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Biology of Reproduction 67, 854-861 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Germ Cell and Dose-Dependent DNA Damage Measured by the Comet Assay in Murine Spermatozoaa after Testicular X-Irradiation1

Grant A. Haines3,,a, Jolyon H. Hendryb, C. Paul Danielc, and Ian D. Morris2,,a

a School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom b CRC Experimental Radiation Oncology Group Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, United Kingdom c Genetics Unit, Westlakes Research Institute, Moor Row, Cumbria CA24 3JY, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The single-cell gel electrophoresis (Comet) assay has been widely used to measure DNA damage in human sperm in a variety of physiological and pathological conditions. We investigated the effects of in vivo radiation, a known genotoxin, on spermatogenic cells of the mouse testis and examined sperm collected from the vas deferens using the neutral Comet assay. Irradiation of differentiating spermatogonia with 0.25–4 Gy X-rays produced a dose-related increase in DNA damage in sperm collected 45 days later. Increases were found when measuring Comet tail length and percentage of tail DNA, but the greatest changes were in tail moment (a product of tail length and tail DNA). Spermatids, spermatocytes, differentiating spermatogonia, and stem cell spermatogonia were also irradiated in vivo with 4 Gy X-rays. DNA damage was indirectly deduced to occur at all stages. The maximum increase was seen in differentiating spermatogonia. DNA damaged cells were, surprisingly, still detected 120 days after stem cell spermatogonia had been irradiated. The distribution of DNA damage among individual sperm cells after irradiation was heterogeneous. This was seen most clearly when changes in the Comet tail length were measured when there were discrete undamaged and damaged populations. After increasing doses of irradiation, an increasing proportion of cells were found in the damaged population. Because a proportion of undamaged sperm cells remains after all but the highest dose, the possibility of normal fertility remains. However, fertilization with a spermatozoa carrying high amounts of DNA damage could lead to effects as diverse as embryonic death and cancer susceptibility in the offspring.

male reproductive tract, sperm, spermatogenesis, testis, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spermatogenesis is a complex process in which stem cell spermatogonia develop through rounds of cellular multiplication (mitosis and meiosis) and differentiation, eventually resulting in the production of mature spermatozoa. Due to the diverse nature of this process and the wide variety of different cell types within the testis, many potential targets exist for disruption of spermatogenesis and subsequent effects on reproduction. Chemicals, drugs, and physical agents impair or ablate this process [1–4]. The production of genetically competent spermatozoa by testes is central to the process of reproduction because they deliver the male haploid genome to the oocyte. In humans, it has been estimated that approximately 50% of conceptions do not lead to a viable fetus [5], which may be consistent with the presence of genetic damage in the spermatozoa. Men exposed to radiation may become temporarily or permanently sterile [6–9]. Within the testis, proliferating spermatogonia are the most radio-sensitive cell type, whereas spermatozoa and stem cell spermatogonia are relatively radio-resistant. Therefore, expression of the effects of radiation is both dependent on duration of time since exposure as well as dose exposure.

In addition to cytotoxic effects on germ cells and subsequent decreases in sperm counts, it has also been demonstrated that exposure of germ cells to radiation may have mutagenic consequences [6, 7, 10, 11]. Pregnancies resulting from mating with an irradiated male are associated with impaired fetal development, pregnancy loss, and abnormal somatic development in the fetus. Exposure of sperm to radiation in vitro followed by fertilization of oocytes has demonstrated significantly higher frequencies of abnormalities in sperm-derived chromosomes of pronuclear embryos [12]. Evidence also exists that paternal irradiation is associated with higher tumor incidence in offspring [13–15]. These studies suggest that genetic damage induced by radiation in germ cells may not be effectively repaired, and that it manifests itself in mature spermatozoa. This may have potentially harmful consequences if fertilization occurs with sperm that carry a genetically abnormal haploid genome. There is concern for the children of men who have been conceived after men have been exposed to genotoxic agents such as chemotherapy and radiation [16, 17]. Although studies have not proven a link in the human population [18–20], the overwhelming evidence from animal studies suggest that their conclusions should be viewed with caution and further research should be undertaken.

