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Molecular Effects of Polymorphism in the 3'UTR of Unc-5 homolog C Associated with Conception Rate in Holsteins.

Sugimoto M, Gotoh Y, Kawahara T, Sugimoto Y - PLoS ONE (2015)

Bottom Line: To enhance our understanding of the genes involved in conception rates, we conducted a genome-wide association study (GWAS) using 822 Holsteins and identified a single-nucleotide polymorphism (SNP) associated with conception rate: A+169G in the 3' untranslated region (UTR) of unc-5 homolog C (UNC5C).G9a-knockout mice have previously been demonstrated to be subfertile, and we found that UNC5C, G9a, and Netrin-1 expression levels increased from the 4-cell stage to the blastocyst stage in fertilized murine embryos, whereas Rhox5 expression decreased.This study provides novel insights into the role of UNC5C during embryonic development.

View Article: PubMed Central - PubMed

Affiliation: National Livestock Breeding Center, Nishigo, Fukushima, Japan.

ABSTRACT
Conception rates among dairy cows in Japan have declined in recent decades. To enhance our understanding of the genes involved in conception rates, we conducted a genome-wide association study (GWAS) using 822 Holsteins and identified a single-nucleotide polymorphism (SNP) associated with conception rate: A+169G in the 3' untranslated region (UTR) of unc-5 homolog C (UNC5C). Cows with higher conception rates carried the A polymorphism in the UNC5C 3'UTR. Luciferase assays and quantitative analysis of allele ratios revealed that UNC5C transcripts with the A polymorphism were expressed at higher levels than those carrying the G polymorphism. UNC5C transmits either pro- or anti-apoptotic signals depending on the availability of its ligand, Netrin-1. UNC5C expression is negatively regulated by reproductive homeobox X-linked 5 (Rhox5), and the Rhox5 locus is methylated by G9a methyltransferase. G9a-knockout mice have previously been demonstrated to be subfertile, and we found that UNC5C, G9a, and Netrin-1 expression levels increased from the 4-cell stage to the blastocyst stage in fertilized murine embryos, whereas Rhox5 expression decreased. Repression of UNC5C, G9a, or Netrin-1 or forced expression of Rhox5 in the anterior nucleus stage inhibited development to the blastocyst stage, suggesting that cows carrying the G polymorphism in UNC5C might have lower conception rates because of the poor development of preimplantation embryos. This study provides novel insights into the role of UNC5C during embryonic development.

No MeSH data available.


G9a decreases Rhox5 expression.A. DNA methylation at the Rhox5 promoter as assessed by PCR following digestion with the methylation-insensitive MspI (M) or methylation-sensitive HpaII (H) restriction enzymes of the genomic DNA from OV2944-HM-1 transfected with siG9a or negative control (NC). “-”represents undigested DNA. “Mr” is a DNA size marker. B. Representative immunoblots with anti-UNC5C, anti-Rhox5, anti-G9a, and anti-actin antibodies for OV2944-HM-1 transfected with negative control (NC) or siG9a.
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pone.0131283.g005: G9a decreases Rhox5 expression.A. DNA methylation at the Rhox5 promoter as assessed by PCR following digestion with the methylation-insensitive MspI (M) or methylation-sensitive HpaII (H) restriction enzymes of the genomic DNA from OV2944-HM-1 transfected with siG9a or negative control (NC). “-”represents undigested DNA. “Mr” is a DNA size marker. B. Representative immunoblots with anti-UNC5C, anti-Rhox5, anti-G9a, and anti-actin antibodies for OV2944-HM-1 transfected with negative control (NC) or siG9a.

Mentions: Because Rhox5 expression is controlled by DNA methylation [18] and the methylation of Rhox loci requires the histone methylase G9a in mouse embryonic fibroblasts [19], the level of G9a expression might influence methylation of the Rhox5 locus in the murine ovary. To explore this possibility, we performed methylation-sensitive PCR to assess murine OV2944-HM-1 transfected with siG9a, which represses the expression of endogenous g9a in mice, or that transfected with a negative control. We found that the Rhox5 locus was hypomethylated in the siG9a-transfected cells (Fig 5A). Furthermore, immunoblotting of OV2944-HM-1 transfected with siG9a revealed that decreased G9a expression increased Rhox5 expression and decreased UNC5C expression (Fig 5B). Therefore, these results imply that G9a expression may affect UNC5C expression through Rhox5 methylation.


