Programmed DNA Damage Response in Human Elongating Spermatids.

Abstract

Spermiogenesis of mammals is characterized by the acquisition of a flagella, the synthesis of an acrosome and a striking reorganization of the nucleus. During this process, spermatidal histones will be sequentially replaced by transition proteins and finally by protamines leading to a compact chromatin of the male gamete. Recent findings suggest that both programmed DNA fragmentation and DNA damage response occur during the chromatin remodeling steps of spermatids in mice and rats. We seek to extend these findings to human spermiogenesis given the similarity in nucleoproteins exchange and the demonstration that DNA fragmentation occurs in normal human spermatids as well. Using confocal microscopy, we performed immunofluorescence on formalin-fixed testis of men with normal spermatogenesis prior to the orchiectomy. Before the incubation with specific antibodies, heat-induced epitope retrieval was achieved by boiling the sections in EDTA or citrate buffer. The TUNEL assay (terminal deoxynucleotidyl transferase dUTP-biotin nick-end labeling) was also done after heat-induced epitope retrieval. TUNEL positivity was confirmed in human spermatids. As observed in the nucleus of elongating spermatids of mice and rats, human elongating spermatids presented nuclear gamma-H2AFX staining, a well known DNA double-strand break marker, coincident with histone H4 hyperacetylation. Human spermatids present a DNA damage response during the chromatin remodeling steps. This study suggests that human spermiogenesis has so far been probably overlooked as an important source of genomic instability. Spermiogenesis may, in fact, be the most crucial period of spermatogenesis because of the haploid character of spermatids that must resolve programmed double-stranded breaks. These results prompt for further investigation on the nature of the DNA damage response involved. Given the limited repair capacity of the oocyte many idiopathic cases of infertility or embryo loss may be understood by a better knowledge of the chromatin steps of spermiogenesis.