The single-cell gel electrophoresis technique (the Comet assay) measures DNA damage, including double-strand and single-strand breaks, in somatic cells after a variety of genotoxic insults, including in vivo and in vitro radiation [21]. This assay has recently has been applied to both animal and human spermatozoa [22, 23]. The aim of the present study was to use our recently developed Comet assay to examine whether sperm DNA damage reflects the known genotoxicity of testicular irradiation. Our hypothesis was that in vivo irradiation of germ cells within the testis would result in the transmission of DNA damage through the process of spermatogenesis, which can then be detected in spermatozoa. If this was the case, then DNA damage should be related to both the dose of radiation and the type of germ cell receiving the insult. This information is important in order to interpret the many studies of sperm DNA damage in fertile and infertile men, and that are as yet unable to indicate how and where sperm acquire DNA damage or to interpret the risk to the offspring by fertilization with these damaged sperm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Male B6D2F1 mice 12 wk of age were obtained from the Animal Services Unit, Paterson Institute for Cancer Research, Manchester, U.K. All animals were housed under standard animal husbandry conditions (12L:12D, lights-on 0700 h) and allowed access to standard commercial diet and tap water ad libitum. All animal studies were performed according to the requirements of the 1986 United Kingdom Animals (Scientific Procedures) Act.

Testicular X-Irradiation

Mice were restrained in plastic tubular containers and the anterior two thirds of the body were covered with lead sheeting, leaving the final third and testes exposed. The radiation dose was delivered using a 300 kVp X-ray machine (dose rate 0.5 Gy/min-1). The control animals were sham-irradiated. Mice were replaced in their cages immediately after irradiation and groups of five were killed 16, 31, 45, and 120 days later. Sperm from testes and vas deferens of irradiated animals were examined at four time points after irradiation in order to investigate the effects caused by irradiation of specific germ cell stages. The duration of germ cell development from spermatogonia to spermatozoa in the mouse is 41 days. The time points for this study were chosen so that sperm collected from the vas deferens would be derived specifically from irradiated spermatids (16 days), spermatocytes (31 days), differentiating spermatogonia (45 days), or spermatogonial stem cells (120 days) [7, 9].

Collection of Sperm Samples

Levels of DNA damage in spermatozoa were examined after different doses of X-rays were delivered to the testes (0, 0.25, 0.5, 1, 2, and 4 Gy). Sperm were collected 45 days after irradiation, and these represented the progeny of irradiated differentiating spermatogonia.

Animals were weighed and killed by cervical dislocation. The male reproductive tissues were removed and weighed. Both cauda vas deferens were dissected free. Sperm were expelled from the isolated vas deferens into 1 ml of PBS pH 7.4 using a watchmakers forceps. The sperm count in a sample of this suspension was estimated using a Neubauer cytometer. The cell suspension was then snap-frozen in liquid nitrogen, and samples were stored at -70°C until Comet analysis was performed.

Sperm Comet Assay

Spermatozoa were suspended in 1% (w/v) low-melting-point agarose (Type VIIA; Sigma-Aldrich, Poole, U.K.) at a concentration of 1 x 104 cells ml-1. One milliliter of this suspension was applied to the surface of a microscope slide to form a microgel and allowed to set at 4°C for 5 min. Microgels were submersed in cell lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris HCl pH 10.0 containing 1% Triton X-100 and 40 mM dithiothreitol) for 1 h at room temperature and protected from light. Following this initial lysis period, proteinase K was added to the lysis solution (final concentration 10 µg/ml) and additional lysis was performed at 37°C for 2.5 h. Following cell lysis, all slides were washed through three changes of deionized water at 20-min intervals to remove salt and detergent from the microgels. Slides were placed in a horizontal electrophoresis unit and were allowed to equilibrate for 20 min with TBE buffer (10 mM Tris containing 0.08 M boric acid and 0.5 M EDTA pH 8.2) before electrophoresis (25 V, 0.01 A) for 20 min. When electrophoresis was complete the slides were rinsed with water, air-dried, and stored protected from light until analysis [22].

