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Chen C.-F.,Ocean University of China | Chen C.-F.,Zhejiang Wanli University | Wen H.-S.,Ocean University of China | He F.,Ocean University of China | And 2 more authors.
Aquaculture Research | Year: 2013

Cytochrome P450c17 (CYP17, 17α-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2102 bp full-length cDNA of P450c17-II (CYP17A2) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 524 amino acids. The putative P450c17-II enzyme shares higher sequence identity with those of teleosts than with P450c17-I of vertebrate. The similarity between the two types of tongue sole P450c17 was 48%.Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was specifically expressed in the ovary and the head kidney. However, temporal expression shows that P450c17-II can be found in the brain. Furthermore, temporal expression pattern of P450c17-II in ovary and brain revealed developmental stage-dependency, and ovary P450c17-II expressed remarkably throughout the whole reproductive cycle. Otherwise, the expression pattern of P450c17-II in head kidney indicated negative ovary development-dependence. In addition, combined with our data on P450c17-I, T and E2 levels, the results further endorse the critical role of P450c17-II during shift in steroidogenesis, suggesting that P450c17-I and -II may act together to this physiological process. Based on the present study, we indicate an important role for P450c17-II during ovarian development. © 2012 John Wiley & Sons Ltd.


Chen C.F.,Ocean University of China | Wen H.S.,Ocean University of China | Wang Z.P.,Ocean University of China | He F.,Ocean University of China | And 9 more authors.
Fish Physiology and Biochemistry | Year: 2010

Cytochrome P450c17 (CYP17, 17a-hydroxylase/17,20-lyase) is a critical enzyme in the production of androgens and estrogens in vertebrates. A 2,469 bp full length cDNA of P450c17-I (CYP17A1) has been isolated from the ovary of half-smooth tongue sole, Cynoglossus semilaevis which encodes 509 amino acids. Additionally, a relatively shorter cDNA (1,742 bp), a likely result of polyadenylation, was also found. The putative P450c17-I enzyme shares high sequence identity with that of the fathead minnow (73%), zebrafish (71%), the Japanese eel (70%), catfish (70%), tilapia (79%), three-spined stickleback (81%), medaka (79%), dogfish (60%), chicken (65%), rat (47%), and human (49%). Semi-quantitative RT-PCR analysis of spatial expression showed the enzyme was predominantly expressed in the ovaries and the brain. P450c17-I was also detected in the stomach, intestine, gill, spleen, kidney, and head kidney, albeit weakly. Further examination of temporal expression pattern of P450c17-I in ovary and brain revealed developmental stage-dependency. In addition to this our data on T and E2 levels further endorse the critical role of P450c17-I during shift in steroidogenesis. Based on the present study we indicate an important role for P450c17-I during ovarian development. However, further studies are needed at transcriptional regulation level for deeper insights into the physiological functions of P450c17-I. © 2010 Springer Science+Business Media B.V.


Wen H.,Ocean University of China | Wen H.,Inner Mongolia Nationality College | Si Y.,Ocean University of China | Zhang Y.,Ocean University of China | And 2 more authors.
Chinese Journal of Oceanology and Limnology | Year: 2015

Follistatin (FST) is a monomeric glycoprotein highly enriched in cysteines and belongs to TGF-β superfamily. FST can suppress the secretion of follicle-stimulating hormone and plays a vital role in the reproduction of vertebrates. We used rapid amplification of cDNA ends technology to clone the FST gene of half-smooth tongue sole, Cynoglossus semilaevis. We characterized its phylogenetic context and expression patterns to elucidate its function in the breeding season. The full-length sequence of FST is 1 455 bp and encodes a protein of 321 amino acids. We investigated the expression pattern of FST in C. semilaevis at different stages of reproduction using reverse transcription-polymerase chain reaction (RTPCR). FST mRNA was expressed in all 13 tissues analyzed, and was expressed at high levels in gonad and at slightly lower levels in gill and brain. During the reproductive cycle of C. semilaevis, the transcript level of FST was the highest in the perinucleolus stage, decreased in the primary yolk stage, slightly increased in the tertiary yolk stage, and then reduced to a minimal level in the atretic follicles stage of the ovary. We concluded that FST suppressed follicle-stimulating hormone, which stimulated oocyte development. However, no significant variation was observed across all stages of testis development, although the expression level in the spermatogenesis stage was relatively low, which may result from the regulation of FST by aromatase. © 2015, Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg.


