Ability of deoxyribonucleic acid damaged sperm to withstand freeze-thaw induced damage during cryopreservation

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1 MALE FACTOR Ability of deoxyribonucleic acid damaged sperm to withstand freeze-thaw induced damage during cryopreservation Satish Kumar Adiga, Ph.D., Zaheer Khan, B.Sc., Dinesh Upadhya, M.Sc., Guruprasad Kalthur, Ph.D., and Pratap Kumar, M.D. Division of Reproductive Medicine, Kasturba Medical College, Manipal University, Manipal, India Objective: To understand the association between sperm DNA damage and the ability of DNA-damaged sperm to withstand the freeze-thaw process during cryopreservation. Design: Experimental prospective study. Setting(s): Embryology research laboratory. Animals: Eight- to 12-week-old Swiss strain male albino mice (Mus musculus). Intervention(s): Sperm carrying a known amount of DNA damage was subjected to cryopreservation and thereafter evaluated for survival and freeze-thaw induced DNA damage. Main Outcome Measure(s): Elucidation of association between the amount of initial sperm DNA damage, cryosurvival, and freeze-thaw induced DNA modification. Result(s): A strong correlation (R ¼ 0.87) was observed between the amount of initial sperm DNA damage and postthaw survival. However, no significant enhancement in DNA damage was observed by the cryopreservation of spermatozoa with various amounts of DNA damage. Conclusion(s): Cryopreservation of DNA-damaged sperm does not deteriorate the DNA quality, but sperm survival is compromised. (Fertil Steril Ò 2009;92: Ó2009 by American Society for Reproductive Medicine.) Key Words: Cryopreservation, DNA damage, sperm DNA, postthaw survival The structural stability of transcriptionally silent paternal chromatin is of vital importance for the fertilization process and early embryonic development. It has been shown that defective spermiogenesis is associated with abnormal remodeling of sperm chromatin and membrane components, which leads to morphologically abnormal spermatozoa. Recently, chromatin structure abnormality and DNA integrity in human and animal sperm, possibly due to cellular damage induced by low-temperature storage of sperm, have been identified by various techniques (1 5). Although numerous studies have reported an increase in the DNA damage (5), apoptosis, and mitochondrial dysfunction (3) due to sperm cryopreservation, there is no information available on the level of DNA damage that can sustain the process of freeze-thawing during cryopreservation. In the present study, we explored the association between the amount of DNA Received June 15, 2008; revised July 4, 2008; accepted July 23, 2008; published online October 1, S.K.A. has nothing to disclose. Z.K. has nothing to disclose. D.U. has nothing to disclose. G.K. has nothing to disclose. P. has nothing to disclose. Supported by a research grant from Kasturba Medical College, Manipal, India. Reprint requests: Satish Kumar Adiga, Ph.D., Division of Reproductive Medicine, Kasturba Medical College, Manipal University, Manipal , India (FAX: ; satish.adiga@manipal.edu). damage present in the sperm and their ability to withstand stress during cryopreservation. Because of ethical restrictions in performing this study in humans, we used mouse sperm as our study model, and g-radiation was used to introduce varying levels of DNA damage. Then we examined the association between sperm DNA damage and the ability of DNA-damaged sperm to withstand the cryopreservation process. MATERIALS AND METHODS Animals Eight- to 12-week-old healthy Swiss albino male mice were used for the experiments. Sperm DNA damage was induced by testicular irradiation with 0-, 2.5-, 5.0-, and 10.0-Gy g-radiation delivered from a cobalt 60 teletherapy unit. At least five animals were used for each data point. The Institutional Animal Ethical Committee s approval was obtained before we performed the experiment. Testicular Irradiation Male mice were anesthetized with use of ketamine 50 mg/kg body weight. The whole body except the testicular area was covered with a lead shield, and the animals were exposed to g-radiation at a rate of 1 Gy/min from the cobalt 60 teletherapy unit /09/$36.00 Fertility and Sterility â Vol. 92, No. 3, September doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 Sperm Extraction and Evaluation Animals were killed by cervical dislocation, and spermatozoa were extracted from the cauda epididymis by squeezing them