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Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol™, a cytoskeleton stabilizer
FERTILITY AND STERILITY威 VOL. 75, NO. 6, JUNE 2001 Copyright ©2001 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol™, a cytoskeleton stabilizer Sung E. Park, M.S.,a Hyung M. Chung, Ph.D.,a Kwang Y. Cha, M.D.,a Woo S. Hwang, D.V.M., Ph.D.,b Eun S. Lee, D.V.M., Ph.D.,c and Jeong M. Lim, D.V.M., Ph.D.b College of Medicine, Pochon CHA University, Seoul, Korea
Received September 5, 2000; revised and accepted January 3, 2001. This work was supported by a grant (no. 1999-2205-002-5) from the Interdisciplinary Research Program of the Korea Science and Engineering Foundation (KOSEF). Reprint requests: Jeong M. Lim, D.V.M., Ph.D., Gamete Biotechnology Laboratory, School of Agricultural Biotechnology, Seoul National University, 103 Seodun-dong, Suwon 441744, Korea (E-mail: [email protected]). a Infertility Medical Center of CHA General Hospital. b School of Agricultural Biotechnology, Seoul National University, Suwon, Korea. c Department of Veterinary Medicine, Kangwon National University, Chunchon, Korea. 0015-0282/01/$20.00 PII S0015-0282(01)01809-X
Objective: To establish an effective cryopreservation method. Design: In vitro model study. Setting: Infertility Medical Center, Pochon CHA University. Animal(s): Four-week-old ICR mice superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. Intervention(s): Vitrified-thawed oocytes were fertilized and subsequently cultured in vitro. Main Outcome Measure(s): Post-thawed development, chromosome/spindle normalities, and blastocyst quality. Result(s): More cumulus-enclosed oocytes were fertilized and developed to the 8-cell stage after vitrification and thawing than denuded oocytes. However, cryopreserved oocytes of both types had lower spindle and chromosome normalities than fresh oocytes, which resulted in reduced developmental competence after thawing. The addition of 1 M of Taxol™, a cytoskeleton stabilizer, to vitrification solution greatly promoted the blastocyst formation of vitrified-thawed oocytes, compared with no addition (24.0% vs. 58.6%). No difference in blastocyst quality, which was evaluated by blastomere and inner cell mass cell numbers and inner cell mass cell per trophoblast ratio, was found between fresh oocytes and oocytes vitrified with Taxol™. Conclusion(s): A vitrification solution consisting of 5.5 M ethylene glycol, 1.0 M sucrose, 10% fetal bovine serum, and 1 M Taxol™ greatly improved post-thawed development of vitrified oocytes. (Fertil Steril威 2001;75:1177– 84. ©2001 by American Society for Reproductive Medicine.) Key Words: Mouse, oocyte, vitrification, Taxol™, cumulus cells
In our previous studies (1–3), we developed an effective culture system for supporting preimplantation development of outbred ICR mouse embryos. This system employs modified preimplantation-1 medium (mP-1), which contains amino acids, ethylenediaminetetraacetic (EDTA) acid, glucose, hemoglobin, and polyvinyl alcohol (PVA). Using this chemically defined medium, more than 65% of in vitro– derived 1-cell embryos could develop to blastocysts with improved quality. This culture system will become not only one of the core factors for a disease model mouse production system, but also a good culture model for improving human in vitro fertilization (IVF)– embryo transfer (ET) programs.
Now we have turned our attention to establishing a mouse oocyte bank, which uses our developed embryo culture system. Selection of an oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. In this study, we attempted to employ a vitrification method using ethylene glycol (EG) and an electron microscope (EM) grid for the cryopreservation of mouse oocytes, because this could yield higher post-thawed survival and subsequent embryo development than conventional slow freezing methods (4 – 5). The vitrification procedure, however, still leaves much room for improvement in fully regaining developmental competence after thawing, because it employs cryoprotectants of 1177
high molar concentration that induce cytoskeletal damage (6 – 8). This damage directly causes abnormal progression of meiotic division and the retardation of embryo development. Stabilizing the cytoskeleton system during vitrification is likely beneficial for improving post-thawed survival and subsequent development of vitrified oocytes.
velopment of stored oocytes. The objective of this study, therefore, was to establish an effective cryopreservation method for the mouse oocyte bank system, which could provide additional information for improving the efficacy of a human oocyte bank.
With this in mind, we first examined the feasibility of the vitrification method in the long-term preservation of ICR mouse oocytes. We evaluated the developmental competence of oocytes vitrified and thawed at the mature stage and subsequently examined the incidence of chromosome and spindle abnormalities after thawing. In the second set of experiments, we examined whether the addition of a cytoskeleton stabilizer, Taxol™, to the vitrification solution could promote the post-thawed survival and subsequent de-
MATERIALS AND METHODS Collection of Oocytes Four-week-old female ICR mice were maintained under controlled lighting conditions (14L: 10D) and superovulated by the injection of 5 IU of PMSG (Folligon; Intervet Co., The Netherlands) followed by the injection of 5 IU of hCG (Chorulon; Intervet) 48 hours apart. Mature oocytes with expanded cumulus cells and with a first polar body were
FIGURE 1 Procedure of mouse oocyte vitrification using electron microscopic grid. (A), Oocyte mounting on EM grid during final equilibration procedure; (B), liquid nitrogen plunging procedure; and (C), a combined structure of cryovial cap and goblet for ordered placement of grid in liquid nitrogen.
Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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TABLE 1 Preimplantation development of cumulus-enclosed or cumulus-free oocytes vitrified and thawed at the mature stage. No. (%)d of inseminated oocytes developed to
No. of oocytes
Treatments
Status of oocytesa
Examined
Survived after thawing (%)b
No. of oocytes fertilized (%)c
4-Cell 48e
8-Cell 72e
Morula 84e
Blastocyst 96e
None Vitrification Vitrification
Cumulus-enclosed Cumulus-enclosed Cumulus-free
77 90 80
77 (100)f 72 (80)g 59 (73.8)g
72 (93.5)f 45 (62.5)g 28 (47.5)h
59 (81.9)f 23 (51.1)g 11 (39.3)g,h
52 (72.2)f 16 (35.6)g 4 (14.3)h
48 (66.7)f 10 (22.2)g 3 (10.7)g,h
46 (63.9)f 10 (22.2)g 2 (7.1)g
Model effect of the treatments, which was indicated as P value, was less than .0001 in each parameter. a ICR mouse oocytes that were mature at the time of retrieval were provided. b Percentage of the no. of oocytes examined. c Percentage of the no. of oocytes survived after the treatments. d Percentage of the no. of oocytes fertilized. e Hours after culture. f,g,h Different superscripts within each column are significantly different, P ⬍ .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
retrieved from superovulated mice 14 hours after hCG injection. Either intact cumulus-oocyte complexes (COCs) or oocytes denuded by repeated pipetting in a 0.1% hyaluronidase solution were provided for this experiment.
