Abstract
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.
Highlights
Holstein dairy cows are the species with the highest amount of milk production and the widest breeding range in China
In order to clearly understand the situation of genetic polymorphisms in the population of Chinese Holstein bulls, PCR-RFLP was performed to genotype of g
Two polymorphisms—g.-556 G> T and g.-692 C> T—in the 5' flanking region of Inner centromere protein (INCENP) were examined using PCR-RFLP [33] in Chinese Holstein bull; g. -556 G> T was closely related to initial sperm motility, thereby suggesting that single nucleotide polymorphisms (SNPs) influence INCENP gene expression by regulating promoter activity [28]
Summary
Various techniques and protocols can be used to evaluate conventional semen quality parameters, including sperm concentration, motility, progressive motility, and deformity rate, which cannot be selected directly because of low heritability [2]. With the continuous development of molecular genetic techniques, the candidate gene approach in genetics and breeding has been widely popularized in the recent decades [3]. The candidate gene approach could provide theoretical guidance for marker-assisted selection by screening polymorphic loci of candidate genes, analyzing the correlation of polymorphic loci with semen quality, and authenticating molecular markers related to sperm quality. Several research teams have identified single nucleotide polymorphism (SNP) sites through a genome-wide association study closely related to semen quality and found that these SNPs affect sperm motility, density, deformity rate, and embryonic development after fertilization [6,7,8]
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