Name: high-resolution respirometry to assess mitochondrial function in human spermatozoa
Uploaded: 2023-06-23T14:35:00.000+00:00
Duration: 8 min 32 s
Description: Watch this Scientific Journal Video about Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function at JoVE.com

We would like to thank the Fertilab Andrology clinic, especially Jos&#233; Mar&#237;a Montes and Andrea Torrents, for allowing us access to donors. Funding: A.C. is supported by grants from Universidad de la Rep&#250;blica (CSIC_2018, Espacio Interdisciplinario_2021). Additional funding was obtained from the Programa de Desarrollo de Ciencias B&#225;sicas (PEDECIBA, Uruguay). P.I. and R.S. are supported by Universidad de la Rep&#250;blica (I+D, CSIC 2014; I+D, CSIC 2016, Iniciaci&#243;n a la Investigaci&#243;n, CSIC 2019 and FMV_1_2017_1_136490 ANII- Uruguay). P.I. is supported by POS_FMV_2018_1_1007814 and CAP-UDELAR 2020. The figures were illustrated using Biorender.com.

The experiments were approved by the Ethics Committee of the Facultad de Medicina de la Universidad de la Rep&#250;blica, Montevideo, Uruguay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65493/65493fig01.jpg" /> 
Figure 1: Workflow for high-resolution respirometry to assess mitochondrial function in intact and permeabilized human sperm. The protocol was divided into four different steps: 1) preparation of the sample, 2) oxygen calibration in the Oroboros instrument, 3) oxygen consumption measurement for intact and permeabilized cells, and 4) data extraction from the equipment and analysis. Abbreviations: CASA = computer-assisted sperm analysis; BWW = Biggers Whitten Whittingham medium; MRM = mitochondrial respiration medium; ADP = adenosine diphosphate; FCCP = carbonyl cyanide -p- trifluoromethoxyphenylhydrazone; AA = antimycin A. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
NOTE: The workflow for measuring the oxygen consumption in sperm cells using HRR is shown in Figure 1. Information on the materials, equipment, and reagents used in the protocol is presented in the Table of Materials.
1. Sample preparation
<ol>
	<li>Sample collection
	<ol>
		<li>Collect freshly ejaculated human semen by masturbation after a recommended 3 day abstinence in a sterile plastic container. Transport the samples immediately to the laboratory.</li>
		<li>Incubate the samples for 30-60 min at room temperature (RT) to liquefy completely24.</li>
		<li>After liquefaction, store the samples at 37 &#176;C until the experiment begins.</li>
	</ol>
	</li>
	<li>Sperm evaluation with computer-aided sperm analysis (CASA)
	<ol>
		<li>Mix the sample, and load 7 &#181;L into a pre-warmed sperm counting chamber.</li>
		<li>Place the chamber on the pre-warmed (37&#160;&#176;C) stage of a direct light microscope.</li>
		<li>Open the computerized sperm analysis software and, enter the motility and concentration module (click on Mot).</li>
		<li>Select the configuration that corresponds to human sperm conditions. 
		NOTE: The configuration must be adapted to the type and depth of the chamber as well as to the sample species and the system CASA.</li>
		<li>Randomly analyze 10 different fields per chamber by clicking on the Analyze button.</li>
		<li>Click on Results to obtain the sample concentration and motility.</li>
	</ol>
	</li>
	<li>Cell preparation 
	NOTE: If the HRR is not calibrated, start with steps 2.1-2.2 before preparing the cells (step 1.3). It is important to measure the oxygen consumption immediately when the sperm cells are resuspended in the medium.
	<ol>
		<li>Centrifuge the samples at 400 x g for 10 min at RT.</li>
		<li>Remove the seminal plasma, and resuspend the spermatozoa in 2 mL of Biggers Whitten Whittingham (BWW) for experiments with intact cells or mitochondrial respiration medium (MRM) for studies with permeabilized cells. The compositions of the media are shown in Table 1.</li>
		<li>Repeat the steps described in step 1.2 for sperm concentration studies.</li>
	</ol>
	</li>
</ol>
2. High-resolution respirometry: OXPHOS analysis
NOTE: HRR integrates highly sensitive oxygraphs (Oxygraph-2 K; Oroboros Instruments GmbH, Innsbruck, Austria) with software (DatLab, version 4.2; Oroboros Instruments GmbH). The experimental data are displayed as the oxygen concentration versus time (as pmol of O2/106 cells&#183;min&#8722;1) and as real-time transformations of these data, allowing the experimenter to track the respiration (oxygen consumption, oxygen flux) of biological and biochemical samples while the experiment is still running. The HRR can be used to follow the respiration of living and motile cells, which is particularly useful for sperm, whose motility is associated with the sperm quality and fertility potential. The laboratory uses an HRR Oroboros Oxygraph2-k, Oroboros Instruments, with two chambers. The steps described in this protocol must be performed independently for both 2 mL chambers.
