Two Types of Assays for Detecting Frog Sperm Chemoattraction

Summary

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 – sperm accumulation assays and sperm tracking assays.

Abstract

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 μm 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 coworkers^2,3. 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.

Protocol

1. Materials and buffers used

1. Oocyte Ringer′s Buffer (1.5 x OR2) contains 124 mM NaCl, 3.75 mM KCl, 1.5 mM CaCl₂, 1.5 mM MgCl₂, 1.5 mM Na₂HPO₄, 10 mM Hepes, pH 7.8. Fertilization Buffer (F-1) contains 41.25 mM NaCl, 1.25 mM KCl, 0.25 mM CaCl₂, 0.06 mM MgCl₂, 0.5 mM Na₂HPO₄, 2.5 mM Hepes, pH 7.8.
2. Xenopus laevis egg water is prepared according Sugiyama et al.⁴. Briefly described, freshly spawned jellied frog eggs are swirled in a small volume of F-1 buffer for 30 minutes and the conditioned medium removed by micropipet....

Discussion

Chemotaxis of cells moving by either amoeboid movement or flagella-powered swimming is found in many biological contexts and study of this phenomenon requires the availability of practical and reliable assays. Some examples of the phenomenon, such as attraction of sperm to a sea urchin egg or gathering of slime mold cells to form a fruiting body, have immediate visual impact. Quantitation of this phenomenon has been undertaken in a variety of ways as described by Eisenbach¹⁸. These assays include use of chemoa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24-well plates	BD Biosciences	35/1147
12 mm outer diameter inserts with 12 μm pore membrane	EMD Millipore	PIXP01250	We previously used Costar-Corning transwell plate #3403 -now discontinued
Zigmond chamber	Neuroprobe	Z02
Silicone oil	GE Healthcare	SF1154	Equivalent to Dow Corning 550 Fluid
Image J software	Wayne Rasband, Research Services Branch, National Institute of Mental Health	Free download at http://rsbweb.nih.gov/ij	Java program that runs on Windows, Linux and Mac
MtrackJ software	Biomedical Imaging Group	Free download at http://www.imagescience.org/meijering/software/mtrackj/	Java program that runs on Windows, Linux and Mac
Virtual Dub software	GNU General Public Licensed	Free download via http://www.virtualdub.org/index.html	Setup instructions at the Image J website under plugins; for Windows only
cellSens software	Olympus Corporation	See website: http://www.olympusamerica.com/seg_section/product.asp?product=1070	Controls and acquires images from a variety of cameras. Also has image processing capability