high-resolution respirometry - oxphos analysis for human spermatozoa

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

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.

Author Spotlight: Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function 

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Our work is focused on male infertility, specifically on sperm mitochondria. Sperm cells lose most of their cytoplast and organelles during the spermatogenesis, but they do retain mitochondria. Besides their main role in ATP production, mitochondria participate in the production of reactive oxygen species, calcium storage, and apoptosis.The role of sperm mitochondria is not completely understood. Understanding mitochondrion function in sperm is key to enlighten a sperm energy metabolism and male infertility. We have demonstrate that human sperm mitochondrial function correlate to spermiogram parameters.Our group show that mitochondria are the main source of superoxide in normal and pathological conditions. Moreover, we have been able to establish metabolic cutoff values using high-resolution respirometry that can differentiate between males with abnormal and normal spermiogram. Spermiogram is the gold standard laboratory study to analyze male infertility.It provides a lot of information, but doesn't reflect septal sperm defects. Although many studies have been proposed, at present, the measurement of mitochondrial function is not included the study. High-resolution respirometry has a higher sensitivity than conventional Clark electrodes.With this experimental approach, we can analyze each step of mitochondrial activity. We determine ATP production, respiratory reserve capacity and compliance status. Moreover, it allows us to study experiment suspension without affecting the movement and function.

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

High-Resolution Respirometry - OXPHOS Analysis for Human Spermatozoa  

To begin, prepare the equipment by turning on the high resolution respirometer and connect it to the respirometry software VatLab for data acquisition and analysis. Replace the 70%ethanol in the oxygraph chamber with double distilled water. Using a magnetic stir bar, stir it continuously at 750 RPM in the chamber.Let it stand for 10 minutes and then aspirate the water. Wash the chamber three times with double distilled water for five minutes each. To calibrate the oxygen sensors, first remove the double distilled water and pipette two milliliters of the cell preparation medium into the chamber.Place the stoppers leaving an air exchange bubble. To record the oxygen calibration values, adjust the settings such as gain for the sensor, polarization voltage, and data recording interval. Then label the experiment and enter the medium.Click on layout and select 01 Calibration Experiment GR3 Temp. Stir the medium with the stir bar at 750 RPM for at least 30 minutes at 37 degrees Celsius, and monitor the performance of the sensor membrane. Holding the left mouse button and the Shift key, drag the mouse to select an area where the change in oxygen concentration is stable.Then click on oxygraph and then O2 Calibration. In air calibration, change the selected mark to the region selected earlier. Then click on calibrate and copy to clipboard.Stop the recording and save by clicking on oxygraph. Followed by OK Control and then save and disconnect. Next, to perform respiratory assessment, open the chamber and aspirate the medium inside.Load sperm cells in a final volume of two milliliters of MRM for permeabilized cell analysis. Load the calibration by double clicking on the POS calib box in the bottom corner and opening the calibration performed earlier. Then stop the experiment.Inject 4.5 microliters of 10 millimolar digitonin and permeabilize the cells for five minutes. Record the respiration of the intact cells for at least five minutes until a stable signal is obtained. Then add the substrates for complex I or complex II.Measure the oxygen consumption until the signal increases and stabilizes.Next, inject five microliters of 0.5 molar ADP and measure the oxygen consumption until the signal increases and stabilizes. Add one microliter of four milligrams per milliliter Oligomycin, which is an ATP synthetase inhibitor. Measure the oxygen consumption until the signal decreases and stabilizes.Titrate by adding one microliter of FCCP in successive steps, starting from 0.1 millimolar to one millimolar until a maximum uncoupled respiration rate is reached. Measure the oxygen consumption until the signal increases and stabilizes. Stop injecting the drug when oxygen consumption begins to decrease.Finally, inject one microliter of one millimolar rotenone for complex I or five millimolar Antimycin A for complex II to discriminate between the mitochondrial and residual oxygen consumption. Measure the oxygen consumption until the signal decreases and stabilizes. To perform data analysis select regions where the oxygen flux per volume correlated is stable after the injection of a substrate or inhibitor, click on marks, then statistics windows, and export the data.Normalize the data obtained per 1 million sperm cells and subtract the non-mitochondrial oxygen consumption from all the values. Calculate the indices using these equations. Successful recording of intact sperm cells from a semen sample was achieved where the blue line showed the oxygen concentration and the red line represented the oxygen flow per volume correlated.Lower sperm cell counts resulted in lower baseline oxygen consumption. Further, oxygen consumption curves specifically for the mitochondrial complex I or complex II were obtained using this protocol.

high-resolution-respirometry-oxphos-analysis-for-human-spermatozoa

Research

JoVE Journal

Medicine

143 visualizações.  Unidad Académica Histología y Embriología. Para começar, prepare o equipamento ligando o respirômetro de alta resolução e conecte-o ao software de respirometria VatLab para aquisição e análise de dados. Substitua o etanol 70% na câmara oxigráfica por água bidestilada. Usando uma barra de agitação magnética, agite-a continuamente a 750 RPM na câmara.Deixe agir por 10 minutos e depois aspirar a água. Lave a câmara três vezes com água bidestilada por cinco minutos cada. Para calibrar os sensores de oxigênio, pri...