Differential distribution of 5-formylcytosine and 5-carboxylcytosine in human spermatogenic cells and spermatozoa

Background. The epigenome of gametes is formed under the control of the developmental programme and the influence of environmental factors. How cytosine oxidation patterns are formed and altered in human spermatogenesis remains obscure so far. The aim of the study was to assess 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) patterns in human spermatogenic cells and spermatozoa. Materials and Methods. The study was performed on testicular biopsy samples of 10 azoospermic patients and ejaculate samples of 5 sperm donors and 8 patients from infertile couples. The microscope slides were prepared for further indirect immunofluorescence to detect 5fC and 5caC and FISH to determine spermatogenic cell ploidy. Results. 5fC and 5caC were undetectable in mitotic and meiotic chromosomes of spermatogenic cells, and was present exclusively in some spermatogonia and spermatid interphase nuclei as well as in some ejaculated spermatozoa. The frequency of spermatozoa with 5fC and 5caC varied in a wide range and was higher in patients than in sperm donors (p=0,007, p=0,028). The increase in frequency of spermatozoa with 5fC and 5caC was accompanied with the decrease in frequency of morphologically normal and progressively motile spermatozoa. Conclusions. 5fC and 5caC are differentially distributed in human spermatogenic cells and spermatozoa. The immunocytochemically detected increase of 5fC and 5caC in individual spermatozoa is most likely induced by oxidative stress caused by effects of internal and external factors rather than developmental programme. The evaluation of 5fC and 5caC in spermatozoa can be potentially used as an additional criterion of ejaculate quality.