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

Melanie Balbach; Jochen Buck; Lonny  R. Levin

doi:10.3791/60815

JoVE Journal > Biochemistry

Bioquímica

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

Published: January 22, 2020

doi:

10.3791/60815

Melanie Balbach¹, Jochen Buck¹, Lonny R. Levin¹

¹Department of Pharmacology,Weill Cornell Medical College

Summary

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

Abstract

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H⁺– and O₂– sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

Introduction

The application of mass spectrometry has revolutionized the study of metabolism. Targeted metabolic profiling and metabolomic tracing allow precise monitoring of changes in energy metabolism. However, performing metabolomics successfully requires extensive training, experienced staff, and expensive, highly sensitive mass spectrometers not readily available to every laboratory. In recent years, using an extracellular flux analyzer, such as the Seahorse XFe96 has grown popular as a surrogate method for measuring changes in energy metabolism in various cell types¹^,²^,³^,⁴^,⁵.

Sperm are highly specialized motile cells; whose task is to deliver the paternal genome to the oocyte. Sperm leaving the male reproductive tract after ejaculation are still functionally immature and cannot fertilize the oocyte because they are unable to penetrate the oocytes' vestments. Sperm acquire fertilization competence as they transit through the female reproductive tract in a maturation process known as capacitation⁶^,⁷. Freshly ejaculated sperm or sperm dissected from the cauda epididymis can be capacitated in vitro by incubation in defined capacitation media containing Ca²⁺, bicarbonate (HCO₃^–) or a cell-permeable cAMP analog (e.g., dibutyryl-cAMP), a cholesterol acceptor (e.g., bovine serum albumin, BSA), and an energy source (e.g., glucose). During capacitation, sperm modify their motility pattern into an asymmetric flagellar beat, representing a swimming mode called hyperactivation⁸^,⁹, and they become competent to undergo the acrosome reaction⁷, where proteolytic enzymes are released that digest the oocytes' vestments. These processes require energy, and similar to somatic cells, sperm generate ATP and other high energy compounds via glycolysis as well as mitochondrial TCA cycle and oxidative phosphorylation (oxphos)¹⁰. While multiple studies demonstrate that glycolysis is necessary and sufficient to support sperm capacitation¹¹^,¹²^,¹³^,¹⁴, the contribution of oxphos is less clear. Contrary to other cell types where glycolysis is physically coupled to the TCA cycle, sperm are highly compartmentalized and are thought to maintain these processes in separate flagellar compartments: the midpiece concentrates the mitochondrial machinery, whereas the key enzymes of glycolysis appear to be restricted to the principal piece¹⁵^,¹⁶. This compartmentalization results in an ongoing debate about whether pyruvate produced in the principal piece by glycolysis can support mitochondrial oxphos in the midpiece, and whether ATP produced by oxphos in the midpiece would be able to diffuse sufficiently rapidly along the length of the flagellum to support the energy requirements in distal parts of the principal piece¹⁷^,¹⁸^,¹⁹. There is also support of a role for oxphos in sperm capacitation. Not only is oxphos more energetically favorable than glycolysis, generating 16 times more ATP than glycolysis, but midpiece volume and mitochondrial content are directly correlated with reproductive fitness in mammalian species which exhibit greater degrees of competition between males for mates²⁰. Addressing these questions requires methods for examining the relative contributions of glycolysis and oxphos during sperm capacitation.

Tourmente et al. applied a 24-well extracellular flux analyzer to compare the energy metabolism of closely related mouse species with significantly different sperm performance parameters²¹. Instead of reporting the basal ECAR and OCR values of non-capacitated sperm, here, we adapt their method using a 96-well extracellular flux analyzer to monitor changes in energy metabolism during mouse sperm capacitation in real-time. We developed a method that allows simultaneously monitoring glycolysis and oxphos in real-time in sperm with beating flagella in up to twelve different experimental conditions by measuring the flux of oxygen (O₂) and protons (H⁺) (Figure 1A). Due to the breakdown of pyruvate to lactate during glycolysis and the production of CO₂ via the TCA-cycle, non-capacitated and capacitated sperm extrude H⁺ into the assay media which are detected by the extracellular flux analyzer via H⁺-sensitive fluorophores immobilized to the probe tip of a sensor cartridge. In parallel, O₂ consumption by oxidative phosphorylation is detected via O₂-sensitive fluorophores immobilized to the same probe tip (Figure 1B). Effective detection of the released H⁺ and consumed O₂ requires a modified sperm buffer with low buffering capacity without bicarbonate or phenol red. Thus, to induce capacitation in the absence of bicarbonate, we adopted the use of a cell-permeable cAMP analog injected together with the broad-range PDE inhibitor IBMX²². Three additional independent injection ports allow the injection of pharmacological activators and/or inhibitors, which facilitates real-time detection of changes in cellular respiration and glycolysis rate due to experimental manipulation.

