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>

<ol>
	<li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparameter+metabolic+analysis+reveals+a+close+link+between+attenuated+mitochondrial+bioenergetic+function+and+enhanced+glycolysis+dependency+in+human+tumor+cells.">Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells.</a> American Journal of Physiology - Cell Physiology. 292 (1), C125-C136 (2007).</li><li>Amo, T., Yadava, N., Oh, R., Nicholls, D. G., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+assessment+of+bioenergetic+differences+caused+by+the+common+European+mitochondrial+DNA+haplogroups+H+and+T.">Experimental assessment of bioenergetic differences caused by the common European mitochondrial DNA haplogroups H and T.</a> Gene. 411 (1-2), 69-76 (2008).</li><li>Choi, S. W., Gerencser, A. A., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+analysis+of+isolated+cerebrocortical+nerve+terminals+on+a+microgram+scale:+spare+respiratory+capacity+and+stochastic+mitochondrial+failure.">Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure.</a> Journal of Neurochemistry. 109 (4), 1179-1191 (2009).</li><li>de Groof, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+OXPHOS+activity+precedes+rise+in+glycolytic+rate+in+H-RasV12/E1A+transformed+fibroblasts+that+develop+a+Warburg+phenotype.">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><li>Chao, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+resistance+and+altered+systemic+glucose+metabolism+in+mice+lacking+Nur77.">Insulin resistance and altered systemic glucose metabolism in mice lacking Nur77.</a> Diabetes. 58 (12), 2788-2796 (2009).</li><li>Chang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertilizing+capacity+of+spermatozoa+deposited+into+the+fallopian+tubes.">Fertilizing capacity of spermatozoa deposited into the fallopian tubes.</a> Nature. 168 (4277), 697-698 (1951).</li><li>Austin, C. R., Bishop, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+rodent+acrosome+and+perforatorium+in+fertilization.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+flagellar+movement+in+hyperactivated+and+acrosome-reacted+golden+hamster+spermatozoa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+hyperactivation+in+sperm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+substrates+exhibit+differential+effects+on+functional+parameters+of+mouse+sperm+capacitation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+relationships+between+capacitation-dependent+cell+signaling+and+compartmentalized+metabolic+pathways+in+murine+spermatozoa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+tyrosine+phosphorylation+in+sperm+during+gamete+interaction+in+the+mouse:+the+influence+of+glucose.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoglycerate+kinase+2+(PGK2)+is+essential+for+sperm+function+and+male+fertility+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyceraldehyde+3-phosphate+dehydrogenase-S,+a+sperm-specific+glycolytic+enzyme,+is+required+for+sperm+motility+and+male+fertility.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glyceraldehyde+3-phosphate+dehydrogenase+is+bound+to+the+fibrous+sheath+of+mammalian+spermatozoa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+ATP+in+sperm+flagella.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+transport+of+ATP+within+the+motile+sperm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme+termini+of+a+phosphocreatine+shuttle.+Purification+and+characterization+of+two+creatine+kinase+isozymes+from+sea+urchin+sperm.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+mammalian+lineages+respond+differently+to+sexual+selection:+metabolic+rate+constrains+the+evolution+of+sperm+size.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+ATP+generation+via+glycolysis+and+oxidative+phosphorylation+and+relationships+with+sperm+motility+in+mouse+species.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitation+of+mouse+spermatozoa.+II.+Protein+tyrosine+phosphorylation+and+capacitation+are+regulated+by+a+cAMP-dependent+pathway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+adenylyl+cyclase+defines+a+unique+signaling+molecule+in+mammals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacitation+of+mouse+spermatozoa.+I.+Correlation+between+the+capacitation+state+and+protein+tyrosine+phosphorylation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+PKA+inhibition+using+a+chemical+genetic+approach+and+its+application+to+studies+on+sperm+capacitation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methodology+for+the+detection+and+quantification+of+the+acrosome+reaction+in+mouse+spermatozoa.">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 oxidative phosphorylation in real-time during sperm capacitation. The detection of changes in extracellular H+ and O2 with fluorophores immobilized to a sensor probe is minimally invasive and the four individually operated injection ports allow manipulation with pharmacological inhibitors or activators at distinct time points before or during the capacitation process. This protocol gives only one example of a mouse sperm capacitation experiment. To simplify the interpretation of the results we chose the show an exemplary experiment where glucose was used as the only energy source. The conditions are variable depending on the goal of the experiment, and up to 12 different conditions (i.e., different energy sources like glucose vs. glucose and pyruvate) can be measured in parallel. Additionally, four independent injection ports allow the injection of pharmacological activators and/or inhibitors at any desired time point before or during capacitation. This opens the possibility to use the extracellular flux analyzer as a semi high-throughput screening device. Similar to mouse sperm, in other species like human or bovine it is still enigmatic how sperm change their metabolism during capacitation. The protocol can be easily adapted; thus, we recommend optimizing the sperm concentration each time before starting a real experiment.
