Kubota Y, Sasaki S, Kubota H, Tatsura H, Kohri K
Department of Urology, Nagoya City University Medical School.
Nihon Hinyokika Gakkai Zasshi. 2001 Jan;92(1):13-22. doi: 10.5980/jpnjurol1989.92.13.
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.
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.
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.
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.
输精管结扎术可能导致生精功能受损。对此种损伤有多种解释，包括生精小管内压力升高和自身免疫反应。最近，有报道称输精管结扎术可通过凋亡诱导生殖细胞死亡。然而，这种输精管结扎术诱导生殖细胞凋亡的确切机制尚不清楚。为阐明此机制，我们设计了输精管结扎大鼠模型，并从生化和显微组织病理学方面研究了睾丸的变化及凋亡性退变。特别地，我们分析了输精管结扎术后睾丸中一氧化氮合酶（NOS）和核因子κB（NF-κB，其在诱导诱导型NOS基因中起关键作用）的表达，以深入了解生殖细胞凋亡与这些因子之间的关联。
对40只10周龄的Wistar大鼠在单侧（左侧）输精管结扎术后1、2、5和10周进行研究。体重290至310克的Wistar大鼠分为2组，在乙醚麻醉下分别接受单侧输精管结扎术或假手术。对双侧睾丸进行仔细的生化和组织病理学观察。通过原位末端标记技术（检测细胞DNA片段化）和电子显微镜检测凋亡。使用每种NOS单克隆抗体，通过蛋白质免疫印迹法和免疫组织化学研究检测神经元型NOS（nNOS）、内皮型NOS（eNOS）和诱导型NOS（iNOS）蛋白。为证实生殖细胞中细胞DNA片段化与每种NOS的共定位，检查了每组连续的睾丸切片（一张用于细胞DNA片段化染色，其他用于每种NOS染色）。使用NF-κB p65多克隆抗体通过免疫组织化学检查NF-κB蛋白的表达。
输精管结扎术后5周和10周，结扎侧左侧睾丸明显比未结扎侧右侧睾丸和假手术睾丸轻。此时，输精管结扎侧睾丸的生精小管严重受损，表现为管径变窄、细胞排列紊乱、生殖细胞减少以及局部间质纤维化。与假手术睾丸相比，输精管结扎术后5周和10周，输精管结扎侧睾丸每横截面积的凋亡生殖细胞数量显著增加（p < 0.01）。电子显微镜显示凋亡的生殖细胞，每个细胞核染色深。蛋白质免疫印迹法和免疫组织化学研究表明，随着输精管结扎术后时间的推移，iNOS蛋白在输精管结扎侧睾丸中的表达更强。对输精管结扎侧睾丸连续切片的检查显示，显示细胞DNA片段化阳性染色的明显凋亡生殖细胞对eNOS和iNOS也有强烈染色。NF-κB p65蛋白在输精管结扎侧睾丸生殖细胞核中的表达比假手术睾丸更强。
我们发现输精管结扎术导致成年大鼠生精功能受损，可能诱导生殖细胞凋亡，并且iNOS和NF-κB可能在输精管结扎术后的生殖细胞凋亡中起关键作用。
