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Laboratory of Animal Reproduction,3 Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Swedish University of Agricultural Sciences,4 Uppsala, 750-07 Sweden
Research Farm,5 Miyagi Agricultural College, Sendai 982-0231, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, bovine, embryo, gamete biology, GV oocytes, nylon mesh, oocyte development, ovum, vitrification
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Besides of the ordinal cryopreservation, vitrification has been widely developed to apply to cryopreservation of mammalian embryos. In efforts to increase cooling and warming rates during vitrification, modification of the methods has been approved especially to develop various containers. Unfortunately, neither of the containers developed appears to apply to vitrification of bovine immature oocytes because all of them have aimed to cryopreserve single embryos or oocytes with a smaller area for holding it, whereas bovine immature GV oocytes are collected as cumulus-oocytes complexes (COCs) for IVM, indicating that vitrification of bovine GV oocytes needs a container having an appropriate area for holding them. For this purpose, recently, we developed a nylon-mesh holder that was useful to vitrify large quantities of bovine GV oocytes [6]. After IVM of bovine GV oocytes vitrified using the nylon-mesh holder, however, we found that both maturation and cleavage rates were not satisfactory compared with those of the nonvitrified control.
To solve the problem of low rates for IVP, it must be considered that vitrification requires a highly concentrated solution of cryoprotectants such as ethylene glycol that may have possible toxic effect and may produce irreversible damage to a cytoskeleton of the samples [7, 8]. Thus, an adequate approach should be developed to minimize toxic, osmotic, and other injuries during cryopreservation by vitrification. Inclusion of nontoxic cryoprotectants, such as sugars, seems to be one approach. For example, trehalose has been considered beneficial either in counteracting the osmotic effect or specifically interacting with membrane phospholipids [9]. Compared with sucrose, Kim et al. [10] reported exogenous trehalose improved the viability of mouse morulae frozen ultrarapidly. Alternatively, another approach for alleviating the toxic or major volume expansion effects exerted by cryoprotectants at high concentrations may be attained by employment of a stepwise addition of cryoprotectants during the procedure [11].
To improve IVP rates after vitrification of bovine GV oocytes using a nylon-mesh holder, the aims of the present study were i) to compare ethylene glycol-trehalose with ethylene glycol-sucrose as cryoprotectants, ii) to determine the effect of a stepwise exposure to cryoprotectants on damage in the organelles after vitrification, and iii) to assess the developmental ability of the hereby vitrified GV oocytes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bovine offal ovaries of Japanese Black were collected at a local slaughterhouse and transported to the laboratory on a thermos flask at approximately 37°C. GV-COCs were aspirated from visible follicles (2–8 mm in diameter) by a 10-ml syringe with an 18-gauge needle. After three washes, about 20 GV-COCs each were put into each of 100-µl droplets of a maturation medium consisting of TCM199 supplemented with 5% fetal calf serum (FCS) [6] in a humidified atmosphere of 5% in air at 39°C under a layer of mineral oil. After culturing for 1 h for stabilizing the state of the GV-COCs [6], they were applied to the experiments, allotting into two groups, e.g., nonvitrified (control) and vitrified oocytes (treatment). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) except for the ones specifically described. The present study was approved by the Ethics Committee for Care and Use of Laboratory Animals for Biomedical Research of the Graduate School of Agricultural Science, Tohoku University.
Vitrification and Thawing
The GV-COCs were vitrified using a nylon-mesh holder described in the previous report [6]. In brief, nylon-mesh (pore size 60 µm; DIN100, Sefar Inc., Ruschlikon, Switzerland) was cut into triangular holds, with each base of approximately 10 mm. For easy handling, a cotton-covered polyester thread (10 mm in length) was attached to the one corner of the holders. Before use, the holders were sterilized with ethylene oxide.
