Figure 1.

PIWIs are expressed in hamster oocytes. (A) Expression patterns of PIWI families in male and female hamsters. Transcriptome data were obtained from Illumina HiSeq2000. The x-axis suggests the normalized expression level (TPM). (B) Protein homology of PIWIL3 in hamster and human. Approximately 74.6% of the PAZ domain and 83.3% of the PIWI domain in hamster PIWIL3 are conserved in human PIWIL3. The large extension of the amino-terminal portion of the ORF, which contains 14 repeats of nucleotide sequences encoding the amino acid sequences QLQSPGAGPPRSGA is presented in hamster PIWIL3. (C) PIWIL1 (upper panel) and PIWIL3 (lower panel) levels from an ovary. Immunostaining with PIWIL1 and PIWIL3 are shown: (green) PIWIL1 and PIWIL3; (blue) Hoechst. The signal in the zona pellucida at upper panel is the autofluorescence. (D) Western blotting was performed from hamster whole ovaries, MII oocytes, and 2-cell embryos with anti-PIWIL3, anti-PIWIL1, and anti-β-TUBULIN antibodies. The size of the PIWIL3 protein largely shifted by approximately 40 kDa in MII oocytes. The anti-MARWI (PIWIL1) antibody (1A5) and anti-PIWIL3 antibody (3E12) detected a single band in each sample except for MII oocytes. (E) Western blotting was performed from hamster MII oocytes with (+) or without (−) CIP treatment. A discrete band migrated to the estimated molecular weight of 130 kDa in the CIP-treated sample, demonstrating that PIWIL3 is heavily phosphorylated in MII oocytes.