Study On Differentiation Of Gonad And The Characterization Of Mtnrs,kiss/kissr And GnRH During Breading Season Of Turbot(Scophthalmus Maximus)

As a flatfish,the turbot(Scophthalmus maximus)is one of the most important farmed fish species with great commercial value.The turbot was firstly introduced to China at the year of 1992.However,in recent years,the efficiency of aquaculture has been brought down for the reccession of genetic resource for the rapid development of the industry.In this study,we investigated the formation and differentiation of turbot gonad and evaluated the localizations of expressions and the temporal transcription patterns of melatonin receptors,kiss/kissr and GnRH throughout their reproductive cycle.This study will provide basic knowledge in the regulation of reproductive endocrine and artificial reproduction.(1)Turbot larvae were reared at the temputer of 18-19 oC.The Histology showed that the primordial germ cells(PGCs)underwent a migration after embryo development,and PGCs arrived at genital ridge at 15 dph.The primary gonad of turbot formed by the PGCs and the aggregation of somatic cells at 20 dph.The cytological and morphological gonadal differentiation was observed at 36 dph.One type of gonad showed lance-shape with fewer germ cells,while the other type showed club-like shape with larger amount of germ cells.According to the morphology of gonad and the proliferation of germ cells,we speculated the gonad with lance-shape could develop into testes,and the gonad with club-like shape could develop into ovary.At about 60 dph,with the presentation of ovarian cavity,the differentiation of ovary finished.About 90 dph,spermatogonial cysts appeared and the differentiation of testes finished.(2)The full-length cDNA of turbot lymphocyte antigen 75(smly75)homologue were cloned.In situ hybridization,smly75 mRNA were exclusively detected in A-SG.Thus,the smly75 could be a new and convincing molecular marker on identification of turbot A-SG.Through histology,we found that testes from 8 to 14 month old males contained high rate of A-SG.And the smly75 expressed highly at these gonadal development stages.Thus,testes of 8 to 14 month old males could be used for further isolation and purification of type A-SG.(3)Three forms turbot Melatonin receptor(Mtnr)genes: Mtnr1,Mtnr2 and Mtnr1 c were cloned and sequenced by isolating the cDNAs.The expression patterns by qPCR indicated Mtnr mRNAs were all highly expressed in brain and the expression of Mtnr1 and Mtnr2 were relatively higher than Mtnr1 c.Meanwhile,in hypothalamus,Mtnr1 mRNA concentrations were significantly higher than Mtnr2.With the gonadal development throughout the reproductive cycle(II-VI),the expression of Mtnr1 gradually dreased and reached the lowest level at mature stage(V).Therefore,Mtnr1 could be play an importment role in the regulation of reproduction.(4)Turbot kiss1,kiss2 and kiss receptor(kissr)cDNA were obtained.Multiple alignments of deduced amino acid indicated that turbot kissr and Japanese flounder(Paralichthys olivaceus)kissr4 are homologous.The results of qPCR showed that kiss1,kiss2 and kissr were all strongly expressed in brain,and kiss2 mRNA was restricted to hypothalamus.Meanwhile,kiss2 and kissr mRNA were significantly higher than kiss1 in hypothalamus.Through the reproductive cycle,the expression of kiss2 and kissr in brain showed a variation of decreasing firstly and then increasing.Therefore,turbot kiss2 and kissr could be involved in the reproductive activity and gonadal development.(5)Three forms turbot GnRH genes: sbGnRH,cGnRH-II,and sGnRH were cloned and sequenced by isolating the cDNAs.The expression of three mRNAs by qPCR showed that all the three GnRH mRNAs were highly expressed in brain,and sbGnRH and sGnRH mRNAs increased during the immature stage and reached the highest level at breading stage(V),then decreased at the degradation stage.Moreover,the expression of sbGnRH was significant higher than sGnRH.All the results indicated the critical role of sbGnRH on the regulation of GtH and sGnRH functionally compensates for sbGnRH.In addition,the evaluation of Mtnr1,kiss2/kissr and sbGnRH throughout the reproductive cycle showed that,at breading stage(V),the expression of Mtnr1 and kiss2/kissr reached to the lowest level,which was opposite with the expression of sbGnRH.Meanwhile,in consideration of the variation of serum hormones concentrations of melatonin,GnRH,FSH and LH in male and female turbot,it is suggested that melatonin could induce the mature of gonad and regulate the reproduction by driving the expression of Mtnr1,kiss2/kissr down to elevate the sbGnRH mRNA.