Figure 1.
Spermatogenic cell-specific intronless genes are expressed in testis but not in any other tissues.
Semiquantitative RT-PCR for the 51 intronless genes identified in Table S1 was carried out using total RNA isolated from adult mouse tissues. Thirty-nine genes were exclusively detected in testis and chosen for further study. The intensity of the β-actin band was used as a quantitative standard. The absence of genomic DNA in RNA samples is shown in Figure S1.
Table 1.
Identified spermatogenic cell-specific intronless genes.
Figure 2.
Expression levels of 39 spermatogenic cell-specific intronless genes are increased in later stages of spermatogenesis.
Semiquantitative RT-PCR was carried out using total RNA isolated from D7, D14, D21, and D35 testes. The number of PCR cycles increases from left to right in each panel, as described in Table S2. The β-actin band represents equal expression levels for each stage. The absence of genomic DNA in RNA samples is shown in Figure S2.
Figure 3.
Methylation profiles of spermatogenic cell-specific intronless genes are classified three groups in juvenile mouse testis.
COBRA was carried out to examine the methylation status of the selected genes, the CpG island in the Oaz1 gene, and constitutively methylated endogenous IAP retroviruses in genomic DNA isolated from D7, D14, D21, and D35 testes and adult liver. Sodium bisulfite-treated DNA was amplified with specific primers (details in Table S3) and digested with HpyCH4 IV (recognition site: ACGT), Taq I (TCGA) or Acc II (CGCG).
Figure 4.
Methylation profiles of spermatogenic cell-specific intronless genes are classified three groups in male germ cells.
COBRA was carried out to examine the methylation status of the selected genes, the CpG island in the Oaz1 gene, and constitutively methylated IAP in genomic DNA isolated from 15.5 dpc embryonic (E15.5) and neonatal (D0–1) gonocytes, spermatogonia (SG), pachytene spermatocytes (PS), round spermatids (RS), epididymal spermatozoa (SZ), and adult liver. The sodium bisulfite reaction and restriction enzyme digestion were carried out as described in Figure 3. The COBRA results are also represented by bar graphs.
Figure 5.
Methylation levels of the three categorized genes depend on their CpG contents.
The number of CpG sites within a 200-bp segment containing the COBRA restriction sites in the 20 intronless genes described in Figures 3 and 4 was determined, and the results plotted as a histogram. Each dot indicates a gene in each group classified according to the methylation profiles shown in Figures 3 and 4. Hyper, constitutive hypermethylated; Hypo, constitutive hypomethylated; G-Hypo, germ cell-specific hypomethylated.