Name: Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation (Video) | JoVE Journal
Uploaded: 2020-01-22T15:00:00.000+00:00
Duration: 8 min 22 s
Description: Learn to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation using an extracellular flux analyzer. This method reveals metabolic differences between non-capacitated and capacitated sperm.

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5 minutes

The authors wish to acknowledge support from Dr. Lavoisier Ramos-Espiritu at the Rockefeller High Throughput and Spectroscopy Resource Center.

Sperm are collected from 8-16-week-old CD-1 male mice. Animal experiments were approved by Weill Cornell Medicine&#39;s Institutional Animal Care and Use Committee (IACUC).
1. Day prior to assay
<ol>
	<li>Preparation of sensor cartridge and extracellular flux analyzer calibrant
	<ol>
		<li>To hydrate the sensor cartridge, remove the sensor cartridge from the XFe96 Extracellular Flux Assay Kit and place the sensor cartridge upside down next to the utility plate.</li>
		<li>Fill a solution reservoir with 25 mL of double-distilled H2O using a multichannel pipette, add 200 &#181;L of H2O to each well of the utility plate. Place the sensor cartridge back into the utility plate and incubate overnight in a 37 &#176;C non-CO2 incubator. 
		NOTE: Cartridge needs to be hydrated for at least 4 h.</li>
		<li>Aliquot 25 mL of the extracellular flux analyzer calibrant into a 50 mL conical tube and incubate it overnight in a 37 &#176;C non-CO2 incubator.</li>
	</ol>
	</li>
	<li>Preparation of ConA-coated microplates
	<ol>
		<li>Dissolve 2.5 mg of ConA in 5 mL of double-distilled H2O to prepare a 0.5 mg/mL (w/v) stock.</li>
		<li>Using a multichannel pipette, fill each well of an extracellular flux analyzer 96-well plate with 50 &#181;L of the 0.5 mg/mL Con A solution. Leave the lid open and let the plate dry overnight at room temperature. 
		NOTE: Multiple plates can be coated at once and stored at 4 &#176;C for up to four weeks until ready for use.</li>
	</ol>
	</li>
	<li>Preparation of sperm buffer
	<ol>
		<li>Prepare 250 mL of extracellular flux analyzer TYH buffer with low HEPES containing 138 mM NaCl, 4.7 mM KCl, 1.7 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.6 mM glucose, and 1 mM HEPES. Heat the buffer to 37 &#176;C and adjust the pH to 7.4.</li>
	</ol>
	</li>
</ol>
2. Day of the assay
<ol>
	<li>Preparation for cartridge calibration
	<ol>
		<li>Replace the H2O in the utility plate with 200 &#181;L of XF calibrant per well and incubate for at least 1 h in a non-CO2 37 &#176;C incubator. 
		NOTE: Instead of calibrant, sterile-filtered PBS, pH 7.4 can be used.</li>
		<li>Heat 50 mL of TYH buffer at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Generating a wave template for the extracellular flux analyzer
	<ol>
		<li>Turn the extracellular flux analyzer on and allow the temperature to stabilize to 37 &#176;C.</li>
		<li>Open the wave software and design a new template by opening a blank template (see Supplemental File 1, wave template).</li>
		<li>Open the Group definitions tab. Define assay media as TYH; pH 7.4 and cell type as mouse sperm, leave injection strategies and pretreatments blank. Create different groups for each assay condition; in this example, there are 6 different groups (TYH, TYH + db-cAMP/IBMX, 2-DG, 2-DG + db-cAMP/IBMX, Ant/Rot, Ant/Rot + db-cAMP/IBMX); dibutyryl cAMP (db-cAMP) and 3-Isobutyl-1-methylxanthine (IBMX) to induce capacitation, 2-deoxyglucose (2-DG) as glycolysis inhibitor, antimycin A (antA) and rotenone (rot) as inhibitor of complex III and complex I of the electron transport chain. 
		NOTE: To account for variability between wells, at least 7-8 wells per condition should be measured in parallel.</li>
		<li>Open the Plate map tab and define the plate map by assigning groups to specific wells. 
		NOTE: The four corner wells (A1, A12, H1, H12) will be filled with TYH buffer (no sperm) and will later be used for background subtraction.</li>
		<li>Open the Protocol tab, add 4 different measurement cycles, select the respective port and edit the measurement details (Figure 2, Table 1). Highlight measure after injection and do not highlight equilibrate.</li>
		<li>Save the assay template, fill out the project summary and define the saving location for the results file.</li>
	</ol>
	</li>
	<li>Preparation of compounds to load into sensor cartridge
	<ol>