The protocol&#39;s biggest limitation is that high-quality results can only be achieved in the absence of bicarbonate. Bicarbonate in seminal fluid is the physiological signal that initiates the sperm&#39;s capacitation signaling cascade following ejaculation. Bicarbonate activates the soluble adenylyl cyclase (sAC; ADCY10), which catalyzes the conversion of ATP into cAMP23. The increase in cAMP then drives a signaling cascade mediated by Protein Kinase A, which ultimately leads to downstream tyrosine phosphorylation of target proteins (e.g., ion channels, metabolic enzymes, and structural proteins24,25). This restriction against bicarbonate is overcome by injecting the product of bicarbonate-activated sAC, cAMP. We use 5 mM of the cell-permeable cAMP analog db-cAMP in parallel with the broad-specificity phosphodiesterase inhibitor IBMX, which prevents rapid degradation of db-cAMP by phosphodiesterases. This combination effectively initiates the cAMP-regulated capacitation signaling pathway post sAC activation with a similar kinetic as bicarbonate (Supplementary Figure 1). In parallel to bicarbonate, a cholesterol acceptor (e.g., BSA) is used to in vitro capacitate freshly ejaculated sperm or sperm dissected from the cauda epididymis. Albumin cannot be injected because it clogs the injection port and, therefore, needs to be added to the sperm buffer before plating the cells. Performing the experiment in the presence or absence of BSA revealed that the increase in ECAR and OCR during sperm capacitation is independent of the cholesterol acceptor. However, the presence of BSA in the sperm buffer decreased fluctuations in the detected ECAR and OCR values between different wells and experiments; thus, we highly recommend including BSA in the sperm buffer to increase reproducibility.
Isolating sperm from the cauda epididymis results in the contamination of sperm with epididymal fluid. To avoid artificial results due to seminal fluid components, we recommend washing sperm two times before using them for an experiment. Sperm concentration and plating is another critical factor determining the success of the experiment. For reliable results, the manufacturer recommends initial ECAR values to be larger than 10 and OCR values to be larger than 20. The sperm concentration used in this protocol was optimized so that the average basal ECAR and OCR values of the 7-8 wells measured per condition are above 10 and 20, respectively. Freely moving sperm disturb the detection of changes in extracellular H+ and O2. Thus, it is crucial to adhere all sperm with their head to the bottom of the plate. We found success adhering sperm by coating the plate with ConA, a plant lectin that specifically interacts with the outer acrosomal membrane and is commonly used for acrosome assays26, and by gently spinning the plate (see step 2.7.3). With this method, sperm are localized to the bottom of the well solely via their head so they can still freely move their flagella and change their flagellar beating pattern during capacitation.
Sperm constantly extrude H+ and O2 in both, the non-capacitated and the capacitated state. To determine the initial ECAR and OCR as accurately as possible, it is crucial to start the experiment as quickly as you can after the last washing step. This can be accomplished by loading the sensor cartridge while the sperm are swimming out and by starting the method in the extracellular flux analyzer before the first washing step. Calibrating the instrument takes approximately the same time as washing and plating the cells and spinning the plate. The manufacturer recommends an equilibration phase to allow the system to stabilize before the first real data point is measured. Since the protocol includes 8 measurement cycles before capacitation is initiated, to save time, the equilibration step is excluded from this protocol.
The ability to inject solutions during the assay and to observe their effects on respiration and glycolytic rate in real-time is a key feature of the extracellular flux analyzer. Loading the sensor cartridge is one of the critical steps in the protocol and should be carried out carefully. To ensure proper injection into all wells, each series of ports needs to contain the same volume, including the background wells. Loading the ports with a multichannel pipette requires some practice but decreases variability and loading time considerably. We highly recommend using the port loading guide but to inject only four ports simultaneously. It is also important to appreciate that during loading, the injection volumes are gradually increased to compensate for the increasing volume in the well. While loading the sensor cartridge, it is important to not fully insert the tips into the port. This might prematurely push injection solution through the port orifice. While establishing the method we found that injecting liquid into a sperm well causes unwelcome injection artifacts, probably due to dilution of the sperm in the well and/or displacing sperm from the well bottom. The first injection causes the largest injection artifact, so we included a mock injection with sperm buffer into all wells at the beginning of the protocol.