Analysis and Scoring of Comet Slides

Microgels were rehydrated with double-distilled water, and the DNA fluorochrome SYBR Green (1:10 000 dilution; Molecular Probes Europe, Leiden, The Netherlands) was applied for 1 h. Slides were rinsed briefly with double-distilled water and coverslips were applied before image analysis. Cells were visualized at 200x using an epifluorescent microscope (Optiphot 2; Nikon, Kingston upon Thames, U.K.). Each cell had the appearance of a comet, with a brightly fluorescent head and a tail to one side formed by the DNA, which contained strand breaks that were drawn away during electrophoresis. Quantitative image analysis was performed using an intensified solid state CCD camera (Sony CCD-IRIS; I.S.S. Group, Manchester, U.K.) attached to the microscope and linked to the Comet Assay II image analysis software (Perceptive Instruments, Haverhill, Suffolk, U.K.). Samples were run in duplicate, and 50 cells were randomly analyzed per slide for a total of 100 cells per sample and scored for Comet tail parameters as previously defined by Olive [24]. Comet tail length is the maximum distance the damaged DNA migrates from the center of the cell nucleus, the percentage of tail DNA is total DNA that migrates from the nucleus into the comet tail, and the tail moment is a product of the tail length and the percentage of tail DNA, which gives a more integrated measurement of overall DNA damage in the cell.

Statistical Analysis

Significance of differences were determined using ANOVA and the Bonferroni post hoc test, except sperm Comet parameters, which were evaluated using Kruskal-Wallis ANOVA and the Mann-Whitney U-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Dose-Response Effects of Testicular X-Rays

Testicular X-irradiation had no significant effects on body weights of the mice 45 days after treatment. Significant dose-dependent decreases after 45 days of irradiation, however, were evident when testis weights and vas deferens sperm counts were analyzed (Table 1). The first significant decrease in testis weight was found after 1 Gy, although the vas deferens sperm count was reduced after the lowest dose investigated, 0.25 Gy. Testicular X-irradiation with 4 Gy, the highest dose studied, reduced vas deferens sperm count to 16% of control.


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  		TABLE 1. Effects of increasing doses of testicular X-rays on body weight, individual testis weights, and vas deferens sperm content measured 45 days postirradiation. Data shown represent the group means ± SEM (n = 5). Asterisks denote results that are significantly different from control (*P < 0.05, **P < 0.01)

Irradiation of the testis with X-rays between 0 and 4 Gy produced dose-dependent increases in the levels of DNA damage present in spermatozoa collected from the vas deferens (Fig. 1A and Table 2). Statistically significant increases in Comet tail moment, tail length, and percentage of tail DNA were detected after 0.5 Gy X-rays. Irradiation with 1, 2, and 4 Gy resulted in 3-fold, 7-fold, and 10-fold increases in Comet tail moment, respectively. Similar changes were seen for percentage of tail DNA and tail length, although the magnitudes were not as great (e.g., 4 Gy increased the percentage of tail DNA and tail length by 5.7-fold and 2-fold, respectively). Using the data for Comet tail moment and linear regression, a doubling dose (the dose of radiation required to double the background level of DNA damage) of 0.35 Gy was calculated for the induction of DNA damage in spermatozoa after testicular irradiation (Fig. 1B).



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  		FIG. 1. A) Dose-response effects of X-rays on testicular DNA damage, assessed by the Comet tail moment, in vas deferens spermatozoa 45 days after irradiation. Data shown represent means ± SEM for each experimental group (n = 5). Asterisks denote values that are significantly different from control (*P < 0.05, **P < 0.01). B) Linear regression line of dose-response data in order to calculate the radiation doubling dose on sperm DNA damage measured by the Comet assay; n = 5 per treatment. Each data point represents the mean Comet tail moment for one experimental animal after scoring 100 Comets


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  		TABLE 2. Effects of increasing doses of testicular X-rays on sperm Comet measurements, tail length, and % tail DNA, 45 days postirradiation. Data shown represent group means ± SEM (n = 5; 100 cells scored per animal). Asterisks denote results that are significantly different from control (*P < 0.05, **P < 0.01)

