Differential expression of <i>Gas6</i> during oocyte maturation and early embryogenesis.

<p>(<b>A</b>) Typical pattern of <i>Gas6</i> expression during oocyte maturation. The mRNA equivalent to a single oocyte taken after culture for 0, 2, 8, and 16 hours, corresponding to GV, GVBD, MI, and MII stages, respectively, was used for each lane. <i>H1foo</i> used as an internal control. <i>GFP</i> was used as an external control to measure equal recovery. (<b>B</b>) Expression of <i>Gas6</i> during early embryogenesis. Relative gene expression of <i>Gas6</i> in a single oocyte and single embryo throughout the developmental stages was measured by quantitative real-time PCR. Relative expression levels of <i>Gas6</i> were calculated from C_T values and normalized to added <i>GFP</i> synthetic RNA, and the expression ratio was calculated against <i>Gas6</i> expression in the GV oocyte. Experiments were repeated at least three times, and data were expressed as mean ± SEM.</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

Microinjection of <i>CSN3</i> or <i>CSN5</i> dsRNA into GV oocytes resulted in MI arrest.

<p>Microphotographs of oocytes (A) and maturation rates (B) after <i>in vitro</i> culture for 16 h. Control and buffer-injected groups were MII after 16 h in culture, but injected oocytes were arrested at the MI stage. Bars = 50 µm. Asterisks indicate statistically significant differences compared to that of control or buffer group (p<0.05).</p

<i>in vitro</i> maturation of mouse oocytes after <i>CSN3</i> or <i>CSN5</i> RNAi.

<p>*Values are statistically significant at p<0.05.</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

In vitro maturation of mouse oocytes after injection of <i>Gas6</i> dsRNA into GV oocytes.

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

Efficiency of RNAi-mediated knockdown of <i>CSN3</i> and <i>CSN5</i>.

<p>(A) Determination of the critical time point for complete knockdown of <i>CSN3</i> and <i>CSN5</i> after RNAi. Oocytes were collected every 2 h after RNAi, and <i>CSN3</i> and <i>CSN5</i> mRNA was assessed by RT-PCR. (B) Specific suppression of <i>CSN3</i> or <i>CSN5</i> mRNA by RNAi. The other CSN subunits were not affected by RNAi treatment. <i>H1foo</i> was used as an internal control of oocytes, and exogenous <i>GFP</i> mRNA was used as an external control. (C) Western blot analysis of CSN3 and CSN5. The blot was incubated with each antibody using 100 oocytes. Numbers on the left side of the band indicate the sizes (kDa) of the protein markers, while arrows indicate the specific proteins. β-Actin was used as a loading control. (D) Protein knockdown after RNAi. Levels of CSN3 and CSN5 protein in RNAi-treated oocytes were determined using Western blot analysis. Proteins were extracted from 100 MI oocytes for each lane. (E, F) Bar graphs show the relative mRNA and protein levels after RNAi. The mRNA level was calculated with quantitative real-time RT-PCR using single-equivalent oocyte cDNA, while protein level was calculated by measuring the density and area of the bands. The mRNA and protein levels are presented relative to those of control oocytes. Asterisks indicate statistically significant differences compared to that of control or buffer group (p<0.05).</p

Sequences of oligonucleotide primers and their expected RT-PCR product sizes.

a<p>F, forward; R, reverse.</p>b<p>Primers were used for preparation of dsRNA.</p>c<p>Primers were used for confirmation the knockdown of target mRNA after RNAi.</p

<i>Gas6</i> RNAi impaired reactivation of MPF after emission of the first polar body.

<p>(<b>A</b>) To assess the activities of MPF and MAPK after <i>Gas6</i> RNAi, phosphorylation of the substrates histone H1 and MBP, reflecting the kinase activities of MPF and MAPK, respectively, was determined. Each lane contained one MII stage oocyte. Control, uninjected oocytes; Buffer, buffer-injected for sham control oocytes; <i>Gas6</i> RNAi, <i>Gas6</i> dsRNA-injected oocytes. (<b>B</b>) Dual kinase activity analysis to determine the phosphorylation levels of the substrates histone H1 and MBP was performed every 2 hours after <i>Gas6</i> RNAi injection. C, uninjected control oocytes; R, <i>Gas6</i>-depleted oocytes. (<b>C</b>) The amount of phosphorylated histone H1 was calculated, and relative amounts were presented in line graphs. Asterisks represent statistical significance at <i>p<</i>0.05. Intact line with open circles, Control, uninjected oocytes; Dotted line with closed circles, <i>Gas6</i> RNAi, <i>Gas6</i>-silenced oocytes. (<b>D</b>) Western blot analysis of cyclin B1, p34^cdc2, and p34^cdc2 p-Tyr15 in MII oocytes after <i>Gas6</i> RNAi treatment. Depletion of GAS6 protein after <i>Gas6</i> RNAi treatment affected MPF activity. Protein lysates of 250 MII oocytes were loaded per lane. α-Tubulin was used as a loading control. Control, uninjected control MII oocytes; <i>Gas6</i> RNAi, <i>Gas6</i>-silenced MII oocytes.</p

Down-regulation of <i>CSN3</i> or <i>CSN5</i> caused meiotic spindle disassembly.

<p>Spindle structure was observed noninvasively using Polscope microscopy. (A) Control MI oocytes cultured for 8 h showed normal barrel-shaped spindles. (B, C) After RNAi treatment followed by 16 h culture, oocytes were arrested at the MI stage and showed no spindle structure. Left panel, bright field; middle panels, dark field; right panels, magnified view of boxed area from the middle panels. Original magnifications ×200 (Left and middle panels). Bar s = 20 µm.</p