Name: evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa
Uploaded: 2018-03-11T13:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

We thank AQUAGAMETE FA 1205 COST Action. This work was financially supported by AGL201568330-C2-1-R project (MINECO/FEDER). David G. Valcarce was funded by Junta de Castilla y Le&#243;n (EDU1084/2012) and Fondo Social Europeo. Authors acknowledge Dr. Ana Riaza and Stolt Sea Farm S.A.,&#160;Dr. Paulino de Paz, Dr. Ignacio Mart&#237;nez Montero and&#160;Jos&#233; Ram&#243;n Gui&#233;rrez. We also thank&#160;Paula Fern&#225;ndez Colado for videography.

NOTE: Fluorochrome incubation and confocal analysis will take at least 2-3 h for a control and a treated sample. Data processing is not included in this time calculation. Required materials can be found in the Table of Materials. This protocol can be applied to fresh or cryopreserved spermatozoa. Solea senegalensis is a fish species that spawns in cold water, work always under cold conditions (4-7 &#176;C). See Figure 1 for a general view of the protocol.
1. Preparatory Work Before the Experiment
<ol>
	<li>Prepare a 1 mM mitochondria stain stock solution in analytical grade DMSO.</li>
	<li>Prepare a 1 &#956;g/mL 4&#8242;,6-diamidino-2-phenylindole dihydrochloride (DAPI) stock solution in deionized water.</li>
	<li>Prepare a 200 mOsm/kg Ringer solution (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.7)</li>
	<li>Prepare a 4 mM 2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (DCFH-DA) stock solution in analytical grade methanol. 
	NOTE: Keep the stock solutions in aliquots at -20 &#176;C until needed.</li>
	<li>Set the microcentrifuge at 4-7 &#176;C.</li>
</ol>
2. Sample Preparation Before Fluorochrome Incubation
NOTE: Gentle handling of spermatozoa is desirable when pipetting and resuspending.
<ol>
	<li>If working with cryopreserved cells, prepare a water bath for thawing at 40 &#176;C for Solea senegalensis spermatozoa10. Immerse the straw samples in the water bath for 7 s.</li>
	<li>Using scissors to cut, empty the sample into a microfuge tube and centrifuge at 1000 x g, 4-7 &#176;C for 1 min.</li>
	<li>Remove seminal plasma or seminal plasma with cryoprotectants from the cryopreserved sample by pipetting to discard any component that could interfere with the staining protocol.</li>
	<li>Resuspend the cells in 50-100 &#956;L of 4 &#176;C 200 mOsm/kg Ringer solution.</li>
	<li>Determine the sperm concentration with a Neubauer or a similar counting chamber under a stereoscopic microscope.</li>
	<li>Dilute the cell suspension up to 1-2 x 106 cells/mL in a final volume of 0.5 mL in 4 &#176;C Ringer solution.</li>
</ol>
3. Fluorochrome Incubation
NOTE: Fluorochromes must be handled in low light conditions, especially when in solution.
<ol>
	<li>Add 3.125 &#956;L of DCFH-DA stock solution (final concentration in the sample: 25 &#956;M) to the working sample dilution. Incubate at 4-7 &#176;C for 40 min in darkness10.</li>
	<li>After 30 min, add 0.5 &#956;L of the DAPI stock solution (final concentration in the sample: 2 &#956;M) and 0.5 &#956;L of the mitochondria stain stock solution (final concentration in the sample: 100 nM) to the sample. Incubate both fluorochromes for 10 min in darkness.</li>
</ol>
4. Sample Preparation for Confocal Microscopy
<ol>
	<li>After the incubation time, centrifuge the cell suspension at 1000 x g, 4-7 &#176;C for 1 min.</li>
	<li>Discard the supernatant carefully by pipetting.</li>
	<li>Add a 10-20 &#956;L of 200 mOsm/kg Ringer solution.</li>
	<li>Place 5 &#956;L of a concentrated cell suspension drop on a slide.</li>
	<li>Carefully, put a cover slide on the preparation and seal it with polish. Once the polish is dry, the preparation is ready for confocal microscopy.</li>
</ol>
5. Confocal Setup Prior to the Experiment
NOTE: Depending on the microscope, DCFH-DA could be &#34;burnt&#34;. Establish the best conditions with a non-valuable sample first.
<ol>
	<li>Turn on the microscope, lasers, camera and the computer running the microscope.</li>
	<li>Set excitation lasers taking into account the excitation and emission maximums of each fluorochrome:
	<ol>
		<li>For DAPI, use an excitation maximum of 358 nm and an emission maximum of 465 nm.</li>
		<li>For the mitochondrial stain, use an excitation maximum of 644 nm and an emission maximum of 662 nm.</li>
		<li>For DCFH-DA, use an excitation maximum of 504 nm and an emission maximum of 525 nm.</li>
	</ol>
	</li>
	<li>Start working with 25X objective to focus on the spermatozoa. Choose the DAPI channel to focus because this fluorochrome reports the highest intensity. Once the cells are focused, use a 63X or 100X objective for a thorough evaluation because Solea senegalensis spermatozoa size is around 2 &#956;m.</li>
	<li>For each channel, optimize the pinhole, the gain and the voltage. In the same way, adjust the digital offset to reduce background. Once all parameters are optimized for the fluorochrome used, save the settings. For a routine experiment for a single Solea senegalensis sperm cell we used the following (these parameters may change in each experiment): pinhole of 1 and the gain voltage of 750 V.</li>
</ol>
6. Acquisition of Images
<ol>
