Epidermal growth factor-mediated mitogen-activated protein kinase3/1 pathway is conducive to in vitro maturation of sheep oocytes.

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo's capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10-6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes

Microscopic characteristics of sheep oocyte maturation stained with aceto-orcein (200× magnification).

<p>(A) Oocyte at GV stage, arrow points to germinal vesicle; (B) oocyte at GVBD stage, arrow points to condensed chromosomes; (C) oocyte at late spindle stage, arrow points to spindle fibers; and (D) oocyte at middle MII stage, arrow points to the first polar body.</p

The α-tubulin distribution around chromosomes in sheep oocytes after 22 h of <i>in vitro</i> maturation.

<p>Red indicates chromosomes and green indicates α-tubulin. (A, 10×40) Disorderly distribution of chromosomes; (B, 10×40) little α-tubulin distributed around chromosomes; (C,10×20) nearly no α-tubulin distributed around chromosomes; (D, 10×40) α-tubulin distributed on a spindle; and (E, F, 10×40) formation of microtubules and extrusion of polar bodies at meiosis II.</p

The recipes of reagents.

<p>The recipes of reagents.</p

The α-tubulin distribution around chromosomes in sheep oocytes after different periods of <i>in vitro</i> maturation.

<p>Red indicates chromosomes and green indicates α-tubulin. (A1-C1, 4 h) Oocytes show GVBD in the control group (group I, A1, 10×40) and the 10⁻⁴ mol/l U0126 group (group III, C1, 10×20), but remain in the germinal vesicle stage in the 10⁻⁶ mol/l U0126 group (group II, B1,10×40); (A2-C2, 8 h) chromosome condensation (A2, 10×40), beginning of chromosome condensation (B2,10×20), and GVBD (C2, 10×40); (A3-C3, 12 h) telophase of meiosis I (A3, 10×20; B3, 10×40), and chromosome condensation (C3, 10×40); (A4–C4, 24 h) meiosis II (A4, B4, 10×40), and telophase of meiosis I (C4, 10×20).</p

Statistics of the first polar body (PB1) extrusion rate of sheep oocytes in different treatment groups after 24 h of <i>in vitro</i> maturation.

<p>Note: The same superscript letters in the same column indicate no statistically significant differences (<i>p</i> > 0.05); different superscript letters in the same column indicate statistically significant differences (<i>p</i> < 0.05).</p><p>Statistics of the first polar body (PB1) extrusion rate of sheep oocytes in different treatment groups after 24 h of <i>in vitro</i> maturation.</p