Dysregulated methylation of the human BOLL promoter hinders germ cell development from spermatogonia to spermatids

BOLL, a highly conserved gene crucial for meiosis in spermatogenesis, is epigenetically regulated through DNA methylation in various species. This study aimed to determine whether BOLL promoter methylation contributes to its downregulation in azoospermic men with hypospermatogenesis (HS) and to explore its potential regulatory association with human spermiogenesis.

We conducted pyrosequencing to assess methylation levels at the putative BOLL promoter on testicular samples from azoospermic men with HS versus normal spermatogenesis (NR) and evaluate correlations with spermatogenic severity and gene expression. In silico CpG island (CGI) prediction and luciferase reporter assays were used to confirm methylation-sensitive regulatory regions. Pathway enrichment analysis identified biological processes associated with BOLL. Functional assays using BOLL overexpression in HEK293T cells and GC2 cells, as well as demethylation using 5-Aza-2'-deoxycytidine (5-AZA) in GC2 cells, were conducted to explore downstream transcriptional effects.

Eight CpG sites (CpGs) within the BOLL promoter CGI were significantly hypermethylated in HS samples, with seven exhibiting a significant inverse correlation with the spermatogenic score, and three with BOLL transcript levels. The region from - 1434 to + 180 was confirmed as a methylation-sensitive promoter. Gene ontology analysis indicated that spermatid development and differentiation were the top BOLL-associated pathways. BOLL overexpression by plasmid transfection in HEK293T cells and GC2 cells upregulated spermatid-specific genes (TNP2 and GAPDHS), while concurrently suppressing spermatogonia-associated markers (ID4 and PIWIL4). In GC2 cells, 5-AZA treatment enhanced Boll expression and induced downstream spermatid-specific genes, including Prm2, which was not responsive to BOLL overexpression alone.

We identified specific BOLL promoter CpGs that are hypermethylated in HS and associated with reduced gene expression. These findings suggest that promoter methylation may contribute to BOLL silencing and impaired spermatid differentiation, supporting its regulatory role in human spermiogenesis.