	<li>Select the desired quality conditions for the image acquisition. Define acquisition settings like bit depth, image format, light sheet thickness, and choose single sided illumination. For a routine experiment for a single Solea senegalensis spermatozoa we used the following (these parameters may change in each experiment): frame size of 1024 px; bits per pixel of 16; scan speed of 2-4).</li>
	<li>Open and center the scan area to begin and take an image of the field.</li>
	<li>Using the crop tool, select the region of interest and press live.</li>
	<li>With the help of the range indicator tool, adjust the digital offset to reduce the background. Do this for each channel and take the image.</li>
	<li>Check the quality of individual channels for each fluorochrome and their combinations.</li>
</ol>
7. Creating a 3D Image Video
<ol>
	<li>Z-stack acquisition settings.
	<ol>
		<li>Choose the Z-stack option in the software.</li>
		<li>Select the single cell, group of cells or region of interest and focus it.</li>
		<li>Delimit the Z-stack with the &#39;First Slice&#39; and &#39;Last Slice&#39; options and establish the z step to interval to 0.2 &#181;m with the help of the fine focus. Choose DAPI channel to focus because this fluorochrome may report the highest intensity and you can easily select the whole spermatozoa head. Choose the optimal acquisition parameters for each channel and run the experiment.</li>
	</ol>
	</li>
	<li>Run the Z-stack experiment. Split the channels and observe the localization of each signal within the cells.</li>
	<li>Z-stack process.
	<ol>
		<li>Volume render: Once the experiment is finished, select the options of the Z-stack processing software and choose the volume render option. Select the number of steps and the rotation degrees (360&#176;). Save the file with a video extension.</li>
		<li>Stereo anaglyph: Choose stereo anaglyph in the options process window. This tool allows to create a 3D image video with split channels.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Amaral, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+functionality+and+chemical+compound+action+on+sperm+function.">Mitochondrial functionality and chemical compound action on sperm function.</a> Curr Med Chem. , (2016).</li><li>Morielli, T., O'Flaherty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+impairs+function+and+increases+redox+protein+modifications+in+human+spermatozoa.">Oxidative stress impairs function and increases redox protein modifications in human spermatozoa.</a> Reproduction. 149 (1), 113-123 (2015).</li><li>G&#252;rler, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+sperm+viability,+synthesis+of+reactive+oxygen+species,+and+DNA+damage+of+bovine+sperm.">Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm.</a> Theriogenology. , (2016).</li><li>Zhu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+E+Analogue+Improves+Rabbit+Sperm+Quality+during+the+Process+of+Cryopreservation+through+Its+Antioxidative+Action.">Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.</a> PLoS One. 10 (12), e0145383 (2015).</li><li>Fang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+ROS+production+through+mitochondria-targeted+antioxidant+and+mitochondrial+uncoupling+increases+post-thaw+sperm+viability+in+yellow+catfish.">Inhibition of ROS production through mitochondria-targeted antioxidant and mitochondrial uncoupling increases post-thaw sperm viability in yellow catfish.</a> Cryobiology. 69 (3), (2014).</li><li>Thomson, L. K., Fleming, S. D., Aitken, R. J., De Iuliis, G. N., Zieschang, J. A., Clark, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation-induced+human+sperm+DNA+damage+is+predominantly+mediated+by+oxidative+stress+rather+than+apoptosis.">Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis.</a> Hum Reprod. 24 (9), 2061-2070 (2009).</li><li>Kim, S. H., Yu, D. H., Kim, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+phosphatidylserine+translocation,+intracellular+hydrogen+peroxide,+and+DNA+integrity+in+canine+sperm.">Effects of cryopreservation on phosphatidylserine translocation, intracellular hydrogen peroxide, and DNA integrity in canine sperm.</a> Theriogenology. 73 (3), (2010).</li><li>Guthrie, H. D., Welch, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+reactive+oxygen+species+on+sperm+function.">Effects of reactive oxygen species on sperm function.</a> Theriogenology. 78 (8), 1700-1708 (2012).</li><li>Aitken, R. J., Jones, K. T., Robertson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+and+sperm+function--in+sickness+and+in+health.">Reactive oxygen species and sperm function--in sickness and in health.</a> J Androl. 33 (6), (2012).</li><li>Valcarce, D. G., Robles, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+captivity+and+cryopreservation+on+ROS+production+in+Solea+senegalensis+spermatozoa.">Effect of captivity and cryopreservation on ROS production in Solea senegalensis spermatozoa.</a> Reproduction. 152 (5), (2016).</li><li>Gibb, Z., Lambourne, S. R., Aitken, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradoxical+relationship+between+stallion+fertility+and+oxidative+stress.">The paradoxical relationship between stallion fertility and oxidative stress.</a> Biol Reprod. 91 (3), (2014).</li><li>Cabrita, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+enhancing+fish+sperm+quality+and+emerging+tools+for+sperm+analysis.">Factors enhancing fish sperm quality and emerging tools for sperm analysis.</a> Aquaculture. 432, 389-401 (2014).</li></ol>