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

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 depending on the goal of the experiment. Non-capacitated mouse sperm in BSA/TYH were attached to the bottom of a ConA-coated transient microchamber via their head. In this example, basal ECAR and OCR values on average between all the detected wells were 12.76 &#177; 2.75 mpH/min and 23.64 &#177; 2.78 pmol/min, respectively.
After a mock injection with TYH buffer, followed by injection of 2-DG and ant/rot to inhibit glycolysis and oxidative phosphorylation, respectively, sperm capacitation was induced by injection of db-cAMP/IBMX.
The representative results show that in the presence of glucose, capacitation is accompanied by a 7-fold increase in Extracellular Acidification Rate (ECAR), which is inhibited by blocking glycolysis with 2-DG (Figure 4A). Capacitated sperm show a 20-fold increase in Oxygen Consumption Rate (OCR) compared to non-capacitated sperm (Figure 4B), demonstrating that mouse sperm enhance both glycolysis and oxidative phosphorylation to support the increasing energy demand during capacitation. The rise in ECAR during sperm capacitation is inhibited by the glycolysis inhibitor 2-DG, but not affected by the oxidative phosphorylation inhibitors antimycin A and rotenone (Figure 4C), indicating that the change in ECAR is mainly driven by H+ release from glycolysis. The increase in OCR is, as expected, blocked by antimycin A and rotenone (Figure 4D), but it is also inhibited by 2-DG (Figure 4B) revealing that the increase in oxphos during sperm capacitation is dependent on glycolytic activity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60815/60815fig1.jpg" /> 
Figure 1: Principle of the extracellular flux analyzer. (A) Due to the breakdown of glucose to lactate during glycolysis and the generation of CO2 via the TCA cycle, changes in glycolysis and oxphos are accompanied by H+ excretion into the extracellular media. The XFe96 Analyzer detects these changes in extracellular H+ concentration as ECAR. In parallel, changes in extracellular O2 concentration due to O2 consumption by oxidative phosphorylation is measured as OCR. Blocking glycolysis with 2-deoxyglucose (2-DG) or respiration with the complex I and complex III inhibitors rotenone and antimycin A reveals which metabolic pathways support the increasing energy demand during sperm capacitation. (B) Mouse sperm are attached via their heads to the bottom of a ConA-coated microchamber; their flagella are freely moving. While changes in the extracellular H+ and O2 concentration are detected by H+- and O2-sensitive fluorophores immobilized to a sensor probe, up to four different compounds can be injected sequentially. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/60815fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60815/60815fig2a.jpg" /> 
Figure 2: Schematic representation of the exemplary experiment. Changes in ECAR (mpH/min) and OCR (pmol O2/min) are detected in non-capacitated and capacitating sperm using an extracellular flux analyzer. Cycle 1: Basal ECAR and OCR values. Cycles 2-5: System stabilization after TYH mock injection. Cycles 6-8: Drug incubation. Cycles 9-27: Sperm capacitation. Arrows indicate injections. 2-DG: final concentration 50 mM, AntA/Rot: final concentration 0.5 &#181;M, db-cAMP: final concentration 1 mM, IBMX: final concentration 500 &#181;M. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/60815fig2alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60815/60815fig3a.jpg" /> 
Figure 3: Data analysis. (A) Raw data of changes in ECAR during mouse sperm capacitation. (B) Data after removal of the first 7 data points. (C) Data normalized to the data point before cAMP/IBMX injection. Data is shown as mean of 7-8 wells &#177; S.E.M. Injections are indicated with an arrow. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/60815fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60815/60815fig4a.jpg" /> 
Figure 4: Changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation. (A) Normalized ECAR in non-capacitated and capacitating mouse sperm in the presence and absence of 50 mM 2-DG. (B) Normalized OCR in non-capacitated and capacitating mouse sperm in the presence and absence of 50 mM 2-DG. (C) Normalized ECAR in non-capacitated and capacitating mouse sperm in the presence and absence of 0.5 &#181;M antimycin A and rotenone. (D) Normalized OCR in non-capacitated and capacitating mouse sperm in the presence and absence of 0.5 &#181;M antimycin A and rotenone. Data is shown as mean &#177; S.E.M normalized to the data point before db-cAMP/IBMX injection; n = 6. Injections are indicated with an arrow. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/60815fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Port</td>
			<td>Number of cycles</td>
			<td>Mix (min)</td>
			<td>Wait (min)</td>
			<td>Measure (min)</td>
		</tr>
		<tr>
			<td>A: Basal ECAR/OCR</td>
			<td>no port</td>
			<td>1</td>
			<td>2:00</td>
			<td>0:00</td>
			<td>3:00</td>
		</tr>
		<tr>
			<td>B: Mock injection</td>
			<td>1</td>
			<td>4</td>
			<td>2:00</td>
			<td>0:00</td>
			<td>3:00</td>
		</tr>
		<tr>
			<td>C: Drug injection</td>
			<td>2</td>
			<td>3</td>
			<td>2:00</td>
			<td>0:00</td>
			<td>3:00</td>
		</tr>
		<tr>
			<td>D: Capacitation</td>
			<td>3</td>
			<td>18</td>
			<td>2:00</td>
			<td>0:00</td>
			<td>3:00</td>
		</tr>
	</tbody>
</table>
Table 1: Measurement Details.
<img alt="Supplemental Figure 1" src="/files/ftp_upload/60815/60815suppfig1a.jpg" /> 
Supplementary Figure 1: Capacitation of mouse sperm in extracellular flux analyzer TYH buffer. Phosphorylation of tyrosine residues of mouse sperm detected at different time points during capacitation (0 - 90 min) after incubation in (A) TYH with 25 mM HCO3-, 3 mg/mL BSA and 20 mM HEPES or in (B) Extracellular flux analyzer TYH with 5 mM db-cAMP, 500 &#181;M IBMX, and 1 mM HEPES, detected with an &#945;-phosphotyrosine antibody. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/60815suppfig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Wave assay template to detect changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation. The wave desktop software can be downloaded for free after filling out a registration form (<a href="http://www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/wave-desktop-2-6" target="_blank">www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/wave-desktop-2-6</a>) and installed on windows 7, 8 or 10, (Mac OSx 10.11 (or higher) with Parallels 12 (or higher). Thereby, wave templates can be generated independently from the extracellular flux analyzer, exported and then imported into the wave software of any extracellular flux analyzer. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/069a7839-997e-4e69-9ab6-8fe217bd07c6" target="_blank">Please click here to view this file (Right click to download).</a>
Supplemental File 2: Graph pad prism file exported from wave software with exemplary data analysis. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60815/supplemental_file2.pzfx" target="_blank">Please click here to view this file (Right click to download).</a>

