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

लाइव माउस शुक्राणु का अध्ययन करने के लिए एक उत्तेजक वितरण प्रणाली के साथ इमेजिंग का संयोजन

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.

लेखक स्पॉटलाइट: माउस शुक्राणु में कैल्शियम सिग्नलिंग और एक्रोसोम एक्सोसाइटोसिस का अध्ययन करने के लिए एक लाइव सेल इमेजिंग तकनीक 

लाइव माउस शुक्राणु में एक्रोसोमल कैल्शियम गतिशीलता और एक्सोसाइटोसिस की वास्तविक समय इमेजिंग

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  

संग्रह और वास्तविक समय इमेजिंग के लिए AcroSensE माउस मॉडल से शुक्राणु की धुलाई

कोटा एपिडीडिमस से शुक्राणु एकत्र करके शुरू करें, एक ट्रांसजेनिक मॉडल माउस से अलग एक्रोसेंस व्यक्त करते हैं। 37 डिग्री सेल्सियस पर 15 मिनट की तैरने की प्रक्रिया की अनुमति देकर 0.5 मिलीलीटर मेगावाट मध्यम युक्त 3.5 सेंटीमीटर टिशू कल्चर डिश में शुक्राणु संग्रह करें। फिर ठीक संदंश का उपयोग, ध्यान से बाहर तैरना संग्रह पकवान से किसी भी epididymal ऊतक को हटा दें.शुक्राणु युक्त माध्यम को 15 मिलीलीटर शंक्वाकार ट्यूब में स्थानांतरित करें और एक झूलती बाल्टी रोटर का उपयोग करके कमरे के तापमान पर एक मिनट के लिए 100 ग्राम पर निलंबन को अपकेंद्रित्र करें। एक प्लास्टिक विंदुक की मदद से, सकल ऊतक मलबे के किसी भी गोली को त्यागने और एक दौर नीचे ट्यूब के लिए हस्तांतरण. 37 डिग्री सेल्सियस के लिए पहले से गरम मेगावाट मध्यम के 500 माइक्रोलीटर जोड़कर सतह पर तैरनेवाला मात्रा को एक मिलीलीटर में समायोजित करें।एक गोल नीचे ट्यूब में कमरे के तापमान पर आठ मिनट के लिए 400 ग्राम पर इस मिश्रण अपकेंद्रित्र. फिर ध्यान से सतह पर तैरनेवाला हटा दें, जबकि ढीले गोली शुक्राणु के पीछे छोड़ने. एक बड़े बोर ट्रांसफर पाइप का उपयोग करके, धीरे से ट्यूब को टैप करके शुक्राणु को फिर से निलंबित करें जिसे मैन्युअल रूप से उंगली फ्लिकिंग के रूप में जाना जाता है।एक बार जब शुक्राणु समान रूप से फिर से निलंबित हो जाते हैं, तो उन्हें एक नई गोल नीचे की ट्यूब में स्थानांतरित करें।

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 ...