international journal of andrology, 27:140–146 (2004)
The neutral comet assay detects double strand DNA damage in selected and unselected human spermatozoa of normospermic donors
R. J. VAN KOOIJ,* P. DE BOER, J. M. T. DE VREEDEN-ELBERTSE,* N. A. GANGA,* N. SINGHà and E. R. TE VELDE* *Division of Perinatology and Gynaecology, University Medical Center, Utrecht, The Netherlands, Department of Obstetrics and Gynaecology, University Medical Center St Radboud, Nijmegen, The Netherlands, and àDepartment of Bioengineering, University of Washington, Seattle, USA
Summary The occurrence of DNA breaks in human sperm is of concern to genetic safety in artificial reproduction techniques. Here, we have explored the neutral comet assay (NCA) for evaluating the frequency of spermatozoa with double strand (ds) DNA breaks in normospermic donors. The NCA results into DNA tail formation by fibre extension and by the separation of DNA fragments. Gamma-irradiated native, lysed and lysed plus RNA and protein degraded human sperm nuclei have been used to assess sensitivity and specificity of fragment formation as an indication for ds DNA breaks. At 5 and 10 Gy gamma irradiation, the sensitivity increases in the order: native, lysed, lysed plus RNA and protein degraded. At 10 Gy, a uniform response between donors was obtained. For technical and biological reasons, the NCA underestimates the true incidence of ds DNA breaks by an unknown factor. Semen samples of six healthy normospermic donors were differentiated by swim up and by Percoll density centrifugation, followed by the NCA. In native semen, percentages of sperm nuclei with ds DNA breaks ranged from 15 to 25%. Swim up and selection for high-density sperm nuclei (high Percoll fraction) reduced the frequency of sperm with ds DNA breaks by about one third, whereas an increased frequency was found in the low Percoll fraction. In conclusion, the response to gamma irradiation of DNA fragment formation indicates the NCA to demonstrate ds DNA breaks which is in keeping with theory and experimental results from somatic cells. Ds DNA breaks are a characteristic of the sperm population of normal donors. Current sperm selection procedures reduce the fractions of sperm with ds DNA breaks, yet are not effective in eliminating these cells. Keywords: comet assay, DNA-damage, human sperm, irradiation, sperm selection Introduction The recent increasing popularity of assisted reproduction techniques (ART) including intra cytoplasmic sperm injection (ICSI) demands sensitive estimation of sperm DNA
Correspondence: P. de Boer, Department of Obstetrics and Gynaecology, UMC St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail:
[email protected]
integrity in order to ensure the genetic health of resultant offspring. Full sperm DNA integrity usually is defined as the absence of DNA nicks or single strand (ss) breaks, double strand (ds) breaks and chemical modifications of the DNA. Of these, the ds DNA break is the most mutagenic lesion, because in the pronucleus stage zygote, the two genomes are separated, hence DNA template information for error free repair of the ds breaks is absent. Also, in the G1 stage zygote, 2004 Blackwell Publishing Ltd.
Double strand DNA breaks in human sperm
non-homologous endjoining (NHEJ) which is essentially error-prone, likely prevails in the repair of ds DNA breaks (Hoeijmakers, 2001). Four main methods are currently used to detect the integrity of DNA in individual spermatozoa: in situ nick translation, the terminal deoxynucleotidyl transferase assay (TdTA or TUNEL), the sperm chromatin structure assay (SCSA) and the comet assay. In situ nick translation utilizes DNA polymerase I to incorporate labelled nucleotides in a template specific manner (Manicardi et al., 1995). The TdTA or TUNEL detects DNA strand breaks where 3¢ OH groups are available (in ss and ds DNA breaks). On human sperm smears, nick translation and TUNEL assays are equally sensitive (Manicardi et al., 1998). A negative association between DNA damage and fertilization rate in IVF (Sun et al., 1997) and ICSI (Sakkas et al., 1996; Lopes et al., 1998) has been found using TUNEL as a measure of sperm DNA integrity. The SCSA indirectly measures DNA stability via the relative amount of red acridine orange fluorescence indicating ss DNA (Evenson et al., 1980). The SCSA has been previously correlated with fertility in an ART setting (Evenson et al., 1999; Larson et al., 2000; Evenson et al., 2002). The comet assay (microgel electrophoresis technique) also detects ss and ds DNA strand breaks (Singh et al., 1989; Hughes et al., 1996; McKelvey Martin et al., 1997). The comet assay has previously been used to correlate sperm DNA damage with implantation success after ICSI (Donnelly et al., 2000; Morris et al., 2002). Aravindan and co-workers (1997) have compared the SCSA with a version of the neutral comet assay (NCA) and the TUNEL assay and found the correlation between SCSA and comet assay to be superior to that between SCSA and TUNEL assay. Because of the superior correlation between the comet assay and the SCSA (Aravindan et al., 1997), we have adopted the NCA of Singh & Stephens (1998) for the assessment of ds DNA breaks as the most relevant DNA lesion. The assay involves mixing the sperm with agarose, making a microgel on microscopic slides from the spermagarose mixture, lysing the embedded sperm to remove membranes and some proteins, treating the sperm with various enzymes to degrade RNA and proteins, electrophoresing the DNA in microgels, staining with the sensitive fluorescent dye YOYO-1 and lastly, visualizing and analysing the fluorescent images. By focussing the comet analysis on free DNA fragments, we have investigated the frequency of sperm with ds DNA breaks in normospermic donors.
Materials and methods Chemicals Chemicals (DNase free) were obtained from Sigma (St Louis, MO, USA) unless indicated otherwise. 2004 Blackwell Publishing Ltd, International Journal of Andrology, 27, 140–146
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Radiation experiments Sperm samples were irradiated at room temperature with a Gamma Cell apparatus, using a Co source, at doses of 5 and 10 Gy¢ and a dose rate of 0.065 Gy/min. Semen samples Ejaculates were obtained from six men, between 20 and 40 years of age and attending the AI donor programme of the infertility clinic at the University Medical Center (Utrecht). Samples selected for this study had concentration and progressive motility in the normal range [WHO guidelines (World Health Organization, 1999)] and were produced in a facility at the hospital. Semen samples were gently shaken on a rollerbank for 20 min to allow rapid and uniform liquefaction. Preparation of sperm samples The samples were diluted 1 : 1 with human tubal fluid medium (HTF, BioWhittakerEurope, Verviers, Belgium) (Quinn et al., 1985), supplemented with 10% pasteurized plasma proteins (Red Cross Blood Transfusion Laboratory, Amsterdam, The Netherlands). Thereafter the samples were divided into three parts. Part 1 was diluted once with HTF, centrifuged at 300 g for 10 min and adjusted to a concentration of about 20.106 sperm/ mL, optimal for comet analysis. This population was assumed to reflect the native sample. Part 2 was gently overlaid with HTF and left in a 5% CO2 incubator for about 1 h at 37 C. Swim up sperm were harvested and checked for motility. Part 3 was diluted once with HTF, layered on a discontinuous Percoll gradient (consisting of 40 and 80% Percoll, Pharmacia, Uppsala, Sweden) (Mortimer, 1990) and centrifuged for 15 min at 300 g. Both high density and low density fractions of the gradient were washed with HTF and used for comet analysis. Single cell electrophoresis ÔNeutralÕ single cell electrophoresis (Singh & Stephens, 1998) was performed with only minor modifications. Briefly, a small sample of sperm (
