| The Hypothalamus-Pituitary-Gonadal axis is an important regulatory axis in the neuroendocrine system of fish.Gonadotropin-releasing hormone(GnRH)is secreted by the hypothalamus,stimulating the pituitary gland to secrete luteinizing hormone(LH)and follicle stimulating hormone(FSH).Therefore,the key elements of the signaling path of the LH and FSH receptor are activated.They act on the gonadal sex steroid hormone synthesis to promote gonadal maturation and lead to discharge of sperms and eggs.In this study,red crucian carp,triploids,alltetraploid,blunt snout bream,triploid hybrids,tetraploid hybrids as experimental materials were used,and it was the first time that gene cloning,Western Blot and RT-PCR technique have been used to comparatively analyze the expression difference among GnRH2,LH?,FSH?.This paper outlines the main results.The main results we obtained can be presented as follows:(1)The intron sequences of GnRH2 were cloned and analyzed in the different ploidy fishes.Differences and similarities in GnRH2 intron sequences among these different ploidy fishes were found.The length of the second intron sequences are different:the carp was 545bp,the diploid red crucian carp was 422bp,the triploids was 837bp and the allotetraploid was 837bp;the length of the third intron sequences were also different:the carp was 11 Obp,the diploid red crucian carp was 120bp,the triploids was 128bp and the allotetraploid was 128bp.However,the similarities that both of the intron sequences were start with "GT".and end with"AG".In addition,it is presented that offsprings are inherited partial gene sequences from their parents and are inserted a certain new nucleotide sequences by the comparison of their parents.Also it is speculated that a certain degree of gene recombination is observed.Consequently,offsprings not only inherit the good traits of their parents but also.produce various heterosis.(2)The expression of GnRH2 in different tissues of different ploidy fishes was comparative studied.From the aspect of mRNA and protein level,GnRH2,which was expressed in midbrain and pituitary in different ploidy fishes presented that the expression in the midbrain was higher than that in the pituitary.It is suggested that gene.transcription and translation primarily present in the midbrain tissue and plays a role in passing signal in the GnRH transduction pathway which involved in the regulation of other downstream factor gonadotropic hormone.By western blot analyzing,the highest expression level of GnRH2 was in tetraploids,with the lowest in triploids in the breeding season,however,that of triploids was the highest in the non-breeding season.It can be speculated that the abnormal regulation of GnRH signal due to presence of some kind of downstream negative signal paths inhibit that the expression of GnRH2 and results in sexual gland is abnormal in triploids that is sterile.(3)The expression of GnRH2 in different tissues of different ploidy fishes was comparative studied.From the point of view of mRNA and protein level,the expression of GnRH2 was higher in the midbrain than in the pituitary from different ploidy fishes.It is suggested that gene transcription and translation are primarily present in the midbrain tissue.By using western blot and RT-PCR analysis,the expression level of GnRH2 is highest in tetraploids and lowest in triploids during the breeding season.On the contrary,the expression level of GnRH2 in triploids is the highest during the non-breeding season.It is indicated that the abnormal regulation of GnRH signal exist due to some kind of downstream negative signal paths which inhibit the expression of GnRH2,so the triploids are sterile.(4)The expression of LH? in different tissues and developmental stages in the allotetraploid was studied.From the point of view of mRNA and protein level LH? as expressed only in the pituitary gland of tetraploid,however,almost no expression in other tissues.It is suggested that LH is mainly secreted in the pituitary GTH cells.In addition,from the aspect of mRNA level,LH? was variously expressed at different developmental stages of tetraploid.With fish aged,the amount of LH?expression was increased.It can be indicated that LH? plays an important role in the development of gametes and final maturation process of gonadal.Furthermore,LH? was expressed in the early stages,which related in tetraploids sexual maturity in advance.(5)The expression of FSH? in different tissues and developmental stages in the allotetraploid was studied.From the aspect of mRNA and protein level,FSH? was variously expressed in different developmental stages of tetraploid.It can be indicated that FSH? plays an important role in the development of gametes and final maturation process of gonadal.In addition,FSH? was expressed in the early stages of sexual maturation,suggested that it was related to the sex-maturity age advanced oftetraploids.In conclusion,this study indicates that the expression of GnRH2,LH?,FSH? in the different ploidy fishes are related to the fertility of tetraploids and the sterility of triploids.It is possible to prove that related genes of HPG axis play a critical role in reproductive development and regulation of the reproductive process in fish.This study helps us take further to explore the functions of gene in lower-class vertebrate animal on the basis of molecular biology. |
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