The putative promoters of germ cell-specific genes and Nanog are hypomethylated in chicken sperm

Abstract

Germ cell-specific genes such as Ddx4, Dnd1, and Dazl play critical roles in the proliferation and survival of germ cells. However, the methylation state of the promoter in mature germ cells is still unknown. Here, we investigated the methylation levels of these genes and the pluripotency marker gene Nanog in chicken sperm as compared with the Alb gene in the liver. CpG islands and/or promoter motifs such as TATA box, GC box and CAAT box were found within the putative promoter regions that we identified. By using the bisulfite reaction, CpG sites in the putative promoters were converted, and they were analyzed by sequencing. The putative promoters of Ddx4, Dnd1, Dazl and Nanog showed very low methylation levels in sperm, but they were highly methylated in the liver. Conversely, the Alb gene promoter was highly methylated in sperm and hypomethylated in the liver. However, no transcripts of Ddx4, Dnd1, Dazl and Nanog were detected in sperm or the liver. Also, no transcripts of Dnmt1 and Dnmt3a were detected in sperm. Our present results may indicate that these germ cell-specific genes and the pluripotency marker gene are ready to express any time after fertilization. Our findings showing that low methylation and selective DNA methylation of specific genes are present in chicken sperm contribute to our understanding of fertilization and embryogenesis of birds.

Fig. 1.

Structure of Ddx4, Dnd1, Dazl, Nanog, and Alb in the 2000-bp region including the downstream regions of the putative promoter regions in the chicken. Vertical line: exon. Shadow: CpG island. White triangle: TATA and TATA-like motifs. Gray triangle: GC and GC-like motifs. Black triangle: CAAT and CAAT-like motifs.

Fig. 2.

Analyses of the expression of Dnmts in chicken sperm and liver samples. Sperm and liver mRNA samples were subjected to RT-PCR for expression of Dnmt1, Dnmt3a, and Dnmt3b. Gapdh was used as an internal control.

Fig. 3.

Methylation of CpG sites within the putative promoter region containing the region 500 bp upstream from the first exon. (A) Localization of CpG sites and a CpG island within the putative promoter region. Ddx4, Nanog and Alb lack CpG islands. Vertical line: CpG site. Shadow: CpG island. (B) Methylated CpG sites in the putative promoter regions of Ddx4, Dnd1, Dazl, Nanog and Alb in chicken sperm and liver samples. Left: sperm; right: liver. Filled circles and open circles represent the methylated and unmethylated CpG sites, respectively. Each line represents an examined clone. The localizations of the CpG sites in each gene shown in A (vertical line) and B (circle) represent the same positions.

Fig. 4.

Analyses of the expression of Ddx4, Dnd1, Dazl, Nanog and Alb in chicken sperm and liver samples. Sperm and liver mRNA samples were subjected to RT-PCR for expression of Ddx4, Dnd1, Dazl, Nanog and Alb. Gapdh was used as an internal control.