Name: Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging
Uploaded: 2023-10-13T14:20:00
Description: Watch this Scientific Journal Video about Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging at JoVE.com

collection and washing of sperm from the acrosense mouse model for real-time imaging

Combinando imagens com um sistema de entrega de estimulantes para estudar espermatozoides vivos de camundongos

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&#39;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 &#181;M 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 &#34;channel&#34; 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.

Autor em destaque: uma técnica de imagem de células vivas para estudar a sinalização de cálcio e exocitose acrossômica em espermatozoides de camundongos 

Imagem em Tempo Real da Dinâmica do Cálcio Acrossomal e Exocitose em Espermatozoides Vivos de Camundongos

Our research goal is to better understand the molecular mechanisms that regulate sperm function, including the processes such as capacitation and fertilization. Specifically, we're exploring how membrane lipids activate calcium signaling and acrosome exocytosis. This protocol addresses the gap in our understanding of the interplay between capacitation, sperm activation, calcium signaling, and acrosome exocytosis.The advantages of this protocol include imaging sperm under physiological conditions, as well as its ability to simultaneously monitor both calcium signaling and acrosome exocytosis without the need for complicated preloading or standing procedures.

Watch this Scientific Journal Video about Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging at JoVE.com

Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging  

Coleta e Lavagem de Espermatozoides do Modelo de Camundongo AcroSensE para Imagem em Tempo Real

Comece coletando espermatozoides do epidídimo da cota, isolados de um camundongo modelo transgênico expressando AcroSensE. Realizar a coleta de espermatozoides em uma placa de cultura de tecidos de 3,5 centímetros contendo 0,5 mililitros de meio MW, permitindo um procedimento de nado de 15 minutos a 37 graus Celsius. Em seguida, usando pinças finas, remova cuidadosamente qualquer tecido epididimal da placa de coleta de natação.Transfira o meio que contém os espermatozoides para um tubo cônico de 15 mililitros e, usando um rotor de balde oscilante, centrifugar a suspensão a 100 G por um minuto à temperatura ambiente. Com a ajuda de uma pipeta de plástico, colete o sobrenadante MW contendo os espermatozoides enquanto descarta qualquer pelota de detritos de tecido grosseiro e transfira-o para um tubo de fundo redondo. Ajuste o volume sobrenadante para um mililitro adicionando 500 microlitros de MW médio pré-aquecido a 37 graus Celsius.Centrifugar esta mistura a 400 G durante oito minutos à temperatura ambiente num tubo de fundo redondo. Em seguida, remova cuidadosamente o sobrenadante, deixando para trás os espermatozoides soltos. Usando um tubo de transferência de furo grande, ressuspenda o esperma batendo suavemente o tubo manualmente conhecido como movimento do dedo.Uma vez que os espermatozoides são uniformemente ressuspensos, transferi-los para um novo tubo de fundo redondo.

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JoVE Journal

Developmental Biology

Collection and Washing of Sperm from the AcroSensE Mouse Model for Real-Time Imaging

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

Acrosome exocytosis (AE), in which the sperm&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...