combining microscopy with a stimulant delivery system to monitor live sperm from an acrosense model mouse

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 Combining Microscopy with a Stimulant Delivery System to Monitor Live Sperm from an AcroSensE Model Mouse at JoVE.com

Combining Microscopy with a Stimulant Delivery System to Monitor Live Sperm from an AcroSensE Model Mouse  

Combinazione della microscopia con un sistema di somministrazione di stimolanti per monitorare gli spermatozoi vivi di un topo modello AcroSensE

Inizia rivestendo i vetrini coprioggetti da 35 millimetri con Poly-D-Lisina o PDL. Per fare ciò, erogare una gocciolina da 0,5 microlitri di PDL al centro di un piatto di vetrino coprioggetti. Utilizzando una pipetta graduata da 10 microlitri, spalmare la goccia PDL sul vetrino coprioggetto.Lasciare asciugare il vetrino coprioggetto rivestito a 37 gradi Celsius per 10 minuti. Per l'imaging dello sperma appena raccolto da un modello murino che esprime AcroSensE, aggiungere 80 microlitri della sospensione spermatica preparata al centro del vetrino coprioggetto rivestito in PDL. Quindi, aggiungi lentamente tre millilitri di terreno di base Whitten integrato e modificato preriscaldato a 37 gradi Celsius al piatto.Procedere all'esecuzione dell'imaging utilizzando una combinazione di un microscopio con la configurazione di somministrazione di stimolanti a singola cellula. Per prima cosa, montare la capsula contenente lo sperma sul microscopio. Quindi, utilizzando il micromanipolatore, posizionare la punta capillare del sistema di rilascio dello stimolante a circa 100 micron di lato e da cinque a 10 micron sopra il piano della cellula di interesse.Posizionare il capillare entro 100 micron dalla testa dello spermatozoo. Quindi, avviare la sequenza di imaging sul microscopio. Immagine di spermatozoi a una frequenza di fotogrammi elevata, preferibilmente superiore a 10 fotogrammi al secondo.10 secondi dopo aver avviato l'acquisizione dell'immagine, attivare il sistema di rilascio a cellula singola per erogare un soffio di stimolante di 10 secondi. Continuare a catturare immagini di cellule per una durata di 10-15 minuti. In modo simile, è possibile creare l'immagine di più celle da una singola parabola in base alle posizioni mostrate sullo schermo.

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Research

JoVE Journal

Developmental Biology

Combining Microscopy with a Stimulant Delivery System to Monitor Live Sperm from an AcroSensE Model Mouse

52 Views. Inizia rivestendo i vetrini coprioggetti da 35 millimetri con Poly-D-Lisina o PDL. Per fare ciò, erogare una gocciolina da 0,5 microlitri di PDL al centro di un piatto di vetrino coprioggetti. Utilizzando una pipetta graduata da 10 microlitri, spalmare la goccia PDL sul vetrino coprioggetto.Lasciare asciugare il vetrino coprioggetto rivestito a 37 gradi Celsius per 10 minuti. Per l'imaging dello sperma appena raccolto da un modello murino che esprime AcroSensE, aggiungere 80 microlitr...

Video: Combining Microscopy with a Stimulant Delivery System to Monitor Live Sperm from an AcroSensE Model Mouse  

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 ...