The authors would like to acknowledge the lab of Timo Str&#252;nker for supervision of AR and DLE during their stays at his lab. Furthermore, we would like to thank our colleagues at the Department of Growth and Reproduction, Copenhagen University Hospital, Rigshospitalet for their assistance with setting up these two assays. This project was supported by the Danish Environmental Protection Agency as a project under Centre on Endocrine Disrupters and by grants from the Innovation Fund Denmark (grant numbers 005-2010-3 and 14-2013-4).

The collection and analysis of human semen samples in the protocols follows the guidelines of the Research Ethics Committee of the Capital Region of Denmark. All semen samples have been obtained after informed consent from volunteer donors. After delivery, the samples were fully anonymized. For their inconvenience each donor received a fee of 500 DKK (about $75 US dollars) per sample. The samples were analyzed on the day of delivery and then destroyed immediately after the laboratory experiments.
NOTE: The medium throughput Ca2+-signaling assay is described in steps 4-5 and the medium throughput acrosome reaction assay in steps 6-7. The protocol to prepare human tubal fluid (HTF+) medium is described in step 1 and the purification of sperm cells for the assays is described in steps 2-3.
1. Preparation of Human Tubal Fluid (HTF+) Medium
NOTE: Use volumetric flasks of appropriate sizes, a 1 L measuring cylinder, a magnetic stirrer, salts as listed in Table 1 and Table 2, purified water, a 0.2 &#181;m pore filter, and 50 mL plastic tubes.
<ol>
	<li>Prepare stock solutions of salts in purified water (Table 1).
	<ol>
		<li>Add the amount of salt listed in Table 1 to a volumetric flask of the appropriate size, add purified water to a bit below the desired volume, and mix well using a magnetic stirrer.</li>
		<li>When salt is fully dissolved, remove the magnet and fill with purified water to the desired final volume.</li>
	</ol>
	</li>
	<li>Transfer stock solution to a bottle and store until use. 
	NOTE: Stock solutions can be kept and reused for longer periods of time: glucose and HEPES at -20 &#176;C and the other stock solutions at room temperature (RT).</li>
	<li>Prepare 1 L of HTF+ medium from stock solutions (Table 2).
	<ol>
		<li>Ensure that all stock solutions are completely dissolved and well mixed before use.</li>
		<li>Add the amount of stock solution and salt listed in Table 2 to a 1 L measuring cylinder and add purified water to 900 mL.</li>
		<li>Mix well using a magnetic stirrer and adjust pH to 7.35 with NaOH solution.</li>
		<li>Remove the magnet and add purified water to 1,000 mL.</li>
	</ol>
	</li>
	<li>Sterile filtrate HTF+ medium through a 0.2 &#181;m pore filter, aliquot HTF+ medium to 50 mL plastic tubes, and store at 4 &#176;C until use.</li>
	<li>Just before running an experiment, add 165 &#181;L of Na-pyruvate to the 50 mL aliquot to get a final Na-pyruvate concentration of 0.33 mM. 
	NOTE: Human tubal fluid medium can also be obtained from various commercial providers. When using the 4 mM HCO3- medium, samples can be incubated with closed lids in an incubator without extra CO2 in the air without large drifts in pH. If a CO2 incubator is available, the corresponding amount of CO2 can be calculated using the Henderson-Hasselbalch equation and used. If using the 25 mM HCO3- medium, samples should be incubated with open lids in a CO2 incubator with air containing a corresponding amount of CO2 (depends on atmospheric pressure, usually between 5-10%) to avoid a drift in pH. The exact amount of CO2 can be calculated using the Henderson-Hasselbalch equation.</li>
</ol>
2. Purification of Motile Sperm Cells via Swim-up (Figure 1)