Vitrification and Thawing of Oocytes COCs or denuded oocytes were pre-equilibrated for 2.5 minutes in 2 mL of Dulbecco’s phosphate buffered saline (DPBS; Gibco BRL, Grand Island, NY) supplemented with 1.5 M of EG (E-9129; Sigma, St. Louis, MO) and 10% (v/v) fetal bovine serum (FBS; Gibco BRL), with and without 1 M Taxol™, at 37°C. Oocytes were then placed for the final equilibration in the same volume of DPBS supplemented with 5.5 M of EG, 1.0 M of sucrose, and 10% FBS, with and without 1 M Taxol™, for 20 seconds. In the meantime, 10 –20 oocytes were mounted on an EM grid (Gilder, Westchester, PA) using a fine pipette, and excess cryoprotectant solution was removed with the underlying sterilized filter paper. The grids containing oocytes were immediately plunged into liquid nitrogen, and a cryovial cap and goblet
were used for ordered placement of the grid. The grids were then stored for 1 hour to 7 days. For thawing, the grids were sequentially transferred to culture dishes containing 2 mL of 10% (v/v) FBS-containing DPBS, to which 1.0, 0.5, 0.25, 0.125, or 0 M of sucrose was added, at intervals of 2.5 minutes at 37°C. After washing 4 – 6 times, thawed oocytes were cultured for 2 hours and used in each experiment. Oocyte mounting onto the EM grid during the equilibration, the liquid nitrogen plunging procedure, and the combined goblet and cryovial cap structure for the storage of the grid are depicted in Figure 1.
In Vitro Fertilization Either fresh or vitrified-thawed oocytes were inseminated in vitro with 1.0⫻106 epididymal spermatozoa/mL in Tyrode’s 6 medium supplemented with fatty acid-free BSA (cat. no. A-4161, 15 mg/mL; Sigma). Inseminated oocytes were freed from spermatozoa at 4 hours after in vitro insemination.
TABLE 2 Spindle normality of cumulus-enclosed or cumulus-free oocytes after vitrification and thawing at the mature stage. No. of oocytes
Treatments
Examineda
Analyzed
With normal spindle (%)b
No. of abnormal oocytes
Cumulus-enclosed Cumulus-enclosed Cumulus-free
120 142 130
104 124 109
83 (79.8)c 84 (67.7)d 66 (60.6)d
21 40 43
Status of oocytes None Vitrified and thawed Vitrified and thawed
Model effect of the treatments on the spindle normality (indicated as P value) was .0069. a ICR mouse oocytes that maintained normal morphology after thawing were provided. b Percentage of the no. of oocytes analyzed. c,d Different superscripts within the same parameter are significantly different, P ⬍ .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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FIGURE 2 Chromosomes and spindle configurations of mouse mature oocytes. (A), Normal barrel-shaped spindle with chromosome. (B), Abnormal appearance of a disorganized spindle with chromosome. Arrows indicate chromosomes.
Sigma), EDTA (0.1 mM), PVA (0.05 mg/ml), 10 g/mL gentamycin, and 5 mg/ml phenol red. This medium does not contain glucose, inorganic phosphate, or protein, and the osmolarity and pH of the medium were within the ranges of 295–305 mOsm and 7.34 –7.38, respectively. Media used for embryo culture were equilibrated in such an atmosphere for at least for 3 hours before culture, and the droplets of equilibrated medium were covered with warm mineral oil (BDH Co., Poole, England). A group of 10 –15 1-cell embryos were cultured in a 5-L droplet of mP-1 at 37°C, 5% CO2 in air atmosphere, and embryos were transferred to the same volume of glucosecontaining mP-1 at 48 hours after culture. The number of embryos developed to the 4-cell, 8-cell, morula, and blastocyst stages were monitored under a stereo microscope at 48, 72, 84, and 96 hours after culture, respectively.
Chromosome Analysis Chromosome preparation for analyzing the numeric normality of oocyte chromosome was performed by the method of Tarkowski (9). Briefly, oocytes were mounted onto a grease-free slide after gradual fixation using water–methanol– glacial acetic acid solutions of different mix ratios. After drying for 1 hour, fixed oocytes were subsequently stained with 10% (v/v) Giemsa solution. The number of chromosomes in each spread was counted under an inverted microscope.
Immunostaining for Tubulin Fresh or vitrified-thawed oocytes were freed from the zona pellucida by treatment in 0.5% (v/v) acid Tyrode’s solution. These zona-free oocytes were treated in microtubule-stabilizing buffer containing 25% (v/v) 25 mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 0.5 mM MgCl2, 10 mM ethylene glycerol-bis (-aminoethyl ether)-N, N, N, N-tetraacetic acid, 50 mM KCl, 25 M phenylmethyl sulfonyl fluoride, and 2% (v/v) Triton X-100 for 60 minutes. Subsequently, the oocytes were attached to siliconized slides and fixed with absolute methanol at ⫺20°C for 7 minutes.
Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
Culture of Embryos and Assessment of Preimplantation Development The basic medium used for the culture of embryos was mP-1 (3), which consists of 101.6 mM NaCl, 4.69 mM KCl, 2.04 mM CaCl2 䡠 2H20, 0.2 mM MgSO4 䡠 7H2O, 0.33 mM sodium pyruvate, 21.4 mM sodium lactate, 25 mM NaHCO3, 0.15 mg/mL sodium citrate, MEM nonessential (0.5%, v/v) and essential (1%, v/v) amino acid solutions (Gibco BRL), hemoglobin (1 g/mL, methemoglobin, cat. no. H-7379, 1180 Park et al.
