We thank all the fieldworkers for their help and the interviewees for their cooperation.

NOTE: Workflow for the detection of biomarkers for sperm function damage by flow cytometry includes (1) cell preparation, (2) staining with fluorescent reagents, and (3) flow cytometric analysis and data interpretation (Figure 1). The protocol follows the guidelines of the Ethics Committees of the Army Medical University (1.0/2013.4-12).
1. Cell preparation
<ol>
	<li>Obtain human semen samples by masturbation in plastic clinical specimen jars, preferably after 3-5 days of abstinence.</li>
	<li>Immediately incubate the semen samples in a 37 &#176;C incubator and completely liquefy the samples (up to 1 h).</li>
</ol>
2. Sperm cells counted using computer-aided sperm analysis (CASA)
<ol>
	<li>Mix the liquefied semen samples thoroughly.</li>
	<li>Add 10 &#181;L of semen into a sperm counting chamber (see the Table of Materials).</li>
	<li>Scan the slides in the Sperm Class Analyzer (see the Table of Materials), and count at least six areas and 400 spermatozoa for the estimation of sperm concentration. 
	&#8203;NOTE: Semen samples must be fresh, not frozen, for sperm apoptosis and MMP analysis.</li>
</ol>
3. Staining sperm cells
<ol>
	<li>Detection of sperm apoptosis using Annexin V-FITC/PI staining
	<ol>
		<li>Add 1 &#215; 106 sperm cells to a 1.5 mL centrifuge tube.</li>
		<li>Wash the cells with 500 &#181;L of cold phosphate-buffered saline (PBS).</li>
		<li>Centrifuge the sperm cell suspension at 300 &#215; g for 7 min and discard the supernatant.</li>
		<li>Resuspend the sperm cells in 200 &#181;L of 1x Binding Buffer.</li>
		<li>Add 2 &#181;L of FITC Annexin V and 2 &#181;L of PI (see the Table of Materials).</li>
		<li>Gently vortex the cells, incubate for 15 min at room temperature (25 &#176;C) in the dark, and place them in the flow cytometer immediately for analysis.</li>
	</ol>
	</li>
	<li>Analysis of mitochondrial membrane potential using JC-1 probe
	<ol>
		<li>Add 2 &#215; 106 sperm cells to a 1.5 mL centrifuge tube and centrifuge at 600 &#215; g for 5 min.</li>
		<li>Resuspend the sperm cell suspension in 1 mL of JC-1 working solution (see the Table of Materials).</li>
		<li>Gently vortex the cells and incubate for 20 min at 37 &#176;C in the dark.</li>
		<li>Centrifuge the suspension at 600 &#215; g for 5 min and discard the supernatant.</li>
		<li>Wash the pellet twice with 1 mL of PBS at a speed of 600 &#215; g for 5 min each.</li>
		<li>Resuspend the dyed sperm cells in 1 mL of PBS in flow cytometer tubes, and place the tubes in the flow cytometer immediately for analysis.</li>
		<li>Use carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a positive control. Resuspend the sperm cells (as previously described in step 3.2.1) in 1 mL of CCCP working solution (see the Table of Materials), and gently vortex the cells and incubate for 20 min at room temperature. Centrifuge the suspension at 600 &#215; g for 5 min and discard the supernatant. Repeat steps 3.2.2-3.2.6.</li>
	</ol>
	</li>
	<li>Sperm chromatin structure assay
	<ol>
		<li>Place an aliquot of liquefied raw semen (0.25 mL) into a cryotube, and immediately freeze the semen samples in liquid nitrogen (-196 &#176;C) until ready for the SCSA.</li>
		<li>Thaw the semen samples in a 37 &#176;C water bath and dilute them with TNE buffer (see the Table of Materials) to a concentration of 1-2 &#215; 106 cells/mL.</li>
		<li>Add 200 &#181;L of the diluted sample to a flow cytometer test tube and mix it with 400 &#181;L of acid solution (see the Table of Materials) for 30 s.</li>
		<li>Stain the samples with 1.2 mL of AO staining solution (see the Table of Materials) and place in the flow cytometer immediately for analysis.</li>
		<li>Use a reference sample as an internal standard to flow cytometer set-up and calibrations. Dilute the reference sample with 4 &#176;C TNE buffer to a concentration of 1-2 &#215; 106 cells/mL. Repeat steps 3.3.1-3.3.4. 
		NOTES: All solutions and buffers are stored at 4 &#176;C. (1) The semen should not be diluted before flash freezing. Raw semen samples should be thawed and diluted with TNE buffer at the time of flow cytometry measurements. (2) For infertility clinics, ~0.25 mL of raw semen should be flash-frozen in an ultracold freezer or a LN2 tank. These frozen aliquots can then be sent to an SCSA diagnostic laboratory on dry ice or in a LN2 dry shipper. (3) A human ejaculate sample that demonstrates heterogeneity of DNA integrity (e.g., 15% DNA fragmentation index [DFI]) was used as an internal standard reference.</li>
