| The formation of allotetraploid population derived from the red crucian carps(Carassius auratus red var.,♀,2n=100) and common carps (Cyprinus carpio L.,♂,2n=100) played an important role in biological evolution and fish breeding.The sterile triploid crucian carp was produced by mating the males of allotetraploids with the females of Japanese crucian carp(Carassius cuvieri).Both tetraploids and triploids provided a biological platform for the comparative studies of different ploidy level fishes.In fishes,reproductive activities are primarily controlled by an endocrine system consisting of the brain-pituitary-gonad axis.Gonadotropin-releasing hormone(GnRH) produced in the brain stimulates the production and release of gonadotropin(GTH) in the pituitary.Then GTH acts on the receptors of gonads to stimulate the production of sex steroids,which regulate gonadal development.The pituitary is an important endocrine organ,which plays an important role in regulating the secretion of hormones related to the growth and development.In this paper,we firstly observed the histological and ultra-structure of endocrine cells in different ploidy fishes,and comparatively analyzed the molecular and expression characterizations of Gnrh,Gth and Gthr genes in different ploidy fishes.The major results were presented as follows:1.The histological structure and the endocrine cells' size of pituitary in different ploidy fishes were comparatively studied.For each type of endocrine cells,the cell size was increased gradually with the increasing ploidy level.In breeding season,the proportion of gonadotropin cells (GTH) in tetraploids was the highest,while that in triploids was the lowest.It might be related to the earlier sexual maturity of tetraploids and sterility of triploids.However,the proportion of somatotropin(STH) cells in triploids was the highest,whereas that in tetraploids was the lowest.It might be connected with the faster growth rate of triploids and slower growth rate of tetraploids.2.The ultra-structures of GTH and STH cells in different ploidy fishes were observed.These two types of endocrine cells in diploids and tetraploids had normal biological activities.But the number of endocrine particles in GTH cells of triploids was fewer than that of diploids and tetraploids within breeding season,and only a few cavities could be observed after breeding season.It presumed that some factors restrained the releasing of endocrine particles,which then led to the sterility.3.The full lengths of Gnrh2 cDNAs in different ploidy fishes were obtained and their sequences were comparatively analyzed.It was indicated that Gnrh2 gene had high homology among the cyprinids.The results of amino acid sequences among different ploidy fishes also revealed that the GnRH2 decapeptide and proteolytic cleavage site were absolutely consistent.The differences in signal peptide and GAP between the diploids and tetraploids were fewer than those between diploids and triploids.The possible reason was that the red crucian carp was the original maternal parent of tetraploids,so they had closer relationship.4.The expression patterns of Gnrh2 in different ploidy fishes were comparatively studied.In these three kinds of fishes,Gnrh2 was not only expressed in midbrains,but also expressed in pituitaries and gonads.And Gnrh2 mRNA was mainly located in posterior end of midbrain.It was indicated that Gnrh2 might mainly act as the neurotransmitter and neuromodulator,and it also might participate in the regulating of other hormones releasing,such as GTH and STH.In addition,the expression level of Gnrh2 was the highest in tetraploids and the lowest in triploids within the breeding season,while that of triploids was the highest and that of tetraploids was the lowest after breeding season.It might be related to the abnormal gonadal development of triploids within breeding season and the Gnrh2 expression might be less down-regulated after breeding season.5.The full length of Fshβand LhβcDNAs in different ploidy fishes were obtained and the sequence homology was then analyzed.Both of the two genes had high homology among the cyprinids.In these three fishes, Fshβhad 13 cysteine residues,12 of which formed 6 conserved disulfide bridges by utilizing the cleavage sites between first and second cysteine residue.In addition,the lack of second glycosylation sites in Fshβof these three fishes might influence the spacial structure and biological activities.6.The expression patterns of Fshβand Lhβin different ploidy fishes were comparatively studied.Both of these two genes only expressed in the pituitaries of these three fishes,and they mainly located in the meso-adenohypophysis, which indicated that the gonadotropins were secreted by GTH cells in meso-adenohypophysis.Within breeding season,the Lhβgene in three fishes had higher expression level and wider distribution range than that of Fshβ,which suggested that Lhβwas important for inducing final gonadal maturity.In addition,Fshβand Lhβexpression levels in tetraploids were a little higher than that of diploids for its earlier sexual maturation.While Fshβand Lhβin triploids had higher expression levels than in other two fishes after breeding season for restraining by the feedbake regulation of downstream factors.7.The expression patterns of Fshr and Lhr in different ploidy fishes were comparatively studied.Both of these two genes only expressed in the ovaries and testis of three fishes,and they mainly located in the follicle cells,granulose cells,outside layer of zona radiate,Sertoli cells and Leydig cells.It was indicated that Fshr and Lhr gene might stimulate the development and maturation of gonads.However,the hybridization signals of Fshr and Lhr gene in triploids were less than other two fishes. It was suggested that the triploids had fewer GTHR on target cell surfaces which couldn't interacting with the GTH sufficiently,so the gonad could not produce enough sex steroids and finally led to sterility.In a word,all the results indicated that the differences of the structure of pituitary endocrine cells and the expression of Gnrh,Gth and Gthr genes in different ploidy fishes were related to the sterility of triploids and fertility of tetraploids.It also provided cytological and molecular evidences for the endocrine mechanism of the fertile tetraploids and sterile triploids.
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