Studies On Cloning And Expression Of Inhibin And Activin Subunit In Different Ploidy Cyprinid Fishes

The allotetraploid population was successfully produced by distant hybridization of red crucian carp(Carassius auratus red var.)(2n=100,♀)×common carp(Cyprinus carpio L.)(2n=100,♂).The sterile allotriploid crucian carp was produced by mating the males of improved allotetraploids with the females of red crucian carps(Xiangyun crucian carp II).The gonadal developmental mechanism of vertebrates has been a hot topic in the field of biology over the years.Some researches on inhibin and activin related to the gonadal development of teleosts have been reported,but fewer studies have been reported about these in different ploidy fishes.Studies have shown that inhibin and activin are involved in many physiological processes,especially in the reproductive process.Inhibin and Activin involvement in the regulation of the hypothalamic-pituitary-gonadal(HPG)axis.Within the reproductive system,inhibin and activin have been found to regulate the release of gonadotropin releasing hormone(GnRH)from the hypothalamus and the expression of follicle stimulating hormone(FSH)and gonadotropin releasing hormone receptor(Gn RHR)from the pituitary.In ovaries,they can regulate the development of follicular and maturation of the oocyte.The sterile allotriploid crucian carp provide a good experimental material for studying the function and mechanism of fertility.The main contents and results are as follows:1.The full lengths of inhibinαcDNAs in diploid red crucian carp and allotriploid crucian carp were obtained and their sequences were comparatively analyzed.The full-length sequence in diploid red crucian carp is 1642 bp including a 5’untranslated region(UTR)of 170 bp,a 3’UTR of 428 bp,and an ORF of 1044 bp that encodes proteins of 347amino acids.The full-length sequence in allotriploid crucian carp is 1632bp,which consists of a 5’untranslated region(UTR)of 165 bp,a 3’UTR of 423 bp,and an ORF of 1044 bp that encodes proteins of 347 amino acids.2.The expressions of inhibinαmRNA in different tissues of diploid and allotriploid fish were then analyzed by Real-time PCR.The result showed inhibinαis high expressions in the gonad.We speculated the main function of Inhibin is related to the reproductive process of fish,which is consistent with previous research on mammalian and zebrafish.Then the expressions of the ovary in diploid red crucian carp and allotriploid crucian carp were detected,which showed that inhibinαwas elevated expressed in the ovary of allotriploids.The change of the expression level may be related to the sterility of allotriploids.In the ovary,elevated expression of inhibinαin allotriploids may inhibit the gonad development by blocking the effect of Activin,resulting in the sterility of allotriploids.3.Based on the full-length cDNA of activinβ_A and activinβ_B genes in different ploidy crucian carp have been obtained at the earlier study,we researched the expression of activinβ_A and activinβ_B genes in different tissues from diploid and allotriploid fish.The result showed activinβ_A and activinβ_B were widely expressed in different tissues,which suggested that Activin acted as an autocrine and paracrine modulator.When we detected the expressions of activinβ_A and activinβ_B in diploid red crucian carp and allotriploid crucian carp and different period of development,we found activinβ_A and activinβ_B were expressed differently in the pituitaries.Lower activinβ_A expression was observed in the pituitary of diploid fish during both both the non-breeding and breeding seasons.However,activinβ_B expression was higher in diploid fish.According to the previous studies,we speculated that activinβ_A andβ_B may be differentially regulated in the pituitary and play different roles.In the ovary,activinβ_A and activinβ_B were elevated expressed of allotriploids during both periods.According to the result,inhibinαwas elevated expressed in the ovary of allotriploids,we speculated that inhibinαcan affect the formation of activin dimer by competitive binding activinβ_A and activinβ_B and suppress the development of gonad.4.The immunohistochemistry of Activinβ_A andβ_B were analyzed in ovaries of diploid and allotriploid fish and different period of development,which indicated that Activinβ_A andβ_B mainly located at the granulosa cells.Besides,we found Activinβ_A andβ_B were strongly expressed in the small oogonium-like cells of allotriploid.In this study,we analyzed the expressions of inhibin and activin in fertile diploid red crucian carp and sterile allotriploid crucian carp,which further revealed the molecular mechanism of reproductive regulation in allotriploid crucian carp and provided a theoretical basis for the research on fish reproductive physiology and breeding.