Liu Yuxin, Sun Qihao, Du Kun, Long Houtao, Zhang Daofeng, Zheng Junhao, Zhao Yong, Zhang Haiyang
Department of Urology, Shandong Provincial Hospital, Shandong University, Jinan, China.
Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China.
Transl Androl Urol. 2024 Aug 31;13(8):1405-1415. doi: 10.21037/tau-24-158. Epub 2024 Aug 26.
Cryptorchidism, the failure of testes to descend into the scrotum, exposes the testes to higher temperature and external pressure. Scholars from Razi University found through research conducted at different pressure gradients (0, 25, 50, and 100 mmHg) and time gradients (2 and 4 h) that high hydrostatic pressure may lead to sperm apoptosis. In this work, we investigated the effect of external pressure on spermatogonia, exploring a new mechanism of male infertility caused by cryptorchidism.
Various pressure gradients (0, 25, 50, and 100 mmHg) were applied to spermatogonia for different durations (0, 2, and 4 h) in the Cell Counting Kit-8 (CCK8) experiment. Morphological changes, cell ultrastructure, apoptosis rates, and the expression of apoptosis-related proteins (bax, bcl-2, caspase-3, and caspase-9) were assessed through immunofluorescence, electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, flow cytometry, immunohistochemistry, real-time quantitative polymerase chain reaction (qPCR), and western blot.
The cell viability assay showed that higher external pressure had a greater negative time-dependent impact on cell viability. Immunofluorescence results indicated that external pressure stimuli altered the morphology of spermatogonia. The results of TUNEL assay and flow cytometry demonstrated that external pressure stimuli induced apoptosis in spermatogonia. Transmission electron microscopy (TEM) observations showed the generation of apoptotic bodies, mitochondrial swelling, vacuolization, and mitochondrial cristae fusion. The results of immunohistochemistry indicated that pressure induced the expression of caspase-3 and caspase-9 proteins. qPCR and western blot analyses revealed an increased ratio of bax/bcl-2 and expression of caspase-3 and caspase-9. Methazolamide (cytochrome C inhibitor) blocked the pressure-induced cell apoptosis and inhibited the activation of caspase-3 while Z-IETD-FMK (caspase-8 inhibitor) did not.
External pressure promotes spermatogonia apoptosis through the intrinsic apoptosis pathway, which may be one of the mechanisms of male infertility induced by cryptorchidism.
隐睾症是指睾丸未能降至阴囊，使睾丸暴露于更高温度和外部压力之下。拉齐大学的学者通过在不同压力梯度（0、25、50和100 mmHg）和时间梯度（2小时和4小时）下进行的研究发现，高静水压力可能导致精子凋亡。在本研究中，我们研究了外部压力对精原细胞的影响，探索隐睾症导致男性不育的新机制。
在细胞计数试剂盒-8（CCK8）实验中，对精原细胞施加不同压力梯度（0、25、50和100 mmHg）并持续不同时间（0、2和4小时）。通过免疫荧光、电子显微镜、末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记（TUNEL）检测、流式细胞术、免疫组织化学、实时定量聚合酶链反应（qPCR）和蛋白质印迹法评估形态变化、细胞超微结构、凋亡率以及凋亡相关蛋白（bax、bcl-2、caspase-3和caspase-9）的表达。
细胞活力测定表明，较高的外部压力对细胞活力具有更大的负时间依赖性影响。免疫荧光结果表明，外部压力刺激改变了精原细胞的形态。TUNEL检测和流式细胞术结果表明，外部压力刺激诱导精原细胞凋亡。透射电子显微镜（TEM）观察显示凋亡小体的产生、线粒体肿胀、空泡化和线粒体嵴融合。免疫组织化学结果表明，压力诱导caspase-3和caspase-9蛋白表达。qPCR和蛋白质印迹分析显示bax/bcl-2比值增加以及caspase-3和caspase-9表达增加。甲醋唑胺（细胞色素C抑制剂）可阻断压力诱导的细胞凋亡并抑制caspase-3的激活，而Z-IETD-FMK（caspase-8抑制剂）则无此作用。
外部压力通过内源性凋亡途径促进精原细胞凋亡，这可能是隐睾症导致男性不育的机制之一。
