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Abstract

Medicine

高分辨率呼吸测定法评估人类精子的线粒体功能

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

精液质量通常通过常规精液分析进行研究,常规精液分析是描述性的,通常没有定论。男性不育症与精子线粒体活性的改变有关,因此精子线粒体功能的测量是精子质量的指标。高分辨率呼吸测量法是一种测量密闭腔系统中细胞或组织耗氧量的方法。该技术可用于测量人类精子的呼吸,并提供有关精子线粒体质量和完整性的信息。高分辨率呼吸测量法允许细胞自由移动,这在精子的情况下是一个 先验 的优势。该技术可以应用于完整或透化的精子,并允许研究完整的精子线粒体功能和单个呼吸链复合物的活性。高分辨率氧谱仪使用传感器测量氧气浓度,并结合灵敏的软件来计算氧气消耗量。这些数据用于根据耗氧率计算呼吸指数。因此,这些指数是两种耗氧率的比例,并在内部归一化为细胞数量或蛋白质质量。呼吸指数是精子线粒体功能和功能障碍的指标。

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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