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CLINICAL ASSISTED REPRODUCTION
Cryopreservation of Human Cumulus Cells for Co-cultures and Assessment of DNA Damage After Thawing Using the Comet Assay ELISA M. LINDLEY,1 JOHN D. JACOBSON,1 JOHANNAH CORSELLI,1 ALAN KING,1 and PHILIP J. CHAN1,2,3
Submitted: January 22, 2001 Accepted: April 4, 2001
More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.
Purpose: Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation. Methods: Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures ( N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. Results: The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability. Conclusions: The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw.
KEY WORDS: apoptosis; comet assay; cumulus granulosa; single cell gel electrophoresis.
INTRODUCTION The use of cumulus oophorus cells in co-cultures have been shown to result in better morphology embryos (1) and improved blastocyst formation (2). In the ovarian follicle, cumulus cells are derived from granulosa cells but may be distinguished by the location of these cells immediately surrounding the corona radiata cell layer on each oocyte (3), tight-junction intercellular communication, matrix expansion following oocyte maturation, formation of structural passageways for sperm-egg interaction, secrete progesterone to stimulate sperm acrosome reaction, have the ability to affect the rate of oocyte apoptosis (4), improve implantation (5), and give rise to clone offsprings after nuclear transfer (6). Studies of cumulus cell DNA integrity after cryopreservation have been lacking. The information from such studies will be helpful for the efficient cryopreservation of cumulus cells for co-cultures. In this study, the null hypothesis was that all cryoprotectants were equally efficacious for the cryopreservation of cumulus cells. The objective was to use the modified fixed cell comet assay to analyze for DNA damage in cumulus cells that had been frozen by one of three different methods. The name of the assay was based on the comet-like appearance of damaged DNA loops (single and double-stranded) unwinding from a relaxed supercoiled nucleus (7–8). A unique feature here was the use of the comet assay for fixed
1
Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, California 92350. 2 Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, California 92350. 3 To whom correspondence should be addressed at Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, 11370 Anderson Street Suite 3950, Loma Linda, California 92354; e-mail:
[email protected].
C 2001 Plenum Publishing Corporation 1058-0468/01/1000-0534$19.50/0 °
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cells instead of living cells with obvious advantages including convenience, simplicity, and low cost. MATERIALS AND METHODS Collection of Cumulus Cells The cumulus cells were obtained from discarded follicular aspirates that did not contain oocytes and were no longer needed for the in vitro fertilization procedure (IVF). The identities of the patients were unknown and the specimens were anonymous. The research was approved by the Institutional Review Board. Ovulation induction (9) was carried in each female patient (N = 4 cases) after several days of leuprolide acetate suppression by administering recombinant follicle stimulating hormone (average of 4 ampules in split doses daily, 75 I.U. per ampule) for about 9 to 12 days followed by 10,000 units of human chorionic gonadotropin (hCG), generally given on the 10th day where the estradiol was over 500 pg/mL and the largest follicular diameter was over 18 mm. Patients on leuprolide acetate cycles followed a similar induction protocol with the exception that 0.5 mg leuprolide acetate was administered beginning on Day 21 of the previous cycle and ending on the last day of stimulation (9). At about 34–36 h after hCG administration, follicular aspiration was performed by transvaginal ultrasound techniques and the aspirates examined for oocytes. The oocytes were pipetted out and used for the IVF procedure. The darkened sheets of membrana granulosa cells were not used. The remaining expanded cumulus cells from large follicles were pooled and washed through several changes of phosphate buffered saline (PBS; Irvine Scientific, Santa Ana, CA). A small aliquot of the cells was removed and examined for viability using the 0.5% Eosin stain method (10). The cells were divided and transferred into cryogenic vials (Nalgene Co., Rochester, NY) at room temperature (23◦ C) containing either 12:20% glycerol-egg yolk cryoprotectant (sperm freezing medium, Irvine Scientific, Santa Ana, CA), 28% glycerol hypoosmolar medium (sperm maintenance medium, Irvine Scientific, Santa Ana, CA), 40% ethylene glycol, and 0.5 M sucrose dissolved in PBS medium (11), or plain PBS medium (control). The cumulus cells in the control vials were immediately submerged in liquid nitrogen and frozen. The cells in ethylene glycol were kept at room temperature for
