Real-Time Imaging of Acrosomal Calcium Dynamics and Exocytosis in Live Mouse Sperm

Summary

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.

Abstract

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.

Introduction

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 in^2,3). 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 line^7,8,9.

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.

Protocol

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

1. Collect cauda epididymal sperm (more details on this procedure are provided in¹¹) from AcroSensE mice. Allow 15 min swim-out procedure in a 3.5 cm tissue-culture dish containing 0.5 mL of Modified Whitten's medium (MW) at 37 °C. Dish should be covered to reduce evaporation of the MW, and should be tilted so that the medium has enough depth to cover the cauda epididymides.
   NOTE: Modified Whitten's medium (MW) includes 22 mM HEPES, 1.2 mM MgCl₂, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid hemi-Ca²⁺ salt, pH 7.35. Glucose (5.5 mM), NaHCO₃ (10 mM), and 2-hydroxypropyl--cyclodextrin (CD; 3 mM) are supplemented (see Table of Materials) as needed for induction of capacitation, and concentrations can be modified for specific experiments. In addition, alternative sterol acceptors could be used instead of CD (e.g., bovine serum albumin or high density lipoproteins).
2. Using fine forceps, remove any epididymal tissue from the swim-out collection dish. Transfer the medium containing the sperm into a 15 mL conical tube, and centrifuge the suspension at 100 x g for 1 min in a swinging bucket rotor at room temperature. Using a plastic pipette, remove the MW supernatant containing the sperm from any gross tissue debris that will have pelleted at this step.
3. Bring the MW supernatant containing the sperm to a total volume of 3 mL by adding MW at 37 °C. Centrifuge at 400 x g for 8 min (at room temperature) in a round-bottomed tube. Slowly remove the supernatant of MW from the loosely-pelleted sperm.
4. If viable, the sperm should appear as a "fluffy"-looking pellet. Gently resuspend the sperm via trituration with a large-bore transfer pipette or via gentle manual taps on the tube ("finger flicking"). Once resuspended evenly, transfer them into a new round-bottom tube. Use 10-20 µL of the sperm suspension to count and assess motility. Dilute sperm in MW for use.
   NOTE: For most experiments involving mouse sperm capacitation, a final concentration of 5-10 million sperm/mL MW is utilized. The imaging protocol described here does not require the capacitation of sperm, although it is necessary for sperm to acquire the ability to undergo physiologically-relevant acrosome exocytosis. One could study pathological exocytosis or exocytosis induced through reagents such as a calcium ionophore without capacitating the sperm. Additionally, one could explore potential changes in acrosomal calcium in non-capacitated cells in response to various stimuli.
5. Use the sperm within 3-5 h from collection, keeping the time as short as possible and consistent among experimental trials.
6. Perform all steps of collection and washing at 37 °C using MW medium, employing methods to minimize membrane damage. This includes the use of large-bore plastic transfer pipettes or large-orifice pipette tips, which are recommended for use during all stages of the procedure.

2. Poly-D-lysine-coating of coverslip dishes for imaging

NOTE: Poly-D-lysine (PDL) dishes should be prepared fresh before each experiment.

1. Dispense a 0.5 μL droplet of PDL (0.5 mg/mL, see Table of Materials) at the center of a coverslip dish.
2. Using a 10 μL graduated tip, smear the PDL droplet across the coverslip (crisscross pattern as shown in Figure 1B).
3. Allow to dry at 37 ˚C for 10 min. Keep PDL-coated dishes covered and at 37 ˚C until use.

3. Capillary pulling, loading for puffing

1. Pull borosilicate glass capillaries (O.D, 2 mm, I.D, 1.56 mm, see Table of Materials) using settings as follows- Heat: 330, Pull: 250, Velocity: 250, and Time: 70. This step should be optimized for the specific puller in use.
2. Before each experiment, load a single capillary with the stimulating solution containing 3-5x the concentration of the stimulant (to account for the rapid diffusion of the solution upon puffing). Before filling, use fine tweezers to break open the capillary tip at approximately 1-2 mm from the end. This should produce a ∼5 µm opening diameter. Heat polishing is not required.
3. Using a thin plastic transfer pipette, slowly inject the stimulating solution into the capillary until three-fourths of its length is full.
4. Remove any air bubbles formed during filling by gentle flicking of the capillary.
5. Mount filled capillary on the micromanipulator (see Table of Materials).

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.

1. Set up the single-cell delivery system settings to provide a 10 s pulse at 5 psi.
2. Connect the borosilicate capillary to the single-cell delivery system pipette holder. Ensure seals are tight.
3. While observing the capillary tip, apply a short puff of 2 s at 20 psi to ensure that the solution is indeed dispensed through the tip's end (a small drop should be obvious at the end of the tip).

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.

