| In this study, HIBADH and TNP1genes are selected as the research object. At the levels ofSNPs, promoter and miRNA, we screen the functional molecular markers which are related tosemen quality traits of Chinese Holstein bulls and attempt to explore their possible molecularregulatory mechanisms.This is helpful to bull breeding with high reproduction traits in the future.1. Analysis of promoter activity, functional SNPs and methylation level in promoter regionof HIBADH geneThe HIBADH transcribed and expressed in testis, caput epididymis, corpus epididymis,cauda epididymis, and sperm by western blotting analysis. We detected the immunoreactivity inthe seminiferous epithelium, including pachytene spermatocytes, primary spermatocytes, andspermatids and the whole bull epididymis through immunohistochemistry (IHC). Further,immunofluorescence assay (IFA) showed that HIBADH was expressed in the neck-andmid-piece of bull spermatozoa. We found that the transcriptional level of HIBADH is reduced inbull poor-motility spermatozoa. Considering sperm mitochondria are located in the mid-pieceand produce ATP through aerobic respiration. In view of this, HIBADH may be involved inregulating sperm motility.Then, we screened the SNPs in the5’ flankling region of HIBADHgene. One SNP g.-165T>C was detected. The association analysis between this SNP and spermquality traits showed that bulls with TT genotype had higher initial sperm motility than that of CC(P<0.05). By predicting, we found that the SNP was located in the core promoter of HIBADHgene. The SNP may affect the activity of the promoter and change the expression of HIBADHgene.We constructed different fragment length into pGL3-Basic, transient transfected intoMLTC-1cell lines and determined dual luciferase activity. The results showed a878(-703bp~+175bp) fragment constitutesd the core promoter region.Then, two genotypes TT and CC, whichhad significant association with the initial sperm motility, were transfected into MLTC-1celllines.The result showed that TT genotype had the maximal activity. The g.-165T>C increased E47transcription factor binding site. As an important transcription factor of eukaryotes, the E47factor could bind to their response element in the gene promoter to regulate the expression ofdifferent target genes and could be involved in the promotion of cell aging. Therefore, the SNPg.-165T>C may regulate the expression of bovine HIBADH gene via transcription factor E47and may cause significant potential phenotype diversity. In addition, we detected a CpG-richdomain in the core promoter. Using the quantitative bisulfite sequencing method, we amplifiedthe175bp fragment (-233bp to-59bp) with17CpG sites. Sixty selected clones (10clones perbull) and a total of1020CpGs were analyzed. We did not find any significant difference in thefrequency of DNA methylation of the HIBADH promoter between the high-performance andlow-performance bulls (P>0.05), and all clones were hypomethylated. However, a very specificmethylation pattern was observed in the7thCpG site in the HIBADH core promoter. The7thCpGsite exhibited higher methylation level in the high-motility group than in the low-motility group(P <0.01). In conclusion, we speculated that the g.-165T>C rather than methylation in the5’-flanking region can affect the bovine sperm motility by regulating the transcriptional activityof the HIBADH gene.2. Relevant research between bta-miR-204, bta-miR-532and TNP13’UTRTwo SNPs loci were found in the bovine TNP1gene, respectively was g.442A> G andg.528G> A. Both were located in the3’UTR region. Through the correlation analysis showedthat the g.528G> A and sperm deformity rate were significantly correlated. In this position,bulls with GG genotype had lower sperm deformity rate than that of GA and AA (P<0.05).Whereas, the g.442A> G had no significantly relationship with bovine sperm quality traits(P>0.05). We combined the two SNPs loci and built a total of8haplotype combinations.Statistical analysis revealed that H1H1and H1H3haplotype combinations with sperm deformityrate was lower that with other haplotype combinations (P<0.05). Therefore, H1H1and H1H3were excellent haploidtype combinations and may be used for assisted breeding.Both the two SNPs located in the3’UTR region, we suspect that they may play a regulatoryrole by corresponding microRNAs. microRNAs can lead to the degradation of mRNA andtranslational repression by binding to the3’UTR of target gene. This is a kind of regulation at theposttranscriptional level. Using online miRNA prediction software, we detected that bta-miR-204and bta-miR-532couldbind to the TNP1-3’UTR in Chinese Holstein bulls. The SNP g.442A>Gå'Œg.528G>A, whichhave association with the sperm deformity rate, were respectively located within the region ofmiRNA (bta-miR-204and bta-miR-532) binding sites. Based on the SNP g.442A>G loci, AA andGG genetypes of3’UTR were respectively constructed into the luciferase report vector(PMIR-ReportTM), transfected MLTC-1cells with bta-miR-204expression vector. In terms of theSNPg.528G>A loci, GG and AA genetypes of3’UTR were respectively inserted into thePMIR-ReportTM, transfected MLTC-1cells with bta-miR-532expression vector. The dualluciferase report system analysis demonstrated that the intensities of Fluorescence were bringdown compared with the control. Bta-miR-204and bta-miR-532could bind to the TNP13’UTRand reduce the expression of gene. The g.442A>Gå'Œg.528G>A mutation increased thebinding affinity on the3’UTR of TNP1gene. In addition, four haplotypes (H1:AG H2:AAH3:GG H4:GA) of3’UTR were respectively inserted into the PMIR-ReportTM, transfectedMLTC-1cells with bta-miR-204and bta-miR-532expression vector. The result revealed that thehaplotype H4had the highest binding capacity with miRNAs. Q-PCR was used to compare therelative expression of TNP1mRNA in bull sperm cells with four haplotype combinations. Therelative expression of TNP1mRNA with the combined haplotype H1H1was significantly higher(P<0.05) than that with H4H4. The results of Q-PCR and haplotype test were consistent.Thesedatas suggested that g.442A> G and g.528G> A in TNP1are functional SNPs in bta-miR-204and bta-miR-532target sites. g.442A> G and g.528G> A in TNP1are also genes with stronga priori biological relevance; these genes are possibly implicated in spermatogenesis and innormal morphological characteristics of Holstein bull sperm. We propose that SNPs on the3′-UTR of TNP1may help select semen quality trait in Chinese Holstein bulls in the dairyindustry.3. Polymorphisms of HIBADH gene and their correlation with semen quality traits inChinese Holstein bullsWe found two SNPs loci in the bull HIBADH gene, respectively was g.26736T>C andg.90209C>T. The g.26736T>C located in intron4and the g.90209C>T in exon5. The g.90209C>T was identified as a synonymous mutation (Alaâ†'Ala). SAS statistical analysis showed that the g.26736T>C-TC had a higher initial sperm motility than that of TTand CC (P<0.05). The g.90209C>T had a significant effect on post-thaw cryopreserved sperm motility. In this position, bulls withCC genotype had a higher post-thaw cryopreserved sperm motility than that of TT (P<0.05).Using SHEsis software, four haplotypes (H1, H2, H3, H4) and nine haplotype combinationswere found. Correlation analysis revealed H1H3haplotype combination with initial sperm motility,sperm density, and post-thaw cryopreserved sperm motility was higher than those with otherhaplotype combinations.Therefore, H1H3haplotype combination may be used as an effectivemolecular marker in MAS of bulls breeding programs. |
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