Mouse oocyte vitrification using Taxol™
After rehydration in phosphate-buffered saline (PBS; Gibco BRL), oocytes were incubated with monoclonal antitubulin antibody for 60 minutes at 37°C followed by washing in PBS supplemented with 0.01% (v/v) Triton X-100. The intensity of tubulin staining was amplified by incubating the oocytes with tetramethylrhodamine isothiocyanate– conjugated goat anti-mouse immunoglobulin G (1/200; Biodesign, ME) in PBS for 60 minutes at 37°C. Hoechst 33258 (40 g/mL; Sigma) was used for counterstaining chromosomes. Oocytes were then examined under an uprighted microscope with epifluorescent apparatus (Leiz, Wetzlar, Germany). Hoechst 33258 staining was visualized by using a UVA filter (BP, 340 –380 nm), and a rhodamine signal was observed by using an N2.1 filter (BP, 515–560 nm). Vol. 75, No. 6, June 2001
Assessment of the Number of Blastocysts and Inner Cell Mass Cells To evaluate the quality of blastocysts, total blastomere, trophoblast, and inner cell mass (ICM) cell numbers were counted by the method of Hardy et al. (10) with a slight modification. The zona pellucida of blastocysts was removed by 0.5% (v/v) protease solution (cat. no. P-8811, Sigma), and the blastocysts were placed in 15 mM trinitrobenzene sulfonic acid (cat. no. P-2297, Sigma) for 15 minutes at 4°C. Blastocysts were then incubated for 10 minutes in Tyrode’s lactate solution supplemented with 25 mM HEPES and 0.1 mg/mL anti-dinitrophenol-BSA (cat no. 61-007-1, ICN, Irvine, CA) at 39°C. Subsequently, they were treated with 0.01 mg/mL propidium iodide (cat. no. P-4170, Sigma) and incubated with 15% (v/v) guinea pig complement (cat. no. S-1639, Sigma) for 20 –30 minutes at 39°C. The blastocysts were then placed in absolute ethanol solution supplemented with 0.05 mM fluorochrome bisbenzimide (cat. no. B-2261, Sigma) overnight at 4°C. After washing in absolute ethanol, blastocysts containing cells stained with different fluorescent dyes were mounted on a glass slide and cell numbers were examined under an upright microscope with an epifluorescent apparatus (Leiz).
Experimental Design In experiment 1, either COCs or denuded oocytes were vitrified, and the survival and subsequent development of the oocytes after thawing were compared with those of fresh oocytes. In experiments 2 and 3, both chromosome and spindle normalities of vitrified oocytes were examined at 2 hours after thawing, respectively, and those of fresh oocytes were also evaluated as a control. In experiment 4, COCs were vitrified with a cryoprotectant solution supplemented with and without 1 M Taxol™, and survival and subsequent development were monitored. In experiment 5, the number of blastomeres and ICM cells, and the ratio of ICM cell per trophoblast in blastocysts derived from either fresh oocytes or oocytes vitrified and thawed with Taxol™ were examined at 96 hours after culture.
Statistical Analysis Each experiment was replicated four (experiments 1, 3, 4, and 5) or five (experiment 2) times. The embryos that developed to the 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages were scored and subjected to analysis of variance using the generalized linear model (PROC-GLM) in the SAS program (11). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least-squares method. P⬍.05 was considered statistically significant.
RESULTS Experiment 1 As shown in Table 1, a significant (P⬍.0001) model effect was found in all experimental parameters. COCs had FERTILITY & STERILITY威
TABLE 3 Chromosome normality of cumulus-enclosed or cumulusfree oocytes after vitrification and thawing at the mature stage. No. of oocytes
Stature of oocytes None Vitrified/thawed Vitrified/thawed
Treatments Cumulus-enclosed Cumulus-enclosed Cumulus-free
With normal chromosome Examineda Analyzed (%)b 95 110 104
46 58 55
37 (80.4)c 39 (67.2)d 36 (65.5)d
Model effect of the treatments on the chromosome normality (indicated as P value) was .004. a ICR mouse oocytes that maintained normal morphology after thawing were provided. b Percentage of the no. of oocytes analyzed. c,d Different superscripts within the same parameter are significantly different, P .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
higher developmental competence after vitrification and thawing than denuded oocytes, and the statistical significances were found in the rates of fertilization (47.5% vs. 62.5%) and development to the 4-cell (39.3% vs. 51.1%) and 8-cell (14.3% vs. 35.6%) stages. In spite of these improvements, higher rates of fertilization (93.5%) and development to the 4-cell (81.9%), 8-cell (72.2%), morula (66.7%), and blastocyst stages (63.9%) were observed in fresh oocytes than in vitrified oocytes of any category.
Experiment 2 The rates of post-thawed spindle normality were 79.8% in fresh oocytes, 67.7% in vitrified-thawed COCs, and 60.6% in vitrified-thawed denuded oocytes (Table 2), and there was a significant difference in the spindle normality between fresh and vitrified oocytes (model effect ⫽ .0069). Normal spindles appeared as fine microtubules traversing the metaphase plate and forming the classic barrel shape (Fig. 2). The spindle structure was frequently observed in the peripheral region adjacent to the polar body, and the chromosomes were aligned on the metaphase plate (Fig. 2A). A disorganized spindle in any part of the ooplasm or spindles of non-barrel shape were considered abnormal (Fig. 2B).
Experiment 3 As shown in Table 3, a significant decrease in chromosome normality, which was indicated as a high model effect (P ⫽ .004), was found. The incidence of chromosome normality was lower in vitrified oocytes with or without cumulus cells (65.5%– 67.2%) than in fresh oocytes (80.4%).
Experiment 4 Significant (P⬍.0002) model effects were found in the number of oocytes developed beyond the 4-cell stage. More oocytes developed to the 4-cell (48% vs. 84.4%), 8-cell 1181
TABLE 4 Effects of the addition of Taxol™ (1 g/mL), a cytoskeleton stabilizer, to cryoprotectant solution on the preimplantation development of ICR mouse cumulus-enclosed oocytes vitrified and thawed at the mature stage. No. (%)c of inseminated oocytes developed to
No. of oocytes Cryoprotectant with (⫹) or without (⫺) Taxol™ ⫺ ⫹
Examined
Survived after thawing (%)a
No. of oocytes fertilized (%)b
4-cell 48d
8-cell 72d
76 83
63 (82.8) 74 (89.1)
50 (79.3) 58 (78.3)
24 (48.0)e 49 (84.4)f
17 (34.0) 41 (70.6)f
e
Morula 84d
Blastocyst 96d
13 (26.0)e 37 (63.7)f
12 (24.0)e 34 (58.6)f
Based vitrification solution was a 10% (v/v) fetal bovine serum– containing preimplantation-1 medium supplemented with 5.5 M ethylene glycol and 1.0 M sucrose. Model effects of the treatments indicated as P value were .2561, .8890, .0001, .0001, .0001, and .0002 in the no. of oocytes survived, fertilized, and developed to the 4-cell, 8-cell, morula, and blastocyst stages, respectively. a Percentage of the no. of oocytes examined. b Percentage of the no. of oocytes that survived after the treatments. c Percentage of the no. of oocytes fertilized. d Hours after culture. e,f Different superscripts within each column are significantly different, P .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
(34% vs. 70.6%), morula (26% vs. 63.7%), and blastocyst (24% vs. 58.6%) stages after the addition of Taxol™ to the cryoprotectant than after no addition (Table 4). Blastocysts derived from oocytes vitrified with Taxol™ could develop to the expanded blastocyst stage and had normal morphology (Fig. 3).
cells to trophoblasts (0.25 ⫾ 0.01 to 0.26 ⫾ 0.01) were similar between the two groups.