in 1 ml of Earle s balanced salt solution (EBSS) medium. The sperm suspension was divided into two parts. The first part was used to analyze motility and viability and for terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay to identify the DNA-damaged sperm. The second part was used for cryopreservation. FIGURE 1 The incidence of sperm DNA damage in mouse testicular sperm after exposure to varying doses of g-radiation. The number of TUNEL-positive sperm in the 5- and 10-Gy groups was significantly higher than in the 0-Gy (P<.001) and 2.5-Gy (P<.01) groups. Deoxyribonucleic Acid Damage Analysis One drop of sperm suspension was placed on a poly-l-lysine coated coverslip and was allowed to dry at room temperature. The cells were fixed in 4% paraformaldehyde solution for 30 minutes. The sperm membranes were permeabilized by treating with 0.2% Triton X-100 for 30 minutes and then washed three times with phosphate-buffered saline solution (PBS). The coverslip was incubated in terminal deoxynucleotidyl transferase and nucleotide mix (Apoalert DNA fragmentation assay kit, catalog No ; Clontech, Mountain View, CA) for 1 hour at 37 C in a humidified incubator. The cells were washed three times with PBS, counterstained with propidium iodide solution (10 mg/ml), and mounted on a glass slide. Cells that were positive on TUNEL assay exhibited a strong nuclear green fluorescence that was observed under a fluorescence microscope (Imager-A1; Zeiss, Gottingen, Germany) equipped with a 490 nm excitation filter. A total of 1,000 spermatozoa were assessed per animal in random fields. Deoxyribonucleic acid damage was expressed as percentage of TUNEL-positive spermatozoa. Cryopreservation and Thawing Sperm suspension was mixed with an equal amount of cryoprotective medium containing glycerol, egg yolk, sodium citrate, and fructose. The medium was added dropwise with proper mixing and transferred to a cryogenic vial (Laxbro, Pune, India). The Cryovial was placed for 10 minutes at 4 C, followed by 10 minutes at 0 C, then held in liquid nitrogen vapor for 1 minute, and finally plunged into liquid nitrogen ( 196 C) for storage. After 7 days, the Cryovial was taken from liquid nitrogen and immediately held in running tap water for 5 minutes to accomplish rapid thawing of the sample. The thawed suspension was mixed with 2 ml of EBSS medium, and the cryoprotective medium was completely removed by repeated washing and centrifugation. The sperm pellet was resuspended in 0.5 ml of EBSS medium and evaluated for sperm viability and DNA damage. Statistical Analysis The values were expressed as mean SEM. One-way analysis of variance was done to determine significance levels. Comparisons between the fresh and postthaw samples were performed with post hoc tests. A P value <.05 was considered as statistically significant. RESULTS Influence of Varying Doses of g-irradiation on the Induction of Sperm DNA Damage The amount of sperm DNA damage was quantified by TUNEL assay. A minimum of 1,000 spermatozoa were screened for the incidence of DNA-damaged sperm, and the data were expressed as percent TUNEL-positive cells (mean SEM). The incidence of baseline TUNEL-positive sperm in the 0-Gy group was 0.38% 0.07%. The amount of DNA damage induced by the lowest level of radiation (2.5 Gy) was approximately 1.5-fold higher than in the 0-Gy group (Fig. 1). However, the difference in DNA damage between the 0-Gy and the 2.5-Gy groups was not statistically significant. The incidence of TUNEL-positive sperm was doubled in the 5.0-Gy group compared with the 2.5-Gy group and approximately 3.6 times higher when compared with the 0-Gy group (Fig. 1). The highest dose of radiation used in this study, 10 Gy, resulted in 1.74% DNA-damaged sperm, which was approximately 3.4-fold and 2.0-fold higher than the damage introduced by 0- and 2.5-Gy g-rays, respectively (P<.001). However, the differences between the 5.0- and 10-Gy groups were not statistically significant. Ability of the DNA-Damaged Sperm to Survive After Cryopreservation In our study, the overall DNA damage induced from varying doses of radiation was between 0.38% and 1.74%. On the 960 Adiga et al. Cryopreservation of DNA-damaged sperm Vol. 92, No. 3, September 2009

