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N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures
Cryobiology xxx (2016) 1e7
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N-acetyl cysteine prolonged the developmental ability of mouse twocell embryos against oxidative stress at refrigerated temperatures Yuka Horikoshi, Toru Takeo, Naomi Nakagata* Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2016 Received in revised form 25 April 2016 Accepted 4 May 2016 Available online xxx
Cold storage of two-cell embryos at refrigerated temperatures is a useful means to ship genetically engineered mice. We previously reported that M2 medium maintained the developmental ability of twocell embryos for 48 h at 4 C, and offspring were obtained from embryos transported by a courier service under refrigerated temperatures. The limitation of 48 h practically restricts the shipping destination of the embryos. To enhance the applicability of the cold-storage technique, prolonging the time to maintain developmental ability of the embryos is required. Oxidative stress may be a cause of the declining developmental ability of cold-stored embryos. However, the effect of oxidative stress on developmental ability of embryos has not been investigated. We examined intracellular glutathione (GSH) levels of coldstored two-cell embryos to evaluate the effect of oxidative and investigated the efficacy of adding Nacetyl cysteine (NAC) to the preservation medium on the developmental ability of cold-stored embryos and transported two-cell embryos at refrigerated temperatures. Intracellular GSH levels of two-cell embryos decreased by cold storage for longer than 72 h, whereas NAC recovered this reduction and improved the developmental ability of embryos cold-stored for 96 h. In the transport experiment, the developmental rate of transported two-cell embryos to offspring was increased by adding NAC to the preservation medium. We found that NAC prolonged the storage period of two-cell embryos and maintained the developmental ability by alleviating the reduction of intracellular GSH. These findings will improve the technique of cold-storage of two-cell embryos to facilitate efficient transport of genetically engineered mice worldwide. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cold storage Two-cell embryo Mouse N-acetyl cysteine Intracellular GSH
1. Introduction Mouse resource banks that produce, preserve, and supply genetically engineered mice have been established worldwide [5,30]. At present, more than 32,000 strains of genetically engineered mice that have been archived in mouse resource banks are available from banks via the International Mouse Strain Resource website [7,8]. Advances in reproductive technology made it possible to efficiently manage genetically engineered mice in mouse resource banks [11,50]. In vitro fertilization and embryo transfer have enabled the efficient production of genetically engineered animals [44,45,48,49]. Cryopreservation of embryos, oocytes, and sperm is a common method to preserve mouse lines and also reduces labor,
* Corresponding author. E-mail address: [email protected] (N. Nakagata).
space, and cost [28,29,32]. In addition, transportation of fresh embryos and sperm at refrigerated temperatures is useful for the shipment of genetically engineered mice alternative to the shipment of live animals [6,43,46,47,51]. In the shipment of genetically engineered mice, many factors must be considered, such as various laws and regulations, animal loss, microbiological control, working efficiency, and cost [33,55]. Transport of live animals should basically be avoided because of stress to animals, risk of death or escape of animals, and microbial infection during transport [41]. Experience with different freezing and thawing protocols is necessary to transport cryopreserved embryos and sperm, and special containers are required to keep the specimens at a constant temperature of 80 or 196 C [39]. On the other hand, fresh embryos and sperm can be transported without the above risks and drawbacks [46,51]. Hence, shipment of fresh embryos or sperm is the simplest and most suitable method for transport of genetically engineered mice. Early stages of mouse embryos maintain developmental ability
