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,
From the * Molecular Toxicology Program,
Center for Occupational and Environmental
Health, School of Nursing, and
Department of Biostatistics, University of
California, Los Angeles; || Fertility Solutions
Inc., Cleveland, Ohio; and ¶ National Health and
Environmental Effects Research Laboratory, Office of Research and Development,
Reproductive Toxicology Division US EPA (MD-72), Research Triangle Park, North
Carolina.
| Correspondence to: Dr Wendie A Robbins, Room 5-254 Factor Building, Mail Code 956919, University of California, Los Angeles, Los Angles, CA 90095-6919 (phone: 310-825-8999; fax: 310-206-3241; e-mail: wrobbins{at}sonnet.ucla.edu). |
| Received for publication February 14, 2003; accepted for publication June 16, 2003. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Key words: Snap-freezing, sperm-FISH, TUNEL, neutral comet assay
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A pooled standard reference for sperm DNA/chromatin quality evaluation was created by combining semen from 3 healthy men. Samples were delivered to the UCLA laboratory within 1 hour of ejaculation. Routine semen analysis was conducted according to World Health Organization guidelines (World Health Organization, 1999). The pooled specimen was aliquoted into 2 groups, 1 of which was immediately frozen at -80°C (negative control) and the other X-irradiated (ex vivo) at room temperature with a dose of 15 Gy then frozen (positive control). X-ray irradiation was done at a dose rate of 535 cGy/minute (Mark 1-30 x-ray irradiator; Shephard & Associates, San Fernando, Calif).
Sperm-FISH Analysis for Chromosome Breakage
We used tandem-label sperm-FISH for chromosome breakage, as described in
Rupa and Eastmond (1997) and
modified by Robbins et al
(2001), for buccal cell
analysis. The probe mixture was 26.4 µL Oncor hybrizol hybridization buffer
(Oncor Inc, Gaithersburg, Md), 1.2 µL direct fluorescein isothiocyanate
(FITC)-labeled alpha satellite probe (Vysis Inc, Dowers Grove, Ill) targeting
a small centromeric region of chromosome I, and 2.4 µL direct labeled-Cy3
probe (provided by Dr. D. A. Eastmond, University of California, Riverside)
targeting the adjacent classical satellite II region in the heterochromatin of
chromosome I. Posthybridization washes were conducted according to the method
of Robbins et al (2001).
Slides were counterstained with 9 µL of 0.025 µg/mL DAPI (Sigma
Chemical, St Louis, Mo), mounted in VectaShield mounting media (Vector
Laboratories, Burlingame, Calif), covered by a 22 x 22 mm cover slip,
and stored at 4°C until scoring.
Sperm nuclei were observed via fluorescence microscopy using a Zeiss Axiophot microscope with DAPI/FITC/Texas Red triple-bandpass filter setup (61002) for simultaneous visualization of DAPI plus fluorochromes FITC and Cy3 (Chroma Technology, Brattleboro, Vt). A single-bandpass emission filter for green (41001; exciter 480/40, and emitter 535/50) or for red (41004; exciter 560/55 and emitter 645/75) were used to confirm discrimination of only green or only red signals during scoring. A single scorer systematically analyzed 10 000 distinct sperm cells per sample for aberrations in the 1cen-1q12 region. Hybridized regions with juxtaposed alpha (green) and classical (red) satellite signals were scored as an intact 1cen-1q12 region of chromosome I. A clear separation of greater than or equal to the diameter of the probe region was scored as a break when separated signal intensities were equal. Sperm cells were scored for breaks within the classical satellite region or between the classical and alpha satellite region.
TUNEL Assay for DNA Fragmentation
We conducted the TUNEL assay using the Fluorescein FragEL DNA Fragmentation
Detection Kit (Oncogene Research Products, Cambridge, Mass). In brief, sperm
samples were centrifuged at 1000 x g for 5 minutes at 4°C,
resuspended in 4% formaldehyde (1 x phosphate-buffered saline [PBS]) at
a cell density of 1 x 106 cells/mL, and incubated at room
temperature for 10 minutes. At the end of incubation, samples were centrifuged
at 1000 x g for 5 minutes (at 4°C), resuspended in 80%
ethanol, and stored at 4°C. On storage removal, 10 µL of well-mixed
specimen was smeared onto a clean microscope slide and allowed to air dry at
room temperature for 24 hours. Slides were immersed in 1x Tris-buffered
saline (TBS; FisherBiotech) for 15 minutes at room temperature. Two micrograms
of proteinase K (Amresco, Solon, Ohio; 2 mg/mL proteinase K diluted 1 : 100 in
10 mM Tris [pH 8]) was added to each sample prior to a 5-minute incubation at
room temperature. Sperm was rinsed in 1x TBS and dried. Once dry,
samples were incubated in 35 mL of 1x TdT equilibration buffer
(Fluorescein FragEL) for 30 minutes at room temperature in a 75-mL coplin jar.
