Study Of Genes Related To Pig Sperm Tail

Sperm tail take shape during spermiogenesis after mitosis, meiosis of spermatogonial stem cells located on testicular seminiferous tubules epithelium. Sperm tail, the metabolic and mobile organ of sperm, has very close connection with the sperm mobility which is important for sperm to accomplish insemination. Low mobility, sperm tail abnormality and disorder of sperm movement may lead to sterility. The study on molecular mechanism of sperm tail development is very crucial to understand the development process of sperm and the primary abnormality of sperm tail structure. At present, the process of spermiogenesis can’t be duplicated in vitro. The genes with very important function during the process of sperm tail development were specially focused on present study. The studies on sequence features, characters of tissue expressions, subcellular localization and impact of overexpression of genes on spermatogonias cultured in vitro would help to understand the molecular mechanism of spermatogensis. The results for this study are shown as follows.By RT-PCR and RACE, complete sequences of Odf2, Odf3, Tektin4and three isforms of Cabyr gene were cloned. Bioinformatics analysis on these sequences was performed. The results showed that the length of Odf2gene including complete CDS was3300bp, encoding a polypeptide of810amino acids which was spiral protein. The complete length of Odf3was1154bp, encoding a polypeptide of193amino acids which contained5conservative PXP locuses among those of human, cattle, dog, mouse and rat by multiple sequence alignment. The length of pig Tektin4CDNA was1500bp, encoding a polypeptide of447amino acids, which had the conservative TEKTIN family domain of four Coiled coils. Between spirals helix2A and helix2B, the characterized sequences RPNVELCRD for TEKTIN family were present. Cabyr genes contained three isoforms in Meishan boar testis. Cabyr1and Cabyr2encoded a polypeptide of433amino acids, and Cabyr3encoded a polypeptide of382amino acids. RⅡα domain presented at N ends of two polypeptides. By CLUSTAL W, pig Odf2and Odf3amino acids were significantly homologous to other mammalian according to homologous alignment which were84-97%. The homology of Tektin4was78-89%and that of Cabyrl was58-78%which was relatively low. On phylogenetic tree created by DNAMAN, all species in tree was in accordance with the biological classification.With semi-quantitative PCR, the expression of Odf2, Odf3, Tektin4and Cabyr on tissues and organs of Day150Meishan boar and sow were detected. The results showed Odf2and Odf3genes were testis-specific expression genes. Tektin4had strong expressive abundance in testis and weak abundance in boar pituitary gland, sow uterus corners and oviduct. The expression of Cabyr can be detected in all tissues. The expression pattern of Odf2, Odf3, Tektin4and Cabyr genes in the testis of Day2, Day30, Day60, Day90and Day150Meishan boars were analyzed through realtime PCR. The results showed that mRNA of Odf2, Odf3, Tektin4and Cabyr can’t found on Day2and Day30. The expression of four genes emerged from Day60when the sperm cells appeared on Day60testis tissue and cauda epididymis, which showed that the expression of Odf2, Odf3, Tektin4and Cabyr were consistent with the occurrence of boar spermatogensis. The expression of Odf2and Odf3were significantly higher (P<0.01) on Day90and150than those on Day60. There was no significantly difference between the expression on Day90and Day150(P>0.05). The expression of Tektin4and Cabyr on Day150were significantly higher (P<0.01) than those on Day60and90. There was no significantly difference between the expression on Day60and Day90(P>0.05). From60days to90days, Meishan boar initiated puberty and produced sperm. Maybe Odf2and Odf3had possible function on the process of sexual development. Subcellular localization of Odf2, Odf3, Tektin4and Cabyr were studied by fusion protein localization and indirect immunofluorescence. The recombinant eukaryotic expression vector pEGFP-C1-Odf2, pEGFP-C1-Odf3, pEGFP-C1-Tektin4and pEGFP-C1-Cabyr were constructed and transfected to3T3cells. After24hours, fluorescence of fusion protein of Odf2, Odf3and Cabyr can be seen in both nucleus and cytoplasm, while that of Tektin4was in nucleus by inverted flurescence microscopy. The eukaryotic expression vectors pcDNA3.1-Odf2, pcDNA3.1-Odf3,pcDNA3.1-Tektin4and pcDNA3.1-Cabyr were constructed and purified plasmid were prepared for mouse immune to produce antibody. Dilution rate of serum from mouse was1:50,1:100,1:200,1:300. Subcellular localization was implemented by indirect immunofluorescence and four proteins were located in cytoplasm which consistent with the prediction. The ployclone antiserum could recognize Odf2, Odf3, Tektin4and Cabyr protein and the appropriate dilution rate of serum was1:50.The expression of Odf2, Odf3, Tektin4and Cabyr in mature sperm RNA was detected by RT-PCR. The results showed that only Cabyr mRNA expressed in mature sperm. Localizations of Odf2, Odf3, Tektin4and Cabyr in sperm were studied by indirect immunofluorescence. The results showed only Odf3located in the tail of mature sperm which implied Odf3had exclusive function in sperm tail formation. Odf2protein was located on acrosome and equator of head, neck and tail of sperm. Tektin4were on acrosome, equator and tail of sperm. Maybe Odf2and Tektin4had other function during spermatogensis. Cabyr protein could be found in the surface of whole sperm which may relate to energy supplement and capacity. The pig SSCs were cultured by differential attachment and identified. The results showed that the cells from newborn piglet testis have morphological characters of SSCs and the AKP stain was positive. The Oct4and CD9mRNA were found. It could be concluded that these cells were pig SSCs.The impact of overexpression of Odf2、Odf3、Tektin4and Cabyr on pig SSCs meiosis initiation were studied by semi-quantitative PCR. The specific gene Sycp3for meiosis can be detected by overexpression of Odf2to pig SSCs. This implied that Odf2can initiate meiosis. When four genes plasmids were transfected simultaneously, the other specific gene Dmcl can be detected. Maybe there were genes have synergistic impact on meiosis initiation.By realtime PCR, the impact of overexpression of Odf2、Odf3、Tektin4and Cabyr after RA induction on mouse SSCs line meiosis were also studied. The results showed the appropriate concentration of RA was10-5M because the expression of Dazl was the lowest (P<0.05) and Sycp3was the highest (P<0.05) than that of control and10-6M group. After induction of10-5M, the overexpression of Odf2made the expression of Dazl decreased significantly (P<0.01) and the cell formation had an apparent changes. These can be concluded that the overexpression of Odf2made the meiosis of mSSCs further development.