Studies On Reproduction Physiological Function Of Four Cytochrome P450 Enzymes In Half-smooth Tongue Sole, Cynoglossus Semilaevis

The full length of P450c17-Ⅰand P450c17-ⅡcDNA is cloned from half-smooth tongue sole Cynoglossus semilaevis, and the spatial and temporal expression of P450c17(-Ⅰ,-Ⅱ) are well studied, as well as P450c19(A,B).The results of the spatial and temporal expression of the four enzymes are firstly linked to ovarian development, serum sexual steroid hormones testosterone(T) and estradiol-17β(E2) level, so as to work over the enzymes' molecular physiological mechanism in female and male half-smooth tongue sole.1. Studies on ovarian development and spawn type of cultured female half-smooth tongue sole Cynoglossus semilaevisSeasonal variations of ovarian development and the relationship with Gonadosomatic index (GSI),hepatosomatic index (HSI) was studied in the cultured half-smooth tongue sole Cynoglossue semilaevis. The results show that ovarian development consists of phaseⅡ,Ⅲ,Ⅳ,Ⅴ,Ⅵ, then cycles to recrudescent phaseⅡ; under artificial culture, most fish has phaseⅡovarium from March to May, and the oocytes begin to accumulate vitellin granule from June, then the whole cytoplasm is stuffed with vitellin granule ranging from September to November, suggesting the oocytes growth to mature, after spawning, the ovarium come to assimilative phaseⅥor recrudescent phaseⅡ;GSI reaches to an peak during reproduction, then dramatically drop after spawning or recrudescent phaseⅡwhile HSI significantly increases to maximum; there is only one breeding season in the half-smooth tongue sole, and it belongs to batch-spawning fish.2. Study on the seasonal changes of serum sexual steroid hormones in half-smooth tongue sole Cynoglossus semilaevisSerum sexual steroid hormones testosterone(T) and estradiol-17β(E2) levels are measured by 125I radioimmunoassay(RIA). Serum E2 and T levels are shown to be accord with ovarian developmental phase (GSI) in female hale-smooth tongue sole. During the reproductive period, serum E2 and T levels significantly increase, and reach to the highest level throughout the whole year(P<0.05). After spawn, the females live through winter, and both of serum E2 and T levels decline. In the period of artificially enriching and culturing, serum E2 level remarkably increase(P<0.05), but the content of serum T drops markedly to the value that can't be detected. The results show that seasonal changes of female half-smooth tongue sole serum E2 level is closely correlated with ovarian development and vitellus' formation; and as the precursor of E2, the changes of T level has some relationship with ovarian development, furthermore it is essential for certain ovarian developmental phase.Serum T and E2 levels are also shown to be accord with testis development(GSI) in male half-smooth tongue sole. The peak of serum T and E2 levels both occur at reproductive season; from November to January of next year, both of the contents drop rapidly(P<0.05); during February to April, serum T level slightly increase, but the content of serum E2 still decrease; in the period of May to July, serum T and E2 levels both significantly increase(P<0.05). The results show that T directly effects on the growth of sperm cell, and can boost sperm' formation and maturation.3. Spatial and temporal expression analysis of half-smooth tongue sole Cynoglossus semilaevis P450c19sThe spatial expression of P450aromA and P450aromB show that P450aromA only expresses in gonad, and P450aromB only expresses in brain and gill. Further, P450aroms express in the gonad and brain throughout the whole gonad development in female and male C. semilaevis, respectively. The temporal expression pattern of P450aromA is consistent with the changes of serum E2 and T level in ovaries, indicating that it plays an important role in ovarian development and oocyte growth. However, the pattern of P450aromA in testis is not related with the changes of serum E2 and T level. The results of P450aromB'temporal expression shows that, in female tongue sole of atretic follicles stage, P450aromB expression in brain increases significantly and reaches to the peak; the similar result occurs in male tongue sole brain after spermiation. These results suggested the reproductive cycle of female tongue sole is not only regulated by CYP19A, but also regulated by CYP19B. 4. Programming Design of Degenerate Primers and Cloning Half-smooth Tongue Sole Cynoglossus semilaevis CYP17 partial cDNAA new method of designing degenerate primers-CODEHOP is introduced to clone half-smooth tongue sole Cynoglossus semilaevis CYP17 gene. With Bioinformatics Resources such as NCBI,Blastp,BlockMaker,CODEHOP and biological software such as DNAMAN,Oligo6.0, the CODEHOP primer is programming designed, and finally we succeeded to clone the gene from the ovaries of half-smooth tongue sole Cynoglossus semilaevis. The fragment of the gene has high identities with the CYP17 family of other species, and it is clustered into the CYP17 gene group of all the other vertebrates, especially into the fish subgroup. The experiment results showed that compared with traditional degenerate primers, programming designed primers had more Specific ,lower positive rate, and more benefit for accomplishing the experiment.5. Cloning, spatial and temporal expression analysis of half-smooth tongue sole Cynoglossus semilaevis P450c17-ⅠA full length cDNA of P450c17-Ⅰhas been isolated from half-smooth tongue sole (Cynoglossus semilaevis) ovary. It is 2469 bp in length, encoding 509 amino acids. Also a relative shorter cDNA (1742bp) was found, likely due to a polyadenylation. The putative P450c17-Ⅰenzyme shares high sequence identity with that of fathead minnow, zebrafish, the Japanese eel, tilapia, three spined stickleback, medaka dogfish, chicken, rat, and human. Semi-quantitative RT-PCR analysis of spatial expression shows that it was predominantly expressed in the ovary, followed by brain, and faint signals were also detected in stomach, intestine, gill, spleen, kidney, head kidney. Further examination of temporal expression pattern of P450c17-Ⅰin 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-Ⅰduring shift in steroidogenesis.6. Cloning, spatial and temporal expression analysis of half-smooth tongue sole Cynoglossus semilaevis P450c17-ⅡA full length cDNA of P450c17-Ⅱhas been isolated from half-smooth tongue sole (Cynoglossus semilaevis) ovary. It is 2102bp in length, encoding 524 amino acids. The putative tongue sole P450c17-Ⅱenzyme shares higher sequence identity with that of other animals, than the identity of P450c17-Ⅰenzyme of the same species. Semi-quantitative RT-PCR analysis of spatial expression shows that it was specially expressed in the ovary, brain and head kidney. Further examination of temporal expression pattern of P450c17-Ⅱin ovary and brain revealed developmental stage-dependency. However, the expression pattern of P450c17-Ⅱin testis shows negative relationship with testis development. P450c17-Ⅱexpression in gonad, brain and head kidney is studied, combined with serum E2 and T levels, so as to find out the relationship between P450cl7-Ⅱand gonad development, and provide theory foundation of flatfish reproductive physiology.