West China School of Basic Medical Sciences & Forensic Medicine, Sichuan University, Chengdu, China.
Reproductive & Women-Children Hospital, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Andrology. 2024 Feb;12(2):338-348. doi: 10.1111/andr.13474. Epub 2023 Jun 19.
The ubiquitin ligase HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 is essential for the establishment and maintenance of spermatogonia. However, the role of HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 in regulating germ cell differentiation remains unclear, and clinical evidence linking HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 to male infertility pathogenesis is lacking.
This study aims to investigate the role of HUWE1 in germ cell differentiation and the mechanism by which a HUWE1 single nucleotide polymorphism increases male infertility risk.
We analyzed HUWE1 single nucleotide polymorphisms in 190 non-obstructive azoospermia patients of Han Chinese descent. We evaluated HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulation by retinoic acid receptor alpha using chromatin immunoprecipitation assays, electrophoretic mobility shift assays, and siRNA-mediated RARα knockdown. Using C18-4 spermatogonial cells, we determined whether HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participated in retinoic acid-mediated retinoic acid receptor alpha signaling. We performed luciferase assays, cell counting kit-8 assays, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting. We quantified HUWE1 and retinoic acid receptor alpha in testicular biopsies from non-obstructive azoospermia and obstructive azoospermia patients using quantitative real-time polymerase chain reaction and immunofluorescence.
Three HUWE1 single nucleotide polymorphisms were significantly associated with spermatogenic failure in 190 non-obstructive azoospermia patients; one (rs34492591) was in the HUWE1 promoter. Retinoic acid receptor alpha regulates HUWE1 gene expression by binding to its promoter. HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 participates in retinoic acid/retinoic acid receptor alpha signaling pathway and regulates the expression of germ cell differentiation genes STRA8 and SCP3 to inhibit cell proliferation and reduce γH2AX accumulation. Notably, significantly lower levels of HUWE1 and RARα were detected in testicular biopsy samples from non-obstructive azoospermia patients.
An HUWE1 promoter single nucleotide polymorphism significantly downregulates its expression in non-obstructive azoospermia patients. Mechanistically, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 regulates germ cell differentiation during meiotic prophase through its participation in retinoic acid/retinoic acid receptor alpha signaling and subsequent modulation of γH2AX. Taken together, these results strongly suggest that the genetic polymorphisms of HUWE1 are closely related to spermatogenesis and non-obstructive azoospermia pathogenesis.
泛素连接酶 HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 对于精原细胞的建立和维持至关重要。然而，HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 在调节生殖细胞分化中的作用尚不清楚，并且缺乏将 HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 与男性不育发病机制联系起来的临床证据。
本研究旨在探讨 HUWE1 在生殖细胞分化中的作用以及 HUWE1 单核苷酸多态性增加男性不育风险的机制。
我们分析了 190 名汉族非梗阻性无精子症患者的 HUWE1 单核苷酸多态性。我们使用染色质免疫沉淀分析、电泳迁移率变动分析和 siRNA 介导的 RARα 敲低来评估视黄酸受体 α 对 HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 的调节作用。使用 C18-4 精原细胞，我们确定 HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 是否参与视黄酸介导的视黄酸受体 α 信号通路。我们进行了荧光素酶测定、细胞计数试剂盒-8 测定、免疫荧光、实时定量聚合酶链反应和 Western blot。我们使用实时定量聚合酶链反应和免疫荧光定量检测非梗阻性无精子症和梗阻性无精子症患者睾丸活检组织中的 HUWE1 和视黄酸受体 α。
三个 HUWE1 单核苷酸多态性与 190 名非梗阻性无精子症患者的生精失败显着相关；其中一个（rs34492591）位于 HUWE1 启动子内。视黄酸受体 α 通过结合其启动子来调节 HUWE1 基因表达。HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 参与视黄酸/视黄酸受体 α 信号通路，并调节生殖细胞分化基因 STRA8 和 SCP3 的表达，以抑制细胞增殖并减少 γH2AX 积累。值得注意的是，在非梗阻性无精子症患者的睾丸活检样本中，明显检测到 HUWE1 和 RARα 的水平较低。
HUWE1 启动子单核苷酸多态性显着下调非梗阻性无精子症患者的表达。从机制上讲，HECT、UBA 和 WWE 结构域包含 E3 泛素蛋白连接酶 1 通过参与视黄酸/视黄酸受体 α 信号通路并随后调节 γH2AX，调节减数分裂前期的生殖细胞分化。总之，这些结果强烈表明，HUWE1 的遗传多态性与精子发生和非梗阻性无精子症发病机制密切相关。