<ol>
	<li>Equipment preparation
	<ol>
		<li>Turn on the HRR, and connect it to the respirometry software (DatLab) for data acquisition and analysis.</li>
		<li>Replace the 70% ethanol in the oxygraph chamber with ddH2O. Stir it continuously with the magnetic stir bar in the chamber at 750 rpm. Let it stand for 10 min, and aspirate the double-distilled (dd) H2O afterward.</li>
		<li>Wash the chamber three times with ddH2O for 5 min each time. 
		NOTE: This step is necessary to remove the remaining ethanol from the chambers. Sperm cells are very sensitive to ethanol. The recording could be compromised if this step is omitted.</li>
	</ol>
	</li>
	<li>Calibration of the oxygen sensors 
	NOTE: The calibration procedure varies slightly depending on the instrument. Perform an air calibration of the polarographic oxygen sensor as described by the manufacturer25. In this section, the calibration protocol is explained briefly.
	<ol>
		<li>Remove the ddH2O, and pipette 2 mL of the same medium used for the cell preparation into the chamber. Place the stoppers, leaving an air exchange bubble. 
		NOTE: It is important to know the volume of the chamber to determine the exact volume of medium needed.</li>
		<li>Record the oxygen calibration values (click on Layout &#62; 01 Calibration Exp. Gr3-Temp) to monitor the performance of the sensor membrane by stirring the medium with the stir bar at 750 rpm for at least 30 min at 37 &#176;C. Use the other settings as mentioned: gain for sensor: 2; polarization voltage: 800 mV; data recording interval: 2.0 s. 
		&#8203;NOTE: It is expected to obtain an O2 slope uncorrected (red line) within &#177;2 pmol&#8729;s&#8722;1&#8729;mL&#8722;1 with a stable signal from the polarographic sensor.</li>
		<li>Drag the mouse while holding down the left mouse button and the shift key to select an area where the change in oxygen concentration (Y1 O2 Concentration, blue line) is stable.</li>
		<li>Open the O2 Calibration window (click on Oxygraph &#62; O2 Calibration). In Air Calibration, change the selected mark to the region selected in step 2.2.3. Finish by clicking on Calibrate and Copy to Clipboard.</li>
		<li>Stop the recording, and save by clicking on Oxygraph &#62; Ok Control &#62; Save and Disconnect. 
		NOTE: This dataset must be saved so that it can be used in all of the day&#39;s experiments. The calibration is only performed once per day for each medium.</li>
	</ol>
	</li>
	<li>Digitonin-permeabilization titration
	<ol>
		<li>Open the chamber, and aspirate the medium inside.</li>
		<li>Load in the chamber at least 24 x 106 and no more than 70 x 106 sperm cells in a final volume of 2 mL of MRM. 
		&#8203;NOTE: It is important to measure the number of cells in the chamber in order to adjust the oxygen consumption at the end of the experiment. A lower number of cells than recommended cannot be measured.</li>
		<li>Close the chamber by pushing the stoppers all the way in, and aspirate the remaining liquid at the top. Start the experiment with the same settings as for the calibration: stirring speed: 750 rpm; temperature: 37 &#176;C; gain for sensor: 2; polarization voltage: 800 mV; and data recording interval: 2.0 s.</li>
		<li>To load the calibration, double-click on the Pos Calib box in the bottom corner. Open the calibration performed in step 2.2 (click on Oxygraph &#62; O2 Calibration &#62; Copy from File), and click on Calibrate and Copy to Clipboard. 
		NOTE: The POS Calib box will change from yellow to green. The data are displayed in oxygen flow corrected per volume charts (Layout 05 Flux per Volume uncorrected). Different layouts are available in Oxygraph &#62; Layout.</li>
		<li>Add 5 &#181;L of 0.5 M adenosine diphosphate (ADP), 10 &#181;L of 2 M glutamate, and 2.5 &#181;L of 0.4 M malate (final concentrations: 1.25 mM, 10 mM, and 0.5 mM). Measure the oxygen consumption until the signal stabilizes. 
		NOTE: Precision Hamilton micro-syringes are used for injection through the loading port in the stopper. Use one syringe per drug to avoid cross-contamination. Click on F4 to register, and mark in the oxygen register when a treatment is added. 
		NOTE: The substrates are prepared in ultrapure water and stored at &#8722;20 &#176;C for 3 months.</li>
		<li>Tritate by adding 1 &#181;L of 10 mM digitonin in successive steps until the oxygen consumption reaches a maximal level. 
		NOTE: Thorough washing with water, 70% ethanol, and 100% ethanol is essential if the same chamber is used for two experiments on the same day. 
