使用细胞外通量分析仪测量小鼠精子发作期间糖解和氧化磷酸化的变化
Summary
我们描述了细胞外通量分析仪在小鼠精子增生期间实时监测糖解和氧化磷酸化变化的应用。
Abstract
哺乳动物精子在女性生殖道中获得受精能力，这个过程称为卵子。与电容相关的过程需要能量。关于产生ATP的来源，促进精子渐进性、充能性、超活化和杂技反应的来源，仍有持续的争论。在这里，我们描述了细胞外通量分析仪的应用，作为分析小鼠精子细胞内能量代谢变化的工具。使用H+– 和O2-敏感荧光草，该方法允许在非电容和电容精子中实时监测糖解和氧化磷酸化。在存在不同能量基质和/或药理活化剂和/或抑制剂的情况下使用此测定，可以深入了解不同代谢途径的贡献以及精子加速期间信号级联和代谢之间的交集。
Introduction
质谱学的应用彻底改变了代谢学的研究。靶向代谢特征分析和代谢追踪可以精确监测能量代谢的变化。然而，成功实施代谢组学需要大量的培训、经验丰富的工作人员和昂贵、高度敏感的质谱仪，而每个实验室都不容易获得。近年来，使用细胞外通量分析仪，如海马XFe96，作为测量各种细胞类型1、2、3、4、5能量代谢变化的替代方法，已经越来越流行。
精子是高度专业化的活细胞;其任务是把父系基因组传送给卵母细胞。射精后离开男性生殖道的精子在功能上仍然不成熟，不能受精卵母细胞，因为它们不能穿透卵母细胞的背心。精子在成熟过程中通过女性生殖道，称为”生育能力6，7″，获得受精能力。从 cauda 表皮解剖的新鲜射精精子或精子可在体外孵育，在定义的电容培养基中孵育，其中含有 Ca2+、碳酸氢盐 （HCO3–） 或细胞渗透 cAMP 模拟（例如，二丁基林-cAMP）、胆固醇接受器（例如，牛血清白蛋白、BSA）和能量源（如葡萄糖）。在细胞发育过程中，精子将运动模式改变为不对称的鞭状节拍，代表一种称为超活化8、9的游泳模式，它们能够进行acrosome反应7，其中蛋白水解酶被释放，消化卵母细胞的背心。这些过程需要能量，并且类似于体细胞，精子通过糖解以及线粒体TCA周期和氧化磷酸化（牛磷酸化）10生成ATP和其他高能化合物。虽然多项研究表明糖解是必要的，足以支持精子的上限11，12，13，14，但牛磷的贡献则不那么明显。与其他细胞类型不同，糖解在物理上与TCA周期结合，精子高度分裂，被认为能将这些过程保持在单独的鞭节内：中间部分浓缩线粒体机械，而糖解的关键酶似乎仅限于主要部分15，16。这种分割导致一场持续的争论，关于糖解在主要部分产生的丙酮酸盐是否可以支持线粒体牛磷在中间部分，以及ATP产生的牛磷在中间是否能够足够迅速地扩散沿旗杆的长度，以支持能量需求在主要部分17，18，19。也有支持牛磷在精子能力中的作用。牛磷不仅比糖解更有利，产生的ATP比糖解多16倍，而且中片体积和线粒体含量与哺乳动物物种的生殖适应性直接相关，而哺乳动物之间对配偶20的竞争程度更大。解决这些问题需要检查糖解和牛磷在精子发作期间的相对贡献的方法。
Tourmente等人应用了24井细胞外通量分析仪，比较了与精子性能参数明显不同的小鼠物种的能量代谢21。在这里，我们使用96井细胞外通量分析仪来实时监测小鼠精子吸收过程中能量代谢的变化，而不是报告非电容精子的基础ECAR和OCR值。我们开发了一种方法，通过测量氧的通量（O2）和质子（H+）（图1A），在多达12个不同的实验条件下，通过实时监测精子中的糖解和牛磷，在多达12个不同的实验条件下击败鞭状藻。由于在糖解过程中乳酸分解为乳酸，并通过TCA循环生产CO2，非电容和容积的精子挤出H–进入检测介质，由细胞外通量分析仪通过H+敏感荧光管固定到传感器盒的探针尖端检测到。同时，通过氧化磷酸化检测O2消耗，通过O2-敏感荧光道固定到同一探针尖端（图1B）。有效检测释放的H+和消耗的O2需要经过修饰的精子缓冲液，缓冲能力低，不含碳酸氢盐或苯酚红。因此，为了在没有碳酸氢盐的情况下诱导电容，我们采用了一种可渗透的cAMP模拟注射与广泛PDE抑制剂IBMX22一起。三个额外的独立注射端口允许注射药理活化剂和/或抑制剂，这有助于实时检测细胞呼吸和糖解率因实验操作而发生的变化。
Protocol
Representative Results
Discussion
在缺乏某些代谢基质或关键代谢酶的情况下，精子的丧失表明能量代谢是支持成功受精的关键因素。细胞激活过程中的代谢开关是其他细胞类型的一个既定概念，然而，我们才刚刚开始了解精子如何适应细胞加速过程中日益增长的能源需求。我们使用细胞外通量分析仪开发了一种易于应用的工具，用于实时监测精子增生过程中糖解和氧化磷酸化的变化。在细胞外H+和O2的变化检测?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者希望感谢洛克菲勒高通量和光谱资源中心的拉沃西尔·拉莫斯-埃斯皮里图博士的支持。
Materials
| Reagents | |||
| 2-Deoxy-D-glucose | Sigma-Aldrich | D8375 | 2-DG |
