Studies On The Function Of The Reproductive Endocrine And Cloning Partial Sequence Of GH In Hexagrammos Otakii Jordan And Starks

Hexagrammos otakii Jordan and Starks mainly distributing in Bohai, and Yellow Sea in China,is a kind of commercial Scorpaeniformes fish.In this paper, development of the gonad , sexual steroid hormone, steroid hormone recepters and aromatase are studied by the methods of histological staining mehods, immunohistochemical techniques and radioimmunoassay mehods in details。In the study, molecular biological technique was also used to cloning partial sequence of GH in Hexagrammos otakii。the main results and conclusions are as follows:1 Histological study on the development and annual change in the gonad of Hexagrammos otakiiThe double testes of Hexagrammos otakii is lobular type in appearance. The immature testis is filament, half clarity while the mature one is big velvet, milkiness in color. The histological staining techniques shows that spermary of Hexagrammos otakii is lobular type. The non-germinal cells, both Leydig and Sertoli cell, were observed in testes of Hexagrammos otakii. The testis development at the age of sex maturity could be divided into the following stages: (1) the recover stage (Ⅲ); (2) the spermiogenesis stage (Ⅳ); (3) the maturity stage (Ⅴ); (4) the stage after spawning (Ⅵ). It begins spermatogenesis in late August. It's reproductive period is from November to late January ,Ⅵstage is from February to April andⅢstage is from May to July.There is a pair of triangular ovaries in Hexagrammos otakii. The color of immature ovary is light yellow or rosiness and that of the mature one is maize or mahogany. The development of ovary can be divided into five stages and the oocytes be divided into five phases. The cytoplasm can be easily dyed to deep blue in phase 2 oogyte and vitellin starts to accumulate in phase 3 oogyte; There are two follicular cells in phase 4 oogyte; The nuclear deflexion and formation of embryo radicel occur in the telophase of phase 4 ovary; The nuclear disappears in phase 5 oogyte when the oogyte becomes dissociating. In the stageⅥ, vitellin granule starts to inosculate into big granule, and degenerate, be assimilated subsequently. Great changs take place in the nucleolus, follicular cells and cell volume in different phases.2 Study on the changes of sex hormone in Hexagrammos otakii in season and the reproductive cycle .Blood samples were collected by injectors from Hexagrammos otakii every season , Plasma testosterone and 17β-Estradiol in female and male was detected with radio immunoassay(RIA) .The results show that the level of Plasma steroid hormone displayed seasonal variation . The main value of steroid hormone went up to peak in reproductive period from November to late January. In male, the plasma T level increase significantly to 21.2625ng/dl, E2 level increase significantly to 13.36083pg/ml;in female ,plasma T level and E2 respectively is 39.381 ng/dl and 13.36083pg/ml. The plasma steroid hormone began to significantly drop from later February to August.In female the plasma T level was not detected from May to July.Before spawning the level of Plasma steroid hormone began to rise slowly from August to October.3 Immunohistochemical study on the gonad of Hexagrammos otakiiIn female,at the early developmental stage of ovary,the cytoplasm of Oocyte shows strong immunopositive reaction to AR and ER antibodies. immunopositive reaction to AR and ER antibodies in cytoplasm of phase III oocyte weakened obviously .At large growth stage ,the cytoplasm of oocyte showed negative reactinon,the nucleus of oocyte shows weak immunopositive reaction to ER antibodies and oocyte membrane shows weak immunopositive reaction to AR and ER antibodies. at the early developmental stage of Oocyte,the cytoplasm,nucleoplasm and nucleolus all shows strong immunopositive reaction to aromatase antibodies, At large growth stage shows immunopositive reaction weaken. In phase IV oocyte,only membrane and nucleoplasm shows weak immunopositive reaction to aromatase antibodies.In male, immunopositive ER receptor is located in the The non-germinal cells, both Leydig and Sertoli cell in testis, show strong immunopositive reaction. Immunopositive AR receptor is located only in the Sertoli cell ,and show medium positive reaction. The recover stage (Ⅲ) of testis show strong immunopositive reaction to aromatase antibodies, especially is spermatogonia and spermatocyte.The immunopositive reaction in spermatocyte to aromatase antibodies weaked gradually during the development and maturation of testis,the spermatid and sperm showed negative immunopositive reactinon. These result s provide a new morphological proof that sex steroid hormones and aromatase participate in the regulation of gonadal development and functional maturation of reproductive endocrine control axis in Hexagrammos otakii.4 Cloning partial sequence of GH in Hexagrammos otakiiAccording to comparative genomics theory, we have designed a pair of primer by software Oligo 6.0. About 915bp sequence, located in a intron, was cloned successfully.