		NOTE: Digitonin is prepared in ultrapure water and stored at &#8722;20 &#176;C for 3 months.</li>
	</ol>
	</li>
	<li>Routine respiratory assessment protocol for intact and permeabilized sperm cells (complex I or complex II)
	<ol>
		<li>Open the chamber, and aspirate the medium inside.</li>
		<li>Load in the chamber at least 24 x 106 and no more than 70 x 106 sperm cells in a final volume of 2 mL of BWW (intact cell analysis) or MRM (permeabilized cell analysis).</li>
		<li>Start the experiment with the same settings as for the calibration (this is described in step 2.3.3).</li>
		<li>Load the calibration performed in step 2.2 as described in step 2.3.4.</li>
		<li>Record the respiration of the cells for at least 5 min until a stable signal is obtained. This measurement corresponds to basal respiration in intact cells.</li>
		<li>If the experiment is with intact cells, proceed to step 2.4.9. For permeabilized cells, inject 4.5 &#181;L of 10 mM digitonin (final concentration: 22.5 &#181;M). Permeabilize the cells for 5 min.</li>
		<li>Add the substrates: 10 &#181;L of 2 M glutamate and 2.5 &#181;L of 0.4 M malate (final concentrations: 10 mM and 0.5 mM, respectively) for complex I or 20 &#181;L of 1 M succinate (final concentration: 10 mM) for complex II. Measure the oxygen consumption until the signal increases and stabilizes. This is state 4, which means basal complex I or basal complex II supported respiration in the absence of ADP. 
		NOTE: The substrates are prepared in ultrapure&#160;water and stored at &#8722;20 &#176;C for 3 months.</li>
		<li>Inject 5 &#181;L of 0.5 M ADP (final concentration: 1.25 mM). Measure the oxygen consumption until the signal increases and stabilizes. The addition of ADP increases the signal corresponding to the maximum oxygen consumption through complex I or complex II (state 3, in permeabilized cells).</li>
		<li>Add 1 &#181;L of 4 mg/mL oligomycin (final concentration: 2 &#181;g/mL), an ATP synthetase inhibitor. Measure the oxygen consumption until the signal decreases and stabilizes. 
		NOTE: Oligomycin is prepared in ethanol and stored at &#8722;20 &#176;C for 3 months.</li>
		<li>Titrate by adding 1 &#181;L of 0.1 mM to 1 mM carbonyl cyanide-P- trifluoromethoxy-phenylhydrazone (FCCP) in successive steps until a maximum uncoupled respiration rate is reached. Measure the oxygen consumption until the signal increases and stabilizes. 
		NOTE: FCCP is prepared in ethanol and stored at &#8722;20 &#176;C for 3 months.</li>
		<li>The final concentration of FCCP is sample-dependent. Stop injecting the drug when oxygen consumption begins to decrease.</li>
		<li>Finally, inject 1 &#181;L of 5 mM antimycin A (2.5 &#181;M final concentration). This is a complex III inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 &#181;L of 1 mM rotenone (0.5 &#181;M final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes. 
		&#8203;NOTE: The drugs are prepared in ethanol and stored at &#8722;20 &#176;C for 3 months.</li>
	</ol>
	</li>
</ol>
3. Data extraction and analysis
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65493/65493fig2v2.jpg" /> 
Figure 2: Acquisition of respiratory parameters from a high-resolution respirometry experiment. (A,B) Schematic representations of graphs obtained, as described in Figure 1, for intact and permeabilized cells, respectively. These parameters have been previously described15. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Drag the mouse by pressing the left mouse button and the shift key to select regions where the oxygen flux per volume correlated (Y2 O2 slope uncorr., red line) is stable after the injection of a substrate or inhibitor. Figure 2 shows the different parameters obtained from the register previously described15. 
	NOTE: The parameters depend on the experiment; all of them are as follows: basal respiration in intact cells and respiration in the presence of glutamate/malate or succinate (state 4), ADP (state 3), oligomycin (proton leak), FCCP (maximum respiration rate), rotenone/AA (non-mitochondrial respiration). In permeabilized cells, basal respiration corresponds to state 3.</li>
	<li>Click on the Marks &#62; Statistics windows, and export the data.</li>
	<li>Normalize the data obtained per 1 million sperm cells. The units of the slopes are pmolO2&#183;s&#8722;1&#183;mL&#8722;1&#183;10&#8722;6 cells.</li>
	<li>Subtract the non-mitochondrial oxygen consumption from all the values before calculating the indices.</li>
	<li>Calculate the indices using the various equations previously described15: 
	<img alt="Equation 1" src="/files/ftp_upload/65493/65493eq01v3.jpg" style="margin: auto;" /></li>
</ol>