Slides were removed, and 60 µL of TdT labeling reaction mix (57 µL
Fluorescein FragEL TdT labeling reaction mix and 3 µl TdT enzyme) was
applied to each slide; the slides were covered with parafilm and incubated at
37°C for 60 minutes in a humidified chamber. After incubation, the
parafilm was removed, and slides were rinsed twice in fresh 1x TBS at
room temperature for 1 minute. A 22 x 22 mm cover slip was mounted using
Fluorescein FragEL Mounting Media, and slides were stored at 4°C. Each
sample was treated in the absence of TdT to serve as its own control. A single
scorer systematically analyzed 2000 cells per sample using a fluorescence
microscope (Zeiss Inc, Thornwood, NY) equipped with a FITC filter (41001;
exciter 480/40 and emitter 535/50) and categorized each cell as bright (a high
concentration of TUNEL-positive signal within the sperm head), light (a low
concentration of signal within the sperm head), or dark (no signal within the
sperm head).
Comet Assay for DNA Fragmentation
Modifications for the comet assay were based on existing methods described
by McKelvey-Martin et al
(1993), Hughes et al
(1996), and Singh and Stephens
(1998). All steps were
conducted under reduced light, to prevent further DNA damage. Semen samples
were thawed at room temperature, and approximately 1 x 105
cells were suspended in 500 µL 0.65% normal melting point (NMP) agarose
(FisherBiotech) and Ca2+- and Mg2+-free PBS at 45°C.
Partially frosted microscope slides were covered with 60 µL of 0.7% NMP
agarose in 1x PBS, to permit the rigid attachment of subsequent agarose
layers. A volume of 50 µL of the sperm cell suspension was layered on top
and covered with a 24 x 50 mm cover slip. After solidification of the
second layer, the cover slip was removed, and a third layer of 150 µL of
0.65% NMP agarose at 45°C was deposited and covered with a 24 x 50
mm cover slip. After solidification of the third layer and the removal of the
cover slip, the slides were incubated in freshly prepared cold lysing solution
(2.5 M sodium chloride, 10 mM Tris base, 100 mM EDTA disodium salt, 1% sodium
lauryl sarcosine, and 1% Triton X-100, all Sigma Chemical [pH 10], with 3.5
mg/35 mL proteinase K, Amresco) for 4 hours at 4°C in a 75-mL coplin jar.
Slides were drained and placed equidistant in a horizontal electrophoresis
unit (Midi-Horizontal System; Fisher Scientific, Pittsburgh, Penn) filled with
fresh, cold 1x neutral electrophoresis running buffer (pH 9; 10x
stock solution consisted of 3 M sodium acetate from FisherBiotech and 1 M Tris
base) to a level 0.25 cm above the slides. Slides remained immersed for 20
minutes, to allow DNA to unwind
(McKelvey-Martin et al, 1993).
Microgel electrophoresis was done using Electrophoresis Power Supply-EPS 601
(Amersham Pharmacia Biotech Inc, Piscataway, NJ) at 5°C for 20 minutes
(final parameters, 20 V, 31 mA, and 1 W). Slides were transferred to the DNA
fixing solution (5 ml 10x stock solution ammonium acetate;
FisherBiotech) in 45 ml of reagent alcohol (90% anhydrous ethyl alcohol, 5%
methyl alcohol, and 5% isopropyl alcohol [v/v]; FisherBiotech). After 15
minutes, slides were placed in 50 mL of reagent alcohol (for 30 minutes) then
air-dried at room temperature. Samples were run in duplicate.
