Haishen Wen

Cloning and expression analysis of a HSP70 gene from Korean rockfish (Sebastes schlegeli)

The gene encoding HSP70 was isolated from Korean rockfish Sebastes schlegeli by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of HSP70 cDNA was composed of 2259xa0bp and encoded a polypeptide of 639 amino acids. BLAST analysis showed that HSP70 of S.xa0schlegeli shared high identities with those of the Lates calcarifer, Oreochromis niloticus, Seriola quinqueradiata HSP70s (88-89%). Our current study also revealed that HSP70 of Korean rockfish was expressed in many tissues by RT-PCR under unstressed condition. Quantitative real-time PCR showed that the expression patterns of Korean rockfish HSP70 were developmental stage-dependency. The expression of HSP70 was measured by quantitative real-time PCR after different oxygen treatments. The results showed that expression of HSP70 increased significantly after exposure to hypoxia for 30xa0min in gill and ovary, and then decreased for 60xa0min, and the level in spleen and liver gradually increased and reached the highest at 60xa0min. In addition, in gill, spleen and liver, the HSP70 mRNA level reached the maximum in hypoxia group after one hour different oxygen concentration stress. Increased amounts of serum thyroxine (T4), and triiodothyronine (T3) were also found during 30xa0min hypoxia treatment and 60xa0min normoxia group in our study. All of the results provide information to further study the mechanism of physiology and immune function under stress conditions of ovoviviparous teleosts.

DNA methylation status of cyp17-II gene correlated with its expression pattern and reproductive endocrinology during ovarian development stages of Japanese flounder (Paralichthys olivaceus).

Cytochrome P450c17-II (cyp17-II, 17α-hydroxylase) is responsible for the production of steroid hormones during oocyte maturation in vertebrates. The comparative expression pattern of cyp17-II gene during the gonadal development stages will provide important insights into its function of gonadal development. In addition, epigenetic modification especially DNA methylation plays a vital role in regulation of gene expression. The adult female Japanese flounder at different ovarian development stage (from stages II to V) was obtained in this experiment. The expression of cyp17-II gene in the ovary of Japanese flounder during the gonadal development stages was measured by quantitative PCR. Reproductive traits included gonadosomatic index (GSI), plasma estradiol-17β (E2) and testosterone (T) were also measured. Moreover, whole CpG dinucleotides methylation status of the two CpG rich regions in cyp17-II coding region was detected by bisulfate sequencing. In the ovary, the cyp17-II gene had the lowest mRNA expression at the early ovarian development stage, but then increased afterward. The variation trends of T and E2 level were consistent with the cyp17-II expression pattern in ovary. In contrast, the whole methylation levels of each CpG rich region (exon 4 and 6) in cyp17-II coding region were declined from stages II to IV, then increased at stage V. The methylation levels of whole CpG sites in each CpG rich region were inversely correlated with the values of ovarian cyp17-II gene expression, T and E2 level, and GSI. Based on the present study, we proposed that cyp17-II may regulate the level of steroid hormone, and then stimulate the oocyte growth and maturation. The cyp17-II gene transcriptional activity was possibly affected by the methylation level of CpG rich regions in coding region. These findings will help in the study of the molecular mechanism of fish reproduction and endocrine physiology.

Polymorphism in exons CpG rich regions of the cyp17-II gene affecting its mRNA expression and reproductive endocrine levels in female Japanese flounder (Paralichthys olivaceus)

Cytochrome P450c17-II (cyp17-II) gene is an important factor affecting the growth, gonad differentiation and development, and other reproductive traits of fish. There are three CpG rich regions in the coding region of cyp17-II gene in Japanese flounder (Paralichthys olivaceus). The aim of this study was to understand whether mutations in exons of the cyp17-II gene occured at CpG sites, and mutations and methylation status of those CpG sites were involved in regulation of the expression level of cyp17-II gene and the reproductive endocrine of Japanese flounder. The results showed that three single nucleotide polymorphisms (SNPs) were identified. SNP1 [(c. G594A (p.Gly 188Arg)] located in exon 4 of L1 locus, and SNP2 (c.A939G) and SNP3 (c.C975T) of L2 locus located in CpG rich region of the exon 6 of cyp17-II gene. Furthermore, the A to G transition at 939bp position added a new methylation site to the cyp17-II coding region. According to multiple-comparison analysis, two loci (L1 and L2) were significantly associated with serum testosterone (T) level (P<0.05) and the expression of cyp17-II in ovary (P<0.01). Intriguingly, individuals with GG genotype of L2 locus containing eight CpG methylation sites had significantly lower serum testosterone level and cyp17-II mRNA expression than those with AA genotype containing seven CpG methylation sites. Moreover, the CpG site was highly methylated (≥77.8%) at 938 bp position of individuals with GG genotype of L2 locus. These implied that the mutation and methylation status of the coding region of cyp17-II could influence the gene expression and the reproductive endocrine levels in female Japanese flounder and L2 locus could be regarded as a candidate genetic or epigenetic marker for Japanese flounder breeding programs.