The GV-COCs were randomly divided into either a stepwise group or a single-step group. In the stepwise group, the GV-COCs were exposed first for 7 min to 200-µl droplets of solution A, which was composed of 10% (v/v) ethylene glycol, 4.5% (w/v) Ficoll-70, and 0.075 M sucrose or trehalose in PB1 [6], then for 2 min to 200 µl droplets of solution B, which was composed of 20% (v/v) ethylene glycol, 9.0% (w/v) Ficoll-70, and 0.15 M sucrose or trehalose in PB1, and finally for 1 min to 200-µl droplets of solution C, which was composed of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll-70, and 0.3 M sucrose or trehalose in PB1 (designated as EFS40 or EFT40, respectively) in 35-mm culture dishes (SUMILON; Sumitomo Bakelite Co., Ltd., Tokyo, Japan). In the single-step group, the GV-COCs was exposed for 1 min only to 200-µl droplets of solution C in the culture dishes. After equilibrium, 20–40 GV-COCs each were transferred onto a nylon-mesh holder, and the excess of the solution was removed by placing the holder on a filter paper. Immediately after removing, the holder was directly plunged into a 2-ml polypropylene tube (MS-4503; Sumitomo Bakelite Co., Ltd.) followed directly be plunging into LN2 by holding for 40–60 sec. After 1 h of storage in LN2, the GV-COCs were ultrarapidly thawed, and the cryoprotectants were removed in a stepwise manner at 37°C: the nylon-mesh holder was transferred from LN2 into 300-µl droplets of the warm PB1 with a sequential series of 0.5, 0.25, and 0.125 M sucrose or trehalose by keeping for 1 min in each solution, and finally transferred into PB1 for 5 min in the culture dishes. After removing, the GV-COCs were cultured for 21–22 h in 100-µl droplets of the fresh maturation medium.
Examination of IVM in the GV Oocytes Vitrified
After culture for IVM, the oocytes were denuded of cumulus cells in phosphate-buffered saline supplemented with 0.1% hyaluronidase by putting in and out, then using a fine-bore pipette. The cumulus-free oocytes were fixed in acetic acid/ethanol (1/3, v/v) for 3 days and then stained with 1% orcein (v/v) in 45% acetic acid. The nuclear configuration in each oocyte was examined by a phase-contrast microscopy (Olympus, Tokyo, Japan).
IVF of the Oocytes Vitrified
Straws containing frozen bull semen of Japanese Black cattle (Livestock Improvement Association of Japan, Tokyo, Japan) were thawed at 37°C in a water bath. After warming, the spermatozoa were washed twice by centrifugation at 600 x g for 8 min in Brackett and Oliphant (BO) medium [12] containing 2.5 mM theophylline. The spermatozoa washed were extended in the appropriate volume of a fertilization medium to give a concentration of 5 x 106 cells/ml. Ten to 20 COCs matured each were coincubated with washed sperm in 100-µl droplets of the fertilization medium consisting of BO medium with 3 mg/ml bovine serum albumin and 2.5 mM theophylline for 18 h in a humidified atmosphere of 5% CO2 in air at 39°C under a layer of mineral oil.
In Vitro Culture of Embryos and Embryo Transfer
In vitro culture (IVC) of embryos was carried out in a modified synthetic oviduct fluid medium (SOF) supplemented with 22 amino acids (1 mM glutamine, 5 mM glycine, 2 mM taurine, essential amino acids for basal medium Eagle, and nonessential amino acids for minimum essential medium), 10 µg/ml insulin, and 1 mg/ml polyvinyl alcohol in a humidified atmosphere of 5% CO2:5% O2:90% N2 at 39°C under a layer of mineral oil [12]. Eighteen hours after insemination, presumptive zygotes were denuded of cumulus cells by pipetting in and out the COCs. The zygotes were then washed three times with SOF and cultured for in vitro development to the blastocyst stage. After examination, four blastocysts obtained at Day 8 postculture were transferred nonsurgically to three synchronized recipient cows (1–2 blastocysts per animal).
