Sequence analysis of choriogenin H gene of medaka (Oryzias latipes) and mRNA expression

Abstract

Zona radiata proteins of medaka (Oryzias latipes) consist of two major subunit groups, ZI-1,2 and ZI-3. The precursor of ZI-1,2, which is a glycoprotein with a molecular weight of 74,000 to 76,000 Da, is called choriogenin H, and the precursor of ZI-3, with a molecular weight of 49,000 Da, is called choriogenin L. The precursor proteins are synthesized in the liver in response to estrogen in sexually mature female medaka. However, they are also induced in the male medaka when fish are exposed to estrogenic chemicals. Therefore, choriogenin is known as a possible sensitive biomarker for endocrine disruption in fish. In this study, the choriogenin H cDNA sequence was reanalyzed and the genomic DNA sequence was newly analyzed. This was done for the selection of proper reverse transcription-polymerase chain reaction (RT-PCR) primers to measure the choriogenin mRNA induction by estrogenic chemicals. In the results, the full cDNA sequence was found to be 2,109 bp long and the size of the open reading frame (ORF) was found to be a total of 1,998 bp encoding 666 predicted amino acids, which was found to be different from previously reported cDNA sequence of medaka choriogenin. In the choriogenin H cDNA, a repetitive domain and a nonrepetitive domain were shown. Regarding the repetitive domain, seven complete repeats of the 45 bp of 5'-ccc cag tac cca tca aag cct cag ccc cct cag aat cct cag gtc-3' encoding PQYPSKPQPPQNPQV were found. Medaka choriogenin H gene was found to possess seven exons and six introns, and the total length was 2,643 bp long. The seven repetitive nucleotide residues described above existed in exon 1, which was found to be 1,033 bp long. Based on the full sequence information, proper primers were synthesized for RT-PCR to detect choriogenin H mRNA induction in male and juvenile medaka by 17alpha-ethinylestradiol, and this type of measurement system was found to be effective as a simple tool for the screening of endocrine-disrupting chemicals.