Experiment 5
The results of this study clearly demonstrate that the addition of Taxol™, a cytoskeleton stabilizer, significantly improved the post-thaw development of cumulus-enclosed ICR mouse oocytes vitrified at the mature stage. Compared with no treatment, Taxol™-treated COCs doubled the rate of blastocyst formation after vitrification and thawing (24% to 58.6%). This result further suggests that damage in the oocyte cytoskeleton system is one of the main cryoinjuries in a vitrification program.
As shown in Table 5, no significant model effect was found in all experimental parameters (P⬎.2403). The mean cell number of blastocysts (65.8 ⫾ 3.9 to 69.6 ⫾ 3.1) and ICM cells (13.6 ⫾ 0.9 to 13.9 ⫾ 0.7) and the ratio of ICM
FIGURE 3 Morphology of blastocysts derived from oocytes vitrified with Taxol™. Fully expanded blastocele with distinct inner cell mass is visible (arrow), ⫻200.
DISCUSSION
TABLE 5 Effects of the addition of Taxol™ (1 g/mL), a cytoskeleton stabilizer, to cryoprotectant solution on the quality of blastocysts derived from oocytes vitrified and thawed at the mature stage. No. of blastocysts
Cell nos. (mean ⫾ SE) of blastocysts
ICM cell/ Successfully Total trophoblast Treatments Examined stained (%) blastomeres ICM cells ratio None Vitrification
51 34
38 (74.5) 24 (70.6)
69.6 ⫾ 3.1 13.9 ⫻ 0.7 0.25 ⫾ 0.01 65.8 ⫾ 3.9 13.6 ⫾ 0.9 0.26 ⫾ 0.01
Model effect (P value) in the cell numbers of total blastomere and inner cell mass (ICM) cells, and the ratio of ICM cells to trophoblasts were .7059, .9567, and .2403, respectively. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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It is debatable whether cumulus cells, which encompass the outermost layer of oocytes, are beneficial for enhancing post-thaw survival and subsequent development of oocytes after cryopreservation (12, 13). In our study, intact COCs had a higher developmental competence than denuded oocytes. The results of this study suggest that the vitrification of COCs is a good cryopreservation strategy for promoting oocyte survival after thawing. Ashwood-Smith et al. (12) hypothesized that cumulus cells might have a protective action against the cryoprotectant. They may help oocytes to maintain a rigid structure, which prevents morphologic damage during cryopreservation. Nevertheless, the post-thaw developmental competence of vitrified-thawed COCs was significantly lower than that of fresh COCs (Table 1). Furthermore, a high incidence of spindle and chromosome abnormalities was detected in thawed oocytes even after vitrification with cumulus cells (Tables 2–3). These results suggest that survival oocytes after vitrification and thawing may still have a high risk of abnormal completion of oocyte meiosis and fertilization events, which results from cytoskeletal and chromosome abnormalities (6, 7). Thus, a vitrification procedure causing abnormal cytoskeleton dynamics reduces the developmental competence of oocytes after thawing. Most interestingly, the addition of a cytoskeleton stabilizer, Taxol™, significantly improved the post-thaw development of vitrified oocytes (Table 4). Oocytes that were vitrified-thawed with this cytoskeletal stabilizer did not show any decrease in blastocyst quality, compared with fresh oocytes (Table 5). These results clearly support the idea that the stabilization of the cytoskeletal system during vitrification is effective for improving the post-thaw developmental competence. Taxol™, paclitaxel, is a microtubule stabilizer and is currently being used as an anticancer drug. It increases the rate of polymerization by reducing the critical concentration of tubulin that is needed for polymerization (14). At relatively high doses, Taxol™ stabilizes microtubules by causing a tighter linkage between ␣- and -tubulin dimmers and by enhancing microtubular cross-linking after changes in the conformation and binding of high molecular weight microtubule–associated proteins (15). In Experiment 5, we retrieved a total of 176 COCs from superovulated mice and subsequently fertilized and cultured in vitro with or without vitrification using Taxol™ and, as shown in Table 5, there was no significant difference in the quality of blastocysts between the two groups. Nevertheless, the rate of blastocyst formation in COCs vitrified and thawed with Taxol™ was more than 10% lower than that in fresh COCs (data not shown). These supplementary data suggest that further improvement of oocyte development after thawing may be achieved by additional modification of the vitrification procedure. The use of different types of cytoskeletal stabilizers, permeable cryoprotectants, and macroFERTILITY & STERILITY威
molecules may be alternatives for such a purpose. Dobrinsky et al. (16) reported the positive effect of the other cytoskeleton stabilizer (cytochalasin B) on promoting the postthawed development of porcine embryos. It is necessary to evaluate the viability of blastocysts derived from oocytes vitrified and thawed with Taxol™ after transfer to the recipient mice. Furthermore, our data lack some information on the response of oocytes with the different concentrations of Taxol™ and on post-thawed cytoskeleton dynamics in Taxol™-treated oocytes. All of the information contributes to confirming the safety of our established cryopreservation program, and we are currently undertaking a series of experiments for providing such information. Once a full set of experimental procedures is completed, we will immediately apply this technique to the human vitrified IVF-ET cycle. However, our developed vitrification system is currently being applied to a mouse oocyte bank system to assist in transgenic mouse production.
Acknowledgments: The authors thank the Ministry of Education for the graduate fellowship provided through the BK21 program.
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13. Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8:1101–9. 14. Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M, Albertini DF. Taxol™-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mut Res 1999;423:79 –90.
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15. Albertini DF, Herman B, Sherline P. In vivo and in vitro studies on the role of HMW-MAPs in Taxol™-induced microtubule binding. Eur J Cell Biol 1984;33:3695–702. 16. Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000;62:564 –70.
