Transcriptional and epigenetic changes associated with lead exposure in spermatozoa

Lead (Pb) exposure is a risk factor of male infertility, while the transcriptional and epigenetic changes associated with lead exposure in spermatozoa are poorly understood. Our previous findings revealed significant changes in DNA methylation of the calcium (Ca) homeostasis pathway of human spermatozoa in men with a blood Pb level over 5 µg dl-1, which was associated with decreased sperm motility. In this study, we explored the effects of Pb exposure on expression of differentially methylated genes (DMGs) by analyzing semen samples from six healthy, non-smoking, and non-drinking men (aged 20-40 years). Using methylated DNA immunoprecipitation sequencing (MeDIP-seq) and RNA sequencing (RNA-seq), we compared DNA methylation and RNA abundance patterns between two groups: three men with blood Pb level 0-2.5 µg dl-1 and three men with blood Pb level 5-10 µg dl-1. Additionally, we experimentally validated the regulatory function of the differentially methylated regions associated with 11 hub genes using dual-luciferase reporter assays. We revealed differences in promoter activity between methylated and unmethylated promoter regions of seven cloned genes, namely calcium voltage-gated channel subunit alpha1 H (CACNA1H), calcium voltage-gated channel subunit alpha1 G (CACNA1G), calcium voltage-gated channel subunit alpha1 I (CACNA1I), calcium/calmodulin dependent protein kinase II gamma (CAMK2G), ATPase sarcoplasmic/endoplasmic reticulum Ca²+ transporting 3 (ATP2A3), solute carrier family 8 member A2 (SLC8A2), and glutamate ionotropic receptor NMDA type subunit 2D (GRIN2D). Our results of Pb exposure-induced expression changes of essential genes associated with the calcium signaling pathway, particularly CACNA1H, SLC8A2, and GRIN2D, in spermatozoa, may be a potential cause of low sperm quality.