Effects of E2 and E2-BSA on the activation of PKCδ, PKCα and PKA in Ishikawa cells.

<p>A and B. Serum starved Ishikawa cell was treated with 10 nM E2 or 10 nM E2-BSA for the indicated time points. Protein extracts were prepared and used for Western blot analysis to measure levels of PKCδ phosphorylation. Protein levels of total PKCδ were also examined as controls. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C. Serum starved Ishikawa cell was treated with 10 nm E2 for the indicated time points and cells were lysed for western blot analysis to measure levels of PKCα phosphorylation. Protein levels of total PKCα were measured as controls. D, Serum starved Ishikawa cell was treated with 0, 0.1,1, 10, 100 and 1,000 nM E2 for 20 min. Protein extract was prepared for Western blot analysis to measure levels of PKCα phosphorylation and total PKCα. E and F, Serum starved Ishikawa cells were treated with 10 nM E2 or 10 nM E2-BSA for indicated time points, 20 µM Forskolin (F) was added for 15 min as a positive control, after which the cells were lysed and tested in the PepTag PKA assay. Samples were separated on an agarose gel. The lower band represents phosphorylated peptide, and the upper band represents the remaining unphosphorylated peptide.</p

ER-α36 mediates E2-stimulated PKCδ activation.

<p>A and B. ER-α36 expression in Ishikawa/V and Ishikawa/RNAiER36 cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Ishikawa/V cells. C. Ishikawa/V and Ishikawa/RNAiER36 cells were treated with 10 nm E2 or 10 nm E2-BSA for 10 min, and PKCδ phosphorylation was analyzed by Western blot. Total levels of PKCδ were measured as controls, and each bar repents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. #, P<0.05 compared to E2- or E2-BSA- treated Ishikawa/V cells. D and E. ER-α66 expression in Ishikawa/V and Ishikawa/RNAiER66 cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Ishikawa/V cells. F. Ishikawa/V and Ishikawa/RNAiER66 cells were treated with 10 nM E2 or 10 nM E2-BSA for 10 min, and then PKCδ phosphorylation was assessed with Western blot. Total PKCδ was measured as controls. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. G, Hec1A/V and Hec1A/RNAiER36 cells were treated with 10 nM E2 or 10 nM E2-BSA for 10 min, and PKCδ phosphorylation was analyzed by Western blot. Total PKCδ was measured as controls. Each bar represents mean value ± SEM (n = 3).*, P<0.05 compared to untreated cells. #, P<0.05 compared to E2- or E2-BSA-treated Hec-1A/V cells.</p

Maturation of oocytes after preservation at 27.5°C in different media.

<p>Data are presented as means ± SEM from three replicated experiments.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p

Effect of different media on CG distribution in porcine oocytes after preservation.

<p>A. a, b, CGs perinuclear area distribution within the porcine oocytes; a, most of the CGs aggregate in the perinuclear area, with a few migrating to the central inner cytoplasm or cortical area; b is intermediate pattern between a and c, with most of the CGs still remaining in perinuclear area and some migrating to the central cytoplasm; c, CGs distributed uniformly in the inner cytoplasmic area, and a few migrated to the cortical area to form a discontinuous ring; d, CGs cortical area distribution; almost all of the CGs have migrated into the cortical area, and a few still remained in the central cytoplasm. Green, cortical granules. Scale bar = 10 µm. (White arrows pointing at the white circle denotes the germinal vesicle). B. Ratios of different CG distribution patterns within the porcine oocytes after preservation in different media. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, and then CG distribution was stained with FITC-labeled peanut agglutinin. Three categories of CG distribution were detected: perinuclear area; inner cyoplasmic area; cortical area. The graph shows the mean ± SEM of three independent experiments. The number “n” in the bracket means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values that differ significantly in every categories of CG distribution (P<0.05).</p

ER-a36 mediates E2 stimulated cell proliferation.

<p>A. Ishikawa/V and Ishikawa/RNAiER36 cells were treated with 10 nM E2-BSA for 24 h, 48 h, 72 h and 96 h. MTT assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015408#s4" target="_blank">materials and methods</a>. Results of three independent experiments were averaged and mean value ± SEM are shown. *, P<0.05 compared to E2-BSA treated Ishikawa/V cells respectively. B. Ishikawa cells were treated with 10 nM E2-BSA alone or together with 5 µM rottlerin, a PKCδ specific inhibitor, or 5 µM bisindolylmaleimide, a pan-PKC inhibitor, or HBDDE, a PKCα specific inhibitor, or 5 µM H89, a PKA specific inhibitor for 72 h, and MTT assays were then performed. Results of three independent experiments were averaged and mean value ± SEM are shown. *, P<0.05 compared to control cells.</p

Morphologic changes of porcine cumulus-oocyte complexes (COCs) after preservation for 3 d.

<p>a1, a2, b1, b2, c1, c2, When preserved in TCM-199, pFF and FCS at 4°C or 27.5°C, cumulus cells attached tightly to oocytes and displayed no expansion; a3, c3, After preservation in TCM-199 and FCS at 38.5°C, almost all of the cumulus have shed from the oocytes; b3, After pFF preservation at 38.5°C, the whole COCs degenerated and showed black appearance; b2, c2, After preservation in pFF and FCS at 27.5°C, oocytes displayed dark ooplasm with compact cumulus cells. Scale bar  = 200 µm.</p

Effects of different preservation media on spindle configuration in porcine oocytes after IVM.

<p>A. a, represents a normal spindle in porcine oocyte. b and c represent abnormal spindle. In b1, c1 arrows indicate abnormal spindle organization distribution. In b2, arrows indicate fragmented and displaced chromosomes. Green, tubulin; Red, chromatin. Scale bar  = 10 µm. B. Normal ratios of spindle configuration in porcine oocytes after preservation in different media following IVM. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, then spindle was stained with FITC-α-tubulin after culture for 44 h <i>in vitro</i>. Normal and abnormal spindle configuration were detected. The graph shows the mean ± SEM of three independent experiments. The number “n” over the bars means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values of normal tubulin configuration that differ significantly between groups (P<0.05).</p

Effect of different media on actin configuration in porcine oocytes after preservation.

<p>A. a, represents the normal actin pattern in porcine oocytes (white arrow pointing at the white circle denotes the germinal vesicle). b, c, and d represent abnormal actin patterns. In b, actin fragments formed an incomplete ring with large punctiform aggregation. In c, small punctiform actin aggregated as an incomplete ring. In d, only some actin aggregated near oocyte membrane. Green, microfilaments; Red, chromatin. Scale bar  = 10 µm. B. Ratios of oocytes with normal actin configuration in control group and different preservation groups. Porcine oocytes were preserved in TCM-199, pFF and FCS at 27.5°C for 3 d, then the actin configuration was stained with FITC-phalloidine. Normal and abnormal actin configuration were detected. The graph shows the mean ± SEM of three independent experiments. The number “n” over the bars means the total treated oocyte in every group. The superscripts ^{a, b} over the bars represent values of normal actin configuration that differ significantly (P<0.05).</p

Effects of different preservation media on GSH level after preservation at 27.5°C for 3 d.

<p>Data are presented as means ± SEM from three replicated experiments.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p

Effect of preserving medium on early development of IVM-IVF porcine oocytes after preservation at 27.5°C for 3 d.

<p>Data are presented as means ± SEM from three replicated experiments.</p><p>The values “n” in bracket represent the number of the oocytes cleaved at 48 hr and forming blastocysts at 7 d after IVF.</p>1<p>Percentages are based on the total number of oocytes examined.</p>a, b, c<p>Values with different superscripts are significantly different in each column (P<0.05).</p