The authors have nothing to disclose.

It is well known that mitochondria are key organelles for sperm motility and function. These organelles are concurrently directly involved in ROS production. Interestingly, controlled levels of ROS are needed for proper sperm function1. Positive relationships between fertility and oxidative stress have been shown in mammals11 but excessive levels affect sperm quality12. One crucial factor that could be decisive towards a positive or negative effect is not only ROS levels but also ROS intracellular localization. One of the deleterious effects of ROS is producing DNA damage9 and therefore the nuclear presence of ROS could be an indicator of potential damage in the nucleus whereas ROS levels could be normal in mitochondria. It is necessary to supplement existing quantitative methods (e.g., flow cytometry) with techniques like confocal microscopy that could provide information about ROS intracellular localization.
Confocal microscopy is an optimal option to visualize the intracellular localization of ROS. The use of specific fluorochromes such as a live mitochondria stain and DAPI allows the visualization of mitochondria and nucleus respectively and the combination of these molecules with DCFH-DA allows the intracellular localization of peroxide.
The critical steps within the protocol are fluorochrome incubation and confocal setup. Both steps should be optimized and carefully performed to obtain reproducible and consistent results. Methodology modifications should be performed at these two levels depending on the seminal plasma used. Therefore, fluorochrome incubation should be adapted. Temperatures and storage conditions significantly differ among sperm samples, and most of the protocols are optimized for mammals. 
Here, a protocol for teleost sperm samples, particularly for Solea senegalensis spermatozoa, is described (Figure 1). Successful labeling of nuclei and mitochondria have been performed using the described protocol and ROS presence has been reveal using DCFH-DA (Figure 2). Results indicate that co-localization of H2O2 have been found in the nuclei or mitochondria depending on the sperm sample (Figure 3). As previously explained, those samples with a high percentage of spermatozoa displaying high levels of ROS within the nucleus would be prone to DNA damage. This protocol has a unique limitation, the requirement of expensive equipment (confocal microscope), but it could have future utility in selecting sperm samples with low presence of ROS within the nucleus for cryopreservation purposes.