Inspection of frequency histograms showed that changes in DNA tail lengths represented the redistribution of sperm from a low DNA damage group into one with high DNA damage (Fig. 2). The figure shows that DNA damage in the control group had a mean tail length between 40 and 50 µm, and as the irradiation dose increased, the fraction of sperm with a short tail length decreased, and there was a corresponding increase in sperm with a length of between 90 and 100 µm. A similar distribution pattern was observed for the tail moment (Fig. 2). However, redistribution from the control population of sperm with greater DNA damage did not appear to result in such a discrete sperm population. The fraction of sperm with abnormal DNA therefore increased with the dose, and for the 0 and 4 Gy groups, was calculated by considering the percentage of sperm with tail lengths longer than 70 µm or with moments greater than 12. For tail lengths at 0 Gy, the fraction of abnormal sperm was 0% (0–4), whereas after 4 Gy this increased to 90% (82–94), and for tail moments it was 0% (0–4) and 80% (78–96), respectively (median and range [n = 5] samples of 100 sperm each).



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  		FIG. 2. Frequency distribution of sperm DNA damage expressed as the Comet assay tail moment. A) Arbitrary units or the Comet assay tail length. B) Micrometers measured 45 days after increasing doses of testicular X-irradiation (0–4 Gy). Mean ± SEM, n = 5. Characteristics of the distribution in each mouse were determined using 100 sperm

Effects of Time after 4 Gy Testicular Irradiation

At all time points after irradiation, the body weights of X-irradiated animals were not significantly different from those of time-matched control animals. Testicular X-irradiation resulted in significant decreases in individual testis weights (Table 3). At the earliest time point (16 days after irradiation), testis weights from irradiated animals had decreased by approximately 55% compared with time matched controls. These decreases in testis weights were sustained through 31 days, but at 45 days after irradiation testis weights showed signs of recovery, although they were still significantly lower than those of control animals. At the final time point (120 days after irradiation), despite further recovery in testis weight by irradiated animals, a significant weight reduction was measured compared with control animals. Testicular irradiation with 4 Gy X-rays also produced marked reductions in vas deferens sperm counts (Tables 1 and 3). This was first evident 31 days after irradiation when sperm counts were approximately 33% of control levels, which resulted from spermatocytes having been killed by irradiation. Further decreases in sperm counts were observed after 45 days when levels were 25% of control sperm counts, when this resulted from the radiation cytotoxicity on differentiating spermatogonia. At 120 days after X-irradiation, sperm arising from spermatogonial stem cells had recovered to numbers that were similar to those from age-matched control animals.


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  		TABLE 3. Effects of 4 Gy testicular X-rays on body weight, individual testis weights, and vas deferens sperm content. Data shown represents group means ± SEM (n = 5). Asterisks denote results that are significantly different from control (*P < 0.05, **P < 0.01)

Sperm recovered from the vas deferens of irradiated animals were analyzed for DNA strand breaks using the Comet assay. Baseline levels of DNA damage in control animals did not alter significantly over the course of the experimental period (Fig. 3 and Table 4). Higher levels of DNA strand breaks were detected in sperm from animals at all time points after irradiation. After 16 days, there were small but significant increases in the values for Comet tail moment (1.3 ± 0.1 control; 3.6 ± 0.8 irradiated) and percentage of tail DNA. At the next time point, 31 days after irradiation, levels of DNA damage in sperm were again significantly higher than those of time matched controls. All Comet parameters were greater than those observed previously at 16 days after irradiation. Sperm recovered from the vas deferens 45 days after testicular exposure to 4 Gy X-rays showed the highest levels of DNA strand breaks detected at any time point. This was reflected in the measurements of Comet tail moment (a 21-fold increase), tail length (a 2.1-fold increase), and the percentage of tail DNA (a 10.5-fold increase; 1.4 ± 0.1 for controls, 30.0 ± 1.4 for irradiated animals). By 120 days after irradiation, levels of DNA damage in spermatozoa were significantly lower than those observed at earlier time points, indicating signs of testicular recovery. However, small but significant differences were detectable between sperm from control and irradiated animals for both Comet tail moment (2.4-fold) and percentage of tail DNA (2-fold), but not tail length.



