preparation of human spermatozoa for high-resolution respirometry

Determinación de la respiración en espermatozoides humanos mediante respirometría de alta resolución

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 intermitochondrial linkers and anchored to the cytoskeleton by ordered protein arrangements on the outer mitochondrial membrane4,5. This structure makes it particularly difficult to isolate sperm mitochondria. Therefore, most studies of sperm mitochondrial function use in situ analyses or demembranated sperm6.
Sperm mitochondrial structure and function have been consistently linked to male infertility7,8,9,10,11, suggesting that analysis of the structure and function of these organelles may be a good candidate for inclusion in sperm analysis.
Mitochondria play an important role in cellular energy metabolism, particularly by using oxygen to produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). In spermatozoa, in particular, the source of ATP (glycolysis vs. OXPHOS) is disputed, and much of the data remains controversial and depends on different experimental approaches4,12,13. Measurements of respiration by oximetry offer significant insights into the mitochondrial respiratory capacity, mitochondrial integrity, and energy metabolism of the cell14,15,16. Traditionally, this technique has been performed using the Clark oxygen electrode-an instrument that has been used to measure mitochondrial respiration for more than 50 years17,18. In addition, sperm mitochondrial oxygen consumption has been analyzed using the classic Clark oxygen electrode19,20,21. High-resolution respirometry (HRR) using oxygraphs (Oroboros) provides higher sensitivity than using classical respirometry devices22. The oxygraphs are composed of two chambers with injection ports, and each chamber has a polarographic oxygen sensor. With this technique, it is possible to analyze tissue slides, cells, and isolated mitochondrial suspensions. The specimen is continuously stirred in the chamber, and during the experiment, the oxygen consumption is measured, and the oxygen rates are calculated using specific software. The chambers show reduced oxygen leakage, which is an advantage over the conventional oxygen electrode devices14,23.
As with other cells, in the case of spermatozoa, the sensitivity of HRR equipment is higher than for conventional respirometry, meaning that HRR equipment can be used for the analysis of a limited number of intact or permeabilized sperm cells. There are two main strategies for assessing sperm mitochondrial function by HRR: (a) measuring the oxygen consumption in intact cells, which involves reproducing the respiratory function in a medium containing substrates such as glucose, or (b) measuring the oxygen consumption in permeabilized cells using one of the OXPHOS complexes, with the addition of specific substrates to monitor each function separately.
In the present study, we describe the use of HRR to determine mitochondrial respiration in human sperm cells.

Autor destacado: Avance en la investigación de la infertilidad masculina al desentrañar el metabolismo de los espermatozoides y la función mitocondrial 

Respirometría de alta resolución para evaluar la función mitocondrial en espermatozoides humanos

Watch this Scientific Journal Video about Preparation of Human Spermatozoa for High-Resolution Respirometry at JoVE.com

Preparation of Human Spermatozoa for High-Resolution Respirometry  

Preparación de espermatozoides humanos para respirometría de alta resolución

Para empezar, recoge las muestras e invuélvalas durante 30 a 60 minutos a temperatura ambiente para que se licúen por completo. Luego, mezcle la muestra y cargue siete microlitros en una cámara de recuento de espermatozoides precalentada a 37 grados centígrados. Coloque la cámara en una platina precalentada de un microscopio de luz directa.A continuación, abra el software de análisis de espermatozoides computarizado y haga clic en mot para ingresar al módulo de motilidad y concentración. Seleccione la configuración que corresponda a las condiciones de los espermatozoides humanos. Analice aleatoriamente 10 campos diferentes por cámara haciendo clic en el botón Analizar.A continuación, haga clic en Resultados para obtener la concentración y la motilidad de la muestra. Para preparar las células para la respirometría de alta resolución, primero centrifugue las muestras a 400 a 500 G durante 10 minutos a temperatura ambiente. A continuación, se extrae el plasma seminal y se resuspenden los espermatozoides en dos mililitros de medio adecuado.Repita la evaluación de espermatozoides con un software de análisis de espermatozoides asistido por computadora para estudios de concentración de espermatozoides.

preparation-of-human-spermatozoa-for-high-resolution-respirometry

Research

JoVE Journal

Medicine

Preparation of Human Spermatozoa for High-Resolution Respirometry

127 vistas. Para empezar, recoge las muestras e invuélvalas durante 30 a 60 minutos a temperatura ambiente para que se licúen por completo. Luego, mezcle la muestra y cargue siete microlitros en una cámara de recuento de espermatozoides precalentada a 37 grados centígrados. Coloque la cámara en una platina precalentada de un microscopio de luz directa.A continuación, abra el software de análisis de espermatozoides computarizado y haga clic en mot para ingresar al módulo de motilidad y conc...

Video: Preparation of Human Spermatozoa for High-Resolution Respirometry  

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

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.

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 ...