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

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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+mitochondrial+activity+upon+CatSper+channel+activation+is+required+for+mouse+sperm+capacitation.">Increased mitochondrial activity upon CatSper channel activation is required for mouse sperm capacitation.</a> Redox Biology. 48, 102176 (2021).</li><li>Irigoyen, P., 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=Mitochondrial+metabolism+determines+the+functional+status+of+human+sperm+and+correlates+with+semen+parameters.">Mitochondrial metabolism determines the functional status of human sperm and correlates with semen parameters.</a> Frontiers in Cell and Developmental Biology. 10, 926684 (2022).</li></ol>

The authors have nothing to disclose.

HRR critically depends on several steps: (a) the equipment maintenance, (b) accurate calibration of the oxygen sensors, (c) the uncoupler titration26, and finally, (d) the adequate use of indices representing the mitochondrial function. The equipment maintenance is crucial. It is recommended to replace the membranes of the polarographic oxygen sensor regularly and to correct the instrumental background. Extensive washing after the collection of spermatozoa from the chambers is essential to obtain good replicates, especially in laboratories in which the instruments are frequently used to analyze various tissues or cells. Spermatozoa are sensitive to even small amounts of rotenone, AA, oligomycin, and FCCP, so the washing step must be performed carefully (at least three times with ethanol and another three times with distilled water). Calibration is a crucial step; it must be performed every day and for each of the media used (see protocol step 2.2). The uncoupler titrations must be performed carefully, as the optimal uncoupler concentrations must be used to achieve the maximum stimulation of flux and avoid over-titration, which paradoxically inhibits respiration27. The optimal uncoupler concentration depends on the cell type, cell concentration, and medium and is different for permeabilized cells compared to intact cells14. Thus, in the case of sperm samples, in which the sperm cells may be heterogeneous, the uncoupler must be adapted to each sample28,29,30. After respirometry, several parameters are obtained that must be corrected for the number of cells before analysis. Three indices are calculated that allow for the interpretation of mitochondrial function, and these have the advantage of being independent of the number of mitochondria and cells. We highlight the use of respiratory control ratio (RCR), which indicates the efficiency of mitochondrial coupling and is sensitive to multiple potential sites of dysfunction7. However, other parameters and indices may be used depending on the experiment31.
The limitations of HRR are as follows: (a) the device is not automated, so it requires the constant presence of an operator and is time-consuming; (b) the HRR device has only two chambers, and only two assays can be performed simultaneously; (c) the chambers are not single-use and can be contaminated with inhibitors or another type of tissue; (d) although the sensitivity of the instrument is very high, our team found that at least 12 million/mL of sperm are required to detect variations in oxygen consumption, so we could not measure sperm from oligozoospermic males; and (e) the operator must be aware that the measurements are for all the cells present in the samples. Semen samples are heterogeneous and contain other cell types (e.g., leukocytes). To avoid contamination, it is necessary to use samples with low concentrations of leukocytes7. Alternatively, an additional sperm purification step can be performed prior to the oximeter experiments (e.g., swim-up or swim-down)24.
An alternative to HRR is the use of the extracellular flux analyzer. The advantages of the extracellular flux analyzer over the high-resolution oxygraph for measuring sperm metabolism are as follows: (a) the extracellular flux analyzer is a largely automated instrument; (b) it can measure the oxygen consumption in 24-well and 96-well plates for high-throughput screening, meaning it requires smaller amounts of biological samples; and (c) it permits the additional measurement of sperm glycolytic flux. Some advantages of HRR over the extracellular flux analyzer are as follows: (a) with HRR, the cost of the instrument and consumables is lower; (b) with HRR, three to four replicates of each measurement are not required as in the extracellular flux analyzer, for which these replicates are needed to address the high fluorescence variability; (c) the feasibility of the substrate uncoupler inhibitor titration protocols with HRR is high; and (c) the cells are monitored in motion and not attached. Oximetry can analyze the oxygen consumption of motile (live) and intact (non-permeabilized) spermatozoa7,31,32. This is an advantage in the case of spermatozoa since sperm adhesion to the plastic surface may potentially alter their movement patterns and function.
HRR is more sensitive than conventional respirometry and is suitable for medical use in human spermatozoa, for which the number of cells from each semen sample cannot be increased (such as cultured cells). This technique can be used to monitor sperm respiration from most semen samples, both from normal and infertile men. RCR can be used as a good indicator of human sperm functional status, and a cut-off value of 3.15 (sensitivity and specificity of 73% and 61%, respectively) distinguishes between sperm samples with normal and abnormal conventional semen analysis32. HRR could be used to understand sperm mitochondrial metabolism, which may be responsible for male infertility, and be a complement to standard semen analysis.
The latest new generation of Oroboro instruments allows the analysis of oxygen consumption in conjunction with other sperm functions, such as the production of H2O2, and could help to understand sperm function.