| 3-Isobutyl-1-methylxanthine | Sigma-Aldrich | I7018 | IBMX; prepare a 500 mM stock solution in DMSO (111.1 mg/ml) and store in small aliquots |
| Antimycin A | Sigma-Aldrich | A8674 | AntA; prepare a 5 mM stock solution in DMSO (2.7 mg/ml) and store in small aliquots |
| Bovine serum albumin | Sigma-Aldrich | A1470 | BSA |
| Calcium chloride | Sigma-Aldrich | C1016 | CaCl2 |
| Concanacalin A, Lectin from Arachis hypogaea (peanut) | Sigma-Aldrich | L7381 | ConA |
| Glucose | Sigma-Aldrich | G7528 | |
| Hepes | Sigma-Aldrich | H0887 | |
| Isothesia | Henry Schein Animal Health | 1169567761 | Isoflurane |
| Magnesium sulfate | Sigma-Aldrich | M2643 | MgSO4 |
| N6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt | Sigma-Aldrich | D0627 | db-cAMP |
| Potassium chloride | Sigma-Aldrich | P9333 | KCl |
| Potassium dihydrogen phosphate | Sigma-Aldrich | P5655 | KH2PO4 |
| Rotenone | Cayman Chemical Company | 13995 | Rot; prepare a 5 mM stock solution in DMSO (2mg/ml) and store in small aliquots |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | NaHCO3- |
| Sodium chloride | Sigma-Aldrich | S9888 | NaCl |
| Equipment and materials | |||
| 12 channel pipette 10-100 μL | eppendorf | ES-12-100 | |
| 12 channel pipette 50-300 μL | vwr | 613-5257 | |
| 37 °C, non-CO2 incubator | vwr | 1545 | |
| 5 mL cetrifuge tubes | eppendorf | 30119380 | |
| 50 mL conical centrifuge tubes | vwr | 76211-286 | |
| Centrifuge with plate adapter | Thermo Scientific | IEC FL40R | |
| Dissection kit | World Precision Instruments | MOUSEKIT | |
| Inverted phase contrast microscope with 40X objective | Nikon | ||
| OctaPool Solution Reservoirs, 25 ml, divided | Thomas Scientific | 1159X93 | |
| OctaPool Solution Reservoirs, 25 mL, divided | Thomas Scientific | 1159X95 | |
| Seahorse XFe96 Analyzer | Agilent | ||
| Seahorse XFe96 FluxPak | Agilent | 102416-100 | Also sold as XFe96 FluxPak mini (102601-100) with 6 instead of 18 cartidges. |
Referências
- Wu, M., et al. Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. American Journal of Physiology – Cell Physiology. 292 (1), C125-C136 (2007).