		<li>Prepare 2 mL of 50 mM db-cAMP by dissolving 9.8 mg of compound in TYH buffer and add IBMX to a final concentration of 5 mM. 
		NOTE: IBMX has the tendency to precipitate after being diluted in TYH buffer. Precipitates can be dissolved by vortexing and incubating the TYH/db-cAMP/IBMX solution in a 37 &#176;C heat block for 5 min.</li>
		<li>Prepare 1 mL of 500 mM 2-DG by dissolving 82.1 mg of compound in TYH buffer.</li>
		<li>Prepare 1 mL of 5 &#181;M of AntA/Rot by diluting both drugs in TYH buffer. 
		NOTE: Compounds are diluted 10-fold by injection into the extracellular flux analyzer cartridge; therefore, be sure that injection solutions are 10x more concentrated.</li>
	</ol>
	</li>
	<li>Isolation of mouse sperm
	<ol>
		<li>Anesthetize 3 male mice between 8 and 16 weeks by isoflurane and sacrifice the mice by cervical dislocation after no response to toe pinching was detected. Remove the cauda epididymides and vasa deferentia.</li>
		<li>Place each cauda in 500 &#181;L of prewarmed TYH buffer in 24-well plate well, immobilize the cauda to the bottom of the plate using a pair of forceps and open the tissue by making 5-7 small incisions with feather scissors.</li>
		<li>Transfer the plate immediately into a non-CO2 37 &#176;C incubator and let the sperm disperse for 15 min.</li>
	</ol>
	</li>
	<li>Loading of the sensor cartridge
	<ol>
		<li>Two port-loading guides for port A and D and port B and C are provided in the Extracellular Flux kit. For loading a specific port, remove the lid from the sensor cartridge and align the letter of the respective port loading guide with the upper left corner of the cartridge. While injecting, use the fingertips of the non-injecting hand to hold the port loading guide in place and insert the pipette tips vertically into the port loading guide holes.</li>
		<li>Port A: Using a multichannel pipette, inject 20 &#181;L of the TYH buffer into every column.</li>
		<li>Port B: Inject 22 &#181;L of TYH buffer into column 1-4, inject 22 &#181;L of 500 mM 2-DG into column 5-8, and 25 &#181;L of 5 &#181;M AntA/Rot into column 9-12.</li>
		<li>Port C: Inject 25 &#181;L of TYH buffer into every column with odd numbers, inject 25 &#181;L of 10 mM db-cAMP/ 500 &#181;M IBMX into every column with even numbers.</li>
		<li>Place the sensor cartridge with the calibration plate into the extracellular flux analyzer and start the assay. Calibration takes 10 - 15 min.</li>
	</ol>
	</li>
	<li>Preparation of sperm plates
	<ol>
		<li>Dissolve 45 mg of BSA in 15 mL of TYH buffer (3 mg/mL w/v) and prepare three aliquots of 3 mL BSA/TYH solution each in 5 mL centrifuge tubes.</li>
		<li>Combine two sperm wells each in one 1.5 mL centrifuge tube, count the sperm using a hematocytometer and dilute the sperm to a concentration of 2 x 107 sperm/mL. 
		NOTE: Use cut pipette tips for pipetting sperm to avoid damaging the cells.</li>
		<li>Centrifuge the sperm for 3 min at 700 x g, remove the supernatant and add 1 mL of TYH buffer. Repeat the centrifugation step, remove the supernatant, and transfer each sperm suspension aliquot into one 5 mL centrifuge tube with 3 mL of BSA/TYH.</li>
		<li>Place 180 &#181;L of TYH buffer into each corner well (A1, A12, H1, H12) of a ConA-coated plate. Place 180 &#181;L of sperm suspension into each empty well of the ConA-coated plate (1.2 x 106 sperm/well).</li>
		<li>Centrifuge the sperm plate at 250 x g for 1 min, rotate the plate by 180&#176;, and centrifuge again at 250 x g for 1 min (lowest braking rate 1).</li>
		<li>Remove the calibration plate from the extracellular flux analyzer and add the sperm plate. Continue the assay.</li>
	</ol>
	</li>
	<li>Data extraction and analysis
	<ol>
		<li>After finishing the assay, remove the cartridge, open the results file, click the Export tab and export the completed run as a GraphPad Prism file (Supplemental File 2: Primary example data used for Figure 3).</li>
		<li>Duplicate the ECAR and OCR file by clicking on the New tab and then the Duplicate Family&#8230; tab (Figure 3A).</li>
		<li>Delete the first 7 rows of data (Figure 3B).</li>
		<li>Normalize to the data point before the cAMP/IBMX injection by baseline subtracting row 1. Click on the Analyze tab, then on the Remove baseline and column math tab. Highlight Selected row(s): First Row, Calculation: Ratio:Value/Baseline, and Subcolumns: Ignore Subcolumn. Click OK (Figure 3C).</li>
	</ol>
	</li>
</ol>

