Role of DNase Activity in Human Sperm DNA Fragmentation.

In this clinical era of intracytoplasmic sperm injection (ICSI), where a single spermatozoon is chosen for fertilization, the diagnostic functionality of the classical parameters typically associated with fertilization, such as sperm concentration, sperm motility, acrosome integrity, and mitochondria, is perhaps becoming less critical. In contrast, the contribution of sperm DNA quality to our understanding of the impact of male fertility within the context of ICSI is gaining increasing interest and importance. Even with respect to natural conception, high levels of sperm DNA fragmentation (SDF) in the ejaculate can adversely affect reproductive outcomes. However, the precise origin of SDF pathology in sperm cells is often ambiguous and most likely to be multifactorial. Hence, the genetic makeup of an individual, unbalanced REDOX processes, enzymatic activity, environmental and lifestyle factors, and even damage during sperm handling in the laboratory all operate in a unique and often synergistic manner to produce or induce sperm DNA damage. Surprisingly, the contribution of active enzymes as potential agents of SDF has received much less attention and, therefore, is likely to be underrated. This review highlights the roles of different enzymes related to the degradation of sperm DNA as possible effectors of DNA molecules in spermatozoa.

Bacterial DNase activity as a putative inductor of sperm DNA fragmentation in infected bull frozen-thawed semen samples.

The aim of this study was to investigate the relationship between DNase activity associated with bacterial contamination of incubated bovine frozen-thawed spermatozoa and elevated sperm DNA fragmentation. Electrophoresis analysis of plasmid PBR322 incubated for 30 min at 37 °C with the supernatant of the diluent of frozen-thawed centrifuged bovine semen straws infected with bacteria showed clear evidence of DNase activity when compared to plasmid incubated in similarly prepared non-infected bovine diluent supernatant (Experiment 1). This DNase activity was subsequently found to be time dependent (0-60 min) and its activity prevented in the presence of EDTA (10 and 20 mM; Experiment 2). Semen straws infected (n = 10) and not infected (n = 10) with bacteria where incubated at 37 °C for up to 48h post-thaw. Semen infected with bacteria showed an exponential increase in bacterial growth and a corresponding increase in sperm DNA fragmentation. Non-infected semen samples showed no change in the incidence of sperm DNA fragmentation over the same period of incubation (Experiment 3). Our experiments reinforce the idea that exogenous DNases present in the semen should be considered as one of the primary contributing causes of sperm DNA fragmentation post ejaculation. In the case of the bull, post-thaw incubation of commercial straws contaminated with bacteria, resulted in increased levels of sperm DNA fragmentation, most likely associated with DNase activity (potentially restriction endonucleases) derived from the bacteria. Such adverse changes in sperm DNA fragmentation, as described here in vitro, may be also operative after insemination in the female reproductive tract (in vivo) and highlight the importance of implementing high levels of hygiene practice during semen processing, especially in light of future trends of bacterial resistance to the common antibiotics used in semen diluents.

Sperm DNA damage in men with spinal cord injury: the relative incidence of single- and double-strand DNA breaks.

BACKGROUND: Men with spinal cord injury (SCI) show a high proportion of sperm DNA damage in their ejaculate but the underlying pathology remains elusive. OBJECTIVE: To investigate the relative incidence of single (SSBs) and double-strand DNA breaks (DSBs) and DNase activity in men with SCI. MATERIALS AND METHODS: This study included ejaculates of 20 men with SCI and 27 normozoospermic (sperm donors). A TwoTails comet assay (TTComet) allowed visualization of three categories of sperm DNA damage corresponding to SSBs, DSBs and those with a combination of SSBs and DSBs, facilitating accurate calculation of the total proportion of SSBs and DSBs. A subset of 15 individuals (sperm donors and SCI patients) was used to test for DNase activity in the seminal plasma. RESULTS: While the proportion of DSBs in men with SCI (median-57.5%) was higher (P = 0.000) than normozoospermic samples (median-4.6%), the proportion of SSBs was higher (P = 0.022) in the normozoospermic ejaculates (median-6.0%) compared to men with SCI (median-2.5%). The relative proportion of the total DSBs with respect to the total SSBs was 3.3× in men with SCI but 0.9× in normozoospermic samples. We further confirmed the high DNase activity in the seminal plasma of men with SCI. DISCUSSION: The TTComet assay provided new insights to the pathology of sperm DNA in men with SCI and may have diagnostic value in developing sperm selection methodologies to reduce DSBs prior to ART. CONCLUSION: Men with SCI are characterized by a high proportion of sperm with DSBs and high levels of DNase activity in the seminal plasma compared to normozoospermic men.

Free circulating DNA and DNase activity in the ejaculates of men with spinal cord injury.