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/65493">High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa</a>. The Protocol and Representative Result&#160;sections were&#160;updated.
Step 2.4.12 of the Protocol was updated from:
Finally, inject 1 &#181;L of 5 mM antimycin A (2.5 &#181;M final concentration). This is a complex II inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 &#181;L of 1 mM rotenone (0.5 &#181;M final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes.
to:
Finally, inject 1 &#181;L of 5 mM antimycin A (2.5 &#181;M final concentration). This is a complex III inhibitor to discriminate between the mitochondrial and residual oxygen consumption (non-mitochondrial respiration). For the analysis of complex I, add 1 &#181;L of 1 mM rotenone (0.5 &#181;M final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes.
Figure 3 in the Representative Results section was updated from:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65493/65493fig03.jpg" /> 
Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 &#176;C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means &#177; standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65493/65493fig3v2.jpg" /> 
Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 &#176;C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means &#177; standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/65493">High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa</a>. The Protocol and Representative Result&#160;sections were&#160;updated.

Erratum: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Male infertility is estimated to account for 40%-50% of all cases of infertility in couples1. Conventional semen analysis plays a crucial part in determining male fertility; however, approximately 15% of infertile men have normal sperm parameters2. In addition, routine semen analysis provides limited information about sperm function and does not reflect subtle sperm defects3.
Sperm mitochondria have a special structure, as they are arranged as a helical sheath around the flagella. The mitochondrial sheath contains a variable number of mitochondria connected by intermitochondrial linkers and anchored to the cytoskeleton by ordered protein arrangements on the outer mitochondrial membrane4,5. This structure makes it particularly difficult to isolate sperm mitochondria. Therefore, most studies of sperm mitochondrial function use in situ analyses or demembranated sperm6.
Sperm mitochondrial structure and function have been consistently linked to male infertility7,8,9,10,11, suggesting that analysis of the structure and function of these organelles may be a good candidate for inclusion in sperm analysis.
Mitochondria play an important role in cellular energy metabolism, particularly by using oxygen to produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). In spermatozoa, in particular, the source of ATP (glycolysis vs. OXPHOS) is disputed, and much of the data remains controversial and depends on different experimental approaches4,12,13. Measurements of respiration by oximetry offer significant insights into the mitochondrial respiratory capacity, mitochondrial integrity, and energy metabolism of the cell14,15,16. Traditionally, this technique has been performed using the Clark oxygen electrode-an instrument that has been used to measure mitochondrial respiration for more than 50 years17,18. In addition, sperm mitochondrial oxygen consumption has been analyzed using the classic Clark oxygen electrode19,20,21. High-resolution respirometry (HRR) using oxygraphs (Oroboros) provides higher sensitivity than using classical respirometry devices22. The oxygraphs are composed of two chambers with injection ports, and each chamber has a polarographic oxygen sensor. With this technique, it is possible to analyze tissue slides, cells, and isolated mitochondrial suspensions. The specimen is continuously stirred in the chamber, and during the experiment, the oxygen consumption is measured, and the oxygen rates are calculated using specific software. The chambers show reduced oxygen leakage, which is an advantage over the conventional oxygen electrode devices14,23.
As with other cells, in the case of spermatozoa, the sensitivity of HRR equipment is higher than for conventional respirometry, meaning that HRR equipment can be used for the analysis of a limited number of intact or permeabilized sperm cells. There are two main strategies for assessing sperm mitochondrial function by HRR: (a) measuring the oxygen consumption in intact cells, which involves reproducing the respiratory function in a medium containing substrates such as glucose, or (b) measuring the oxygen consumption in permeabilized cells using one of the OXPHOS complexes, with the addition of specific substrates to monitor each function separately.
In the present study, we describe the use of HRR to determine mitochondrial respiration in human sperm cells.