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<li>de Groof, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increased+OXPHOS+activity+precedes+rise+in+glycolytic+rate+in+H-RasV12%2FE1A+transformed+fibroblasts+that+develop+a+Warburg+phenotype%5BTitle%5D)+AND+%22Molecular+Cancer%22%5BJournal%5D)">Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype.</a> Molecular Cancer. 8, 54(2009).</li>
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<li>Austin, C. R., Bishop, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+rodent+acrosome+and+perforatorium+in+fertilization%5BTitle%5D)+AND+%22Proceedings+of+the+Royal+Society+of+London.+Series+B%2C+Biological+Sciences%22%5BJournal%5D)">Role of the rodent acrosome and perforatorium in fertilization.</a> Proceedings of the Royal Society of London. Series B, Biological Sciences. 149 (935), 241-248 (1958).</li>
<li>Ishijima, S., Baba, S. A., Mohri, H., Suarez, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+analysis+of+flagellar+movement+in+hyperactivated+and+acrosome-reacted+golden+hamster+spermatozoa%5BTitle%5D)+AND+%22Molecular+Reproduction+and+Development%22%5BJournal%5D)">Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa.</a> Molecular Reproduction and Development. 61 (3), 376-384 (2002).</li>
<li>Suarez, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+hyperactivation+in+sperm%5BTitle%5D)+AND+%22Human+Reproduction+Update%22%5BJournal%5D)">Control of hyperactivation in sperm.</a> Human Reproduction Update. 14 (6), 647-657 (2008).</li>
<li>Goodson, S. G., Qiu, Y., Sutton, K. A., Xie, G., Jia, W., O'Brien, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+substrates+exhibit+differential+effects+on+functional+parameters+of+mouse+sperm+capacitation%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation.</a> Biology of Reproduction. 87 (3), 75(2012).</li>
<li>Travis, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+relationships+between+capacitation-dependent+cell+signaling+and+compartmentalized+metabolic+pathways+in+murine+spermatozoa%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Functional relationships between capacitation-dependent cell signaling and compartmentalized metabolic pathways in murine spermatozoa.</a> Journal of Biological Chemistry. 276 (10), 7630-7636 (2001).</li>
<li>Urner, F., Leppens-Luisier, G., Sakkas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+tyrosine+phosphorylation+in+sperm+during+gamete+interaction+in+the+mouse%3A+the+influence+of+glucose%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Protein tyrosine phosphorylation in sperm during gamete interaction in the mouse: the influence of glucose.</a> Biology of Reproduction. 64 (5), 1350-1357 (2001).</li>
<li>Danshina, P. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phosphoglycerate+kinase+2+%28PGK2%29+is+essential+for+sperm+function+and+male+fertility+in+mice%5BTitle%5D)+AND+%22Biology+of+Reproduction%22%5BJournal%5D)">Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice.</a> Biology of Reproduction. 82 (1), 136-145 (2010).</li>
<li>Miki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glyceraldehyde+3-phosphate+dehydrogenase-S%2C+a+sperm-specific+glycolytic+enzyme%2C+is+required+for+sperm+motility+and+male+fertility%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility.</a> Proceedings of the National Academy of Sciences. 101 (47), 16501-16506 (2004).</li>
<li>Mori, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+spermatogenic+cell-specific+type+1+hexokinase+%28mHk1-s%29+transcripts+are+expressed+by+alternative+splicing+from+the+mHk1+gene+and+the+HK1-S+protein+is+localized+mainly+in+the+sperm+tail%5BTitle%5D)+AND+%22Molecular+Reproduction+and+Development%22%5BJournal%5D)">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.</a> Molecular Reproduction and Development. 49 (4), 374-385 (1998).</li>
