Fig 1.
Protein expression of RGS2 in oocytes during maturation in vitro.
RGS2 protein expression in mature GV (A), GVBD (B), metaphase I (C), and metaphase II (D) oocytes. RGS2 protein was initially present in the GV ooplasm (A), and 2 h after GVBD it was localized near condensing chromosomes (B). During the first meiotic division, RGS2 accumulated on the spindle (C). In metaphase II oocytes, RGS2 could be observed on the microtubules of spindles and the first polar body (D). Nuclei were stained in blue and RGS2 is shown in green. Scale bar, 20 μm.
Fig 2.
Localization of RGS2 proteins in ICSI oocytes and pronuclear zygotes.
RGS2 accumulated on the spindle and sperm DNA (arrow) during meiosis II did not change 30 min after a sperm head was injected into a MII oocyte. By 1 h later, the oocyte had entered the anaphase/telophase stage and RGS2 had become ‘stretched’ to the opposite direction (B). At 2.5 h after ICSI, the second polar body was extruded, and RGS2 was strongly stained in the spindle of fertilized eggs and the second polar body (C). By 5 h after the sperm entered the oocyte, two pronuclei had formed and RGS2 was only present in the zygote cytoplasm and second polar body, but not in pronuclei. Nuclei are stained blue and RGS2 is shown in red. Scale bar, 20 μm.
Fig 3.
Co-localization of RGS2 and β-tubulin in the meiotic spindle.
RGS2 and β-tubulin were co-expressed in spindles of metaphase oocytes (A), anaphase/telophase oocytes (B), and both in telophase oocytes and their polar bodies (C). RGS2 and β-tubulin co-localized on all meiotic spindles. The image shows staining for RGS2 in red, β-tubulin in green, and nuclei in blue. Scale bar, 20 μm.
Table 1.
Effects of antibody-mediated inhibition of the binding of RGS2 and β-tubulin on GVBD and first polar body extrusion oocyte maturation.
Fig 4.
Antibody-mediated inhibition of the binding of RGS2 and β-tubulin in GV oocytes leads to spindle defects during maturation.
All stages of control rabbit IgG-injected oocytes were cultured for 1 h (A), 4 h (B), 6 h (C), or 9 h (D) after GVBD, and all oocytes showed normal morphologies. Anti-Rgs2 antibody-injected oocytes were inhibited and exhibited various spindle morphologies (Fig 4E–4L), including microtubule accumulation near the chromatin condensing region, but almost no β-tubulin polymerization in the spindle (Fig 4E). Although a few oocytes were assembled into a normal MI spindle (Fig 4J), most oocytes were blocked at the metaphase I to anaphase I stage (Fig 4F–4K) and were accompanied by spindle defects, such as non-integral spindles (Fig 4G, 4I and 4K) and chromosomal migration failure (Fig 4F, 4H and 4J). Some MII oocytes even extruded first polar bodies that remained morphological abnormal in spindle (Fig 4L, arrow points to a muti-polar spindle). Images show staining of nuclei in blue and RGS2 in green. Scale bar, 20 μm.
Fig 5.
Antibody-mediated inhibition of the binding of RGS2 and β-tubulin reduced the expression of p-MEK1/2 in meiotic spindles during oocyte maturation.
(A) p-MEK1/2 was localized in the microtubule-organizing center of spindles in rabbit IgG-injected oocytes. However, in anti-rgs2 antibody-injected oocytes, the p-MEK1/2 signal was very week or not visible. p-MEK1/2 was labeled with Alexa Fluor 594 (red), while β-tubulin was stained with FITC (green) and nuclei were stained by DAPI (blue). (B) Fluorescence intensities of p-MEK1/2 protein in control and anti-Rgs2 antibody-injected oocytes were assessed. Images were obtained using the same laser confocal microscope using the same exposure parameters (HV 520 V; Gain X1; offset 8%). Mean fluorescence intensities were significantly different (t-test, p<0.01) between the control IgG- and anti-Rgs2 antibody-injected oocytes; 10 oocytes were measured per group. (C) Western blot analysis of p-MEK1/2 in anti-rgs2 antibody and rabbit IgG-injected oocytes. Blot is representative of 3 independent replicates; each sample includes 50 oocytes. (D) Quantitation of p-MEK1/2 blot. Data are integrated density of blot (mean gray value x blot area) ±SD from three independent experiments. Difference is considered to be significant at p<0.05.