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 Tecnolog&iacute;a (CONACyT-Mexico) (99333 and 128566 to CT); Direcci&oacute;n General de Asuntos del Personal Acad&eacute;mico/ Universidad Nacional Aut&oacute;noma de M&eacute;xico (IN202212-3 to CT).

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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L., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+activates+the+principal+Ca2++channel+of+human+sperm.">Progesterone activates the principal Ca2+ channel of human sperm.</a> Nature. 471, 387-391 (2011).</li><li>Xia, J., Ren, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BSA-induced+Ca2++influx+during+sperm+capacitation+is+CATSPER+channel-dependent.">The BSA-induced Ca2+ influx during sperm capacitation is CATSPER channel-dependent.</a> Reprod. Biol. Endocrinol. 7, 119 (2009).</li><li>Galantino-Homer, H. L., Florman, H. M., Storey, B. T., Dobrinski, I., Kopf, G. 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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 ...

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 limits through different cellular mechanisms that continuously buffer, sequester, compartmentalize, and/or accumulate Ca2+. Changes in the concentration of this ion can occur in highly localized areas within the cell 1, and deciphering such fluctuations is essential for gaining a deeper understanding of (1) their role in the signaling mechanism, (2) their physiological significance, and (3) general mechanisms of cell signaling. Ca2+-mediated signaling is of particular importance in sperm physiology 2. Sperm motility is one of the most important functions for fertilization success, and in fact, several sperm motility defects can cause sterility 3-5. The importance of Ca2+ in flagellar movement has been long recognized 6; however, the mechanism of how Ca2+ controls the specific form of flagellar bending is not fully understood.
Before fusing with the egg, spermatozoa must undergo capacitation, a complex process dependent on sperm residence inside the female tract. During capacitation, the sperm membrane's lipid architecture and organization are modified, mainly as a result of cholesterol removal from the plasma membrane. Additionally, several proteins are tyrosine-phosphorylated 7. Importantly, during capacitation there is an increase in intracellular pH (pHi) and in [Ca2+]i, and the membrane potential hyperpolarizes in some species 2. Capacitation only takes place in a subpopulation of spermatozoa (20-40%), and the mechanisms involved in all these cellular changes are far from clear. It is generally accepted that only a subpopulation of capacitated sperm undergo the acrosome reaction (AR) when exposed to physiological inductors. The AR is also a Ca2+-regulated event required for fertilization in all species possessing an acrosome (specialized organelle with outer and inner membranes). During this process the outer acrosomal membrane fuses with the sperm's plasma membrane, releasing hydrolytic enzymes that allow the sperm cell to penetrate the glyco-proteinaceous matrix surrounding the egg (zona pellucida, or ZP). The AR also exposes a new fusogenic sperm cell surface that interacts with the egg plasma membrane for the final fusion of both gametes. There are several cellular ligands that induce the AR, progesterone being one of the most studied among them.
In this work we present four different techniques involving the use of a Ca2+-sensitive fluorescent dye to measure [Ca2+]i changes in human sperm triggered by progesterone (except for flow cytometry, in which we measured the [Ca2+]i increase induced during the in vitro capacitation process). In this particular case we used Fluo-3 AM (Life Technologies, Grand Island, NY), a membrane-permeable dye with a Kd = 325 nM. In vitro we monitored fluorescence changes as a function of time with three of the methodologies, and with the fourth technique we measured fluorescence values at a single given point in time. These different approaches complement each other, since altogether they provide spatial and temporal resolution at both the single cell and cell population levels.
Cell Population or Bulk Experiments
Bulk techniques are extensively used not only because the instruments they require are readily available, but also because they are simple, well established, and allow for the averaging of information from measurements performed on millions of cells in a single experiment.
Technique #1. Conventional Fluorometry 
This technique monitors changes in fluorescence as a function of time; the experiments are performed in glass cuvettes with sample volumes ranging from 200 to 1,000 &mu;l. Proper mixing of added reagents requires magnetic stirring, and therefore the temporal resolution obtained is in the order of seconds. The typical cell concentration range of the samples analyzed is 105-108 cells/ml.
Technique #2. Stopped Flow Fluorometry 
This technique also monitors changes in fluorescence as a function of time, but the reagents are rapidly mixed together (using pressure) into a recording cuvette containing a very small sample volume (ranging from 25-100 &mu;l). Therefore, homogenization of reagents is instantaneous, enabling a high temporal resolution in the order of milliseconds. Analysis of the resulting fluorescence vs. time traces are suitable for determining reaction rates, elucidating the complexity of the reaction mechanism, obtaining information on short-lived reaction intermediates, etc. The common cell concentration range of the samples analyzed is 105-107 cells/ml.
Single Cell Experiments
Bulk experiments report the average behavior of a large number of cells; however, a population may frequently exhibit heterogeneous properties that are overlooked during such type of measurements. Single cell techniques are thus used to complement the information obtained with cell population experiments.