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  		FIG. 3. DNA damage assessed by the Comet assay in vas deferens spermatozoa after testicular irradiation with 4 Gy X-rays. Effects on Comet tail moment 0, 16, 31, 45, and 120 days after irradiation are shown by closed bars; sham-irradiated controls are shown by open bars. Data shown represent means ± SEM for each experimental group (n = 5). Asterisks denote values that are significantly different from time-matched controls. *P < 0.05, **P < 0.01


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  		TABLE 4. Effects of 4 Gy testicular X-rays on sperm Comet measurements, tail length, and % tail DNA. Data shown represent group means ± SEM (n = 5; 100 cells scored per animal). Asterisks denote results that are significantly different from control (*P < 0.05, **P < 0.01)

The tail length frequency histograms of the time-related DNA damage changes confirmed the dose-response results; there were two DNA-damage populations, the control population was depleted as the damaged population increased (Fig. 4). This redistribution was observed at all times, including 120 days after X-radiation. The maximum increase in the DNA-damage population was observed 45 days after irradiation with 4 Gy. As in the dose-response study, the population with high levels of DNA damage was less discrete when the data were expressed as tail moment (Fig. 4).



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  		FIG. 4. Frequency distribution of sperm DNA damage expressed as the Comet assay tail moment. A) Arbitrary units or the Comet assay tail length. B) Micrometers measured 16, 31, 45, and 120 days after testicular X-irradiation with 4 Gy. The control group, 0 Gy, was sham-irradiated. Means ± SEM, n = 5. Characteristics of the distribution in each mouse were determined using 100 sperm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Paternal exposure to ionizing radiation can produce dramatic effects on reproduction resulting in a decrease in fertility, which may be associated with a drop in sperm production, and an increase in dominant lethal mutations in offspring [9]. Evidence from animal studies suggests that an elevated risk of transmissible defects exists that results from increases in the frequency of germ line mutations [11, 25, 27], predisposing future generations to risks such as cancer [13, 15, 27]. We have previously demonstrated the dose-related induction of DNA strand breaks in spermatozoa following in vitro exposure to ionizing radiation, although this occurs at much higher doses than those used for this in vivo study [22]. We support our previous findings that in vivo exposure of the testis to external radiation at known genotoxic doses damages the DNA in spermatozoa [28]. The current study clearly demonstrates that the magnitude of DNA damage is related to both the dose administered and germ cell types exposed to radiation in situ.

The Comet assay has been widely used to measure DNA damage in human spermatozoa, and some associations between damage and clinical infertility of unknown etiology have previously been shown [29–32]. However, the reasons why DNA damage appears in sperm have not been established. Suggestions have been made that DNA damage may arise from abortive apoptosis within the germ cells of the testis [33], or that this is a posttesticular effect of free radicals generated within the reproductive tract [34]. We have previously reported that DNA damage, measured by the Comet assay, is higher in human sperm obtained during but not before genotoxic chemotherapy for cancer. This may suggest that genotoxins could also play a role in the generation of sperm DNA damage [30]. Experiments to further define the in vivo influences that induce sperm DNA damage have not been reported, with the exception of preliminary experiments from our laboratory. These have shown that exposure of mice testes to a known genotoxin, internal radionuclide and external irradiation, produces a 12-fold increase in sperm DNA damage as measured by the Comet assay [28].

DNA Damage and Radiation Dose

In the present experiment, sperm were collected from the vas deferens 45 days after exposure, and these were differentiating spermatogonia at the time of irradiation. The dose-dependent inhibition of testicular growth and consequential decreases in sperm count are consistent with many previous observations and are a direct consequence of radiation-induced apoptosis of premeiotic germ cells and subsequent maturation depletion of the later cells [2, 6, 7, 35–37]. It is interesting that induction of DNA damage takes place over a very similar range of radiation doses. The first significant change in DNA damage was observed after 0.5 Gy, whereas testis weight was not changed until a dose of 1 Gy had been reached. Testicular weight changes precede changes in sperm count and the latency depends on the germ cells affected, which makes association between cause and effect more difficult to make. However, it is useful to consider that although a drop in vas deferens sperm count to 88% of control was noted after 0.25 Gy, a significant increase in DNA damage was not measured. This could indicate that a cohort of differentiating spermatogonia exist in which the DNA damage efficiently induces apoptosis so that the defective cell is eliminated from the testis [38]. At higher doses, the germ cells with radiation-induced DNA damage survive. Cell death is also increased as evidenced by the further reduction in sperm count after the higher doses. However, in some cells, the apoptotic mechanism is not engaged and damage is carried through spermatogenesis into the spermatozoa. Whether this reflects a subpopulation of spermatogonia that are more radio-resistant or with a less efficient DNA repair process is not clear, although spermatogonial heterogeneity in response to radiation is well described [7].

