.

Recruiting testicular torsion introduces an azoospermic mouse model for spermatogonial stem cell transplantation.

To investigate the long-term effect of testicular torsion on sperm parameters and testis structure in order to introduce a novel mice azoospermic model for spermatogonial stem cell transplantation. Unilateral testicular torsion was created. The animals were divided into two groups each containing 15 mice. They underwent 2 and 4 hours of unilateral testicular ischemia, respectively. All animals in this experiment were aged matched. The experimental (n = 5) groups were studied 2, 4 and 10 weeks after testicular ischemia reperfusion. Moreover, the left testes and epididymis were removed for sperm analysis and for weight and histopathological evaluation. Finally isolated spermatogonial stem cells were transplanted in the testes that underwent 2 hours of ischemia reperfusion, two weeks post-surgery. All the investigated parameters demonstrated a sharp decline at 2, 4 and 10 weeks after testicular torsion, whereas 2-hour ischemia was found to be less injurious in testicular tissue structure. Two months after xenotransplantation, the transplanted cells were localized in the basal of the seminiferous tubules of the recipient ischemic testes. Torsion can cause permanent azoospermia in mouse. Also Testicular torsion 2 weeks after the 2 hours ischemia reperfusion may prove useful for recipient preparation for SSCs transplantation in mouse.

Beneficial effects of (−)-epigallocatechin gallate on ischemia-reperfusion testicular injury in rats

Background/PurposeThe purpose of the study was to determine the effect of (−)-epigallocatechin gallate (EGCG) on testicular ischemia-reperfusion injury in rats. MethodsForty male Wistar rats were assigned to 5 groups. A sham operation was performed on the animals in group 1. In group 2, after 4 hours of unilateral testicular ischemia, 4 hours of testicular reperfusion was performed with EGCG administered 1 hour before reperfusion. In group 3, the same surgical procedure as in group 2 was performed, but without EGCG. Serum superoxide dismutase activity, creatine kinase, and lactate dehydrogenase were then measured in blood samples from groups 1 to 3. In group 4, after 4 hours of unilateral testicular ischemia, testicular reperfusion was performed. In group 5, the same procedure as in group 4 was performed, but with EGCG administered 1 hour before reperfusion. For groups 4 and 5, bilateral orchiectomy was performed for histologic examination 4 weeks after reperfusion was started. ResultsSerum superoxide dismutase activity was significantly higher in group 2 than in group 3. The ratios of bilateral testicular weight, mean seminiferous tubule diameter, and germinal epithelial cell thickness were significantly higher in group 5 than in group 4. ConclusionsTherapy with EGCG before reperfusion might exert protective effects via antioxidant activities in a rat experimental model of testicular ischemia-reperfusion injury.

Protective effect of sivelestat, a neutrophil elastase inhibitor, on ipsilateral and contralateral testes after unilateral testicular ischaemia–reperfusion injury in rats

To investigate the effect of a neutrophil elastase inhibitor, sivelestat sodium hydrate, on testicular ischaemia-reperfusion (IR)-injury. Eight-week-old male Sprague-Dawley rats were divided into four groups: sham-operated control rats; IR rats (group IR); and IR rats that received intra-abdominal administration of 15 mg/kg or 60 mg/kg sivelestat (group IR15 and group IR60, respectively). Right testicular vessels were clamped for 90 min in groups IR, IR15 and IR60. Sivelestat had been administered 45 min after the induction of the ischaemia in groups IR15 and IR60. In subpopulations of IR, IR15 and IR60 rats, reperfusion was performed after ischaemia for 2 h (groups IR-A, IR15-A and IR60-A, respectively) or 48 h (groups IR-B, IR15-B and IR60-B, respectively). At the end of the reperfusion period, blood samples were aspirated from both spermatic veins of each rat and testosterone was evaluated. Then both testes from all rats were collected and tissue levels of malondialdehyde (MDA), myeloperoxidase (MPO), and heat-shock protein-70(HSP-70) were evaluated. Testicular tissue samples were also processed for histological evaluation and TUNEL staining. MDA, MPO and HSP-70 levels in the ischemic testis were significantly higher in the IR group compared with the control group. MDA and HSP-70 in the contralateral testis were significantly higher in the IR group compared with the control group. Bilateral testosterone levels were lower in all rat groups in comparison with the control group. Bilateral testicular samples in group IR showed extensive histopathologic degenerative alterations and increased percentage of apoptotic cells. Sivelestat treatment lowered the MDA concentration and the percentage of apoptotic cells bilaterally and ameliorated the testicular histological pattern bilaterally. Unilateral testicular ischaemia causes significant contralateral testicular damage. Sivelestat may be a novel adjunct tool for reducing oxidative stress and partially preventing bilateral testicular damage.