Vol. 75, No. 6, June 2001
Cryopreservation of ICR mouse oocytes: improved post-thawed preimplantation development after vitrification using Taxol™, a cytoskeleton stabilizer Sung E. Park, M.S.,a Hyung M. Chung, Ph.D.,a Kwang Y. Cha, M.D.,a Woo S. Hwang, D.V.M., Ph.D.,b Eun S. Lee, D.V.M., Ph.D.,c and Jeong M. Lim, D.V.M., Ph.D.b College of Medicine, Pochon CHA University, Seoul, Korea
Received September 5, 2000; revised and accepted January 3, 2001. This work was supported by a grant (no. 1999-2205-002-5) from the Interdisciplinary Research Program of the Korea Science and Engineering Foundation (KOSEF). Reprint requests: Jeong M. Lim, D.V.M., Ph.D., Gamete Biotechnology Laboratory, School of Agricultural Biotechnology, Seoul National University, 103 Seodun-dong, Suwon 441744, Korea (E-mail: [email protected]). a Infertility Medical Center of CHA General Hospital. b School of Agricultural Biotechnology, Seoul National University, Suwon, Korea. c Department of Veterinary Medicine, Kangwon National University, Chunchon, Korea. 0015-0282/01/$20.00 PII S0015-0282(01)01809-X
Objective: To establish an effective cryopreservation method. Design: In vitro model study. Setting: Infertility Medical Center, Pochon CHA University. Animal(s): Four-week-old ICR mice superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. Intervention(s): Vitrified-thawed oocytes were fertilized and subsequently cultured in vitro. Main Outcome Measure(s): Post-thawed development, chromosome/spindle normalities, and blastocyst quality. Result(s): More cumulus-enclosed oocytes were fertilized and developed to the 8-cell stage after vitrification and thawing than denuded oocytes. However, cryopreserved oocytes of both types had lower spindle and chromosome normalities than fresh oocytes, which resulted in reduced developmental competence after thawing. The addition of 1 M of Taxol™, a cytoskeleton stabilizer, to vitrification solution greatly promoted the blastocyst formation of vitrified-thawed oocytes, compared with no addition (24.0% vs. 58.6%). No difference in blastocyst quality, which was evaluated by blastomere and inner cell mass cell numbers and inner cell mass cell per trophoblast ratio, was found between fresh oocytes and oocytes vitrified with Taxol™. Conclusion(s): A vitrification solution consisting of 5.5 M ethylene glycol, 1.0 M sucrose, 10% fetal bovine serum, and 1 M Taxol™ greatly improved post-thawed development of vitrified oocytes. (Fertil Steril威 2001;75:1177– 84. ©2001 by American Society for Reproductive Medicine.) Key Words: Mouse, oocyte, vitrification, Taxol™, cumulus cells
In our previous studies (1–3), we developed an effective culture system for supporting preimplantation development of outbred ICR mouse embryos. This system employs modified preimplantation-1 medium (mP-1), which contains amino acids, ethylenediaminetetraacetic (EDTA) acid, glucose, hemoglobin, and polyvinyl alcohol (PVA). Using this chemically defined medium, more than 65% of in vitro– derived 1-cell embryos could develop to blastocysts with improved quality. This culture system will become not only one of the core factors for a disease model mouse production system, but also a good culture model for improving human in vitro fertilization (IVF)– embryo transfer (ET) programs.
Now we have turned our attention to establishing a mouse oocyte bank, which uses our developed embryo culture system. Selection of an oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. In this study, we attempted to employ a vitrification method using ethylene glycol (EG) and an electron microscope (EM) grid for the cryopreservation of mouse oocytes, because this could yield higher post-thawed survival and subsequent embryo development than conventional slow freezing methods (4 – 5). The vitrification procedure, however, still leaves much room for improvement in fully regaining developmental competence after thawing, because it employs cryoprotectants of 1177
high molar concentration that induce cytoskeletal damage (6 – 8). This damage directly causes abnormal progression of meiotic division and the retardation of embryo development. Stabilizing the cytoskeleton system during vitrification is likely beneficial for improving post-thawed survival and subsequent development of vitrified oocytes.
velopment of stored oocytes. The objective of this study, therefore, was to establish an effective cryopreservation method for the mouse oocyte bank system, which could provide additional information for improving the efficacy of a human oocyte bank.
With this in mind, we first examined the feasibility of the vitrification method in the long-term preservation of ICR mouse oocytes. We evaluated the developmental competence of oocytes vitrified and thawed at the mature stage and subsequently examined the incidence of chromosome and spindle abnormalities after thawing. In the second set of experiments, we examined whether the addition of a cytoskeleton stabilizer, Taxol™, to the vitrification solution could promote the post-thawed survival and subsequent de-
MATERIALS AND METHODS Collection of Oocytes Four-week-old female ICR mice were maintained under controlled lighting conditions (14L: 10D) and superovulated by the injection of 5 IU of PMSG (Folligon; Intervet Co., The Netherlands) followed by the injection of 5 IU of hCG (Chorulon; Intervet) 48 hours apart. Mature oocytes with expanded cumulus cells and with a first polar body were
FIGURE 1 Procedure of mouse oocyte vitrification using electron microscopic grid. (A), Oocyte mounting on EM grid during final equilibration procedure; (B), liquid nitrogen plunging procedure; and (C), a combined structure of cryovial cap and goblet for ordered placement of grid in liquid nitrogen.
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TABLE 1 Preimplantation development of cumulus-enclosed or cumulus-free oocytes vitrified and thawed at the mature stage. No. (%)d of inseminated oocytes developed to
No. of oocytes
Treatments
Status of oocytesa
Examined
Survived after thawing (%)b
No. of oocytes fertilized (%)c
4-Cell 48e
8-Cell 72e
Morula 84e
Blastocyst 96e
None Vitrification Vitrification
Cumulus-enclosed Cumulus-enclosed Cumulus-free
77 90 80
77 (100)f 72 (80)g 59 (73.8)g
72 (93.5)f 45 (62.5)g 28 (47.5)h
59 (81.9)f 23 (51.1)g 11 (39.3)g,h
52 (72.2)f 16 (35.6)g 4 (14.3)h
48 (66.7)f 10 (22.2)g 3 (10.7)g,h
46 (63.9)f 10 (22.2)g 2 (7.1)g
Model effect of the treatments, which was indicated as P value, was less than .0001 in each parameter. a ICR mouse oocytes that were mature at the time of retrieval were provided. b Percentage of the no. of oocytes examined. c Percentage of the no. of oocytes survived after the treatments. d Percentage of the no. of oocytes fertilized. e Hours after culture. f,g,h Different superscripts within each column are significantly different, P ⬍ .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
retrieved from superovulated mice 14 hours after hCG injection. Either intact cumulus-oocyte complexes (COCs) or oocytes denuded by repeated pipetting in a 0.1% hyaluronidase solution were provided for this experiment.