	</ol>
	</li>
</ol>
4. Flow cytometer setup
NOTE: Before starting the flow cytometer, the instrument&#39;s system check should be performed to verify analytical performance. Run the samples after all checks have been completed and passed.
<ol>
	<li>Open the flow cytometer software and start the cytometer.</li>
	<li>Collect sample data.
	<ol>
		<li>First, load a control sample onto the flow cytometer, and click RUN to start data collection (adjust settings if needed). Wait for the instrument to begin aspirating the sample and provide a real-time preview of detected events. 
		NOTE: The control sample: a negative control (unstained sperm cells) for sperm apoptosis; a positive control (CCCP-treated cells) for MMP; a reference sample for SCSA.</li>
		<li>Create dot plots for viewing data and select the x/y-axis parameters (such as FSC, SSC) and linear to specify data are displayed. Select and apply a polygonal gate to delineate the sperm population for analysis, and so that the instrument plots detected events in real-time.</li>
		<li>Analyze the sperm events within the region.
		<ol>
			<li>For sperm apoptosis, set the FL1 as the x-axis and the FL2 as the x-axis. To isolate the live, apoptotic, or necrotic cells, tap and drag the quadrant gate to subset the sperm populations down to four specific populations.</li>
			<li>For MMP, set the FL1 as the x-axis and the FL2 as the y-axis. Create a polygonal gate to subset the sperm populations down to two specific populations.</li>
			<li>For SCSA, set the FL4 as the x-axis (red fluorescence, 125/1,024 flow cytometry channels) and the FL1 as the y-axis (green fluorescence, 475/1,024 flow cytometry channels). Draw the gates at a 45&#176; angle to exclude cellular debris signals.</li>
		</ol>
		</li>
		<li>Set a run limit to indicate when to stop collecting data. Calculate a total of 10,000 sperm cells for each sample for sperm apoptosis, MMP, and SCSA analyses.</li>
		<li>Set the fluidics rate (slow, medium, and fast, or a custom fluidics rate), depending on the cell numbers. 
		NOTES: For SCSA analysis, to determine sperm concentrations, thaw the frozen raw sample and run it on the FCM. If the flow rate is &#62;250/s, dilute the sample to the correct flow rate. Measure all samples independently twice, and calculate the mean values. A reference sample was tested when every 10-15 samples were measured to ensure standardization and stability of the instrument from day-to-day.</li>
		<li>Set the threshold to eliminate debris and noise from cell samples. Apply these settings for all samples in the experiment.</li>
		<li>If needed, set the color compensation to correct fluorescence spillover.</li>
		<li>Gently back-pipette the samples before loading them onto the flow cytometer, name the sample, and run the samples.</li>
		<li>Once all samples have been run, save the sample data as FCS files by giving them a file name for further software analysis. Save the work template of the current experiment for retrieval in future runs (optional).</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Import the data into the data analysis software for the flow cytometer (see the Table of Materials).</li>
	<li>Double-click the first sample in workspace. Wait until a Graph window opens, showing a plot of events along FSC versus SSC parameters. Create a gate that isolates the sperm population included within the gate, producing a &#39;child&#39; population from the parent set of events.</li>
	<li>Double-click within the gated area, and open a new Graph window containing only the sperm events. Set the x- and y-axis parameters to select fluorescent channel.</li>
	<li>Select the Gate tool. Click within the plot to make the gates appear, so that the created gates isolate the different cell populations.</li>
	<li>Right-click (or control-click) any of the highlighted rows, and select Copy analysis to group. Apply the gating tree to all samples within the group.</li>
	<li>Save and export the data table.</li>
</ol>