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30 s and then plunged into liquid nitrogen. The cells in glycerol-egg yolk or glycerol medium were kept at 4◦ C for 1 h before being stored in liquid nitrogen. The experiment was repeated with cumulus cells from the remaining patients. After 2 days in the frozen state, the cumulus cells were thawed at room temperature for 10 s followed by warming in a water bath at 37◦ C for 5 min. Samples of cumulus cells from each of the vials were pipetted out, smeared on glass slides and air-dried. The air-dried smears were submerged in 50% methanol solution (Fixative Solution, Diff-Quik, Dade Behring Inc., Newark, DE) for 15 s and air-dried. The remaining cells in each vial were analyzed for viability using the 0.5% eosin method (10). Each analysis was repeated and the mean percent viability recorded. Modified Comet Assay for Fixed Cumulus Cells The procedure was same as previously reported (12). The smears of cumulus cells were stained for 5 min in freshly prepared acridine orange solution (13) consisting of 0.2 mg acridine orange (United States Biochemical Corp., Cleveland, Ohio) in 1.0 mL purified water. The slides were washed with water to remove excess stain and air-dried. Following this, selfadhesive sticker labels (Catalog No. 43-540, Preaply, Dennison Co., Framingham, MA) were cut and pasted to form a rectangular frame around the stained cells on each glass slide. The rectangular frame served to anchor the mini-agarose gel layer (14). The 0.8% agarose was prepared by mixing 0.8 g agarose (Catalog No. 15510-019, Life Technologies, GIBCO BRL, Grand Island, NY) in 100 mL of the 1 × TBE solution consisting of 13.5 g Tris-HCl pH 7.5 (Life Technologies, GIBCO BRL, Grand Island, NY), 6.8 g boric acid (Sigma Chemical Co. St. Louis, MO), and 5 mL of 0.5 M EDTA (Sigma Chemical Co. St. Louis, MO) in 1250 mL purified water, and then heating until dissolved and cooling to 45◦ C. The agarose was pipetted on to the surface of each glass slide placed at a slight angle to the bench surface and excess agarose allowed to run off the edges. The agarose gel layer should be made thin for easy viewing. A cover slip was not used for this procedure. Each slide was placed horizontal and once the agarose had solidified, the slides were placed in 4◦ C alkaline lysis buffer consisting of 1% N-lauroylsarcosine (Sigma Chemical Co. St. Louis, MO), 1.0 M TrisHCl pH 7.5 (GIBCO BRL Life Technologies, Grand Island, NY), 0.5 M EDTA (Sigma Chemical Co.,
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Fig. 1. The modified comet assay for cryopreserved-thawed cumulus cells. The dried cumulus cell smear was fixed, stained in acridine orange, layered over with a mini-agarose gel, lysed, and electrophoresis performed. Lighter nuclear DNA with a trailing mass of DNA material forming a “tail” (see arrows) was indicative of DNA damage. Analyses of the fluorescent images showed (A) greatest DNA damage in the control cells with no cryoprotectant, (B) minimal damage with trailing DNA in ethylene glycol, (C) little or no damage in glycerol egg-yolk medium, and (D) glycerol hypoosmolar medium.
St. Louis MO), 0.3 M mercaptoethanol (Sigma Chemical Co., St. Louis, MO) and pH adjusted to >10 with sodium hydroxide pellets. The slides were in the lysis buffer for 1 h to release and unwind the DNA in the cells. The slides were rinsed and placed submerged (1–2 mm from the solution surface) in 1 × TBE solution (pH 10) in the electrophoresis chamber for 10 min to presoak the agarose layer. Constant voltage at 50 V, 0.02 A was then applied for 40 min. After electrophoresis, excess solution was blotted off and each slide was examined using an epifluorescent microscope at 250× magnification. Over 25 cells were analyzed for each slide. The 640 × 480 pixels images (Fig. 1) were recorded using a QuickCam Pro camera (Logitech Inc., Fremont, CA) placed over one of the microscope eyepieces (12). The color of the recorded images were inverted, converted to gray-scale and the pixel intensities analyzed using Paint Shop Pro 6 software (Jasc Software, Inc., Eden Prairie, MN). Variability between images due to changes in the settings was corrected by subtracting the background intensity from the image intensity. The tail moment, a calculated index of DNA damage for each cell, was determined by multiplying the mean length of the tail (measured from the center of the bright nucleus to the end of the trailing tail of DNA material) by the mean fluorescent intensity of the DNA in the trailing tail (15). The tail moment was used instead of the percentage of cells with damaged DNA because the tail moment was more sensitive and resolved subpopulations with subtle variations (15,16).
