Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Summary

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.

Abstract

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.

Introduction

Ca²⁺ is a universal second messenger of signal transduction pathways in eukaryotic cells. Intracellular Ca²⁺ (Ca²⁺_i) participates in the regulation of many fundamental physiological processes in both excitable and non-excitable cells. The importance and universality of Ca²⁺ as second messenger during signal transduction events is derived from its spatio-temporal versatility in the transmission of information within the cell. While Ca²⁺ cannot be synthesized de novo or degraded within the cell, its intracellular concentration ([Ca²⁺]_i) is maintained within very strict limit....

Protocol

In this paper we report the use of the four aforementioned techniques to measure [Ca²⁺]_i changes in human sperm cells. We used progesterone to trigger a Ca²⁺ response, as it is well established that this steroid produces a transient [Ca²⁺]_i increase in spermatozoa. Particularly, in human sperm, progesterone directly activates a Ca²⁺ channel (namely CatSper) expressed exclusively in the plasma membrane of sperm cells ^10,11. We also measured resting [Ca²⁺]_i before and after capacitation given that it is also widely accepted that an increase in [Ca²⁺]_i....

Results

Figure 1. Schematic diagram of the experimental protocol for sperm sample preparation by the swim-up method. The major steps for separation of motile sperm and for adjustment of.......

Discussion

Intracellular signaling is vital for most cellular activities; Ca²⁺ is a ubiquitous messenger that accompanies mammalian cells throughout their entire lifespan, from their origin at fertilization, to the end of their life cycle. In response to different stimuli, [Ca²⁺]i increases, oscillates and decreases with spatio-temporal codification; accordingly, diverse processes are activated, modulated or terminated by Ca²⁺-encoded messages. Intracellular Ca²⁺ dynamics are very .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ham's F-10	Sigma-Aldrich	N-6013
Bovine Serum Albumin	Sigma-Aldrich	A-7906
Calcium Chloride Dihydrate approx. 99%	Sigma-Aldrich	C-3881
Makler Counting Chamber	SEFI Medical Insruments LTD	SEF-MAKL
Fluo-3 AM	Invitrogen	F-1242	20 vials/50 μg each
Ionomycin	Alomone	I-700
Progesterone	Sigma-Aldrich	P0130
Sodium chloride	Sigma-Aldrich	S-9888	Reagents for human sperm medium (HSM)
Potassium chloride	Sigma-Aldrich	P-3911	Reagents for human sperm medium (HSM)
Sodium bicarbonate	JT Baker	3506	Reagents for human sperm medium (HSM)
Magnesium chloride	Sigma-Aldrich	M-2670	Reagents for human sperm medium (HSM)
Calcium chloride anhydrous	Sigma-Aldrich	C-1016	Reagents for human sperm medium (HSM)
HEPES	Sigma-Aldrich	H-3125	Reagents for human sperm medium (HSM)
D-Glucose	JT Baker	1906-01	Reagents for human sperm medium (HSM)
Sodium pyruvate	Sigma-Aldrich	P-2256	Reagents for human sperm medium (HSM)
Sodium L-lactate (aprox. 99%)	Sigma-Aldrich	L- 7022	Reagents for human sperm medium (HSM)
Propidium Iodide	Invitrogen	L-7011	Component B
Triton X-100 (t-Octylphenoxypolyethoxyethanol)	Sigma- Aldrich	X-100	2.4 mM solution in water
Round coverslip	VWR	48380 080	25 mm diameter
Poly-L-lysine solution	Sigma-Aldrich	P8920
Manganese chloride	Sigma-Aldrich	M-3634
Attofluor; Cell Chamber, for microscopy	Life technologies	A-7816
Dimethyl Sulphoxide	Sigma-Aldrich	D2650	5x5 ml