Techniques for Imaging Ca²⁺ Signaling in Human Sperm

Abstract

Fluorescence microscopy of cells loaded with fluorescent, Ca 2+ -sensitive dyes is used for measurement of spatial and temporal aspects of Ca 2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca 2+-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 Ca 2+ -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 Ca 2+ response as % change in fluorescence is obtained. Video LinkThe video component of this article can be found at http://www.jove.com/video/1996/ ProtocolSperm from healthy fertile males, with a normal semen analysis, are normally prepared for imaging as follows. .8 Na lactate) supplemented with 0.3% charcoal de-lipidated/fatty acid free Fraction V BSA (quality of the BSA is crucial for successful capacitation of sperm). 1 ml of sEBBS is pipetted into each of a series of 5 ml tubes and gently underlayered with 0.3 ml of semen. After incubation for 1 hour (37°C; 6% CO 2 ) the top 0.7 ml is gently removed from each tube and pooled. 10 μl of the sperm suspension is diluted with 90 μl of 1% (v/v) formalin to immobilize the cells, then sperm are counted in a Neubauer chamber. Cell density in the suspension is then adjusted (with sEBSS) to 6 million cells/ml. 2. The sample is then divided into aliquots of 200 μl in loosely-capped tubes and incubated (37°C; 6% CO 2 ) in for 5-6 h to allow capacitation. 3. Coverslips (22x50 mm) have previously been treated with poly-D-lysine. 10 μl of poly-D-lysine solution (10% w/v) is applied as a number of small drops to the centre of the coverslip. The poly-D-lysine is then allowed to air dry. This can be on a heated stage and should be to complete dryness. A coverslip is attached with vacuum grease to an enclosed, purpose-built, perfusable, polycarbonate imaging chamber (dimensions 35 mm x 20 mm x 5 mm; capacity ≈ 180 μl) similar to the Warner RC20 chamber .The poly-D-lysine-coated coverslip forms the base ...

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm*

“…Loading with Oregon Green BAPTA 1 (OGB) and time lapse fluorescence imaging was as described previously (33). Fluorescence of OGB shows negligible pH sensitivity over the range pH 6–9, making it suitable for the experiments in which pH i is varied.…”

“…Fast (9–60-Hz) imaging was as above, but a ×60 oil objective was used with the Andor camera. Fluorescence from the sperm posterior head/neck was background-corrected and normalized as described previously (33) using Δ F = (( F − F rest )/ F rest ) × 100%, where Δ F is percentage change in intensity, F is fluorescence intensity at time t , and F rest is the mean of ≥10 determinations of F during the control period. Mean Δ F of all cells in the experiment (Δ F mean ) was used to compare responses between experiments, and n values represent the number of experiments.…”

Cell-penetrating peptides, targeting the regulation of store-operated channels, slow decay of the progesterone-induced [Ca2+]isignal in human sperm

“…Loading of cells with Oregon Green BAPTA 1 (Invitrogen) and time-lapse fluorescence imaging was done as described previously ( Nash et al , 2010 ). All experiments were performed at 25 ± 0.5°C in a continuous flow of medium (sEBSS).…”

Fluorescence Technique Description for Measuring Ca<sup>2+</sup> in Mice Sperm

“…With considering choosing an area where sperm has an adequate amount of fluorescent intensity, to prevent affects the quality and sincerity of data through signal reception of from adjacent cells. In addition, where sperms have fixed, however, flagellar activity is discernible [29]. The imaging contrast phase, should be saved, after selection the area of imaging, visible light of microscope should be closed, then turn to switch the microscope to fluorescence mode (emission 491 nm) and time of exposure to obtain a clear fluorescence image here we selected time per 5 seconds (Figure 2).…”

“…However in current studies some authors showed the section of material method in their published papers in briefly without details. So that, we depended on this experiment on reviewing our materials method according to reference [26] [27] for preparing HS medium and [28] for imaging system and ∆F/F 0 (%) as describes in [29], unless otherwise details is our current study as describe later. …”