Effects of 3-aminobenzamide on unilateral testicular ischemia–reperfusion injury: What is the role of PARP inhibition?

The therapeutic effects of poly(adenosine diphosphate-ribose) polymerase inhibition by 3-aminobenzamide (3-AB) were investigated in testicular ischemia-reperfusion (I/R) injury, using sperm analysis and histopathological and biochemical examinations, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities and reduced glutathione (GSH) levels. Male rats were divided into 3 groups: sham (n = 12), I/R (n = 12), and I/R with 3-AB (I/R-3-AB) (n = 12). The left testicular artery was occluded for 1 h, followed by 24 h (for biochemical and histopathological examinations) and 30 days (for sperm analysis) of reperfusion. 3-AB treatment intraperitoneally 10 min prior to and 1 h after reperfusion increased the I/R-induced decrease in sperm motility in both testes and reduced the increased abnormal sperm rates in the ipsilateral testis. However, 3-AB treatment failed to prevent the I/R-induced decrease in sperm concentration in both testes. SOD and CAT activities did not change in any group. GSH-Px activity and GSH levels were increased by I/R. 3-AB treatment reversed the I/R-induced increase in GSH-Px activity, similar to the level in sham rats, but did not alter GSH levels. 3-AB treatment significantly increased the I/R-induced decrease in histopathologic score. In conclusion, 3-AB treatment has potential biochemical and histopathological benefits beyond improving sperm quality and may have the potential to decrease damage from testicular torsion.

Protective effect of ischaemic post-conditioning on ipsilateral and contralateral testes after unilateral testicular ischaemia-reperfusion injury

The aim of this study was to investigate the effect of ischaemic post-conditioning (IPostC) against ischaemia-reperfusion (IR) injury on bilateral testes after unilateral testicular ischaemia in the rat. Eight-week-old male Sprague-Dawley rats were divided into control group; IR group (60 min ischaemia-24 h reperfusion); IPostC1 × 10 group (60 min ischaemia followed by one cycle of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC3 × 10 group (three cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion); IPostC5 × 10 group (five cycles of 10 sec reperfusion-10 sec ischaemia; then 24 h reperfusion) and IPostC3 × 30 group (three cycles of 30 sec reperfusion-30 sec ischaemia; then 24 h reperfusion). In the IR and IPostC groups, the right testicular vessels were clamped using a special vascular clip. Malondialdehyde (MDA) and myeloperoxidase (MPO) levels were measured in testicular tissue samples bilaterally. Additionally, bilateral testicular tissue samples were processed for histological evaluation including haematoxylin-eosin, 4-hydroxy-2-nonenal (4-HNE) and TdT-mediated dUTP Nick End Labelling (TUNEL) staining. The levels of MDA and MPO as well as the positive cells per seminiferous tubule in TUNEL and 4-HNE stain in bilateral testes from the IR group were significantly higher compared with the control group. IPostC3 × 30 protocol significantly ameliorated the aforesaid parameters in both testes compared with the IR group. For the first time, we have demonstrated that IPostC protects both testes after unilateral testicular ischaemia-reperfusion. IPostC3 × 30 protocol offered the most effective protection.

Constitutive Expression of Hypoxia-Inducible Factor-1 Regulatory Proteins in the Normoxic and Ischemic Testis.