<ol>
	<li>Agarwal, A., Mulgund, A., Hamada, A., Chyatte, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+view+on+male+infertility+around+the+globe.">A unique view on male infertility around the globe.</a> Reproductive Biology and Endocrinology. 13 (1), 1-9 (2015).</li><li>Nachtigall, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+disparities+in+access+to+infertility+services.">International disparities in access to infertility services.</a> Fertility and Sterility. 85 (4), 871-875 (2006).</li><li>Agarwal, A., Allamaneni, S. S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+DNA+damage+assessment:+a+test+whose+time+has+come.">Sperm DNA damage assessment: a test whose time has come.</a> Fertility and Sterility. 84 (4), 850-853 (2005).</li><li>Pe&#241;a, F. J., Ortega Ferrusola, C., Mart&#237;n Mu&#241;oz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+flow+cytometry+approaches+in+equine+andrology.">New flow cytometry approaches in equine andrology.</a> Theriogenology. 86 (1), 366-372 (2016).</li><li>Oosterhuis, G. J., 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=Measuring+apoptosis+in+human+spermatozoa:+a+biological+assay+for+semen+quality.">Measuring apoptosis in human spermatozoa: a biological assay for semen quality.</a> Fertility and Sterility. 74 (2), 245-250 (2000).</li><li>Koopman, G., 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=Annexin+V+for+flow+cytometric+detection+of+phosphatidylserine+expression+on+B+cells+undergoing+apoptosis.">Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.</a> Blood. 84 (5), 1415-1420 (1994).</li><li>Freitas, M. J., Vijayaraghavan, S., Fardilha, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+mechanisms+in+mammalian+sperm+motility.">Signaling mechanisms in mammalian sperm motility.</a> Biology of Reproduction. 96 (1), 2-12 (2017).</li><li>Lehti, M. S., Sironen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+function+of+sperm+tail+structures+in+association+with+sperm+motility+defects.">Formation and function of sperm tail structures in association with sperm motility defects.</a> Biology of Reproduction. 97 (4), 522-536 (2017).</li><li>Durairajanayagam, D., Singh, D., Agarwal, A., Henkel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+sperm+mitochondrial+dysfunction.">Causes and consequences of sperm mitochondrial dysfunction.</a> Andrologia. 53 (1), 13666 (2021).</li><li>Agnihotri, S. K., 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+membrane+potential+(MMP)+regulates+sperm+motility.">Mitochondrial membrane potential (MMP) regulates sperm motility.</a> In Vitro Cellular and Developmental Biology. Animal. 52 (9), 953-960 (2016).</li><li>Smiley, S. T., 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=Intracellular+heterogeneity+in+mitochondrial+membrane+potentials+revealed+by+a+J-aggregate-forming+lipophilic+cation+JC-1.">Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (9), 3671-3675 (1991).</li><li>Evenson, D. P., Darzynkiewicz, Z., Melamed, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+mammalian+sperm+chromatin+heterogeneity+to+fertility.">Relation of mammalian sperm chromatin heterogeneity to fertility.</a> Science. 210 (4474), 1131-1133 (1980).</li><li>Evenson, D. P., Djira, G., Kasperson, K., Christianson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+between+the+age+of+25,445+men+attending+infertility+clinics+and+sperm+chromatin+structure+assay+(SCSA&#174;)+defined+sperm+DNA+and+chromatin+integrity.">Relationships between the age of 25,445 men attending infertility clinics and sperm chromatin structure assay (SCSA&#174;) defined sperm DNA and chromatin integrity.</a> Fertility and Sterility. 114 (2), 311-320 (2020).</li><li>Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+chromatin+structure+assay+(SCSA&#174;).">Sperm chromatin structure assay (SCSA&#174;).</a> Methods in Molecular Biology. 927, 147-164 (2013).</li><li>Agarwal, A., Said, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+sperm+chromatin+abnormalities+and+DNA+damage+in+male+infertility.">Role of sperm chromatin abnormalities and DNA damage in male infertility.</a> Human Reproduction Update. 9 (4), 331-345 (2003).</li><li>Ricci, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+human+sperm:+its+correlation+with+semen+quality+and+the+presence+of+leukocytes.">Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes.</a> Human Reproduction. 17 (10), 2665-2672 (2002).</li><li>Zhang, H. -. B., 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=Early+apoptotic+changes+in+human+spermatozoa+and+their+relationships+with+conventional+semen+parameters+and+sperm+DNA+fragmentation.">Early apoptotic changes in human spermatozoa and their relationships with conventional semen parameters and sperm DNA fragmentation.</a> Asian Journal of Andrology. 10 (2), 227-235 (2008).</li><li>. WHO laboratory manual for the examination and processing of human semen Available from: <a target="_blank" href="https://www.who.int/publications/i/item/9789240030787">https://www.who.int/publications/i/item/9789240030787</a> (2022)</li><li>Evenson, D. P., Wixon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+toxicants+cause+sperm+DNA+fragmentation+as+detected+by+the+Sperm+Chromatin+Structure+Assay+(SCSA).">Environmental toxicants cause sperm DNA fragmentation as detected by the Sperm Chromatin Structure Assay (SCSA).</a> Toxicology and Applied Pharmacology. 207 (2), 532-537 (2005).</li><li>Wang, Y. X., 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=Phthalate+exposure+in+association+with+serum+hormone+levels,+sperm+DNA+damage+and+spermatozoa+apoptosis:+A+cross-sectional+study+in+China.">Phthalate exposure in association with serum hormone levels, sperm DNA damage and spermatozoa apoptosis: A cross-sectional study in China.</a> Environmental Research. 150, 557-565 (2016).</li><li>Ni, W., 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=Diurnal+variation+in+sperm+DNA+fragmentation:+analysis+of+11,382+semen+samples+from+two+populations+and+in+vivo+animal+experiments.">Diurnal variation in sperm DNA fragmentation: analysis of 11,382 semen samples from two populations and in vivo animal experiments.</a> Chronobiology International. 36 (11), 1455-1463 (2019).</li></ol>

