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Abstract

Medicine

ヒト精子のミトコンドリア機能を評価するための高分解能肺活法

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 …

Abstract

精液の質は、ルーチンの精液分析によって研究されることが多いが、これは記述的であり、しばしば決定的ではない。男性不妊症は精子のミトコンドリア活動の変化と関連しているため、精子のミトコンドリア機能の測定は精子の質の指標です。高分解能肺活量測定は、密閉チャンバーシステム内の細胞または組織の酸素消費量を測定する方法です。この技術は、ヒト精子の呼吸を測定するために実装することができ、精子ミトコンドリアの品質と完全性に関する情報を提供します。高解像度の肺活法により、細胞は自由に動くことができ、これは精子の場合の 先験的な 利点です。この技術は、無傷または透過処理された精子に適用でき、無傷の精子のミトコンドリア機能および個々の呼吸鎖複合体の活性の研究を可能にします。高解像度の酸素写真機器は、センサーを使用して酸素濃度を測定し、高感度のソフトウェアを組み合わせて酸素消費量を計算します。このデータは、酸素消費比に基づいて呼吸指数を計算するために使用されます。したがって、指数は2つの酸素消費率の比率であり、細胞数またはタンパク質量に内部的に正規化されます。呼吸指数は、精子のミトコンドリア機能と機能障害の指標です。

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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