The transcription factor hypoxia inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis in cells. HIF-1 consists of a hypoxia-dependent alpha (α) subunit and an oxygen-independent beta (β) subunit. HIF-1 activates transcription of over 100 genes involved in a wide range of cellular and molecular responses to ischemia and hypoxia including angiogenesis, glucose transport, glycolysis, antiapopotic and proapoptotic pathways, and cellular proliferation. In most tissues, HIF-1α is ubiquitinated and rapidly degraded under normoxic conditions, but stabilized from degradation and activated by hypoxia. Previously we have demonstrated that HIF-1 is abundant in Leydig cells and that, unlike HIF-1 in other tissues, testicular HIF-1 exists in a non-ubiquitinated form that is constitutively expressed in both the normoxic and hypoxic rat testis. We hypothesize that HIF-1 plays key roles in protecting Leydig cells from apoptosis and that HIF-1 is important for regulating oxygen microenvironments in the testis. Studies in our laboratory have focused on the role of HIF-1 in the normoxic testis and in conditions of testis ischemia such as testicular torsion. Prolyl hydroxylases 1 and 2 (PHD1 and PHD2) and Factor Inhibiting HIF (FIH1) are oxygen-dependent enzymes that hydroxylate the HIF-1α subunit under normoxic conditions. Hydroxylated HIF-1α binds to the von Hippel-Lindau (pVHL) tumor suppressor protein, a ubiquitin ligase that targets HIF-1α for ubiquitination and subsequent degradation by the proteasome. The purpose of this study was to examine expression of PHDs, FIH1, and pVHL in the rat testis as an initial step towards understanding the regulatory mechanisms responsible for constitutive expression of testicular HIF-1. Experimental unilateral testicular ischemia (I) was created by 720° of testicular torsion for 1h followed by 4h or 24h of reperfusion (I/R). Cytoplasmic and nuclear proteins were isolated from sham-operated and ischemic testes and steady-state levels of PHD1, PHD2, FIH-1, and pVHL proteins detected by immunoblot analysis. PHD1 and 2 were constitutively expressed in normoxic and ischemic testes (P<0.05), and PHD2 was the predominant isoform detected. FIH1 and pVHL were also abundant in the testis and the levels of these proteins showed no significant changes following I or I/R (P<0.05). In conclusion, constitutive expression of HIF-1 and HIF-1 regulatory proteins in the normoxic and ischemic testis suggest that stabilization and activation of HIF-1 in the testis may occur through pathways that are fundamentally different from HIF-1 activation mechanisms in other tissues. Further studies on the functions of testicular HIF-1 and HIF-1 regulatory pathways will advance an understanding of the role of Leydig cells in oxygen homeostasis of the testis and cellular and molecular responses to testis ischemia and hypoxia. Supported by NIH grant R15HD046451 to M.A.P.

The protective effect of ischemic preconditioning on rat testis

BackgroundIt has been demonstrated that brief episodes of sublethal ischemia-reperfusion, so-called ischemic preconditioning, provide powerful tissue protection in different tissues such as heart, brain, skeletal muscle, lung, liver, intestine, kidney, retina, and endothelial cells. Although a recent study has claimed that there are no protective effects of ischemic preconditioning in rat testis, the protective effects of ischemic preconditioning on testicular tissue have not been investigated adequately. The present study was thus planned to investigate whether ischemic preconditioning has a protective effect on testicular tissue.MethodsRats were divided into seven groups that each contained seven rats. In group 1 (control group), only unilateral testicular ischemia was performed by creating a testicular torsion by a 720 degree clockwise rotation for 180 min. In group 2, group 3, group 4, group 5, group 6, and group 7, unilateral testicular ischemia was performed for 180 min following different periods of ischemic preconditioning. The ischemic preconditioning periods were as follows: 10 minutes of ischemia with 10 minutes of reperfusion in group 2; 20 minutes of ischemia with 10 minutes of reperfusion in group 3; 30 minutes of ischemia with 10 minutes of reperfusion in group 4; multiple preconditioning periods were used (3 × 10 min early phase transient ischemia with 10 min reperfusion in all episodes) in group 5; multiple preconditioning periods were used (5, 10, and 15 min early phase transient ischemia with 10 min reperfusion in all episodes) in group 6; and, multiple preconditioning periods were used (10, 20, and 30 min early phase transient ischemia with 10 min reperfusion in all episodes) in group 7. After the ischemic protocols were carried out, animals were sacrificed by cervical dislocation and testicular tissue samples were taken for biochemical measurements (protein, malondialdehyde, nitric oxide) and histological examination.ResultsAlthough decreased tissue malondialdehyde levels were detected in the groups of 2, 3, 4, and 5 compared to group 1, significant decreases were observed in only group 2 and group 5 (p < .05). Nitric oxide levels were numerically decreased in all groups compared to the control group but was statistically significant only in group 5 (p < .05). Histopathological examination demonstrated that all groups subjected to ischemic preconditioning had less tissue damage than group 1 (p < .05).ConclusionThese results suggest that ischemic preconditioning provides tissue protection in testicular tissue.

