A sperm-specific proteome-scale metabolic network model identifies non-glycolytic genes for energy deficiency in asthenozoospermia

About 15% of couples experience difficulty in conceiving a child, of which half of the cases are thought to be male-related. Asthenozoospermia, or low sperm motility, is one of the frequent types of male infertility. Although energy metabolism is suggested to be central to the etiology of asthenozoospermia, very few attempts have been made to identify its underlying metabolic pathways. Here, we reconstructed SpermNet, the first proteome-scale model of the sperm cell by using whole-proteome data and the mCADRE algorithm. The reconstructed model was then analyzed using the COBRA toolbox. Genes were knocked-out in the model to investigate their effect on ATP production. A total of 78 genes elevated ATP production rate considerably of which most encode components of oxidative phosphorylation, fatty acid oxidation, the Krebs cycle, and members of the solute carrier 25 family. Among them, we identified 11 novel genes which have previously not been associated with sperm cell energy metabolism and may thus be implicated in asthenozoospermia. We further examined the reconstructed model by <i>in silico</i> knock out of currently known asthenozoospermia implicated-genes that were not predicted by our model. The pathways affected by knocking out these genes were also related to energy metabolism, confirming previous findings. Therefore, our model not only predicts the known pathways, it also identifies several non-glycolytic genes for deficient energy metabolism in asthenozoospermia. Finally, this model supports the notion that metabolic pathways besides glycolysis such as oxidative phosphorylation and fatty acid oxidation are essential for sperm energy metabolism and if validated, may form a basis for fertility recovery. <b>Abbreviations</b>: mCADRE: metabolic context-specificity assessed by deterministic reaction evaluation; ATP: adenosine triphosphate; RNA: ribonucleic acid; FBA: flux balance analysis; FVA: flux variability analysis; DAVID: database for annotation, visualization and integrated discovery; OXPHOS: oxidative phosphorylation; ETC: electron transfer chain; SLC: solute carrier; DLD: dihydrolypoamide dehydrogenase; DLST: dihydrolypoamide S-succinyl transferase; OGDH: oxoglutarate dehydrogenase; CS: citrate synthase; FH: fumarate hydratase; IDH: isocitrate dehydrogenase; SUCLG1: succinate-CoA ligase; SD: succinate dehydrogenase; HADHA: hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, subunit A; HADHB: hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, subunit B; PPA2: pyrophosphatase (inorganic) 2; PP_i: inorganic phosphate; GALT: galactose-1-phosphate uridylyltransferase

约15%的夫妇存在受孕困难问题，其中半数病例被认为与男性因素相关。弱精子症（asthenozoospermia），又称精子活力低下，是男性不育的常见类型之一。尽管已有研究提示能量代谢是弱精子症病因的核心，但目前针对其潜在代谢通路的探索仍十分有限。本研究利用全蛋白质组数据与mCADRE算法，构建了首个精子细胞蛋白质组规模的代谢模型SpermNet。随后通过COBRA工具箱对该重构模型展开分析。通过在模型中敲除基因以探究其对ATP生成的影响。共计78个基因可显著提升ATP生成速率，其中多数编码氧化磷酸化、脂肪酸氧化、三羧酸循环相关组分，以及溶质载体25家族成员。在这些基因中，我们鉴定出11个此前未被报道与精子细胞能量代谢相关的全新基因，其可能与弱精子症的发生存在关联。我们进一步通过<i>in silico</i> 虚拟敲除当前已知但未被本模型预测到的弱精子症相关基因，对重构模型进行验证。敲除这些基因所影响的通路同样与能量代谢相关，印证了此前的研究结论。因此，本模型不仅可预测已知的代谢通路，还可鉴定出弱精子症能量代谢缺陷中若干非糖酵解相关基因。最后，该模型支持了这一学术观点：除糖酵解外，氧化磷酸化与脂肪酸氧化等代谢通路对精子能量代谢至关重要；若该模型得到实验验证，可为男性生育力恢复提供理论基础。<b>缩略语</b>：mCADRE：基于确定性反应评估的代谢情境特异性分析（metabolic context-specificity assessed by deterministic reaction evaluation）；ATP：三磷酸腺苷（adenosine triphosphate）；RNA：核糖核酸（ribonucleic acid）；FBA：通量平衡分析（flux balance analysis）；FVA：通量变异性分析（flux variability analysis）；DAVID：注释、可视化与集成发现数据库（database for annotation, visualization and integrated discovery）；OXPHOS：氧化磷酸化（oxidative phosphorylation）；ETC：电子传递链（electron transfer chain）；SLC：溶质载体（solute carrier）；DLD：二氢硫辛酰胺脱氢酶（dihydrolypoamide dehydrogenase）；DLST：二氢硫辛酰胺S-琥珀酰转移酶（dihydrolypoamide S-succinyl transferase）；OGDH：α-酮戊二酸脱氢酶（oxoglutarate dehydrogenase）；CS：柠檬酸合酶（citrate synthase）；FH：延胡索酸水合酶（fumarate hydratase）；IDH：异柠檬酸脱氢酶（isocitrate dehydrogenase）；SUCLG1：琥珀酰-CoA连接酶（succinate-CoA ligase）；SD：琥珀酸脱氢酶（succinate dehydrogenase）；HADHA：羟酰辅酶A脱氢酶/3-酮酰辅酶A硫解酶/烯酰辅酶A水合酶亚基A（hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, subunit A）；HADHB：羟酰辅酶A脱氢酶/3-酮酰辅酶A硫解酶/烯酰辅酶A水合酶亚基B（hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, subunit B）；PPA2：无机焦磷酸酶2（pyrophosphatase (inorganic) 2）；PP_i：无机磷酸（inorganic phosphate）；GALT：半乳糖-1-磷酸尿苷酰转移酶（galactose-1-phosphate uridylyltransferase）