Rapamycin Influences the Efficiency of Fertilization and Development in the Mouse: A Role for Autophagic Activation

The mammalian target of rapamycin (mTOR) regulates cellular processes such as cell growth, metabolism, transcription, translation, and autophagy. Rapamycin is a selective inhibitor of mTOR, and induces autophagy in various systems. Autophagy contributes to clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified-warmed mouse oocytes show acute increases in autophagy during warming, and suggested that it is a natural response to cold stress. In this follow-up study, we examined whether the modulation of autophagy influences survival, fertilization, and developmental rates of vitrified-warmed mouse oocytes. We used rapamycin to enhance autophagy in metaphase II (MII) oocytes before and after vitrification. The oocytes were then subjected to in vitro fertilization (IVF). The fertilization and developmental rates of vitrified-warmed oocytes after rapamycin treatment were significantly lower than those for control groups. Modulation of autophagy with rapamycin treatment shows that rapamycin-induced autophagy exerts a negative influence on fertilization and development of vitrified-warmed oocytes

The transcription factor Egr3 is a putative component of the microtubule organizing center in mouse oocytes.

The early growth response (Egr) family of zinc finger transcription factors consists of 4 members. During an investigation of Egr factor localization in mouse ovaries, we noted that Egr3 exhibits a subcellular localization that overlaps with the meiotic spindle in oocytes. Using Egr3-specific antibodies, we establish that Egr3 co-localizes with the spindle and cytosolic microtubule organizing centers (MTOCs) in oocytes during meiotic maturation. Notably, the Egr3 protein appears to accumulate around γ-tubulin in MTOCs. Nocodazole treatment, which induces microtubule depolymerization, resulted in the disruption of spindle formation and Egr3 localization, suggesting that Egr3 localization is dependent on the correct configuration of the spindle. Shortly after warming of vitrified oocytes, growing arrays of microtubules were observed near large clusters of Egr3. An in vitro microtubule interaction assay showed that Egr3 does not directly interact with polymerized microtubules. Egr3 localization on the spindle was sustained in early preimplantation mouse embryos, but this pattern did not persist until the blastocyst stage. Collectively, our result shows for the first time that the Egr3 a transcription factor may play a novel non-transcriptional function during microtubule organization in mouse oocytes

The formin protein mDia2 serves as a marker of spindle pole dynamics in vitrified-warmed mouse oocytes.

The mouse diaphanous 2 (mDia2) protein belongs to the formin family and has been shown to nucleate actin filaments and stabilize microtubules, thus indicating a role in cytoskeleton organization. Our previous study, which showed that mDia2 specifically localizes to spindle poles of metaphase I mouse oocytes and NIH3T3 cells, provided the first evidence of its spindle pole-associated cellular function. In the present study, we aim to determine whether spindle pole proteins, such as mDia2 and pericentrin, can be used to monitor the status of spindle poles in cryopreserved mouse oocytes. We show herein that mDia2 exhibits an overlapping distribution with pericentrin, which is a crucial component of centrosomes and microtubule organizing centers (MTOCs). In vitrified-warmed oocytes, the overlapping distribution of mDia2 and pericentrin was immediately detected after thawing, thereby suggesting that mDia2 maintains a tight association with the spindle pole machinery. Interestingly, we observed that microtubules extend from mDia2 clusters in cytoplasmic MTOCs after thawing. This result suggests that mDia2 is a major MTOC component that is closely associated with pericentrin and that it plays a role in microtubule growth from MTOCs. Collectively, our results provide evidence that mDia2 is a novel marker of spindle pole dynamics before and after cryopreservation

Immunofluorescence staining of Egr3 and tubulin proteins in MI oocytes.

<p>PI oocytes were cultured in vitro for 8% formaldehyde, and subjected to immunofluorescence staining with anti-Egr3 (Santa Cruz antibody in upper panel and Abcam antibody in low panel), anti-α-tubulin, or anti-γ-tubulin (Sigma-Aldrich) antibody. All the antibodies were used at 2 μg/ml. DNA was counter-stained with TO-PRO-3-iodide. Red scale bar, 30 μm; white scale bar, 5 μm. Green, Egr3; red, α-tubulin or γ-tubulin; blue, DNA. The arrows indicate the localization of Egr3 protein near γ-tubulin-positive MTOCs.</p

Immunofluorescence staining of Egr3 in preimplantation mouse embryos.

<p>Embryos at different developmental stages were fixed in 4% paraformaldehyde and subjected to immunofluorescence staining with anti-Egr3 (Santa Cruz) and anti-α-tubulin antibodies. The arrows indicate mitotic spindles in the blastomeres. Green, Egr3; red, α-tubulin; blue, DNA.</p

Egr3 localization is associated with microtubule organization in mouse oocytes.

