Studys On Physiological Function Of Gonadotropins In Reproductive Cycle Of The Female Cynoglossus Semilaevis Günther

We take cultured Cynoglossus semilaevis Günther as the experiment fish and researchon its ovary development, GtH related genes cloning, and the expression level of GtHhormone by means of histological methods, Endocrinology method, radio immunoassayand molecular biology, in order to interpret the physiological function of GtH in further.And the result may be valuable to the improving of the industrial aquacultured fish’sreproductive regulation. The main researches and contribution are as follows:1. Cloning and mRNA spatio-temporal expression pattern of common glycoprotein αsubunit in half smooth tongue sole Cynoglossus semilaevis GüntherFull-length cDNA encoding CGα is firstly cloned from half smooth tongue sole C.Günther by means of RACE. The whole cDNA length of tongue sole CGα gene is685bp, consisting of a384bp open reading frame (ORF). The predicted mature tonguesole CGα consisted of127amino acids preceded by a signal peptide of33residues. Thestructure of mature CGα in tongue sole CGα was similar with those in the vertebrateCGα, including10conserved Cysteine residues and2N-linked glycosylation sites.Sequence comparison shows that tongue sole CGα mature peptide has the highestidentity to their orthologs of percomorphs (perciforms and pleuronectiforms;60-70%identity).The CGα mRNA expression characterization in tissues of tongue sole is determinedby quantitative Real-time PCR (RT-PCR) method. The mRNA expression of tong soleCGα gene is detected in all the examined tissues. The highest expression level is inpituitary, followed by gill, kidney, muscle, ovary and brain, and the low level in othertissues including heart, head kidney, liver and spleen.A determination in the expression of the genes encoding CGα is an important first step in the understanding of molecular control of the reproductive cycle in fish, and thehighest expression level of CGαmRNA of female C.semilaevis determined by RT-PCRappared on the stage of Ⅳ、Ⅴ.2. Full length cDNA cloning and spatio-temporal expression of FSH from half-smoothtongue sole C. Günther and periodic variation analyze of plasma FSHFSH is cloned by using RT-PCR and RACE technology for the first time. ThecDNA of FSH is541bp, and the open reading frame393bp encoding peptides of130amino acid. One N-glycosylation site is found in the deduced amino acid sequence.Multiple amino acid sequences alignments shows that half-smooth tongue sole FSHmature peptie is most similar to their orthologs of Pleuronectiformes and Perciformeswhich is42-49%identity.Real-time PCR analysis shows that FSH mRNA is expressed with the highest levelin pituitary. Also strong amplification signals are detected in all other tissues, especiallybrain and gonad. FSH mRNA of half-smooth tongue sole expressed widely innon-pituitary tissues may suggest it has the more physiological functions; Adetermination using RT-PCR techonolgy in the expression of FSH mRNA of thereproductive cycle in female C.semilaevis. And the results shows that the highestexpression level of FSH mRNA appares on the stage of Ⅳ.3.Full length cDNA cloning and spatio-temporal expression of LH from half-smoothtongue sole C. Günther and periodic variation analyze of plasma LHThe full length cDNA of LH has been cloned from pituitary of half smooth tonguesole C. Günther using RACE technology for the first time. The cDNA of LH is670bp,and the open reading frame is477bp encoding peptides of158amino acid. Multipleamino acid sequences alignments are performed among vertebrates show that tonguesole LH mature peptie is the most similar to their orthologs of Pleuronectiformes andPerciformes which is58-68%identity. And12conserved cysteine residues and1N-linked glycosylation site are found in the deduced amino acid sequence.Real-time PCR analysis showed that LH mRNA is expressed in all twelve tissues including brain, pituitary, gill, heart, head kidney, kidney, liver, spleen, stomach,intestine, ovary and muscle. The highest expression level is in pituitary. And thestronger amplification signals are detected in the brain and gonad than all othernon-pituitary tissues. It suggest that LH may have the more physiological functionsbecause LH mRNA of tongue sole expressed widely in non-pituitary tissues; Adetermination using RT-PCR techonolgy in the expression of LH mRNA of thereproductive cycle in female C.semilaevis. And the results show that the highestexpression level of LH mRNA appared on the stage of Ⅳ、Ⅴ.4.The determination of the plasma GtH concentration and its changes in C.semilaevisthoughtout an annual reproductive cycleRadio immunoassay (RIA) marked by125I is used to detect the concentration ofplasma GtH in C.semilaevis.The results show that the biggest plasma FSHconcentraction appares on the stage of Ⅳ while LH appares on stageⅤ. FSH and LHhave the same variation trend in C.semilaevis thoughtout an annual reproductive cycle,and with a elevation on September. Statistical analysis show that there is significantpositive correlation between FSH/LH and water temperature, which suggest thatcontrolling warter temperatue produces an effect on C.semilaevis’s reproductiveendocrine mechanism.