Cloning And Expression Characterization Of Dmc1 And Ly75 Gene During Spermatogenesis In Acipenser Dabryanus

Dabry’s sturgeon(Acipenser dabryanus)is endemic to China,which is the pure freshwater settled fish.It was used to be one of economic fish in the upper reaches of the Yangtze River.But in the late of 20 th century,due to overfishing,construction of water conservancy projects,waterway regulation engineering and other human activites,the survival environment of Dabry’s sturgeon was severely damaged,and the population resource was declined rapidly.Dabry’s sturgeon was nearly extinct.In 1988,it was listed as the First Class Protected Animal by the national government of China.Thus,the works about species protection and stock enhancement are urgent to be paid more attention,especially in the field of reproductive biology.With the development of fish germ cell transplantation in the past decade,this technique has been expanded across a variety of teleost,which could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species rehabilitating the stocks in the nature.Recently,the research of germ cells of Dabry’s sturgeon culture in vitro and transplantation had achieved initial progress,but efficient transplantation depends on the in vitro culture of spermatogonial stem cells.We have accomplished research on spermatagonial cells in vitro of Dabry’s sturgeon in early period,but we haven’t done enough for phenotypic characterization on spermatogonia.At present,Dabry’s sturgeon germ cell marker genes which have been identified are dnd and vasa,however they expressed not only in the spermatogonia,but also in spermatocyte.Therefore,it is necessary to identify the germ cells-specific marker,thus laying the foundation for the identification of spermatogonia in vitro.In this study,we identified two germ cell-specfic genes such as dmc1 and ly75.RT-PCR was applied to clone the coding sequence of dmc1 and ly75.The predicted protein secquence and phylogenetic relationships were analyzed by bioinformatics methods.qRT-PCR was used to analyze the expression in various tissues of male and female Dabry’s sturgeon,on the other hand combined with the HE staining results,we explored the gene expression characteristics in different stages of development of testis,we finally confirmed the localization of genes in the testis by the means of in situ hybridization.The main results of this thesis are summarized as the following:1.The HE staining results of testes in 1to 3 years old of male Dabry ’s sturgeon: the stage of 0,the testis had not formed the seminiferous lobule,a few number of primordial germ cells could be found.When testis stayed in the stage of Ⅰ,a large number of type A spermatogonia could be observed as in the stage of spermatogenesis proliferation.With the differentiation of spermatogonia,we could find some type B spermatogonia,but type A spermatogonia occupied most amount in the stage of II,whereas in the stage of III,the testis entered into the meiosis,most cells differerntiated into primary spermatocytes,a little number of secondary spermatocyte could be found,then in the stage of IV,the cells appeared in many types,including a large number of spermatocytes,spermatid and few spermatozoa.The V stage of testis were full of spermatozoa.2.Disrupted meiotic cDNA(dmc1)is a meiotic-specific gene,which encodes a protein required for homologous chromosome synapsis during the prophase of meiosis Ⅰ.In this investigation,we obtained the partial cDNA of Acipenser dabryanus dmc1 from the gonad transcriptome database.RT-PCR was applied to clone the cDNA of dmc1 gene,which contained an ORF of 1029 bp that encoded a protein of 342 amino acid.A comparison of the deduced amino acid sequence of Addmc1 with that of other fishes and tetrapods,it showed that the Dmc1 shared highly identity more than 87% among any species,and they mainly contain two motifs as GEFRTGKT and LLIID which are combined with nucleic acid.RT-PCR analysis revealed that Addmc1 was only detected in the testis and ovary.The expression pattern of dmc1 in testis of Acipenser dabryanus at different stage of development was also studied by Real-time PCR,which showed no expression of Dmc1 at the testis of 0 to Ⅰ stage,increased gradually from the stage II to Ⅳ testis,reached the peak at the stage Ⅳ testis,and decreased dramatically at the stage Ⅴ testis(P<0.05).And the result of ISH showed the Addmc1 mRNA located in primary spermatocyte and secondary spermatocyte.To sum up,it suggested that dmc1 may be specific to meiosis.3.Although ly75 gene express in many tissues of fishes,it specifically expresses in germ cells of gonad.To characterize the expression of ly75 in tissues,firstly RT-PCR was applied to clone the cDNA of ly75 gene,which contains an ORF of 5175 bp that encodes a protein of 1724 amino acid.Bioinformatics analysis showed that the protein sequences of the clone isolated in this study have a predicted N-terminal signal sequence and single-pass TM domain.In addition,the sequence contains a cysteine-rich domain,a single fibronectin type II domain,and 10 C-typelectin-like domains(CTLDs).A comparison of the deduced amino acid sequence of AdLy75 with that other species indicated it shares low identity between 42.7% to 47.5%,while a phylogenetic tree analysis compared the candidate protein with related proteins from other species(including Ly75 homologs and other members of the mannose receptor family)revealed that the candidate gene belongs to the Ly75 protein subfamily of mannose receptor proteins and showed strong association with teleosts Ly75 proteins.qRT-PCR analysis revealed that Adly75 was detected in all tissues examined,furthermore the expression level was higher than that in ovary,and it suggested that Ly75 mainly play a role in testis.The ly75 expression level in different stage of development in testis showed that little expression in early developmental stages,increasing with the spermatogonia proliferation which got a highest level in stage of II,following the expression gradually decreased during the stage of Ⅲ to Ⅳ as the differentiation of spermatogonia.On the other hand,Adly75 mRNA mainly expressed in spermatogonia by the means of in situ hybridization.It suggested that Adly75 is a specific marker for spermatogonia.