| Half-smooth tongue sole (Cynoglossus semilaevis Günther) is a Chinese inshore species with great potential for aquaculture. This paper mainly study on cultured male Cynoglossus semilaevis Günther, from which the testis, sexual steroid hormone of blood plasma and their receptors are taken and studied by the methods of histological staining, immunohistochemical techniques, radioimmunoassay and molecular methods in details. The main results and conclusions are as follows:1. Histological study on the development and annual change in the testis of cultured male Cynoglossus semilaevis Günther.Cynoglossus semilaevis Günther has a pair of testical. Like other teleosts, the testitis of Cynoglossus semilaevis Günther belong to lobular type, but has unconspicuous spermatogenic cyst. Male Cynoglossus semilaevis Günther has a seminal vesicle, which is the same length with testis, extended forward from the back out of the testis. Several cavities which is full of mature sperms may be observed in the seminal vesicle, they are surrounded by smooth muscle and connective tissue. The efferent duct together the side of the testis, and mature sperm can transfer from the efferent duct to seminal vesicle.Sperm experiences 5 typical steps during its life: spermatogonium, spermatocyte, secondary spermatocyte, spoermatid, sperm, however, sperm cells in the same testis may not be synchronized. Cultured Cynoglossus semilaevis Günther's testis develop later than those who lived in wild. From the beginning of October the testicle start to breed, and it last to February, March to June is convalescent period, after which the sperm happens.2. Immunohistochemical study on the testis of Cynoglossus semilaevis Günther including AR, ERαand ERβ.2.1 Immunohistochemical study on ERαin testis and sperm cells The estrogen receptorα(ERα) expresses at spermatogonium, spermatocyte, spermid and space between folioles in each period of development, but the nucleolus of spermatogonium, karyon of spermatocyte and sperm assume immunonegative reaction to ERα.2.2 Immunohistochemical study on ERβin testis and sperm cellsThere are ERβimmunopositive reaction at spermatogonium, spermatogonium, spermid and space between folioles in each period of development, but the nucleolus of spermatogonium and sperm are immunonegative to ERβ.2.3 Immunohistochemical study on AR in testis and sperm cellsVaring levels of immunopositive reaction are found at spermatogonium, spermatogonium, spermid and space between folioles in each period of development. These results indicate that ER and AR play an important role in the multiplication and development of germ cell, as well as a proof of the important function of sexual steroid hormone in testis growth and matureness.3. Cloning and expression analysis of male Cynoglossus semilaevis Günther's androgen receptor (AR) genePartial androgen receptor (AR) gene (434bp in length) has been cloned from Cynoglossus semilaevis. Tissue expression analysis showed that AR mRNA expressed in all eleven tissues including testis, liver, stomach, spleen, kidney, head kidney, intestines, gill, heart, brain and muscle, the highest expression level was in kidney and the lowest in gill, however.Plasma testosterone (T)'s seasonal changes is detected by radio immunoassay (RIA).The result shows that the plasma testosterone significantly change following the development o f the testis in a reproductive period. The peak of a year is between February and April, then it descends and touches the lowest point in November.It is the fist study to analyze the seasonal expression change of AR mRNA in testis, liver, spleen, kidney and brain. The results show that AR gene expression change seasonally in the five tissues. AR'expression and distribution in Cynoglossus semilaevis can be opened out by plasma testosterone, immunohistochemical study and RT-PCR methods. 4. Cloning and expression analysis of male Cynoglossus semilaevis Günther's estrogen receptorα(ERα) genePartial estrogen receptorα(ERα) gene (454bp in length) has been cloned from Cynoglossus semilaevis. Tissue expression analysis showed that ERαmRNA expressed in all tissues excluding gill and muscle, including testis, liver, stomach, spleen, kidney, head kidney, intestines, heart and brain, the highest expression level was in liver and the lowest in head kidney, however.Plasma 17β-Estradiol (E2)'s seasonal changes is detected by radio immunoassay (RIA).The result shows that the plasma testosterone significantly change following the development o f the testis in a reproductive period. The peak of a year is between August and October, then it significantly descends and touches the lowest point, it ascends when May comes.It is the fist study to analyze the seasonal expression change of AR mRNA in testis, liver, spleen, kidney and brain by RT-PCR method. The results show that AR gene expression change seasonally in the five tissues. The trend in testis, liver and spleen is similar to HSI curve, but contrary to GSI and E2 curve.5. Cloning and expression analysis of male Cynoglossus semilaevis Günther's estrogen receptorβ(ERβ) genePartial estrogen receptorβ(ERβ) gene (525bp in length) has been cloned from Cynoglossus semilaevis. Tissue expression analysis showed that ERβmRNA expressed in all eleven tissues including testis, liver, stomach, spleen, kidney, head kidney, intestines, heart, gill, muscle and brain, the highest expression level was in liver and the lowest in head kidney, however.The seasonal analysis for ERβgene expression in testis, liver, spleen, kidney and brain show that the ERβgene expression is similar to the ERα's. |
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