Molecular cloning and expression analysis of estrogen receptor betas (ERβ1 and ERβ2) during gonad development in the Korean rockfish, Sebastes schlegeli

Estrogen receptors (ER) play a crucial role in mediation of estrogen activities. Here we report the isolation and expression analysis of ERβ1 and ERβ2 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, ERβ1 (588 amino acids) and ERβ2 (659 amino acids) were identified using reverse-transcriptase PCR (RT-PCR) and rapid amplification of cDNA ends procedures. Structural analysis showed both ERβs contain six typical nuclear receptor-characteristic domains. Phylogenetic analysis indicated that Korean rockfish ERβs were highly conserved among teleost. RT-PCR confirmed that the ERβs 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 KrERβ1 and KrERβ2. For female, the expressions of KrERβ1 and KrERβ2 were significantly higher in the ovary at the early-oocyte stage. Cloning these two ERβ 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.

Cloning and expression analysis of the cytochrome P450c17s enzymes during the reproductive cycle in ovoviviparous Korean rockfish (Sebastes schlegeli).

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.

Polymorphisms and DNA methylation level in the CpG site of the GHR1 gene associated with mRNA expression, growth traits and hormone level of half-smooth tongue sole (Cynoglossus semilaevis)

The objectives of the present study were to estimate the GHR1 gene mutations and methylation status of CpGs, and whether those mutations and methylation were involved in the regulation of GHR1 gene expression, hormone level and growth traits in half-smooth tongue sole (Cynoglossus semilaevis). Identification of single-nucleotide polymorphisms was performed on 43 male fish. Through polymerase chain reaction-single-strand conformation polymorphism and sequencing, two SNPs were found. SNP1 [c.G1357A (p.Val376Ile)] creating one CpG site located in exon 8 was named L1 locus, and SNP2 (c.G1479A) located in exon 9 was named L2 locus. Individuals were divided into three genotypes, AA, AG and GG according to L1 locus (GG genotype had one more CpG site because of the mutation), and into two genotypes, AA- and GG-based on L2 locus. The results showed that only L1 locus was significantly associated with body weight (Pxa0<xa00.01), gonad weight (Pxa0≤xa00.05), triiodothyronine (T3) level (Pxa0≤xa00.05) and mRNA expression (Pxa0<xa00.01). At L1 locus, newly created CpG site in GG genotype was highly methylated (93.3xa0%), while there was no difference of methylation level in the other two CpG sites among three genotypes. AA genotype and AG genotype having higher T3 level were significantly different (Pxa0≤xa00.05) from GG genotype. There were significant differences among body weights of AA, AG and GG genotypes (Pxa0<xa00.01). Gonad weights of AA genotype and AG genotype were significantly lower than GG genotype. The GHR1 mRNA expression of GG genotype was significantly lower than AA and AG genotypes (Pxa0<xa00.01). These implied that mutations and methylation status of GHR1 gene might influence the hormone level, growth traits and gene expression in male half-smooth tongue sole and the L1 locus could be regarded as a potential candidate genetic and epigenetic marker in half-smooth tongue sole selection.

Cloning and expression analysis of vasa during the reproductive cycle of Korean rockfish, Sebastes schlegeli

Vasa, which is a conserved member of the DEAD-box protein family, plays an indispensable role in primordial germ cell proliferation. However, the expression of vasa gene during the reproductive cycle in ovoviviparous fish has not been documented. In this study, the full-length sequence of vasa was obtained from the ovary of Korean rockfish (Sebastes schlegeli) using reverse transcription-PCR and rapid amplification of cDNA ends. The Vasa with a mature protein of 650 amino acids showed greatest homology (84%) with giant gourami (Osphronemus goramy) and Pacific bluefin tuna (Thunnus orientalis). The expression of vasa mRNA in Korean rockfish was detected in gonads only, suggesting its specific role in gonadal development. In addition, seasonal changes in the vasa expression levels were examined in gonads by quantitative real-time PCR. The vasa transcript levels in adult testis were found higher during spermatogenesis than during spermiation. The vasa transcript levels remained relatively high at the early ovary stage but declined during ovary maturation in adult female fish. These results suggest that the vasa gene play an important role in spermatogenesis and early oogenesis during the reproductive cycle of Korean rockfish.

Mutations in exons of the CYP17 -II gene affect sex steroid concentration in male Japanese flounder ( Paralichthys olivaceus )

As a specific gene of fish, cytochrome P450c17-II (CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI (P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.