Effect of DNA methylation regulation on male infertility and its mechanism

doi:10.3877/cma.j.issn.1674-0785.2021.01.002

Abstract

Abstract:

Objective

To investigate the effect of epigenetic regulation on male infertility and its underlying mechanism.

Methods

From February to December 2020, outpatients at Department of Reproductive Health and Infertility of Guangdong Maternal and Child Health Hospital were selected and divided into either a control group (n=50) or a sterility group (n=50). Semen samples were collected for real-time PCR and Western blot detection of DNMT1, ERp29, PTEN, and TSC2 mRNA and protein. DNA methylation immunoprecipitation sequencing (MeDIP-seq) was used to detect genes with significant methylation differences. GC1, GC2, and TM4 sperm cell lines were divided into a blank control group (con group), ERp29 silencing group (shERp29 group), and ERp29 overexpression group (ERp29^OEgroup). The demethylating drug 5-AZA-DC was used to treat cells, and methylation-specific PCR (MSP), CCK-8, and flow cytometry were then performed to detect cell proliferation and apoptosis.

Results

Compared with the control group, DNMT1 mRNA (5.25±1.12 vs 8.22±0.67, P＜0.05) and protein (3.33±0.98 vs 7.31±0.55, P＜0.05) expression was up-regulated, while ERp29 mRNA ([5.17±0.98 vs 2.27±0.33, P＜0.05) and protein (6.12±1.03 vs 2.59±0.43, P＜0.05), PTEN mRNA (4.58±0.77 vs 1.97±0.28, P＜0.05) and protein (6.21±1.04 vs 2.28±0.36, P＜0.05), and TSC2 mRNA (6.39±0.83 vs 3.16±0.52, P＜0.05) and protein (6.56±0.67 vs 3.03±0.17, P＜0.05) expression were down-regulated. The MeDIP-Seq results showed significant differences in methylation levels of ERp29, PTEN, and TSC2 promoter regions in the sterility group (P＜0.05). MSP confirmed the DNA methylation of CPG islands in the promoter region of PTEN gene in the GC1 cell line. Before 5-AZA-DC intervention, the shERp29 and ERp29^OE group had increased methylation, and after intervention, the two groups had decreased methylation. After intervention of GC1, GC2, and TM4 cell lines with 5-AZA-DC, cell proliferation in the shERp29 and ERp29^OE groups was significantly enhanced and apoptosis significantly decreased (P＜0.05), although there was no significant difference in the con group (P＞0.05).

Conclusion

ERp29 is an important gene that causes male infertility and can be a potential target for epigenetic treatment of male infertility.

Key words: Epigenetics, DNA methylation, Male infertility

Peng Du, Xiaoli Zhang, Xiaoyong Zhao. Effect of DNA methylation regulation on male infertility and its mechanism[J]. Chinese Journal of Clinicians(Electronic Edition), 2021, 15(01): 10-16.

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Figures/Tables 8

References 15

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组别	例数	ERp29	PTEN	TSC2
对照组	50	10.53±1.21	9.38±1.04	9.53±1.49
不育组	50	17.69±1.88	13.67±1.12	13.84±1.51
t值		2.883	2.815	2.941
P值		0.023	0.026	0.021

组别	例数	ERp29	PTEN	TSC2
对照组	50	10.53±1.21	9.38±1.04	9.53±1.49
不育组	50	17.69±1.88	13.67±1.12	13.84±1.51
t值		2.883	2.815	2.941
P值		0.023	0.026	0.021

组别	5-AZA-DC（-）			5-AZA-DC（+）
组别	GC1	GC2	TM4	GC1	GC2	TM4
Con组	7.21±1.03	7.23±1.02	7.20±1.01	7.22±1.01	7.20±1.03	7.22±1.02
shERp29组	17.24±1.53	15.26±1.68	15.33±1.49	10.25±1.35^*	13.34±1.28^*	13.32±1.29^*
ERp29^OE组	15.29±1.52	13.24±1.31	13.26±1.32	8.26±1.05^*	9.37±1.14^*	9.38±1.15^*

组别	5-AZA-DC（-）			5-AZA-DC（+）
组别	GC1	GC2	TM4	GC1	GC2	TM4
Con组	7.21±1.03	7.23±1.02	7.20±1.01	7.22±1.01	7.20±1.03	7.22±1.02
shERp29组	17.24±1.53	15.26±1.68	15.33±1.49	10.25±1.35^*	13.34±1.28^*	13.32±1.29^*
ERp29^OE组	15.29±1.52	13.24±1.31	13.26±1.32	8.26±1.05^*	9.37±1.14^*	9.38±1.15^*

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