Name: Preparation of Human Spermatozoa for High-Resolution Respirometry
Uploaded: 2023-06-23T14:35:00
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preparation of human spermatozoa for high-resolution respirometry

קביעת נשימה בתאי זרע אנושיים על ידי ספירומטריה ברזולוציה גבוהה

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.

מחבר זרקור: קידום מחקר פוריות הגבר על ידי חשיפת חילוף החומרים של הזרע ותפקוד המיטוכונדריה 

רספירומטריה ברזולוציה גבוהה להערכת תפקוד המיטוכונדריה בזרעונים אנושיים

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.

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Preparation of Human Spermatozoa for High-Resolution Respirometry  

הכנת זרע אנושי לרספירומטריה ברזולוציה גבוהה

כדי להתחיל, לאסוף את הדגימות לדגור אותם במשך 30 עד 60 דקות בטמפרטורת החדר כדי לנזול לחלוטין. לאחר מכן, ערבבו את הדגימה והעמיסו שבעה מיקרוליטרים לתוך תא ספירת זרע שחומם מראש בטמפרטורה של 37 מעלות צלזיוס. הניחו את החדר על במה שחוממה מראש במיקרוסקופ אור ישיר.לאחר מכן, פתח את תוכנת ניתוח הזרע הממוחשבת ולחץ על mot כדי להיכנס למודול התנועתיות והריכוז. בחר את התצורה המתאימה לתנאי זרע אנושיים. נתח באופן אקראי 10 שדות שונים לכל תא על ידי לחיצה על כפתור ניתוח.לאחר מכן לחץ על תוצאות כדי לקבל את ריכוז הדגימה ואת תנועתיות. כדי להכין את התאים לרספירומטריה ברזולוציה גבוהה, תחילה צנטריפוגו את הדגימות ב 400 עד 500 גרם במשך 10 דקות בטמפרטורת החדר. לאחר מכן, להסיר את פלזמה הזרע להשעות מחדש את הזרעונים בשני מיליליטר של המדיום המתאים.חזור על הערכת הזרע באמצעות תוכנת ניתוח זרע בעזרת מחשב למחקרי ריכוז זרע.

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

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JoVE Journal

Medicine

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 ...

To begin, collect the samples and incubate them for 30 to 60 minutes at room temperature to liquefy completely. Then, mix the sample and load seven microliters into a prewarmed sperm counting chamber at 37 degrees Celsius. Place the chamber on a prewarmed stage of a direct light microscope.Next, open the computerized sperm analysis software and click on mot to enter the motility and concentration module. Select the configuration that corresponds to human sperm conditions. Randomly analyze 10 different fields per chamber by clicking on the Analyze button.Then click on Results to obtain the sample concentration and motility. To prepare the cells for high-resolution respirometry, first centrifuge the samples at 400 to 500 G for 10 minutes at room temperature. Then, remove the seminal plasma and resuspend the spermatozoa in two milliliters of appropriate medium.Repeat the sperm evaluation with computer-aided sperm analysis software for sperm concentration studies.

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.