Observation of the Oocytes by Transmission Electron Microscopy
After cryopreservation, representative numbers of COCs (vitrified and controls) were fixed in 2.5% glutaraldehyde in a sodium cacodylate buffer (0.067 M, pH 7.2–7.4) and stored at 4°C until processed. After postfixation for 5 min in 2% (w/v) osmium tetraoxide in the cacodylate buffer, the specimens were embedded in Agar100 resin (Agar Scientific Ltd., Cambridge, UK). Semithin sections were cut for light microscopy and stained with toluidine blue for further sectioning of areas. Thereafter, ultrathin sections were cut and stained with uranyl acetate and lead citrate, and examined by transmission electron microscopy (TEM; Philips 420 electron microscope; Einhoven, The Netherlands) at 80 kV.
Statistical Analysis
Data were compared using chi-square test by StatView software (Abacus Concepts, Inc., Berkeley, CA). The data for cleavage and blastocyst rates were analyzed using one-way ANOVA with Fisher protected least significant difference (PLSD). Differences were considered significant at a level of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cleavage and developmental rates are shown in Table 2. Vitrification resulted in a significant reduction in the cleavage rate in any vitrified groups compared with the nonvitrified control. Strikingly, the oocytes vitrified by stepwise exposure to either EFS40 or EFT40 showed significantly higher cleavage rates than the corresponding rates in the single-step procedure (37.7% ± 10.5% or 22.2% ± 5.2% versus 20.8% ± 5.3% or 0%, respectively; P < 0.05). Only 8.0% ± 4.5% of oocytes vitrified in the stepwise group exposed to EFS40 developed to the blastocyst stage, whereas, unexpectedly, none of them in the EFT40 developed to blastocysts. After transfer of some of these blastocysts, one Japanese Black female weighting 32 kg was born at 288 days after transfer.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The injury to the cells in the process of cryopreservation can be due to osmotic effects accompanying saturation with permeable cryoprotectants [13, 14]. The reduction in subsequent developmental competence caused by exposure to cryoprotectant solutions has been shown to be more severe in the GV oocytes than in the MII ones [15–17]. One explanation for the greater osmotic sensitivity in the GV oocytes may be their recorded twofold lower hydraulic conductivity compared with that found in IVM MII oocytes [15]. Fuku et al. [17] and Kasai [18] proposed that the supplementation of saccharides such as sucrose in the vitrification medium could reduce toxicity to the embryos by reducing the extracellular concentration of the cryoprotectant. The osmometric behavior of mouse oocytes in the presence of different extracellular sugars is an important variable factor when considering optimization of cryopreservation protocols using sugars [19].
Trehalose has been found to have a protective action related to an osmotic effect and to specific interactions with membrane phospholipids [10]. Compared with sucrose, Kim et al. [10] reported the use of trehalose to improve the viability of mouse morulae ultrarapidly frozen. The present study demonstrated the effect of the addition of trehalose instead of sucrose into the vitrification medium on subsequent developmental ability of bovine GV oocytes to the MII and blastocyst stages. Although the oocytes vitrified by stepwise exposure to either EFS40 or EFT40 showed significantly higher IVM and cleavage rates than the corresponding rates vitrified by the single step, there was no difference in effects between EFS 40 and EFT 40 on maturation and cleavage rates. Kuleshova et al. [19] reported that raffinose with an equal concentration, estimated by a glass transition point, has proven effective in increasing the survival rate after exposure to either monosaccharide or disaccharide, suggesting that the damage to the cell may be reduced depending on the kind of saccharides added to the vitrification medium. Considering that, in the present study, blastocysts were obtained in the GV oocytes vitrified by EFS40 but not by EFT40, it may be possible to speculate that, in bovine GV oocytes, the difference between osmometric behaviors of different extracellular sugars may affect cytoplasmic maturation important for subsequent developmental ability. Further study seems to be needed to get information on how the osmotic property inside the bovine GV oocyte cytoplasm would change when extracellular sugars are used as cryoprotectants.
