Studies On Reproductive Physiological Characterstics And Molecular Cloning Of CGnRH-Ⅱ In Tongue Sole, Cynoglossus Semilaevis G(?)nther

The physiological characteristics, sex steroids (17β-estrodial, testosterone) variation during annual ovary maturation cycle of captive Cynoglossus semilaevis Günther were studied by using light microscopy, morphometric methods and Immuno-histo-chemical localization. The physiological roles of sex steroids and its receptors in gonadal sex differentiation were also investigated as well as the enhancement of spermiation in males induced by exogenous hormones. In addition, cGnRH-Ⅱin the brain was cloning and analyzed. The results are as follows: 1. Study on physiological characteristics during annual gonadal maturation cycle of Cynoglossus semilaevis GüntherThe temporal dynamics of oocyte growth, ovary development were examined during a 12 month ovarian maturation cycle in captive Cynoglossus semilaevis Günther using light microscopy and morphometric methods. Results showed that the oocyte growth could be divided into 6 phases, and the ovary maturation cycle was divided into 6 stages accordingly. In addition, two or three distinct batches of vitellogenic oocytes coexisted in Cynoglossus semilaevisovary at one development stage, indicating that this species is a non-synchronized multiple batch spawners. The ovaries are in stageⅠwhen the fish are less than 6 months old and only appears once in their life.As fish grew, the ovaries enter into stageⅡwhen they are 9 to 12 months old. The stageⅢwhich were characterised by cortical alveoli accumulation was attained when the fish were 12 months old and maintained until 24 months old. Deposition of lipids and vitellogenesis began to happen in ovaries of stageⅣwhen the fish was over 3 years old. The ovaries could mature (stageⅤ) under artificial photothermal control from May to August and fish spawned between September and November. Hydration happened and observed in oocytes indicating that they were completely matured for ovulation. After spawning period, the ovaries retrogressed to stageⅢvia stageⅥ, then continued regressed to stageⅡand maintained until next reproductive season. Gonadosomatic index (GSI), hepatosomatic index (HSI) and condition factor (CF) were all found to increase prior to, or during the peak phase of vitellogenic growth corresponding to the gonad development, which are important indices for evaluating the reproductive condition of the parental fish. Cynoglossus semilaevis Günther appears to strategically utilize episodes of high feeding activity to accrue energy reserves early in the reproductive cycle prior to its deployment during periods of rapid ovarian growth. In the developing ovaries (mainly after stageⅣ), some post-vitellogenic follicles failed to undergo final maturation, resulting in widespread preovulatory atresia, which may depress the fecundity or be an indicator of capacity of multiple spawns.2. Relationships between serum sex steroids levels, gonadal development and photothermal regulation during the annual maturation of captive Cynoglossus semilaevis GüntherThe annual serum sex steroids (17β-estrodial, testosterone) expression and gonadal development parameters [gonadosomatic index (GSI), hepatosomatic index (HSI) and condition factor (CF)] of Cynoglossus semilaevis Günther were studied during annual maturation cycle. In addition, the relationships between GSI, CF, HSI, sex steroids and temperature and photoperiod were investigated using statistical analysis. The serum estradiol level increased in June (stageⅢ) and peaked in August (stageⅣ, P<0.05), just before spawning. After the spawning, it dropped to a relative low level, and back to the lowest level after November until May. The T level ascended in July and peaked in September (P<0.05), a month later than that of estrodiol. During the year round development, the serum estrodiol level was always higher than testosterone; and a positive relationship was found between them (r = 0.733, P<0.05). The E₂/T ratio increased in November, and peaked in next January, then decreased to low level until October. Temperature imposed significant effect on E₂ and T levels and the positive relationship between them; while photoperiod showed no significant effect (r<0.3, P>0.05). These results highlight the temperature may be the primary environmental cue to activate BPG axis. In addition, E₂ levels were positively correlated with the HSI values, but negatively with the CF values. Moreover, the T level was significantly correlated with the HSI values. In conclusion, based on the relationship between sex steroids and gonadal development, the sex steroids level could serve as a useful tool to determine the timing for hormone induced-spawning in farmed female C. semilaevis.3. Studyies on immunohistochemical location and possible roles of E₂, ERαand AR in gonad differentiation and development of tongue soleImmuno-histo-chemical localization of 17β-estradiol (E₂), receptors of testosterone (AR) and esrogenα(ERα) were investigated for the first time in the gonads during differentiation and development of Cynoglossus semilaevis Günther using polyclonal antibodies. The results indicated that E₂, AR and ERαcoexisted in the primary gonad before differentiation. Oogonia, oocytes, spermatogonia, primary spermatocytes and secondary spermatocytes all showed positive reaction to E₂, AR and ERαantibodies, whereas in spermatid and spermatozoa, E₂ and its classical receptor ERαwere not expressed expecr for AR. AR existed and strongly expressed in the gonad at all different differentiation and developmental stages, which indicated that sex steroid hormone may regulate the maturation of gametes through paracrine way. The sex steroid hormone and their receptors were expressed in cytomembrane, cytoplasm, nuclear membrane or nuclear plasm at different developmental stages with different intensity respectively, which showed their special functional characters, these results make it clear that E₂, AR and ERαmay physiologically functioned differently at different developmental stages of gonad. 4. Effects of exogenous hormones on enhancement of spermiation in male Cynoglossus semilaevisThe effects on exogenous hormones including salmon gonadotropin releasing hormone analogue (sGnRHa) and Luteinizing Hormone Releasing Hormone( LHRH) on enhancement of spermiation in male Cynoglossus semilaevis were investigated in the present study. Results showed that exogenous hormone can greatly improve the milt volume, sperm motility. Meanwhile, the serum sex steroids level elevated and remained at a relatively higher level after the exogenous hormone treatment. The sGnRH induction showed better effects. This study can provide useful information on male maturation cycle and be beneficial for artificial insemination in Cynoglossus semilaevis culture.5. Molecuar Cloning of cDNA encoding cGnRH-Ⅱin the brain of Cynoglossus semilaevis GüntherThe total RNA was extracted from tongue solebrain.The GnRH cDNA was amplifled by RT-PCR method using isolated total RNA as template.The cGnRH-ⅡcDNA is 566bp in length that contains an open reading fram (ORF) of 258bp,which encodes 85 amino acids residues , a GnRH decapeptide, a GnRH-associated peptide(GAP) linked to GnRH decapeptide by the cleavage site Gly-Lys-Arg sequence.The cGnRH cDNA encoded the GAP of 49 amino acid residues.The sequence structure of amino acids precursor encoded by cGnRH-ⅡcDNA is identical to all others species reported to date.The result showed that the coding region of cGnRH-ⅡcDNA are highly conserved and the untranslated regions are markely divergent in nucleotide sequence in the listed species.Sequence and phylogenic analyses showed that the putative cGnRH-Ⅱprecursor of tongue sole shared over 88% ofidentity with that of perciformes, and shared 85.9% of similarity with pleuronectiformes. It showed that cGnRH-Ⅱwas highly conserved during evolution of fish. When compared with amphibians, the similarity dropped to to 56.6%, wheareas the similarity dramatically dropped to less than 25% when compared with birds and mammals. These results showed that cGnRH-Ⅱof tongue sole may evolve faster that that of birds and mammals or evolve divergently. Phylogenetic analysis revealed that the cGnRH-Ⅱprecursor of tongue sole clustered into one clade with those from pleuronectiformes, perciformes and Cypriniformes.