The authors declare that there are no conflicts of interest.

Chromatin integrity, MMP, and apoptosis of human spermatozoa have been found to be valuable predictors of reproductive outcomes. The latest-released WHO laboratory manual for the examination and processing of human semen (sixth edition) also highlighted some of these indicators (chromatin integrity and apoptosis) as extended examinations beyond the basic examination of routine parameters such as sperm count18. Recent publications also suggest that these indicators may be more sensitive markers of response to external hazardous exposures, indicating their potential for the identification of reproductive damage at an earlier stage19,20. Combining these indicators with routine examinations may also provide a more comprehensive understanding about the profile of sperm dysfunction and the complexity of male reproductive damage in the population.
There are several key issues that substantially determine the success of sperm analysis with FCM. First, the measurement of sperm MMP and apoptosis requires immediate examination after the semen is liquefied. To minimize the time cost, two technicians can separately take charge of the pretreatment of MMP and apoptosis analysis of a sample. As the pretreatment of apoptosis is simpler, it would transfer to the flow-cytometric step faster than the MMP analysis. For the SCSA analysis, fresh semen samples should be cryopreserved immediately upon availability after liquefaction. The sperm samples can then be stored in liquid nitrogen for a relatively long period and do not need immediate examination.
Accordingly, the in-site time cost of the experiment can be compressed to 40 min, which is the duration of MMP analysis plus the flow-cytometric step of sperm analysis. Second, the setup of the gating in the flow cytometric platform must be decided for each batch of samples separately. The sperm samples with clear cell subgroups in the scatter plot can be selected as the reference to draw the gating area. Third, as there are no widely accepted clinical reference values for the indicators of this experiment, historic results may be used as an important tool for quality control. Positive and negative controls are also encouraged to be set in each batch of measurements, especially for SCSA and MMP.
The representative results of sperm analysis provided here are derived using an Accuri C6. However, the technique could be applied to other commercial platforms with minor modification. Usually, a total of 5 &#215; 106 sperm cells would be needed to meet the requirement for the measurement of all the indicators. If the sperm concentration of a sample is much lower than the average level, the need for the volume of semen would increase.
Similar to routine sperm parameters, there is also noticeable intraindividual variation in the chromatin integrity, MMP, and apoptosis of human spermatozoa21. Accordingly, multiple tests of samples collected at different times may provide a more accurate estimation of the sperm function damage level. Although there is no recommended period of abstinence before sperm sampling for the flow cytometric analysis, 2-7 days of ejaculatory abstinence, suggested for routine sperm analysis by WHO, may be adopted. It may help to improve the comparability of the results among laboratories and among different samples collected from the same men.
A major limitation of this method is that two of the three tested biomarkers - the apoptosis and mitochondrial membrane potential - require fresh semen samples. This may limit their application to men who can provide semen samples to the laboratory. For the test of DNA damage (SCSA), reference samples should be prepared before the experiment to calibrate the flow cytometer. Of note, the application of the present protocol requires an FCM instrument in the lab, which is still a considerable challenge for many clinical laboratories, although cooperation with a third-party service may be an alternative choice in some regions.
Additionally, the expense of the dyes and other reagents is also a financial factor for males who may need the examination. Lastly, the protocol may need modification if different brands or versions of FCM are used to examine the biomarkers. In summary, this paper presents a practical approach to estimate three biomarkers of human spermatozoa damage by a single flow cytometry machine. This may provide valuable information about male reproductive health and complement the routine sperm examination.