A fresh 50-µL application of 0.1% YOYO-1 iodide (Molecular Probes, Eugene, Ore) in 10% DMSO (FisherBiotech) and 2% sodium dihydrogen phosphate (Sigma) served as the intercalating DNA stain for comet analysis. Slides were viewed using a fluorescence microscope (Zeiss) equipped with a FITC filter combination (41001; exciter 480/40 and emitter 535/50) at x250 magnification (x20 objective, 10 eyepiece, and 1.25 optovar). A single scorer randomly selected and captured 50 cells using the LAI Automated Comet Assay Analysis System (LACAAS) (Loats Associates Inc, Westmister, Md) from each replicate slide (i.e., 100 cells for a given sperm population). LACAAS calculated a corrected image by scanning the uninterrupted background signal above and below the comet being analyzed, to generate an approximation of the actual shape and magnitude of the background field and subtracted that value from the total image to define the comet. Quantitative measurements included comet tail length (the number of pixels from the back of the comet head to the end of the tail), tail moment (the product of migration distance and normalized intensity integrated over the tail length), and percentage of DNA in the tail (the integrated tail intensity x 100 divided by the total integrated cell intensity).
Statistical Evaluation
The hypotheses that chromosome breakage and DNA fragmentation did not
differ between fresh frozen (FF) semen and semen frozen after simulated
shipping (F24) were tested using the 2-sided paired Student's t test.
The 50 comet observations per subject, within each condition, were averaged
prior to using the paired t test. Sperm-FISH data and comet data
(tail moment and tail length) were transformed [reciprocal square root and
log(x + 1), respectively]. TUNEL data were normally distributed.
Within- and between-subject variability was determined using repeated-measures
analysis of variance. Data are presented as group mean ± SE unless
otherwise stated. Calculations were done using Microsoft Excel, Splus
(Insightful, Seattle, Wash), and STATA7 (Stata Press, College Station, Tex)
statistical software packages. P 
.05 was considered to be
statistically significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Pooled standard reference specimens, exposed to x-irradiation (1, 5, 10, and 15 Gy) and measured by neutral comet assay, exhibited dose-dependent DNA damage (Table 2). Interslide variation between the means of control slides showed coefficients of variation ranging from 0.10% to 0.86% in irradiated cells and 1.6% to 3.5% in nonirradiated cells. (Table 3).
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The reproducibility of the comet software was studied using the irradiated and nonirradiated semen references. Twenty cells were chosen at random from positive (Figure 1) and negative (Figure 2) control slides. Each cell was analyzed 10 times (Hughes et al, 1997). Coefficients of variation less than 2.4% for repeated comet tail-length analysis of individual irradiated cells demonstrated reproducibility of the image analysis software for tail length. Coefficients of variation ranged 2.9% to 13.0% for percentage of DNA in the tail and 11.0% to 55.0% for tail moment, which suggests these measures may be less reproducible than comet tail length. Coefficients of variation for tail length in nonirradiated cells averaged 10.2% (data not shown).
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The tandem-label sperm-FISH assay also detected dose-dependent DNA damage in the pooled standard reference exposed ex vivo to x-irradiation. (Table 2). Induced breakage was detected at a lower dose by comet than by sperm-FISH assay. However, both assays demonstrated statistically significant increases in DNA damage with increasing doses of x-irradiation.
Immediate Versus Delayed Freezing
Tandem-label sperm-FISH detected no significant difference in chromosome
breakage between FF and F24 cells; neither breaks within the classical
satellite region (P = .22, Student's t test), nor breaks
between classical and alpha satellite region (P = .71, Student's
t test). Mean frequency of total chromosome breakage in FF sperm was
10.5 ± 1.3 breaks per 10 000 cells scored versus 11.2 ± 1.1
breaks per 10 000 cells in F24 sperm (P = .69, Student's t
test). (Table 4). Sperm-FISH
hybridization efficiency was 99.5%.
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DNA fragmentation detected by TUNEL increased for F24 compared with FF, but this did not reach statistical significance. (Table 5). The mean frequency of DNA strand breakage in FF sperm was 136 ± 29 TUNEL-positive cells per 2000 cells scored versus 213 ± 28 TUNEL-positive cells per 2000 cells in F24 sperm (P = .07, Student's t test). Differences between cells exhibiting a slight signal (light; P = .15, Student's t test) and no signal (dark; P = .07, Student's t test) also did not reach statistical significance as based on sample age.
