Kusari Fitore, Backova Lenka, Panek Dalibor, Benda Ales, Trachtulec Zdenek
Laboratory of Germ Cell Development, Division BIOCEV, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic.
Present address: Department of Meiosis, Max Planck Institute for Multidisciplinary Sciences, Göttingen, Germany.
BMC Biol. 2025 Mar 24;23(1):85. doi: 10.1186/s12915-025-02167-1.
Sperm metabolic pathways that generate energy for motility are compartmentalized within the flagellum. Dysfunctions in metabolic compartments, namely mitochondrial respiration and glycolysis, can compromise motility and male fertility. Studying these compartments is thus required for fertility treatment. However, it is very challenging to capture images of metabolic compartments in motile spermatozoa because the fast beating of the flagellum introduces motion blur. Therefore, most approaches immobilize spermatozoa prior to imaging.
Our findings indicate that immobilizing sperm alters their metabolic profile, highlighting the necessity for measuring metabolism in spermatozoa during movement. We achieved this by encapsulating mouse epididymis in a hydrogel followed by two-photon fluorescence lifetime imaging microscopy for imaging motile sperm in situ. The autofluorescence of endogenous metabolites-FAD, NADH, and NADPH-enabled us to visualize sperm metabolic compartments without staining. We trained machine learning for automated image segmentation and generated metabolic fingerprints using object-based phasor analysis. We show that metabolic fingerprints of spermatozoa and the mitochondrial compartment (1) can distinguish individual males by genetic background, age, or fecundity status, (2) correlate with fertility, and (3) change with age likely due to increased oxidative metabolism.
Our approach eliminates the need for sperm immobilization and labeling and captures the native state of sperm metabolism. This technique could be adapted for metabolism-based sperm selection for assisted reproduction.
为精子运动产生能量的代谢途径在鞭毛内是分隔的。代谢区室功能障碍，即线粒体呼吸和糖酵解功能障碍，会损害精子活力和男性生育能力。因此，生育治疗需要研究这些区室。然而，捕获活动精子中代谢区室的图像极具挑战性，因为鞭毛的快速摆动会产生运动模糊。因此，大多数方法在成像前会使精子固定。
我们的研究结果表明，固定精子会改变其代谢谱，突出了在精子运动过程中测量其代谢的必要性。我们通过将小鼠附睾包裹在水凝胶中，然后进行双光子荧光寿命成像显微镜检查来对活动精子进行原位成像，从而实现了这一点。内源性代谢物——黄素腺嘌呤二核苷酸（FAD）、烟酰胺腺嘌呤二核苷酸（NADH）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的自发荧光使我们能够在不染色的情况下可视化精子代谢区室。我们训练机器学习进行自动图像分割，并使用基于对象的相量分析生成代谢指纹。我们表明，精子和线粒体区室的代谢指纹（1）可以根据遗传背景、年龄或生育状态区分个体雄性，（2）与生育能力相关，（3）可能由于氧化代谢增加而随年龄变化。
我们的方法无需固定和标记精子，并能捕捉精子代谢的天然状态。该技术可用于辅助生殖中基于代谢的精子筛选。
