Novel biomarker ZCCHC13 revealed by integrating DNA methylation and mRNA expression data in non-obstructive azoospermia

The objective of this study was to identify genes regulated by methylation that were involved in spermatogenesis failure in non-obstructive azoospermia (NOA). Testis biopsies of patients with NOA and OA (with normal spermatogenesis) were evaluated by microarray analysis to examine DNA methylation and mRNA expression using our established integrative approach. Of the coordinately hypermethylated and down-regulated gene list, zinc-finger CCHC-type containing 13 (ZCCHC13) was present within the nuclei of germ cells of testicular tissues according immunohistochemistry, and there was decreased protein expression in men with NOA compared with OA controls. Mechanistic analyses indicated that ZCCHC13 increased c-MYC expression through the p-AKT and p-ERK pathways. To confirm the changes in ZCCHC13 expression in response to methylation, 5-aza-2′-deoxycitidine (5-Aza), a hypomethylating agent, was administered to mouse spermatogonia GC-1 cells. We demonstrated that 5-Aza enhanced protein and mRNA expression of ZCCHC13 epigenetically, which was accompanied by activation of p-AKT and p-ERK signaling. Our data, for the first time, demonstrate that ZCCHC13 is an important signaling molecule that positively regulates the AKT/MAPK/c-MYC pathway and that methylation aberrations of ZCCHC13 may cause defects in testis development in human disease, such as NOA.