Effect of allopurinol on germ cell apoptosis following testicular ischemia–reperfusion injury in a rat

Recent evidence suggests that apoptosis is involved in germ cell loss following testicular ischemia-reperfusion (IR) injury. Allopurinol (Allo) is as a free radical scavenger which prevents tissue damage caused by reperfusion and oxygenation after ischemia; however, its effect on apoptosis in this type of injury has not been studied. To examine the effect of allopurinol on germ cell apoptosis following testicular IR in a rat. Forty rats were divided randomly into 4 experimental groups of 10 rats each: group A (Sham)-Sham operated animals; group B (Sham-Allo)-Sham operated rats treated with allopurinol given PO (by gavage) at a dose of 200 mg/kg, once daily, immediately before and 24 h following operation; group C (IR)-rats underwent 90 min of unilateral testicular ischemia and 48 h of reperfusion; group D (IR-Allo)-rats underwent IR and were treated with allopurinol similar to group B. The ipsilateral and contralateral testes were harvested 48 h following operation. Johnsen's criteria and the number of germinal cell layers were used to categorize spermatogenesis. TUNEL assay was used to determine germ cell apoptosis. Statistical analysis was performed using one-way ANOVA test, with P < 0.05 considered statistically significant. Testicular ischemia in rats led to histological damage in the ipsilateral testis. In the contralateral testis minimal damage was observed. Treatment with allopurinol increased significantly Johnsen's score in both the ischemic (7.3 +/- 0.5 vs 5.6 +/- 0.5, P < 0.05) and contralateral (8.9 +/- 0.1 vs 8.3 +/- 0.2, P < 0.05) testis, compared to IR-animals. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. Treatment with allopurinol resulted in a significant decrease in germ cell apoptosis in the ipsilateral testis, expressed as the number of positive tubules per 100 tubules (AI-1, (apoptotic index) threefold decrease, P < 0.005) and the number of apoptotic cells per 100 tubules (AI-2, fivefold decrease, P < 0.005) as well as a significant decrease in germ cell apoptosis in the contralateral testis (AI-1, 3.5-fold decrease, P < 0.05, AI-2- sixfold decrease, P < 0.005) compared to IR animals. In a rat model of testicular IR, treatment with allopurinol decreases germ cell apoptosis in both ischemic and contralateral testes and improves spermatogenesis.

METHYLENE BLUE INCREASES CONTRALATERAL TESTICULAR ISCHAEMIA–REPERFUSION INJURY AFTER UNILATERAL TESTICULAR TORSION

1. Testicular ischaemia-reperfusion injury is commonly seen in childhood. Infertility occurs in 25% of patients after unilateral testicular ischaemia. It is has been reported that methylene blue has a positive effect in the reparation of ischaemia-reperfusion injury in different tissues. Therefore, we hypothesized that methylene blue may prevent the hazardous effects of ischaemia-reperfusion injury in testicular tissue after unilateral testicular torsion. 2. Thirty-two prepubertal Wistar-albino rats were divided into four groups. Testicular torsion was created by rotating the right testis 720 degrees in a clockwise direction for 5 h in all groups except for Group C, which was the sham control group. In Group T, bilateral orchiectomy was performed following the torsion period. In Group TD, both testes were removed 5 days after the torsion period. In Group MB, methylene blue (1 mg/kg, i.p.) was administered 40 min before detorsion and once daily over 5 days; then, both testes were harvested. Tissue levels of malondialdehyde (MDA), serum levels of creatine kinase (CK), mean testicular biopsy score (MTBS) and mean seminifer tubule diameter (MSTD) were determined. 3. There was a significant difference in MTBS between Groups T and TD (P < 0.05) in both ipsilateral and contralateral testes. In the contralateral testis, treatment with methylene blue decreased MTBS and MSTD (P < 0.05) and increased MDA levels (P < 0.05). In Group T, mean serum CK concentrations were higher than in any of the other groups (P < 0.05). 4. After 5 h of unilateral testicular torsion and a 5 day reperfusion period, serious tissue damage occurred on both the ipsilateral and contralateral sides. Serum CK concentrations may be an indicator for ischaemia, but not for ischaemia-reperfusion injury. Contrary to our hypothesis, methylene blue increased contralateral testicular damage after unilateral testicular torsion and exacerbated oxidative events.

