[Epigenetic regulation for spermatogenic dysfunction-related infertility in C57BL/6J male mice: An experimental study].

Objective: To investigate the effect of epigenetic regulation on spermatogenic dysfunction-related infertility (SDI) in C57BL/6J male mice.

Methods: Sixty C57BL/6J male mice were randomly divided into a normal control and an SDI model group and the SDI model was established using the epididymis-targeting polypeptide CSA combined with indocyanine green-loaded free nanoparticles (ICG-NPS), busufan and dimethyl sulfoxide (DSMO). After intervention with 5-AZA-DC, the epididymides were collected from the mice for measurement of the rates of sperm DNA fragmentation (SDF), sperm acrosome integrity (SAI) and spontaneous acrosome reaction (SAR), amplification of the ERp29 gene by FISH, determination of the mRNA and protein expressions of DNMT1, ERp29, PTEN and TSC2 by quantitative real-time PCR and Western blot, and analysis of the ERp29, PTEN and TSC2 genes by methylated DNA immunoprecipitation sequencing (MeDIP-seq).

Results: After 5-AZA-DC intervention, statistically significant differences were observed between the normal control and the SDI model groups in the rates of SDF (［15.67 ± 1.33］% vs ［30.15 ± 2.87］%, P < 0.05) and SAI (［65.33 ± 7.14］% vs ［47.16 ± 3.45］%, P < 0.05), but not SAR (［11.52 ± 2.31］% vs ［11.48 ± 2.27］%, P > 0.05). FISH confirmed evident amplification of the ERp29 gene in the SDI model but not in the normal control group. Compared with the baseline, the SDI model mice showed significant decreases after intervention in the mRNA and protein expressions of DNMT1 (［9.33 ± 1.15］ vs ［7.01 ± 1.14］, P < 0.05; ［15.66 ± 1.45］ vs ［12.33 ± 1.27］, P < 0.05), but increases in those of ERp29 (［3.04 ± 1.13］ vs ［6.54 ± 1.18］, P < 0.05; ［4.37 ± 1.02］ vs ［6.95 ± 1.03］, P < 0.05), PTEN (［3.25 ± 1.01］ vs ［5.85 ± 1.04］, P < 0.05; ［3.54 ± 1.01］ vs ［5.17 ± 1.02］, P < 0.05) and TSC2 (［4.27 ± 1.16］ vs ［6.98 ± 1.13］, P < 0.05; ［3.83 ± 1.12］ vs ［6.98 ± 1.13］, P < 0.05). No statistically significant differences, however, were found in the above parameters in the normal control group before and after intervention (P > 0.05). MeDIP-seq manifested 18 significantly differential genes were highly expressed and another 25 lowly expressed in the epididymal tissue of the model mice, all the former 18 down-regulated and all the latter 25 up-regulated after intervention, particularly ERp29, PTEN and TSC2. But there were no statistically significant differences in the expressions of the above genes in the control group (P > 0.05). MeDIP-seq also showed significant differences in the regional methylation levels of the Erp29, PTEN and TSC2 promoters in the epididymal tissue of the model mice (P < 0.05), but not in that of the normal controls after intervention (P > 0.05).

Conclusions: A stable and efficient animal model provided valuable experimental evidence for the diagnosis and treatment of spermatogenic dysfunction-related infertility. ERp29 is an important gene involved in infertility and can be used as a potential target for epigenetic regulation in the treatment of infertility.