High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Summary

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.

Abstract

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.

Introduction

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 intermito....

Protocol

The experiments were approved by the Ethics Committee of the Facultad de Medicina de la Universidad de la República, Montevideo, Uruguay.

Figure 1: Workflow for high-resolution respirometry to assess mitochondrial function in intact and permeabilized human sperm. The protocol was divided into four different step.......

Discussion

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 .......

Materials

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