The reactive oxygen species production has been linked with sperm quality recently1. Although ROS production in mitochondria can be considered a normal physiological process, oxidative stress by an excess of ROS production is a clear cause of damage in spermatozoa at different levels. In humans, oxidative stress is associated with male infertility, altering motility and the ability to undergo capacitation2; in mammals, change of DNA integrity in frozen sperm samples has been also related to synthesis of H2O23.
Cryopreservation is a common technique for gene banking in aquaculture. This technology is particularly important in species with reproductive problems such as Solea senegalensis. This valuable species in the market shows reproductive dysfunction in individuals born in captivity due to a lack of courtship. This fact makes sperm cryopreservation necessary to have sperm availability for artificial fertilization. However, cryopreservation could be a source of oxidative stress that could be detrimental for spermatozoa4 as studies have reported a beneficial effect of antioxidant supplementation. ROS inhibition through mitochondrial-targeted antioxidant was reportedly beneficial for sperm cryopreservation in yellow catfish5.
Therefore, the levels of ROS in sperm samples are important to know, particularly after cryopreservation6,7 because these molecules have been recognized as a drawback for sperm survival and fertility8. Moreover, studying the distribution of ROS within the cell could be crucial to infer the level of potential damage. As an example, low levels of ROS in the mitochondria could be assumed normal and compatible with sperm function, but high levels of ROS within the nucleus could be indicators of spermatozoa DNA damage. H2O2 is one of the most relevant ROS that could be released from the mitochondria and penetrate the nucleus because it is a small and charge-less molecule9. Dichlorofluorescein diacetate (DCFH-DA) can specifically reveal intracellular peroxide emitting green fluorescence. In this article, a detailed protocol for detecting H2O2 intracellular localization in Solea senegalensis sperm using confocal microscopy is presented.

<table><tbody><tr><td>2&amp;prime;,7&amp;prime;-Dichlorodihydrofluorescein diacetate (DCFH-DA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D6883</td><td></td></tr><tr><td>4&amp;prime;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C1016&amp;nbsp;</td><td></td></tr><tr><td>Confocal Microscopy</td><td>Zeiss</td><td>LSM800</td><td></td></tr><tr><td>Cover slips</td><td>Thermo Fisher Scientific</td><td>12-541B</td><td></td></tr><tr><td>DMSO, Analytical Grade</td><td>Sigma-Aldrich</td><td>W387520</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Methanol, Analytical Grade</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>MitoTrackerDeep Red&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>M22426</td><td></td></tr><tr><td>Microcentrifuge (refrigerated)</td><td>Thermo Fisher Scientific</td><td>75002441</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S7653&amp;nbsp;</td><td></td></tr><tr><td>Neubauerchamber</td><td>Sigma-Aldrich</td><td>BR717810</td><td></td></tr><tr><td>Slides</td><td>Thermo Fisher Scientific</td><td>10143562BEF</td><td></td></tr></tbody></table>

evaluation of intracellular location of reactive oxygen species in solea senegalensis spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2&#8242;,7&#8242;-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the co-localization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.

