Table 1.
Primer used for real-time PCR.
Figure 1.
The level of free cytoplasmic zinc in oocytes increases during maturation.
(A) Representative images of oocytes after 0, 20, 28, and 44 h of IVM. (B) The fluorescence intensity of fluozin-3-AM relative to that in oocytes after 0 h of IVM (set at 1) was analyzed using Photoshop. Data are expressed as mean values ± standard error of the mean (SEM) of three independent experiments. White lines indicate the peripheries of oocytes. Different letters above the bars denote statistically significant differences (P<0.05).
Figure 2.
Effects of different concentrations of TPEN on meiotic resumption in porcine oocytes.
Nuclear distribution was assessed after 25(0, 0.1, 1.0, 2.0, 2.5, and 3.0 µM). The percentages of oocytes at the GV, GVBD, and MI stages are shown. TPEN inhibits oocyte maturation in a dose-dependent manner. Data are presented as mean values of three independent experiments. Ctrl, control group.
Figure 3.
Effects of different concentrations of TPEN on the maturation of porcine oocytes.
(A) TPEN inhibits oocyte maturation in a dose-dependent manner. Oocytes were cultured in IVM media containing various concentrations of TPEN, and the maturation rate was determined. The EC50 of TPEN in oocytes is about 2.7 µM. (B) Oocytes underwent IVM in the presence of TPEN for 20 h and were then transferred to media lacking TPEN. The maturation rate was determined. Data are presented as mean values ± SEM of three independent experiments. ⧫ Ctrl, control group, ▪ TPEN rescue group. *, P<0.05; **, P<0.01.
Figure 4.
TPEN treatment affects the expression of maternal genes in porcine oocytes.
TPEN treatment significantly decreases the mRNA levels of the MPF pathway-related genes Cdc2 and CyclinB1 and the MAPK pathway-related gene C-mos. Data are expressed as mean values ± SEM of three independent experiments. Ctrl, control group. **, P<0.01; ***, P<0.001.
Figure 5.
TPEN controls meiotic resumption in a PKC-dependent manner.
(A) Nuclear distribution was examined after 25 h of IVM and treatment with various concentrations of staurosporine. The percentages of oocytes at the GV, GVBD, and MI stages are shown. (B) Nuclear distribution was examined after 25 h of IVM and treatment with TPEN and/or PMA. The percentages of oocytes at the GV, GVBD, and MI stages are shown. (C) Change in the level of free zinc following PMA treatment. Zinc content was analyzed using Photoshop and was normalized against the level in the control group (set at 1). Ctrl, control group; TPEN, oocytes matured in the presence of 2.7 µM TPEN; T+P, oocytes matured in the presence of TPEN and PMA; PMA, oocytes matured in the presence of PMA only. PMA (in figure C), GV-stage oocytes treated with PMA for 1 h. White dashed lines indicate the peripheries of oocytes. Data are expressed as mean values ± SEM of three independent experiments. *, P<0.05.
Figure 6.
TPEN inhibits phosphorylation of PKC substrates in porcine oocytes.
(A) TPEN downregulates phosphorylation of PKC substrates and MAPK in porcine oocytes, while PMA increases the phosphorylation of these proteins, even in the presence of TPEN. The density of band were digitalized. Oocytes were treated with TPEN, PMA, or TPEN plus PMA (T+P). A sample containing 400 oocytes was loaded per lane. (B) Oocytes were treated with various concentrations of TPEN (0, 0.1, 1.0, and 3.0 µM) or staurosporine (STA). Phosphorylation of PKC substrates was examined after 25 h of IVM. A sample containing 200 oocytes was loaded per lane. (A′) and (B′) Western blots were analyzed using Gel-pro software. The IOD of each treatment group was normalized against that of the control group. Ctrl, control group. Data are expressed as mean values ± SEM of three independent experiments. *, P<0.05; **, P<0.01.
Figure 7.
TPEN treatment reduces p34cdc2 kinase activity in porcine oocytes.
p34cdc2 kinase activity was examined in oocytes after 25 h of IVM in the absence (Ctrl, control group) or presence of TPEN, PMA, or TPEN plus PMA (T+P). Twenty oocytes were examined per group. Data are expressed as mean values ± SEM. Different letters above the bars denote statistically significant differences (P<0.05).