Dose-dependent effects of stress inducers on localization of XBP-1.

<p>A. Two-cell stage embryos cultured with activator or inhibitor of ER stress were examined using a specific anti-XBP-1 antibody (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm. B. Active XBP-1 in two-cell embryos in the presence of activator or inhibitor of ER stress was analyzed using Western blotting. β-actin served as the control. Each experiment was repeated three times. (*) indicates a statistically significant difference from control (P<0.05).</p

Effect of TUDCA on apoptosis in mouse blastocyst.

<p>Apoptosis in blastocysts was evaluated using the TUNEL assay. The original magnification was approximately × 200. A. Images of mouse blastocysts cultured with stress inducers in the presence or absence of TUDCA. The green color indicates DNA fragmentation, and nuclei are stained in red; Scale bar, 20 μm. B. Mean values ± SEM of number of DNA fragments in blastocysts cultured with stress inducers in the presence or absence of TUDCA. Each experiment was repeated three times. (*) indicates a statistically significant difference from control (P<0.05).</p

Detection of endogenous XBP-1 in mouse oocytes and preimplantation embryos <i>in vivo</i>.

<p>A. A specific anti-XBP-1 antibody was used to detect localization of XBP-1 in mouse oocytes via immunostaining (green). Nuclei were stained with DAPI (blue); Scale bar, 20 µm. B. Confocal immunofluorescence images of mouse pre-implantation embryos. The XBP-1 protein was detected using a specific antibody (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue); Scale bar, 20 μm.</p

All primer sequences used in the experiments.

<p>All primer sequences used in the experiments.</p

Induction of active XBP-1 in one-cell stage embryos.

<p>A. <i>XBP-1</i> mRNA was spliced to produce the spliced and unspliced forms in the presence of TM and sorbitol. B. Immunofluorescence micrographs of two-cell stage embryos with TM or sorbitol. Active XBP-1 protein was detected in nuclei in the presence or absence of stress inducers (green). Negative control embryos were probed directly with the secondary antibody. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. C. Active and inactive XBP-1 proteins were detected in the presence and absence of stress inducers using Western blotting. β-actin served as a control. D. Quantification of the Western blot analysis in C. The data were presented as means ± SD from three independent experiments.</p

Effects of stress inducers on mouse embryo development <i>in vitro</i>.

<p>A. Two-cell embryos collected at 44 h phCG were cultured in M16 containing 0, 1, 2, 5 and 10 μg/ml TM, for 60 h. B. Two-cell embryos were cultured in M16 containing 0, 10, 25, 50, 75 mM sorbitol, for 60 h. (*) indicates a statistically significant difference (P<0.05) and (**) indicates a statistically significant difference from control (P<0.01).</p

Engineering of polyketide synthases: How close are we to the reality?

<p>(A) Oocytes and parthenogenetic embryos were immunostained with the anti-H3K36me3 antibody, which was then localized with a FITC-conjugated secondary antibody (green). DNA was stained with DAPI (blue). NSN and SN are two types of GV stage oocytes. MI, metaphase I stage oocytes; MII, metaphase II stage oocytes. Scale bar = 20 µm. (B) Relative intensities of fluorescence signals from H3K36me3. Total 23 oocytes and 47 embryos were analyzed in triplicate in this experiment. Bars represent least-squares showed the standard error in each group. P-values <0.05 were considered statistically significant. ^a,b,c Values represent least-squares with different superscripts are significantly different (P<0.05).</p

Extrusion rate (A) and morphology (B) of PB1s.

<p>The number of oocytes examined in each group is given in parentheses. G1, G2, G3, G4, and G5 represent Grade 1, Grade 2, Grade 3, Grade 4, and Grade 5, respectively.</p

Changes in H3K36me3 status in porcine IVF and SCNT embryos.

<p>(A) Sperm, MII stage oocytes coincubated with sperm in mTBM medium for 1 h (IVF 1 h), and IVF embryos cultured in PZM-3 medium for 18 h (IVF) were immunostained with the anti-H3K36me3 antibody. (B) A single fetal fibroblast cell injected into the perivitelline space of a denuded oocyte without activation (Embedded), a nuclear-transferred embryo cultured in PZM-3 for 1 h post activation (1h), a nuclear-transferred embryo cultured in PZM-3 for 18 h after activation (18h), and other stages of SCNT embryos were immunostained with the anti-H3K36me3 antibody. The antibody was localized with a FITC-conjugated secondary antibody (green). DNA was stained with DAPI (blue). The arrowhead indicates sperm and the arrow indicates the nuclei of donor cell. Scale bar = 20 µm. (C) Relative intensities of fluorescence signals from H3K36me3. Total 41 embryos were analyzed in triplicate in this experiment. Bars represent least-squares showed the standard error in each group. P-values <0.05 were considered statistically significant. ^a,b,c Values represent least-squares with different superscripts are significantly different (P<0.05).</p

Morphological analysis identifies five categories of porcine first polar bodies.

<p>Grade 1: Round or ovoid PB1 with intact smooth membrane (A). Grade 2: Round or ovoid PB1 with intact membrane (B). Grade 3: Broken PB1 with a small PB1 fragment (C). Grade 4: Broken PB1 with a big PB1 fragment (D). Grade 5: Broken PB1 completely (E). Grade 1, 2 were considered as good quality. Black arrows: Polar bodies. White arrows: Fragments. </p