| The animal reproductive technologies,including artificial insemination,in vitro fertilization(IVF)and semen cryopreservation etc,play an important role in rapid reproduction and breed improvement of the large livestock(cattle,pigs,sheep and horse),as well as in rescuing endangered animals and human infertility.Guangxi is a large province of buffalo breed,with the improvement of agricultural mechanization level,buffalo labor trait has been gradually turned to meat and milk trait,Guangxi local buffalo breed improvement is close related to buffalo production.During buffalo semen collection and production process,it was found that some bull sperm motility was lower,they could not been used in buffalo practical production.Currently,the regulation fuction and their mechanism of the important genes related to buffalo sperm motility were unclear.Based on the results of sperm motility related research of mouse,human and cattle,the important genes and their expression patterns which maybe affect buffalo sperm motility were preliminarily studied.The results would establish a foundation to elucidate these genes function during buffalo spermatogenesis and their influences on buffalo sperm motility.The obtained results are as followed:Cloning and analysis of buffalo PHB,CAPZB and TEKT2 genes:By using buffalo testicular tissue as material,specific primers were designed according to cattle related gene sequences,the gene encoding region fragments of buffalo PHB,CAPZB and TEKT2 were cloned using RT-PCR method respectively.The cloned fragments were sequenced and analyzed.The results showed that buffalo PHB gene was 948 bp,contained 819 bp ORF,encoded 272 amino acids.CAPZB gene was 958 bp,contained 906 bp ORF,encoded 301 amino acids.Buffalo TEKT2 gene was 1449 bp,contained 1293 bp ORF,encoded 430 amino acids.Multiple sequence comparison analysis results showed that buffalo PHB,CAPZB and TEKT2 genes shared more than 90%similarity with nucleotide sequence of Bos Taurus,Sus scrofa,Ovis aries,Pan troglodytes and Hom sapiens.Buffalo CAPZB shared 100%similarity with amino acid sequence of Bos Taurus,Sus scrofa,Pan troglodytes and Hom sapiens,99%with Ovis aries and Mus musculus,which displayed high conservation during evolution;Buffalo PHB and TEKT2 amino acid sequences were also highly conservative in different species.The results of Phylogenetic tree analysis showed that PHB,CAPZB and TEKT2 genes had similar trend,buffalo,cattle and Ovis aries were grouped together,in accordance with the biological classification.The result of protein structure prediction showed that the isoelectric point of buffalo PHB,CAPZB and TEKT2 proteins was 5.58,6.22 and 6.17,respectively,they were slightly acidic protein.They didn't have the signal peptide sequence,not secretion proteins.The result of cell electronic location showed that buffalo PHB possibly located in the mitochondria,CAPZB possibly located in the cytoskeleton,TEKT2 possibly located in the cytoplasm.The result of transmembrane structure prediction showed that PHB protein contained a transmembrane structure,CAPZB and TEKT2 protein didn't.The result of protein domain analysis showed that PHB protein had a domain,CAPZB and TEKT2 didn't.Expression patterns of PHB,CAPZB,TEKT2 in buffalo different tissues:The result of QRT-PCR showed that PHB gene had a higher expression in the testis,kidney,liver,ovary and pituitary gland,lower expression in the brain,the minimal expression was in the muscle.CAPZB gene had a very high expression in the testis,significantly higher than kidney,ovary,pituitary gland and brain(p<0.05).TEKT2 gene had the most abundant expression in the testis,significantly higher than kidney,liver,ovary,pituitary gland and brain,almost no expression in muscle(p<0.01).The immunohistochemical result displayed that PHB,CAPZB and TEKT2 all expressed in the buffalo testis:TEKT2 expression was the highest,followed by CAPZB,PHB expression was the lowest,which was similar to the QRT-PCR result.PHB,CAPZB and TEKT2 genes all were located on the convoluted seminiferous tubule,enriched in spermatogenic cells of different development stages.Microstructure observation of buffalo sperm samples with high and low motility:Buffalo semen with high sperm motility(sm:0.74)and low sperm motility(sm:0.37)was collected respectively,the difference was significant(p<0.05).Normal buffalo sperm consisted of 3 parts under transmission electron microscope:head,neck and tail.The shape of head was oval,the tail was composed of the middle,main and end segment;the middle consisted of the axial filament and peripheral spiral mitochondrial sheath,the main segment was surrounded by vagina fibrosa,the middle and main segment had a clear "9x2+2" structure,the end axial filament was wrapped by cell membrane.The high sperm motility sample had lots of sperms which owned normal structures and“9x2+2”axoneme.The low sperm motility sample had various kinds of abnormal sperms,such as double tails,and broken tail,mitochondrial dysfunction,nuclear vacuolization,acrosome deficiency,necrospermia,head abnormities.Expression analysis of PHB,CAPZB and TEKT2 in buffalo sperm samples with high and low motilityThe result of QRT-PCR showed that PHB had a different degree expression in high and low sperm motility sample,the relative expression level in the high sperm motility sample(11.81 ± 1.94)was significantly higher than that in the low sperm motility sample(1.0 ± 0.36)(P<0.05);CAPZB and TEKT2 expression in both samples also had a significant difference(P<0.05),the expression in the high motility sperm sample was significantly higher than that in the low motility sample(P<0.05).The result of Western-blot showed that the ?-actin protein band in high and low sperm motility sample was clear and accordant.PHB,CAPZB and TEKT2 expressions were down-regulated in the low sperm motility sample,which was consistent with the detection result of mRNA level.Conclusion:PHB,CAPZB and TEKT2 genes were highly conservative among different species,they mainly located on the convoluted seminiferous tubule,enriched in spermatogenic cells of different development stages,the expression level had a high positive correlation with sperm motility. |
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