Journal: Reproduction (Cambridge, England)

Article Title: Mechanisms underlying disruption of oocyte spindle stability by bisphenol compounds

doi: 10.1530/REP-19-0494

Figure Lengend Snippet: Inhibition of Aurora Kinase A (AURKA) limits bisphenol-mediated spindle pole disruption. (A) Western blot (5 replicates) of phosphorylated/active AURKA (pT288AURKA), total AURKA and control β-tubulin protein levels in control, BPA or BPF-treated oocytes lysates (50 oocytes/lane) either alone or together with MLN8337. (B) The expression levels of active pT288AURKA compared between treatment groups and the control (media alone), which was normalized to 1.0. (C) Representative fluorescence microscopy images of oocytes from control as well as BPA or BPF treatment (50μg/ml) groups following a 4h culture with or without a specific AURKA inhibitor, MLN8237 (500nM). Oocytes were double labeled with anti-acetylated tubulin (green) and pT288 AURKA (red). Chromosomes (grey) were counterstained with DAPI. Scale bar: 10μm. Quantitative analysis (mean ± s.e.m) of the (D, F) meiotic spindle length (pole-to-pole distance) and (E, G) average spindle pole width assessed in BPA or BPF-treated oocytes (50-80 total oocytes per group, from 3 replicates) –either alone (solid bars) or together with MLN8237 (hatched bars). Control oocytes were cultured in either media alone or with MLN8237. Different letters denote statistical significance at P<0.05.

Article Snippet: Immunofluorescence analysis Oocytes were fixed and immunolabeled with specific antibodies as previously described ( Baumann & Viveiros 2015 ) including, anti-TPX2 (1/500; Novus Biological, Centennial, CO), anti-PCNT (1/1000; BD Biosciences, San Jose, CA or Covance, Princeton, NJ), anti-acetylated α-tubulin (1/1000; Sigma Aldrich) and anti-pT288AURKA (1/1000; Cell Signaling Technology, Beverly, MA).

Techniques: Inhibition, Western Blot, Expressing, Fluorescence, Microscopy, Labeling, Cell Culture