The AcroSensE mouse model and live cell imaging methods described here provide a new approach to studying calcium dynamics in the subcellular compartment of the sperm acrosome and how they regulate intermediate steps leading to membrane fusion and acrosome exocytosis.
Acrosome exocytosis (AE), in which the sperm's single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process essential for fertilization. However, our understanding of how calcium signaling regulates AE is still incomplete. In particular, the interplay between intra-acrosomal calcium dynamics and the intermediate steps leading to AE is not well-defined. Here, we describe a method that provides spatial and temporal insights into acrosomal calcium dynamics and their relationship to membrane fusion and subsequent exocytosis of the acrosome vesicle. The method utilizes a novel transgenic mouse expressing an Acrosome-targeted Sensor for Exocytosis (AcroSensE). The sensor combines a genetically encoded calcium indicator (GCaMP) fused with mCherry. This fusion protein was specifically designed to enable the concurrent observation of acrosomal calcium dynamics and membrane fusion events. Real-time monitoring of acrosomal calcium dynamics and AE in live AcroSensE sperm is achieved using a combination of high frame-rate imaging and a stimulant delivery system that can target single sperm. This protocol also provides several examples of basic methods to quantify and analyze the raw data. Because the AcroSensE model is genetically encoded, its scientific significance can be augmented by using readily available genetic tools, such as crossbreeding with other mouse genetic models or gene-editing (CRISPR) based methods. With this strategy, the roles of additional signaling pathways in sperm capacitation and fertilization can be resolved. In summary, the method described here provides a convenient and effective tool to study calcium dynamics in a specific subcellular compartment-the sperm acrosome-and how those dynamics regulate the intermediate steps leading to membrane fusion and acrosome exocytosis.
Sperm acquire the ability to fertilize during a process called capacitation1. One endpoint of this process is that the sperm acquire the ability to undergo AE. Over two decades of data support the presence of a complex, multi-step model of AE in mammalian sperm (summarized in2,3). However, studying AE in live sperm is challenging, and currently available methods to monitor this process with adequate resolution are cumbersome and require multiple preparation steps4, are limited to the detection of the final step of AE (e.g., using PNA5), are limited to measurements of changes in cytosolic calcium (in contrast to acrosomal calcium dynamics), or are limited to measurements of either cytosolic calcium dynamics or AE6.
To overcome some of the key limitations of real-time AE studies under physiological conditions and to enable investigation of the interplay between calcium dynamics and AE, a unique mouse model was generated. In this mouse model, a fusion protein composed of the genetically encoded Ca2+-sensor (GCaMP3) and mCherry is expressed and targeted to the acrosome using an acrosin promoter and signaling peptide2. The targeted dual GCaMP3-mCherry sensor enables simultaneous real-time measurements of calcium concentrations and the status of the acrosomal contents in live sperm under physiological conditions using microscopy and a single-cell stimulant delivery system (Figure 1). As a component of the acrosomal matrix, membrane fusion, and AE would result in the loss of the photostable and pH-insensitive mCherry fluorescence from the sperm, as this protein diffuses out of the acrosome vesicle. In this regard, the model's ability to reflect the timing and occurrence of AE is akin to the benefits of the acrosome-targeted GFP mouse line7,8,9.
The GCaMP3 variant used in this transgenic mouse line has an approximate KD of 400 µM and a dynamic range for Ca2+ of 10-4-10-3 M10, which is suitable for this vesicle. We showed that this combination of features of GCaMP3 could reveal fusion pore formation between the plasma membrane and the outer acrosomal membrane (OAM)2. The fusion pore detection is a result of the pore size being too small to allow the AcroSensE protein to disperse out of the acrosome (via loss of acrosome content) while providing a membrane "channel" that enables the influx of Ca2+ ions into the acrosome lumen, leading to an increase in fluorescence intensity of the GCaMP3.
The bright, monomeric, non-calcium-sensitive fluorescent protein mCherry supports visualization of the acrosome while the GCaMP3 signal is faint (e.g., prior to Ca2+ binding, Figure 2), and importantly, it also allows for the identification of acrosome-intact sperm cells suitable for imaging.
The following protocol describes the utilization of the unique AcroSensE mouse model and the methods for microscopy used experimentally to study AE and sperm calcium dynamics with high spatial and temporal resolution.