Protocol

Sperm are collected from 8-16-week-old CD-1 male mice. Animal experiments were approved by Weill Cornell Medicine's Institutional Animal Care and Use Committee (IACUC).

1. Day prior to assay

Preparation of sensor cartridge and extracellular flux analyzer calibrant
1. 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.
2. Fill a solution reservoir with 25 mL of double-distilled H₂O using a multichannel pipette, add 200 µL of H₂O to each well of the utility plate. Place the sensor cartridge back into the utility plate and incubate overnight in a 37 °C non-CO₂ incubator.
  NOTE: Cartridge needs to be hydrated for at least 4 h.
3. Aliquot 25 mL of the extracellular flux analyzer calibrant into a 50 mL conical tube and incubate it overnight in a 37 °C non-CO₂ incubator.
Preparation of ConA-coated microplates
1. Dissolve 2.5 mg of ConA in 5 mL of double-distilled H₂O to prepare a 0.5 mg/mL (w/v) stock.
2. Using a multichannel pipette, fill each well of an extracellular flux analyzer 96-well plate with 50 µL 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 °C for up to four weeks until ready for use.
Preparation of sperm buffer
1. 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 °C and adjust the pH to 7.4.

2. Day of the assay

Preparation for cartridge calibration
1. Replace the H₂O in the utility plate with 200 µL of XF calibrant per well and incubate for at least 1 h in a non-CO₂ 37 °C incubator.
  NOTE: Instead of calibrant, sterile-filtered PBS, pH 7.4 can be used.
2. Heat 50 mL of TYH buffer at 37 °C.
Generating a wave template for the extracellular flux analyzer
1. Turn the extracellular flux analyzer on and allow the temperature to stabilize to 37 °C.
2. Open the wave software and design a new template by opening a blank template (see Supplemental File 1, wave template).
3. 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.
4. 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.
5. 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.
6. Save the assay template, fill out the project summary and define the saving location for the results file.
Preparation of compounds to load into sensor cartridge
1. 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 °C heat block for 5 min.
2. Prepare 1 mL of 500 mM 2-DG by dissolving 82.1 mg of compound in TYH buffer.
3. Prepare 1 mL of 5 µM 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.
Isolation of mouse sperm
1. 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.
2. Place each cauda in 500 µL 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.
3. Transfer the plate immediately into a non-CO₂ 37 °C incubator and let the sperm disperse for 15 min.
Loading of the sensor cartridge
1. 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.
2. Port A: Using a multichannel pipette, inject 20 µL of the TYH buffer into every column.
3. Port B: Inject 22 µL of TYH buffer into column 1-4, inject 22 µL of 500 mM 2-DG into column 5-8, and 25 µL of 5 µM AntA/Rot into column 9-12.
4. Port C: Inject 25 µL of TYH buffer into every column with odd numbers, inject 25 µL of 10 mM db-cAMP/ 500 µM IBMX into every column with even numbers.
5. Place the sensor cartridge with the calibration plate into the extracellular flux analyzer and start the assay. Calibration takes 10 – 15 min.
Preparation of sperm plates
1. 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.
2. 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.
3. 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.
4. Place 180 µL of TYH buffer into each corner well (A1, A12, H1, H12) of a ConA-coated plate. Place 180 µL of sperm suspension into each empty well of the ConA-coated plate (1.2 x 10⁶ sperm/well).
5. Centrifuge the sperm plate at 250 x g for 1 min, rotate the plate by 180°, and centrifuge again at 250 x g for 1 min (lowest braking rate 1).
6. Remove the calibration plate from the extracellular flux analyzer and add the sperm plate. Continue the assay.
Data extraction and analysis
1. 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).
2. Duplicate the ECAR and OCR file by clicking on the novo tab and then the Duplicate Family… tab (Figure 3A).
3. Delete the first 7 rows of data (Figure 3B).
4. 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).

Representative Results

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 ± 2.75 mpH/min and 23.64 ± 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.

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. Please click here to view a larger version of this figure.