In the single-step method, as in the previous report [20], both treatments of EFS40 and EFT40 showed that the GV-COCs after vitrification had partly dispersed cumulus cells and oocytes with shrunken cytoplasm, in particular in the GV-COCs vitrified with EFT40, accompanied with ultrastructural damage in the mitochondria. Some studies revealed a loss of cumulus cells from the COCs frozen following slow cooling and thawing, after which cumulus cells were no longer connected to the oocytes [20], suggesting that the cumulus cells of the COCs may be disrupted physically by damage due to ice formation in the slow cooling method. The connection between cumulus cells and the oocyte in the GV-COCs is shown to be of utmost importance for completion of normal oocyte maturation in vitro [21]. In the present study, we found the decrease of IVM rate in the single step with EFS40 and EFT40, indicating that, during vitrification of bovine GV-COCs, the disconnection between cumulus cells and the oocyte may be caused by the same phenomenon explained in the reports mentioned above. In addition, in the present study, the time for exposing oocytes to the final cryoprotectant was only 1 min, which seems to provide insufficient penetration of the cryoprotectant into the COCs in the single-step group, although the prolongation of treated time resulted in a lower viability by toxic effect of cryoprotectant [22].
Because vitrification is a nonequilibrium cryopreservation method that needs relatively high concentrations of cryoprotectants, a stepwise addition of cryoprotectants may reduce the toxic effect of cryoprotectants and be considered to minimize damage due to extreme cell-volume expansion [11]. In fact, for vitrification of bovine blastocysts derived from IVP, two-step exposure to the cryoprotectants showed less damage compared with the single-step procedure [23]. Kuwayama et al. [13, 24] reported high survival rates of bovine blastocysts after vitrification using a 16-step method for saturation of embryos with permeable cryoprotectants. They suggested that osmotic injury to cells occurring in the blastocysts is due to the osmotic stress accompanying saturation with permeable cryoprotectants. The results obtained in the present study are substantially consistent with these previous reports.
Despite improvement by the stepwise exposure in the maturation rate to the MII stage and production of viable blastocysts, both cleavage and developmental rates were still low compared with the nonvitrified control. The cooling of the GV oocytes to 0 or 4°C strongly affected the subsequent meiotic spindle formation and polar body extrusion, although the telophase I spindle could divide normally into two chromosome sets [3]. We observed that, in some vitrified oocytes, the polar body was not normally extruded even after the chromosomes separated, which in turn indicated that vitrified oocytes having the ability to mature normally to telophase I stage may be damaged in some key regulatory factors that are involved in the extrusion of the polar body [25–27]. Although the ultrastructural figures in the stepwise group were similar to those in the control, the metabolic activity of mitochondria may be different between them. Furthermore, Kim et al. [28] reported the changes in the lipid content (mainly phospholipids) and fatty acid composition were also observed in frozen-thawed immature oocytes. The mitochondria and lipid alteration after cryopreservation may affect subsequent fertilization and development processes. The shape and intercellular distribution of mitochondria are known to relate to the level of cell metabolism, proliferation, and differentiation for generation of the essential energy required in crucial periods of the cell cycle [29]. Thouas et al. [30] reported that oocyte mitochondria can make a necessary physiological contribution to the cytoplasmic regulation of preimplantation embryo development.
In conclusion, we demonstrated the feasibility of a nylon-mesh holder for vitrification of bovine GV-COCs, producing the same IVM rate of vitrified GV oocytes as the control by using a stepwise exposure to EFS40 and showing the integrity of organelles, which allowed for successful development to the blastocyst stage. After nonsurgical embryo transfer of blastocysts, one Japanese Black calf was born at 288 days after transfer. Considered that a nylon mesh holder can easily handle large numbers of bovine GV-COCs, the vitrification of GV oocytes using this holder would be expected to facilitate gamete storage for further application to assisted reproductive technologies, such as in vitro fertilization, cloning, and stem cell biology.
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FOOTNOTES |
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2 Correspondence: Yasuyuki Abe, Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan. FAX: 81 22 717 8687; abe-y{at}bios.tohoku.ac.jp
Received: 15 October 2004.
First decision: 9 November 2004.
Accepted: 27 January 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  K. M. Lowther, V. N. Weitzman, D. Maier, and L. M. Mehlmann Maturation, Fertilization, and the Structure and Function of the Endoplasmic Reticulum in Cryopreserved Mouse Oocytes Biol Reprod, July 1, 2009; 81(1): 147 - 154. [Abstract] [Full Text] [PDF]
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