[Impact of the expression of DNA methyltransferase 1 on the differentiation of spermatogonial stem cells in mice].

Objective: To investigate the influence of the expression of DNA methyltransferase 1 (DNMT1) on the differentiation of spermatogonial stem cells (SSC) in mice.

Methods: SSCs were isolated from the testis tissue of 1-week-old BALB/c male mice by two-step enzyme digestion. DNMT1-siRNA and negative control siRNA (NC-siRNA) were transfected into the third-generation SSCs after isolation and purification, and the untransfected cells were used as the control. At 24 hours after transfection, the mRNA and protein expressions of DNMT1 were detected by real-time quantitative PCR (RT-qPCR) and Western blot, respectively, and the methylation level of DNMT1 was determined. The SSCs were induced to differentiate into spermatocytes using the stem cell growth factor, and the expressions of the germ cell proliferation-related protein (Nanos2), promyelocytic leukemia zinc finger protein (PLZF) and retinoic acid-stimulated protein 8 (Stra8) were measured by RT-qPCR and Western blot after 48 hours of differentiation.

Results: At 24 hours after transfection, the relative mRNA and protein expressions of DNMT1 and the DNA methylation level were significantly decreased in the DNMT1-siRNA group compared with those in the control and DNMT1-NC groups (P < 0.05), but showed no statistically significant difference between the latter two (P > 0.05). The relative mRNA and protein expressions of Nanos2 and PLZF were also decreased while those of Stra8 increased in the DNMT1-siRNA group in comparison with those in the control and DNMT1-NC groups after 48 hours of differentiation (P < 0.05), but none exhibited any statistically significant difference between the control and DNMT1-NC groups (P > 0.05).

Conclusion: Knockdown of DNMT1 promotes the differentiation of SSCs into spermatocytes in mice, which may be related to the reduction of the genome methylation level, inhibition of the expressions of Nanos2 and PLZF, and promotion of the expression of Stra8.