Zap70 and downstream <i>RanBP2</i> are required for the exact timing of the meiotic cell cycle in oocytes

<p>In previous studies, we observed that Zeta-chain-associated protein kinase 70 (Zap70) regulates spindle assembly and chromosome alignment in mouse oocyte and that Ran binding protein 2 (RanBP2) is a highly associated gene with Zap70 based on a microarray analysis. Because RanBP2 is related to nuclear envelope breakdown (NEBD) during mitosis, the aim of the present study was to elucidate the molecular mechanism of Zap70 with respect to RanBP2 in the germinal vesicle breakdown (GVBD) of oocytes. Results indicated that RanBP2 expression was regulated by Zap70 and that depletion of RanBP2 using <i>RanBP2</i> RNAi manifested comparable phenotypes to those observed in <i>Zap70</i> RNAi-treated oocytes, which presented faster processing of GVBD. Additionally, <i>Zap70</i> RNAi-treated oocytes showed faster meiotic resumption with premature activation of maturation-promoting factor (MPF), premature division of chromosomes at approximately 6–8 h and more rapid degradation of securin. In conclusion, we report that Zap70 is a crucial factor for controlling the exact timing of meiotic progression in mouse oocytes.</p

<i>Txnip</i> RNAi-mediated degradation of <i>Txnip</i>.

<p>Specific depletion of (<b>A</b>) <i>Txnip</i> mRNA and (<b>B</b>) TXNIP protein after RNAi. <i>H1foo</i> was used as an internal control for oocytes, while α-Tubulin was used as a loading control. Protein lysates of 50 oocytes were loaded per lane for Western blotting. Experiments were repeated at least three times.</p

<i>Txnip</i> RNAi-treated oocytes arrested at the MI stage during <i>in vitro</i> maturation.

a<p>Values with statistical significance (<i>p</i><0.05) compared to control or buffer-injected groups.</p

Results of 2-NBDG treatment to visualize glucose uptake into oocytes.

<p>DOs were incubated in M16 medium containing 200 µM 2-NBDG for 20 minutes followed by <i>in vitro</i> maturation in M16 medium for 16 hours, and oocytes were imaged for fluorescence quantification using time lapse microcopy. Fluorescence intensities of 2-NBDG in <i>Txnip</i> RNAi-treated oocytes and high concentration lactate-treated oocytes were stronger than control oocytes. Bars = 100 µm.</p

<i>Txnip</i> RNAi treatment resulted in MI arrest and granule formation.

<p>Microphotographs of (<b>A</b>) control oocytes and (<b>B</b>) <i>Txnip</i> RNAi-treated oocytes after <i>in vitro</i> culture for 16 hours in M16 medium following 8 hours incubation in IBMX-supplemented M16 medium. Bars = 100 µm.</p

<i>Txnip</i> RNAi affects meiotic cell cycle by changing glucose metabolism and cytoplasmic streaming in mouse oocyte.

<p>In control oocytes, actin flow (green arrow) leads to dynamic cytoplasmic streaming (blue arrow) and both of them cause spindle migration in MI stage necessary to the first polar body extrusion in completing the meiosis I <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070708#pone.0070708-Li1" target="_blank">[41]</a>. In the case of <i>Txnip</i> RNAi-treated oocytes, however, glucose uptake and lactate production was increased and as a result, intracellular granule formation was increased. This increased granule formation disturbed actin flow and cytoplasmic streaming, and led to abnormal spindle assembly (green dots), aggregated chromosomes (red figure), and finally, MI arrest of the meiotic cell cycle of the oocytes. GV: germinal vesicle; MI: metaphase I; MII: metaphase II oocytes.</p

Lactate production was increased in <i>Txnip</i> RNAi-treatment oocytes.

<p>Lactate production was measured by a lactate colorimetric assay kit after droplet culture in 20 µl of M16 medium. The y-axis indicates the concentration of lactate from a total of 250 oocytes used for each group. Asterisk indicates statistically significant difference compared to the control (p<0.05).</p

Down-regulation of <i>CSN3</i> or <i>CSN5</i> caused chromosome aggregation.

<p>(A, B) Control MI oocytes showing typical chromosome configuration. (C, D) <i>CSN3</i> and (E, F) <i>CSN5</i> RNAi-treated oocytes which were arrested at MI showed abnormally aggregated chromosomes. Upper panel (A–F), view of oocytes by optic microscopy (×400); Lower panel (A′–F′), Magnified view of upper panel. These microphotographs were digitally processed to increase magnification. Bars = 25 µm.</p

Primer sequences and RT-PCR conditions used for <i>Gas6</i> RNAi.

<p>*For  =  Forward; Rev  =  Reverse.</p><p>**Primer set-A was used for the preparation of dsRNA, whereas set-B was used to confirm the gene-specific knockdown after RNAi.</p

Development after parthenogenetic activation by Sr²⁺ stimulation.

a, b, c, d<p>Different letters indicate significant difference at <i>p<</i>0.05.</p