Infertility has become a public health problem, with male infertility contributing to 40%-50% of all cases1,2. Although conventional semen quality analysis plays an important role in determining male fertility potential, approximately 15% of patients with infertility have normal sperm parameters such as sperm count, motility, and morphology3. Moreover, routine sperm examinations have poor repeatability, provide limited information on sperm function, and cannot give an accurate evaluation of male fertility or reflect the subtle defects of sperm. Multiple techniques have been developed to test sperm function, such as the hemizona assay (HZA), the sperm penetration assay, the hypo-osmotic swelling test, the antisperm antibody testing, but these methods are time-consuming and vulnerable to subjective influences of the operators. Therefore, it is necessary to develop rapid and accurate methods for sperm function analysis.
FCM is a rapid, single-cell analysis technology developed in the 1970s, which has been widely used in various fields of cell biology and medicine. FCM is a robust tool for the evaluation of sperm function that analyzes spermatozoa labeled with a specific fluorescence probe4. The spermatozoa pass through single- or multi-channel lasers, and scattered light and emitted fluorescence are produced by a laser beam. The scattered light includes forward scattered light (FSC) and side scattered light (SSC), which reflects the size of the tested cell and the cell granularity or internal structure. These signals are collected, displayed, and analyzed by the computer system, and, consequently, a series of characteristics from the spermatozoa are measured quickly and accurately. Therefore, FCM is a rapid, objective, multi-dimensional, and high-throughput technique that has gained increasing attention in the field of sperm analysis. The application of FCM can make up for shortcomings in traditional methods and provides a new approach for the detection of sperm internal structure and function.
Sperm apoptosis is closely related to male fertility5. Detection of sperm apoptosis is an important index to evaluate sperm function at a molecular level. FCM is widely recognized as a reliable and sensitive method to detect sperm apoptosis using Annexin V-FITC/PI staining. The basic principle is that phosphatidylserine (PS) is transferred from the inner layer of the cell membrane to its outer layer in the early stage of apoptosis. Annexin V is a Ca2+-dependent phospholipid protein (usually labeled by FITC) that has a high affinity for PS, thus, detects PS exposed to the outer surface of the cell membrane6. Necrosis and apoptosis can be distinguished in combination with Pl staining. Therefore, the method of Annexin V-FITC/PI double staining is widely used, as it is rapid, simple, and easy to detect different apoptotic sperm cells.
A mature mammalian sperm contains approximately 72-80 mitochondria7, suggesting a biological reason for the retention of sperm mitochondria8. Sperm mitochondria have been found to play important roles in sustaining sperm motility and fertility9. The JC-1 stain, which can be used as an indicator of MMP in a variety of cell types, is one of the most popular fluorochromes for sperm mitochondrial activity assessment10. JC-1 is a cationic dye that potential-dependently accumulates in the mitochondria. JC-1 has a maximum fluorescence emission at 525 nm (green) and forms J-aggregates11 when binding to membranes with a high potential (&#916;&#936;m, 80-100 mV), resulting in a shift in fluorescence emission to ~590 nm (orange-red). Consequently, sperm mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio, and FCM can be used to detect MMP levels in human semen samples.
The SCSA methodology was invented by Evenson et al.12, and is considered a precise and repeatable test with unique dual-parameter data (red vs green fluorescence) on a 1,024 &#215; 1,024 channel scale13. In damaged sperm, DNA and altered proteins in sperm nuclei are labeled by AO, and breakages of the DNA strand are measured by red fluorescence, while intact double strands emit green fluorescence in FCM14. Nowadays, many methods have been developed to estimate sperm DNA integrity. However, unlike SCSA, these assays are often labor-intensive and lack the ability for a diagnosis of male infertility. The SCSA test results significantly corelate with human DNA pregnancy and miscarriage, provide compelling evidence that SCSA may be useful in human semen analysis, and may serve as a valuable tool to infertility clinicians15.
Chromatin integrity, MMP, and apoptosis reflect different aspects of sperm function, and thus, the combination of these biomarkers may provide a more comprehensive insight into sperm status. FCM can be used for separate measurements of either chromatin integrity, MMP, or apoptosis in human semen specimens. Although the expense of FCM and the cost of dyes have limited the wide application of these techniques in clinical laboratories for reproductive health, their value for fecundity estimation is being accepted.
However, as each of the experiments requires pretreatment of sperm samples, and all pretreated samples need to be tested as soon as possible, simultaneous measurement of all the three biomarkers for a sample with a single FCM may lead to a long waiting time for some of the experiments and obstruct the reliability of the results. This is because the sperms receive additional damage during the waiting process, if the protocol is not properly arranged taking all the three experiments entirely into consideration. This paper presents a protocol that realizes the fluent measurement of all the three biomarkers in a single cytometry without substantial compromise of experiment quality induced by prolonged waiting times.

