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

Summary

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

Abstract

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

Introduction

The application of mass spectrometry has revolutionized the study of metabolism. Targeted metabolic profiling and metabolomic tracing allow precise monitoring of changes in energy metabolism. However, performing metabolomics successfully requires extensive training, experienced staff, and expensive, highly sensitive mass spectrometers not readily available to every laboratory. In recent years, using an extracellular flux analyzer, such as the Seahorse XFe96 has grown popular as a surrogate method for measuring changes in energy metabolism in various cell types^1,2,3,

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

1. 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 ....

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 .......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.