Shi D.,Ocean University of China | Wen H.S.,Ocean University of China | He F.,Ocean University of China | Li J.F.,Ocean University of China | And 8 more authors.
Steroids | Year: 2011

This paper revealed the expression pattern of ERα in the ovoviviparous teleost, Sebastes schlegeli. In this paper, we isolated the cDNA encoding for estrogen receptor alpha of black rockfish (S. schlegeli) from its ovary, named as black rockfish ERα (brfERα). The cDNA sequence of brfERα consists of 2972 bp with an open reading frame encoding a 624 amino acid putative protein which exhibits high identities with other teleosts'. The tissue distribution of brfERα mRNA was examined using RT-PCR. BrfERα showed generally expressions in most tissues of female black rockfish, besides, the higher degree of expressions were seen in ovary, liver, duodenum and fat, whereas it had a more restricted distribution in male fish. In ovary, the expression level of brfERα was as similar as the serum levels of E 2 and P in female. However, it was a different situation in male, where the serum concentration of E 2 showed higher levels after spermiation and Serum concentration of P did not show any significant changes during a year. Based on the present study, it is supposed that brfERα plays an important role in ovary and other target organs during the reproductive cycle, Further studies will focus on the transcriptional regulation and localization of brfERα in gonad in order to get a better understand of the physiological function of brfERα in ovoviviparous teleost. This study indicates that the black rockfish may be a good candidate for understanding the mechanism of estrogen in ovoviviparous fish. © 2011 Elsevier Inc. All rights reserved.


Mu W.J.,Ocean University of China | Wen H.S.,Ocean University of China | He F.,Ocean University of China | Li J.F.,Ocean University of China | And 5 more authors.
Gene | Year: 2013

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. © 2012 Elsevier B.V.


Mu W.J.,Ocean University of China | Wen H.S.,Ocean University of China | He F.,Ocean University of China | Li J.F.,Ocean University of China | And 4 more authors.
Fish Physiology and Biochemistry | Year: 2013

Full-length cDNA sequences encoding the receptors for follicle-stimulating hormone (FSHR) and luteinizing hormone (LHR) were isolated from ovary of Korean rockfish (Sebastes schlegeli) using reverse transcription-polymerase chain reaction (PCR) and rapid amplification of cDNA ends procedures. The cDNA of the KrFSHR encodes a predicted protein of 703 amino acids that showed the greatest homology with European seabass (Dicentrarchus labrax) (78 %) and gilthead seabream (Sparus aurata) (73 %). The cDNA of the KrLHR encodes a predicted protein of 703 amino acids and exhibited the highest homology with European seabass (Dicentrarchus labrax) (79 %) and gilthead seabream (Sparus aurata) (76 %). Besides the gonads, expressions of GTHRs mRNA were also obtained in extra gonadal tissues. Seasonal changes in the gonads expression profiles of KrGTHRs mRNA were examined by quantitative real-time PCR, and the present results suggest that levels for KrFSHR mRNA increase during gonadal growth, whereas KrLHR shows high levels during the late gamete generation period. Our study provides molecular characterization of the GTHRs and expressions profile during reproductive cycles, reinforcing previous knowledge of GTHRs important role in the reproductive endocrine regulation of Korean rockfish. © 2012 Springer Science+Business Media B.V.


Mu W.,Ocean University of China | Wen H.,Ocean University of China | He F.,Ocean University of China | Li J.,Ocean University of China | And 5 more authors.
Journal of Ocean University of China | Year: 2013

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. © 2013 Science Press, Ocean University of China and Springer-Verlag Berlin Heidelberg.


Jin G.X.,Ocean University of China | Wen H.S.,Ocean University of China | He F.,Ocean University of China | Li J.F.,Ocean University of China | And 8 more authors.
Fish Physiology and Biochemistry | Year: 2012

P450c17, a key steroidogenic enzyme, plays important roles in the production of sex steroid and cortisol. In teleost, there are two types of P450c17, P450c17-I possessing 17α-hydroxylase and 17, 20-lyase activities, and P450c17-II only possessing 17α-hydroxylase activity. This work describes the molecular cloning of the cDNA encoding the barfin flounder (Verasper moseri) P450c17-I and P450c17-II by means of RT-PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) analyses and mRNA expression profiles analyzing by semiquantitative RT-PCR. Respectively, P450c17-I and P450c17-II mRNA levels in the testes correlated with serum testosterone (T) level, as well as gonadosomatic index (GSI) of males during specific stages of spermatogenesis. P450c17-I and P450c17-II mRNA were expressed in the testis and ovary, suggesting that both of them participate in the production of sex steroid in barfin flounder gonads. P450c17-I mRNA was undetectable; in contrast, P450c17-II mRNA was detected at the highest level in the head kidney, meaning that only P450c17-II is involved in the production of cortisol in barfin flounder. The results demonstrated that both of P450c17-I and P450c17-II participate in the production of sex steroid in male barfin flounder gonads. © 2011 Springer Science+Business Media B.V.


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