| The medaka，Oryzias latipes，it is popuiar as a pet fish.because ofit’s prolifical reproducatio short reproductive cycle and large, hightransparency, easy to identify fertilization of the eggs and the embryo isfast development, sensitive to water quality, has proven to be an excellentexperimental vertebrate model system for Developmental biology,molecular biology and toxicology.it’s a perfect material to Study theproliferation of germ cell, the genetic mutations and diseases,and thetoxicity on medaka. To get more detailed information of sexdetermination and control in medakaof. In this study, firstly we use aspecific primer to identify the genetic sex of medaka; second, throughparaffin section and hematoxylin-eosin staining, Observated thehistological sexual diferentiation and development of gonad; thirdly,combined with results of sexual diferentiation and development of gonad,we analyse the relation between the different expression of DMY andDmrt1based on RT-QPCR and the sex determination. The main resultswere as follows:1. Now,it has been many reports about the identify the genetic sex ofmedaka,however, the existing methords all needed two primer pairs.We used this primer, designed by Jawahar, permitting gender identificationaccurately of both immature and adult fish. Howover，using this primer,we optimized a different result from the previous (males had two bands,one diagnostic band of933bp, another was1906bp; female only yield aspecial diagnostic band of1906bp), after analysised, we believed that ourresults are more accurate.This technique will be useful in both ecologicaland biomedical researches that employ medaka and rely on geneticsexing.2. In this study, Medaka gonad developmental process from the earliestrecognizable stage, through differentiation, to maturation, was observedunder light microscope after paraffin section and hematoxylin-eosinstaining. The results showed that Gonads could be observed on the rightside of dorsal abdominal cavity of5-10days’juvenil. In which germ cellswere significantly bigger than the surrounding somatic cells; At10dayafter hatching (dah), clear sex differences were observed between femaleand male individuals, germline cysts derived from oogonium divisioncould be seen in the gonads of10dah.Subsequently, we observed theoogenesis and spermatogenesis in female and male individuals; In50dah,we observed the ovarian cavity and mature sperm firstly. In addition, wealso observed that two sex characteristics coexisted in a male individualin the process of development. These observations will provide valuableinformation and precious basic materials to the developmental biology and Genetics Biology.3. Now, it is popular that the DMY gene is a "switch" of sexdetermination in medaka；Another "stars"about the sex-control gene isDMER1, both of two genes are controling factors of male sexdevelopment and differentiation in Medaka.However, until now,itremains unclear how DMY controls XY gonads sex determination anddifferentiation,and we also don’t know howDmrt1regulated the male sexdetermination and differentiation.In this study, combined with results ofsexual diferentiation and development of gonad, we analyse the relationbetween the different expression of DMY and Dmrt1based on RT-QPCRand the sex determination. We found that,as for DMYin male,basallevels of expression were detectable until20daf, the expression levelsdrastic increased,when the sex differentiation begun,(20day afterfertilization) the expression levels did not differ between XX and XYindividuals;forDmrt1, in XY male,the expression levels alsoincreased,although the expression levels was not so obvious as DMY.Inaddition,before the sex differention, the differention of Dmrt1expressionis not obvious.until the spermatogenesis begun, the expression levels ofDmrt1was drastic increased. All the evdances suggest that the DMY genemay induce the development and differentiation of the male testes;Addition to the regulation spermatogenesis,the Dmrt1gene may alsoparticipate in the formation of embryonic organs in medaka. |
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