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. Author manuscript; available in PMC: 2023 Jan 24.
Published in final edited form as: Dev Cell. 2022 Jan 13;57(2):197–211.e3. doi: 10.1016/j.devcel.2021.12.011

Figure 1: Two different sets of MTOCs are present in acentriolar oocytes.

Figure 1:

(A) Time-lapse confocal microscopy of MTOCs in live oocytes. Full-grown prophase-I oocytes were injected with cRNAs encoding H2b-mCherry (red) and Aurka-Gfp (pseudo white), incubated in milrinone-containing CZB medium for 3 h, followed by in vitro maturation. Shown are representative images (Z-projection of 16 sections every 3 μm) from a time course (see Video S1). Lower panels represent a 3D reconstruction of MTOCs from oocyte shown in upper panels (see Video S1). Fluorescence images were captured every 20 min. The scale bar represents 10 μm. The white arrowhead represents cytoplasmic MTOC that participate in spindle pole formation as a pMTOC. The white arrow represents cytoplasmic MTOC that remains in the cytoplasm during Met I (mcMTOC). (B,D) Quantification of average MTOC number and MTOC volume over time during meiosis I, respectively. Error bars show S.D. Dashed blue lines represent the time of Anaphase I onset. (C,E). Quantification of average MTOC number and MTOC volume, respectively, during metaphase I (Met I) and anaphase I/telophase I (Ana I/Telo I) stages. The data are expressed as mean ± SEM. Student t-test was used to analyze the data. Values with asterisks vary significantly, ****P < 0.0001. The total number of analyzed oocytes (from three independent replicates) is specified above each graph. (F) Fully grown prophase-I-arrested oocytes were in vitro matured for 0 h (prophase I, Pro I), 8 h (Met I), 9 h (Ana I/Telo I) or 16 h (Met II) prior to fixation and immunocytochemistry using γ-tubulin and α-tubulin antibodies to label MTOCs (magenta) and microtubules (green). DAPI was used to detect DNA (grey). (G) Fully grown prophase-I-arrested oocytes were in vitro matured for 8 h (Met I) followed by fixation and immunostaining using CEP192 (green) and pericentrin (magenta) antibodies to label MTOCs. DAPI was used to detect DNA (blue). Shown are representative confocal Z-projections. Arrowheads represent mcMTOCs. A total of 184 oocytes were examined. Scale bars represent 10 μm (zoomed panels, 2 μm).