Confocal microscopy is an ideal method for intracellular ROS evaluation in teleost sperm. The combination of the three fluorochromes (DAPI, a mitochondria stain and DCFH-DA) presented in this study (Figure 1) provides many useful information that can be applied in basic research and can have applications in improving procedures used in industrial aquaculture plants, such as cryopreservation protocols. Different types of analysis may be carried out in order to correlate intracellular ROS presence and other parameters: motility, viability, different patterns between good and bad breeders, activation of motility or seasonal sperm variations among many others. In the present work, two different patterns of intracellular H2O2 distribution within the spermatozoa are shown: collocated with mitochondrion or spread in the nucleus (Figures 2, 3). Those spermatozoa showing low potential damage produced by ROS showed DCFH-DA labelling only in the mitochondria whereas those suffering DNA damage showed DCFH-DA labelling also in the nuclei. Confocal microscopy software allows to create useful videos and provide easy and fast colocalization graphs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55323/55323fig1.jpg" /> 
Figure 1. General overview of the protocol. <a href="/files/ftp_upload/55323/55323fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55323/55323fig2.jpg" /> 
Figure 2. Example of confocal image acquisition. A. Scheme of the sample B. DAPI channel (labelling dsDNA). C. Mitochondria stain channel (labelling mitochondrion). D. Mitochondria stain channel merged with DAPI channel. E. DCFA-DH channel (labelling intracellular H2O2). F. DCFA-DH channel merged with DAPI channel. G. Merge of the three channels. Abbreviations: n: nuclei, mt: mitochondria, f: flagellum and mb: membrane. Scale bar: 5 &#956;m. <a href="/files/ftp_upload/55323/55323fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55323/55323fig3.jpg" /> 
Figure 3. Different patterns of intracellular H2O2 in Solea senegalensis spermatozoa (Volume render). Resulting channels of spermatozoa showing intracellular ROS in the nuclear fossa and mitochondrion (A, C, E). Resulting channels of spermatozoa showing intracellular H2O2 also within the nuclei (B, D, F). A and B: DAPI. C and D: DCFH-DA. E and F: Mitochondria stain. <a href="/files/ftp_upload/55323/55323fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa at JoVE.com

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

This protocol describes a detailed methodology for detecting H2O2 localization within Solea senegalensis spermatozoa using a sensitive fluorochrome DCFH-DA for ROS, a live mitochondria stain for mitochondria, and DAPI for nuclei visualization, respectively. The protocol is designed to be performed within 2 h with either fresh or thawed spermatozoa.

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) ...

evaluation-intracellular-location-reactive-oxygen-species-solea

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.2K Views.  University of León. Dit protocol beschrijft een gedetailleerde methodologie voor het opsporen van H2O2 localisatie binnen Solea senegalensis spermacellen met behulp van een gevoelige fluorescerende DCFH-DA ROS, een levende mitochondriën vlek voor mitochondria en DAPI voor kernen visualisatie, respectievelijk. Het protocol is ontworpen om te worden uitgevoerd binnen de 2 uur met verse of ontdooide spermacellen.

Video: Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Detection of Total Reactive Oxygen Species in Adherent Cells by 2',7'-Dichlorodihydrofluorescein Diacetate Staining

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative stress by measuring total reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in colorectal cancer cell lines as an example. This protocol describes detailed steps including preparation of DCFH-DA solution, incubation of cells with DCFH-DA solution, and measurement of normalized intensity. DCFH-DA staining is a simple and cost-effective way to detect ROS in cells. It can be used to measure ROS generation after chemical treatment or genetic modifications. Therefore, it is useful for determining cellular oxidative stress upon environment stress, providing clues to mechanistic studies.

Here, we present a protocol to detect total cellular reactive oxygen species (ROS) using 2&#39;,7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA). This method can visualize cellular ROS localization in adherent cells with a fluorescence microscope and quantify ROS intensity with a fluorescence plate reader. This protocol is simple, efficient and cost-effective.