NOTE: Use a clean wide-mouthed plastic container for the semen sample, an incubator, 50 mL plastic tubes, a rack for placing 50 mL plastic tubes at a 45&#176; angle, HTF+ medium (prepared in step 1 or obtained from commercial provider), a centrifuge, and human serum albumin (HSA).
<ol>
	<li>Obtain a freshly donated human semen sample produced through masturbation and ejaculated into a wide-mouthed clean plastic container. Only use semen samples that fulfill the parameters established by the WHO laboratory manual for examination and processing of human semen.</li>
	<li>Incubate the sample at 37 &#176;C for 15-30 min to allow it to liquefy.</li>
	<li>Heat up 50 mL of HTF+ medium with 4 mM HCO3- to 37 &#176;C.</li>
	<li>Place clean 50 mL plastic tubes in a rack at a 45&#176; angle (to increase the surface area between liquids). Use 1 tube per mL of raw semen.</li>
	<li>Add 4 mL of the preheated HTF+ medium to each of the tubes.</li>
	<li>Carefully pipette 1 mL of the liquefied semen sample to the bottom of each tube containing 4 mL of preheated HTF+. Avoid release of air bubbles.</li>
	<li>Close the lids and incubate the tubes at 37 &#176;C for 1 h.</li>
	<li>Gently aspirate and transfer as much of the upper fraction (HTF+ with motile sperm cells) as possible to a new 15 mL plastic tube, while paying attention that nothing from the bottom fraction (semen with immotile and dead cells) is included. Generally, it is safe to transfer 3.5 mL of the top fraction, without disturbing the bottom fraction. To avoid suction turbulence, cut off the outermost 1-2 mm of the pipette tip at a 45&#176; angle to obtain a larger opening.</li>
	<li>Fill up the 15 mL plastic tube with HTF+ to wash cells and centrifuge the tube at 700 x g for 10 min at RT.</li>
	<li>Gently aspirate and remove the supernatant and resuspend the sperm cell pellet in 2 mL of HTF+.</li>
	<li>Transfer 20 &#181;L of the sperm suspension to two small plastic tubes for assessment of sperm concentration (see step 3).</li>
	<li>Fill up the 15 mL plastic tube with HTF+ to wash cells and centrifuge the tube at 700 x g for 10 min at RT.</li>
	<li>Gently aspirate and remove the supernatant and resuspend the sperm cell pellet to 10 x 106 cells/mL (based on the cell concentration assessed in step 2.11).
	<ol>
		<li>For experiments using un-capacitated sperm cells (routine Ca2+-signaling assay)
		<ol>
			<li>Resuspend cells in HTF+ with 4 mM HCO3-.</li>
			<li>Add 30 &#181;L of human serum albumin (HSA) (100 mg/mL) per mL HTF+ to get a final concentration of 3 mg/mL.</li>
			<li>Close lids and incubate for &#8805;1 h at 37 &#176;C.</li>
		</ol>
		</li>
		<li>For experiments using capacitated sperm cells (acrosome reaction assay)
		<ol>
			<li>Resuspend cells in HTF+ with 25 mM HCO3-.</li>
			<li>Add 30 &#181;L of HSA (100 mg/mL) per mL HTF+ to get a final concentration of 3 mg/mL.</li>
			<li>Attach lids loosely and incubate for &#8805;3 hours at 37 &#176;C with the corresponding amount of CO2 (see step 1, usually between 5-10% CO2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Counting of Sperm Cells
NOTE: Sperm cells can either be counted manually50 or using an image cytometer51, as described here. Use an image cytometer, a vortexer, S100 buffer, a SP1-cassette, swim-up purified sperm cells (prepared in step 2).
<ol>
	<li>Dilute a 20 &#181;L aliquot to a factor of 10, 20, 30, 50 or 90 in S100 buffer using a round bottom 2 mL tube according to the expected concentration (evaluated by manual inspection of the pellet in step 2.10). Use the dilution that is closest to the concentration expected (i.e., if a concentration of 15 x 106 sperm cells per mL after swim-up is expected then dilute the 20 &#181;L sperm sample with 380 &#181;L of S100 buffer [dilution factor 20]).</li>
	<li>Mix well for 10 s using a vortex mixer at the maximal 700 rpm, immerse the SP1-cassette into the sample and press the white plunger fully down to aspirate an aliquot of the sample into the cassette.</li>