STUDY DESIGN: Retrospective descriptive study. OBJECTIVES: To study the presence of cell-free DNA (cfDNA) and DNase activity in males with spinal cord injury (SCI) with elevated sperm DNA fragmentation. SETTING: Hospital in Toledo, Spain; University-based Genetics laboratory in Madrid, Spain. METHODS: Semen was collected from 15 males with spinal cord injury and elevated sperm DNA fragmentation (SDF). The presence and concentration of cfDNA was assessed using standard gel electrophoresis and microfluidic electrophoresis. DNase activity was evaluated in seminal plasma and under the presence of EDTA and EGTA to control the response of enzyme activity. cfDNA fragments were mapped on the sperm genome using FISH. All results were compared to 15 normozoospermic fertile donors. RESULTS: Standard gel electrophoresis revealed a cfDNA band of ~150 bp in all samples from males with SCI; this band was ocasionally accompanied by another band of ~90 bp. These bands were not observed in normozoospermic donors. Microfluidid electrophoresis only identified the equivalent band of 150 bp. No correlation was observed between the intensity of the two bands and the level of SDF in males with SCI. Although DNase activity was present in the seminal plasma of both normozoospermic donors and men with SCI it did not digest cfDNA. cfDNA fragments were found to be hybridized all over the sperm genome. CONCLUSIONS: SCI patients with elevated sperm DNA fragmentation showed a 150 bp DNA band of cfDNA in the seminal plasma, which appeared resistant to DNase activity.

Co-incubation of spermatozoa with human follicular fluid reduces sperm DNA fragmentation by mitigating DNase activity in the seminal plasma.

PURPOSE: To examine the effect of co-incubating spermatozoa with human follicular fluid (HFF) on the rate of sperm DNA fragmentation. METHODS: This prospective study used semen (n = 23) and HFF from oocyte donors (n = 23). Liquified semen was divided into four aliquots: (1) neat semen (NEAT), (2) seminal plasma removed and replaced with sperm media (HTF) containing 0% (FF0), (3) 20% (FF20), or (4) 50% (FF50) HFF. Sperm motility and DNA fragmentation (SDF) were assessed following 24 h of incubation at 37 °C. Pro-oxidant capacity of HFF and seminal plasma and the effect of HFF on seminal plasma DNase activity was assessed in a sub-sample of 10 ejaculates. RESULTS: Sperm motility was higher after 3 h of incubation in media that contained HFF compared to the NEAT sample or when sperm was diluted in media without HFF. r-SDF (increase of SDF per time unit) values after 24 h of incubation for NEAT, FF0, FF20 and FF50 were 0.91, 0.69, 0.25 and 0.36, respectively. While pro-oxidant capacity of seminal plasma samples showed large variation (mean: 94.6 colour units; SD 65.4), it was lower and more homogeneous in FF samples (mean: 29.9 colour units; SD: 6.3). Addition of HFF to seminal plasma appeared to inhibit DNase activity. CONCLUSION: While differences exist in the pro-oxidant capacity of seminal plasma of patients, sperm DNA integrity was preserved with addition of HFF to sperm media, irrespective of the level of pro-oxidant capacity. DNase activity in the original seminal plasma was abolished after HFF co-incubation.

Characterization of DNA cleavage produced by seminal plasma using leukocytes as a cell target.

Numerous studies have shown the presence of DNA lesions in human spermatozoa affecting sperm quality. However, the nature of this anomaly and its relationship with patient etiology are poorly understood since different mechanisms can be involved in the formation of these novel DNA configurations including the action of a seminal plasma nuclease activity. The objective of this study was to assess the capacity of seminal plasma for producing endogenous DNA cleavage using nuclei of peripheral blood leukocytes as external targets. For this purpose, we used seminal plasma from fertile males with normal semen parameters to produce DNA cleavage in a sample of leukocytes. Three different tests were performed to visualize DNA cleavage: (a) DNase activity detection, (b) DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH), and (c) Two-dimensional comet assay (Two-tail comet assay). Our results demonstrate that: (i) the seminal plasma is able to cleave DNA compacted with histones in the leukocytes; (ii) this DNA cleavage can be associated with DNase activity and (iii) DNA damage mainly corresponds to single-strand DNA breaks. In conclusion, capacity of seminal plasma for producing DNA cleavage represents a solid contribution to expand the analysis of the standard seminal profile and could constitute a putative diagnostic tool for evaluating male infertility.Abbreviations: ALS: alkali labile sites; ART: Assisted Reproduction Technologies; DBD-FISH: DNA Breakage Detection-Fluorescence In Situ Hybridization; DNA: deoxyribonucleic acid; DSBs-DNA: double-strand DNA; FITC: Fluorescein IsoThioCyanate; GEDA: Gravity Enforced Diffusion Assays; PBS: phosphate-buffered saline; ROS: Reactive Oxigen Species; SSBs-DNA: single-strand DNA; SSC: saline-sodium citrate.