Journal: International Journal of Molecular Sciences

Article Title: Dynamic Changes in pStat3 Are Involved in Meiotic Spindle Assembly in Mouse Oocytes

doi: 10.3390/ijms21041220

Figure Lengend Snippet: Dynamic changes of pStat3 localization during oocyte maturation. ( A ) Time course observation of pStat3 localization after germinal vesicle break down (GVBD). Immediately after GVBD, the accumulated pStat3 in the GV declines remarkably (0.5 h) and appears at microtubule asters (0.5 h, 2 h, and thereafter). The pStat3 emerges at the spindle (4 h) and microtubule-organizing centers (MTOCs) in the MI spindle (6 h), and it then disappears in the anaphase/telophase (7 h). The pStat3 localizes again at the MTOCs in the MII spindle (15h). Arrows indicate first polar body (PB). ( B ) The pStat3 localizes at the MTOCs in the M-phase (left figures), but not in the telophase (right figures) in the 1-cell stage. ( C ) The pStat3 exhibits a ring-shape at the MTOCs. The right-hand figures are views from the MTOC. ( D ) The ring-shape localization of pStat3 was also confirmed by a reconstructed 3D image with Imaris software 8.4. ( E ) pStat3 co-localizes with γ-tubulin at MTOCs in the MI (upper figure) and MII spindles (lower figure). The γ-tubulin exhibits a diffused pattern of expression. ( F ) Though pericentrin (Pcnt) is not detected in the GV, it emerges after GVBD and co-localizes with pStat3 during maturation. Different colors indicate pStat3 (green), α-tublin or γ-tublin (red), pericentrin (magenta), and DNA (blue).

Article Snippet: The samples were washed with PBS-X and then incubated with Alexa Fluor 488-anti-rabbit secondary antibody (1:500, A-11070, TFS) in PBS-XB at room temperature for 1 h. After washing with PBS-X, the samples were incubated anti-α-Tublin-Alexa Fluor 594 antibody (1:200, M175-A59, MBL, Nagoya, Japan) in PBS-XB at room temperature for 1 h. As a negative control, the samples were incubated with the secondary antibody alone.

Techniques: Software, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dynamic Changes in pStat3 Are Involved in Meiotic Spindle Assembly in Mouse Oocytes

doi: 10.3390/ijms21041220

Figure Lengend Snippet: Phenotype of Stat3 −/− oocytes. ( A ) Schematic strategy of conditional knock out mouse showing the positions of primers (arrows). Parentheses indicate the PCR product size for genotyping. ( B ) Genotyping PCR of oocytes with the primer set F1L/R2L. The Stat3 −/− oocytes (lower panel) were obtained from Stat3 f/f (f/−) ; Gdf9-iCre female mice (upper panels). ( C ) RT-qPCR of oocytes with the primer set 148F/148R. In the Stat3 −/− oocyte, the expression level of stat3 is approximately 40% that of the Stat3 +/+ oocyte. * P < 0.05. ( D ) Western blotting of oocytes. Arrows indicate the wild type (88 kDa) and truncated (77 kDa) Stat3 and pStat3. Both proteins were detected only in the Stat3 −/− oocytes. The middle panel shows quantification of the Western blotting signal for Stat3. The Stat3 +/+ oocyte (5 oocytes) was used as a reference. The amount of the Stat3 protein from 100 oocytes show approximately twice compared to that from 50 oocytes. Relative amounts of the Stat3 protein after calculation are shown, in which the total amount of Stat3 protein in the Stat3 −/− oocyte is approximately 18% of the Stat3 protein in the Stat3 +/+ oocyte. Numbers (5, 50, and 100) show the oocyte numbers loaded per lane. ( E ) Immunocytochemical analysis shows proper localization of the pStat3 in the Stat3 −/− oocyte. Note that the pStat3 signals (green) are weaker compared to those of the wild type shown in the . Different colors indicate pStat3 (green), α-tublin (red), pericentrin (Pcnt) (magenta), and DNA (blue).

Article Snippet: The samples were washed with PBS-X and then incubated with Alexa Fluor 488-anti-rabbit secondary antibody (1:500, A-11070, TFS) in PBS-XB at room temperature for 1 h. After washing with PBS-X, the samples were incubated anti-α-Tublin-Alexa Fluor 594 antibody (1:200, M175-A59, MBL, Nagoya, Japan) in PBS-XB at room temperature for 1 h. As a negative control, the samples were incubated with the secondary antibody alone.

Techniques: Knock-Out, Quantitative RT-PCR, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Dynamic Changes in pStat3 Are Involved in Meiotic Spindle Assembly in Mouse Oocytes

doi: 10.3390/ijms21041220

Figure Lengend Snippet: Representative features of abnormal MII oocytes treated with either Stat3 inhibitors or anti-pStat3 antibody. ( A – C ) These oocytes appear normal under bright field microscopy. ( A ) The spindle elongation and chromosomes aberration. ( B ) Two spindles involving chromosomes. A chromosome aggregation without spindle body is also seen. ( C ) Shortened and improper spindle formation. String-shaped microtubules are observed to extend from the polar body. ( D ) A larger polar body with expanded spindle assembly. ( E ) Two polar bodies with no spindle formation. ( F ) No polar body and spindle formation. Condensed chromosome with pericentrin is observed. ( G , H ) Higher magnification of abnormal spindles and chromosome mis-location. Note that the expression of pStat3 is either very faint and at an incorrect location (allows shown in A , B , G , H ) or negative ( C – F ). Different colors indicate pStat3 (green), α-tublin (red), pericentrin (Pcnt) (magenta), and DNA (blue).

Article Snippet: The samples were washed with PBS-X and then incubated with Alexa Fluor 488-anti-rabbit secondary antibody (1:500, A-11070, TFS) in PBS-XB at room temperature for 1 h. After washing with PBS-X, the samples were incubated anti-α-Tublin-Alexa Fluor 594 antibody (1:200, M175-A59, MBL, Nagoya, Japan) in PBS-XB at room temperature for 1 h. As a negative control, the samples were incubated with the secondary antibody alone.

Techniques: Microscopy, Expressing