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 µM, db-cAMP: final concentration 1 mM, IBMX: final concentration 500 µM. Please click here to view a larger version of this figure.

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 ± S.E.M. Injections are indicated with an arrow. Please click here to view a larger version of this figure.

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 µM antimycin A and rotenone. (D) Normalized OCR in non-capacitated and capacitating mouse sperm in the presence and absence of 0.5 µM antimycin A and rotenone. Data is shown as mean ± S.E.M normalized to the data point before db-cAMP/IBMX injection; n = 6. Injections are indicated with an arrow. Please click here to view a larger version of this figure.

	Port	Number of cycles	Mix (min)	Wait (min)	Measure (min)
A: Basal ECAR/OCR	no port	1	2:00	0:00	3:00
B: Mock injection	1	4	2:00	0:00	3:00
C: Drug injection	2	3	2:00	0:00	3:00
D: Capacitation	3	18	2:00	0:00	3:00

Table 1: Measurement Details.

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 µM IBMX, and 1 mM HEPES, detected with an α-phosphotyrosine antibody. Please click here to view a larger version of this figure.

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 (www.agilent.com/en/products/cell-analysis/cell-analysis-software/data-analysis/wave-desktop-2-6) 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. Please click here to view this file (Right click to download).

Supplemental File 2: Graph pad prism file exported from wave software with exemplary data analysis. Please click here to view this file (Right click to download).

Discussion

The loss of sperm capacitation in the absence of certain metabolic substrates or critical metabolic enzymes revealed energy metabolism as a key factor supporting successful fertilization. A metabolic switch during cell activation is a well-established concept in other cell types, however, we are just beginning to understand how sperm adapt their metabolism to the increasing energy demand during capacitation. Using an extracellular flux analyzer, we developed an easily applicable tool to monitor changes in glycolysis and 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'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'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.

Declarações

The authors have nothing to disclose.

Acknowledgements

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

Materials

Reagents
2-Deoxy-D-glucose	Sigma-Aldrich	D8375	2-DG
3-Isobutyl-1-methylxanthine	Sigma-Aldrich	I7018	IBMX; prepare a 500 mM stock solution in DMSO (111.1 mg/ml) and store in small aliquots
Antimycin A	Sigma-Aldrich	A8674	AntA; prepare a 5 mM stock solution in DMSO (2.7 mg/ml) and store in small aliquots
Bovine serum albumin	Sigma-Aldrich	A1470	BSA
Calcium chloride	Sigma-Aldrich	C1016	CaCl2
Concanacalin A, Lectin from Arachis hypogaea (peanut)	Sigma-Aldrich	L7381	ConA
Glucose	Sigma-Aldrich	G7528
Hepes	Sigma-Aldrich	H0887
Isothesia	Henry Schein Animal Health	1169567761	Isoflurane
Magnesium sulfate	Sigma-Aldrich	M2643	MgSO4
N6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate sodium salt	Sigma-Aldrich	D0627	db-cAMP
Potassium chloride	Sigma-Aldrich	P9333	KCl
Potassium dihydrogen phosphate	Sigma-Aldrich	P5655	KH2PO4
Rotenone	Cayman Chemical Company	13995	Rot; prepare a 5 mM stock solution in DMSO (2mg/ml) and store in small aliquots
Sodium bicarbonate	Sigma-Aldrich	S5761	NaHCO3-
Sodium chloride	Sigma-Aldrich	S9888	NaCl
Equipment and materials
12 channel pipette 10-100 μL	eppendorf	ES-12-100
12 channel pipette 50-300 μL	vwr	613-5257
37 °C, non-CO₂ incubator	vwr	1545
5 mL cetrifuge tubes	eppendorf	30119380
50 mL conical centrifuge tubes	vwr	76211-286
Centrifuge with plate adapter	Thermo Scientific	IEC FL40R
Dissection kit	World Precision Instruments	MOUSEKIT
Inverted phase contrast microscope with 40X objective	Nikon
OctaPool Solution Reservoirs, 25 ml, divided	Thomas Scientific	1159X93
OctaPool Solution Reservoirs, 25 mL, divided	Thomas Scientific	1159X95
Seahorse XFe96 Analyzer	Agilent
Seahorse XFe96 FluxPak	Agilent	102416-100	Also sold as XFe96 FluxPak mini (102601-100) with 6 instead of 18 cartidges.

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Balbach, M., Buck, J., Levin, L. R. Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation. J. Vis. Exp. (155), e60815, doi:10.3791/60815 (2020).