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.

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.