Supplementary file 2_RGS14 binds to GNAI3 and regulates the proliferation and apoptosis of human spermatogonial stem cells by affecting PLPP2 expression and MAPK signaling.csv

BackgroundNon-obstructive azoospermia (NOA) represents a severe form of male infertility, characterized by the absence of sperm in the ejaculate due to impaired spermatogenesis. Spermatogonial stem cells (SSCs), which ensure continuous sperm production, are critical for maintaining male fertility. Despite their importance, the molecular mechanisms governing SSC fate determination and their role in NOA pathogenesis remain incompletely understood. This study investigates the regulatory networks underlying SSC dysfunction in NOA patients.
ResultsUsing single-cell RNA sequencing, we identified significant downregulation of RGS14 in SSCs of NOA patients compared to normal testes. Immunofluorescence validation confirmed RGS14 localization primarily in SSCs. Functional assays demonstrated that RGS14 knockdown in SSC lines markedly suppressed cell proliferation and induced apoptosis. RNA-sequencing analyses revealed that RGS14 deficiency inhibited PLPP2 expression and MAPK signaling activation. Notably, PLPP2 overexpression rescued the phenotypic defects caused by RGS14 depletion. Protein-protein interaction assays and co-immunoprecipitation experiments further established that RGS14 physically interacts with GNAI3 to coordinately regulate cell proliferation and PLPP2 expression. Expression validation in NOA testes demonstrated concurrent downregulation of GNAI3 and PLPP2 in NOA patients, implicating their dysregulation in spermatogenic failure.
ConclusionOur findings uncover a novel RGS14-GNAI3-PLPP2 regulatory axis critical for SSC homeostasis. The dysregulation of these molecules contributes to SSC dysfunction and NOA pathogenesis. These data not only elucidate RGS14's role in SSC fate determination but also identify RGS14 and its interactome as promising therapeutic targets for restoring spermatogenesis in male infertility.

非梗阻性无精子症（Non-obstructive azoospermia, NOA）是一类严重的男性不育表型，其特征为因精子发生障碍导致精液中无精子存在。精原干细胞（Spermatogonial stem cells, SSCs）是维持持续精子生成的核心细胞群，对男性生育力的维持至关重要。尽管其功能意义重大，但调控SSC命运决定的分子机制，以及其在NOA发病机制中的具体作用仍未完全明确。本研究旨在探究NOA患者体内SSC功能异常背后的调控网络。
本研究通过单细胞RNA测序发现，相较于正常睾丸组织，NOA患者的SSCs中RGS14的表达水平显著下调。免疫荧光验证实验证实，RGS14主要定位于SSCs中。功能实验结果显示，在SSC细胞系中敲低RGS14会显著抑制细胞增殖并诱导细胞凋亡。RNA测序分析表明，RGS14的缺失会抑制PLPP2的表达以及MAPK信号通路的激活。值得注意的是，过表达PLPP2可挽救RGS14缺失所引发的表型缺陷。蛋白质相互作用实验与免疫共沉淀实验进一步证实，RGS14可与GNAI3发生物理相互作用，协同调控细胞增殖与PLPP2的表达。对NOA睾丸组织的表达验证结果显示，NOA患者体内GNAI3与PLPP2的表达均出现同步下调，提示二者的表达失调参与了精子发生障碍的病理过程。
本研究揭示了一条调控SSC稳态的全新RGS14-GNAI3-PLPP2信号轴。上述分子的表达失调会导致SSC功能异常，并参与NOA的发病进程。本研究不仅阐明了RGS14在SSC命运决定中的作用，还将RGS14及其互作组鉴定为恢复男性不育患者精子生成能力的潜在治疗靶点。