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.
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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 &#181;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>CaCl2</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 NaHCO3 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>KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</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>NaHCO3</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&#176; 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 ...

Our assays can be used to test compounds for their effects on human sperm cell function. Specifically, these methods can be used to quickly and easily screen large numbers of compounds for their effects on calcium signaling and acrosome reaction in human sperm cells. Visual demonstrations of this method will make it easier for others to set up similar experiments in their own labs.Begin by placing one 50 milliliter tube per one milliliter of raw semen sample into a tube rack at a 45 degree angle and adding four milliliters of 37 degree Celsius Human Tubal Fluid or HTF positive medium to each tube. Next, carefully pipette one milliliter of liquified semen sample to the bottom of each tube and place the tubes at 37 degrees Celsius for one hour. At the end of the incubation, use a modified pipette tip held at a 45 degree angle to gently transfer as much of the upper fraction of motile sperm cell containing HTF positive medium as possible into a new 15 milliliter plastic tube.To count the collected sperm cells, dilute a 20 microliter aliquot to a factor of 10, 20, 30, 50, or 90 in S100 buffer in a round bottom two milliliter tube according to the expected concentration. Vortex the diluted sperm cells for 10 seconds at 700 rotations per minute. Next, immerse an SP1 cassette into the sample and fully depress the white plunger to aspirate an aliquot of the sample into the cassette.Then place the cassette in the image cytometer tray and run the PI stained human sperm cell assay according to the applied dilution factor. To measure changes in the free intracellular calcium concentration, gently pipette the sperm sample up and down one time and use an automatic repeater pipette to aliquot 50 microliters of fluorophore-stained sperm cells to 24 wells of one row of a 384 micro well plate. Place the micro well plate in the fluorescence plate reader at 30 degrees Celsius and select the appropriate protocol for fluorophore.Select a well in the row of sperm cells and adjust the gain to a target value of 20%Then start the measurement. After 10 baseline cycles, pause the experiment and eject the drawer with the micro well plate. Using a 12 channel pipette, quickly add 25 microliters of the prepared solutions of compounds and controls of interest into the 12 duplicate wells in the row and immediately return the plate to the reader to continue the measurement as quickly as possible.Any changes in fluorescence in response to the addition of the compounds or controls will be captured by the instrument. For acrosome reaction analysis, after incubating the capacitated sperm cells with the appropriate fluorescent dyes, compounds or controls, mix the samples by pipetting and add 50 microliters of each test sample to 100 microliters of immobilizing solution. Mix the samples again and load the chambers of one A2 slide per sample with approximately 30 microliters of sample per chamber.Then place the first loaded slide onto the tray of the image cytometer. Select the FlexiCyte Protocol and press Run. Here representative results from an experiment testing the effect of four compounds, a positive progesterone control and a negative buffer control using the medium throughput calcium signaling assay as demonstrated can be observed.In this graph, a dose response curve of progesterone derived from peak percents changes in the intracellular free calcium concentration induced by serially diluted concentration of progesterone is shown. These data illustrate the effects of one negative or one of two positive controls on the induction of an acrosome reaction in capacitated human sperm cells in a medium throughput acrosome reaction assay. To avoid missing the peaks of the induced signals, it is critical to add the chemicals quickly to the replicate wells and to immediately continue the measurement thereafter.By changing the type of fluorophores and dyes, our assays can easily be modified to answer different scientific questions. These assays have already been used in several studies to test large groups of compounds for their effects on human sperm function.

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Research

JoVE Journal

Biology

8.0K Görüntüleme.  Copenhagen University Hospital. Bizim tahliller insan sperm hücresi fonksiyonu üzerindeki etkileri için bileşikleri test etmek için kullanılabilir. Özellikle, bu yöntemler hızlı ve kolay insan sperm hücrelerinde kalsiyum sinyalizasyon ve akrozom reaksiyonu üzerindeki etkileri için bileşiklerin çok sayıda ekran için kullanılabilir. Bu yöntemin görsel gösterimleri, başkalarının kendi laboratuvarlarında benzer deneyler ayarlamalarını kolaylaştırır.45 derecelik açıyla bir tüp raf içine h...