METHODS: We analyzed differentially expressed genes (DEGs) based on RNA sequencing of nine patients with SCOS and three patients with obstructive azoospermia and normal spermatogenesis. We further explored the identified genes using ELISA and immunohistochemistry.
RESULTS: In total, 9406 DEGs were expressed (Log2|FC|≥ 1; adjusted P value < 0.05) in SCOS samples, and 21 hub genes were identified. Three upregulated core genes were found, including CASP4, CASP1, and PLA2G4A. Thus, we hypothesized that testis cell pyroptosis mediated by CASP1 and CASP4 might be involved in SCOS occurrence and development. ELISA verified that CASP1 and CASP4 activities in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenesis. Immunohistochemical results showed that CASP1 and CASP4 in the normal spermatogenesis group were mainly expressed in the nuclei of spermatogenic, Sertoli, and interstitial cells. CASP1 and CASP4 in the SCOS group were mainly expressed in the nuclei of Sertoli and interstitial cells because of the loss of spermatogonia and spermatocytes. CASP1 and CASP4 expression levels in the testes of patients with SCOS were significantly higher than those in patients with normal spermatogenisis. Furthermore, the pyroptosis-related proteins GSDMD and GSDME in the testes of patients with SCOS were also significantly higher than those in control patients. ELISA also showed that inflammatory factors (IL-1 β, IL-18, LDH, and ROS) were significantly increased in the SCOS group.
CONCLUSIONS: For the first time, we found that cell pyroptosis-related genes and key markers were significantly increased in the testes of patients with SCOS. We also observed many inflammatory and oxidative stress reactions in SCOS. Thus, we propose that testis cell pyroptosis mediated by CASP1 and CASP4 could participate in SCOS occurrence and development.
方法:我们基于RNA测序分析了9例SCOS患者和3例梗阻性无精子症和正常精子发生患者的差异表达基因(DEGs)。我们使用ELISA和免疫组织化学进一步探索了鉴定的基因。
结果:总计,在SCOS样品中表达了9406个DEGs(Log2|FC|≥1;调整后的P值<0.05),并鉴定了21个hub基因。发现了三个上调的核心基因,包括CASP4、CASP1和PLA2G4A。因此,我们推测CASP1和CASP4介导的睾丸细胞焦亡可能参与了SCOS的发生和发展。ELISA证实,SCOS患者睾丸中的CASP1和CASP4活性明显高于精子发生正常患者。免疫组织化学结果显示,CASP1和CASP4在正常生精组中主要表达于生精细胞核,Sertoli,和间质细胞。由于精原细胞和精母细胞的丢失,SCOS组的CASP1和CASP4主要在支持细胞和间质细胞核中表达。SCOS患者睾丸中CASP1和CASP4的表达水平明显高于正常生精患者。此外,SCOS患者睾丸中的焦亡相关蛋白GSDMD和GSDME也明显高于对照组.ELISA还显示炎症因子(IL-1β,IL-18,LDH,和ROS)在SCOS组中显著增加。
结论:第一次,我们发现,在SCOS患者的睾丸中,细胞焦亡相关基因和关键标志物显著增加.我们还在SCOS中观察到许多炎症和氧化应激反应。因此,我们认为,CASP1和CASP4介导的睾丸细胞焦亡可能参与了SCOS的发生和发展。