ABSTRACT
Estrogen receptors (ER) play a crucial role in mediation of estrogen activities. Here we report the isolation and expression analysis of ERß1 and ERß2 from ovary Korean rockfish (Sebastes schlegeli). were isolated using reverse transcription-polymerase chain reaction (PCR) and rapid amplification of cDNA ends procedures. The cDNA of this study, ERß1 (588 amino acids) and ERß2 (659 amino acids) were identified using reverse-transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends procedures. Structural analysis showed both ERßs contain six typical nuclear receptor-characteristic domains. Phylogenetic analysis indicated that Korean rockfish ERßs were highly conserved among teleost. RT-PCR confirmed that the ERßs were widely distributed in both gonads and extra gonadal tissues. Further, we analyzed the expression patterns of male and female S. schlegeli during the reproductive cycle using quantitative real-time PCR (qRT-PCR). The results showed that the highest expression levels were observed in testis at immature sperm stage for both of KrERß1 and KrERß2. For female, the expressions of KrERß1 and KrERß2 were significantly higher in the ovary at the early-oocyte stage. Cloning these two ERß subtypes in the Korean rockfish, together with the information on expression levels in adult fish has given us the foundation to investigate their possible role in brain-pituitary-gonad neuroendocrine axis in future studies.
Subject(s)
Estrogen Receptor beta/metabolism , Fishes/growth & development , Ovary/metabolism , Testis/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , DNA, Complementary/genetics , DNA, Complementary/metabolism , Estrogen Receptor beta/classification , Estrogen Receptor beta/genetics , Female , Fishes/genetics , Fishes/metabolism , Gene Expression Regulation , Male , Molecular Sequence Data , Ovary/cytology , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Reproduction , Reverse Transcriptase Polymerase Chain Reaction , Seasons , Species Specificity , Testis/cytology , TranscriptomeABSTRACT
Cytochrome P450c17 (CYP17, 17α-hydroxylase/17, 20-lyase) plays a critical role in the production of androgens and estrogens in vertebrates. We isolated the full length cDNAs of P450c17-I and P450c17-II from Sebastes schlegeli. The cDNA sequences of P450c17-I and P450c17-II encoded 515 and 533 amino acid residues respectively. The putative P450c17-I and P450c17-II enzymes of Korean rockfish share high sequence identity with that of Japanese flounder (92% and 81%) respectively. Our current study describes that P450c17s of Korean rockfish are mainly expressed in gonads, head kidney and kidney by RT-PCR. Quantitative real-time PCR showed that the expression patterns of Korean rockfish P450c17s were developmental stage-dependency. In addition, the testosterone (T) and gonadosomatic index (GSI) levels further support the important role of P450c17-I during shift in steroidogenesis. Taken together, this study provides information about the Korean rockfish P450c17s characterization and mRNA expression as such helps in further understanding of its function in gonadal development.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , Perciformes/metabolism , Steroid 17-alpha-Hydroxylase/biosynthesis , Animals , Cloning, Molecular , DNA, Complementary/genetics , Fish Proteins , Fishes , Isoenzymes/biosynthesis , Isoenzymes/genetics , Organ Specificity/physiology , Perciformes/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Steroid 17-alpha-Hydroxylase/geneticsABSTRACT
The cytochrome P450c17-I (CYP17-I) is one of the enzymes critical to gonadal development and the synthesis of androgens. Two single nucleotide polymorphisms (SNPs) were detected within the coding region of the CYP17-I gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). They were SNP1 (c.C445T) located in exon2 and SNP2 (c.T980C (p.Phe307Leu)) located in exon5. Four physiological indices, which were serum testosterone (T), serum 17ß-estradiol (E2), Hepatosomatic index (HSI), and Gonadosomatic index (GSI), were studied to examine the effect of the two SNPs on the reproductive endocrines of Japanese flounder. Multiple comparisons revealed that CT genotype of SNP1 had a much lower T level than CC genotype (p<0.05) and the GSI of individuals with CC genotype of SNP2 was higher than those with TT genotype (p<0.05). Four diplotypes were constructed based on the two SNPs and the diplotype D3 had a significantly lower T level and GSI. In conclusion, the two SNPs were significantly associated with reproductive traits of Japanese flounder.