	<li>Open the image cytometer, place the cassette in the tray and run the assay Count of PI Stained Human Sperm Cells Assay according to the applied dilution factor (chosen in step 3.1).</li>
	<li>Repeat step 3.1-3.3 with another 20 &#181;L sample, using a dilution factor closest to the measured concentration obtained from the first 20 &#181;L sample.</li>
	<li>Calculate mean sperm cell concentration from the two measurements.</li>
	<li>Multiply mean sperm cell concentration with the original volume to obtain the total sperm cell count (in step 2, this original volume is 2 mL).</li>
</ol>
4. Measurement of Changes in the Free Intracellular Ca2+-concentration ([Ca2+]i) Using Ca2+-fluorimetry (Figure 2)
NOTE: Use a fluorescence plate reader, 384 multi-well plates, Fluo-4 AM, swim-up purified sperm cells (prepared in step 2), compounds of interest as well as positive and negative controls, a automatic repeater pipette, and a 12-channel pipette.
<ol>
	<li>Prepare a 2 mM Fluo-4 AM stock by adding 22.7 &#181;L of dimethyl sulfoxide (DMSO) to a vial of 50 &#181;g Fluo-4 AM and mix the tube well.</li>
	<li>Add an aliquot of 700 &#181;L swim-up recovered sperm suspension (capacitated or un-capacitated as outlined in step 2) to a new test tube. 700 &#181;L of sperm sample is the amount needed to analyze one row of 24 wells in the 384-microwell plate (used to test 3 controls and 9 compounds in doublets).</li>
	<li>Add 3.5 &#181;L of Fluo-4 AM stock solution to the 700 &#181;L of sperm suspension to obtain a final concentration of 10 &#181;M Fluo-4 AM.</li>
	<li>Incubate sample for 45 min at 37 &#176;C, protected from light.</li>
	<li>Centrifuge sample at 700 x g for 10 min, aspirate and discard the supernatant to remove excess Fluo-4.</li>
	<li>Resuspend stained pellet in HTF+ to 5 x 106 cells/mL (i.e., use 2x the volume of cell suspension taken in step 4.2)</li>
	<li>Prepare dilutions of compounds and controls (can be done during the incubation and centrifugation periods in step 4.4 and 4.5, respectively).
	<ol>
		<li>Dilute compounds, as well as a negative control (buffer with vehicle) and a positive control (progesterone) in HTF+. Dilute compounds and controls to 3x the desired final concentration, as they are diluted 1:3 when they are added to the sperm cells in step 4.16.</li>
		<li>Place the tubes with dilutions in a rack with a distance between tubes corresponding to the distance between the pipette tips of the 12-channel pipette used in step 4.16.</li>
	</ol>
	</li>
	<li>Use an automatic repeater pipette (24 steps of 50 &#181;L).
	<ol>
		<li>Mix Fluo-4 stained sperm sample gently by pipetting the entire amount up and down once.</li>
		<li>Add aliquots of 50 &#181;L to the 24 wells of a row in the 384-microwell plate.</li>
	</ol>
	</li>
	<li>Place the microwell plate in a fluorescence plate reader at 30 &#176;C and close the drawer.</li>
	<li>Select the appropriate protocol for Fluo-4. Excite fluorescence at 480 nm and record emission at 520 nm. Use the fastest cycle time possible with the setting. 
	NOTE: Use at least 10 flashes per well and bottom optics, if possible.</li>
	<li>Select a well in the row and adjust gain to a target value of 20%.</li>
	<li>Start the measurement.</li>
	<li>Control the baseline during the first 5 cycles.
	<ol>
		<li>If the fluorescent readout is increasing or decreasing over time, stop the experiment, readjust the gain and start the experiment again.</li>
		<li>If the baseline differs a lot between individual wells in a row, either the pipetting in step 4.8 has been unprecise or the sample was not mixed well enough before pipetting. Discard the experiment.</li>
		<li>If the fluorescent readout is comparable between the wells in the row and steady during the 5 first cycles, continue to the next step.</li>