<p>(A) MI oocytes were treated with taxol (microtubule stabilizer, 1 μM) or nocodazole (microtubule depolymerizer, 10 μg/ml) for 1–2 h and were subjected to immunofluorescence staining with anti-Egr3 antibody (Santa Cruz). Red scale bar, 30 μm; white scale bar, 10 μm. Green, Egr3; red, γ-tubulin; blue, DNA. (B) Localization of Egr3 is shown in MI oocyte treated with CytoD (actin depolymerizer, 10 μM) and in <i>Fmn2</i>-/- oocyte. <i>Fmn2</i>-/- oocyte was co-stained with anti-Egr3 (Santa Cruz) and anti-γ-tubulin antibodies. Red scale bar, 30 μm. Green, Egr3; red, γ-tubulin; blue, DNA. (C) The localization of Egr3 and microtubule at thawing of vitrified oocytes. Vitrified MII oocytes were stored in LN₂ for 2 weeks. Oocytes were taken out from LN₂, incubated in decreasing concentrations of sucrose, and then fixed immediately. These oocytes were subjected to immunofluorescence staining with anti-Egr3 (Abcam) and anti-α-tubulin antibodies. Arrows indicate the growing arrays of microtubules at the site of Egr3 accumulation. Green, Egr3; red, microtubule (MT).</p

The expression of <i>Egr3</i> transcripts and proteins in mouse tissues and cultured cells.

<p>(A) An RT-PCR analysis of <i>Egr3</i> expression in various tissues. Expression was compared to that of ribosomal protein L7 (<i>rpl7</i>) or <i>Gapdh</i>. Oocytes at PI (130 oocytes) or MI stage (120 oocytes) were pooled and subjected to Egr3 RT-PCR and nested PCR. Cu, cumulus cells; -, no RT. (B) Western blot analysis of Egr3 in various tissue and cell types. Upper panel: 293 T cells transfected with CMV-GFP plasmid (GFP), empty plasmid (-), pcDNA3.1/myc-his-NM plasmid (NM), or pcDNA3.1/myc-his-BC plasmid (BC) were analyzed using Western blots probed with anti-Egr3 antibody (Santa Cruz). Fifty micrograms of each cell lysates were run on a 10% SDS-PAGE gel. Bottom panel: MI oocyte (268 GV oocytes cultured for 8 h in vitro), PI oocyte (112 oocytes), ovary, brain, and MCF7 cell (with or without estrogen treatment) lysates were analyzed by Western blotting. Two hundred micrograms of tissue extract was loaded in each lane of a 12% SDS-PAGE gel. The blots were stripped and tubulin was detected using an anti-α-tubulin antibody.</p

Subcellular localization of Egr3 protein in meiotic spindle of mouse oocytes.

<p>(A) Egr3 localization in a mouse oocyte within a growing follicle. Immunofluorescence staining of Egr3 was performed on ovarian cryosection fixed in acetone. The rabbit polyclonal anti-Egr3 antibody (Santa Cruz) was used at 2 μg/ml. Primary antibody was probed with goat anti-rabbit IgG-Alexa Fluor 488 antibody. The DNA was counter-stained with TO-PRO-3-iodide. White scale bar, 100 μm. Mock control, rabbit IgG. Green, Egr3; red, DNA. (B) Egr3 is localized to the meiotic spindle of mouse oocytes at all stages of maturation. Germinal vesicle (GV) stage oocytes were obtained by ovary puncture and were cultured in M16. Oocytes were fixed in 3.7% formaldehyde and were subjected to immunofluorescence staining with anti-Egr3 antibody (Santa Cruz). Oocytes in the left panel are shown at 60X and enlarged images of chromosome-containing areas are shown in the right panel. Mock control, rabbit IgG. PI, prophase I; PMI, prometaphase I (cultured for 3 h); MI, metaphase I (cultured for 8 h); MII, metaphase II (cultured for 12 h). Red scale bar, 30 μm; white scale bar, 10 μm. Green, Egr3; blue, DNA.</p

Microtubule interaction assay using in vitro translated Egr3 isoforms.

<p>To examine whether polymerized microtubules interact directly with Egr3 protein, we performed a microtubule-interaction assay. Tubulin from the bovine brain (5 μg/μl) was polymerized at 37°C and diluted to 0.2 μg/μl. Four microliters of in vitro translated lysates (NM or BC) and 50 μl of diluted MTs were incubated at room temperature for 30 min. The reaction was centrifuged at 100,000×g for 30 min to pellet the polymerized MTs. The supernatant (S) and pellet (P) were run on 10% SDS-PAGE and Western blotting was performed with anti-Egr3 or anti-Myc antibody which detects the epitope in the pcDNA3.1/myc-his vector. The antibodies were used at 1∶5000. The blots were stripped and tubulins were detected with anti-tubulin antibody. The data show that the presence of microtubules does not affect the quantity of Egr3 products in the supernatant and pellet, suggesting that there is no direct interaction between microtubules and Egr3.</p

Expression of in mouse gonads and its localization and function in oocytes

Objective The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (ΔEgr3) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5′untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-ΔEgr3-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-ΔEgr3-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice