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This study investigated the biochemical composition of ovarian fluid and its effect on the fertilization capacity of turbot Scophthalmus maximus during the spawning season. The fertilization rate and pH of ovarian fluid varied throughout the spawning season, with the highest values recorded at the mid-season. Positive correlations were found between the fertilization rate and the ovarian fluid pH. The composition of major inorganic ions (Na(+) , K(+) , Ca(2+) and Cl(-) ) showed no significant changes during the spawning season. Alkaline phosphatase (AKP) activity was significantly higher during mid-season than other seasons. The lowest levels of protein, acid phosphatase (ACP) and aspartate aminotransferase (AAT) were in the ovarian fluid released at the mid-season. Moreover, significant relationships were observed between the fertilization rate and the levels of protein, ACP, AKP and AAT. These observations suggest that the biochemical profile of ovarian fluid affects the insemination microenvironment as well as the fertilization capacity of S. maximus eggs. Determination of such profiles may prove to be a useful strategy to improve S. maximus breeding techniques.

A new ovarian cell line, CSO, was established from half-smooth tongue sole Cynoglossus semilaevis. Primary culture of CSO cells was initiated from digestion of ovarian tissues pieces by trypsin solution and cultured at 24° C in Dulbecco's modified Eagle's medium-F12 medium (DMEM-F12, 1:1) (pH 7·0), supplemented with 20% foetal bovine serum, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and human chorionic gonadotropin (HCG). The cultured CSO cells, fibroblastic in morphology, proliferated to 100% confluency 3 days later and had been subcultured to passage 80. Chromosome analyses indicated that the CSO cells exhibited chromosomal aneuploidy with a modal chromosome number of 42 that displayed the normal diploid karyotype of C. semilaevis [2n = 42 t, fundamental number (NF ) = 42]. Reverse transcription polymerase chain reaction revealed that CSO cells could express ovarian somatic cell functional genes p450armo, foxl2 and sox9a but not ovary germ cell marker gene vasa and male-specific gene dmrt1. Transfection experiment demonstrated that CSO cells transfected with pEGFP-N3 plasmid could express green fluorescence protein (GFP) with higher transfection efficiency. The CSO cell line might serve as a valuable tool for studies on the mechanism of sex determination and oogenesis of ovary in flatfish.

Estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase is the terminal enzyme in steroidogenic pathway that converts androgens into estrogens. In most of vertebrates,P450arom is the product of the CYP19 gene,which exists as a single copy per haploid genome. In contrast,there are two isoforms of aromatase in teleosts,which are encoded by two different genes,CYP19a and CYP19b,as well as have different distribution. This work describes the molecular cloning of the cDNA encoding the barfin flounder(Verasper moseri) gonad cytochrome P450 aromatase(CYP19a) by means of reverse transcriptase polymerase chain reaction(RT-PCR),degenerate primers PCR amplification and 5'-and 3'-rapid amplification of cDNA ends(RACE) analyses. We cloned the complete cDNA sequence of CYP19a gene and the length is 2 167 bp,encoding a protein of 523 amino acids. Via Blastn,the CYP19a cDNA displayed high identity with Atlantic halibut(Hippoglossus hippoglossus),Japanese flounder(Paralichthys olivaceus) and southern flounder(Paralichthys lethostigma). Alignment analysis using barfin flounder CYP19a gene deduced animo acid sequence and animo acid sequences of other species showed that there were transmembrane helix region,I-helix region,Ozol's peptide region,aromatase-specific conserved region and heme-binding region in the deduced animo acid sequence. Phylogenetic analysis suggested the occurrence of a gene duplication for cytochrome P450 aromatase in the teleost lineage after its divergence from the tetrapods. Furthermore,the higher identity between barfin flounder P450aromA and P450aromAs of other teleosts suggested that they have the same origin. A semiquantitative PCR was developed to measure mRNA expression levels of CYP19a gene of barfin flounder. Expression analysis in different tissues demonstrated that P450aromA transcripts were highly abundant in brain,ovary and testis,less abundant in intestine,liver and kidney,showing that not only gonads can produce steroids but also other tissues have steroidogenic capability. And we analyzed the relative mRNA expression level of CYP19a in testes at different stages of reproductive cycle,there were the highest level at stageⅡ and the lowest level at stageⅤ. These changes in expression profiles may imply differences in the functionality of the enzyme between different stages,suggesting an important role for P450aromA in barfin flounder testes development.