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Method Article
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 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 μl. 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 μl). 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 http://www.bdbiosciences.com/resources/protocols/setting_compensation.jsp). 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.
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 occurs during capacitation. For techniques requiring a positive control we used a Ca2+ ionophore -ionomycin- to induce maximal Ca2+ uptake into the cell, and thus, maximal fluorescence response; for the minimal fluorescence value, we used Mn2+ to quench fluorescence.
1. Sperm Sample Preparation by the Swim-up Method (See Figure 1)
Use only ejaculated samples (obtained by masturbation) whose characteristics fulfill the parameters established by the latest edition of the World Health Organization laboratory manual (available at http://whqlibdoc.who.int/publications/2010/9789241547789_eng.pdf ) for the examination and processing of human semen.
2. Fluorescent Dye Loading for Ca2+ Measurements
There are several fluorescent dyes available to measure intracellular Ca2+; the appropriate one must be selected according to its Kd, and its emission and excitation wavelengths (for qualitative and quantitative measurements, single and double emission and excitation wavelengths, respectively, must be used) (visit http://es-mx.invitrogen.com/search/global/searchAction.action?query=ion+indicators&resultPage=1&resultsPerPage=15 for more information). For the present qualitative application we used Fluo-3 AM, a cell-permeant dye with a Kd = 325 nM, and single emission and excitation wavelengths of 506/526 nm, respectively 12.
3. Technique #1. Conventional Fluorometry (Average Information from a Large Cell Population)
Equipment: For our sperm population [Ca2+]i measurements we use an SLM Aminco spectrofluorometer operated by Olis software (Bogart, GA, USA) with magnetic stirrer control (SIM Aminco), and coupled to a Blue LED (Luxeon Star LXHL-LB3C, from LUMILEDS) and a 465-505 nm band-pass filter (Chroma Technology Corp.) for Fluo-3 AM excitation. The LED is controlled by a custom-built power supply (700 mA). Emission light is measured by setting the emission wavelength (λEm) to 525 nm on the spectrofluorometer's monochromator.
4. Technique #2. Stopped Flow Fluorometry (Information with High Temporal Resolution from a Large Cell Population)
Equipment: Intracellular [Ca2+] changes are measured with high temporal resolution using a SFM-20 stopped-flow mixer coupled to a MOS-200 rapid kinetics optical system, both from BioLogic science instruments (Grenoble, France). All data are analyzed with Bio-Kine32 software from the same company.
5. Technique #3. Flow Cytometry (Single Cell Information Obtained from a Large Number of Cells)
Equipment: This technique allows the simultaneous measurement of several parameters in a single moment in time, but unlike the previous techniques, it does not measure changes over time; rather it provides the parameter values at the time of measurement. Therefore, instead of adding Pg to trigger the response, in this case we measured intracellular Ca2+ levels in sperm cells before and after inducing capacitation. We used a FACSCanto Cytometer (Becton Dickinson) and data were analyzed with FlowJo software (Tree Star 9.3.3).
6. Technique #4. Single Cell Imaging (Single Cell Information with High Spatial Resolution)
Equipment: Custom-built Imaging set-up. Our imaging set-up is composed of an inverted Nikon Diaphot 300 microscope equipped with a temperature controller (Medical System Corp., Greenvale, N.Y.), a Nikon PlanApo 60X (1.4 NA oil immersion) objective. Fluorescence illumination is provided by a Luxeon V Star Lambertian Cyan LED part # LXHL-LE5C (Lumileds Lighting LLC, San Jose, CA) attached to a custom-built stroboscopic control box. The LED was mounted into a FlashCube40 assembly with dichroic mirror M40-DC400 (Rapp Opto Electronic, Hamburg, Germany) (bandwidths: excitation 450-490 nm, dichroic mirror 505 nm, and emission 520-560 nm). LED output was synchronized to the Exposure Out signal of a Cool Snap CCD camera via the control box to produce a single flash of 2 msec duration per individual exposure. The camera exposure time was set equivalent to the flash duration (2 msec). Images are collected every 250 msec (or may be adjusted according to the desired temporal resolution) using IQ software (Andor Bioimaging, Wilmington, NC).
Technique #1. Conventional Fluorometry
Progesterone is one of the known AR inducers and, as expected, it does provoke a transient [Ca2+]i increase in human sperm (shown in Figure 2). Addition of a calcium ionophore (ionomycin) causes the maximum [Ca2+]i increase, which does not return to basal levels.
Technique #2. Stopped Flow Fluorometry
The progesterone-induced [Ca2+]i increase was measured as before (conventional fluorometry), but this time with greater temporal resolution; in this case the frequency of acquisition was 0.1 Hz. As shown in Figure 3, both progesterone (transient, red line) and ionomycin (sustained, blue line) caused a very fast [Ca2+]i increase. The absence of a delay in the progesterone-induced [Ca2+]i increase is consistent with previous reports suggesting that progesterone directly activates the Ca2+ channel CatSper, without intermediate signaling 10,14.
Technique #3. Flow Cytometry
[Ca2+]i was measured in capacitated and non-capacitated human sperm. As previously reported in mouse 15, bovine sperm 16 and human sperm 17, we also observed increased [Ca2+]i in capacitated compared to non-capacitated human sperm. Baldi, et al. (1991) 17 reported higher basal [Ca2+]i in capacitated than in non-capacitated human sperm using conventional fluorometry. In this work we used flow cytometry to measure [Ca2+]i before and after in vitro capacitation. Flow cytometry enables us to see that the distribution of fluorescence values for capacitated sperm (Figure 4D, blue trace) is shifted to higher values compared to non-capacitated sperm (Figure 4D, red trace). The fluorescence values for each individual cell can be observed in the two-dimensional dot plots shown in Figure 4G; importantly, the signal arising from dead cells (15% approximately) can be eliminated (Figure 4G, upper quadrants).
Technique #4. Single Cell Imaging
The progesterone-induced [Ca2+]i change was measured in single sperm cells. Progesterone addition causes an increment in [Ca2+]i both in the sperm head and in the flagellum. As observed in population experiments, single cell analysis revealed a transient and a sustained increase for progesterone and ionomycin, respectively.
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.
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 ...
We have nothing to disclose.
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).
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 |
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