<table><tbody><tr><td>Acid free- Bovine serum albumine</td><td>Sigma Aldrich</td><td>A8806</td><td></td></tr><tr><td>Adenosine 5&#039;-diphosphate monopotassium salt dihydrate</td><td>Sigma Aldrich</td><td>A5285</td><td></td></tr><tr><td>Animycin A from streptomyces sp.</td><td>Sigma Aldrich</td><td>A8674</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C4901</td><td></td></tr><tr><td>carbonyl cyanide-P- trifluoromethoxy-phenylhydrazone</td><td>Sigma Aldrich</td><td>C2920</td><td></td></tr><tr><td>DatLab sofware version 4,2</td><td>Oroboros Instruments GmbH</td><td>N/A</td><td></td></tr><tr><td>D-glucose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Digitonin</td><td>Sigma Aldrich</td><td>D141</td><td></td></tr><tr><td>EGTA</td><td>Sigma Aldrich</td><td>E4378</td><td></td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td></td></tr><tr><td>L glutamic acid</td><td>Sigma Aldrich</td><td>G1251</td><td></td></tr><tr><td>L malic acid</td><td>Sigma Aldrich</td><td>M1000</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sigma Aldrich</td><td>M7506</td><td></td></tr><tr><td>Microliter Syringes</td><td>Hamilton</td><td>87900 or 80400</td><td></td></tr><tr><td>Microscope camera</td><td>Basler</td><td>acA780-75gc</td><td></td></tr><tr><td>Microscope Eclipse E200 with phase contrast 10X Ph+</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>MOPS</td><td>Sigma Aldrich</td><td>M1254</td><td></td></tr><tr><td>Oligomycin A</td><td>Sigma Aldrich</td><td>75351</td><td></td></tr><tr><td>Oxygraph-2 K</td><td>Oroboros Instruments GmbH</td><td>N/A</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P3911</td><td></td></tr><tr><td>Power O2k-Respirometer</td><td>Oroboros Intruments</td><td>10033-01</td><td></td></tr><tr><td>Rotenone</td><td>Sigma Aldrich</td><td>R8875</td><td></td></tr><tr><td>Saccharose</td><td>Sigma Aldrich</td><td>S0389</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Sodium lactate</td><td>Sigma Aldrich</td><td>L7022</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma Aldrich</td><td>P2256</td><td></td></tr><tr><td>Sperm class analyzer 6.3.0.59 Evolution-SCA Research</td><td>Microptic</td><td>N/A</td><td></td></tr><tr><td>Sperm Counting Chamber DRM-600</td><td>Millennium Sciences CELL-VU</td><td>N/A</td><td></td></tr><tr><td>Succinate disodium salt</td><td>Sigma Aldrich</td><td>W327700</td><td></td></tr></tbody></table>

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.

Determination of the optimal concentration of digitonin in sperm cells 
In this protocol, we present the use of HRR to monitor real-time changes in OXPHOS in human sperm cells. Since the method can be used to analyze intact or digitonin-permeabilized sperm, we first present the standardization of digitonin concentration required to permeabilize sperm cells (Figure 3).
Digitonin is used for chemical permeabilization, which allows substrates to enter cells, and for measuring mitochondrial activity. This compound must be titrated in intact cells to obtain the optimal concentration required to ensure cell membrane permeabilization without compromising mitochondrial membrane integrity. We performed a titration in the presence of ADP, glutamate, and malate. Respiration rates were measured at baseline and after the gradual addition of digitonin (Figure 3A). Oxygen consumption rises with increasing detergent concentration and reaches a point where the integrity of the outer mitochondrial membrane starts to be compromised (red arrow in Figure 3A). Figure 3B shows the mean &#177; standard error of 4 representative experiments. The result shows that permeabilization is optimal at a digitonin concentration of 22.5 &#181;M (Figure 3A,B). Mitochondrial activity decreases when too high amounts of digitonin are used.
Representation of successful and suboptimal register of sperm respiration by HRR 
During monitoring sperm mitochondrial respiration, the register can be visualized as O2 concentration (blue line) and O2 flow per volume correlated (red line) calculated with the DatLab 4 analysis software in both permeabilized and non-permeabilized cells (Figure 4). Figure 4 shows representative curves of intact (Figure 4A,B) and digitonin-permeabilized (Figure 4C,D) sperm cells from a semen sample. Figure 4A represents a successful recording of intact sperm cells from a semen sample, where the effects of drugs modulating oxygen consumption are observed, and it is possible to extract the parameters for calculating the indices. Basal respiration is recorded in intact cells by oxygen consumption in the presence of exogenous substrates. Then, the non-phosphorylating respiration rate was measured by adding an ATP synthase inhibitor (oligomycin). Subsequently, the protonophore FCCP was injected at different concentrations and the maximal mitochondrial uncoupled respiration rate was determined. This drug leads to an increase in proton permeability of the inner membrane, which allows the passive movement of protons to dissipate the chemiosmotic gradient that causes an increase in oxygen consumption. Finally, AA was added to inhibit mitochondrial respiration.
The quality of the data obtained is closely related to the cell number. It is important to have a high basal oxygen consumption to obtain a good record. The subsequent analysis depends on subtle changes in O2 consumption that may not be detected if the basal line is not high enough. Cell counts lower than recommended could lead to a result like Figure 4B.
Representative curves obtained using the protocol for complex I or II specific substrates are shown in Figure 4C and Figure 4D, respectively. To study oxygen consumption through the mitochondrial complex I or complex II, the substrates malate and glutamate or succinate were added after permeabilization of the cells. Then, ADP is added for conversion to ATP (state 3, active complex I or II dependent respiration). Finally, rotenone or AA is administered to inhibit complex I or II, respectively.
For interpretation of the results, it is important to consider that cellular respiration inhibited by oligomycin is a direct measurement of proton leakage rate across the mitochondrial membrane in intact cells, which is comparable to the state 4 of permeabilized cells (without ADP) (Figure 2B). In the case of intact cells (non-permeabilized), this amount indicates the fraction of basal mitochondrial oxygen consumption that is independent of ATP synthesis. The maximum respiration rate is reached after the addition of several doses of FCCP. In intact cells depends on two factors; the activity of the electron transport complexes and the number of mitochondria in each cell. State 3 of permeabilized cells after the addition of ADP resembles basal respiration of the intact cell at saturating concentrations of exogenous substrates (Figure 2B)7. Table 2 shows an example of the indices calculated from the records in Figure 4.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65493/65493fig3v2.jpg" /> 
Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 &#176;C in MRM medium with glutamate, malate, and adenosine diphosphate. (A) Representative respiratory trace. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. (B) Mitochondria respiration rate means &#177; standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig3v2large.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/65493/65493fig04.jpg" /> 
Figure 4: Representative respiratory traces for the oxygen consumption rate obtained using the Oroboros instrument. The respiration rates were measured at 37 &#176;C in (A,B) intact and (C,D) permeabilized cells. The blue line is the O2 concentration, and the red line represents the O2 flow per volume correlated. The rates in A, C, and D were measured with more than 30 x 106 sperm cells, while the rate in B was measured with 14 x 106 sperm cells. Abbreviations: Oligo = oligomycin; FCCP = carbonyl cyanide -p- trifluoromethoxyphenylhydrazone; Glu/Mal = glutamate/malate; ADP = adenosine diphosphate; AA = antimycin A; Rot = rotenone. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/65493fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Composition of BWW and MRM media for experiments with intact or permeabilized cells, respectively. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Representative examples of HRR indices. Data were obtained from the traces shown in Figure 4. The indices were calculated according to Figure 2 and protocol section 3. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65493/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function at JoVE.com