Oxidative stress is an important event under both physiological and pathological conditions. In this study, we demonstrate how to quantify oxidative ...

Biochemistry

Using an Extracellular Flux Analyzer to Measure Changes in Glycolysis and Oxidative Phosphorylation during Mouse Sperm Capacitation

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes require energy. There remains an ongoing debate about the sources generating the ATP which fuels sperm progressive motility, capacitation, hyperactivation, and acrosome reaction. Here, we describe the application of an extracellular flux analyzer as a tool to analyze changes in energy metabolism during mouse sperm capacitation. Using H+- and O2- sensitive fluorophores, this method allows monitoring glycolysis and oxidative phosphorylation in real-time in non-capacitated versus capacitating sperm. Using this assay in the presence of different energy substrates and/or pharmacological activators and/or inhibitors can provide important insights into the contribution of different metabolic pathways and the intersection between signaling cascades and metabolism during sperm capacitation.

We describe the application of an extracellular flux analyzer to monitor real-time changes in glycolysis and oxidative phosphorylation during mouse sperm capacitation.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. Capacitation-associated processes ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. Thiols are especially important for illuminating the redox status of cells via their reduced dithiol and oxidized disulfide ratios. Engineered cysteine-containing fluorescent proteins open a new era for redox-sensitive biosensors. One of them, redox-sensitive green fluorescent protein (roGFP), can easily be introduced into cells with adenoviral transduction, allowing the redox status of subcellular compartments to be evaluated without disrupting cellular processes. Reduced cysteines and oxidized cystines of roGFP have excitation maxima at 488 nm and 405 nm, respectively, with emission at 525 nm. Assessing the ratios of these reduced and oxidized forms allows the convenient calculation of redox balance within the cell. In this method article, immortalized human triple-negative breast cancer cells (MDA-MB-231) were used to assess redox status within the living cell. The protocol steps include MDA-MB-231 cell line transduction with adenovirus to express cytosolic roGFP, treatment with H2O2, and assessment of cysteine and cystine ratio with both flow cytometry and fluorescence microscopy.

This protocol describes the assessment of subcellular compartment-specific redox status within the cell. A redox-sensitive fluorescent probe allows convenient ratiometric analysis in intact cells.

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Fluorimetric Techniques for the Assessment of Sperm Membranes

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4&#39;,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5&#39;,6,6&#39;-tetra-chloro-1,1&#39;,3,3&#39;-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Here, we present methodologies to evaluate spermatozoan membrane integrity, a cellular feature associated with sperm fertilization competence. We describe three techniques for the fluorimetric assessment of sperm membranes: simultaneous staining with specific fluorescent probes, fluorescence microscopy, and advanced sperm-dedicated flow cytometry. Examples of combining the methodologies are also presented.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, ...

Detecting, Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H2O2 production, respectively.

We adapted a set of protocols for the measurement of reactive oxygen species (ROS) that can be applied in various amoeba and mammalian cellular models for qualitative and quantitative studies.

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or ...

Biology

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Quantification of Reactive Oxygen Species Using 2&#8242;,7&#8242;-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in M&#252;ller Glial Cells

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the modification of proteins, lipids, and DNA, which finally may lead to alteration in cellular function and cell death. Therefore, it is beneficial for cells to increase their antioxidant defense in response to detrimental insults, either by activating an antioxidant pathway like Keap1/Nrf2 or by improving redox scavengers (vitamins A, C, and E, &#946;-carotene, and polyphenols, among others). Inflammation and oxidative stress are involved in the pathogenesis and progression of retinopathies, such as diabetic retinopathy (DR) and retinopathy of prematurity (ROP). Since M&#252;ller glial cells (MGCs) play a key role in the homeostasis of neural retinal tissue, they are considered an ideal model to study these cellular protective mechanisms. In this sense, quantifying ROS levels with a reproducible and simple method is essential to assess the contribution of pathways or molecules that participate in the antioxidant cell defense mechanism. In this article, we provide a complete description of the procedures required for the measurement of ROS with DCFH-DA probe and flow cytometry in MGCs. Key steps for flow cytometry data processing with the software are provided here, so the readers will be able to measure ROS levels (geometric means of FITC) and analyze fluorescence histograms. These tools are highly helpful to evaluate not only the increase in ROS after a cellular insult but also to study the antioxidant effect of certain molecules that can provide a protective effect on the cells.

