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Fig. 6. Vegetal-to-animal translocation of the proteasome during the oocyte-to-embryo transition. (A) Real-time RT-PCR showing the expression of psme1, psme2, psme3 and psme4 during Xenopus development. (B) In situ hybridization showing the expression of psme1, psme2, psme3, psme4, psmc6, psma2, dnd1, pgat, eIF4A1 and eIF4E in hemi-sectioned oocytes, ovulated eggs, and embryos at the 2-cell stage. Images shown here are representative images from at least 15 samples. (C) Immunofluorescence showing the subcellular distribution of 20S CP in oocytes, ovulated eggs and embryos at the 2-cell stage. We stained oocytes, eggs and embryos with two different anti-proteasome 20S CP antibodies (see Materials and Methods) and obtained essentially the same results. Results shown here, which were obtained using the antibody from Enzo Life Sciences, are representative images from 23 samples. (D) Quantification of proteasome components in the animal and vegetal hemispheres of 1-cell-stage embryos by mass spectrometry. The histogram shows the average protein abundance across all conditions and replicates in the y-axis against log2 of protein abundance ratio between animal and vegetal hemispheres. Of note, higher abundance of proteins enables more confident quantifications. n=5 replicates of animal and vegetal. Error bars represent s.e.m. |
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Figure S2. Overexpression of F110M had no effect on PGC development. (A) In situ
hybridization showing the expression of pgat in control, and F110M (1 ng) injected embryos.
RNA was injection into the vegetal pole at the 1-cell stage. (B) Quantification of results
shown in A. The number of pgat-positive PGCs from each embryo was counted and plotted
on the graph. There is no statistically significant difference between control and F110M
overexpressed embryos. |
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Fig. 7. Ubiquitin-independent proteasomes inhibit germline development. (A) In situ hybridization showing the expression of pgat in control, and embryos injected with psme1+psme2, psme3, psme4, or all four psme RNAs. RNAs were injected into the vegetal pole at the 1-cell stage. (B) Quantification of the results shown in A. The number of pgat-positive PGCs from each embryo was counted and plotted on the graph. Two-tailed t-tests were performed. *P<0.05; **P<0.01; ****P<0.0001. Data are mean±s.d. (C) Western blot showing that overexpression of PSME4 reduced the expression of myc-Trim36, but not myc-Dazl or myc-Nanos1. Hsc70 served as a loading control. Experiments were performed four times. (D) Working hypothesis of ubiquitin-independent proteasome function in controlling germline development. Ubiquitin-independent proteasome forms an animal-to-vegetal gradient in fully grown oocytes. In the vegetal hemisphere, it promotes degradation of germline determinants such as Dnd1. During the oocyte-to-embryo transition, RNAs coding for proteasome components are translocated to the animal hemisphere. Consequently, the proteasomal degradation rate is decreased in the vegetal hemisphere of the embryo. Reduced protein turnover in the vegetal pole creates a permissive environment, allowing rapid accumulation of germline determinants, which facilitates PGC development. |
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Figure S3. Asymmetric distribution of RNAs encoding ubiquitin-independent proteasome
activator. (A) An intact stage VI oocyte and an oocyte that was dissected into animal and
vegetal halves. (B) RT-PCR results showing the expression of psme1, psme2, psme3, psme4,
and pgat in animal and vegetal halves of dissected oocytes. pgat was used as a marker for the
vegetal hemisphere. |
