Cpg Methylation Participates in Regulation of Hepatitis B Virus Gene Expression in Host Sperm and Sperm-Derived Embryos

Figures & data

Figure 1.  CpG site methylation in islands II and III in the hepatitis B virus genome in sperm transfected with an unmethylated plasmid and in sperm-derived embryos, as detected by bisulfite sequencing PCR.

(A) Detection of CpG site methylation in island II. (A1) in plasmid; (A2) in sperm; and (A3) in sperm-derived embryo. (B) Detection of CpG site methylation in island III. (B1) in plasmid; (B2) in sperm; and (B3) in sperm-derived embryo. The methylation statuses of 14 clones for each sample are presented; each column represents one CpG position in island II or III, and each circle in the column indicates either cytosine (open circles) or methyl cytosine (filled circles).

Figure 2.  CpG site methylation in islands II and III in the hepatitis B virus genome in sperm transfected with a methylated plasmid and in sperm-derived embryos, as detected by bisulfite sequencing PCR.

(A) Detection of CpG site methylation in island II. (A1) in plasmid; (A2) in sperm; and (A3) in sperm-derived embryo. (B) Detection of CpG site methylation in island III. (B1) in plasmid; (B2) in sperm; and (B3) in sperm-derived embryos.

Figure 3.  Transcription of the HBV X and HBV S genes in sperm transfected with a methylated plasmid and in sperm-derived embryos.

(A) In sperm. M: marker DL2000; 1: X gene; 2: S gene; 3: -RT; 4: positive control; 5: β-actin; and 6: -T. (B) In sperm-derived embryos. M: marker DL2000; 1: X gene; 2: S gene; 3: -T; 4: -RT; 5: positive control; 6: β-actin.

Figure 4.  For sperm from the hepatitis B virus-infected patients and controls, aliquots of the bisulfite sequencing PCR amplified products were subjected to electrophoresis through a 1.5% agarose gel and stained with ethidium bromide.

Methylation-specific bands for CpG sites in island II (A) and island III (B) in the hepatitis B virus genome were observed for the patient sperm but not for the control sperm.

C1–C11: Sperm from controls; M: Marker; P1–P11: Sperm from the patients.

Figure 5.  Correlation between the methylation percentages (mCpG%) of CpG sites in islands II and III in the hepatitis B virus genome and the transcriptional levels (2^-△△Ct) of the hepatitis B virus X and S genes in patient sperm and sperm-derived embryos.

The transcriptional levels of the X and S genes increased with decreases in the methylation percentages of CpG sites in islands II and III, respectively, and strong negative correlations were observed. (A) X gene/island II correlation in sperm (Spearman’s r = -1; p < 0.0001); (B) X gene/island II correlation in sperm-derived embryos (Spearman’s r = -0.9623; p < 0.0001); (C) S gene/island III correlation in sperm (Spearman’s r = -0.9909; p < 0.0001); and (D) S gene/island III correlation in sperm-derived embryos (Spearman’s r = -0.9745; p < 0.0001).

Figure 6.  Effects of S-adenosylmethionine on the methylationof CpG sites in islands II and III in embryos.

(A1) Island II with S-adenosylmethionine (SAM); (A2) island II without SAM; (B1) island III with SAM; and (B2) island III without SAM. The results showed that the methylation percentages of CpG sites in islands II and III in the embryos markedly increased following treatment with SAM.

Table 1.  Effects of the methylation statuses of CpG sites on the transcriptional levels of the hepatitis B virus X and S genes in human sperm transfected with an unmethylated and a methylated plasmids and in sperm-derived embryos, respectively.

Samples	HBV X (2^-△△CT)			HBV S (2^-△△CT)
	M	Un-M	FCV	M	Un-M	FCV
Sperm	1.2 × 10^-4***	1.00	-8333	0.086***	1.00	-11.63
Sperm-derived embryo	0.96*	1.00	0.96	0.96*	1.00	0.96

*p > 0.05.

***p < 0.0001.

Real-time qRT-PCR and the 2^-△△CT methods were used to evaluate effects of the methylation statuses of CpG sites on the transcriptional levels of the X and S genes in human sperm transfected with an unmethylated or a methylated plasmid and in sperm-derived embryos, respectively. The data were presented as an FCV in transcription of the X and S genes in the M normalized to internal control gene (GAPDH) and relative to those in the Un-M. The individual data were converted into a linear form using 2^-△△CT calculation and then subjected to a paired-sample t test to determine whether there was a significant difference in average transcriptional levels of the X and S genes between the methylated and unmethylated groups. A p-value of less than 0.05 was considered significant.

FCV: Fol change value; M: Methylated group; Un-M: Unmethylated group.

Table 2.  Methylation percentages (mCpG%) of CpG sites in ilands II and III, and the transcriptional levels of the hepatitis B virus X and S genes in the patient sperm and sperm-derived embryos.

Patients	CpG sites in island II/HBV X gene				CpG sites in island III/HBV S gene
	Sperm		Two-celled embryos		Sperm		Two-celled embryos
	mCpG%	2^-ΔΔCT	mCpG%	2^-ΔΔCT	mCpG%	2^-ΔΔCT	mCpG%	2^-ΔΔCT
2#	95.54	1.00	8.04	1.00	96.09	1.00	7.81	1.00
5#	91.96	1.35	6.25	1.16	93.75	1.32	6.25	1.08
7#	89.29	1.81	5.36	1.17	92.19	1.59	5.47	1.13
8#	88.39	2.10	4.46	1.18	89.84	1.93	4.69	1.15
1#	83.93	2.47	2.68	1.20	86.72	2.25	3.13	1.15
10#	64.29	4.58	2.68	1.21	71.09	4.21	2.34	1.17
11#	44.64	6.38	1.79	1.22	55.47	5.76	1.56	1.17
3#	25.89	12.56	1.79	1.23	40.63	11.67	1.56	1.18
4#	17.86	21.66	1.79	1.23	25.00	20.59	0.78	1.18
9#	11.61	24.04	0.89	1.23	24.22	19.57	0.78	1.19
6#	9.82	34.81	0.89	1.23	10.16	28.56	0.78	1.19
Mean	56.66 ± 35.27	10.25	3.33 ± 2.37	1.19	62.43 ± 32.42	8.95	3.20 ± 2.48	1.14

Using real-time qRT-PCR and the 2^-ΔΔCT methods, the data were presented as a fold change in transcriptional levels of the X or S genes in the sperm from the 11 patients and in the sperm-derived embryos normalized to an endogenous reference gene (GAPDH) and relative to those of the X or S gene in the sperm and sperm-derived embryo with the highest level of CpG sitemethylation, respectively.

HBV: Hepatitis B virus.

Table 3.  Transcriptional levels of the hepatitis B virus X and S genes in the embryos treated and untreated with S-adenosylmethionine.

Genes	Treatment with SAM 2^-ΔΔCT	Treatment without SAM 2^-ΔΔCT	Fold change value
HBV X gene	0.47 (0.43–0.51)	1 (0.67–1.33)	-2.13
HBV S gene	0.49 (0.45–0.53)	1 (0.74–1.26)	-2.04

Real time qRT-PCR and the 2^-ΔΔCT methods were used to evaluate the effects of SAM treatment on expression of the X and S genes in the embryos. The data were presented as the fold change value in expression of the X and S genes in SAM-treated group normalized to internal control gene (GAPDH) and relative to those in SAM-untreated group.

HBV: Hepatitis B virus; SAM: S-adenosylmethionine.