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

Impact of cytoskeleton on nuclear shape and condensate dynamics in mouse oocytes(생쥐 난모세포의 핵 형태 및 응축물 역학에 대한 세포골격의 영향)

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.

저자 스포트라이트: Elucidating the Dynamics of Mechano-Transduction and Nuclear Agitation in Mouse Oocytes

공간 규모에 걸쳐 마우스 난모세포핵의 세포골격 기반 교반 캡처

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.

Oocyte Collection, Preparation, and Microinjection in Mouse for Live Imaging Studies(생모세포 수집, 준비 및 생쥐 미세주입 연구)

작업 플랫폼에 실험에 필요한 모든 재료를 배열하는 것으로 시작하십시오. 생쥐에서 난소를 추출한 후 미리 데워진 M2 플러스 소 혈청 알부민 배지에 마이크로몰 밀리논 1개가 보충된 배지에 넣습니다. 수술용 바늘로 난포를 찔러 난포에서 자라는 난모세포를 방출합니다.마이크로피펫을 사용하여 실험에 필요한 크기의 난모세포를 채취합니다. 그런 다음 난모세포를 신선한 배지가 있는 접시에 옮깁니다. 마이크로피펫을 사용하여 한 방울에서 한 방울로 난모세포를 옮깁니다.이를 통해 난모세포에서 난모세포를 기계적으로 분리할 수 있습니다. 난모세포를 섭씨 37도에서 1시간 동안 안정화시킵니다. cRNA 부분 표본을 원심분리하여 YFP-Rango 및 SRSF2 GFP를 코팅합니다.마이크로 인젝터를 사용하여 따뜻한 M2 플러스 소 혈청 알부민 배지에 있는 난모세포의 세포질에 cRNA를 주입하고 밀리논을 보충합니다. cRNA 번역을 위해 섭씨 37도의 배양 배지에서 2시간 동안 난모세포를 배양합니다. 다음으로, 난모세포를 미네랄 오일로 덮인 커버 유리 바닥 챔버가 있는 35mm 조직 배양 접시의 배양 배지 방울에 증착합니다.

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

Research

JoVE Journal

Biology

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

99 조회수.  Collège de France, CNRS, INSERM, Université PSL. 작업 플랫폼에 실험에 필요한 모든 재료를 배열하는 것으로 시작하십시오. 생쥐에서 난소를 추출한 후 미리 데워진 M2 플러스 소 혈청 알부민 배지에 마이크로몰 밀리논 1개가 보충된 배지에 넣습니다. 수술용 바늘로 난포를 찔러 난포에서 자라는 난모세포를 방출합니다.마이크로피펫을 사용하여 실험에 필요한 크기의 난모세포를 채취합니다. 그런 다음 난모세포를 신?...

비디오: Oocyte Collection, Preparation, and Microinjection in Mouse for Live Imaging Studies(생모세포 수집, 준비 및 생쥐 미세주입 연구)

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 ...