| Zebrafish (Danio rerio) originates in India and Bangladesh etc. It belongs to Cyprinidae section and Danio genus. It is widely used for embryology, developmental biology, toxicology and molecular biology research. It has been called the ideal vertebrate model for molecular biology and immunology research and it has very high scientific value and wide range of potential applications. It is one of four pattern life-form. This paper studies on zebrafish, and uses histology, immunohistochemistry and sexual steroid hormone radioimmunoassay method to research its gonads and gonadal steroids hormones in extraction of gonadal. Through manipulating environmental factors (for example: temperature, salinity and pheromone) to observe the influences for its reproductive. The results and conclusions are as follows:1. Histological study on the development in the gonad of zebrafishThe double spermaries of zebrafish is in the celiac back . The color of the mature spermary is milky white. The histological staining techniques shows that spermary of zebrafish is lobular type. The non-germinal cells (Leydig and Sertoli cell) and spermatogenic cysts are observed in study of zebra fish. The spermary contains all budding reproductive cells in spermary of sexual mature male zebrafish. The reproductive cells is synchronized in one spermatogenic cysts but not in different spermatogenic cysts. On condition that there is appropriate temperature, enough light and nutrients ,the male zebra fish can reproduce consecutively. The reproductive cycle of male zebrafish is five days.Ovary is the same as spermary and it is in pairs. The color of ovary in the breeding season is white. Oocyte development is divided into five phases through histological observation. At stageⅡ, cell diameter of oocyte is the smallest, and cell cytoplasm dyed by hematoxylin is dark purple. At stageⅢ, oocyte yolk starts to accumulate, and vacuoles and radiation belts began to appear. At stageⅣ, oocytes volume continues to increase, and the egg yolk rapidly accumulates .The vacuole is pressed to the cytoplasm around the oocyte and the radiation belts is obvious. In the anaphase, the nuclear begins to deflect and the embryo appears. At stageⅤ, mature oocyte nuclear disappears and it assumes a free state. At stageⅥ, oocytes in ovary discharge and follicular membrane is remained. The oocyte nucleoli number, cell membrane thickness and cell volume are great diversification in various periods. Zebrafish is the type of eggs in batches of spawning. The female reproductive cycle is about six days.2 .Study on the changes of hormones in extraction of gonadal in the reproductive cycle in zebrafishThe gonad of zebrafish were collected to extraction in continuous 7 days. Both the level of testosterone (T) and 17β-Estradiol (E2) in the extraction of gonad in female and male are detected with radio immunoassay(RIA). The results show that after reproduction both the level of T and E2 increase rapidly, and present significant changes (P <0.05). The level of E2 shows the peak value at the 5th day in both male and female fish. The peak mean of E2 in male gonad is 3195.75 pg / g while in female gonad it is 965.13pg/g. The level of T shows the peak value at the 5th day in both male and female fish. The peak mean of T in male gonad is 1522.72ng/mg while in female gonad it is 413.66 ng/mg. After reproduction both the level of T and E2 show the lowest value in original 3 days. For the male fish, E2 can't be checked in original 2 days. After that, the level of hormones would hoist rapidly and reach to the peak value before it falls.3. Immunohistochemical study on the gonad of zebrafishIn female, at the early developmental stageⅡof ovary, the cytoplasm of oocyte and follicular membrane show strong immunopositive reaction to AR and aromatase antibodies. Karyoplasm shows immunonegative reaction to ER and strong immunopositive reaction to aromatase antibodies. Oocyte at the stageⅡs hows immunonegative reaction to AR. At stage III, strong immunopositive reaction to AR and aromatase antibodies of oocyte cytoplasm, follicular membrane and karyoplasm decreases significantly. At the late stage III of ovary, the karyotheca and vacuole inclusion show weak immunopositive reaction to AR. At the stage IV of ovary, the karyon and yolk granules show immunonegative reaction to ER ,AR and aromatase antibodies while karyotheca, follicular membrane and vacuole inclusion of amphithecium show weak immunopositive reaction to them.In male, immunopositive ER and AR antibody is located in the non-germinal cells (Leydig and Sertoli cell) and spermatogenic cysts wall show strong immunopositive reaction. Immunopositive AR antibody shows weaker positive reaction than ER antibody. Karyon , cytoplasm and cell membrane of spermatogonia and spermatocyte show strong immunopositive reaction to aromatase antibodies while the non-germinal cells and germ cells show immunonegative reaction. These results provide a new proof that sex steroid hormones and aromatase participate in the regulation of gonadal development and functional maturation of reproductive endocrine control axis in zebrafish.4.Influence to reproductive of environmental genes in zebrafishThrough manipulating environmental factors (for example: temperature, salinity and pheromone) in zebrafish, it proved that hatch rate of oosperm is the highest under the circumstance of 25-29℃( temperature) and 1‰～3‰(salinity). The groping study for pheromone reveals that one component of methyltestosterone may be the pheromone of male zebra fish while one component of medroxypogesterone acetate may be the pheromone of female zebra fish. |
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