Name: Preparation of Human Spermatozoa for High-Resolution Respirometry
Uploaded: 2023-06-23T14:35:00
Description: 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

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 Preparation of Human Spermatozoa for High-Resolution Respirometry at JoVE.com

Preparation of Human Spermatozoa for High-Resolution Respirometry  

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.

Research

JoVE Journal

Medicine

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...