Statistical Analysis The number of cells to analyze for each slide was based on previous work by Singh and colleagues (8). The pixel data for each type of cryoprotectant as well as cell viability data were stored in a Microsoft Excel spreadsheet and significance tested using the Student’s t-test statistic. Mean difference with P < 0.05 was considered significant. RESULTS The tail moment parameter of DNA from thawed cumulus cells that had been cryopreserved in each of the three different cryoprotectants were significantly lower than that of the control (Table I). In this study, lower values were associated with reduced DNA damage and the cells typically had darker nuclei and shorter or absent comet tails (Fig. 1B–D) when compared with the control (Fig. 1A). The two cryoprotectants that contained glycerol had the best Table I. Comet Assay Analyses of DNA Damage in Cumulus Cells After Cryopreservation in Different Cryoprotectant Media. Higher Tail Moment Values Reflect Greater DNA Damage Treatment
N
No cryoprotectant control 128 Ethylene glycol medium 119 Glycerol–egg-yolk medium 86 Glycerol hypoosmotic medium 98 a
Tail moment of cumulus cell (Mean pixels2 ± SEM) 0.36 ± 0.02 0.23 ± 0.01a 0.21 ± 0.01a 0.20 ± 0.01a
Different from control, P < 0.05.
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Table II. The Viability of Cumulus Cells Assessed by the Eosin Uptake Method After Cryopreservation and Thawing in Different Cryoprotectant Media Treatment Before freezing After freeze-thaw No cryoprotectant control Ethylene glycol medium Glycerol–egg-yolk medium Glycerol hypoosmotic medium a
N
% live (Mean ± SEM)
% loss
246
81.2 ± 0.7
0.0
172 181 168 234
8.5 ± 0.8 51.9 ± 0.9 68.7 ± 2.1∗ 66.2 ± 2.2∗
89.5 36.1 15.4a 18.5a
Different from control and ethylene glycol, P < 0.05.
protection against DNA damage during cryopreservation as shown by low tail moments. When postthaw cell viability was assessed, the two cryoprotectants containing glycerol were associated with significantly higher cumulus cell viability than the ethylene glycol or control treatment (Table II). However, the combination of glycerol and egg-yolk cryoprotectant had the highest cell viability. DISCUSSION The results showed that the cryoprotectants that contained glycerol had the best cell viability and the least DNA damage suggesting that glycerol protected important components of the cumulus cells during the freeze-thaw process. There is a need to preserve, store, and recover as many as possible, viable cumulus cells with normal DNA for co-culture applications as these cells have been shown to be beneficial for zygotes to develop to blastocysts (2). In addition, a recent report suggested that relaxin secreted from these cells enhanced each embryo’s ability to implant in the endometrium (5). In terms of viability, the cryoprotectant containing both glycerol and egg-yolk gave the highest cell viability and least DNA damage suggesting that the glycerol–egg-yolk cryoprotectant was optimal for freezing cumulus cells. This finding was consistent with a recent study that demonstrated the beneficial effect of egg-yolk on cumulus survival postthaw, although the design of that study did not include an assessment of DNA damage (11). Nevertheless, this study confirmed the beneficial effect of egg-yolk on DNA integrity. It was speculated that the mechanism involved the egg-yolk proteins stabilizing the cumulus cell membrane preventing gelation during rapid cooling (11). Surprisingly, the ethylene glycol cryoprotectant was not the best here although this cryoprotectant has been frequently used for freezing embryos (17). This suggested that a high percentage of cumulus cells Journal of Assisted Reproduction and Genetics, Vol. 18, No. 10, 2001