It is important to note that DNA damage is not uniformly higher in sperm populations, and our original observation of two populations of sperm carrying low or high DNA damage in sperm obtained from irradiated mice was confirmed and extended to other radiation doses and exposed germ cells [28]. This pattern is similar to our observations in human sperm during cancer chemotherapy [30]. The relationship between DNA damage and radiation is normally stochiometric and gradual progression of a single population from low to high DNA damage levels would have been expected [39, 40]. This is the response of somatic cells to radiation and is also seen in spermatozoa after radiation ex vivo [22]. Heterogeneity in the DNA damage response of individual somatic cells to in vitro irradiation has been previously reported and may arise from differences in hypoxia, DNA packing, or repair; however, the presence of two populations in somatic cells was not observed [39]. Although the reasons for the differences in the DNA damage susceptibility of the two germ cell populations are unclear, damage susceptibility may arise from the heterogeneity of the response of spermatogonia to irradiation, most probably depending on the cytoplasmic bridges between cells that influence the induction or repair of DNA damage [7, 25].

Susceptibility of Spermatogenic Cells

The temporal effect of 4 Gy irradiation to the testis has produced further information on the in vivo susceptibility of germ cells to DNA damage. Large doses of radiation (25 Gy and higher) are required to produce significant levels of DNA damage in spermatozoa irradiated ex vivo [12, 22, 23]. This reflects the high radio-resistance of mature spermatozoa, which is a function of the extremely condensed nature of and lack of repair processes in sperm chromatin [41]. It is well established that induction of cell killing requires higher radiation doses to spermatozoa and spermatids, followed by spermatocytes and then differentiating spermatogonia, and this order was recapitulated in the appearance of DNA damage in spermatozoa, suggesting that similar mechanisms determine the response. Studies of DNA minisatellite mutation rates in the offspring of paternally irradiated mice also have suggested that the highest increases in germ line mutation were observed after irradiation of spermatogonia [42]. Spermatozoa from mice 120 days after irradiation originate from germ cells that have undergone approximately four spermatogenic cycles after X-ray exposure. Therefore, spermatozoa collected from these animals must be the progeny of surviving spermatogonial stem cells that were responsible for postirradiation repopulation of the spermatogenic epithelium. Quiescent stem cells are the most radio-resistant spermatogonia [9], and in the present experiment, cell killing in this compartment was not apparent to any significant extent because sperm counts were unchanged and testis weight was only marginally lower. It was surprising that some sperm in these mice contained damaged DNA, albeit not as many as were seen after irradiation of differentiating spermatogonia. This is the first time that DNA damage has been directly measured in sperm arising from the spermatogonial stem cell population, although indirect indications such as the specific locus test show that some paternally transmitted radiation-induced genetic damage is permanent [25].

Significance of Sperm DNA Damage

Single-strand breaks are measured in the alkaline Comet assay and double-strand breaks are measured in the neutral Comet assay that was used in this study [24, 43]. It is of course unlikely that double-strand breaks persist through many rounds of mitosis and meiosis as the spermatogonial stem cells multiply and transform into the haploid spermatozoa. Double-strand breaks are lethal unless they are repaired in a process that may also produce mutations or chromosome damage [44]. However, the Comet assay will detect structural changes in the DNA, which can be changed into breaks during the in vitro measurement because harsh chemical treatments are required to release the DNA from the nuclear matrix of the sperm before electrophoresis [24, 43, 45]. Structural changes in irradiated germ cell DNA may also be more susceptible to the endogenous DNases, which break the strands to aid rearrangement of chromatin during packing of the male genome in the elongating spermatid [46]. Whatever the explanation, the present data suggest that genetic damage induced in testicular germ cells can be transmitted through the process of spermatogenesis and measured by the Comet assay. This has important implications for fertilization and the genetic integrity of offspring.