Protective Effects of Trimetazidine on Testicular Ischemia-Reperfusion Injury in Rats

Introduction: We tried to prove the effectiveness of trimetazidine (TMZ) on testicular torsion-detorsion injury. Materials and Methods: 15 male rats were equally divided into three groups: group 1 was the sham-operated control group; group 2 had 2 h of unilateral testicular ischemia followed by 3 days of reperfusion, and group 3 had 2 h of unilateral testicular ischemia followed by 3 days of oral TMZ treatment (5 mg/kg, bid) during reperfusion. In the removed testicles, tissue reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), malondialdehyde (MDA) levels and pathological modified Johnson scores (MJS) were evaluated. Mann-Whitney U test was used for statistical evaluations. Results: In group 2, on the ipsilateral side, GSH were significantly lower and MDA were higher than in groups 1 and 3, though GSH and MDA were not statistically different between groups 1 and 3. On the other hand, GPx in the control testicles of group 3 was significantly lower compared to those in the counterparts of both groups 1 and 2. Among three groups, GR determined in both testicles were not statistically different. On the ipsilateral side, MJS in group 3 were lower than in the sham group, but significantly higher than in group 2. Conclusions: According to this study, TMZ has an antioxidant effect on testicular torsion-detorsion injury, though the protective effect of TMZ seems to decrease in control testicles. Consequently it has been considered that TMZ can be only used in torsion patients with a healthy contralateral testicle after further studies have been conducted.

Effect of testicular ischemia-reperfusion on recruitment of neutrophils, E-selectin expression and germ cell apoptosis in the contralateral testis in a rat

Recent evidence suggests that neutrophil recruitment may initiate germ cell apoptosis in the ischemic testis. The purpose of the present study was to examine the relationship between germ cell apoptosis and neutrophil recruitment in the contralateral testis following testicular ischemia-reperfusion (IR) injury in a rat. Adult male Sprague-Dawley rats were divided randomly into two experimental groups: Group A: Sham operated animals; Group B: IR rats underwent 90 min of unilateral testicular ischemia following by 96 h of reperfusion. The rats were sacrificed and testes were harvested. Johnsen's criteria and the number of germinal cell layers were measured to categorize the spermatogenesis. TUNEL assay was used to determine germ cell apoptosis in both the ischemic and contralateral testis. The recruitment of neutrophils was calculated per 100 venules. Expression of E-selectin was determined using immunohistochemical analysis. Statistical analysis was performed using Student's t test, with P less than 0.05 considered statistically significant. Germ cell apoptosis in both the ischemic and the contralateral testis increased significantly after IR. E-selectin expression was significantly greater in ischemic testis from IR rats compared to sham animals. The small increase in E-selectin expression and the concomitant increase in neutrophil recruitment in the contralateral testis of the IR rats (vs. sham animals) were not statistically significant. In conclusion, testicular ischemia causes an increase in germ cell apoptosis in the contralateral testis. Mechanisms other than neutrophil recruitment apparently initiate this process.

Automated sonographic evaluation of testicular perfusion

Contrast-enhanced ultrasound (US) imaging is potentially applicable to the investigation of vascular disorders of the testis. We investigated the ability of two automated computer algorithms to analyse contrast-enhanced pulse inversion US data in a rabbit model of unilateral testicular ischaemia and to correctly determine relative testicular perfusion: nonlinear curve fitting of the US backscatter intensity as a function of time; and spectral analysis of the intensity time trace. We compared (i) five metrics based on the algorithmic data to testicular perfusion ratios obtained with radiolabelled microspheres, a reference standard; (ii) qualitative assessment of the US images by two independent readers blinded to the side of the experimental and control testes to the radiolabelled microsphere perfusion ratios; and (iii) results of the algorithmically-derived metrics to the qualitative assessments of the two readers. For the curve fit method, the algorithmically-derived metrics agreed with the reference standard in 54% to 68% of all cases. For the spectral method, the results agreed in 70% of all cases. The two readers agreed with the reference standard in 40% and 35% of all cases, respectively. These results suggest that automated methods of analysis may provide useful information in the assessment of testicular perfusion.

Simultaneous increase in germ cell apoptosis and oxidative stress under acute unilateral testicular ischaemia in rats.

