oocyte collection, preparation, and microinjection in mouse for live imaging studies

Fare Oositlerinde Hücre İskeletinin Nükleer Şekil ve Kondensat Dinamiği Üzerine Etkisi

Nuclear positioning is essential for multiple cellular and developmental functions1,2,3,4,5. Mammalian female germ cells named oocytes remodel their cytoplasm to position the nucleus at the cell center despite undergoing an asymmetric division in size, which relies on subsequent chromosome off-centering6 (Figure 1). This centering of the nucleus predicts successful oocyte development in mice and humans7, 8, and thus, their embryogenic potential (Figure 1).
Cytoplasmic remodeling in mouse oocytes is driven primarily by the actomyosin cytoskeleton9 (Figure 2). Its activity generates mechanical forces that agitate, reposition, and penetrate the nucleus10 (Figure 2). Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear messenger RNA-processing organelles named nuclear speckles11, one of several membrane-less organelles in the nucleus known as biomolecular condensates12,13,14,15,16 (Figure 2).
Live imaging has been decisive in deciphering the functional implications of nuclear agitation. Movies of nuclear migration over hours, as well as high-temporal resolution movies of the actin mesh and the bulk cytoplasm, largely contributed to the elaboration of a theoretical model for nuclear positioning, linking different timescales9. Also, high temporal resolution movies of the cytoplasm, nuclear outline and nuclear components such as chromatin and nuclear condensates, highlighted the role of cytoskeleton-based agitation of the nucleus on RNA-processing and gene expression in mouse oocytes, bridging different spatiotemporal scales within the cell10,11. Altogether, such a scale-crossing approach based on live imaging provided the first rationale linking cytoskeletal agitation of the nucleus to the developmental success of oocytes.
The protocol provides the imaging and image analysis pipeline used to study the transmission of cytoplasmic forces (generated primarily by F-actin and partly by microtubules) to the nucleus and its internal components in mouse oocytes. The outcome of these experiments is to capture the continuum of forces across spatial scales, from the cytoskeleton in the cytoplasm to the nuclear interior via high temporal resolution movies as shown in two recent studies10,11, that established the link between cytoplasmic active movements, fluctuations of the nuclear outline, as well as movement and surface fluctuations of a single type of nuclear biomolecular condensates: nuclear speckles. The same approach may be applied to other model systems where cytoplasmic forces are expected to change, such as in the context of malignant cancer cells17.

Yazar Spot Işığı: Fare Oositlerinde Mekano-Transdüksiyon ve Nükleer Ajitasyon Dinamiklerinin Aydınlatılması

Fare Oosit Çekirdeğinin Hücre İskeleti Tabanlı Ajitasyonunun Uzamsal Ölçeklerde Yakalanması

The lab aims to better understand the mechanisms involved in the acquisition of female gamut quantity, which is essential for the reproduction of sexual species using the mouse oocytes model system. Recently, we identified a novel mechanical transduction process that promotes agitation of the nucleus and its content, leading to the fine regulation of maternal RNAs in mouse oocytes. The imaging and image analysis pipeline presented in this protocol allows us to highlight the transmission of cytoskeletal forces as a continuum across scales from the cytoplasm to the nucleus and its components, including nuclear RNA processing bodies in mouse oocytes.This protocol provides simple tools that allow us to address first transmission across multiple cellular scales in a single pipeline for the first time. Complemented with biophysical modeling, this protocol constitutes a non-invasive technique to address changes in nuclear mechanics and the dissipation of energy across cellular compartments.

Canlı Görüntüleme Çalışmaları için Farede Oosit Toplama, Hazırlama ve Mikroenjeksiyon

Deney için gerekli tüm malzemeleri çalışma platformuna yerleştirerek başlayın. Yumurtalıkları fareden çıkardıktan sonra, bunları önceden ısıtılmış M2 artı bir mikromolar milrinon ile desteklenmiş sığır serum albümin ortamına yerleştirin. Antral foliküllerden büyüyen oositleri serbest bırakmak için yumurtalık foliküllerini cerrahi iğnelerle delin.Bir mikropipet kullanarak, deneyler için gereken büyüklükteki oositleri toplayın. Daha sonra oositleri taze orta dereceli bir kaba aktarın. Mikropipet kullanarak oositleri damladan damlaya aktarın.Bu, oositlerin antral foliküler hücrelerden mekanik olarak ayrılmasına izin verir. Oositi 37 santigrat derecede bir saat stabilize edin. YFP-Rango ve SRSF2 GFP için kaplama olan cRNA alikotlarını santrifüjleyin.Bir mikro enjektör kullanarak, cRNA'yı milrinon ile desteklenmiş ılık bir M2 artı sığır serum albümin ortamında oositlerin sitoplazmasına enjekte edin. cRNA translasyonu için oositleri 37 santigrat derecede bir kültür ortamında iki saat inkübe edin. Daha sonra, oositleri, mineral yağ ile kaplı bir kapak camı alt haznesi olan 35 milimetrelik bir doku kültürü kabında kültür ortamı damlacıklarına bırakın.

oocyte-collection-preparation-microinjection-mouse-for-live-imaging

Research

JoVE Journal

Biology

Oocyte Collection, Preparation, and Microinjection in Mouse for Live Imaging Studies

99 Görüntüleme.  Collège de France, CNRS, INSERM, Université PSL. Deney için gerekli tüm malzemeleri çalışma platformuna yerleştirerek başlayın. Yumurtalıkları fareden çıkardıktan sonra, bunları önceden ısıtılmış M2 artı bir mikromolar milrinon ile desteklenmiş sığır serum albümin ortamına yerleştirin. Antral foliküllerden büyüyen oositleri serbest bırakmak için yumurtalık foliküllerini cerrahi iğnelerle delin.Bir mikropipet kullanarak, deneyler için gereken büyüklükteki oositleri toplayın. Daha sonra oositler...

Video: Canlı Görüntüleme Çalışmaları için Farede Oosit Toplama, Hazırlama ve Mikroenjeksiyon

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named oocytes. Their development is marked by cell growth and subsequent divisions, two critical phases that prepare the oocyte for fusion with sperm to initiate embryogenesis. During growth, oocytes reorganize their cytoplasm to position the nucleus at the cell center, an event predictive of successful oocyte development in mice and humans and, thus, their embryogenic potential. In mouse oocytes, this cytoplasmic reorganization was shown to be driven by the cytoskeleton, the activity of which generates mechanical forces that agitate, reposition, and penetrate the nucleus. Consequently, this cytoplasmic-to-nucleoplasmic force transmission tunes the dynamics of nuclear RNA-processing organelles known as biomolecular condensates. This protocol provides an experimental framework to document, with high temporal resolution, the impact of the cytoskeleton on the nucleus across spatial scales in mouse oocytes. It details the imaging and image analysis steps and tools necessary to evaluate i) cytoskeletal activity in the oocyte cytoplasm, ii) cytoskeleton-based agitation of the oocyte nucleus, and iii) its effects on biomolecular condensate dynamics in the oocyte nucleoplasm. Beyond oocyte biology, the methods elaborated here can be adapted for use in somatic cells to similarly address cytoskeleton-based tuning of nuclear dynamics across scales.

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

A major challenge in understanding the causes of female infertility is to elucidate mechanisms governing the development of female germ cells, named ...