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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), ar....
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
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 .......
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.......
This work was supported by National Institutes of Health grants R01-HD093827 and R03-HD090304 (A.J.T).
....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 |
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