3 FIGURE 2 Sperm viability before (red bars) and after (green bars) cryopreservation in mouse testicular sperm exposed to various doses of g-radiation. The difference in sperm viability between the respective groups was statistically significantly different (P<.01). *P<.05 vs. 0-, 2.5-, and 5-Gy respective groups. FIGURE 3 Regression analysis showing a strong negative correlation (R ¼ 0.87) between the sperm DNA damage and postthaw survival. basis of this observation, the spermatozoa from each group were subjected to cryopreservation by a conventional method as described in the Materials and Methods section. One week later, the postthaw sperm survival in the control group (0 Gy) was approximately 43.2%, and a similar trend was noticed in the 2.5-Gy group (41%) (Fig. 2), rates that are significantly lower (P<.01) than for noncryopreserved spermatozoa of the respective group. In contrast, a maximum decline (approximately 30%, P<.001) in sperm survival was observed in the 5-Gy group although there was no significant difference in the noncryopreserved sperm viability of all three groups (0, 2.5, and 5 Gy). In the 10-Gy group, the postthaw survival was approximately 24%, which was significantly lower (P<.001) than the initial viability of 42% (Fig. 2). Association Between Sperm Survival and DNA Damage An attempt was made to look into the possible association between the amount of DNA damage in fresh sperm and its ability to withstand the freeze-thaw process during cryopreservation. A regression analysis was performed, and the result showed a strong correlation (R value of 0.87), suggesting a poor survival rate when the initial sperm DNA damage is high (Fig. 3). Sperm DNA Damage Before and After Cryopreservation Because our study showed a decline in postthaw sperm viability with increase in DNA damage, the incidence of freeze-thaw induced DNA damage was studied by performing a TUNEL assay on the frozen-thawed spermatozoa. The spermatozoa from the 0-Gy group showed approximately 0.5% TUNEL-positive cells. This was not significantly different from the DNA damage observed in the spermatozoa before cryopreservation. Similarly, the incidence of TUNELpositive spermatozoa was marginally higher in postthaw spermatozoa of the 2.5- and 5-Gy groups, and this was not significantly different from the noncryopreserved spermatozoa of the respective group. However, in the 10-Gy group, the difference in TUNEL-positive cells between fresh and postthaw spermatozoa was completely lacking (Fig. 4). These results suggest that the cryopreservation process itself does not enhance DNA damage when initial sperm DNA damage is high. DISCUSSION Efficient freezing, archiving, and thawing of sperm are essential techniques to support large-scale research programs using mouse models of human disease. The purpose of this study was to investigate the ability of DNA-damaged sperm to withstand the cryopreservation process. Here, first the sperm DNA integrity was quantified in in vivo irradiated mouse sperm by TUNEL assay, and the ability of spermatozoa with varying amounts of DNA damage to survive the freeze-thaw process during cryopreservation was studied. Fertility and Sterility â 961

4 FIGURE 4 The incidence of sperm DNA damage before (red bars) and after (green bars) cryopreservation. The values between respective groups were statistically nonsignificant. The result showed an increase in DNA damage with increase in radiation dose used to induce DNA damage, and the postthaw survival of such spermatozoa declined with the amount of DNA damage they had before cryopreservation. However, our study did not show any significant increase in the amount of DNA damage by the cryopreservation process as such. During spermiogenesis and epididymal maturation in mammals, the sperm DNA undergoes replacement of nuclear histones by transition proteins and protamines, leading to a highly packaged and condensed chromatin, which is resistant to oxidative insults (6). Further stabilization of the sperm chromatin structure is enhanced by the presence of seminal plasma components (7). Moreover, the integrity of the sperm chromatin structure is an important factor in fertilization and embryo development and can be affected by various factors including cryopreservation. In addition to membrane effects, lipid peroxidation also can damage the DNA. Peroxidation of DNA can lead to chromatin cross-linking, base changes, and DNA strand breaks (6, 8). Several researchers have reported DNA damage in human spermatozoa associated with membrane lipid peroxidation (9 11) and oxidative stress (6, 12 14). Freeze-thawing of equine spermatozoa is also associated with an increase in reactive oxygen