Cloning And Expression Characterization Of Several Germ Cell-related Genes In Chinese Sturgeon, Acipenser Sinensis

Chinese sturgeon (Acipense sinensis) is an endangered species, and has beenlisted as a Grade I protected animal in China. It is an anadromous fish, maturing atages between8and18years in males and14and26years in females. Although itscomplete artificial propagation was achieved in2009, it was necessary to cultureparent fish for long term, which would take large amounts of manpower, material,resources and financial powers. Moreover，there are some kinds of risks for thismethod, such as the death of parent fish. Thus, it is necessary to search for othermeasures to protect this species more efficiently. With the improving of biotechnology,germ cell transplantation is believed to be an available and fast method, which hadbeen applied in fish. However, in order to implement this plan, germ cell marker genemust be isolated firstly. Additionally, up to now, there is limited information about sexdifferentiation and molecular mechanisms of reproductive regulation of Chinesesturgeon. Therefore, with the homologous cloning method, pou2, nanos1, dazl andboule, which played important roles in germ cells and/or germline stem cells, werecloned from Chinese sturgeon. The expression patterns of these genes in differenttissues, during embryogenesis and in the ovaries and testes of immuture Chinesesturgeons were examined. Furthermore, a specific polyclonal antibody was preparedfrom the in vitro expressed partial AsNanos1protein. The expression pattern ofAsNanos1proteins in different tissues were analyzed by Western blot. Finally, thecelluar distribution of AsNanos1protein in ovaries was investigated. The main resultsof this thesis are summarized as the following:1. The class V POU family genes, including pou5f1and pou2, encodedtranscription factors critical for the maintenance of pluripotency in embryonic stemcells (ESC) and germ line cells in vertebrates. The full-length cDNA of a pou2ortologue in A.sinensis, Aspou2, was cloned and sequenced. This cDNA sequence was2,853bp in total length, and consisted of489bp5’-untranslated region (UTR), a coding sequence of1,296bp and a1,068bp3’-UTR. A comparison of the deducedamino acid sequence of Aspou2with that of other vertebrate species showed that theywere highly conserved in the POU domain, which shared88and90%identity withthat of zebrafish and medaka, respectively, and was69,67and67%identical to frog,mouse and human, respectively. RT-PCR analysis revealed that Aspou2was detectedin all tissues examined except for the liver, and high mRNA levels of Aspou2werefound in the muscle, pituitary and brain. During the embryogenesis and early larvaldevelopment, the expression level of Aspou2mRNAs decreased gradually apart from1-day larvae which were not observed. Furthermore, Aspou2seemed to rise with thedevelopment of gonads of immuture Chinese sturgeons. These results suggested thepossible involvement of Aspou2in the nonpluripotent cells, pluripotent cells,embryogenesis and gonad development.2. The nanos gene family was essential for germ line development in diverseorganisms. The full-length cDNA of a nanos1homologue in A.sinensis, Asnanos1,was isolated and charactirized. This cDNA sequence without a3’-untranslated region(UTR) was775base pairs (bp) in length and encoded a peptide of228amino acidresidues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1mRNAs wereubiquitously detected in all tissues examined except for the fat, including liver, spleen,heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon,midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonalantibody was prepared from the in vitro expressed partial AsNanos1protein. Westernblot analysis revealed that the tissue expression pattern of AsNanos1was notcompletely coincided with that of its mRNAs, which was not found in fat, muscle andintestines. Additionally, by immunofuoresence localization, it was observed thatAsNanos1protein was in the cytoplasm of primary oocytes and spermatocytes.Additionally, by immunofuoresence localization, it was observed that AsNanos1proteins were in the cytoplasm of primary oocytes and spermatocytes, and with thegrowth of the primary oocytes, it spreaded in the total cytoplasm. The presentedresults indicated that another possible mechanism may exist for Asnanos1localizationand its interaction with other factors; meanwhile, among diverse species, theexpression pattern of Asnanos1was differential conservation and divergence.Moreover, the possible involvement of AsNanos1functioned in the germ celldevelopment, and could be considered as a marker of primary oocytes and spermatocytes.3. The DAZ family genes were gonad-specific expression, which could be usedas markers for germ cells. The full-length cDNA of dazl and boule homologue inA.sinensis, Asdazl and Asboule, were isolated. Asdazl cDNA sequence was2,337bpin total length, and consisted of126bp5’-UTR, a coding sequence of675bp and a1,536bp3’-UTR, while Asboule1cDNA sequence was2,572bp in total length, andconsisted of157bp5’-UTR, a coding sequence of1,062bp and a1,353bp3’-UTR.Two conserved domain, RNA Recognition Motif and DAZ-repeat, were both found inAsDazl and AsBoule1. Alignments and identities of the deduced amino acid sequenceof AsDazl and AsBoule1were compared with that of other species. The similarity ofthe putative amino acid of AsDazl with that of fish was about60%, while its identitywith that of other vertebrates was about40%. However, AsBoule1had about60%amino acid sequence identities to that of mammalian, while merely about30%to thatof other vertebrates and in vertebrates, such as31%to medaka. Moreover, thesimilarity of the putative amino acid of AsDazl with AsBoule1was only25%.According to the results of different subtypes of Asboule and amplification in genome,Asboule had alternative splicing and at least two copies of genes. By RT-PCR analysis,Asdazl and Asboule1mRNA were only detected in gonads; furthermore, theexpression level of Asdazl in female was higher than that in male, while theexpression level of Asboule1in female was lower than that in male.