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Biology

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

Published: May 24th, 2013

DOI:

10.3791/50344

1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología-Universidad Nacional Autónoma de México, 2Math and Sciences Department, Edison State College

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.

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.

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....

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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 resti.......

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Figure 1
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 their concentration are illustrated. The last incubation step is only performed when capacitation is required.

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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 .......

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The authors thank Jose Luis De la Vega, Erika Melchy and Dr. Takuya Nishigaki for technical assistance. This work was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT-Mexico) (99333 and 128566 to CT); Dirección General de Asuntos del Personal Académico/ Universidad Nacional Autónoma de México (IN202212-3 to CT).

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Name Company Catalog Number Comments
Name of Reagent/Material 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

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