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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Published: June 23rd, 2023

DOI:

10.3791/65493

1Departamento de Histología y Embriología, Unidad Académica Histología y Embriología, 2Departamento de Bioquímica, Unidad Académica Bioquímica, 3Centro de Investigaciones Biomédicas (CEINBIO), Facultad de Medicina, Universidad de la República

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

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

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....

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

Figure 1
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.......

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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.......

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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 .......

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We would like to thank the Fertilab Andrology clinic, especially José María Montes and Andrea Torrents, for allowing us access to donors. Funding: A.C. is supported by grants from Universidad de la República (CSIC_2018, Espacio Interdisciplinario_2021). Additional funding was obtained from the Programa de Desarrollo de Ciencias Básicas (PEDECIBA, Uruguay). P.I. and R.S. are supported by Universidad de la República (I+D, CSIC 2014; I+D, CSIC 2016, Iniciación a la Investigación, 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....

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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

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Erratum

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

An erratum was issued for: High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa. The Protocol and Representative Result sections were updated.

Step 2.4.12 of the Protocol was updated from:

Finally, inject 1 µL of 5 mM antimycin A (2.5 µM 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 µL of 1 mM rotenone (0.5 µM final concentration), an inhibitor of this complex, instead of AA. Measure the oxygen consumption until the signal decreases and stabilizes.

to:

Finally, inject 1 µL of 5 mM antimycin A (2.5 µM 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 µL of 1 mM rotenone (0.5 µM 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:

Figure 3
Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °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 ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure.

to:

Figure 3
Figure 3: Determination of the optimal concentration of digitonin for the permeabilization of human sperm cells. The respiration rates were measured at 37 °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 ± standard error, n = 4. The red arrow represents the optimal concentration. Abbreviation: dig = digitonin. Please click here to view a larger version of this figure.

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