Molecular cloning and characterization of gonadotropin subunits (GTHα, FSHß and LHß) and their regulation by hCG and GnRHa in Japanese sea bass (Lateolabrax japonicas) in vivo.

In this study, three cDNA sequences encoding common glycoprotein α subunit (GTHα), follicle-stimulating hormone ß subunit (FSHß) and luteinizing hormone ß subunit (LHß) were isolated from Japanese sea bass (Lateolabrax japonicas). Comparison of the deduced amino acid sequences with other gonadotropic hormones (GTHs) indicated that their cysteine residues and potential N-linked glycosylation sites were highly conserved, and high homology with those of other perciformes was showed in phylogenetic analysis. GTHs transcripts were present highly in the pituitary and brain and weakly in testis and other tissues. During testicular development, GTHs transcriptional levels in pituitary and brain (expect FSHß subunit in brain) were significantly increased at spermiation period, stage V. Subsequently, the effects of hCG and GnRHa on the mRNA levels of GTHs subunits were examined. In brain, both hormones were detected to improve the expression of GTHα subunit mRNA. In pituitary, three GTHs subunits increased parallelly and abruptly in two hormone treatment groups. In testis, hCG was suggested to improve three GTHs subunits expression in Japanese sea bass for the first time. These results suggest that both gonadotropins are probably involved in the control of Japanese sea bass spermatogenesis and provide a framework for better understanding of the mechanisms of hormone-mediated reproduction control in Japanese sea bass and other teleosts.

Cloning and expression analysis of Foxl2 during the reproductive cycle in Korean rockfish, Sebastes schlegeli.

Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to regulate ovarian aromatase, which plays a crucial role in ovarian differentiation. To address the role of Foxl2 in gonads and brain during gonadal development, we isolated the full-length cDNA of Foxl2 and analyzed its spatiotemporal expression patterns in the viviparous teleost Korean rockfish, Sebastes schlegeli. Tissue distribution pattern revealed that the Foxl2 was detected in the liver, fat, gill, brain, and ovary, but could hardly be found in the testis. Reverse transcriptase PCR suggested that Foxl2 in Korean rockfish may involve in ovary development in the study of expression level during gonads development. It also revealed that the stage of highest expression level for Foxl2 was almost much earlier than cyp19a1a and cyp19a1b during the gonadal development stage in gonads and brain except for cyp19a1a in brain. Furthermore, the expression pattern of Foxl2 as well as aromatases may imply the role of Foxl2 in the up-regulation of aromatases not only in the female fish but also in male.

Cloning and expression analysis of follicle-stimulating hormone and luteinizing hormone receptor during the reproductive cycle in Korean rockfish (Sebastes schlegeli).

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.

Molecular cloning, characterization expression of P450c17-I and P450c17-II and their functions analysis during the reproductive cycle in males of barfin flounder (Verasper moseri).

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.

Cloning and expression of P450c17-I (17α-hydroxylase/17,20-lyase) in brain and ovary during gonad development in Cynoglossus semilaevis.

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.

Molecular identification of genes involved in testicular steroid synthesis and characterization of the responses to hormones stimulation in testis of Japanese sea bass (Lateolabrax japonicas).

Testicular steroids are critical hormones for the regulation of spermatogenesis in male teleosts and their productions have been reported to be regulated by gonadotropins and gonadotropin-releasing hormone. In the Japanese sea bass (Lateolabrax japonicas), the reproductive endocrine, particularly regarding the production and regulation of testicular steroids, are not well understood. For this reason, we first cloned and characterized the response of several key genes regulating the production of testicular steroids and, second, we analyzed the changes of mRNA profiles of these genes during testicular development cycle and in the administration of hCG and GnRHa with corresponding testosterone level in serum, GSI and histological analyses. We succeeded in cloning the full-length cDNAs for the fushi tarazu factor-1 (FTZ-F1) homologues (FTZ-F1a and FTZ-F1b), steroidogenic acute regulatory protein (StAR) and anti-Müllerian hormone (AMH) in Japanese sea bass. Multiple sequence alignment and phylogenetic analysis of these proteins clearly showed that these genes in Japanese sea bass were homologous to those of other piscine species. During the testicular development cycle and hCG/GnRHa administration, quantification of jsbStAR transcripts revealed a trend similar to their serum testosterone levels, while a reciprocal relationship was founded between the serum concentrations of testosterone and jsbAMH and the links between gonadal expression of jsbStAR, jsbAMH and jsbFTZ-F1 were also observed. Our results have identified for the first time several key genes involved in the regulation of steroid production and spermatogenesis in the Japanese sea bass testis and these genes are all detected under gonadotropic hormone and gonadotropin-releasing hormone control.

The physiology functions of estrogen receptor α (ERα) in reproduction cycle of ovoviviparous black rockfish, Sebastes schlegeli Hilgendorf.

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 2972bp 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 E2 and P in female. However, it was a different situation in male, where the serum concentration of E2 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.

Association of reproductive performance with SNPs of FOXL2 gene by SSCP in Japanese flounder (Paralichthys olivaceus).

FOXL2 which is a putative winged helix/forkhead transcription factor gene and a sexually dimorphic marker of ovarian differentiation plays an important role in ovarian development, granulosa cell differentiation, and thus the proper maintenance of ovarian function. The aims of this study were to characterize polymorphisms within the FOXL2 gene in a population of 52 female Japanese flounder and analyze the association of FOXL2 polymorphisms with reproductive performance by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Results indicated that five single nucleotide polymorphisms (SNPs), which were SNP1 [c.540A>C (p.Asn102His) and c.591A>G (p.Asn119Asp)], SNP2 [c.864G>A (p.Lys210Glu)and c.875G>A] and SNP3 (c.1169C>A), were identified in the FOXL2 gene. General Linear Model (GLM) analysis showed that SNP1 in the forkhead domain was significantly associated with gonadosomatic index (GSI) (P<0.05). SNP2 in the downstream of forkhead domain was significantly associated with serum 17beta-estradiol (E(2)) level (P<0.05). And SNP3 in the 3'-UTR was significantly associated with hepatosomatic index (HSI) (P<0.05). Moreover, the evaluation of the genetic effects for both Testosterone (T) level of diplotype D3 and GSI of diplotype D5 suggested they were significantly higher than those of other four diplotypes (P<0.05), respectively. These results implied that these SNPs could influence reproductive endocrinology of female Japanese flounder and be also used in marker-assisted selection (MAS) program to reproductive performance in female Japanese flounder in the future.