<li>Westhoff, D., Kamp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glyceraldehyde+3-phosphate+dehydrogenase+is+bound+to+the+fibrous+sheath+of+mammalian+spermatozoa%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Glyceraldehyde 3-phosphate dehydrogenase is bound to the fibrous sheath of mammalian spermatozoa.</a> Journal of Cell Science. 110 (15), 1821-1829 (1997).</li>
<li>Nevo, A. C., Rikmenspoel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusion+of+ATP+in+sperm+flagella%5BTitle%5D)+AND+%22Journal+of+Theoretical+Biology%22%5BJournal%5D)">Diffusion of ATP in sperm flagella.</a> Journal of Theoretical Biology. 26 (1), 11-18 (1970).</li>
<li>Adam, D. E., Wei, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mass+transport+of+ATP+within+the+motile+sperm%5BTitle%5D)+AND+%22Journal+of+Theoretical+Biology%22%5BJournal%5D)">Mass transport of ATP within the motile sperm.</a> Journal of Theoretical Biology. 49 (1), 125-145 (1975).</li>
<li>Tombes, R. M., Shapiro, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enzyme+termini+of+a+phosphocreatine+shuttle.+Purification+and+characterization+of+two+creatine+kinase+isozymes+from+sea+urchin+sperm%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Enzyme termini of a phosphocreatine shuttle. Purification and characterization of two creatine kinase isozymes from sea urchin sperm.</a> Journal of Biological Chemistry. 262 (33), 16011-16019 (1987).</li>
<li>Gomendio, M., Tourmente, M., Roldan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+mammalian+lineages+respond+differently+to+sexual+selection%3A+metabolic+rate+constrains+the+evolution+of+sperm+size%5BTitle%5D)+AND+%22Proceedings+of+the+Royal+Society+of+Biological+Sciences%22%5BJournal%5D)">Why mammalian lineages respond differently to sexual selection: metabolic rate constrains the evolution of sperm size.</a> Proceedings of the Royal Society of Biological Sciences. 278 (1721), 3135-3141 (2011).</li>
<li>Tourmente, M., Villar-Moya, P., Rial, E., Roldan, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differences+in+ATP+generation+via+glycolysis+and+oxidative+phosphorylation+and+relationships+with+sperm+motility+in+mouse+species%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species.</a> Journal of Biological Chemistry. 290 (33), 20613-20626 (2015).</li>
<li>Visconti, P. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacitation+of+mouse+spermatozoa.+II.+Protein+tyrosine+phosphorylation+and+capacitation+are+regulated+by+a+cAMP-dependent+pathway%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway.</a> Development. 121 (4), 1139-1150 (1995).</li>
<li>Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., Levin, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytosolic+adenylyl+cyclase+defines+a+unique+signaling+molecule+in+mammals%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals.</a> PNAS. 96 (1), 79-84 (1999).</li>
<li>Visconti, P. E., Bailey, J. L., Moore, G. D., Pan, D., Olds-Clarke, P., Kopf, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Capacitation+of+mouse+spermatozoa.+I.+Correlation+between+the+capacitation+state+and+protein+tyrosine+phosphorylation%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation.</a> Development. 121 (4), 1129-1137 (1995).</li>
<li>Morgan, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-specific+PKA+inhibition+using+a+chemical+genetic+approach+and+its+application+to+studies+on+sperm+capacitation%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Tissue-specific PKA inhibition using a chemical genetic approach and its application to studies on sperm capacitation.</a> PNAS. 105 (52), 20740-20745 (2008).</li>
<li>Lybaert, P., Danguy, A., Leleux, F., Meuris, S., Lebrun, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa%5BTitle%5D)+AND+%22Histology+and+Histopathology%22%5BJournal%5D)">Improved methodology for the detection and quantification of the acrosome reaction in mouse spermatozoa.</a> Histology and Histopathology. 24 (8), 999-1007 (2009).</li>
</ol>