1. Add 80 μL of diluted sperm to the center of a poly-D-lysine-coated 35 mm coverslip dish. For most experiments involving mouse sperm capacitation, a final concentration of 5-10 million sperm/mL is utilized.
2. Slowly add to the sperm 3 mL of warm (37 ˚C) MW base media supplemented with 10 mM CaCl₂.
   NOTE: One could perform experiments at room temperature to slow cellular processes, but it's important to note that because capacitation is mediated by lipid exchanges and the fluidity of micro- and macro-domains, one must keep in mind that physiologically-relevant capacitation and acrosome exocytosis are temperature-dependent processes.
3. Mount the dish with the sperm on the microscope (pre-heated to 37 ˚C).
4. Excite the GCaMP3 using a 488 nm laser line and view with a 505-550 nm bandpass (BP) filter. Excite the mCherry using a 555 nm laser line and view with a 575-615 nm BP filter.
5. Using a micromanipulator (it is recommended to attach the manipulator to the air-table on which the microscope was set up.), position the tip of the capillary approximately 100 μm to the side and 5-10 μm above the plane of the cell of interest.
   NOTE: The stimulating solutions are made at 3-5x the normal concentration to compensate for the rapid diffusion occurring in the volume between the capillary and the cell.
6. Start the imaging sequence on the microscope.
7. Image sperm cells at a high frame rate, preferably >10 frames/s. Here, a resonant scanner was used that enables imaging at 33 ms/frame.
8. Ten seconds after starting the image acquisition on the microscope, activate the single-cell delivery system to initiate the 10 s puff of stimulant.
9. Image cells for a duration of 10-15 min.
10. Using the single-cell delivery system, image multiple cells from a single dish according to the locations provided in Figure 1B.
    ​NOTE: Untreated/non-stimulated sperm under the microscope should appear mostly red, with a low intensity of the green signal. Sperm heads should be attached to the coverslip, and live sperm can be identified by their moving tails. In sperm that undergo AE, the GCaMP3 green signal will initially increase, and both the mCherry red signal and GCaMP3 green signal will disappear if AE is complete, as illustrated in Figure 2. The reduction of mCherry fluorescence provides a more specific indicator of the time when AE begins because changes in Ca²⁺ concentrations will continually cause changes in the GCaMP3 fluorescence intensity.

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.

1. Depending on the specific microscope system file extension, use the split channel tool (Image > Colors > Split Channels) to generate individual windows for the GCaMP3 and mCherry channels.
2. Synchronize the individual windows using the "Synchronize Window" tool (Analyze > Tools > Synchronize Windows).
3. Select a region of interest (ROI) around the sperm head in the red channel to include the acrosome region (Figure 3A,B). The synchronize tool allows the same area selection to be applied automatically on the green channel, in which the sperm are still too dim to be visible.
4. Choose "Z Profiler" plugin (Plugins > Stacks > Z Profiler) or "Time Series Analyzer" (Plugins > Stacks > Time Series Analyzer) to plot the change in fluorescence intensity as a function of frames/time for each of the two channels.
5. Copy data to a spreadsheet software (e.g., Microsoft Excel) for plotting and further analysis.
   NOTE: Once the data are copied to a spreadsheet, multiple parameters can be extracted for analysis, including the following, as illustrated in Figure 4.
   1. Fusion Pore (FP) start time: measure the time from timepoint 0 to the timepoint where the GCaMP3 signal starts to rise. This parameter indicates the delay between the stimulation and the sperm ACR response.
   2. AE start time: measure the time from timepoint 0 to the timepoint where the mCherry signal starts to decay. This parameter indicates the delay between the stimulation and the sperm AE response. This parameter could also be used to calculate the delay between FP formation and AE response.
   3. Peak FP amplitude: measure the fluorescence signal from the base to the peak of the GCaMP3 signal rise. This parameter indicates the total increase in ACR response.
   4. Rate of loss of mCherry: determine this parameter by a linear or an exponential fit on the section of the mCherry signal loss (as indicated by "slope" in Figure 4B).
      NOTE: Alternatively, determine the decay rate by the residual (R) signal method at various time points (e.g., R60: duration time in seconds to a residual signal of 60% from the baseline of the mCherry signal). This parameter can be used to quantify the rate at which the acrosomal content is dispersed upon AE.
   5. Signal decrease for mCherry loss: determine the difference in amplitude between the baseline mCherry fluorescence intensity and the signal following AE. This parameter indicates the total amount of acrosomal content dispersed upon AE.
   6. Plot the changes in fluorescence (F) for both the red and green channels as raw data, or normalize them by the initial fluorescence (F0) and express them as ΔF/F0. The latter provides better visualization of the changes in both red and green on a single plot (e.g., see Figure 3D and Figure 3H).
      NOTE: Finally, the different measured parameters can be compared between different conditions to determine the relationship between changes in acrosomal calcium concentration and AE. For examples of various experimental condition comparisons, see reference².

Results

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 ...

Discussion

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 capacitat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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