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

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 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  

Canlı Fare Sperminin Gerçek Zamanlı İzlenmesi için Tek Hücreli Uyarıcı Dağıtım Sisteminin Kurulması

Başlamak için, uygun ayarları izleyerek dış çapı iki milimetre ve iç çapı 1.56 milimetre olan borosilikat cam kılcal damarları çekin. Her deneyden önce kılcal damarı doldurmadan önce, kılcal damarın ucunu kırmak için ince cımbız kullanın. Kılcal damarı yüklemek için, uyarıcı çözeltiyi gerekli uyarıcının normal konsantrasyonunun üç ila beş katı kadar kullanın.İnce bir plastik transfer pipeti ile, uyarıcı çözeltiyi, uzunluğunun 3/4'ü dolana kadar kılcal damara yavaşça enjekte edin. Doldurma işlemi sırasında oluşabilecek hava kabarcıklarını kılcal damarı hafifçe vurarak ortadan kaldırın. Ardından, doldurulmuş kılcal damarı mikromanipülatöre monte edin.Tek hücreli dağıtım sistemi ayarlarını, beş psi'de 10 saniyelik bir darbe sağlayacak şekilde yapılandırın. Mikromanipülatöre monte edilmiş borosilikat kılcalını tek hücreli dağıtım sistemi pipet tutucusuna bağlayın. Çözeltinin ucun ucundan dağıtıldığını doğrulamak için kılcal ucu gözlemlerken, 20 psi'lik bir basınçta iki saniye süren kısa bir nefes uygulayın.Kurulum artık tek bir fare sperminin gerçek zamanlı görüntülenmesi için mikroskopi ile kullanıma hazırdır.

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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

63 Görüntüleme.  Cornell University. Başlamak için, uygun ayarları izleyerek dış çapı iki milimetre ve iç çapı 1.56 milimetre olan borosilikat cam kılcal damarları çekin. Her deneyden önce kılcal damarı doldurmadan önce, kılcal damarın ucunu kırmak için ince cımbız kullanın. Kılcal damarı yüklemek için, uyarıcı çözeltiyi gerekli uyarıcının normal konsantrasyonunun üç ila beş katı kadar kullanın.İnce bir plastik transfer pipeti ile, uyarıcı çözeltiyi, uzunluğunun 3/4'ü dolana ...

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 ...

Gelişim Biyolojisi

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

Begin by collecting sperm from the cota epididymus, isolated from a transgenic model mouse expressing AcroSensE. Perform the sperm collection in a 3.5 centimeter tissue culture dish containing 0.5 milliliters of MW medium by allowing a 15 minute swim out procedure at 37 degrees Celsius. Then using fine forceps, carefully remove any epididymal tissue from the swim out collection dish.Transfer the medium containing the sperm into a 15 milliliter conical tube and using a swinging bucket rotor centrifuge the suspension at 100 G for one minute at room temperature. With the help of a plastic pipette, collect the MW supernatant containing the sperm while discarding any pellet of gross tissue debris and transfer it to a round bottom tube. Adjust the supernatant volume to one milliliter by adding 500 microliters of MW medium prewarmed to 37 degrees Celsius.Centrifuge this mixture at 400 G for eight minutes at room temperature in a round bottom tube. Then carefully remove the supernatant while leaving behind the loosely pelleted sperm. Using a large bore transfer pipe, re-suspend the sperm by gently tapping the tube manually known as finger flicking.Once the sperm are evenly re-suspended, transfer them into a new round bottom tube.

Gerçek Zamanlı Görüntüleme için AcroSensE Fare Modelinden Sperm Toplama ve Yıkama

To begin, pull borosilicate glass capillaries with an outer diameter of two millimeters and an inner diameter of 1.56 millimeters, following appropriate settings. Prior to filling the capillary before each experiment, use fine tweezers to break open the tip of the capillary. For loading the capillary, use the stimulating solution at three to five times the normal concentration of the stimulant required.With a thin plastic transfer pipette, slowly inject the stimulating solution into the capillary until 3/4 of its length is filled. Eliminate any air bubbles that may form during the filling process by gently flicking the capillary. Then, mount the filled capillary on the micromanipulator.Configure the single cell delivery system settings to provide a 10-second pulse at five psi. Connect the borosilicate capillary mounted on the micromanipulator to the single cell delivery system pipette holder. Apply a brief puff lasting two seconds at a pressure of 20 psi, while observing the capillary tip to confirm that the solution is being dispensed through the tip's end.The setup is now ready for use with microscopy for real-time imaging of a single mouse sperm.

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

Begin by coating the 35-millimeter coverslip dishes with Poly-D-Lysine, or PDL. To do so, dispense a 0.5 microliter droplet of PDL at the center of a coverslip dish. Using a 10-microliter graduated pipette, smear the PDL droplet across the coverslip.Allow the coated coverslip dish to dry at 37 degrees Celsius for 10 minutes. For imaging the freshly collected sperm from a mouse model expressing AcroSensE, add 80 microliters of the prepared sperm suspension to the center of the PDL-coated coverslip dish. Then, slowly add three milliliters of supplemented, modified Whitten's base media pre-warmed to 37 degrees Celsius to the dish.Proceed to perform the imaging using a combination of a microscope with the single cell stimulant delivery setup. First, mount the dish containing the sperm on the microscope. Then, using the micromanipulator, position the capillary tip of the stimulant delivery system about 100 microns to the side and five to 10 microns above the plane of the cell of interest.Position the capillary within 100 microns from the sperm head. Next, start the imaging sequence on the microscope. Image sperm cells at a high frame rate, preferably greater than 10 frames per second.10 seconds after initiating the image acquisition, activate the single cell delivery system to deliver a 10-second puff of stimulant. Continue capturing images of cells for a duration of 10 to 15 minutes. In a similar manner, image multiple cells from a single dish according to the locations shown on the screen.