Chunhong Yang

TNP1 Functional SNPs in bta-miR-532 and bta-miR-204 Target Sites Are Associated with Semen Quality Traits in Chinese Holstein Bulls.

ABSTRACT Transition nuclear proteins (TNPs), major proteins found in the chromatin of condensing spermatids, have been implicated in spermatogenesis and male fertility. In this study, DNA samples were collected from 404 Chinese Holstein bulls and sequenced to identify genetic variants in the 3′-untranslated region (UTR) of TNP1 and to investigate genetic variations in the TNP1 gene and their common haplotypes. This study was also conducted to determine whether these variations affect bovine semen quality traits and expression levels by PCR-restriction fragment length polymorphism, bioinformatics analyses, quantitative real-time PCR (qPCR), and fluorescence assay. Results showed that one new single-nucleotide polymorphism (SNP; g. 528 G>A, ss1388116558) and one reported SNP (g. 442 A>G, rs110469441) were found in the 3′-UTR of the TNP1 gene. Bioinformatics analysis results revealed that both loci were located in bta-miR-532-binding and bta-miR-204-binding regions, respectively. Association studies revealed that bulls with H1H1 (AGAG) and H1H3 (AGGG) haplotype combinations exhibited a lower deformity rate than those with other haplotype combinations (P < 0.05). The qPCR results showed that the relative mRNA expression of TNP1 in bulls with H1H1 haplotype combination was significantly higher than that in bulls with H4H4 haplotype combination (P < 0.05). MicroRNA qPCR results suggested that bta-miR-532 expression was downregulated by 5-fold in adult bull testicular tissues compared with that in fetal bull testicular tissues; by contrast, bta-miR-204 expression was downregulated by 1.6-fold. Luciferase assay results also indicated that TNP1 expression was directly targeted by bta-miR-532 and bta-miR-204 in murine Leydig tumor cell lines. These results provide the first indication of g. 442 A>G-mediated and g. 528 G>A-mediated translational suppression in which SNPs altered the binding of bta-miR-204 and bta-miR-532 to the 3′-UTR of TNP1; the mediated translational suppression could be involved in the regulation of TNP1 expression and may influence the morphological characteristics of Chinese Holstein bull sperm. We propose that SNPs on the TNP1 3′-UTR may help select semen quality trait in Chinese Holstein bulls in the dairy industry.

Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls

Inner centromere protein (INCENP) plays an important role in mitosis and meiosis as the main member of chromosomal passenger protein complex (CPC). To investigate the functional markers of the INCENP gene associated with semen quality, the single nucleotide polymorphisms (SNPs) g.19970 A>G and g.34078 T>G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A>G in the exonic splicing enhancer (ESE) motif region results in an aberrant splice variant by constructing two minigene expression vectors using the pSPL3 exon capturing vector and transfecting vectors into MLTC-1 cells. INCENP-TV was more highly expressed than INCENP-reference in adult bull testes. The g.34078 T>G located in the binding region of bta-miR-378 could affect the expression of INCENP, which was verified by luciferase assay. To analyze comprehensively the correlation of SNPs with sperm quality, haplotype combinations constructed by g.19970 A>G and g.34078 T>G, as well as g.-692 C>T and g.-556 G>T reported in our previous studies, were analyzed. The bulls with H1H12 and H2H2 exhibited a higher ejaculate volume than those with H2H10 and H9H12, respectively (P < 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P < 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P < 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A>G and g.34078 T>G in INCENP both of which appear to change the molecular and biological characteristics of the mRNA transcribed from the locus may serve as a biomarkers of male bovine fertility by affecting alternative splicing mode and binding affinity with the target bta-miR-378.

Role of an SNP in Alternative Splicing of Bovine NCF4 and Mastitis Susceptibility

