Abstract
E-peptides are proteolytic cleavage products of pre-pro-insulin-like growth factor (Pre-pro-IGF) I and II in posttranslational processing of the pre-pro-hormones. Rainbow trout (rt) Ea4- and human (h) Eb-peptides possess unique anti-tumor activities in established human and fish cancer cells. This paper reports on the effects of rtEa4- and hEb-peptides in fish embryos during early embryonic development. Introduction of either rtEa4 or hEb gene into newly fertilized medaka (Oryzias latipes) eggs by electroporation resulted in abnormal embryonic development of heart, red blood cells, and vasculature. Red blood cells in heart-defective embryos were stained with diaminofluorene (DAF) which revealed a drastic reduction of red blood cells in defective embryos, suggesting that, in addition to heart defects, embryonic red blood cell development may also be disrupted by the E-peptide. The temporal expression patterns of the transcription factor GATA-1 gene, an essential regulator of hematopoiesis during embryonic development, were determined by reverse transcription–polymerase chain reaction (RT-PCR). GATA-1 mRNA was detected at 24 hours postfertilization (hpf), then raised to a higher level at 30 hpf. It was maintained at this level until 100 hpf (>4 days). At 5 days postfertilization (dpf), the expression level of GATA- 1 gene decreased and remained at that level until hatching. The number of GATA-1 transcripts in transgenic embryos at 4 dpf was determined by quantitative real-time RT PCR. Whereas the GATA-1 mRNA in nontransgenic embryos at 4 dpf was 7.15 × 105/3 µg of total RNA, that of the heart defective transgenic embryos at the same stage was 1.35 ×105/3 µg of total mRNA. These results suggest that rtEa4- or hEb-peptide may be responsible for disrupting embryonic hematopoiesis in medaka embryos by reducing the expression of the GATA-1 gene.
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