Du Li, Chen Wei, Li Chunyun, Cui Yinghong, He Zuping
The Key Laboratory of Model Animals and Stem Cell Biology in Hunan Province, School of Medicine, Hunan Normal University; The Manufacture-Based Learning and Research Demonstration Center for Human Reproductive Health New Technology of Hunan Normal University, Changsha, China.
Shanghai Key Laboratory of Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
J Cell Physiol. 2022 Sep;237(9):3565-3577. doi: 10.1002/jcp.30813. Epub 2022 Jun 14.
Studies on gene regulation and signaling transduction pathways of human spermatogonial stem cells (SSCs) are of the utmost significance for unveiling molecular mechanisms underlying human spermatogenesis and gene therapy of male infertility. We have demonstrated, for the first time, that RNF144B stimulated cell proliferation and inhibited the apoptosis of human SSCs. The target of RNF144B was identified as FCER2 by RNA sequencing. We revealed that RNF144B interacted with FCER2 by immunoprecipitation. Consistently, overexpression of FCER2 reversed the phenotype of proliferation and apoptosis of human SSCs caused by RNF144B knockdown. Interestingly, FCER2 pulled down N2ICD (NOTCH2 intracellular domain), while N2ICD could bind to FCER2 in human SSCs. The levels of NOTCH2, FCER2, HES1, and HEY1 were reduced by RNF144B siRNA in human SSCs. Significantly, RNF144B was expressed at a lower level in nonobstructive azoospermia (NOA) patients than in the obstructive azoospermia (OA) patients with normal spermatogenesis, and 52 patients with heterozygous mutations of RNF144B were detected in 1,000 NOA patients. These results implicate that RNF144B promotes the proliferation of human SSCs and suppresses their apoptosis via the FCER2/NOTCH2/HES1 pathway and that the abnormality of RNF144B is associated with spermatogenesis failure. This study thus provides novel molecular mechanisms regulating the fate determinations of human SSCs, and it offers new biomarkers for the diagnosis and treatment of male infertility.
对人类精原干细胞（SSCs）的基因调控和信号转导通路进行研究，对于揭示人类精子发生的分子机制以及男性不育的基因治疗具有至关重要的意义。我们首次证明，RNF144B可刺激人类SSCs的细胞增殖并抑制其凋亡。通过RNA测序将RNF144B的靶标鉴定为FCER2。我们通过免疫沉淀揭示了RNF144B与FCER2相互作用。一致地，FCER2的过表达逆转了由RNF144B敲低引起的人类SSCs增殖和凋亡的表型。有趣的是，FCER2下拉了N2ICD（NOTCH2细胞内结构域），而在人类SSCs中N2ICD可与FCER2结合。在人类SSCs中，RNF144B siRNA降低了NOTCH2、FCER2、HES1和HEY1的水平。值得注意的是，与精子发生正常的梗阻性无精子症（OA）患者相比，非梗阻性无精子症（NOA）患者中RNF144B的表达水平较低，并且在1000例NOA患者中检测到52例RNF144B杂合突变患者。这些结果表明，RNF144B通过FCER2/NOTCH2/HES1途径促进人类SSCs的增殖并抑制其凋亡，并且RNF144B的异常与精子发生失败有关。因此，本研究提供了调节人类SSCs命运决定的新分子机制，并为男性不育的诊断和治疗提供了新的生物标志物。