Vitrification and Thawing of Oocytes COCs or denuded oocytes were pre-equilibrated for 2.5 minutes in 2 mL of Dulbecco’s phosphate buffered saline (DPBS; Gibco BRL, Grand Island, NY) supplemented with 1.5 M of EG (E-9129; Sigma, St. Louis, MO) and 10% (v/v) fetal bovine serum (FBS; Gibco BRL), with and without 1 M Taxol™, at 37°C. Oocytes were then placed for the final equilibration in the same volume of DPBS supplemented with 5.5 M of EG, 1.0 M of sucrose, and 10% FBS, with and without 1 M Taxol™, for 20 seconds. In the meantime, 10 –20 oocytes were mounted on an EM grid (Gilder, Westchester, PA) using a fine pipette, and excess cryoprotectant solution was removed with the underlying sterilized filter paper. The grids containing oocytes were immediately plunged into liquid nitrogen, and a cryovial cap and goblet
were used for ordered placement of the grid. The grids were then stored for 1 hour to 7 days. For thawing, the grids were sequentially transferred to culture dishes containing 2 mL of 10% (v/v) FBS-containing DPBS, to which 1.0, 0.5, 0.25, 0.125, or 0 M of sucrose was added, at intervals of 2.5 minutes at 37°C. After washing 4 – 6 times, thawed oocytes were cultured for 2 hours and used in each experiment. Oocyte mounting onto the EM grid during the equilibration, the liquid nitrogen plunging procedure, and the combined goblet and cryovial cap structure for the storage of the grid are depicted in Figure 1.
In Vitro Fertilization Either fresh or vitrified-thawed oocytes were inseminated in vitro with 1.0⫻106 epididymal spermatozoa/mL in Tyrode’s 6 medium supplemented with fatty acid-free BSA (cat. no. A-4161, 15 mg/mL; Sigma). Inseminated oocytes were freed from spermatozoa at 4 hours after in vitro insemination.
TABLE 2 Spindle normality of cumulus-enclosed or cumulus-free oocytes after vitrification and thawing at the mature stage. No. of oocytes
Treatments
Examineda
Analyzed
With normal spindle (%)b
No. of abnormal oocytes
Cumulus-enclosed Cumulus-enclosed Cumulus-free
120 142 130
104 124 109
83 (79.8)c 84 (67.7)d 66 (60.6)d
21 40 43
Status of oocytes None Vitrified and thawed Vitrified and thawed
Model effect of the treatments on the spindle normality (indicated as P value) was .0069. a ICR mouse oocytes that maintained normal morphology after thawing were provided. b Percentage of the no. of oocytes analyzed. c,d Different superscripts within the same parameter are significantly different, P ⬍ .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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FIGURE 2 Chromosomes and spindle configurations of mouse mature oocytes. (A), Normal barrel-shaped spindle with chromosome. (B), Abnormal appearance of a disorganized spindle with chromosome. Arrows indicate chromosomes.
Sigma), EDTA (0.1 mM), PVA (0.05 mg/ml), 10 g/mL gentamycin, and 5 mg/ml phenol red. This medium does not contain glucose, inorganic phosphate, or protein, and the osmolarity and pH of the medium were within the ranges of 295–305 mOsm and 7.34 –7.38, respectively. Media used for embryo culture were equilibrated in such an atmosphere for at least for 3 hours before culture, and the droplets of equilibrated medium were covered with warm mineral oil (BDH Co., Poole, England). A group of 10 –15 1-cell embryos were cultured in a 5-L droplet of mP-1 at 37°C, 5% CO2 in air atmosphere, and embryos were transferred to the same volume of glucosecontaining mP-1 at 48 hours after culture. The number of embryos developed to the 4-cell, 8-cell, morula, and blastocyst stages were monitored under a stereo microscope at 48, 72, 84, and 96 hours after culture, respectively.
Chromosome Analysis Chromosome preparation for analyzing the numeric normality of oocyte chromosome was performed by the method of Tarkowski (9). Briefly, oocytes were mounted onto a grease-free slide after gradual fixation using water–methanol– glacial acetic acid solutions of different mix ratios. After drying for 1 hour, fixed oocytes were subsequently stained with 10% (v/v) Giemsa solution. The number of chromosomes in each spread was counted under an inverted microscope.
Immunostaining for Tubulin Fresh or vitrified-thawed oocytes were freed from the zona pellucida by treatment in 0.5% (v/v) acid Tyrode’s solution. These zona-free oocytes were treated in microtubule-stabilizing buffer containing 25% (v/v) 25 mM N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 0.5 mM MgCl2, 10 mM ethylene glycerol-bis (-aminoethyl ether)-N, N, N, N-tetraacetic acid, 50 mM KCl, 25 M phenylmethyl sulfonyl fluoride, and 2% (v/v) Triton X-100 for 60 minutes. Subsequently, the oocytes were attached to siliconized slides and fixed with absolute methanol at ⫺20°C for 7 minutes.
Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
Culture of Embryos and Assessment of Preimplantation Development The basic medium used for the culture of embryos was mP-1 (3), which consists of 101.6 mM NaCl, 4.69 mM KCl, 2.04 mM CaCl2 䡠 2H20, 0.2 mM MgSO4 䡠 7H2O, 0.33 mM sodium pyruvate, 21.4 mM sodium lactate, 25 mM NaHCO3, 0.15 mg/mL sodium citrate, MEM nonessential (0.5%, v/v) and essential (1%, v/v) amino acid solutions (Gibco BRL), hemoglobin (1 g/mL, methemoglobin, cat. no. H-7379, 1180 Park et al.