<table><tbody><tr><td>0.1 M citric acid buffer</td><td>Sigma-Aldrich Chemical Co., USA</td><td>251275-5G</td><td>Add 21.01 g/L citric acid monohydrate (FW = 210.14; 0.10 M) to 1.0 L H&lt;sub&gt;2&lt;/sub&gt;O. Store up to several months at 4 &amp;deg;C.</td></tr><tr><td>0.2 M Na&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; buffer</td><td>Sigma-Aldrich Chemical Co., USA</td><td>V900061-500G</td><td>Add 28.4 g sodium phosphate dibasic (FW = 141.96; 0.2 M) to 1.0 L H&lt;sub&gt;2&lt;/sub&gt;O. Store up to several months at 4 &amp;deg;C.</td></tr><tr><td>10 mM CCCP stock solution</td><td>Sigma-Aldrich Chemical Co., USA</td><td>C2759</td><td>Add 20.46 mg CCCP to 10.0 mL DMSO,making up to the final of CCCP in a concentration of 10mM, aliquot and store at &amp;minus;80 &amp;deg;C.</td></tr><tr><td>10 &amp;micro;M CCCP working solution</td><td></td><td></td><td>Add 10 mL of PBS to a 15 mL polypropylene centrifuge tube and add 10 &amp;mu;L CCCP stock solution, making up to the final of CCCP in a concentration of 10 &amp;micro;M.</td></tr><tr><td>1 mM JC-1 stock solution</td><td>Sigma-Aldrich Chemical Co., USA</td><td>T4069</td><td>JC-1 is purchased lyophilized. Add a small quantity of DMSO to the vial and vortex for several minutes until all the dye has dissolved. Transfer the solution to a light-tight tube and rinse the vial with appropriate volume of DMSO, making up to the final of JC-1 in a concentration of 1 mg/mL&amp;nbsp; aliquot and store at &amp;minus;20 &amp;deg;C.</td></tr><tr><td>37 &amp;deg;C incubator</td><td>Thermo Scientific, USA</td><td></td><td></td></tr><tr><td>5 &amp;micro;M JC-1 working solution</td><td></td><td></td><td>Add 10 mL of PBS to a 15 mL polypropylene centrifuge tube and add 50 &amp;mu;L from a thawed JC-1 stock aliquot. Stir gently to assure a homogenous dilution. This solution must be stored in the dark and used promptly.</td></tr><tr><td>Accuri C6 Flow cytometer</td><td>BD Pharmingen, San Diego, CA, United States</td><td></td><td></td></tr><tr><td>Acid solution, pH 1.2</td><td></td><td></td><td>Combine 20.0 mL of 2.0 N HCl (0.08 N), 4.39 g of NaCl (0.15 M), and 0.5 mL of Triton X-100 (0.1%) in H&lt;sub&gt;2&lt;/sub&gt;O for a final volume of 500 mL. Adjust pH to 1.2 with 5 mol/L HCl.</td></tr><tr><td>Acridine Orange (AO) stock solution, 1.0 mg/mL</td><td>Polysciences, Inc, Warrington, Pa</td><td>65-61-2</td><td>Dissolve chromatographically purified AO in dd-H&lt;sub&gt;2&lt;/sub&gt;O at 1.0 mg/mL.</td></tr><tr><td>AO staining solution (working solution)</td><td></td><td></td><td>Add 600 &amp;mu;L of AO stock solution to each 100 mL of staining buffer.</td></tr><tr><td>Biological safety hood</td><td>Airtech, USA</td><td></td><td></td></tr><tr><td>Computer-aided sperm analysis system (CASA )</td><td>Microptic, Barcelona, Spain</td><td></td><td>Sperm Class Analyzer 5.3.00</td></tr><tr><td>Sperm Counting Chamber</td><td>Goldcyto, Spain</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD Biosciences, San Jose, CA</td><td>556547</td><td>Included: (1) FITC Annexin V is bottled at 100 ng/&amp;micro;L; (2) Propidium Iodide (PI):The PI Staining Solution is composed of 50 &amp;micro;g PI/mL in PBS (pH 7.4) and is 0.2 &amp;micro;M sterile filtered; (3) 10x Binding Buffer: 0.1 M Hepes (pH 7.4), 1.4 M NaCl, 25 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. For a 1x working solution, dilute 1 part of the 10x Annexin V Binding Buffer to 9 parts of distilled water. Store at 4 &amp;deg;C and protected from prolonged exposure to light. Do not freeze.</td></tr><tr><td>FlowJo&amp;nbsp;10</td><td>Tree Star, Inc., San Carlos, CA, USA</td><td></td><td></td></tr><tr><td>Horizontal centrifuge</td><td>Thermo Scientific, USA</td><td></td><td></td></tr><tr><td>liquid nitrogen tank</td><td>Thermolyne, USA</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Beyotime, Shanghai, China</td><td>C0221A</td><td>Ready-to-use PBS buffers is purchased and stored at room temperature</td></tr><tr><td>Staining buffer, pH 6.0</td><td></td><td></td><td>Combine 370 mL of 0.10 M citric acid buffer, 630 mL of 0.20 M Na&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; buffer, 372 mg of EDTA (disodium, FW = 372.24; 1 mM), and 8.77 g of NaC1 (0.15 M). Mix overnight on a stir plate to insure that the EDTA is entirely in solution. pH to 6.0 with saturated NaOH solution.</td></tr><tr><td>The reference sample for SCSA analysis</td><td></td><td></td><td>The reference sample was diluted with cold (4 &amp;deg;C) TNE buffer to a working concentration of 1&amp;ndash;2 &amp;times; 10&lt;sup&gt;6&lt;/sup&gt; cells/mL, and used to set the green at 475/1,024 flow cytometry channels and set the red at 125/1,024 flow cytometry channels.</td></tr><tr><td>TNE buffer, 1x, pH 7.4 (working solution)</td><td></td><td></td><td>Combine 60 mL of 10x TNE and 540 mL of H&lt;sub&gt;2&lt;/sub&gt;O. Check pH (7.4).</td></tr><tr><td>TNE buffer, 10x, pH 7.4</td><td>Perfemiker, Shanghai, China</td><td>PM11733</td><td>Ready-to-use buffers is purchased and stored at 4 &amp;deg;C.</td></tr></tbody></table>

