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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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....

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 in.......

Representative 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.......

Acknowledgements

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

....

Materials

NameCompanyCatalog NumberComments
100x oil objective Olympus JapanUPlanApo,
2-hydroxypropyl-b-cyclodextrin SigmaC0926
35 mm coverslip dish, 1.5 thicknessMatTek Corp. P35G-1.5-20-C 
5 mL round-bottomed tubeFalcon352054
Borosilicate glass capilarriesSutter Instrument Co. CA USAB200-156-10
CaCl2SigmaC4901
Confocal microscopeOlympus JapanOlympus FluoView 
Glucose SigmaG7528
Graduated tip TipOne, USA Scientific
HEPESSigmaH7006
ImageJ National Institutes of Healthhttps://imagej.nih.gov/ij/plugins/index.html
KClSigmaP9541
Lactic acidSigmaG5889
Live-Cell Microscope Incubation Systems TOKAI HIT Shizuoka, JapanModel STX
MgCl2SigmaM8266
Micropipette Puller Sutter Instrument Co. CA USAModel P-97
NaClSigmaS3014
NaHCO3SigmaS6297
Plastic transfer pipette FisherBrand 13-711-6M
Poly-D-lysine SigmaP7280
Pyruvic acidSigma107360
Single cell delivery systemParker, Hauppauge, NYPicospritzer III

References

  1. Austin, C. R. Observations on the penetration of the sperm in the mammalian egg. Aust J Sci Res B. 4 (4), 581-596 (1951).
  2. Cohen, R., et al. A genetically targeted senso....

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