Probing human sperm metabolism using 13C-magnetic resonance spectroscopy.
Probing human sperm metabolism using 13C-magnetic resonance spectroscopy.
复制标题
DOI：
10.1093/molehr/gay046
复制
发表时间：
2019-01-01
影响因子：
4
通讯作者：
Pacey AA
中科院分区：
文献类型：
--
作者：
Calvert SJ;Reynolds S;Paley MN;Walters SJ;Pacey AA
选定代谢物的13 C-磁共振波谱（MRS）能否提供有关人类精子代谢以及不同精子群如何利用糖酵解或氧化磷酸化的有用信息？通过密度梯度离心（DGC）制备并与13 Cu-葡萄糖、13 Cu-果糖或13 C1-丙酮酸盐孵育的精子群显示出代谢主要产生乳酸盐和更多间歇性碳酸氢盐的一致证据，并且与来自颗粒的精子相比，从40/80%界面回收的活的或能动的精子从13 Cu-葡萄糖产生显著更多的乳酸盐，这不能用存在的非精子细胞的差异来解释。以前的研究主要集中在人类精子的CO2或其他特定代谢产物的产生上，关于糖酵解和/或氧化磷酸化是否是精子中ATP产生的更重要途径仍然存在相当大的争议。由DGC制备精子群，并进行13 C-MRS以回答以下问题。(i)是否有可能检测人类精子代谢的13 C底物参与能源的产生？(ii)这些反应的动力学是什么？(iii)不同的精子群（例如，“80%”的颗粒精子和“40%”的界面精子）是否以相同的方式利用底物？在这些实验中使用了来自97名男性的精液样本; 52名平行用于目标（i）和（ii），45名用于目标（iii）。使用Percoll/磷酸盐缓冲盐水（PBS）DGC从健康男性的射精中制备精子群，然后与一系列13 C标记的底物孵育（13 Cu-葡萄糖，13 Cu-果糖，13 C1-丙酮酸，13 C1-丁酸，13 C3-乳酸，13 C2，4-D-3-羟基丁酸，13 C5 - 1-谷氨酸，13 C1，2-甘氨酸或13 Cu-半乳糖）与青霉素/链霉素抗生素一起沿着在37°C下处理4小时、24小时或超过48小时，以获得估计的速率常数。测量精子浓度、活力和运动力，对于一组实验，测定非精子细胞浓度。使用9.4 T磁共振波谱仪采集1D 13 C、反向门控1H去耦MRS谱。使用光谱仪软件和Matlab脚本进行光谱处理，以确定每个光谱的峰积分。 13 Cu-葡萄糖、13 Cu-果糖和13 C1-丙酮酸盐一致地转化为乳酸盐，并且在较小程度上转化为碳酸氢盐。精子浓度与13 Cu-葡萄糖和13 Cu-果糖的乳酸峰大小之间存在显著相关性，而13 C1-丙酮酸则未观察到相关性。乳酸峰与非精子细胞浓度（高达6.9 × 106/ml）不相关。13 Cu-葡萄糖、13 Cu-果糖或13 C1-丙酮酸盐的浓度（1.8、3.6、7.2或14.4 mM）在4 h孵育期间对观察到的乳酸盐峰的大小没有影响。13 C1-丙酮酸盐转化为乳酸盐的速率比13 Cu-葡萄糖或13 Cu-果糖快约3倍，这两种物质之间没有显著差异。孵育4小时后，当归一化为总精子浓度时，来自DGC的“40%”界面的精子对13 Cu-葡萄糖、13 Cu-果糖或13 C1-丙酮酸的利用与来自沉淀的那些没有不同。然而，在通过活精子或活动精子浓度进行正常化后，与颗粒精子相比，“40%”界面精子的13 Cu-葡萄糖转化为乳酸的转化率显著增加（活精子= 3.3 ± 0.30 × 106 vs 2.0 ± 0.21 × 106; P = 0.0049;能动性= 7.0 ± 0.75 × 106 vs 4.8 ± 0.13 × 106; P = 0.0032。Mann-Whitney检验P < 0.0055视为统计学显著）。未观察到13 Cu-果糖或13 C1-丙酮酸盐的显著差异。不适用因只有累积到足够高浓度的13 C标记代谢物才能被13 C MRS观察到。因此，如果不使用抑制剂在特定步骤捕获分子，则难以观察代谢链中的中间分子。非精子细胞浓度是一般人群的典型浓度，未发现这些细胞与13 C-乳酸峰的幅度之间存在关联。然而，在其他实验中，高于观察到的最大浓度（6.9 × 106/ml）的浓度可能有助于外源性底物代谢。 13 C-MRS可以提供关于活精子中多个途径的潜在代谢的信息。由于酶受损或缺乏可代谢底物而导致的精子代谢功能障碍，可以通过非破坏性测定在精子中检测到，这可能为改善精子的整体质量和生殖结果提供新的治疗选择。这项工作得到了医学研究理事会拨款MR/M010473/1的支持。作者声明无利益冲突。
Can 13C-Magnetic Resonance Spectroscopy (MRS) of selected metabolites provide useful information about human sperm metabolism and how glycolysis or oxidative phosphorylation are used by different sperm populations? Sperm populations, prepared by density gradient centrifugation (DGC) and incubated with either 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate, showed consistent evidence of metabolism generating principally lactate and more intermittently bicarbonate, and significantly more lactate was produced from 13Cu-glucose by vital or motile sperm recovered from the 40/80% interface compared to those from the pellet, which could not be accounted for by differences in the non-sperm cells present. Previous studies have focused on CO2 or other specific metabolite production by human sperm and there remains considerable debate about whether glycolysis and/or oxidative phosphorylation is the more important pathway for ATP production in sperm. Sperm populations were prepared by DGC and subjected to 13C-MRS to answer the following questions. (i) Is it possible to detect human sperm metabolism of 13C substrates implicated in energy generation? (ii) What are the kinetics of such reactions? (iii) Do different sperm populations (e.g. ‘80%’ pellet sperm and ‘40%’ interface sperm) utilise substrates in the same way? Semen samples from 97 men were used in these experiments; 52 were used in parallel for aims (i) and (ii) and 45 were used for aim (iii). Sperm populations were prepared from ejaculates of healthy men using a Percoll/Phosphate Buffered Saline (PBS) DGC and then incubated with a range