species generation (15); consequently, cryopreservation of spermatozoa may subject spermatozoa to oxidative stress and potential DNA damage. The basic hypothesis of this study was that spermatozoa with an increased amount of DNA damage are more susceptible to freeze-thaw induced damage including chromatin modification during cryopreservation. This is the first report, to our knowledge, that demonstrates the association between the varying amounts of DNA damage in sperm and postthaw survival and DNA integrity. A strong correlation between the number of TUNEL-positive cells in fresh spermatozoa and postthaw survival observed in this study indicates that sperm with DNA damage have poor survival by cryopreservation. It is worth noting that, in the literature, there are still inconsistent reports regarding the relationship between sperm chromatin structure and cryopreservation-induced DNA modification (16, 17). Most of the earlier reports evaluated the basic association between DNA integrity and cryopreservation either in animal models or in human sperm but did not address the amount of sperm DNA damage that can tolerate the process of cryopreservation (18 20). Our recent study using the sperm from a different cohort of patients with male factor infertility demonstrated an increased chromatin modification in patients with teratospermia suggesting that sperm with abnormal morphology has a higher risk of undergoing chromatin modification by cryopreservation (5). To understand the association between the amount of sperm DNA damage on cryosurvival, here we introduced varying levels of sperm DNA damage with different doses of g-irradiation, quantified by TUNEL assay, and then addressed the correlation between the amount of DNA damage and freeze-thaw survival and DNA integrity. In addition to sperm DNA damage, other sperm quality characteristics such as motility and viability were analyzed in the sperm before cryopreservation to get detailed information on the effect of g-radiation on the sperm. We kept a 7- day interval between irradiation and cryopreservation to induce adequate DNA damage in sperm because mature spermatozoa are transcriptionally silent, lack functional repair enzymes, and are resistant to radiation-induced damage (21 23). Hence, short time intervals may not induce adequate damage to spermatozoa. The results have shown that the deterioration in DNA integrity of fresh spermatozoa was concurrent with reduced sperm viability indicating the effect of g-radiation on sperm function. The effects of cryopreservation on the integrity of the sperm nucleus are not well characterized. Hence we tested whether the DNA damage in the fresh sperm is modulated by cryopreservation. It is reported that cryopreservation (freezing and thawing) can increase inappropriate chromatin condensation in sperm (24 27). Recently, Chohan et al. (20) noted that normal chromatin packaging significantly decreases after the freeze-thawing procedure in human sperm. However, in our study, the DNA damage was not significantly enhanced especially when spermatozoa had a high amount of DNA damage introduced with use of 10-Gy g-radiation. This result suggests that when the DNA damage is high in sperm, further deterioration of DNA does not occur by cryopreservation, but the functional capabilities such as survival, motility, and fertilizing ability may be compromised in such spermatozoa. Because these results were obtained from the mouse model where chromatin architecture and sensitivity to radiation are 962 Adiga et al. Cryopreservation of DNA-damaged sperm Vol. 92, No. 3, September 2009

5 different from those of human spermatozoa possibly as a result of difference in protamine mass and DNA mass ratio (28), it is anticipated that further studies with human spermatozoa carrying a wide range of DNA damage need to be evaluated. Moreover, a more in-depth study is needed to evaluate the fertilizing ability of postthaw DNA-damaged spermatozoa and the embryonic developmental potential to understand the impact of cryopreservation on DNA-damaged sperm. REFERENCES 1. Evenson DP, Darzynkiewicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;210: Evenson DP, Thompson L, Jost L. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 1994;41: Martin G, Sabido O, Durand P, Levy R. Cryopreservation induces an apoptosis-like mechanism in bull sperm. Biol Reprod 2004;71: Fraser L, Strzezek J. 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