Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality.

Olga A Efimova,Ilona A Galembo,Oleg S Glotov,Irina S Stepanova,Vladislav S Baranov,Eugene V Daev,Alexander M Gzgzyan,Andrei V Tikhonov,Tatyana V Kuznetzova,Mariia A Mazilina,Olga G Chiryaeva,Svetlana A Shlykova,Mikhail I Krapivin,Evgeniia M Komarova,Irina D Mekina,Igor Yu Kogan,Sergey E Parfenyev,Anna A Pendina

doi:10.18632/oncotarget.18331

Abstract

We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality – normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) – and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.

Highlights

Human spermatozoa have a unique epigenome which is formed through epigenetic changes during a long period of differentiation from primordial germ cells to mature gametes
We address the following questions concerning 5hmC patterns in normal and pathological human spermatogenesis: 1) whether sperm 5hmC patterns show inter-cell and inter-individual variation in sperm donors versus patients from infertile couples; 2) whether sperm 5hmC patterns are associated with semen parameters; and 3) whether 5hmC patterns undergo global changes during differentiation of spermatogenic cells
We evaluated the presence of 5hmC and 5mC in human ejaculated spermatozoa fixed on glass slides

Summary

Introduction

Human spermatozoa have a unique epigenome which is formed through epigenetic changes during a long period of differentiation from primordial germ cells to mature gametes. Establishment, maintenance and alteration of specific 5-methylcytosine (5mC) patterns are the key aspects in cell fate determination through arrangement of DNA-protein interactions and activation / repression of specific gene expression programs [5]. Errors in these processes are associated with various human pathologies [6,7,8,9,10]. Along with 5mC, the DNA methylation/demethylation cycle includes three other forms of modified cytosine: 5-hydroxymethylcytosine (5hmC), 5-formylcytosine and 5-carboxylcytosine. Many current studies discuss the epigenetic effect of 5mC oxidative derivatives, especially of 5hmC, in genome function regulation [13,14,15]

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