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.

Figure 2 shows the measurement of sperm apoptosis using Annexin V-FITC/PI staining. The FITC signal (green fluorescence) was measured in the FL1 channel, and the PI signal (red fluorescence) was measured in the FL2 channel. In the Annexin V/PI bivariate analysis, the quadrant makers identified four distinctive sperm populations. The results were expressed as the percentages of Annexin V-/PI&#8722; sperm (viable or live cells), Annexin V+/PI&#8722; sperm (early apoptotic cells or apoptotic cells), Annexin V+/PI+ sperm (late apoptotic cells), and Annexin V&#8722;/PI+ sperm (necrotic cells)16,17 (Figure 2B). A negative control (unstained sperm cells) was also used to set up compensation and quadrants (Figure 2A).
Figure 3 shows the measurement of MMP using JC-1 probe in human sperm. MMP % is presented as the orange-red/(green + orange-red) fluorescence ratio by analyzing the cytogram. A good-quality semen sample usually shows high orange-red fluorescence and low green fluorescence (active mitochondria, upper-left quadrant) (Figure 3A). Conversely, a poor-quality semen sample usually shows low orange-red fluorescence and high green fluorescence (inactive mitochondria, lower-right quadrant), possibly due to mitochondrial disruption (Figure 3B).
Figure 4 shows the data of SCSA13,14. These typical SCSA cytograms were obtained from two individual samples. Sample A came from a fertile man and sample B from an infertile man. Analysis of the flow cytometric data was carried out using FlowJo software. Cytograms show the source of each component of SCSA data. The x-axis (red fluorescence with 1,024 gradations of red fluorescence) indicates fragmented DNA; the y-axis (green fluorescence with 1,024 gradations) indicates native DNA stainability. The dotted line at y = 750 represents the boundary of normal sperm. Above that line of y = 750 are sperm with partially uncondensed chromatin, which were determined as immature sperm.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63790/63790fig01.jpg" /> 
Figure 1: Workflow for the detection of biomarkers for sperm function damage by flow cytometry. Semen samples were obtained and pretreated as described in protocol step 1. (1) Cell preparation, (2) staining with fluorescent reagents, and (3) flow cytometric analysis and data interpretation. Abbreviations: MMP = mitochondrial membrane potential; SCSA = sperm chromatin structure assay; PBS = phosphate-buffered saline; AO = acridine orange. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63790/63790fig01large.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/63790/63790fig02.jpg" /> 
Figure 2: Representative results of sperm apoptosis using Annexin V-FITC/PI staining. The lower left quadrant contains Annexin V-/PI&#8722; sperm (viable or live cells), the lower right quadrant shows Annexin V+/PI&#8722; sperm (early apoptotic cells or apoptotic cells), the upper right quadrant represents Annexin V+/PI+ sperm (late apoptotic cells), and the upper left quadrant contains Annexin V&#8722;/PI+ sperm (necrotic cells). (A) Negative control (unstained sperm cells); (B) a sample with sperm apoptosis. Abbreviations: FITC = fluorescein isothiocyanate; PI = propidium iodide. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63790/63790fig02large.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/63790/63790fig03.jpg" /> 
Figure 3: Measurement of mitochondrial membrane potential using JC-1 probe. (A) The sperm subpopulation with high MMP. (B) The sperm subpopulation with low MMP. Set FL1-A and FL2-A as the x axis (green fluorescence) and the y-axis (orange-red fluorescence), respectively. Abbreviations: MMP = mitochondrial membrane potential. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63790/63790fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63790/63790fig04.jpg" /> 
Figure 4: Detecting DNA damage in human sperm using sperm chromatin structure assay. (A) A semen sample with normal chromatin structure. (B) A semen sample with high proportion of spermatozoa with abnormal chromatin structure. FlowJo software was used to make computer gates around the cell populations identified as: 1) normal sperm, 2) HDS sperm, 3) DFI sperm, and 4) cell debris, and percentages of HDS and DFI sperm were calculated. Abbreviation: SCSA = sperm chromatin structure assay; HDS = high DNA stainability; DFI = DNA fragmentation index. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63790/63790fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects at JoVE.com

