Abstract
Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2'-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.
Highlights
In the last two decades we have been aware that in human ejaculates there can be high percentages of sperm with DNA fragmentation, representing a threat for male fertility, human reproduction and the health of the offspring
Multicolor Flow Cytometry To study the simultaneous occurrence of apoptosis, immaturity and oxidative damage in sperm with sperm DNA fragmentation (sDF), we used flow cytometry to evaluate specific markers of each of the three processes in sperm samples stained by TUNEL and DAPI
We found an increase (51.4% ± 9.1% in treated samples versus 22.2% ± 1.7% in untreated, p < 0.05, n = 3) of sDF, demonstrating that, in our hands, TUNEL assay is able to reveal DNA fragmentation induced by oxidative stress in vitro
Summary
In the last two decades we have been aware that in human ejaculates there can be high percentages of sperm with DNA fragmentation, representing a threat for male fertility, human reproduction and the health of the offspring. In the era of assisted reproduction techniques (ARTs) which bypass many, if not all, natural barriers to fecundation, the risk that sperm with unresolved DNA damage can fertilize an oocyte [1] appears increased, raising further concerns on the presence of DNA breaks in the sperm genome. The knowledge of the mechanisms responsible for this type of sperm damage is pivotal for the development of effective treatments to prevent the onset of sDF in infertile men. According to one of these proposed mechanisms, the DNA nicks occurring to promote the remodeling of sperm chromatin are not completely repaired due to an impairment of the sperm maturation process [8,9]. According to one of these proposed mechanisms, the DNA nicks occurring to promote the remodeling of sperm chromatin are not completely repaired due to an impairment of the sperm maturation process [8,9]. sDF could reflect a DNA cleav-
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