PURPOSE: Vasectomy may result in damage to spermatogenesis. There are several explanations for this damage, including an increase of pressure in the seminiferous tubules and an autoimmune reaction. Recently, vasectomy has been reported to induce germ cell death by apoptosis. However, the exact mechanism of this vasectomy-induced germ cell apoptosis is unclear. To elucidate this mechanism, we designed a vasectomized rat model and examined the testiscular alterations and apoptotic degeneration biochemically and microhistopathologically. Particularly, we analyzed the expression of nitric oxide synthase (NOS) and nuclear factor kappa B (NF kappa B), which plays a critical role in the induction of the iNOS gene, in the testis after vasectomy to gain insight into the association between germ cell apoptosis and these factors. MATERIALS AND METHODS: The testes of 40 Wistar rats (10-weeks old) were studied at 1, 2, 5 and 10 weeks after unilateral (left) vasectomy. Wistar rats weighting 290 to 310 g were divided into 2 groups and subjected to underwent either unilateral vasectomy or sham surgery under ether anesthesia. Bilateral testes were carefully observed biochemically and histopathologically. Apoptosis was detected by an in situ end-labeling technique (detection of cellular DNA fragmentation) and electron microscopy. Neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) protein was detected by Western blotting and immunohistochemical studies using each NOS monoclonal antibody. To confirm the co-localization of cellular DNA fragmentation in germ cells and each NOS, each set of consecutive testis sections (one stained for cellular DNA fragmentation and the others for each NOS) were examined. Expression of NF kappa B proteins was examined immunohistochemically using a NF kappa B p65 polyclonal antibody. RESULTS: At 5 and 10 weeks after vasectomy, the vasectomized left testis was significantly lighter than the unvasectomized right testis and sham-operated testis. At that time, the seminiferous tubules of vasectomized testes were highly damaged, presenting narrow tubular diameter, disorder of cellular arrangement, depletion of the germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per cross-sectional area compared with sham-operated testes at 5 and 10 weeks after operation (p < 0.01). Electron microscopy revealed apoptotic germ cells each with a darkly stained nucleus. Western blotting and immunohistochemical studies demonstrated that iNOS proteins were more strongly expressed on vasectomized testes as time passed after vasectomy. Examination of consecutive sections from the vasectomized testis revealed that visibly apoptotic germ cells that exhibited positive staining for cellular DNA fragmentation were also intensely stained for eNOS and iNOS. NF kappa B p65 proteins were more strongly expressed in the nucleus of germ cells in the vasectomized testis than in the sham-operated testis. CONCLUSIONS: We found that vasectomy results in damage to spermatogenesis in adult rats, that may induce germ cell apoptosis, and that iNOS and NF kappa B may play a critical role in the germ cell apoptosis after vasectomy.
PURPOSE: Vasectomy may result in damage to spermatogenesis. There are several explanations for this damage, including an increase of pressure in the seminiferous tubules and an autoimmune reaction. Recently, vasectomy has been reported to induce germ cell death by apoptosis. However, the exact mechanism of this vasectomy-induced germ cell apoptosis is unclear. To elucidate this mechanism, we designed a vasectomized rat model and examined the testiscular alterations and apoptotic degeneration biochemically and microhistopathologically. Particularly, we analyzed the expression of nitric oxide synthase (NOS) and nuclear factor kappa B (NF kappa B), which plays a critical role in the induction of the iNOS gene, in the testis after vasectomy to gain insight into the association between germ cell apoptosis and these factors. MATERIALS AND METHODS: The testes of 40 Wistar rats (10-weeks old) were studied at 1, 2, 5 and 10 weeks after unilateral (left) vasectomy. Wistar rats weighting 290 to 310 g were divided into 2 groups and subjected to underwent either unilateral vasectomy or sham surgery under ether anesthesia. Bilateral testes were carefully observed biochemically and histopathologically. Apoptosis was detected by an in situ end-labeling technique (detection of cellular DNA fragmentation) and electron microscopy. Neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) protein was detected by Western blotting and immunohistochemical studies using each NOS monoclonal antibody. To confirm the co-localization of cellular DNA fragmentation in germ cells and each NOS, each set of consecutive testis sections (one stained for cellular DNA fragmentation and the others for each NOS) were examined. Expression of NF kappa B proteins was examined immunohistochemically using a NF kappa B p65 polyclonal antibody. RESULTS: At 5 and 10 weeks after vasectomy, the vasectomized left testis was significantly lighter than the unvasectomized right testis and sham-operated testis. At that time, the seminiferous tubules of vasectomized testes were highly damaged, presenting narrow tubular diameter, disorder of cellular arrangement, depletion of the germ cells, and local interstitial fibrosis. Vasectomized testes demonstrated a significantly increased number of apoptotic germ cells per cross-sectional area compared with sham-operated testes at 5 and 10 weeks after operation (p < 0.01). Electron microscopy revealed apoptotic germ cells each with a darkly stained nucleus. Western blotting and immunohistochemical studies demonstrated that iNOS proteins were more strongly expressed on vasectomized testes as time passed after vasectomy. Examination of consecutive sections from the vasectomized testis revealed that visibly apoptotic germ cells that exhibited positive staining for cellular DNA fragmentation were also intensely stained for eNOS and iNOS. NF kappa B p65 proteins were more strongly expressed in the nucleus of germ cells in the vasectomized testis than in the sham-operated testis. CONCLUSIONS: We found that vasectomy results in damage to spermatogenesis in adult rats, that may induce germ cell apoptosis, and that iNOS and NF kappa B may play a critical role in the germ cell apoptosis after vasectomy.