All animal procedures were performed under the guidelines and approved by the Institutional Animal Care and Use Committee at Cornell University (#2002-0095). 8-10 weeks old AcroSensE mice2 were used for the present study. Requests for information on the availability of the AcroSensE mice can be submitted to the corresponding author.
1. Sperm collection and washing
2. Poly-D-lysine-coating of coverslip dishes for imaging
NOTE: Poly-D-lysine (PDL) dishes should be prepared fresh before each experiment.
3. Capillary pulling, loading for puffing
4. Preparation of the delivery of stimulation for puffing
NOTE: To minimize the risk of user injury, safety goggles should be worn during the operation of the puffing procedure.
5. Microscopy and image acquisition
NOTE: Multiple brands of microscopes are available and can be used; however, a minimum frame rate of 3 frames/s is desirable. Regarding temperature and environmental control, please note that the actual temperature of the medium in the dish should be confirmed, for example, using a non-contact infrared thermometer.
6. Image and data analysis
NOTE: Offline image analysis is conducted using ImageJ (see Table of Materials). Previously, several intermediate steps were reported in the process leading to AE, including acrosomal calcium rise (ACR), and full membrane fusion. The latter leads to the loss of mCherry fluorescence and therefore represents full AE. In some cells, signals akin to pre-spike foot events (PSF) are observed when using amperometric approaches in studies of exocytosis (for more details, please see2). Several optional methods are available for analyzing the AcroSensE raw data to quantify ACR, AE, and its intermediate steps. The following describes some of the basic analytical methods.
Figure 2 provides a simplified illustration showing the sequence of fluorescence changes expected following the successful stimulation of sperm. The top panel of Figure 2 illustrates the changes in GCaMP3 fluorescence intensity, where the signal is initially dim (baseline acrosomal calcium concentrations are lower than GCaMP3 KD), and upon the entry of calcium ions via fusion pores, the fluorescence increases in brightness. Finally, upon AE, there is a loss of signal due to diffusion and the loss of the sensor to the extracellular space. The bottom panel of Figure 2 illustrates the changes in mCherry fluorescence intensity, where the initial signal is bright, and only upon AE and diffusion of the sensor along with other contents of the acrosomal matrix, there is a dimming of the red signal.
Figure 3 provides actual measured data for the stages illustrated above in two live sperm stimulated with 125 µM of the ganglioside GM1 (for more details on how GM1 regulates AE in sperm12), captured in a single field of view. Figure 3A,B, and Figure 3E,F show the red and green signals measured in two sperm at time point 0 (before stimulation), where initially, the mCherry signal is bright, and the GCaMP3 is dim. Figure 3C, Figure 3G, and Figure 3D, Figure 3H provide the Z Profiler analysis for the full duration of the experiment (raw data or F/F0, respectively). Insert panels provide a zoomed-in view of the time window where ACR and AE occur.
Because sperm are both highly heterogeneous and very sensitive to membrane damage due to various handling procedures, Figure 5 provides examples of two types of negative results. Figure 5A,C show several sperm captured within one field of view. The yellow outline highlights two sperm cells that demonstrate a high GCaMP3 signal at time point 0 (before stimulation). In the present experiments, such cells were avoided, as they indicate that the acrosomal vesicle is already going through some membrane fusion process or that they have experienced some membrane damage that allows calcium to leak. The sperm in Figure 5A indicated by the arrow (circle ROI in Figure 5A,C) demonstrates an initial mCherry signal but no response in the red or green channels (Figure 5B,D, respectively, showing the Z-Profiler analysis window as provided by ImageJ) following stimulation with 125 µM GM1. Although a subpopulation of sperm, such as the one provided in this example, shows no response to stimulation, these are included in the final analysis and are calculated in the percent response data analysis2 (Figure 3).
Figure 1: Instrumentation and experimental setup. (A) Live sperm cells on a PDL-coated coverslip dish placed in an incubation chamber at 37 °C. A borosilicate capillary, pulled and attached to a single-cell delivery system, is positioned near the sperm cell in the center of the imaging area for direct stimulant delivery. Both green (GCaMP3) and red (mCherry) channels are continuously monitored and recorded at a high frame rate (e.g., 10 frames/s) during stimulation. (B) Schematic representation of the PDL deposition pattern on the cover-slide dish. (C) Schematic representation of the specific areas on the dish where sperm can be stimulated using the capillary to minimize non-specific stimulation from prior puffs in the same dish. Please click here to view a larger version of this figure.
