calpain and Spermatic-Cord-Torsion

The dynamics of calpain and involvement of apoptosis in sperm formation disorder of the unaffected testis in rats with experimental testicular torsion were investigated.. Using 6-week-old Wistar rats, an experimental unilateral testicular torsion model was prepared. The bilateral testes were excised 1, 3, 5, 7, 14, 35, and 70 days after the left testis was twisted, and the unaffected testes were subjected to immunohistological staining, sodium dodecyl sulfate polyacrilamide gel electrophoresis and Western blotting using anti-calpain antibodies. Apoptosis was detected by the TdT-mediated dUTP biotin nick end-labeling (TUNEL) method.. By immunohistological staining, positive immunostaining by anti-pro mu-calpain antibody was observed in the spermatocyte nucleus, but not with anti-pro m-calpain antibody. The staining was increased until 7 days after testicular torsion, then decreased with progression of sperm formation disorder. By Western blotting, the intensity of staining with anti-pro mu-calpain antibody was increased until 7 days after torsion of the testis. Apoptosis expression in the unaffected testis was significantly inhibited by addition of a calpain inhibitor.. It was suggested that mu-calpain may be involved in apoptosis expression in sperm formation disorder of the unaffected testis in unilateral testicular torsion.

Topics: Animals; Apoptosis; Blotting, Western; Calpain; Immunohistochemistry; Infertility, Male; Male; Rats; Rats, Wistar; Seminiferous Tubules; Spermatic Cord Torsion; Spermatogenesis

The purpose of this study is to determine whether inducible nitric oxide synthase (iNOS) is involved in the pathogenesis of testicular ischemia-reperfusion (I/R) injury in association with germ cell death, through either necrosis or apoptosis. Western blot analysis showed that iNOS expression was markedly increased 1 h after ischemia, and was accompanied by a huge nitric oxide (NO) production, as measured by the Griess method, with a peak at 48 h of reperfusion. Immunohistochemistry showed that iNOS was expressed predominantly in the macrophage-like cells infiltrated in the interstitial tissues of the testis. Intraperitoneal injection of aminoguanidine (AMG) (400 mg/day), the inhibitor of iNOS, reduced NO production by 57.7% at 96 h of reperfusion. Calpain activation and proteolysis of alpha-fodrin induced by I/R were inhibited by AMG. Germ cell apoptosis was demonstrated by in situ TUNEL and DNA fragmentation on agarose gel electrophoresis. Germ cell apoptosis was maximally induced at 24 h of reperfusion, and was not inhibited by AMG. NO produced by iNOS in the delayed phase of reperfusion promoted alpha-fodrin proteolysis, which is closely associated with necrosis. Inducible NOS inhibition combined with calpain inhibition may improve impaired spermatogenesis after testicular torsion.

Topics: Animals; Apoptosis; Calpain; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Enzyme Inhibitors; Guanidines; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Kinetics; Male; Necrosis; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Spermatic Cord Torsion; Spermatogenesis; Spermatozoa; Testis

Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of alpha-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed alpha-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion.

Topics: Animals; Apoptosis; Calpain; Caspase Inhibitors; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Flow Cytometry; Immunohistochemistry; In Situ Nick-End Labeling; Male; Necrosis; Organ Size; Protease Inhibitors; Rats; Rats, Wistar; Reperfusion Injury; Spermatic Cord Torsion; Spermatogenesis; Testis