Name: medium-throughput screening assays for assessment of effects on ca2+-signaling and acrosome reaction in human sperm
Uploaded: 2019-03-01T14:45:00
Description: 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

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 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>
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		<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>
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			<td>CaCl2</td>
			<td></td>
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			<td></td>
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			<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>
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			<td>Incubator</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>KCl</td>
			<td></td>
			<td></td>
			<td></td>
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			<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

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 ...

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 ...

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 ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Toxicological Assays for Testing Effects of an Epigenetic Drug on Development, Fecundity and Survivorship of Malaria Mosquitoes

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making malaria control an omnipresent problem in tropical countries. The emergence of insecticide resistant populations warrants the exploration of novel drug target pathways and compounds for vector mosquito control. Epigenetic drugs are well established in cancer research, however not much is known about their effects on insects. This study provides a simple protocol for examining the toxicological effects of 3-Deazaneplanocin A (DZNep), an experimental epigenetic drug for cancer therapy, on the malaria vector, Anopheles gambiae. A concentration-dependent increase in mortality and decrease in size was observed in immature mosquitoes exposed to DZNep, whereas the compound reduced the fecundity of adult mosquitoes relative to control treatments. In addition, there was a drug-dependent decrease in S-adenosylhomocysteine (SAH) hydrolase activity in mosquitoes following exposure to DZNep relative to control treatments. These protocols provide the researcher with a simple, step-by-step procedure to assess multiple toxicological endpoints for an experimental drug and, in turn, demonstrate a unique multi-prong approach for exploring the toxicological effects of water-soluble epigenetic drugs or compounds of interest against vector mosquitoes and other insects.

A protocol is developed to examine the effects of an epigenetic drug DZNep on the development, fecundity and survivorship of mosquitoes. Here we describe procedures for the aqueous exposure of DZNep to immature mosquitoes and a blood-based exposure of DZNep to adult mosquitoes in addition to measuring SAH hydrolase inhibition.

Insecticidal resistance poses a major problem for malaria control programs. Mosquitoes adapt to a wide range of changes in the environment quickly, making ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...