Ischaemia induced germ cell apoptosis in rat testis was studied in detail to find out (i) spermatogenic stage or seminiferous epithelium region specific involvement of germ cells in apoptosis, (ii) preferential specificity of a particular germ cell type to become apoptotic and (iii) the ratio of live and dead testicular cells isolated in vitro after various period of ischaemic induction. Cell apoptosis, as observed in histological sections increased from 1 to 24 h of ischaemia. Apoptosis was not restricted to any specific germ cell type but was observed simultaneously in all the cell types in the initial hours (1-6 h) of ischaemia. No spermatogenic stage specific preference in apoptotic induction was also observed. However, as the duration of ischaemia progressed, the cell types observed to be most affected in number and morphology were the spermatids followed by spermatocytes. Centrally located tubules of testis were affected first than those located in the periphery. Overexpression of Bax staining was limited to few germ cell nuclei only. More than 95% of the germ cells in the control testis that earlier showed trypan blue dye exclusion were found stained after 12 h of ischaemia. Starting from early hours (1 h), lipid peroxidation rose proportionally with the duration of ischaemia while superoxide dismutase (SOD) and catalase activities were found decreased. Significant (p < 0.05) increase in the activities of glutathion-s-transferase and levels of hydrogen peroxide were observed after 6 h of ischaemia. These findings indicate that the physiological processes of oxidative stress have a direct linkage to the extent of germ cell apoptosis in the seminiferous epithelium.

The effects of unilateral testicular ischemia and hemicastration on contralateral testicular IGF-1 level, histology and lipid peroxidation

Hemicastration is followed by compansatory hypertrophy whereas unilateral testicular torsion is followed by atrophy in contralateral testicle in rats. Insulin-like growth factor (IGF-1) has important roles in testicular paracrine and autocrine functions. In this study it was aimed to compare ischemic parameters and IGF-1 levels in the contralateral testicle in unilateral spermatic cord ligation, testicular torsion, and hemicastratron. 32 wistar rats were equally altocated into sham, ligation, torsion, and hemicastration groups. In ligation group, right spermatic cord was ligated with 3/0 silk suture. In the torsion group, right testis was tcrsed for 720 degrees. In hemicastration group, right orchiectomy was done. 48 hours later left orchiectomy was done in all groups. Malondialdehyde (MDA) and IGF-1 levels were determined in the testicle. Average values of the groups were compared with Anova followed by Dunnett T3 multiple comparison tests. MDA levels were significantly reduced in ligation and torsion groups (p < 0.05). This reduction was more prominent in hemicastration group (p < 0.05). Contralateral testicular IGF-1 levels in ligation and torsion groups were not different compared with the sham group. Left testicular IGF-1 level in the hemicastration group was decreased significantly compared with other groups (p < 0.05). Histological. changes evaluated. Contralateral Johnsen's testicular biopsy scores were significantly decreased in all experimental groups but mean tubular diameter was not changed in all groups.

Stimulation of hypoxia-inducible factor-1 alpha (HIF-1alpha) protein in the adult rat testis following ischemic injury occurs without an increase in HIF-1alpha messenger RNA expression.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor composed of alpha and beta subunits. Stabilized from proteasome degradation and activated by hypoxia, HIF-1 stimulates expression of hypoxia-sensitive genes that mediate oxygen homeostasis in many tissues. Our hypothesis is that HIF-1 is involved in the cellular response to hypoxia in the ischemic testis. Goals of this study were to determine if HIF-1alpha mRNA is expressed in the testis, epididymis, and accessory sex glands of adult Sprague-Dawley rats and to determine if HIF-1alpha mRNA and protein expression in the testis is affected by experimentally induced ischemia. Total RNA from reproductive organs of adult rats was analyzed by relative reverse transcription-polymerase chain reaction (RT-PCR) analysis. HIF-1alpha mRNA showed equal expression in testis, all segments of epididymis, ductus deferens, accessory sex glands, and penis. To examine the effects of ischemia on HIF-1alpha mRNA and protein expression in the testis, rats were subjected to unilateral testicular ischemia by placing a ligature around spermatic artery or ischemia-inducing experimental torsion and reperfusion. RT-PCR revealed that HIF-1alpha mRNA expression at all times of ischemic treatment and reperfusion was unchanged compared with normoxic controls. HIF-1alpha protein was detected by immunoblot analysis of nuclear protein extracts from normoxic testes. Steady-state levels of HIF-1alpha protein were stimulated by 15 min of ischemia and showed a 2-fold increase at 30 min and 1, 3, and 6 h. HIF-1alpha protein was also elevated by experimental torsion and reperfusion compared with normoxic controls. These results support the hypothesis that HIF-1 may play a role in the cellular response to hypoxia in the ischemic testis.