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One semen sample in the study population had a low sperm concentration (11.6 x 106 cells/mL), which was problematic for the assessment of multiple DNA assays; thus, although all 10 samples were analyzed by sperm-FISH and TUNEL, only 9 samples had adequate cells remaining for the comet assay. Selected comet parameters of the study population are shown in Figure 3. Seven semen samples exhibited less DNA damage (depicted as a decrease in the percentage of DNA in the tail and tail moment) and shorter comet tail lengths after 24 hours of exposure to ambient temperature.
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The neutral comet assay detected a statistically significant difference in DNA damage between FF and F24 sperm (Table 5). FF cells revealed more strand breaks. The mean frequency of double-stranded DNA damage (denoted by tail length in µm) for FF sperm was 175.0 ± 15.5 µm per 100 cells scored versus 152.2 ± 17.6 µm per 100 cells in F24 sperm (P = .033, Student's t test). Differences between FF and F24 sperm were also significant as based on the frequency of double-strand breaks expressed as percentage of DNA in the tail (P = .037, Student's t test) and tail moment (P = .006, Student's t test). Within-subject cell-to-cell variability for moment, percentage of DNA in the tail, and length was approximately 4 times as large as the between-subject variability for these same comet parameters.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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No previous studies have evaluated the clinical or research usefulness of DNA damage parameters in semen samples collected at remote sites and delivered to a central laboratory via overnight shipper. In the current study, our hypothesis was that chromosome/DNA damage in ejaculated spermatozoa would not increase significantly if cells remained in seminal fluid at ambient temperature for 24 hours, a first step in assessing the potential use of remote semen collection for DNA biomarker studies.
Tandem-label sperm-FISH is an efficient and reliable method to detect breakage in sperm chromosomes (Robbins et al, 1997, 2001; Sloter et al, 2000; Ong et al, 2002). The method reduces analysis time while enhancing sensitivity to particular regions that are prone to breakage (Rupa et al, 1995). However, hybridization is limited to only one region, and breakage induced at other sites on chromosome I or throughout the genome will go undetected (Rupa et al, 1995), which is a limitation of this assay if other targeted breakage sites are expected. Exposures that induce breakage randomly across the genome would not be affected by this limitation. Estimations are that 85% of chromosome aberrations found in newborns occur de novo in parental germ cells (Mohrenweiser 1991), with the majority of genetic mutations in sperm due to breakage rather than rearrangement (Donnelly et al, 2001). The ability to detect modifications at breakage-prone areas suggests that tandem-label sperm-FISH is a useful method to quantify early chromosomal alterations in human sperm.
No previous investigations comparing chromosome breakage in fresh frozen semen versus aged specimens were identified. In the current study, our focus was on breakage-prone areas in the pericentric and centromeric regions of chromosome I, in an effort to assess whether conditions during transport would induce damage. The number of chromosome breaks (within the classical satellite region and between the classical and alpha satellite regions) were not statistically different between FF and F24 sperm, which suggests that breakage in this region did not increase under these conditions. The mean frequency of total chromosome breakage in our study was consistent with the results of a recent report of one breakage event in chromosome I per 1000 sperm cells scored (Robbins et al, 2001). Finding no difference in chromosome breakage between FF and F24 sperm suggests that remote semen collection and transport to a central laboratory (using a container such as TRANSEM100) would be possible for field studies looking at chromosome breakage at a level detectable by the tandem-label sperm-FISH assay.
TUNEL can be used to quantitate endogenous DNA strand breaks in sperm nuclear chromatin (Sailer et al, 1995; Smith and Haaf, 1998). No previous investigations comparing endogenous DNA breakage in sperm FF versus F24 specimens were identified. Our focus was to assess whether endogenous DNA damage would increase significantly because of conditions during remote collection and transport. TUNEL approached but did not reach a statistically significant difference in endogenous DNA fragmentation between FF and F24 sperm. The percentage of positive cells detected by TUNEL that have been reported in other laboratories ranged 1% to 40% (Aravindan et al, 1997; Sun et al, 1997). The percentage of TUNEL-positive cells in the current study ranged 2.8% to 18.2% across donors and freezing conditions. TUNEL is specific for endogenous breakage, given that undamaged cells and cells in necrosis or that have been exposed to irradiation are only minimally labeled (Gorczyca et al, 1993). The ability to detect endogenous DNA damage suggests that TUNEL is a useful method to quantify DNA breakage in sperm. Because the power to detect a statistically significant difference between groups was only 75%, further investigation using larger sample sizes is necessary to determine the utility of remote collection kits with overnight return to a central laboratory for TUNEL analysis.
