setting up the single cell stimulant delivery system for real-time monitoring of live mouse sperm

Combinazione dell'imaging con un sistema di somministrazione di stimolanti per studiare lo sperma di topo vivo

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.

Autore in primo piano: una tecnica di imaging di cellule vive per studiare la segnalazione del calcio e l'esocitosi dell'acrosomia nello sperma di topo 

Imaging in tempo reale della dinamica del calcio acrosomiale e dell'esocitosi in spermatozoi di topo vivi

Watch this Scientific Journal Video about Setting up the Single Cell Stimulant Delivery System for Real-Time Monitoring of Live Mouse Sperm at JoVE.com

Setting up the Single Cell Stimulant Delivery System for Real-Time Monitoring of Live Mouse Sperm  

Impostazione del sistema di somministrazione di stimolanti a singola cellula per il monitoraggio in tempo reale di spermatozoi di topo vivi

Per iniziare, tirare i capillari in vetro borosilicato con un diametro esterno di due millimetri e un diametro interno di 1,56 millimetri, seguendo le impostazioni appropriate. Prima di riempire il capillare prima di ogni esperimento, utilizzare una pinzetta fine per aprire la punta del capillare. Per caricare il capillare, utilizzare la soluzione stimolante da tre a cinque volte la normale concentrazione dello stimolante richiesto.Con una pipetta di trasferimento in plastica sottile, iniettare lentamente la soluzione stimolante nel capillare fino a riempire i 3/4 della sua lunghezza. Eliminare eventuali bolle d'aria che possono formarsi durante il processo di riempimento muovendo delicatamente il capillare. Quindi, montare il capillare riempito sul micromanipolatore.Configurare le impostazioni del sistema di erogazione a cella singola per fornire un impulso di 10 secondi a cinque psi. Collegare il capillare in borosilicato montato sul micromanipolatore al portapipetta del sistema di erogazione a cella singola. Applicare una breve boccata della durata di due secondi a una pressione di 20 psi, osservando la punta capillare per confermare che la soluzione viene erogata attraverso l'estremità della punta.La configurazione è ora pronta per l'uso con la microscopia per l'imaging in tempo reale di un singolo spermatozoo di topo.

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Research

JoVE Journal

Developmental Biology

Setting up the Single Cell Stimulant Delivery System for Real-Time Monitoring of Live Mouse Sperm

69 Views. Per iniziare, tirare i capillari in vetro borosilicato con un diametro esterno di due millimetri e un diametro interno di 1,56 millimetri, seguendo le impostazioni appropriate. Prima di riempire il capillare prima di ogni esperimento, utilizzare una pinzetta fine per aprire la punta del capillare. Per caricare il capillare, utilizzare la soluzione stimolante da tre a cinque volte la normale concentrazione dello stimolante richiesto.Con una pipetta di trasferimento in plastica sottile, ...

Video: Setting up the Single Cell Stimulant Delivery System for Real-Time Monitoring of Live Mouse Sperm  

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 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.

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&#39;s single exocytotic vesicle fuses with the plasma membrane, is a complex, calcium-dependent process ...