FLIM Measurements of bed bug sperm cells

Data to: Seminal fluid, and sperm diluent, affect sperm metabolism in an insect: evidence from NAD(P)H and FAD autofluorescence lifetime imaging (SemFSpMetab) Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and FAD. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect Medium), sperm showed increased glycolysis compared to a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

本数据集相关研究：昆虫体内精液与精子稀释液对精子代谢的影响——基于NAD(P)H与FAD自体荧光寿命成像的实验证据（数据集标识：SemFSpMetab）
精子代谢是精子活力与雄性生育能力的核心基础。目前精子代谢检测技术尚处于起步阶段，尚无关于是否需要标准化实验室操作流程、以及如何标准化的相关规范建议。
本研究以昆虫——温带臭虫（*Cimex lectularius*）的精子为实验材料，证实针对人工精子储存介质与天然介质（精液），均需开展精子代谢检测的标准化工作。
本研究采用荧光寿命成像显微镜（fluorescence lifetime imaging microscopy, FLIM）结合时间相关单光子计数（time-correlated single-photon counting, TCSPC）技术，基于自体荧光辅酶NAD(P)H与FAD的荧光特性，对精子代谢水平进行定量分析。自体荧光辅酶的游离态与蛋白结合态具有不同的荧光寿命（即衰减时长），二者对寿命信号的相对占比可用于表征细胞的代谢状态。
研究发现，人工储存介质与精液可分别且协同影响精子代谢。在含糖与氨基酸的格雷斯昆虫培养基（Grace's Insect Medium）中，相较于常规储存介质磷酸盐缓冲液（phosphate-buffered saline, PBS），精子的糖酵解水平显著提升。向精子体系中添加精液后，精子的氧化磷酸化水平进一步升高，这或反映了精子激活过程中能量生成需求的提升。
本研究建立了无需依赖精子活力即可检测精子代谢的实验方案，强调需推行精子检测流程的标准化工作，并首次证实精液可调控精子代谢。该研究结果提示，人类精子样本的代谢研究也应遵循等效的实验方案标准化要求。