Figure 2: Changes in fluorescence signal and intensity. Top panel: An increase in GCaMP3 signal signifies calcium influx into the acrosome, followed by signal loss due to diffusion of the AcroSensE fusion protein. Bottom panel: mCherry signal remains stable during initial membrane fusion, with subsequent dimming upon AcroSensE diffusion and acrosomal exocytosis (AE). Please click here to view a larger version of this figure.
Figure 3: Representative data and fluorescence traces. A field containing two sperm cells was imaged. Green/red channel fluorescence was quantified offline using ImageJ and plotted in Excel (as described in step 6). (A–D) Quantification of red (A) and green (B) channel fluorescence intensity for cell #1 over time, calculated for selected ROIs, and then presented as raw data (C) or normalized to the initial signal (D, F/F0). The insert in (D) provides a zoomed-in view of the time frame from 25-125 s. (E–H) Quantification of red (E) and green (F) channel fluorescence intensity for cell #2 over time, calculated for selected ROIs, and then presented as raw data (G) or normalized to the initial signal (H, F/F0). The insert in (H) provides a zoomed-in view of the time frame from 25-125 s. Scale bar = 5 µm. All x-axes represent time in seconds (s). Please click here to view a larger version of this figure.
Figure 4: Data analysis and quantification parameters. (A) Illustration of a typical GCaMP3 fluorescence intensity trace, displaying quantifiable parameters, including Start Time (seconds), Amplitude (fluorescence intensity), and Duration (seconds). (B) Illustration of a typical mCherry fluorescence intensity trace, displaying quantifiable parameters, including Start Time (seconds), Duration (seconds), Slope (F/s), R60 (fluorescence intensity), and Amplitude (fluorescence intensity). Please click here to view a larger version of this figure.
Figure 5: Negative results. (A,C) mCherry (red) and GCaMP3 (green) channels showing multiple sperm cells captured in a single field of view. The arrow in (A) indicates a sperm cell initially exhibiting mCherry fluorescence but no subsequent fluorescence intensity changes in response to 125 µM GM1 stimulation, as observed in the Z-Profiler generated traces provided in (B) (mCherry) and (D) (GCaMP3). Cells within the yellow rectangle exhibit elevated GCaMP3 fluorescence intensity at time point 0, before stimulation. Scale bar = 5 µm. Please click here to view a larger version of this figure.
Here, a microscopy-based method is described to utilize the newly generated AcroSensE mouse model for real-time, single-cell monitoring and analysis of the interplay between acrosomal calcium dynamics and intermediate steps leading to AE. Together with readily available genetic approaches, such as crossbreeding with other mouse genetic models or gene editing, this model and method provide a powerful system to study the role of various components and pathways that take part in sperm signaling pathways related to capacitation, AE, and fertilization.
Critical steps
It is important in all sperm handling steps to use large-bore plastic transfer pipettes or large-orifice pipette tips and to perform all steps of collection and washing at 37 ˚C to minimize membrane damage. Similarly, a swinging-bucket rotor is preferable to a fixed-angle rotor to avoid membrane damage due to cells interacting with the side wall of the micro-centrifuge tube. If Z-position drift correction is available, applying z-position correction hardware/software is highly recommended to avoid the drift of the imaged sperm out of the focal plane during the imaging interval.
Modifications and troubleshooting
The protocol described here was optimized for studies of lipid-mediated signaling2,3 and can be applied to both capacitated and non-capacitated sperm. Different methods for sperm capacitation can be used; for example, either albumin or cyclodextrin can serve as sterol acceptors. In addition, various energy substrates or bicarbonate can be added or omitted. However, note that bicarbonate will form in aqueous media from exposure to air, so testing of truly 'bicarbonate-free' conditions would require careful use of a nitrogen environment in all steps of making/preparing/handling the medium and performing the experiment. The protocol above includes using 10 mM calcium added to the experimental solution; however, calcium ions can be omitted, chelated (e.g., EGTA), or added in lower concentrations for calcium-sensitive experimental applications. Please note that lowering or omitting calcium concentrations will diminish the expected GCaMP3 signal upon AE2. In addition, one can use other media for this protocol (versus the MW used here) that better fit their experimental design and objectives. The provided protocol describes the use of single-cell imaging and single-cell delivery of stimulants; however, a simplified variation is attainable by bulk stimulation, such as by adding the stimulating reagent to the whole dish and not via puffing. In addition, whole-population experiments are also possible using the AcroSensE model via the use of fluorimeters or plate readers with fluorescence capability.
