Fig. 7

hnRNP D, Gemin5, and Dhx9 regulate the translation of Pou5f1 mRNA in mouse embryos. (A) Immunofluorescence of hnRNP D, Gemin5, and Dhx9 in mouse immature (Im) and mature (M) oocytes and two-cell stage embryos (Two-cell). GV, germinal vesicle. (B) IP/RT-PCR analysis of hnRNP D (left), Gemin5 (middle), and Dhx9 (right) with Pou5f1 mRNA. Left: Semiquantitative RT-PCR amplification of Pou5f1 and α-tubulin transcripts of mouse ovary extracts before IP (initial) and IP with control IgG (IgG) or anti–hnRNP D (hnRNP D) antibody. Similar results were obtained from three independent experiments. Middle and Right: Semiquantitative RT-PCR amplification of Pou5f1 and α-tubulin transcripts of mouse two-cell stage embryos before IP (initial) and IP with control IgG (IgG) or anti-Gemin5 (Gemin5) or anti-Dhx9 (Dhx9) antibody. An asterisk indicates nonspecific amplifications. (C) Immunoblotting of oocytes injected without [control (Cont)] or with anti–hnRNP D (left), anti-Gemin5 (middle), or anti-Dhx9 (right) antibody and Trim21 mRNA [knockdown (KD)]. (D) Left: Immunofluorescence of Pou5f1 in embryos injected with IgG (control) or anti–hnRNP D antibody (hnRNP D KD). DNA is indicated at the bottom. Similar results were obtained from three independent experiments. Right: Intensities of Pou5f1 signals were quantified (means ± SD). a.u., arbitrary unit. *P < 0.05, Student’s t test. (E) Left: Immunofluorescence of Pou5f1 in embryos injected with IgG (control) or anti-Gemin5 (Gemin5 KD) and anti-Dhx9 (Dhx9 KD) antibodies. Similar results were obtained from three independent experiments. Right: The intensities of Pou5f1 signals were quantified (means ± SD). **P < 0.01, Dunnett’s test. Scale bars, 20 μm.