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.

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 ...

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1.5K Views.  Unidad Académica Histología y Embriología. 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.

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

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, recovery from SCI typically involves long term rehabilitation. While recovery of walking ability is a primary goal in many patients early after injury, those with a motor incomplete SCI, indicating partial preservation of volitional control, may have the sufficient residual descending pathways necessary to attain this goal. However, despite physical interventions, motor impairments including weakness, and the manifestation of abnormal involuntary reflex activity, called spasticity or spasms, are thought to contribute to reduced walking recovery. Doctrinaire thought suggests that remediation of this abnormal motor reflexes associated with SCI will produce functional benefits to the patient. For example, physicians and therapists will provide specific pharmacological or physical interventions directed towards reducing spasticity or spasms, although there continues to be little empirical data suggesting that these strategies improve walking ability.
In the past few decades, accumulating data has suggested that specific neuromodulatory agents, including agents which mimic or facilitate the actions of the monoamines, including serotonin (5HT) and norepinephrine (NE), can initiate or augment walking behaviors in animal models of SCI. Interestingly, many of these agents, particularly 5HTergic agonists, can markedly increase spinal excitability, which in turn also increases reflex activity in these animals. Counterintuitive to traditional theories of recovery following human SCI, the empirical evidence from basic science experiments suggest that this reflex hyper excitability and generation of locomotor behaviors are driven in parallel by neuromodulatory inputs (5HT) and may be necessary for functional recovery following SCI. 
The application of this novel concept derived from basic scientific studies to promote recovery following human SCI would appear to be seamless, although the direct translation of the findings can be extremely challenging. Specifically, in the animal models, an implanted catheter facilitates delivery of very specific 5HT agonist compounds directly onto the spinal circuitry. The translation of this technique to humans is hindered by the lack of specific surgical techniques or available pharmacological agents directed towards 5HT receptor subtypes that are safe and effective for human clinical trials. However, oral administration of commonly available 5HTergic agents, such as selective serotonin reuptake inhibitors (SSRIs), may be a viable option to increase central 5HT concentrations in order to facilitate walking recovery in humans. Systematic quantification of how these SSRIs modulate human motor behaviors following SCI, with a specific focus on strength, reflexes, and the recovery of walking ability, are missing.
This video demonstration is a progressive attempt to systematically and quantitatively assess the modulation of reflex activity, volitional strength and ambulation following the acute oral administration of an SSRI in human SCI. Agents are applied on single days to assess the immediate effects on motor function in this patient population, with long-term studies involving repeated drug administration combined with intensive physical interventions.

This video demonstrates modulation of reflex activity, volitional strength and ambulation through clinical and quantitative assessments in individuals with motor incomplete SCI as a result of acute oral administration of a serotonin reuptake inhibitor (SSRI).

Spinal cord injury (SCI) is a debilitating disorder, which produces profound deficits in volitional motor control. Following medical stabilization, ...

