Global methylation status of sperm DNA in carriers of chromosome structural aberrations

Abstract

Male infertility might be clearly associated with aberrant DNA methylation patterns in human spermatozoa. An association between oxidative stress and the global methylation status of the sperm genome has also been suggested. The aim of the present study was to determine whether the global sperm DNA methylation status was affected in the spermatozoa of carriers of chromosome structural aberrations. The relationships between the 5-methylcytosine (m 5 C) levels in spermatozoa and chromatin integrity status were evaluated. The study patients comprised male carriers of chromosome structural aberrations with reproductive failure (n = 24), and the controls comprised normozoospermic sperm volunteers (n = 23). The global m 5 C level was measured using thin-layer chromatography (TLC) and immunofluorescence (IF) techniques. The sperm chromatin integrity was assessed using aniline blue (AB) staining and TUNEL assay. The mean m 5 C levels were similar between the investigated chromosome structural aberrations carriers (P) and controls (K). However, sperm chromatin integrity tests revealed significantly higher values in chromosomal rearrangement carriers than in controls (P < 0.05). Although the potential relationship between sperm chromatin integrity status and sperm DNA fragmentation and the m 5 C level juxtaposed in both analyzed groups (P vs K) was represented in a clearly opposite manner, the low chromatin integrity might be associated with the high hypomethylation status of the sperm DNA observed in carriers of chromosome structural aberrations.

Figure 1

Statistical boxes showing the range (s.d.), median and mean (black square) of the values observed in each performed category of examinations; (a) semen parameters: concentration, total count, motility (a: rapid progressive, b: slow or sluggish progressive, c: nonprogressive, and d: immotile), and morphology; according to the WHO guidelines (1999);²⁵ (b) sperm chromatin integrity: DNA fragmentation and chromatin deprotamination; (c) global m⁵C level in spermatozoa. The blue color represents Patients (carriers of chromosome structural aberrations with reproductive failures), while the red color corresponds to the Control group (healthy males with normozoospermia). A value of P < 0.05 indicates a statistically significant difference.

Figure 2

Analysis of the correlations between the global me⁵C level and chromatin integrity features: (a) correlation between two me⁵C measurement methods: TLC versus IF; (b) spermatozoa with deprotaminated chromatin versus spermatozoa with fragmented DNA; (c) me⁵C versus spermatozoa with deprotaminated chromatin; (d) me⁵C versus fragmented DNA. “P” indicates Patients, while “K” corresponds to the Control. A value of P < 0.05 indicates a statistically significant difference.

Figure 3

Representative images of the staining results for spermatozoa. The bar represents 5 µm. (a) aniline blue staining of three populations of spermatozoa: b: deprotaminated, c: semi-deprotaminated and d: normal, with a proper protamine: histone ratio; light microscopy, ×1000 magnification; (b) TUNEL assay involving the two populations of spermatozoa: light-green (with fragmented DNA) and blue (only DAPI as a counterstain, without DNA fragmentation); fluorescent microscopy, ×1000 magnification; (c) examples of the two-dimensional thin-layer cellulose chromatography (TLC) analysis of [5’³²P]-labeled deoxynucleotides obtained after the enzymatic hydrolysis of DNA. dNp – deoxynucleoside monophosphates: A: adenine; C: cytosine; G: guanine; T: thymine and m⁵C: 5-methylcytosine. Three different values for the R coefficient are presented; (d) example of the immunofluorescent staining (IF) of spermatozoa. Left panel: merged DAPI and FITC channels; right panel: FITC panel with cell fluorescence (CF) values calculated for five spermatozoa; fluorescent microscopy, ×1000 magnification.