The biological significance of the changes in DNA damage measured by the Comet assay is not clear. Previous studies that have examined the frequency of dominant lethal mutations following testicular irradiation have suggested that the highest incidences of embryonic lethality are observed following irradiation of early spermatids and late spermatocytes [47, 48]. A substantial study of the genetic damage in offspring from irradiated male mice showed that irradiation of pachytene spermatocytes and early spermatids produced the maximal rate of genetic changes [25]. These results are not consistent with the pattern of increase in DNA damage measured by the Comet assay because the maximal increase occurred after irradiation of differentiating spermatogonia. This suggests that the assay is not predictive for subtle mutational changes such as specific locus mutations. However, DNA damage measured by the Comet assay reflects the incidence of infertility that occurs later after irradiating differentiating spermatogonia and could therefore contribute to the failure of fertilization, implantation, and development, which hitherto has been ascribed to the reduction of sperm numbers to less than 10% of control [48]. DNA damage measured by the Comet assay in human spermatozoa has been shown to be correlated with abnormal embryo development in vitro after intracytoplasmic sperm injection [49].

Other methods have been used to measure DNA damage in rodent spermatozoa after in vivo irradiation of the testis such as alkaline elution [50, 51] and mutation frequency analysis in a Lac-Z transgene [52]. The sperm chromatin structure assay (SCSA) has measured radiation-induced DNA damage over a similar dose range (0.125–4 Gy) and timing (40 days) as the present experiments [53], and the dose-response curve is very similar. It is interesting that the first significant increase in SCSA was reported after 0.25 Gy, compared with 0.5 Gy in the Comet assay. This may indicate that SCSA is more sensitive than the Comet assay when used to detect radiation-induced DNA damage in spermatozoa. However, the known variation in sensitivities of germ cells to radiation between strains of mice precludes such a conclusion [8]. Radiation-induced germ line mutations in minisatellite loci arising in premeiotic germ cells have demonstrated that paternal effects in offspring occur over a similar dose range to those induced by DNA damage that is detectable by the Comet assay [54]. The gonadal doubling dose for this and other paternally transmitted mutational events such as specific locus and dominant cataract range from 0.17 to 0.56 Gy [55]. The doubling dose for sperm DNA damage after irradiation of differentiating spermatogonial cells was 0.35 Gy. Although the damage we have measured may not be causally related to these mutational events, DNA damage measured by the Comet assay appears to be a useful biomarker for genotoxicity in germ cells. The consequence of this type or other mutational damage for the health of offspring is uncertain.

In conclusion, the Comet assay, which has been used extensively to investigate DNA damage of unknown origin in human spermatozoa, has been shown to be able to detect DNA damage in spermatozoa after in vivo treatment of murine testes with radiation, a known DNA damaging agent. These data further establish the utility of the Comet assay for investigating DNA damage in sperm and its biological relevance. This assay should complement data from other DNA damage assays such as SCSA because it does not require large numbers of sperm and expensive, complicated equipment. The level of DNA damage is dependent on both the radiation dose and the type of germ cell, and occurs at doses known to be relevant to testicular and reproductive toxicity. We have also shown that sperm samples from treated animals carry populations with normal or elevated DNA damage. How these are related to reproductive outcome is not clear. If the statistical probability of damaged sperm to fertilize the oocyte and promote early development is unchanged, then we propose that sperm DNA damage profiles will be an important consideration for the investigation of the paternal contribution to normal and abnormal reproduction and development.


   FOOTNOTES
 
First decision: 27 February 2002.

1 G.H. was supported by United Kingdom BBSRC. J.H. was supported by the United Kingdom CRC. Back

2 Correspondence: I.D. Morris, School of Biological Sciences, G38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K. FAX: 0161 275 5600; i.morris{at}man.ac.uk Back

3 Current address: Department of Genetic Toxicology, Huntingdon Life Sciences, Eye Research Centre, Suffolk PE28 4HS, U.K Back

Accepted: April 24, 2002.

Received: February 8, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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