The authors have nothing to disclose.

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 ...

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&#39; 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&#39; 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.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Deoxy-D-glucose</td><td>Sigma-Aldrich</td><td>D8375</td><td>2-DG</td></tr><tr><td>3-Isobutyl-1-methylxanthine</td><td>Sigma-Aldrich</td><td>I7018</td><td>IBMX; prepare a 500 mM stock solution in DMSO (111.1 mg/ml) and store in small aliquots</td></tr><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td>AntA; prepare a 5 mM stock solution in DMSO (2.7 mg/ml) and store in small aliquots</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A1470</td><td>BSA</td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>C1016</td><td>CaCl2</td></tr><tr><td>Concanacalin A, Lectin from Arachis hypogaea (peanut)</td><td>Sigma-Aldrich</td><td>L7381</td><td>ConA</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>Isothesia</td><td>Henry Schein Animal Health</td><td>1169567761</td><td>Isoflurane</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M2643</td><td>MgSO4</td></tr><tr><td>N6,2&#039;-O-Dibutyryladenosine 3&#039;,5&#039;-cyclic monophosphate sodium salt</td><td>Sigma-Aldrich</td><td>D0627</td><td>db-cAMP</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td>KCl</td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Sigma-Aldrich</td><td>P5655</td><td>KH2PO4</td></tr><tr><td>Rotenone</td><td>Cayman Chemical Company</td><td>13995</td><td>Rot; prepare a 5 mM stock solution in DMSO (2mg/ml) and store in small aliquots</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>NaHCO3-</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9888</td><td>NaCl</td></tr><tr><td>&lt;strong&gt;Equipment and materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>12 channel pipette 10-100 &amp;mu;L</td><td>eppendorf</td><td>ES-12-100</td><td></td></tr><tr><td>12 channel pipette 50-300 &amp;mu;L</td><td>vwr</td><td>613-5257</td><td></td></tr><tr><td>37 &amp;deg;C, non-CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>vwr</td><td>1545</td><td></td></tr><tr><td>5 mL cetrifuge tubes</td><td>eppendorf</td><td>30119380</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>vwr</td><td>76211-286</td><td></td></tr><tr><td>Centrifuge with plate adapter</td><td>Thermo Scientific</td><td>IEC FL40R</td><td></td></tr><tr><td>Dissection kit</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td></td></tr><tr><td>Inverted phase contrast microscope with 40X objective</td><td>Nikon</td><td></td><td></td></tr><tr><td>OctaPool Solution Reservoirs, 25 ml, divided</td><td>Thomas Scientific</td><td>1159X93</td><td></td></tr><tr><td>OctaPool Solution Reservoirs, 25 mL, divided</td><td>Thomas Scientific</td><td>1159X95</td><td></td></tr><tr><td>Seahorse XFe96 Analyzer</td><td>Agilent</td><td></td><td></td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Agilent</td><td>102416-100</td><td>Also sold as XFe96 FluxPak mini (102601-100) with 6 instead of 18 cartidges.</td></tr></tbody></table>

using an extracellular flux analyzer to measure changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

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.

This method uses an extracellular flux analyzer to monitor real-time changes in the rate of glycolysis and oxphos during mouse sperm capacitation. Figure 4 shows an exemplary experiment where sperm were capacitated in the presence of glucose as the only energy substrate and 2-DG and antimycin and rotenone as pharmacological modulators. The energy substrate in the extracellular flux analyzer TYH buffer and the pharmacological modulators can be freely selected ...

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

using-an-extracellular-flux-analyzer-to-measure-changes-glycolysis

Research

JoVE Journal

Biochemistry

This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.

Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Proteomic analysis of any cell type is highly dependent on both purity and pre-fractionation of the starting material in order to de-complexify the sample prior to liquid chromatography mass spectrometry (MS). By using back-flushing techniques, pure spermatozoa can be obtained from rodents. Following digestion, phosphopeptides can be enriched using TiO2.

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

This protocol describes a cost-effective way to perform a glycolysis stress test to quantify metabolic reprogramming in primary mouse microglia. After isolation and inflammatory stimulation, extracellular acidification rate (ECAR) is measured using freshly prepared, pH-adjusted reagents, followed by protein normalization to enable accurate and reproducible assessment of glycolytic shifts.

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

Learn to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation using an extracellular flux analyzer. This method reveals metabolic differences between non-capacitated and capacitated sperm.

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation (Video) | JoVE Journal

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

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