Technique #3. Flow Cytometry 
Despite the importance of the information arising from single cell measurements, it is important to analyze a large number of cells in order to prevent the erroneous extrapolation of cell-specific properties to an entire population. For this reason, high-throughput techniques are favored and the most popular method is flow cytometry, in which 10,000 cells per condition are conventionally analyzed. This method enables multi-parametric analysis of heterogeneous populations as it categorizes cells according to their size (forward scatter (FSC)), granularity (side scatter (SSC)) and fluorescence intensity (specific labeling with an antibody, viability marker, etc.), thus providing information on the parameters' distribution for a group of cells. Flow cytometry provides instant rather than time-dependent information 8. Forward and side scatter values are also useful for selecting a gate that includes cells but discriminates cellular debris, dust, etc. For fluorescence measurements, negative and positive fluorescence controls must also be included. If more than one fluorescence channel is used, a process known as compensation must be performed (for details see <a href="http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp" target="_blank">http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp</a>). Compensation allows for spectral overlap discrimination among fluorophores. Flow cytometry also allows discrimination of dead cells, generally by means of propidium iodide staining.
Technique #4. Single Cell Imaging 
Microscopy is another common method to study single cell behavior; it is well suited for time-dependent studies and it also provides spatial resolution. A major drawback is that high-throughput analysis is only in its infancy at the present time 9.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Ham's F-10</td>
 <td>Sigma-Aldrich</td>
 <td>N-6013</td>
 <td></td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A-7906</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride Dihydrate approx. 99%</td>
 <td>Sigma-Aldrich</td>
 <td>C-3881</td>
 <td></td>
 </tr>
 <tr>
 <td>Makler Counting Chamber</td>
 <td>SEFI Medical Insruments LTD</td>
 <td>SEF-MAKL</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-3 AM</td>
 <td>Invitrogen</td>
 <td>F-1242</td>
 <td>20 vials/50 &mu;g each</td>
 </tr>
 <tr>
 <td>Ionomycin</td>
 <td>Alomone</td>
 <td>I-700</td>
 <td></td>
 </tr>
 <tr>
 <td>Progesterone</td>
 <td>Sigma-Aldrich</td>
 <td>P0130</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>S-9888</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Potassium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>P-3911</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>JT Baker</td>
 <td>3506</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Magnesium chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-2670</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Calcium chloride anhydrous</td>
 <td>Sigma-Aldrich</td>
 <td>C-1016</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H-3125</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>D-Glucose</td>
 <td>JT Baker</td>
 <td>1906-01</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium pyruvate</td>
 <td>Sigma-Aldrich</td>
 <td>P-2256</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Sodium L-lactate (aprox. 99%)</td>
 <td>Sigma-Aldrich</td>
 <td>L- 7022</td>
 <td>Reagents for human sperm medium (HSM)</td>
 </tr>
 <tr>
 <td>Propidium Iodide </td>
 <td>Invitrogen</td>
 <td>L-7011 </td>
 <td>Component B</td>
 </tr>
 <tr>
 <td>Triton X-100 (t-Octylphenoxypolyethoxyethanol)</td>
 <td>Sigma- Aldrich</td>
 <td>X-100</td>
 <td>2.4 mM solution in water </td>
 </tr>
 <tr>
 <td>Round coverslip </td>
 <td>VWR</td>
 <td>48380 080</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Poly-L-lysine solution </td>
 <td>Sigma-Aldrich</td>
 <td>P8920</td>
 <td></td>
 </tr>
 <tr>
 <td>Manganese chloride</td>
 <td>Sigma-Aldrich</td>
 <td>M-3634</td>
 <td></td>
 </tr>
 <tr>
 <td>Attofluor; Cell Chamber, for microscopy</td>
 <td>Life technologies</td>
 <td>A-7816</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Sulphoxide</td>
 <td>Sigma-Aldrich</td>
 <td>D2650</td>
 <td>5x5 ml</td>
 </tr>
 </tbody>
</table>

measuring intracellular ca2+ changes in human sperm using four techniques: conventional fluorometry, stopped flow fluorometry, flow cytometry and single cell imaging

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.

<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50344/50344fig1highres.jpg" src="/files/ftp_upload/50344/50344fig1.jpg" /> 
 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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Watch this Scientific Journal Video about Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell 

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

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.

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

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

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20.1K Views.  Instituto de Biotecnología-Universidad Nacional Autónoma de México. Intracellulare di Ca 2 + Dinamiche sono molto importanti in sperma fisiologia e Ca 2 + Sensibili coloranti fluorescenti costituiscono uno strumento versatile per studiarli. Esperimenti di popolazione (fluorometria e fermato il flusso fluorometria) ed esperimenti cella singola (citometria a flusso e l'imaging di singola cellula) sono utilizzati per tenere traccia spazio-temporale [Ca 2 +] Cambiamenti nelle cellule di s...

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