Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation
Summary
We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.
Abstract
Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+– and O2– sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.
Introduction
The application of mass spectrometry has revolutionized the study of metabolism. Targeted metabolic profiling and metabolomic tracing allow precise monitoring of changes in energy metabolism. However, performing metabolomics successfully requires extensive training, experienced staff, and expensive, highly sensitive mass spectrometers not readily available to every laboratory. In recent years, using an extracellular flux analyzer, such as the Seahorse XFe96 has grown popular as a surrogate method for measuring changes in energy metabolism in various cell types1,2,3,4,5.
Sperm are highly specialized motile cells; whose task is to deliver the paternal genome to the oocyte. Sperm leaving the male reproductive tract after ejaculation are still functionally immature and cannot fertilize the oocyte because they are unable to penetrate the oocytes' vestments. Sperm acquire fertilization competence as they transit through the female reproductive tract in a maturation process known as capacitation6,7. Freshly ejaculated sperm or sperm dissected from the cauda epididymis can be capacitated in vitro by incubation in defined capacitation media containing Ca2+, bicarbonate (HCO3–) or a cell-permeable cAMP analog (e.g., dibutyryl-cAMP), a cholesterol acceptor (e.g., bovine serum albumin, BSA), and an energy source (e.g., glucose). During capacitation, sperm modify their motility pattern into an asymmetric flagellar beat, representing a swimming mode called hyperactivation8,9, and they become competent to undergo the acrosome reaction7, where proteolytic enzymes are released that digest the oocytes' vestments. These processes require energy, and similar to somatic cells, sperm generate ATP and other high energy compounds via glycolysis as well as mitochondrial TCA cycle and oxidative phosphorylation (oxphos)10. While multiple studies demonstrate that glycolysis is necessary and sufficient to support sperm capacitation11,12,13,14, the contribution of oxphos is less clear. Contrary to other cell types where glycolysis is physically coupled to the TCA cycle, sperm are highly compartmentalized and are thought to maintain these processes in separate flagellar compartments: the midpiece concentrates the mitochondrial machinery, whereas the key enzymes of glycolysis appear to be restricted to the principal piece15,16. This compartmentalization results in an ongoing debate about whether pyruvate produced in the principal piece by glycolysis can support mitochondrial oxphos in the midpiece, and whether ATP produced by oxphos in the midpiece would be able to diffuse sufficiently rapidly along the length of the flagellum to support the energy requirements in distal parts of the principal piece17,18,19. There is also support of a role for oxphos in sperm capacitation. Not only is oxphos more energetically favorable than glycolysis, generating 16 times more ATP than glycolysis, but midpiece volume and mitochondrial content are directly correlated with reproductive fitness in mammalian species which exhibit greater degrees of competition between males for mates20. Addressing these questions requires methods for examining the relative contributions of glycolysis and oxphos during sperm capacitation.
Tourmente et al. applied a 24-well extracellular flux analyzer to compare the energy metabolism of closely related mouse species with significantly different sperm performance parameters21. Instead of reporting the basal ECAR and OCR values of non-capacitated sperm, here, we adapt their method using a 96-well extracellular flux analyzer to monitor changes in energy metabolism during mouse sperm capacitation in real-time. We developed a method that allows simultaneously monitoring glycolysis and oxphos in real-time in sperm with beating flagella in up to twelve different experimental conditions by measuring the flux of oxygen (O2) and protons (H+) (Figure 1A). Due to the breakdown of pyruvate to lactate during glycolysis and the production of CO2 via the TCA-cycle, non-capacitated and capacitated sperm extrude H+ into the assay media which are detected by the extracellular flux analyzer via H+-sensitive fluorophores immobilized to the probe tip of a sensor cartridge. In parallel, O2 consumption by oxidative phosphorylation is detected via O2-sensitive fluorophores immobilized to the same probe tip (Figure 1B). Effective detection of the released H+ and consumed O2 requires a modified sperm buffer with low buffering capacity without bicarbonate or phenol red. Thus, to induce capacitation in the absence of bicarbonate, we adopted the use of a cell-permeable cAMP analog injected together with the broad-range PDE inhibitor IBMX22. Three additional independent injection ports allow the injection of pharmacological activators and/or inhibitors, which facilitates real-time detection of changes in cellular respiration and glycolysis rate due to experimental manipulation.
Protocol
Representative Results
Discussion
The loss of sperm capacitation in the absence of certain metabolic substrates or critical metabolic enzymes revealed energy metabolism as a key factor supporting successful fertilization. A metabolic switch during cell activation is a well-established concept in other cell types, however, we are just beginning to understand how sperm adapt their metabolism to the increasing energy demand during capacitation. Using an extracellular flux analyzer, we developed an easily applicable tool to monitor changes in glycolysis and …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors wish to acknowledge support from Dr. Lavoisier Ramos-Espiritu at the Rockefeller High Throughput and Spectroscopy Resource Center.
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. |
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