	</ol>
	</li>
	<li>After 10 cycles (used for baseline), pause the experiment.</li>
	<li>Eject the drawer with the microwell plate.</li>
	<li>Using a 12-channel pipette (2 steps of 25 &#181;L), quickly add 25 &#181;L of the prepared solutions of compounds and controls (from step 4.7) to the 12 well duplicates (24 wells) of a row. Solutions will be diluted 1:3, as the wells already contain 50 &#181;L of stained sperm sample.</li>
	<li>Continue the measurement as quickly as possible (drawer will automatically close) to avoid missing any fluorescent signals induced by the added compounds and controls. Changes in fluorescence (&#916;F) after the addition of compounds and controls will now be captured.</li>
	<li>Run the experiment until fluorescent signals have been recorded from the positive control and the compounds of interest.</li>
	<li>Stop the experiment and export the raw fluorescence data to analyze it as in step 5. 
	NOTE: Generally Fluo-4 AM has been used as the Ca2+-sensitive fluorophore in the assay, but other Ca2+-sensitive fluorophores could also be used if excitation and emission spectra fit with filters in the fluorescence plate reader. Depending on the choice of fluorophore, make sure that the gain level is set at a &#8220;safe&#8221; level where the induced fluorescent peaks are within the measuring range of the instrument. This can be tested by adding ionomycin to a single well at a specific gain setting. If this induced a fluorescent signal above the threshold, lower the gain and try again. Some use Pluronic F-127 to aid the loading of Fluo-41,13, but it has been found that this is not necessary2. The maximal number of rows measured simultaneously depends on two factors. 1) The cycle time of the instrument: If the cycle time is too long, induced peaks in [Ca2+]i might be missed. Measure a maximum of two rows simultaneously to keep the cycle time low; 2) The speed of pipetting in step 4.16: Using the 12-channel pipette, it is possible to quickly add 12 different solutions to the 12 duplicate wells (24 wells) of 1 or 2 rows (e.g., one row pre-incubated with vehicle and one row pre-incubated with an inhibitor), without missing the induced Ca2+ peaks. However, it is not recommended to try to add 24 different solutions to two rows for simultaneous measuring, as pipetting tips will have to be replaced in between the two sequential pipetting steps, whereby induced Ca2+ peaks could be missed.</li>
</ol>
5. Analysis of Data from Ca2+-fluorimetry
<ol>
	<li>Open the raw fluorescence data obtained and exported in step 4.</li>
	<li>Calculate the average from duplicate wells. If large differences exist between duplicates, discard data from the specific wells.</li>
	<li>Define baseline as the 10 first cycles.</li>
	<li>Calculate &#916;F/F0 (%) as the percentage change in fluorescence (&#916;F) over time after addition of compounds and controls with respect to the mean basal fluorescence (F0) during the 10 first cycles (baseline).</li>
	<li>If using the data for generation of dose-response curves, subtract the readout of negative control from the other wells, to remove pipetting artifacts.</li>
</ol>
6. Measurement of Acrosome Reaction
NOTE: Use an image cytometer, an incubator, fluorescent dyes: PI, FITC-PSA, and Hoechst-33342, compounds of interest as well as positive and negative controls, an immobilizing solution containing 0.6 M NaHCO3 and 0.37% (v/v) formaldehyde in distilled water, an A2 slide, and capacitated swim-up purified sperm cells (prepared in step 2).
<ol>
	<li>Prepare a staining solution containing PI (5 &#181;g/mL), FITC-PSA (50 &#181;g/mL) and Hoechst-33342 (100 &#181;g/mL) in HTF+, mix it well using a vortex mixer and protect it from light until use.</li>