Watch this Scientific Journal Video about Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

7.8K Views.  Weill Cornell Medical College. We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

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

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

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.

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a ...

Developmental Biology

Combining Imaging with a Stimulant Delivery System to Study Live Mouse Sperm

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

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Author Spotlight: A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm 

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.

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

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

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

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

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.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Biology

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Medicine

Real-Time Measurement of the Mitochondrial Bioenergetic Profile of Neutrophils

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and oxidative burst, and create neutrophil extracellular traps (NETs) for microbial clearance. New insights into the metabolic activities of neutrophils challenge the early concept that they primarily rely on glycolysis. Precise measurement of metabolic activities can unfold different metabolic requirements of neutrophils, including the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle), oxidative phosphorylation (OXPHOS), pentose phosphate pathway (PPP), and fatty acid oxidation (FAO) under physiological conditions and in disease states. This paper describes a step-by-step protocol and prerequirements to measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration on mouse bone marrow-derived neutrophils, human blood-derived neutrophils, and the neutrophil-like HL60 cell line, using metabolic flux analysis on a metabolic extracellular flux analyzer. This method can be used for quantifying the mitochondrial functions of neutrophils under normal and disease conditions.

We describe stepwise protocols measuring the mitochondrial respiration of mouse and human neutrophils and HL60 cells using the metabolic extracellular flux analyzer.

Neutrophils are the first line of defense and the most abundant leukocytes in humans. These effector cells perform functions such as phagocytosis and ...

Immunology and Infection

Phosphopeptide Analysis of Rodent Epididymal Spermatozoa

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

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.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

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.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

Author Spotlight: Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function 

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.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...

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

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

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

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

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

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

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.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...