Human sperm motility is essential for fertilization and among pathologies underlying male infertility is asthenozoospermia. Nevertheless, mechanisms regulating sperm motility are not completely unraveled. This work investigates phosphoproteins underlying human sperm motility by using differential phosphoproteomic in two human sperm subpopulations: high (HM) and low (LM) motility, obtained by centrifugation in a density gradient. Phosphoproteomics (HPLC-MS/MS triple TOF), comparing human LM and HM phosphoproteomes, identified 210 phosphopeptides with different abundance that correspond with 119 sperm proteins. Analysis showed that 40% of phosphoproteins in LM spermatozoa are involved in metabolism, (catabolism, protein transport, lipid biosynthesis), 25% in spermatogenesis and sperm function, 8% in immune system and 6% in DNA repair. In HM spermatozoa, 48% of phosphoproteins are related to spermatogenesis and sperm function (motility), whereas 8% are associated to metabolism. GSK3α resulted one of the most abundant phosphoproteins in HM spermatozoa. Western blot confirmed that GSK3α phosphorylation is higher in HM spermatozoa. Summarizing, this study i) identified phosphoproteins in two human spermatozoa populations, ii) supports that human spermatozoa rely in protein phosphorylation, such as GSK3 α, to regulate sperm motility, iv) raises the challenge of using some identified human sperm phosphorylated proteins (GSK3α) as targets to develop into clinically relevant biomarkers.

Significance

Human sperm phosphoproteome analyzed by nano HPLC-MS/MS triple TOF identifies the differential abundance of sperm phosphoproteins in two human sperm populations exhibiting high motility (HM) and/or low motility (LM) that were isolated from normozoospermic healthy donors. Majority of human phosphoproteins found in LM spermatozoa are involved in sperm metabolism (40%), whereas those in HM spermatozoa are associated to spermatogenesis and sperm function, as motility (48%), and only 8% are associated to metabolism. One of the most abundant phosphoproteins found in HM spermatozoa is GSK3α, kinase directly involved in the regulation of sperm motility that was also validated by western blot. The biological relevance of this study is based in the fact that supports that mature human sperm cells rely in protein phosphorylation to efficiently regulate sperm motility and allows identifying those regulatory human sperm phosphoproteins. This work will clearly impacts the human reproductive field as it raises the challenge of consider identified human sperm phosphoproteins, such as GSK3α, as potential biological targets to develop into relevant biomarkers for the human clinic or assisted reproductive technology.

中文翻译：

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人类精子磷酸化蛋白质组揭示了调节人类精子活力的差异性磷蛋白特征。

人类的精子运动对受精至关重要，而男性不育的潜在病理学则是精子无精症。然而，调节精子活力的机制尚未完全阐明。这项工作通过在两个人类精子亚群中使用差异磷酸化蛋白质组学来研究人类精子运动的潜在磷蛋白：高（HM）和低（LM）运动，通过在密度梯度中离心获得。磷酸蛋白质组学（HPLC-MS / MS三重TOF），通过比较人类LM和HM磷酸蛋白质组，鉴定出210种具有不同丰度的磷酸肽，分别对应于119种精子蛋白。分析表明，LM精子中40％的磷蛋白参与代谢（分解代谢，蛋白质转运，脂质生物合成），25％的精子发生和精子功能，8％的免疫系统和6％的DNA修复。在HM精子中，48％的磷蛋白与精子发生和精子功能（运动）有关，而8％与代谢有关。GSK3α是HM精子中最丰富的磷蛋白之一。Western印迹证实HM精子中GSK3α磷酸化更高。总而言之，这项研究i）在两个人类精子群体中鉴定了磷蛋白，ii）支持人类精子依靠蛋白质磷酸化（例如GSK3α）来调节精子活力，iv）提出了使用某些已鉴定的人类精子磷酸化蛋白（GSK3α）的挑战）作为发展为临床相关生物标志物的靶标。GSK3α是HM精子中最丰富的磷蛋白之一。Western印迹证实HM精子中GSK3α磷酸化更高。总而言之，这项研究i）在两个人类精子群体中鉴定了磷蛋白，ii）支持人类精子依靠蛋白磷酸化（例如GSK3α）来调节精子活力，iv）提出了使用某些已鉴定的人类精子磷酸化蛋白（GSK3α）的挑战）作为发展为临床相关生物标志物的靶标。GSK3α是HM精子中最丰富的磷蛋白之一。Western印迹证实HM精子中GSK3α磷酸化更高。总而言之，这项研究i）在两个人类精子群体中鉴定了磷蛋白，ii）支持人类精子依靠蛋白磷酸化（例如GSK3α）来调节精子活力，iv）提出了使用某些已鉴定的人类精子磷酸化蛋白（GSK3α）的挑战）作为发展为临床相关生物标志物的靶标。

意义

通过纳米HPLC-MS / MS三重TOF分析的人类精子磷酸化蛋白质组学鉴定了从正常精子健康供体中分离出的两个具有高运动性（HM）和/或低运动性（LM）的人类精子群体中精子磷蛋白的差异丰度。LM精子中发现的大多数人类磷蛋白都参与精子代谢（40％），而HM精子中的人类磷蛋白则与精子发生和精子功能相关（如活力）（48％），而只有8％与代谢有关。在HM精子中发现的最丰富的磷蛋白之一是GSK3α，该激酶直接参与精子运动的调节，也已通过Western blot验证。这项研究的生物学相关性是基于这样的事实，即支持成熟的人类精子细胞依靠蛋白质的磷酸化来有效地调节精子的活力，并允许鉴定那些调节性的人类精子磷蛋白。这项工作显然会影响人类生殖领域，因为它提出了将已鉴定的人类精子磷蛋白（例如GSK3α）视为可能发展为人类临床或辅助生殖技术的相关生物标志物的潜在生物靶标的挑战。