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

Canlı Fare Spermini İncelemek için Görüntülemeyi Uyarıcı Dağıtım Sistemi ile Birleştirme

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.

Yazar Spotlight: Fare Sperminde Kalsiyum Sinyalini ve Akrozom Ekzositozunu İncelemek İçin Canlı Hücre Görüntüleme Tekniği 

Canlı fare sperminde akrozomal kalsiyum dinamiği ve ekzositozun gerçek zamanlı görüntülenmesi

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  

Gerçek Zamanlı Görüntüleme için AcroSensE Fare Modelinden Sperm Toplama ve Yıkama

AcroSensE'yi eksprese eden transgenik bir model fareden izole edilen cota epididymus'tan sperm toplayarak başlayın. Sperm toplama işlemini, 0,5 mililitre MW besiyeri içeren 3,5 santimetrelik bir doku kültürü kabında, 37 santigrat derecede 15 dakikalık bir yüzme prosedürüne izin vererek gerçekleştirin. Daha sonra ince forseps kullanarak, herhangi bir epididimal dokuyu yüzme toplama kabından dikkatlice çıkarın.Spermi içeren ortamı 15 mililitrelik konik bir tüpe aktarın ve sallanan bir kova rotoru kullanarak süspansiyonu oda sıcaklığında bir dakika boyunca 100 G'de santrifüjleyin. Plastik bir pipet yardımıyla, herhangi bir brüt doku kalıntısı peletini atarken spermi içeren MW süpernatanı toplayın ve yuvarlak tabanlı bir tüpe aktarın. 37 santigrat dereceye kadar önceden ısıtılmış 500 mikrolitre MW ortamı ekleyerek süpernatan hacmini bir mililitreye ayarlayın.Bu karışımı 400 G'de oda sıcaklığında sekiz dakika boyunca yuvarlak tabanlı bir tüpte santrifüjleyin. Ardından, gevşek bir şekilde peletlenmiş spermi geride bırakırken süpernatanı dikkatlice çıkarın. Büyük çaplı bir transfer borusu kullanarak, parmak hareketi olarak bilinen tüpe hafifçe vurarak spermi yeniden askıya alın.Sperm eşit şekilde yeniden süspanse edildikten sonra, onları yeni bir yuvarlak alt tüpe aktarın.

Research

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 ...