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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

Expression of INCENP gene.(A) Expression of the INCENP gross transcript (INCENP -G) in various tissues in adult bulls using qRT-PCR. (B) Expression of INCENP -G, INCENP—reference and INCENP -TV in testis tissue of adult bulls and calves using qRT-PCR. The vertical bars represent standard errors. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
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pone.0162730.g002: Expression of INCENP gene.(A) Expression of the INCENP gross transcript (INCENP -G) in various tissues in adult bulls using qRT-PCR. (B) Expression of INCENP -G, INCENP—reference and INCENP -TV in testis tissue of adult bulls and calves using qRT-PCR. The vertical bars represent standard errors. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.

Mentions: To evaluate the mechanism that regulates INCENP transcripts, qRT-PCR (quantitative real-time PCR) was conducted to determine the relative expression levels of the INCENP gene in the heart, liver, spleen, lung, kidney, and testis, respectively (Fig 2A). The expression of the bovine INCENP gene in different tissues exhibited variability. Such expression in spleen and testis was significantly higher than in other adult bull tissues. This result suggested that the INCENP gene lacked tissue specificity. We then investigated the mRNA expression of INCENP-reference and INCENP-TV in adult bull testis, as well as calf testis. The results demonstrated that the expression of INCENP-reference and INCENP-TV in the adult bull were higher than that in the calf. In addition, INCENP-TV was more highly expressed than INCENP-reference in the adult bull, whereas INCENP-TV seemed to slightly differ from INCENP-reference in the calf (Fig 2B).


Functional SNPs of INCENP Affect Semen Quality by Alternative Splicing Mode and Binding Affinity with the Target Bta-miR-378 in Chinese Holstein Bulls
Expression of INCENP gene.(A) Expression of the INCENP gross transcript (INCENP -G) in various tissues in adult bulls using qRT-PCR. (B) Expression of INCENP -G, INCENP—reference and INCENP -TV in testis tissue of adult bulls and calves using qRT-PCR. The vertical bars represent standard errors. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036895&req=5

pone.0162730.g002: Expression of INCENP gene.(A) Expression of the INCENP gross transcript (INCENP -G) in various tissues in adult bulls using qRT-PCR. (B) Expression of INCENP -G, INCENP—reference and INCENP -TV in testis tissue of adult bulls and calves using qRT-PCR. The vertical bars represent standard errors. Means with different lowercase superscripts above the error bars are significantly different at P < 0.05.
Mentions: To evaluate the mechanism that regulates INCENP transcripts, qRT-PCR (quantitative real-time PCR) was conducted to determine the relative expression levels of the INCENP gene in the heart, liver, spleen, lung, kidney, and testis, respectively (Fig 2A). The expression of the bovine INCENP gene in different tissues exhibited variability. Such expression in spleen and testis was significantly higher than in other adult bull tissues. This result suggested that the INCENP gene lacked tissue specificity. We then investigated the mRNA expression of INCENP-reference and INCENP-TV in adult bull testis, as well as calf testis. The results demonstrated that the expression of INCENP-reference and INCENP-TV in the adult bull were higher than that in the calf. In addition, INCENP-TV was more highly expressed than INCENP-reference in the adult bull, whereas INCENP-TV seemed to slightly differ from INCENP-reference in the calf (Fig 2B).

View Article: PubMed Central - PubMed

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&gt;G and g.34078 T&gt;G were identified and analyzed. The new splice variant INCENP-TV is characterized by the deletion of exon 12. The g.19970 A&gt;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&gt;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&gt;G and g.34078 T&gt;G, as well as g.-692 C&gt;T and g.-556 G&gt;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 &lt; 0.05). Bulls with H11H11 and H2H10 exhibited higher initial sperm motility than those with H2H2 (P &lt; 0.05). The expression levels of INCENP in bulls with H1H12 and H11H11 were significantly higher than those in bulls with H9H12 (P &lt; 0.05), as determined by qRT-PCR. Findings suggest that g.19970 A&gt;G and g.34078 T&gt;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.

No MeSH data available.


Related in: MedlinePlus