Neutrophil cytosolic factor 4 (NCF4) is component of the nicotinamide dinucleotide phosphate oxidase complex, a key factor in biochemical pathways and innate immune responses. In this study, splice variants and functional single-nucleotide polymorphism (SNP) of NCF4 were identified to determine the variability and association of the gene with susceptibility to bovine mastitis characterized by inflammation. A novel splice variant, designated as NCF4-TV and characterized by the retention of a 48 bp sequence in intron 9, was detected in the mammary gland tissues of infected cows. The expression of the NCF4-reference main transcript in the mastitic mammary tissues was higher than that in normal tissues. A novel SNP, g.18174 A>G, was also found in the retained 48 bp region of intron 9. To determine whether NCF4-TV could be due to the g.18174 A>G mutation, we constructed two mini-gene expression vectors with the wild-type or mutant NCF4 g.18174 A>G fragment. The vectors were then transiently transfected into 293T cells, and alternative splicing of NCF4 was analyzed by reverse transcription-PCR and sequencing. Mini-gene splicing assay demonstrated that the aberrantly spliced NCF4-TV with 48 bp retained fragment in intron 9 could be due to g.18174 A>G, which was associated with milk somatic count score and increased risk of mastitis infection in cows. NCF4 expression was also regulated by alternative splicing. This study proposes that NCF4 splice variants generated by functional SNP are important risk factors for mastitis susceptibility in dairy cows.

PCK1 is negatively regulated by bta‐miR‐26a, and a single‐nucleotide polymorphism in the 3' untranslated region is involved in semen quality and longevity of Holstein bulls

Phosphoenolpyruvate carboxykinase 1 (PCK1) is a multi‐functional enzyme that plays important roles in physiological processes, including reproduction. We previously reported that the PCK1 transcript has five splice variants; PCK1‐AS4, which lacks exon 5, is enriched in the testis of Holstein bulls. In the present study, we profiled select PCK1 transcript variants in the testis, epididymus, and semen of high‐ and low‐performance bulls, and examined the possibility that microRNAs may be involved in single nucleotide polymorphism (SNP)‐mediated modulation of PCK1 expression. PCK1‐AS4 abundance is not significantly different between high‐ and low‐performance bulls. Luciferase reporter assays, however, showed that bovine PCK1 expression is repressed by bta‐miR‐26a in HepG2 hepatocyte cells. One SNP (c. + 2183 G > T) at the miRNA‐binding site of PCK1 does not influence PCK1 expression, but is associated with elevated ejaculation volume, fresh sperm motility, and genomic estimated breeding value of longevity, as well as with reduced values of composite index and calving ease. Collectively, the identified 3′‐untranslated‐region SNP variant highlights the importance of PCK1 in the fecundity of Holstein bulls, and implicates a role for bta‐miR‐26a in regulating PCK1 abundance. Further study is needed to assess the effects of other genetic variants in 5′‐flanking region and exons of PCK1 on enzyme levels in the testis and sperm. Mol. Reprod. Dev. 83: 217–225, 2016.

Splicing-related single nucleotide polymorphism of RAB, member of RAS oncogene family like 2B (RABL2B) jeopardises semen quality in Chinese Holstein bulls

RAB, member of RAS oncogene family like 2B (RABL2B) is a member of a poorly characterised clade of the RAS GTPase superfamily, which plays an essential role in male fertility, sperm intraflagellar transport and tail assembly. In the present study, we identified a novel RABL2B splice variant in bovine testis and spermatozoa. This splice variant, designated RABL2B-TV, is characterised by exon 2 skipping. Moreover, a single nucleotide polymorphism (SNP), namely c.125G>A, was found within the exonic splicing enhancer (ESE) motif, indicating that the SNP caused the production of the RABL2B-TV aberrant splice variant. This was demonstrated by constructing a pSPL3 exon capturing vector with different genotypes and transfecting these vectors into murine Leydig tumour cell line (MLTC-1) cells. Expression of the RABL2B-TV transcript was lower in semen from high- versus low-performance bulls. Association analysis showed that sperm deformity rate was significantly lower in Chinese Holstein bulls with the GG or GA genotype than in bulls with the AA genotype (P<0.05). In addition, initial sperm motility was significantly higher in individuals with the GG or GA genotype than in individuals with the AA genotype (P<0.05). The findings of the present study suggest that the difference in semen quality in bulls with different RABL2B genotypes is generated via an alternative splicing mechanism caused by a functional SNP within the ESE motif.

A g.-1256 A>C in the promoter region of CAPN1 is associated with semen quality traits in Chinese Holstein bulls.

