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), 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.......

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

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