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After rehydration in phosphate-buffered saline (PBS; Gibco BRL), oocytes were incubated with monoclonal antitubulin antibody for 60 minutes at 37°C followed by washing in PBS supplemented with 0.01% (v/v) Triton X-100. The intensity of tubulin staining was amplified by incubating the oocytes with tetramethylrhodamine isothiocyanate– conjugated goat anti-mouse immunoglobulin G (1/200; Biodesign, ME) in PBS for 60 minutes at 37°C. Hoechst 33258 (40 g/mL; Sigma) was used for counterstaining chromosomes. Oocytes were then examined under an uprighted microscope with epifluorescent apparatus (Leiz, Wetzlar, Germany). Hoechst 33258 staining was visualized by using a UVA filter (BP, 340 –380 nm), and a rhodamine signal was observed by using an N2.1 filter (BP, 515–560 nm). Vol. 75, No. 6, June 2001
Assessment of the Number of Blastocysts and Inner Cell Mass Cells To evaluate the quality of blastocysts, total blastomere, trophoblast, and inner cell mass (ICM) cell numbers were counted by the method of Hardy et al. (10) with a slight modification. The zona pellucida of blastocysts was removed by 0.5% (v/v) protease solution (cat. no. P-8811, Sigma), and the blastocysts were placed in 15 mM trinitrobenzene sulfonic acid (cat. no. P-2297, Sigma) for 15 minutes at 4°C. Blastocysts were then incubated for 10 minutes in Tyrode’s lactate solution supplemented with 25 mM HEPES and 0.1 mg/mL anti-dinitrophenol-BSA (cat no. 61-007-1, ICN, Irvine, CA) at 39°C. Subsequently, they were treated with 0.01 mg/mL propidium iodide (cat. no. P-4170, Sigma) and incubated with 15% (v/v) guinea pig complement (cat. no. S-1639, Sigma) for 20 –30 minutes at 39°C. The blastocysts were then placed in absolute ethanol solution supplemented with 0.05 mM fluorochrome bisbenzimide (cat. no. B-2261, Sigma) overnight at 4°C. After washing in absolute ethanol, blastocysts containing cells stained with different fluorescent dyes were mounted on a glass slide and cell numbers were examined under an upright microscope with an epifluorescent apparatus (Leiz).
Experimental Design In experiment 1, either COCs or denuded oocytes were vitrified, and the survival and subsequent development of the oocytes after thawing were compared with those of fresh oocytes. In experiments 2 and 3, both chromosome and spindle normalities of vitrified oocytes were examined at 2 hours after thawing, respectively, and those of fresh oocytes were also evaluated as a control. In experiment 4, COCs were vitrified with a cryoprotectant solution supplemented with and without 1 M Taxol™, and survival and subsequent development were monitored. In experiment 5, the number of blastomeres and ICM cells, and the ratio of ICM cell per trophoblast in blastocysts derived from either fresh oocytes or oocytes vitrified and thawed with Taxol™ were examined at 96 hours after culture.
Statistical Analysis Each experiment was replicated four (experiments 1, 3, 4, and 5) or five (experiment 2) times. The embryos that developed to the 4-cell, 8-cell, morula, blastocyst, and hatched blastocyst stages were scored and subjected to analysis of variance using the generalized linear model (PROC-GLM) in the SAS program (11). When the significance of the main effects was detected in each experimental parameter, the treatment effects were compared by the least-squares method. P⬍.05 was considered statistically significant.
RESULTS Experiment 1 As shown in Table 1, a significant (P⬍.0001) model effect was found in all experimental parameters. COCs had FERTILITY & STERILITY威
TABLE 3 Chromosome normality of cumulus-enclosed or cumulusfree oocytes after vitrification and thawing at the mature stage. No. of oocytes
Stature of oocytes None Vitrified/thawed Vitrified/thawed
Treatments Cumulus-enclosed Cumulus-enclosed Cumulus-free
With normal chromosome Examineda Analyzed (%)b 95 110 104
46 58 55
37 (80.4)c 39 (67.2)d 36 (65.5)d
Model effect of the treatments on the chromosome normality (indicated as P value) was .004. a ICR mouse oocytes that maintained normal morphology after thawing were provided. b Percentage of the no. of oocytes analyzed. c,d Different superscripts within the same parameter are significantly different, P .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
higher developmental competence after vitrification and thawing than denuded oocytes, and the statistical significances were found in the rates of fertilization (47.5% vs. 62.5%) and development to the 4-cell (39.3% vs. 51.1%) and 8-cell (14.3% vs. 35.6%) stages. In spite of these improvements, higher rates of fertilization (93.5%) and development to the 4-cell (81.9%), 8-cell (72.2%), morula (66.7%), and blastocyst stages (63.9%) were observed in fresh oocytes than in vitrified oocytes of any category.
Experiment 2 The rates of post-thawed spindle normality were 79.8% in fresh oocytes, 67.7% in vitrified-thawed COCs, and 60.6% in vitrified-thawed denuded oocytes (Table 2), and there was a significant difference in the spindle normality between fresh and vitrified oocytes (model effect ⫽ .0069). Normal spindles appeared as fine microtubules traversing the metaphase plate and forming the classic barrel shape (Fig. 2). The spindle structure was frequently observed in the peripheral region adjacent to the polar body, and the chromosomes were aligned on the metaphase plate (Fig. 2A). A disorganized spindle in any part of the ooplasm or spindles of non-barrel shape were considered abnormal (Fig. 2B).
Experiment 3 As shown in Table 3, a significant decrease in chromosome normality, which was indicated as a high model effect (P ⫽ .004), was found. The incidence of chromosome normality was lower in vitrified oocytes with or without cumulus cells (65.5%– 67.2%) than in fresh oocytes (80.4%).
Experiment 4 Significant (P⬍.0002) model effects were found in the number of oocytes developed beyond the 4-cell stage. More oocytes developed to the 4-cell (48% vs. 84.4%), 8-cell 1181
TABLE 4 Effects of the addition of Taxol™ (1 g/mL), a cytoskeleton stabilizer, to cryoprotectant solution on the preimplantation development of ICR mouse cumulus-enclosed oocytes vitrified and thawed at the mature stage. No. (%)c of inseminated oocytes developed to
No. of oocytes Cryoprotectant with (⫹) or without (⫺) Taxol™ ⫺ ⫹
Examined
Survived after thawing (%)a
No. of oocytes fertilized (%)b
4-cell 48d
8-cell 72d
76 83
63 (82.8) 74 (89.1)
50 (79.3) 58 (78.3)
24 (48.0)e 49 (84.4)f
17 (34.0) 41 (70.6)f
e
Morula 84d
Blastocyst 96d
13 (26.0)e 37 (63.7)f
12 (24.0)e 34 (58.6)f
Based vitrification solution was a 10% (v/v) fetal bovine serum– containing preimplantation-1 medium supplemented with 5.5 M ethylene glycol and 1.0 M sucrose. Model effects of the treatments indicated as P value were .2561, .8890, .0001, .0001, .0001, and .0002 in the no. of oocytes survived, fertilized, and developed to the 4-cell, 8-cell, morula, and blastocyst stages, respectively. a Percentage of the no. of oocytes examined. b Percentage of the no. of oocytes that survived after the treatments. c Percentage of the no. of oocytes fertilized. d Hours after culture. e,f Different superscripts within each column are significantly different, P .05. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
(34% vs. 70.6%), morula (26% vs. 63.7%), and blastocyst (24% vs. 58.6%) stages after the addition of Taxol™ to the cryoprotectant than after no addition (Table 4). Blastocysts derived from oocytes vitrified with Taxol™ could develop to the expanded blastocyst stage and had normal morphology (Fig. 3).
cells to trophoblasts (0.25 ⫾ 0.01 to 0.26 ⫾ 0.01) were similar between the two groups.