	<li>Prepare solutions of compounds and controls at 10x the desired final concentration, as they are diluted 1:10 in step 6.5. Always include a negative (vehicle) control and two positive controls: Progesterone 10 &#181;M final concentration and ionomycin 2 &#181;M final concentration.</li>
	<li>Transfer aliquots of 240 &#181;L of capacitated sperm cells (prepared in step 2) to plastic tubes.</li>
	<li>Add 30 &#181;L of staining solution to each tube. Final concentration of stains will be: 0.5 &#181;g/mL PI, 5 &#181;g/mL FITC-PSA, and 10 &#181;g/mL Hoechst-33342.</li>
	<li>Add 30 &#181;L of solutions with controls or compounds to each tube (1:10 dilution).</li>
	<li>Mix the samples gently by pipetting up and down a few times or mild vortexing. Due to mechanical stress, avoid excessive mixing.</li>
	<li>Incubate at 37 &#176;C for 30 min.</li>
	<li>Prepare new plastic tubes with 100 &#181;L of an immobilizing solution containing 0.6 M NaHCO3 and 0.37% (v/v) formaldehyde in distilled water, one per test tube.</li>
	<li>After incubation, mix the samples well by pipetting, and add 50 &#181;L of the test sample to a tube with 100 &#181;L of immobilizing solution.</li>
	<li>Before loading the chambers of an A2 slide, mix the sample well by pipetting. Fill the chambers carefully with approximately 30 &#181;L without creating air bubbles. 
	NOTE: During the incubation, the motile sperm cells tend to aggregate. Cell clumps can disturb the measurements (even though they are excluded). Therefore, a thorough mixing by pipetting after incubation is very important. Likewise, air bubbles in the chambers can disturb the measurements. Therefore, fill the chamber slowly and change the angle of the pipette if edges of the liquid are about to meet and create an air bubble.</li>
	<li>Place the loaded slide on the tray of the image cytometer and close the tray.</li>
	<li>Select the FlexiCyte protocol and press Run.
	<ol>
		<li>Choose the following settings for the FlexiCyte protocol: Number of analytical channels: 2; Masking channel: UV (LED365), This is the channel used for image segmentation; Channel 1: Blue (LED475), exposure time 3,000 ms, emission filter 560/35; Channel 2: Green (LED530), exposure time 500 ms, emission filter 675/75; Minimum number of analyzed cells: 5000; Exclude aggregated cells: Yes.</li>
	</ol>
	</li>
	<li>Repeat step 6.9-6.12 until all samples have been measured.</li>
</ol>
7. Analysis of Data from image Cytometry
<ol>
	<li>Open data obtained in step 6.</li>
	<li>Create the two following plots to evaluate the measurements.
	<ol>
		<li>Create a scatter plot of Hoechst-33342 intensity vs. Hoechst-33342 area on biexponential scales. This plot can be used to gate out Hoechst-33342 positive objects that are either too small or too large to be human sperm cells.</li>
		<li>Create a scatter plot of FITC-PSA intensity and PI intensity on biexponential scales. This plot can be used to asses the amount of acrosome reacted sperm cells by use of a quadrant gate that separates the four groups on the plot (Figure 3): 1) a PI positive and FITC-PSA positive group: Acrosome reacted nonviable sperm cells; 2) a PI negative and FITC-PSA positive group: Acrosome reacted viable sperm cells; 3) a PI positive and FITC-PSA negative group: Acrosome intact nonviable sperm cells; and 4) a PI negative and FITC-PSA negative group: Acrosome intact viable sperm cells.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The medium throughput Ca2+ signaling assay is based on measurements of fluorescence from single microwells each containing about 250,000 sperm cells. The captured signal is averaged from all individual sperm cells in the well. The assay thus provides no spatial information about where specifically in the sperm cell [Ca2+]i is changed, in how large a proportion of the sperm cells a change in [Ca2+]i takes place, or how heterogeneous the response is between the individ...