frozen along with the embryos would not survive the freeze-thaw process. The difference in survival rates might be related to cell size, for example, the larger oocyte in comparison to the cumulus cell. Preserving the integrity of the cumulus cell DNA during cryopreservation is essential, especially if the cells are to be used for co-culture (2). Damaged DNA caused by the freezing might result in defective gene products expressed from cumulus cells that would, in turn, affect the survival of embryos in vitro. It is interesting to note that studies on oocyte cryopreservation included freezing both the oocytes and cumulus cells using a single common cryoprotectant. This study suggested that the cumulus cells should be frozen separately from oocytes or embryos, or in the case of frozen ovaries, a cryoprotectant compatible with the survival of both immature oocytes and cumulus-granulosa cells should be used (11,18). This study utilized a modification of the single cell gel electrophoresis or comet assay to assess DNA integrity. Damaged DNA in the form of fragmentation, unwinding, or strand breakages because of the freezing process produced the appearance of a trailing tail of DNA loops from the nucleus after electrophoresis (7,8). Whereas single-stranded DNA breaks could be repaired by the cell, double stranded breaks were considered lethal and were damaging to the cell (19). The comet assay has been modified several times since its introduction and this has been reviewed (15,19–21). A unique feature here was the use of the comet assay to assess cumulus cells dried and fixed on glass slides. Previous studies utilized living cells, particularly lymphocytes, suspended in a miniagarose gel layer (16,21). The advantages of this assay included simplicity, low cost, an intrassay coefficient of variation of 28.2%, a reduction of 3-D visual artefacts associated with suspended cells, and DNA analyses carried out at leisure. Drawbacks included the requirement of a fluorescent microscope, computer, and camera for analyses.
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ln summary, the results suggested that among the three types of cryoprotectant tested, the optimal cryoprotectant for freezing cumulus cells was glycerol– egg-yolk medium. The use of this cryoprotectant resulted in a high percentage of cell viability and DNA integrity. A unique feature in this study was the use of the comet assay to analyze DNA in dried cumulus cells. More studies are still needed to compare the effectiveness of other cryoprotectants such as dimethylsulfoxide (DMSO), and the long-term effect of the cryoprotectants on thawed cumulus cell viability.
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9. Edwards RG, Brody SA: Evaluation and treatment of the infertile woman. In Principles and Practice of Assisted Human Reproduction, RG Edwards, SA Brody (eds), Philadelphia, Saunders, 1996, pp. 150–301 10. Eliasson R, Treschl L: Supravital staining of human spermatozoa. Fertil Steril 1971;22:134–137 11. Isachenko EF, Nayudu PL: Vitrification of mouse germinal vesicle oocytes: Effect of treatment temperature and egg yolk on chromatin and spindle normality and cumulus integrity. Hum Reprod 1999;14:400–408 12. Raman RS, Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A: Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection. Hum Reprod 2001;16:831–835 13. Tejada RI, Mitchell JC, Norman A, Marik JJ, Friedman S: A test for the practical evaluation of male fertility by acridine orange (AO) fluorescence. Fertil Steril 1984;42:87–91 14. Chan PJ, Corselli JU, Patton WC, Jacobson JD, Chan SR, King A: A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes. Fertil Steril 2001;75:1–7 15. Kent CR, Eady JJ, Ross GM, Steel GG: The comet moment as a measure of DNA damage in the comet assay. Int J Radiat Biol 1995;67:655–660 16. Fairbairn DW, Olive PL, O’Neill KL: The comet assay: A comprehensive review. Mutat Res 1995;339:37–59 17. Bautista JA, Kanagawa H: Current status of vitrification of embryos and oocytes in domestic animals: Ethylene glycol as an emerging cryoprotectant of choice. Jpn J Vet Res 1998;45:183– 191 18. Sztein JM, O’Brien MJ, Farley JS, Mobraaten LE, Eppic JJ: Rescue of oocytes from antral follicles of cryopreserved mouse ovaries: Competence to undergo maturation, embryogenesis, and development to term. Hum Reprod 2000;15:567–571 ˇ 19. Collins AR, Dobson VL, Duˇsinska´ M, Kennedy G, Stetina R: The comet assay: What can it really tell us? Mutat Res 1997;375:183–193 20. McKelvey-Martin VJ, Green MHL, Schmezer P, Pool-Zobel BL, De Meo MP, Collins A: The single cell gel electrophoresis assay (comet assay): A European review. Mutat Res 1993;288:47–63 21. Kizilian N, Wilkins RC, Reinhardt P, Ferrarotto C, McLean JRN, McNamee JP: Silver-stained comet assay for detection of apoptosis. Biotechniques 1999;27:926–930
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