Studies On Fertility And Expression Of Related Genes Of HPG Axis In Autotetraploid Fish Derived From Distant Hybridization

Distant hybridization can increase the genetic variation of offspring,and the offspring with different ploidy can be obtained by this method.The autotetraploid fish（4n=200）was obtained by distant hybridization of red crucian carp Carassius auratus red var.（♀,2n=100）×blunt snout bream Megalobrama amblycephala（♂,2n=48）.Until now,the F₂^F₁₃generations of the autotetraploid fish were formed by self-crossing.This is the first report of fertile autotetraploid fish in the world.The gonadal development and gamete maturation in fishes are mainly regulated by the nervous and endocrine system consisting of the hypothalamic-pituitary-gonadaxis（HPG）.Gonadotropin-releasing hormone（GnRH）,gonadotropin hormone（GtH）and gonadotropin receptor（GTHR）are the pivotal signal molecules of the HPG axis,and participate in the entire regulatory process to promote gonadal development and maturation.Autotetraploid fish,which reach sexual maturity at 1 year of age,were derived from the whole genome duplication of red crucian carp.This study is the first to analyze the fertility of autotetraploid fish and the molecular and expression characteristics of HPG axis related genes（Gnrh2,Fshβ,Lhβ,Fshr,Lhr）by using histology,molecular biology and immunofluorescence.The main contents and results are as follows:1.The histological of pituitary structures and gonadal structures at different development stages of the autotetraploid fish and red crucian carp were comparatively analyzed.The results showed that the pituitary and gonad of the autotetraploid fish developed normally,and the autotetraploid fish was basically synchronized with the development of the maternal ancestral red crucian carp.This study revealed that the autotetraploid fish possessed a good fertility basis at the cellular level.2.The cDNA partial sequence cloning and analysis of Gnrh2,Fshβ,Lhβ,Fshr and Lhr genes showed that the similarity of Fshβ,Lhβand Fshr genes coding sequence in the two different fishes was 100%,while the Gnrh2 and Lhr genes have significant base site mutations.This study indicated that the coding sequences of Fshβ,Lhβand Fshr genes are highly conserved in autotetraploid fish and red crucian carp,while Gnrh2 and Lhr genes show obvious gene structure differentiation after autotetraploidization.This change may provide a material basis for diploid-like chromosome pairing,which is conducive to the recovery of fertility.3.The expression of Gnrh2,Fshβ,Lhβ,Fshr,Lhr mRNA in various tissues of autotetraploid fish had determined by RT-PCR.The results revealed that the Gnrh2 gene was not only expressed in the brains but also consistently detected in the pituitaries and gonads.The expression of Fshβand Lhβwas only detected in the pituitaries,and Fshr and Lhr exhibited high expression in gonads.The results in autotetraploid fish were the same as those in red crucian carp.The expressions of Gnrh2,Fshβ,Lhβ,Fshr,Lhr genes in the breeding season and non-breeding season of the autotetraploid fish and red crucian carp were compared and analyzed by Real-time PCR.It was found that the expression of the five target genes was higher for autotetraploid fish than for red crucian carp.The results showed that after autotetraploidization,the main expression characteristics of the five genes did not change significantly,but the level of gene expression was significantly different,and the change of the expression level may be related to the fertility recovery of the autotetraploid fish.4.The localization of Fshr and Lhr in the gonads of autotetraploid fish and red crucian carp was observed by immunofluorescence.Fshr and Lhr were detected in the granulosa cells,follicular cells,Sertoli cells and Leydig cells.The results of Fshr and Lhr in autotetraploid fish were consistent with those of red crucian carp,further indicating that there was no significant change in the expression characteristics of reproductive development-related genes Fshr and Lhr in autotetraploid fish after autotetraploidization.