Experiment 5
The results of this study clearly demonstrate that the addition of Taxol™, a cytoskeleton stabilizer, significantly improved the post-thaw development of cumulus-enclosed ICR mouse oocytes vitrified at the mature stage. Compared with no treatment, Taxol™-treated COCs doubled the rate of blastocyst formation after vitrification and thawing (24% to 58.6%). This result further suggests that damage in the oocyte cytoskeleton system is one of the main cryoinjuries in a vitrification program.
As shown in Table 5, no significant model effect was found in all experimental parameters (P⬎.2403). The mean cell number of blastocysts (65.8 ⫾ 3.9 to 69.6 ⫾ 3.1) and ICM cells (13.6 ⫾ 0.9 to 13.9 ⫾ 0.7) and the ratio of ICM
FIGURE 3 Morphology of blastocysts derived from oocytes vitrified with Taxol™. Fully expanded blastocele with distinct inner cell mass is visible (arrow), ⫻200.
DISCUSSION
TABLE 5 Effects of the addition of Taxol™ (1 g/mL), a cytoskeleton stabilizer, to cryoprotectant solution on the quality of blastocysts derived from oocytes vitrified and thawed at the mature stage. No. of blastocysts
Cell nos. (mean ⫾ SE) of blastocysts
ICM cell/ Successfully Total trophoblast Treatments Examined stained (%) blastomeres ICM cells ratio None Vitrification
51 34
38 (74.5) 24 (70.6)
69.6 ⫾ 3.1 13.9 ⫻ 0.7 0.25 ⫾ 0.01 65.8 ⫾ 3.9 13.6 ⫾ 0.9 0.26 ⫾ 0.01
Model effect (P value) in the cell numbers of total blastomere and inner cell mass (ICM) cells, and the ratio of ICM cells to trophoblasts were .7059, .9567, and .2403, respectively. Park. Mouse oocyte vitrification using Taxol™. Fertil Steril 2001.
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It is debatable whether cumulus cells, which encompass the outermost layer of oocytes, are beneficial for enhancing post-thaw survival and subsequent development of oocytes after cryopreservation (12, 13). In our study, intact COCs had a higher developmental competence than denuded oocytes. The results of this study suggest that the vitrification of COCs is a good cryopreservation strategy for promoting oocyte survival after thawing. Ashwood-Smith et al. (12) hypothesized that cumulus cells might have a protective action against the cryoprotectant. They may help oocytes to maintain a rigid structure, which prevents morphologic damage during cryopreservation. Nevertheless, the post-thaw developmental competence of vitrified-thawed COCs was significantly lower than that of fresh COCs (Table 1). Furthermore, a high incidence of spindle and chromosome abnormalities was detected in thawed oocytes even after vitrification with cumulus cells (Tables 2–3). These results suggest that survival oocytes after vitrification and thawing may still have a high risk of abnormal completion of oocyte meiosis and fertilization events, which results from cytoskeletal and chromosome abnormalities (6, 7). Thus, a vitrification procedure causing abnormal cytoskeleton dynamics reduces the developmental competence of oocytes after thawing. Most interestingly, the addition of a cytoskeleton stabilizer, Taxol™, significantly improved the post-thaw development of vitrified oocytes (Table 4). Oocytes that were vitrified-thawed with this cytoskeletal stabilizer did not show any decrease in blastocyst quality, compared with fresh oocytes (Table 5). These results clearly support the idea that the stabilization of the cytoskeletal system during vitrification is effective for improving the post-thaw developmental competence. Taxol™, paclitaxel, is a microtubule stabilizer and is currently being used as an anticancer drug. It increases the rate of polymerization by reducing the critical concentration of tubulin that is needed for polymerization (14). At relatively high doses, Taxol™ stabilizes microtubules by causing a tighter linkage between ␣- and -tubulin dimmers and by enhancing microtubular cross-linking after changes in the conformation and binding of high molecular weight microtubule–associated proteins (15). In Experiment 5, we retrieved a total of 176 COCs from superovulated mice and subsequently fertilized and cultured in vitro with or without vitrification using Taxol™ and, as shown in Table 5, there was no significant difference in the quality of blastocysts between the two groups. Nevertheless, the rate of blastocyst formation in COCs vitrified and thawed with Taxol™ was more than 10% lower than that in fresh COCs (data not shown). These supplementary data suggest that further improvement of oocyte development after thawing may be achieved by additional modification of the vitrification procedure. The use of different types of cytoskeletal stabilizers, permeable cryoprotectants, and macroFERTILITY & STERILITY威
molecules may be alternatives for such a purpose. Dobrinsky et al. (16) reported the positive effect of the other cytoskeleton stabilizer (cytochalasin B) on promoting the postthawed development of porcine embryos. It is necessary to evaluate the viability of blastocysts derived from oocytes vitrified and thawed with Taxol™ after transfer to the recipient mice. Furthermore, our data lack some information on the response of oocytes with the different concentrations of Taxol™ and on post-thawed cytoskeleton dynamics in Taxol™-treated oocytes. All of the information contributes to confirming the safety of our established cryopreservation program, and we are currently undertaking a series of experiments for providing such information. Once a full set of experimental procedures is completed, we will immediately apply this technique to the human vitrified IVF-ET cycle. However, our developed vitrification system is currently being applied to a mouse oocyte bank system to assist in transgenic mouse production.
Acknowledgments: The authors thank the Ministry of Education for the graduate fellowship provided through the BK21 program.
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13. Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993;8:1101–9. 14. Mailhes JB, Carabatsos MJ, Young D, London SN, Bell M, Albertini DF. Taxol™-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Mut Res 1999;423:79 –90.
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15. Albertini DF, Herman B, Sherline P. In vivo and in vitro studies on the role of HMW-MAPs in Taxol™-induced microtubule binding. Eur J Cell Biol 1984;33:3695–702. 16. Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000;62:564 –70.
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