SURFACTANT ADMINISTRATION REDUCES TESTICULAR ISCHEMIA-REPERFUSION INJURY

Purpose The mechanism of testicular ischemia-reperfusion injury has not been well delineated. We determined the efficacy of a biocompatible surfactant (tetronic 1107) to reduce tissue injury and evaluated cell membrane integrity as reflected by calcium ion permeability in an in vivo animal model of testicular ischemia-reperfusion. Materials and Methods Three groups of male Sprague-Dawley rats (6 per group) were studied. Group 1 was the nonoperative control, and groups 2 and 3 underwent 4 hours of unilateral testicular ischemia followed by 4 hours of reperfusion. Ten minutes after reperfusion 0.4 ml. saline was administered intravenously to group 2 and 180 mg./kg. surfactant tetronic 1107 to group 3. ( 99m) Technetium pyrophosphate was used to monitor calcium ion uptake by the ipsilateral and contralateral testicles. Both testicles were also examined histologically. Results The surfactant treated animals had markedly diminished hemorrhagic discoloration and vascular congestion compared to saline treated animals. These results were confirmed microscopically with improved nuclear chromicity and disarray of germ cell layers of the seminiferous tubules. The surfactant treated group also had a statistically significant (p <0.05) reduction in radiotracer uptake compared to the saline treated animals, confirming a reduction in calcium ion permeability. Conclusions The results of this study suggest that tetronic 1107 is effective in reducing tissue damage in a testicular ischemia-reperfusion animal model.

A study of warm testicular ischaemia and paternity in rats

The effect of testicular ischaemia on fertility as assessed by paternity rate was studied in seven groups of male Sprague-Dawley pubertal rats. Transient unilateral occlusion of the testicular artery and venous outflow was performed for 30, 60, 90, and 120 min using non-crushing microvascular clamps in groups 1–4 (n = 6). Group 5 (n = 12, Fowler-Stephens) had division of both the testicular artery and vein, whereas group 6 (n = 12, Refluo) had division of the testicular artery only. Group 7 (n = 6) consisted of controls upon whom a sham operation was performed. Testicular biopsy 6 weeks after transient testicular ischaemia of 30, 60, and 90 min showed tubular atrophy in 20%–33% of the testes. Ischaemia of 120 min led to tubular atrophy in 84% of testes. Division of only the testicular artery caused tubular atrophy in 58% of testes. Division of both testicular artery and venous outflow was associated with total tubular destruction and necrosis in 78% of testes and 22% had variable tubular atrophy. A paternity rate of 50%–80% was noted in groups 1–4 following transient unilateral testicular ischaemia and contralateral vasectomy. This compared well with the expected 75% paternity rate for a control rat population. Chronic ischaemia (group 5) together with testicular venous interruption was associated with observably lower fertility and a paternity rate of 18%. However, when venous outflow was preserved (group 6), chronic ischaemia after testicular artery division did not affect the paternity rate (50%) significantly.

Long-term study of the immuno-pathological consequences of sympathetic orchiopathia in the rat.

Unilateral testicular ischaemia was induced in Wistar rats by ligation and division of the testicular and deferential vessels. Damage (as assessed by a Johnsen count) to the contralateral testis caused significant spermatogenic suppression only at the equator of the testis at 28 days after operation. Cytotoxic antisperm antibody production increased progressively from 7 to 14 days and became maximal at 28 days after infarction, but after 3 and 6 months antibody production was decreasing. The presence of agglutinating antisperm antibody was noted at 28 days, 3 months and 6 months after infarction. Serum immunoglobulin estimations revealed an increase in IgG and IgM levels at 7 days and IgM levels at 14 days, supporting the contention that an immunological reaction had occurred. It is suggested that unilateral testicular ischaemia in the rat, an animal model intended to mimic torsion of the testis in the human, causes a transient immunological phenomenon (sympathetic orchiopathia) which recovers over the course of time. Caution should be exercised before regarding this relatively common surgical emergency as an aetiology of oligospermia or asthenospermia.