The micromolar calcium-activated neutral protease gene (CAPN1) is a physiological candidate gene for sperm motility. However, the molecular mechanisms involved in regulating the expression of the CAPN1 gene in bulls remain unknown. In this study, we investigated the expression pattern of CAPN1 in testis, epididymis, and sperm at the RNA and protein levels by qRT-PCR, western blot, immunohistochemistry, and immunofluorescence assay. Results revealed that the expression of CAPN1 levels was higher in the sperm head compared with that in other tissues. Moreover, we identified a novel single-nucleotide polymorphism (g.-1256 A>C, ss 1917715340) in the noncanonical core promoter of the CAPN1 gene between base g.-1306 and g.-1012. Additionally, we observed greater sperm motility in bulls with the genotype CC than in those with the genotype AA (P<0.01), indicating that different genotypes were associated with the bovine semen trait. Furthermore, a higher fluorescence intensity of the C allele than that of the A allele at g. -1256 A>C was revealed by transient transfection in MLTC-1 cells and luciferase report assay. Finally, CAPN1 was highly expressed in the spermatozoa with the CC genotype compared with that with the AA genotype by qRT-PCR. This study is the first report on genetic variant g.-1256 A>C in the promoter region of CAPN1 gene association with the semen quality of Chinese Holstein bulls by influencing its expression. g.-1256 A>C can be a functional molecular marker in cattle breeding.

The effect of the SNP g.18475 A>G in the 3′UTR of NCF4 on mastitis susceptibility in dairy cattle

Neutrophil cytosolic factor 4 (NCF4) is a member of the nicotinamide adenine dinucleotide phosphate oxidase subunit. This protein functions as an essential factor in the host defense against the progression of bacterial infection. To explore the variability of the NCF4 gene and the susceptibility of cows to mastitis, NCF4 functional single nucleotide polymorphism (SNP) of the 3′ untranslated region (3′UTR) and its targeted microRNA (miRNA) were identified. One SNP g.18475 A>G in the 3′UTR of NCF4 was found within the binding seed region of bta-miR-2426. We constructed two recombinant pMIR-REPORT™ vectors with the A or G allele in the g.18475 locus and transiently co-transfected the vectors in human embryo kidney 293T (HEK 293T) cells, along with bta-miR-2426 mimics. A luciferase assay indicated that this SNP affects the binding of NCF4 and bta-miR-2426. In addition, the association analysis results showed that cows with the GG genotype in SNP g.18475 A>G had a relatively lower SCS value than cows with the AA genotype. Finally, quantitative real-time PCR (RT-qPCR) results showed that the cows with genotype GG had a relatively higher expression of NCF4 mRNA compared to the cows with genotype AA. NCF4 expression was regulated by the miRNA–mRNA interaction mechanism, and an important role for NCF4 in mastitis susceptibility in dairy cow was suggested.

Functional haplotypes of ARID4A affect promoter activity and semen quality of bulls

The AT-rich interaction domain 4 A (ARID4A) has an important role in regulating Sertoli cell function and male fertility. Its molecular mechanisms, however, remain largely unknown. In this study, two single nucleotide polymorphisms (SNPs) (g.53 G > T, ss 1966531596, and g.826 G > A, rs 210809648) were identified in the promoter region of ARID4A in 215 Chinese Holstein bulls using polymerase chain reaction (PCR)-restriction fragment length polymorphism and created restriction site-PCR. Results revealed that bulls with g.53 G > T-GG and g.826 G > A-G G genotype exhibited higher sperm deformity rate than those with g.53 G > T-TT and g.826 G > A-AA genotype (P <  0.01). Furthermore, three haplotypes (H1 (GG), H3 (TG), H4 (TA)) and six haplotype combinations (H1H1, H1H3, H1H4, H3H3, H3H4, H4H4) were obtained. The bulls with H4H4 exhibited lower sperm deformity rate than those with H1H1 and H1H3 (P <  0.05). In addition, results of bioinformatics analysis revealed that ARID4A has two promoters and that two SNPs of ARID4A are located in transcription factor binding sites. Compared with g.53 G > T-G and g.826 G > A-G allele, there was a greater fluorescence intensity in g.53 G > T-T and g.826 G > A-A allele by transient transfection in MLTC-1 cells and the luciferase report assay. qRT-PCR indicated the ARID4A expression was greater in bull spermatozoa with H4H4 haplotype combination than those with H1H1 haplotype combination (P < 0.05). Results of the present study indicate that g.53 G > T and g.826 G > A are functional mutations that are involved in regulation of ARID4A gene expression by affecting promoter activity and thus semen quality of Chinese Holstein bulls.