Function Laboratory for Marine Science and Food Production Process, Laoshan Laboratory, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China.
School of Fishery, Zhejiang Ocean University, Zhoushan 316022, China.
Int J Mol Sci. 2023 Jul 13;24(14):11430. doi: 10.3390/ijms241411430.
Phosphorylation is a post-translational modification that contributes to versatile protein functions in spermatogenesis, and the variations they generate usually results in abnormal spermatogenesis or sperm dysfunction. The sex-reversal phenomenon exists in Chinese tongue sole under certain conditions such that individuals with a ZW genotype can acquire a male phenotype and are thus called pseudomales. Pseudomale tongue sole can reach sexual maturity but produce only Z-type sperm, and the Z sperm carries paternal epigenetic information. Whether phosphorylation plays a role in the sperm abnormality of pseudomales is unknown. In this study, a phosphoproteomic analysis was performed to compare protein phosphorylation profiles between pseudomale and male testes. Altogether, we identified 14,253 phosphopeptides matching with 4843 proteins, with 1329 differentially phosphorylated peptides corresponding to 1045 differentially phosphorylated proteins (DPPs). Phosphorylation at 781 sites was upregulated and at 548 sites was downregulated. Four motifs were identified among differentially phosphorylated peptides, which were "SP", "SD", "RxxS", and "TP". Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that the cell cycle and DNA/RNA processing were significantly enriched with the genes encoding DPPs. To analyze DPP function in depth, a protein-protein interaction network was constructed, and Ran-binding protein 2 was found to play a central role in spermatogenesis by regulating several processes such as the cell cycle, eukaryotic translation, ubiquitination, and minichromosome maintenance. In kinase-associated network analyses, two "mitogen-activated protein kinase (Mapk)-centered" clusters were identified that may account for abnormal spermatogenesis in pseudomales. One cluster was centered on Mapk6, which predominantly regulated the cell cycle by interacting with several cyclin-dependent kinases, and the other was centered on the "testis-expressed kinase 1-like (Tesk1l)/Pim1l-Mapk4l- testis-expressed 14 (Tex14)" kinase cascade, which might contribute to spermatogenesis by regulating β-catenin. Taken together, these data suggested the new candidates involved in pseudomale sperm abnormalities and provided clues to discover the phosphorylated regulatory mechanism underlying tongue sole spermatogenesis.
磷酸化是一种翻译后修饰，有助于精子发生中多种蛋白质功能的发挥，其产生的变异通常导致异常精子发生或精子功能障碍。在中国舌鳎鱼中存在性反转现象，在某些条件下，ZW 基因型的个体可以获得雄性表型，因此被称为假雄鱼。假雄舌鳎鱼可以达到性成熟，但只能产生 Z 型精子，而 Z 型精子携带父系表观遗传信息。磷酸化是否在假雄鱼的精子异常中发挥作用尚不清楚。在这项研究中，我们进行了磷酸化蛋白质组学分析，以比较假雄和雄性睾丸的蛋白质磷酸化谱。总共鉴定到 14253 个与 4843 个蛋白质匹配的磷酸肽，其中 1329 个差异磷酸化肽对应 1045 个差异磷酸化蛋白质（DPP）。781 个磷酸化位点上调，548 个磷酸化位点下调。在差异磷酸化肽中鉴定到 4 个基序，分别是"SP"、"SD"、"RxxS"和"TP"。基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析表明，细胞周期和 DNA/RNA 处理与编码 DPP 的基因显著富集。为了深入分析 DPP 的功能，构建了蛋白质-蛋白质相互作用网络，发现 Ran 结合蛋白 2 通过调节细胞周期、真核翻译、泛素化和微染色体维持等过程，在精子发生中发挥核心作用。在激酶相关网络分析中，鉴定到两个"丝裂原活化蛋白激酶（Mapk）-中心"簇，可能导致假雄鱼的精子发生异常。一个簇以 Mapk6 为中心，主要通过与几个细胞周期蛋白依赖性激酶相互作用来调节细胞周期，另一个簇以"睾丸表达激酶 1 样（Tesk1l）/Pim1l-Mapk4l-睾丸表达 14（Tex14）"激酶级联为中心，可能通过调节 β-连环蛋白来促进精子发生。总之，这些数据提示了参与假雄鱼精子异常的新候选基因，并为发现舌鳎鱼精子发生中磷酸化调控机制提供了线索。