Here, we propose a systematized, accessible, and reproducible protocol to detect cellular reactive oxygen species (ROS) using 2&#8242;,7&#8242;-dichlorofluorescein diacetate probe (DCFH-DA) in M&#252;ller glial cells (MGCs). This method quantifies total cellular ROS levels with a flow cytometer. This protocol is very easy to use, suitable, and reproducible.

The redox balance has an important role in maintaining cellular homeostasis. The increased generation of reactive oxygen species (ROS) promotes the ...

Neuroscience

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no abnormalities in conventional semen parameters. Additional technologies are needed to explain the idiopathic infertility and detect subtle sperm defects. Currently, biomarkers of sperm function, including sperm apoptosis, mitochondrial membrane potential (MMP), and DNA damage, reveal sperm physiology at the molecular level and are capable of predicting male fertility.
With flow cytometry (FCM) techniques, each of these markers can be rapidly, accurately, and precisely measured in human semen samples, but time costs substantially increase and results could be obstructed if all the biomarkers need to be tested with a single cytometer. In this protocol, after collection and immediate incubation at 37 &#176;C for liquefication, semen samples were further analyzed for sperm apoptosis using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The MMP was labeled with 5,5&#8242;,6,6&#8242;-tetrachloro-1,1&#8242;,3,3&#8242;-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) probe, and DNA damage was assessed using the sperm chromatin structure assay (SCSA) with acridine orange (AO) staining. Thus, flow cytometric analysis of sperm function markers can be a practical and reliable toolkit for the diagnosis of infertility and evaluation of sperm function at both bench and bed.

The present protocol provides a practical solution that allows for the measurement of apoptosis, mitochondrial membrane potential, and DNA damage in human sperm with a single cytometer.

The conventional semen parameter analysis is widely used to assess male fertility. However, studies have found that ~15% of infertile patients show no ...

Medicine

Determining Respiration in Human Sperm Cells by 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.

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

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 ...

Analysis of Semen for Peroxidase-Positive Leukocyte Cells

The Examination of Peroxidase-Positive Leukocytes in Semen

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even affected embryonic development. It is a common andrological disease in clinical practice and one of the important causes of male infertility. When determining whether male reproductive tract inflammation exists, andrologists often choose to examine round cells or seminal plasma elastase in the semen as a clinical diagnostic basis. However, the examination of round cells is easily influenced by sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics. At the same time, the detection process of elastase is relatively complicated, time-consuming, and slow in reporting results, which is not beneficial for early diagnosis and treatment of diseases such as male genital tract infections (MGTIs). We have innovatively applied the examination of peroxidase-positive leukocytes in semen assisted by a computer-assisted semen analysis (CASA) system as a diagnostic criterion for leukocytospermia, successfully solving these problems. This examination only requires the addition of the operating fluid consisting of four reagents into the specimen, and the total reaction time at room temperature can be controlled within 20-30 min. With the subsequent smear and microscopic examination, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, which can be used to diagnose whether the inflammation of the male reproductive tract existed.

Author Spotlight: A Simple and Cost-Effective Method for Leukocyte Analysis in Semen 

This paper presents an economical and efficient protocol for examining peroxidase-positive leukocytes in semen. With the assistance of a computer-assisted semen analysis (CASA) system, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, effectively improving the efficiency of andrology laboratory and andrologists.

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation ...

Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

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