- Amo, T., Yadava, N., Oh, R., Nicholls, D. G., Brand, M. D. Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T. Gene. 411 (1-2), 69-76 (2008).
- Choi, S. W., Gerencser, A. A., Nicholls, D. G. Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. Journal of Neurochemistry. 109 (4), 1179-1191 (2009).
- de Groof, A. J., et al. Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype. Molecular Cancer. 8, 54 (2009).
- Chao, L. C., et al. Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77. Diabetes. 58 (12), 2788-2796 (2009).
- Chang, M. C. Fertilizing capacity of spermatozoa deposited into the fallopian tubes. Nature. 168 (4277), 697-698 (1951).
- Austin, C. R., Bishop, M. W. Role of the rodent acrosome and perforatorium in fertilization. Proceedings of the Royal Society of London. Series B, Biological Sciences. 149 (935), 241-248 (1958).
- Ishijima, S., Baba, S. A., Mohri, H., Suarez, S. S. Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa. Molecular Reproduction and Development. 61 (3), 376-384 (2002).
- Suarez, S. S. Control of hyperactivation in sperm. Human Reproduction Update. 14 (6), 647-657 (2008).
- Goodson, S. G., Qiu, Y., Sutton, K. A., Xie, G., Jia, W., O’Brien, D. A. Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation. Biology of Reproduction. 87 (3), 75 (2012).
- Travis, A. J., et al. Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa. Journal of Biological Chemistry. 276 (10), 7630-7636 (2001).
- Urner, F., Leppens-Luisier, G., Sakkas, D. Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose. Biology of Reproduction. 64 (5), 1350-1357 (2001).
- Danshina, P. V., et al. Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice. Biology of Reproduction. 82 (1), 136-145 (2010).
- Miki, K., et al. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proceedings of the National Academy of Sciences. 101 (47), 16501-16506 (2004).
- Mori, C., et al. Mouse spermatogenic cell-specific type 1 hexokinase (mHk1-s) transcripts are expressed by alternative splicing from the mHk1 gene and the HK1-S protein is localized mainly in the sperm tail. Molecular Reproduction and Development. 49 (4), 374-385 (1998).
- Westhoff, D., Kamp, G. Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa. Journal of Cell Science. 110 (15), 1821-1829 (1997).
- Nevo, A. C., Rikmenspoel, R. Diffusion of ATP in sperm flagella. Journal of Theoretical Biology. 26 (1), 11-18 (1970).
- Adam, D. E., Wei, J. Mass transport of ATP within the motile sperm. Journal of Theoretical Biology. 49 (1), 125-145 (1975).
- Tombes, R. M., Shapiro, B. M. Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm. Journal of Biological Chemistry. 262 (33), 16011-16019 (1987).
- Gomendio, M., Tourmente, M., Roldan, E. R. Why mammalian lineages respond differently to sexual selection: metabolic rate constrains the evolution of sperm size. Proceedings of the Royal Society of Biological Sciences. 278 (1721), 3135-3141 (2011).
- Tourmente, M., Villar-Moya, P., Rial, E., Roldan, E. R. Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species. Journal of Biological Chemistry. 290 (33), 20613-20626 (2015).
- Visconti, P. E., et al. Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. Development. 121 (4), 1139-1150 (1995).
- Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., Levin, L. R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. PNAS. 96 (1), 79-84 (1999).
- Visconti, P. E., Bailey, J. L., Moore, G. D., Pan, D., Olds-Clarke, P., Kopf, G. S. Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. Development. 121 (4), 1129-1137 (1995).
- Morgan, D. J., et al. Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation. PNAS. 105 (52), 20740-20745 (2008).
- Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa. Histology and Histopathology. 24 (8), 999-1007 (2009).