of 13C-labelled substrates (13Cu-glucose, 13Cu-fructose, 13C1-pyruvate, 13C1-butyrate, 13C3-lactate, 13C2,4-D-3-hydroxybutyrate, 13C5-l-glutamate, 13C1,2-glycine or 13Cu-galactose) along with penicillin/streptomycin antibiotic at 37°C for 4 h, 24 h or over 48 h for an estimated rate constant. Sperm concentration, vitality and motility were measured and, for a subset of experiments, non-sperm cell concentration was determined. A 9.4 T magnetic resonance spectrometer was used to acquire 1D 13C, inverse gated 1H decoupled, MRS spectra. Spectrum processing was carried out using spectrometer software and Matlab scripts to determine peak integrals for each spectrum. 13Cu-glucose, 13Cu-fructose and 13C1-pyruvate were consistently converted into lactate and, to a lesser extent, bicarbonate. There was a significant correlation between sperm concentration and lactate peak size for 13Cu-glucose and 13Cu-fructose, which was not observed for 13C1-pyruvate. The lactate peak did not correlate with the non-sperm cell concentration up to 6.9 × 106/ml. The concentration of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate (1.8, 3.6, 7.2 or 14.4 mM) had no influence on the size of the observed lactate peak over a 4 h incubation. The rate of conversion of 13C1-pyruvate to lactate was approximately three times faster than for 13Cu-glucose or 13Cu-fructose which were not significantly different from each other. After incubating for 4 h, the utilisation of 13Cu-glucose, 13Cu-fructose or 13C1-pyruvate by sperm from the ‘40%’ interface of the DGC was no different from those from the pellet when normalised to total sperm concentration. However, after normalising by either the vital or motile sperm concentration, there was a significant increase in conversion of 13Cu-glucose to lactate by ‘40%’ interface sperm compared to pellet sperm (Vital = 3.3 ± 0.30 × 106 vs 2.0 ± 0.21 × 106; P = 0.0049; Motile = 7.0 ± 0.75 × 106 vs 4.8 ± 0.13 × 106; P = 0.0032. Mann–Whitney test P < 0.0055 taken as statistically significant). No significant differences were observed for 13Cu-fructose or 13C1-pyruvate. Not applicable. Only 13C labelled metabolites that accumulate to a sufficiently high concentration can be observed by 13C MRS. For this reason, intermediary molecules in the metabolic chain are difficult to observe without trapping the molecule at a particular step using inhibitors. Non-sperm cell concentration was typical of the general population and no link was found between these cells and the magnitude of the 13C-lactate peak. However, it is possible that higher concentrations than the maximum observed (6.9 × 106/ml) may contribute to exogenous substrate metabolism in other experiments. 13C-MRS can provide information on the underlying metabolism of multiple pathways in live sperm. Dysfunction in sperm metabolism, as a result of either impaired enzymes of lack of metabolisable substrate, could be detected in sperm by a non-destructive assay, potentially offering new treatment options to improve overall sperm quality and outcomes for reproduction. This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.
登录
查看更多内容
影响因子：
4.6
作者：
Reynolds S;Ismail NFB;Calvert SJ;Pacey AA;Paley MNJ
通讯作者：
Paley MNJ
DOI：
10.1016/b978-0-12-372568-4.00015-x
发表时间：
2009-01-01
期刊：
SPERM BIOLOGY: AN EVOLUTIONARY PERSPECTIVE
影响因子：
--
作者：
Pacey, Allan A.
通讯作者：
Pacey, Allan A.
影响因子：
4
作者：
Reynolds S;Calvert SJ;Paley MN;Pacey AA
通讯作者：
Pacey AA
影响因子：
2.9
作者：
du Plessis SS;Agarwal A;Mohanty G;van der Linde M
通讯作者：
van der Linde M
影响因子：
7.4
作者：
Aitken, RJ;Buckingham, DW;Irvine, DS
通讯作者：
Irvine, DS