Temporal and spatial regulation of translation in the mammalian oocyte via the mTOR-eIF4F pathway

Abstract

The fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.

Figure 1. Disruption of the mTOR–eIF4F pathway affects genomic stability in meiosis I and impairs translation of specific mRNAs.

(a,b) Oocytes treated with 4EGI or Rap or microinjected with an eIF4E/eIF4G1 antibody cocktail show aberrant spindle formation. Data are represented as the mean±s.d. Asterisks denote P<0.001; NS, non significant; according to a Student’s t-test; n≥35. Tubulin (green) and DAPI (blue). Scale bar, 5 μm. (c,d) Chromosomal spreads show aneuploidy and loose chromosomes/chromatids in oocytes with a downregulated 4F complex. Representative image from two independent experiments is shown (n≥14); arrow denotes separated chromatid. Scale bar, 10 μm. See also Supplementary Figs 1–3.

Figure 2. The mTOR–eIF4F translational pathway is highly active at the onset of meiosis and downregulated after fertilization.

(a,b) Immunoblot analysis of the key players of the mTOR–eIF4F pathway shows their upregulation after NEBD (3 h PIW). Ratios of the abundance of the phosphorylated form of mTOR, 4E-BP1, S6K, eIF4E end eIF4G1 are presented in the form of a bar chart. Data are represented as the mean±s.d.; values obtained for NEBD stage were set as 100%; asterisk denotes statistically significant differences (Student’s t-test: P<0.05); n≥3; arrow denotes phospho-4E-BP1(T70). See also Supplementary Fig. 6. (c) RNA RL construct with TOP motif (eEF2^TOP) has increased translation after NEBD. Data are represented as the mean±s.d. **P<0.01, according to a Student’s t-test. Data are representative of at least three independent experiments. Values obtained for GV stage were set as 100%. See also Supplementary Fig. 10.

Figure 3. In situ translation shows two distinct hotspots in oocytes.

(a) Oocytes in different stages were cultured in the presence of HPG for 30 min. HPG (red); DAPI (blue). (b) NEBD oocytes cultured for 30 min in HPG. Histogram shows HPG intensity depicted along the white line. The arrowhead and arrow indicates the CTA (dot line) and PTA (uninterupted line), respectively; HPG (red). See also Supplementary Movie 1. (c) ER tracker shows perispindular localization of ER and overlaps with the PTA post NEBD. Polymerized LMN separates the CTA from the PTA. HPG, LMN, (red); ER tracker and DAPI (blue). Data are representative of at least two independent experiments; scale bar, ~10 μm.

Figure 4. mTOR–eIF4F key players are localized at the CTA.

(a) mTOR (green) localizes with HPG signal (red) at the CTA. (b) Immunocytochemistry shows the localization of mTOR–eIF4F pathway components 2 h post NEBD. White line indicates oocyte cortex; representative images of at least three independent experiments are shown; scale bars, 20 μm. See also Supplementary Fig. 6. (c) ER tracker shows perispindular localization of ER and overlaps with the PTA post NEBD. Polymerized LMN separates CTA from PTA. HPG, LMN, MB (red); 4E-BP1(T70), eIF4E(S209) green; ER tracker and DAPI (blue). Data are representatives of at least two independent experiments; white line indicates oocyte cortex; scale bar, ~10 μm.

Figure 5. Downregulation of mTOR–eIF4F does not affect global translation, however, shows decreased level of candidate proteins.

(a,b) 4EGI or Rap treatments during meiotic progression do not affect the overall protein synthesis in the oocytes (data are represented as mean±s.d.; **P<0.01, according to Student’s t-test; n≥3). The overall translation was supressed by puro (puromycin). (c) Translation of TOP motive RL RNA reporter construct is affected in the oocytes with downregulated mTOR–eIF4F pathway (as mean±s.d.; **P<0.01; according to Student’s t-test.

Figure 6. Downregulation of mTOR and 4F abolishes the translation at the CTA.

(a,b) Inhibition of mTOR or 4F decreases HPG fluorescence at the CTA, followed by quantification of HPG fluorescence (bold circle indicates measured area (CTA); thin circle (PTA)). Data are represented as mean±s.d.; **P<0.01 and ***P<0.001, according to a Student’s t-test; n≥20. Scale bar, 35 μm. The overall translation was supressed by puromycin. (c) Measurement of phosphorylation intensity of the 4E-BP1 (T70) shows a decrease at the CTA in the presence of Rap (***P<0.001; n≥19). 4EGI does not affect the phosphorylation intensity significantly (P>0.1; n=20). Bold circle indicates measurement at the CTA and thin circle at PTA/cytoplasm. Data represents the mean ±s.d.; asterisks denote statistically significant differences Student’s t-test. (d,e) Downregulation of the 4F pathway results in decreased translation of selected mRNAs. Data are representative of at least three independent experiments. Values obtained for GV stage were set as 100%. Data are represented as the mean±s.d. **P<0.01, according to a Student’s t-test. (f,g) Immunoblot analysis of the candidate proteins during meiotic maturation. Values obtained for GV stage were set as 100%, n≥3. Data represents the mean ±s.d.; asterisk denotes statistically significant differences Student’s t-test: P<0.05. See also Supplementary Fig. 10.

Figure 7. The oocyte nucleus stores a pool of RNA.

(a) RNA FISH shows the presence of a poly(A)-RNA population in the nucleus and in the vicinity of chromosomes in GV and NEBD stage oocytes. Poly(A) (red); DAPI (Blue). See also Supplementary Fig. 8. Scale bar, 20 μm. (b) MB shows the presence of poly(A)-RNA in the nucleus and in the vicinity of chromosomes in the GV oocyte and 2 h after NEBD. MB (red); H2B-GFP (green fluorescent protein; green). Scale bar, 20 μm. See also Supplementary Movie 2. (c) The oocyte nucleus contains specific mRNAs. RNA isolated from the nuclear (N) and cytosolic (C) fractions shows the presence or absence of specific transcripts by PCR. Data represents at least three independent experiments. Representative images of the isolated nuclei from mouse oocytes are shown. Scale bar, 10 μm. See also Supplementary Fig. 9 and Supplementary Table 1. (d) RNA FISH shows nuclear absence of Camk2a, cMos, Gapdh and nuclear presence of Bub3, Nph1, Survivin, Dazl mRNAs. The white line indicates the border of oocyte cortex; the white dashed line indicates the border of the nucleus; representative images of two independent experiments shown. Bar size, 20 μm.