Methylation Pattern Of Imprinted Genes H19 And IGF2r Of Spermatogenic Cells Of Mouse In Vitro Culture

Objective: To establish a stable and effective culture system of spermatogenic cells of mouse and obstain more haploid spermatids of mouse in vitro by improved the culture conditions. Methods: The mouse spermatogenic cells were isolated by combination of the continuous enzymatic digestion with plating culture method, then put them on the feeder layer of mouse seminiferous tubule cells and cultured in the DMEM/F-12 medium with testosterone (T), FSH, low concentration (10 ng/ml) glial derived neurotrophic factor (GDNF) and epidermis growth factor (EGF). The mRNA expression of pachytene phosphoprotein gene P19 and haploid sperm cell-specific transition proteins gene TP1 were detected by RT-PCR to evaluate the proliferation and differentiation of spermatogenic cells.When massive clones and the round spermatids were observed under microscope, the purity of the haploid spermatids form the cells selected with a micromanipulator was detected by laser scanning confocal microscope (LSCM). Results: In the culture system with the mouse seminiferous tubule cells feeder (MSF), T, FSH, low concentration GDNF and EGF, the massive clones of spermatogonia and the expression of P19 mRNA could be detected on the 7th day of culture, and a plenty of round spermatids and individual mobile sperm could be observed on the 12th day of culture with the expression of TP1 mRNA. The purity of the haploid spermatids from microselected round or elongate spermatids was 82% under LSCM. Conclusions: The culture system established in this research was suitable and effective for cultivating the mouse spermatogenic cells in vitro. LSCM was a simple and useful tool for identifying haploid spermatids. Objective: The imprinting control region (ICR) of imprinted gene H19 and differentially methylated region (DMR) of IGF2r in mouse mature sperm and the hapolid spermatids selected from culture were detected and compared to evaluate its methylation patterns. Methods: The H19 ICR and IGF2r DMR2 of mouse sperm and the hapolid spermatids selected from culture in vitro were detected by bisulfite sequencing PCR (BSP), then the sequences were compared with the correspondence one in GeneBank by DNAman software for judging their methylation patterns. Results: There were 15 CpG sites in mouse, in which 96.67% H19 ICR was essentially methylated and 94.29% IGF2r DMR2 was essentially unmethylated in mouse mature sperm. Meanwhile, 66.33% H19 ICR and 44.29% IGF2r DMR2 were found methylation in the hapolid spermatids selected from culture. There was siginificant difference in methylation patterns between mature sperm and the hapolid spermatids selected from culture (P<0.01). Conclusions: The H19 ICR in mature sperm of KunMing mouse was essentially methylated, whlie the IGF2r DMR2 was essentially unmethylated. The partial loss methylation of H19 ICR and abnormal methylation of IGF2r DMR2 were found in the hapolid spermatids selected from culture.