Numerous reports have been published that have used the comet assay to quantitate double-stranded DNA breaks in somatic cells (Östling and Johanson, 1984; Olive et al, 1991, 1995; McKelvey-Martin et al, 1993; Fairbairn et al, 1995; Collins et al, 1997a,b; Marples et al, 1998; Olive, 1999; Rojas et al, 1999; Tice et al, 2000), but it has only recently been adapted for use in human sperm (Singh et al, 1989; Hughes et al, 1996, 1997, 1999; Duty et al, 2002; Morris et al, 2002). A comparison of DNA damage between somatic and germ cells have shown elongated spermatids, and spermatozoa exhibit more damage (Hughes et al, 1996, 1999; Anderson et al, 1997) because the damage is not repaired (Van Loon et al, 1993) because of the loss of cytoplasm and repair enzymes during spermiogenesis (Sega 1974, 1976; Singh et al, 1989; Sega and Generoso 1990; Genesca et al, 1992). Another consideration when using comet analysis for sperm cells is the lack of stoichiometric staining between DNA complexed in the head, with strands of DNA more accessible to stain because they are pulled out in the comet tail. The comet assay continues to undergo modification for studies of human sperm (Perreault et al, 2003).
The ability to detect sperm DNA integrity without introducing further damage through analytical preparation (as might occur during remote collection and overnight shipper to a central laboratory) suggests that the neutral comet assay (pH 9) may be a robust method for assessing double-strand breaks (Singh and Stephens, 1998; Olive, 1999) and thus led us to use it for this first test of remote collection for DNA outcomes. The current study assessed whether DNA damage significantly increased because of conditions during transport compared with immediate freezing. The neutral comet assay detected a significant difference in DNA fragmentation between FF and F24 sperm, measured as tail moment, tail length, and percentage of DNA in the tail. Tail moment, a function of the percentage of DNA in the tail and tail length, was the most sensitive indicator in this data set. The mean frequency of double-stranded DNA damage was lower in aged specimens than in FF sperm.
The discrepancy between statistically significant findings in the comet versus TUNEL data may be due to the small sample size or may stem from a confounding variable such as an alteration in chromatin stability. Exposure to ambient temperature (for 24 hours) may increase the opportunity for sulfhydryl bond formation, yielding a hyperstabilized nucleus that may resist strand separation during electrophoresis. If hyperstabilization occurs, then the addition of dithiothreitol prior to electrophoresis should reduce the S-S bonds and allow proper migration. An increase in sample stabilization may also be achieved by keeping the samples on ice (4°C; Duty et al, 2002) instead of at ambient temperature.
In the current study, standard reference aliquots were developed from pooled semen samples, and a portion were treated with x-ray (15 Gy) to create a positive external standard for comet analysis. The results of numerous studies have confirmed dose-dependent DNA damage using x-rays (Olive et al, 1990, 1995; Hughes et al, 1996, 1999; Singh, 1996; Duty et al, 2002). In general, when running the sperm comet assay, neighboring comets differ markedly in tail moment, tail length, and percentage of DNA in the tail, which suggests that the majority of variation in parameters is due to inherent cell-to-cell differences within specimens (Singh et al, 1989; Duty et al, 2002; Morris et al, 2002). However, as is seen in Table 3, cell-to-cell variability was lower in the irradiated cells. Interslide variation between the means of control slides suggested that variation in nonirradiated cells is the result of biological differences between cells (ie, live vs dead or mature vs immature cells), which is overcome by the damage induced after exposure to x-irradiation. Measurements between slides were highly reproducible, as has been seen in other studies (Hughes et al, 1997; Morris et al, 2002). We found comet tail length to be the most reproducible measurement, which agrees with the results of a recent study by Duty et al (2002).
The tandem-label sperm-FISH, TUNEL, and neutral comet assays are useful analytical techniques for laboratory-based studies of genomic integrity. DNA damage at a level detectable by TUNEL and neutral comet assay, but not tandem label sperm-FISH, may be affected by the remote collection and overnight return of semen specimens to a central laboratory and will require further study using larger sample sizes before their incorporation into reproductive epidemiology studies of ejaculated semen collected at sites remote from the laboratory.
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Acknowledgments |
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Footnotes |
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