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

Biochemistry

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Biology

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential centrifugation at low- and high-speed is commonly used to isolate mitochondria from tissues and cultured cells. Crude mitochondrial fractions obtained by differential centrifugation are used for respirometry measurements. The differential centrifugation technique is based on the separation of organelles according to their size and sedimentation velocity. The isolation of mitochondria is performed immediately after tissue harvesting. The tissue is immersed in an ice-cold homogenization medium, minced using scissors and homogenized in a glass homogenizer with a loose-fitting pestle. The differential centrifugation technique is efficient, fast and inexpensive and the mitochondria obtained by differential centrifugation are pure enough for respirometry assays. Some of the limitations and disadvantages of isolated mitochondria, based on differential centrifugation, are that the mitochondria can be damaged during the homogenization and isolation procedure and that large amounts of the tissue biopsy or cultured cells are required for the mitochondrial isolation.

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Mitochondria are involved in cellular energy metabolism and use oxygen to produce energy in the form of adenosine triphosphate (ATP). Differential ...

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring mitochondrial function is fundamental in biomedical research. Skeletal muscle is a robust source of mitochondria, particularly in animals with a very high aerobic capacity, such as horses, making them ideal subjects for studying mitochondrial physiology. This article demonstrates the use of high-resolution respirometry with concurrent fluorometry, with freshly harvested skeletal muscle mitochondria, to quantify the capacity to oxidize substrates under different mitochondrial states and determine the relative capacities of distinct elements of mitochondrial respiration. Tetramethylrhodamine methylester is used to demonstrate the production of mitochondrial membrane potential resulting from substrate oxidation, including calculation of the relative efficiency of the mitochondria by calculating the relative membrane potential generated per unit of concurrent oxygen flux. The conversion of ADP to ATP results in a change in the concentration of magnesium in the reaction chamber, due to differing affinities of the adenylates for magnesium. Therefore, magnesium green can be used to measure the rate of ATP synthesis, allowing the further calculation of the oxidative phosphorylation efficiency (ratio of phosphorylation to oxidation [P/O]). Finally, the use of Amplex UltraRed, which produces a fluorescent product (resorufin) when combined with hydrogen peroxide, allows the quantification of reactive oxygen species production during mitochondrial respiration, as well as the relationship between ROS production and concurrent respiration. These techniques allow the robust quantification of mitochondrial physiology under a variety of different simulated conditions, thus shedding light on the contribution of this critical cellular component to both health and disease.

Horses have an exceptional aerobic exercise capacity, making equine skeletal muscle an important tissue for both the study of equine exercise physiology as well as mammalian mitochondrial physiology. This article describes techniques for the comprehensive assessment of mitochondrial function in equine skeletal muscle.

Mitochondrial function-oxidative phosphorylation and the generation of reactive oxygen species-is critical in both health and disease. Thus, measuring ...

High-Resolution Analysis of Mitochondrial Function in Human PBMCs

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes&#160;do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.

Author Spotlight: New Insights into PBMC Mitochondrial Responses Using Fluorespirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these ...

Real-Time Quantification of Mitochondrial Respiration in Muscle Fibers

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle is linked to prevalent metabolic disorders (e.g., diabetes and obesity), muscular dystrophies, and sarcopenia. While there are many techniques to evaluate mitochondrial content and morphology, the hallmark method to assess mitochondrial function is the measurement of mitochondrial oxidative phosphorylation (OXPHOS) by respirometry. Quantification of mitochondrial OXPHOS provides insight into the efficiency of mitochondrial oxidative energy production and cellular bioenergetics. A high-resolution respirometer provides highly sensitive, robust measurements of mitochondrial OXPHOS in permeabilized muscle fibers by measuring real-time changes in mitochondrial oxygen consumption rate. The use of permeabilized muscle fibers, as opposed to isolated mitochondria, preserves mitochondrial networks, maintains mitochondrial membrane integrity, and ultimately allows for more physiologically relevant measurements. This system also allows for the measurement of fuel preference and metabolic flexibility - dynamic aspects of muscle energy metabolism. Here, we provide a comprehensive guide for mitochondrial OXPHOS measurements in human and mouse skeletal muscle fibers using a high-resolution respirometer. Skeletal muscle groups are composed of different fiber types that vary in their mitochondrial fuel preference and bioenergetics. Using a high-resolution respirometer, we describe methods for evaluating both aerobic glycolytic and fatty acid substrates to assess fuel preference and metabolic flexibility in a fiber-type-dependent manner. The protocol is versatile and applicable to both human and rodent muscle fibers. The goal is to enhance the reproducibility and accuracy of mitochondrial function assessments, which will improve our understanding of an organelle important to muscle health.

Author Spotlight: Unveiling Mitochondrial Function and Cellular Metabolic Adaptation in Metabolic Diseases

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Mitochondrial function, a cornerstone of cellular energy production, is critical for maintaining metabolic homeostasis. Its dysfunction in skeletal muscle ...

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

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Chapters in this video

Methods to Quantify Pharmacologically Induced Alterations in Motor Function in Human Incomplete SCI

In Vivo Canine Muscle Function Assay

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-Resolution Fluoro-Respirometry of Equine Skeletal Muscle

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry