High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Irigoyen, Pilar; Sapiro, Rossana; Cassina, Adriana

doi:10.3791/65493

Cited by 1 publication

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“…Oxygen consumption rate (OCR) of 24 × 10 6 sperm NC and CAP was measured by HRR (Oxygraph‐2K; Oroboros Instruments GmbH, Innsbruck, Austria) following the protocol described in Irigoyen et al 36 . Briefly, the two protocols used are described below.…”

Section: Methodsmentioning

confidence: 99%

“…The mitochondrial function of sperm cells was measured in BWW media as previously described 6,36 . Briefly, basal respiration was measured for 10 min before adding 2 μg/mL oligomycin (ATP synthetase inhibitor).…”

Section: Methodsmentioning

confidence: 99%

“…The non‐mitochondrial oxygen consumption was subtracted from all other values before calculating parameters. We compared the different slopes (values were adjusted per million cells), calculated ATP turnover (basal respiration‐proton leak), and the indices as the ratio between different parameters as follows: respiratory control ratio (RCR) = maximal/proton leak, spare respiratory capacity = maximum respiratory rate/basal respiration and coupling efficiency = (basal respiration − proton leak)/basal respiration 24,36,37 …”

Section: Methodsmentioning

confidence: 99%

“…To investigate the role of OXPHOS in human sperm capacitation, we compared the OCR of NC and CAP sperm cells from 19 samples using HRR. Various parameters and three indices representing mitochondrial function, described by Brand et al and Irigoyen et al, 36,37 were obtained (Table S2). The indices are ratios of oxygen consumption and are therefore internally normalized to cell number.…”

Section: Mitochondrial Function Increases In Human Sperm Capacitationmentioning

confidence: 99%

“…The mitochondrial function of sperm cells was measured in BWW media as previously described. 6,36 Briefly, basal respiration was measured for 10 min before adding 2 μg/ mL oligomycin (ATP synthetase inhibitor). Maximum respiration was obtained by subsequent stepwise addition of the uncoupling agent 0.1-1 μM carbonyl cyanide-P-trifluoromethoxy-phenylhydrazone (FCCP).…”

Section: Intact Cellsmentioning

confidence: 99%

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Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players

Irigoyen,

Mansilla,

Castro

et al. 2024

The FASEB Journal

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Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+) increases during capacitation. Herein, we studied mitochondrial function by high‐resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non‐capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2O2) detection was three times higher in CAP than in NC sperm cells. To confirm that H2O2 production depends on mitochondrial superoxide () formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more in the steady‐state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4‐hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2O2 production does not damage sperm and it is necessary for the normal CAP process.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Mitochondrial Function Increases In Human Sperm Capacitationmentioning

confidence: 99%

Section: Intact Cellsmentioning

confidence: 99%

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