The purpose of the two assays described here is to examine effects on Ca2+-signaling and acrosome reaction in human sperm, as has been shown for multiple compounds in several publications employing these assays1,2,3,4,5,6,7. Ca2+-signaling and the acrosome reaction are both vital to normal human sperm cell function and male fertility.
The overall goal of a human sperm cell is to fertilize the egg. To be able to successfully and naturally fertilize the egg, the functions of the sperm cell must be regulated tightly during the journey of the sperm cell through the female reproductive tract8,9. Many of the sperm cell functions are regulated via the intracellular Ca2+-concentration [Ca2+]i (e.g., sperm motility, chemotaxis, and acrosome reaction)10. Also, a maturation process called capacitation, which renders the sperm cell capable of fertilizing the egg, is partly regulated by [Ca2+]i10. Ca2+-extruding Ca2+-ATPase pumps11 maintain an approximately 20.000 fold Ca2+-gradient over the human sperm cell membrane, with a resting [Ca2+]i of 50-100 nM. If Ca2+ is allowed to cross the cell membrane (e.g., through the opening of Ca2+-channels), a sizeable influx of Ca2+ occurs, giving rise to an elevation of [Ca2+]i. However, the sperm cell also carries intracellular Ca2+-stores, which can release Ca2+ and, therefore, also give rise an elevation of [Ca2+]i12. Interestingly, all channel-mediated Ca2+-influx in human sperm cells has so far been found to occur via CatSper (Cationic channel of Sperm), which is only expressed in sperm cells11. In human sperm cells, CatSper is activated by the endogenous ligands progesterone and prostaglandins through distinct ligand binding sites13,14,15, leading to a rapid Ca2+-influx into the sperm cell. Two main sources near the egg provide high levels of these endogenous ligands. One is the follicular fluid that contains high levels of progesterone16. The follicular fluid is released from mature follicles together with the egg at ovulation and mixes with the fluid within the oviducts17. The other main source is the cumulus cells that surround the egg and release high levels of progesterone and prostaglandins. The progesterone-induced Ca2+-influx in the sperm cells has been shown to mediate chemotaxis towards the egg9,18, control sperm motility19,20 and stimulate the acrosome reaction21. Triggering of these individual [Ca2+]i-regulated sperm functions in the correct order and at the correct time is crucial for fertilization of the egg8. In line with this, a suboptimal progesterone-induced Ca2+-influx has been found to be associated with reduced male fertility22,23,24,25,26,27,28,29 and functional CatSper is essential for male fertility26,30,31,32,33,34,35,36.
As the sperm cells reach the egg, a sequence of events must take place for fertilization to occur: 1) The sperm cells must penetrate the surrounding cumulus cell layer, 2) bind to the zona pellucida, 3) exocytose the acrosomal content, the so-called acrosome reaction37, 4) penetrate the zona pellucida, and 5) fuse with the egg membrane to complete fertilization38. To be able to go through these steps and fertilize the egg, the sperm cell must first undergo capacitation11, which begins as the sperm cells leave the seminal fluid containing &#34;decapacitating&#34; factors39 and swim into the fluids of the female reproductive tract with high levels of bicarbonate and albumin37. Capacitation renders the sperm cells able to undergo hyperactivation, a form of motility with a vigorous beating of the flagellum, and acrosome reaction37. Hyperactivated motility is required for penetration of the zona pellucida40, and the acrosome contains various hydrolytic enzymes that aid this penetration process41. Additionally, the acrosome reaction renders the sperm cells capable of fusing with the egg by exposing specific membrane proteins on the sperm surface necessary for sperm-egg fusion42. Consequently, the ability to undergo hyperactivation and acrosome reaction are both required for successful fertilization of the egg40,42. Contrary to what has been seen for mouse sperm cells43,44,45, only human sperm cells that are acrosome-intact can bind to the zona pellucida46. When the human sperm cells are bound to the zona pellucida they have to undergo the acrosome reaction both to penetrate the zona pellucida41 and to expose specific membrane proteins that are needed for the fusion with the egg38. The timing of the acrosome reaction in human sperm is thus critical for fertilization to occur.
As described above, Ca2+-signaling is vital for normal sperm cell function8, and it is, therefore, of great interest to be able to screen large numbers of compounds for effects on Ca2+-signaling in human sperm cells. Similarly, as only human sperm cells that undergo acrosome reaction at the right time and place can penetrate the zona pellucida and fertilize the egg46,47, it is also of great interest to be able to test compounds for their ability to affect the acrosome reaction in human sperm. To this end, two medium-throughput screening assays are described: 1) an assay for effects on Ca2+-signaling in human sperm cells, and 2) an assay for the ability to induce acrosome reaction in human sperm cells.
Assay 1 is a medium-throughput Ca2+-signaling assay. This fluorescence plate reader-based technique monitors changes in fluorescence as a function of time simultaneously in multiple wells. The Ca2+-sensitive fluorescent dye, Fluo-4 has a Kd for Ca2+&#8776; 335 nM and is cell-permeant in the AM (acetoxymethyl) ester form. Using Fluo-4, it is possible to measure changes in [Ca2+]i over time and after the addition of compounds of interest to the sperm cells. The assay was developed by the lab of Timo Str&#252;nker in 201113 and has since been used in several studies to screen compounds for effects on Ca2+-signaling in human sperm1,2,3,4,5. Also a similar method has been used to screen multiple drug candidates48. In addition, this assay is also useful for assessing the pharmacological mode of action1,2,3,4,5, dose-response curves1,2,3,4,5, competitive inhibition1,2, additivity1,2, and synergism3 of compounds of interest.
Assay 2 is a medium-throughput acrosome reaction assay. This image cytometer-based technique measures the amount of viable acrosome reacted sperm cells in a sample, using three fluorescent dyes: propidium iodide (PI), FITC-coupled lectin of Pisum sativum (FITC-PSA), and Hoechst-33342. The assay was modified from a similar flow cytometry-based method by Zoppino et al.49 and has been used in several studies6,7. As for the Ca2+-signaling assay, this acrosome reaction assay could also be used to assess dose-response curves, inhibition, additivity, and synergism of compounds of interest.

