Fibrillarin-GFP Facilitates the Identification of Meiotic Competent Oocytes

Abstract

The nucleolus undergoes significant functional changes and plays important roles during mammalian oocyte meiotic maturation. Fibrillarin (FBL) is the component of nucleolar small nuclear ribonucleoprotein (snRNP) particle and localizes to the dense fibrillar component (DFC) of the nucleolus. We found that FBL-GFP displays an uneven and cloudy localization in the nucleolus of non-surrounded nucleolus (NSN) oocytes, while it distributes evenly and to a few bright dots in the surrounded nucleolus (SN) oocytes. Accordingly, NSN oocytes showed active nascent RNA transcription, while the SN group was transcriptionally quiescent. NSN geminal vesicles also contained more DNA damage marker γH2AX foci. Based on different FBL-GFP patterns in live oocytes, the ones with superior meiotic maturation potential can be identified. Global transcriptome profiling revealed a significant difference in single SN and NSN oocytes. Thus, FBL-GFP can serve as a marker for nucleolus activity, which also correlates with transcription activity and the quality of oocytes.

Keywords: fibrillarin; meiosis; nucleolus; oocyte maturation; transcriptome analysis.

FIGURE 1

Organization of FBL clusters in NSN and SN oocytes. (A,B) FBL exhibits a clustered distribution pattern in fixed NSN GV oocytes. The clusters decrease significantly in fixed SN GV oocytes. Representative z section (A) and 3D projection (B) confocal images of exogenously expressed FBL–GFP and H2B-mCherry in oocytes are shown. Scale bar = 5 μm. (C) Bar graph shows relative FBL signal intensity to H2B signal intensity in fixed NSN and SN GV oocytes. N = 68. **P < 0.01. (D) The intensity distribution of FBL was measured along the arrow lines drawn on the images in (B). Green, NSN; purple, SN. (E,F) FBL distribution patterns in live NSN and SN GV oocytes. Representative z section (E) and 3D projection (F) confocal images of exogenously expressed FBL–GFP and H2B-mCherry in oocytes are shown. Scale bar = 5 μm. (G) The bar graph shows relative FBL signal intensity to H2B signal intensity in live NSN and SN GV oocytes. N = 39. **P < 0.01. (H) The intensity distribution of FBL was measured along the arrow lines drawn on the images in (F). Green, NSN; purple, SN. (I,J) FBL distribution patterns were confirmed with anti-FBL in fixed NSN and SN oocytes. Representative z section (I) and 3D projection (J) confocal images of endogenous FBL localization are shown. Scale bar = 5 μm. (K) The bar graph shows relative anti-FBL signal intensity to DNA signal intensity in fixed NSN and SN GV oocytes. N = 56. ****P < 0.0001. (L) The intensity distribution of anti-FBL was measured along the arrow lines drawn on the images in (J). Green, NSN; purple, SN.

FIGURE 2

The transcription activity of NSN and SN GV oocytes. (A,B) Representative z section images of nascent RNA transcription in NSN and SN GV oocytes. Box–whisker plot shows relative EU signal intensity to DNA signal intensity in NSN and SN GV oocytes. Scale bar = 10 μm. N = 23. ***P < 0.001. (C–E) Representative z section (C) and 3D projection (D) confocal images of nascent RNA transcription in FBL–GFP-classified NSN and SN GV oocytes. Box–whisker plot shows relative EU signal intensity to DNA signal intensity in FBL–GFP-classified NSN and SN GV oocytes. Scale bar = 5 μm. N = 49. ****P < 0.0001. (F–H) Representative live oocyte bright-field images at 0, 4, and 16 h after FBL–GFP classification and Cilostamide release. Scale bars = 100 or 50 μm. The GVBD rate at 4 h was shown in (G). The 1st polar body extrusion rate at 16 h was shown in (H). **P < 0.01.

FIGURE 3

Transcriptome comparison before and after meiotic maturation of NSN and SN oocytes. (A) Schematic illustrating the pipeline of preparing single oocyte RNA-Seq samples. (B) PCA analysis of RNA-Seq data from NSN and SN oocytes at the GV and MII stages. Each dot represents one library, color-coded by oocyte type and stage. (C) Gene expression heatmap of NSN and SN oocytes at the GV stage and MII stage. The blue to red color change represents the transcription level from low to high. (D,E) Volcano plot of DEGs in NSN oocytes vs. SN oocytes at the GV stage and MII stage. The upregulated (fold change >1.5) and downregulated (fold change <–1.5) transcripts in each comparison are labeled by red and blue, respectively (P < 0.05). (F) Gene ontology analysis of the upregulated and downregulated DEGs between NSN and SN MII oocytes. (G) Mfuzz analysis of RNA-Seq data from NSN and SN oocytes at the GV and MII stages. The x axis represents oocyte types and stages, while the y axis represents standardized FPKM. Green to purple colored lines correspond to genes with low to high membership value. (H) Gene ontology analysis of genes in the four clusters from (G) (see also Supplementary Tables 1–3).

FIGURE 4

H2AX phosphorylation dynamics during meiotic maturation. (A) γH2AX staining in NSN and SN oocytes at GV, MI, and MII stage. Scale bars = 10 μm. (B) The scatter plot shows the quantification of γH2AX foci numbers in (A). ****P < 0.0001.