Molecular Effects of Polymorphism in the 3'UTR of Unc-5 homolog C Associated with Conception Rate in Holsteins.

Sugimoto M, Gotoh Y, Kawahara T, Sugimoto Y - PLoS ONE (2015)

G9a decreases Rhox5 expression.A. DNA methylation at the Rhox5 promoter as assessed by PCR following digestion with the methylation-insensitive MspI (M) or methylation-sensitive HpaII (H) restriction enzymes of the genomic DNA from OV2944-HM-1 transfected with siG9a or negative control (NC). “-”represents undigested DNA. “Mr” is a DNA size marker. B. Representative immunoblots with anti-UNC5C, anti-Rhox5, anti-G9a, and anti-actin antibodies for OV2944-HM-1 transfected with negative control (NC) or siG9a.
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Related In: Results  -  Collection

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pone.0131283.g005: G9a decreases Rhox5 expression.A. DNA methylation at the Rhox5 promoter as assessed by PCR following digestion with the methylation-insensitive MspI (M) or methylation-sensitive HpaII (H) restriction enzymes of the genomic DNA from OV2944-HM-1 transfected with siG9a or negative control (NC). “-”represents undigested DNA. “Mr” is a DNA size marker. B. Representative immunoblots with anti-UNC5C, anti-Rhox5, anti-G9a, and anti-actin antibodies for OV2944-HM-1 transfected with negative control (NC) or siG9a.
Mentions: Because Rhox5 expression is controlled by DNA methylation [18] and the methylation of Rhox loci requires the histone methylase G9a in mouse embryonic fibroblasts [19], the level of G9a expression might influence methylation of the Rhox5 locus in the murine ovary. To explore this possibility, we performed methylation-sensitive PCR to assess murine OV2944-HM-1 transfected with siG9a, which represses the expression of endogenous g9a in mice, or that transfected with a negative control. We found that the Rhox5 locus was hypomethylated in the siG9a-transfected cells (Fig 5A). Furthermore, immunoblotting of OV2944-HM-1 transfected with siG9a revealed that decreased G9a expression increased Rhox5 expression and decreased UNC5C expression (Fig 5B). Therefore, these results imply that G9a expression may affect UNC5C expression through Rhox5 methylation.

Bottom Line: To enhance our understanding of the genes involved in conception rates, we conducted a genome-wide association study (GWAS) using 822 Holsteins and identified a single-nucleotide polymorphism (SNP) associated with conception rate: A+169G in the 3' untranslated region (UTR) of unc-5 homolog C (UNC5C).G9a-knockout mice have previously been demonstrated to be subfertile, and we found that UNC5C, G9a, and Netrin-1 expression levels increased from the 4-cell stage to the blastocyst stage in fertilized murine embryos, whereas Rhox5 expression decreased.This study provides novel insights into the role of UNC5C during embryonic development.

View Article: PubMed Central - PubMed

Affiliation: National Livestock Breeding Center, Nishigo, Fukushima, Japan.

ABSTRACT
Conception rates among dairy cows in Japan have declined in recent decades. To enhance our understanding of the genes involved in conception rates, we conducted a genome-wide association study (GWAS) using 822 Holsteins and identified a single-nucleotide polymorphism (SNP) associated with conception rate: A+169G in the 3' untranslated region (UTR) of unc-5 homolog C (UNC5C). Cows with higher conception rates carried the A polymorphism in the UNC5C 3'UTR. Luciferase assays and quantitative analysis of allele ratios revealed that UNC5C transcripts with the A polymorphism were expressed at higher levels than those carrying the G polymorphism. UNC5C transmits either pro- or anti-apoptotic signals depending on the availability of its ligand, Netrin-1. UNC5C expression is negatively regulated by reproductive homeobox X-linked 5 (Rhox5), and the Rhox5 locus is methylated by G9a methyltransferase. G9a-knockout mice have previously been demonstrated to be subfertile, and we found that UNC5C, G9a, and Netrin-1 expression levels increased from the 4-cell stage to the blastocyst stage in fertilized murine embryos, whereas Rhox5 expression decreased. Repression of UNC5C, G9a, or Netrin-1 or forced expression of Rhox5 in the anterior nucleus stage inhibited development to the blastocyst stage, suggesting that cows carrying the G polymorphism in UNC5C might have lower conception rates because of the poor development of preimplantation embryos. This study provides novel insights into the role of UNC5C during embryonic development.

No MeSH data available.