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

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 ...

flow-cytometric-analysis-biomarkers-for-detecting-human-sperm

Nanyang Technological University

Research

JoVE Journal

Medicine

2.2K Views.  Army Medical University (Third Military Medical University). 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.

Video: Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

Step-specific Sorting of Mouse Spermatids by Flow Cytometry

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to the next generation. So far, molecular studies of this morphological transition have been hampered by the lack of a method allowing adequate separation of these important steps of spermatid differentiation for subsequent analyses. Earlier attempts at proper gating of these cells using flow cytometry may have been difficult because of a peculiar increase in DNA fluorescence in spermatids undergoing chromatin remodeling. Based on this observation, we provide details of a simple flow cytometry scheme, allowing reproducible purification of four populations of mouse spermatids fixed with ethanol, each representing a different state in the nuclear remodeling process. Population enrichment is confirmed using step-specific markers and morphological criterions. The purified spermatids can be used for genomic and proteomic analyses.

We describe a sorting strategy for mouse spermatids using flow cytometry. Spermatids are sorted into four highly pure populations, including round (spermiogenesis steps 1-9), early elongating (spermiogenesis steps 10-12), late elongating (spermiogenesis steps 13-14) and elongated spermatids (spermiogenesis steps 15-16). DNA staining, size and granulosity are used as selection parameters.

The differentiation of mouse spermatids is one critical process for the production of a functional male gamete with an intact genome to be transmitted to ...

Developmental Biology

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Biology

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, ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Fish Sperm Assessment Using Software and Cooling Devices

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems for sperm analysis were developed to measure several parameters and one of the most commonly measured is the sperm motility.
Initially, this computer technology was designed for mammalian species, although it can also be used for fish sperm analysis. Fish have specific features that can affect sperm assessment such as a short motility time after activation and, in some cases, adaptation to lower temperatures. Thus, it is necessary to modify both software and hardware components to make motility analysis more efficient for fish sperm analysis. For mammalian sperm, the heating plate is used to maintain optimal temperatures of spermatozoa. However, for some fish species, it is advantageous to use a lower temperature to prolong the duration of motility, since the sperm remain active for less than 2 min. Therefore, cooling devices are necessary to refrigerate samples at constant temperature over the time of analysis, including on the optical microscope. This protocol describes the analysis of fish sperm motility using software for sperm analysis and new cooling devices to optimize the results.

The present protocol describes a procedure of fish sperm assessment using computer-assisted sperm analysis and cooling devices. The software gives a rapid, accurate and quantitative analysis of fish sperm quality based on spermatozoa motility, which can be a useful tool in aquaculture to improve reproduction success.

For gamete quality evaluation, there are innovative, rapid, and quantitative techniques that can provide useful data for aquaculture. Computerized systems ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Flow Cytometric Analysis of Biomarkers for Detecting Human Sperm Functional Defects

Summary

Explore More Videos

Chapters in this video