<table><tbody><tr><td>0.2 &amp;micro;m pore filter</td><td>Thermo Fisher Scientific, USA</td><td>296-4545</td><td></td></tr><tr><td>1 L measuring cylinder</td><td>Thermo Fisher Scientific, USA</td><td>3662-1000</td><td></td></tr><tr><td>1, 4, and 2 mL plastic tubes</td><td>Eppendorf, Germany</td><td>30120086 and 0030120094</td><td></td></tr><tr><td>12-channel pipette</td><td>Eppendorf, Germany</td><td>4861000813</td><td></td></tr><tr><td>384 multi-well plates</td><td>Greiner Bio-One, Germany</td><td>781096</td><td></td></tr><tr><td>15 and 50 mL platic tubes</td><td>Eppendorf, Germany</td><td>0030122151 and 30122178</td><td></td></tr><tr><td>A2-slide</td><td>ChemoMetec, Denmark</td><td>942-0001</td><td></td></tr><tr><td>Automatic repeater pipette</td><td>Eppendorf, Germany</td><td>4987000010</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>Clean wide-mouthed plastic container for semen sample</td><td></td><td></td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td></td><td></td><td></td></tr><tr><td>FITC-coupled lectin of Pisum sativum (FITC-PSA)</td><td>Sigma-Aldrich, Germany</td><td>L0770</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Thermo Fisher Scientific, USA</td><td>F14201</td><td></td></tr><tr><td>FLUOstar OMEGA fluorescence microplate reader</td><td>BMG Labtech, Germany</td><td></td><td></td></tr><tr><td>Glucose anhydrous</td><td></td><td></td><td></td></tr><tr><td>HEPES</td><td></td><td></td><td></td></tr><tr><td>Hoechst-33342</td><td>ChemoMetec, Denmark</td><td>910-3015</td><td></td></tr><tr><td>Human serum albumin (HSA)</td><td>Irvine Scientific</td><td>9988</td><td>For addition to HTF+</td></tr><tr><td>Immobilizing solution containing 0.6 M NaHCO&lt;sub&gt;3&lt;/sub&gt; and 0.37% (v/v) formaldehyde in distilled water</td><td></td><td></td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td></td></tr><tr><td>KCl</td><td></td><td></td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Magnetic stirrer</td><td></td><td></td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>Na-Lactate</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td></td><td></td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>NC-3000 image cytometer</td><td>ChemoMetec, Denmark</td><td>970-3003</td><td></td></tr><tr><td>Pipettes and piptting tips</td><td></td><td></td><td></td></tr><tr><td>propidium iodide (PI)</td><td>ChemoMetec, Denmark</td><td>910-3002</td><td></td></tr><tr><td>Purified water</td><td></td><td></td><td></td></tr><tr><td>Rack for placing 50 mL plastic tubes in 45&amp;deg; angle</td><td></td><td></td><td></td></tr><tr><td>S100 buffer</td><td>ChemoMetec, Denmark</td><td>910-0101</td><td></td></tr><tr><td>SP1-cassette</td><td>ChemoMetec, Denmark</td><td>941-0006</td><td></td></tr><tr><td>Volumetric flasks of appropriate sizes</td><td></td><td></td><td></td></tr><tr><td>Vortexer</td><td></td><td></td><td></td></tr></tbody></table>

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.

Representative results from an experiment testing the effect of 4 compounds (A, B, C, and D) together with a positive (progesterone) and negative (buffer) control on [Ca2+]i in human sperm using the medium-throughput Ca2+-signaling assay can be seen in Figure 4a. In Figure 4b, a dose response curve of progesterone is shown, which was derived from peak &#916;F/F0 (%) data induced by seri...

Watch this Scientific Journal Video about Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm at JoVE.com

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.

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 ...

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Research

JoVE Journal

Biology

8.1K Views.  Copenhagen University Hospital. 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.

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

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

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 ...

Biochemistry

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 ...

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, ...

Developmental Biology

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 ...

Combining Imaging with a Stimulant Delivery System to Study Live Mouse Sperm

Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.

Author Spotlight: A Live Cell Imaging Technique to Study Calcium Signaling and Acrosome Exocytosis in Mouse Sperm 

The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...

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 ...

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

Recording Electrical Currents across the Plasma Membrane of Mammalian Sperm Cells

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

This protocol describes how to perform electrical recordings from mammalian sperm cells in a whole-cell configuration, with the goal of directly recording ion channel activity. The method has been instrumental in describing the electrophysiological profiles of several sperm ion channels and helped to reveal their molecular identity and regulation.

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

Summary

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