Limitations
The method provided here requires the availability, maintenance, and use of an AcroSensE mouse colony. The method also depends on the availability of an advanced imaging system, which is costly and requires trained personnel. For some experimental applications, a possible limitation concerns the inability to concurrently measure cytosolic calcium in AcroSensE sperm, as many available synthetic calcium indicators are of similar wavelength to GCaMP3. Although beyond the scope of this protocol, potential limitations of population-level experiments might include the reading of the mCherry signal after AE and dispersal in the medium or GCaMP3 signal coming from dead/permeabilized cells. To optimize such methods, one might explore employing z-focusing to minimize background noise from secreted mCherry, and/or utilize sham-treated controls to ensure baseline GCaMP quantification from dead or permeabilized sperm.
The significance of the method with respect to existing/alternative methods
Over the last two decades, there have been reports of several methods for real-time studies of AE dynamics in live sperm, including acrosome-targeted GFP transgenic mice7, calcium dyes loaded into the acrosome (e.g., Fluo-412), or using exocytosis-specific indicators such as the synthetic dye FM-6413. However, in comparison to these methods, the AcroSensE model provides the convenience of using minimally treated sperm that do not require any dye loading or other potentially membrane-perturbing procedures. Importantly, there is a great advantage to having sperm that express a dual indicator that can monitor acrosomal calcium dynamics, membrane fusion, and pore formation, as well as AE and the consequent release of the acrosomal content.
Importance and potential applications of the method in specific research areas
Note that the experimental application examples below could be performed as single-cell or population assays, with the latter having significant advantages when high-throughput analysis is desired. Studies of AE dynamics in various mouse models can be conducted by means of crossbreeding with the AcroSensE mouse model, including (1) null or mutated forms of various calcium channels or calcium channel subunits (e.g., crossing with α1E- and CatSper-null models); (2) null or mutated forms of proteins that mediate membrane fusion (e.g., SNAREs). Additionally, studies can be conducted to investigate how various pharmacological agents related to different signaling pathways modulate acrosomal calcium and AE dynamics. Furthermore, research can be conducted on how environmental changes affect AE, including temperature, pH, and the presence or concentration of various ions or small molecules.
The authors have nothing to disclose.
This work was supported by National Institutes of Health grants R01-HD093827 and R03-HD090304 (A.J.T).
100x oil objective | Olympus Japan | UPlanApo, | |
2-hydroxypropyl-b-cyclodextrin | Sigma | C0926 | |
35 mm coverslip dish, 1.5 thickness | MatTek Corp. | P35G-1.5-20-C | |
5 mL round-bottomed tube | Falcon | 352054 | |
Borosilicate glass capilarries | Sutter Instrument Co. CA USA | B200-156-10 | |
CaCl2 | Sigma | C4901 | |
Confocal microscope | Olympus Japan | Olympus FluoView | |
Glucose | Sigma | G7528 | |
Graduated tip | TipOne, USA Scientific | ||
HEPES | Sigma | H7006 | |
ImageJ | National Institutes of Health | https://imagej.nih.gov/ij/plugins/index.html | |
KCl | Sigma | P9541 | |
Lactic acid | Sigma | G5889 | |
Live-Cell Microscope Incubation Systems | TOKAI HIT Shizuoka, Japan | Model STX | |
MgCl2 | Sigma | M8266 | |
Micropipette Puller | Sutter Instrument Co. CA USA | Model P-97 | |
NaCl | Sigma | S3014 | |
NaHCO3 | Sigma | S6297 | |
Plastic transfer pipette | FisherBrand | 13-711-6M | |
Poly-D-lysine | Sigma | P7280 | |
Pyruvic acid | Sigma | 107360 | |
Single cell delivery system | Parker, Hauppauge, NY | Picospritzer III |