http://dx.doi.org/10.1016/j.cryobiol.2016.05.002 0011-2240/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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at refrigeration temperatures [3,12,13,17,18,26,46,47]. Previously, we reported that M2 medium maintained the developmental ability of two-cell embryos for up to 48 h at 4 C and demonstrated that two-cell embryos transported at refrigerated temperatures developed into live young via embryo transfer in receiving laboratories [46,47]. The technique for the cold storage of embryos is useful for short-term storage or shipment of two-cell embryos both domestically and internationally. However, the time limit of 48 h restricts the ability to transport embryos. Oxidative stress has been suggested as a possible cause of inhibition of the developmental ability of two-cell embryos during cold storage. In general, cold storage of cells and organs incur declining intracellular glutathione (GSH) levels because of oxidative stress [54]. Reduction of intracellular GSH plays a role in the decreased developmental ability of early stage mouse embryos [9,15]. However, the relationship between levels of intracellular GSH and the developmental ability of cold-stored two-cell embryos remains unclear. Moreover, the effect of addition of antioxidants, such as N-acetyl cysteine (NAC), to embryo preservation medium on the developmental ability of two-cell embryos has not been examined. In this study, we measured intracellular glutathione (GSH) levels of cold-stored two-cell embryos to monitor oxidative stress during cold storage. In addition, we investigated the effect of NAC on the developmental ability of cold-stored two-cell embryos and transported two-cell embryos at refrigerated temperatures. 2. Materials and methods 2.1. Animals C57BL/6 J mice, purchased from CLEA Japan, were used to obtain sperm (from 12 to 16-week-old male mice) or oocytes (from 8 to 12-week-old female mice). ICR mice (8e12 weeks old) were used as recipients of two-cell embryos. All animals were housed under a 12-h darkelight cycle (light from 07:00 to 19:00) at 22 C ± 1 C with free access to food and water. The Animal Care and Use Committee of Kumamoto University School of Medicine approved the protocols for animal experiments. 2.2. Media Modified Krebs-Ringer bicarbonate solution (TYH) containing 1.0 mg/mL of polyvinyl alcohol and 0.75 mM of methyl-b-cyclodextrin (Sigma-Aldrich) (cTYH) was used as sperm pre-incubation medium [45,52]. Modified human tubal fluid (mHTF) with a high concentration of calcium (5.14 mM) was used as fertilization medium [21,35]. M2 embryo storage medium (Sigma-Aldrich) was used for cold storage of two-cell embryos [34]. To examine the efficacy of NAC, various concentrations (0, 0.5, 1.0, 1.5, 2.0 or 3.0 mM) of NAC were added to M2 cold storage medium before use. Potassium simplex optimized medium (KSOM, embryo culture medium) was used to culture two-cell embryos to the blastocyst stage [1,14]. 2.3. In vitro fertilization Female mice were superovulated by intraperitoneal injection of 7.5 IU of equine chorionic gonadotropin (ASKA Pharmaceutical Co. Ltd.) and then injected with 7.5 IU of human chorionic gonadotropin (hCG; ASKA Pharmaceutical Co. Ltd.) 48 h later. At 14e16 h after hCG injection, the mice were euthanized by cervical dislocation, then their oviducts were quickly removed and transferred to a fertilization dish containing paraffin oil. Under microscopic observation, a needle and forceps were used to remove 8e10
cumuluseoocyte complexes (COCs) from the ampulla of the fallopian tubes from four to five mice. The COCs were then introduced into a 200-mL drop of mHTF and covered with paraffin oil. After male mice were euthanized by cervical dislocation, twotailed caudal epididymides were collected. Each caudal epididymis was transferred into paraffin oil, and the epididymis ducts were cut using a pair of micro-dissecting scissors. Next, using a dissecting needle, the mass of sperm released from the caudal epididymis was introduced into a 100-mL drop of cTYH and incubated at 37 C for 60 min under an atmosphere of 5% CO2/95% air. After incubation, an aliquot of the sperm suspension was added to a 200-mL drop of mHTF containing COCs and co-incubated at 37 C for 60 min in an atmosphere of 5% CO2/95% air. At 3 h after insemination, the oocytes were washed four times in 80-mL drops of mHTF, covered with paraffin oil, and cultured at 37 C in an atmosphere containing 5% CO2/95% air. At 24 h postinsemination, fertilization rates were calculated as the total number of two-cell embryos divided by the total number of inseminated oocytes multiplied by 100. The fertilization rate was more than 90% in all experiments. Two-cell embryos were used for cold storage, in vitro culture, quantification of intracellular GSH, transport experiments, or embryo transfer. 2.4. Cold storage of two-cell embryos Cold storage of two-cell embryos was performed as described previously [46,47]. After in vitro fertilization (IVF), two-cell embryos were transferred, washed three times using an 80-mL drop of M2 medium each time, and then transferred into 0.6-mL tubes (Fisher brand Microcentrifuge Graduated, Flat Cap Tubes; Fisher Scientific, Pittsburgh, PA) containing 600 mL of M2 medium with various concentrations (0, 0.5, 1.0, 1.5, 2.0, or 3.0 mM) of NAC. The tubes were packed in a CARD Cold Transport Kit (Kyudo Co. Ltd) and stored in a refrigerator at 4.0 C ± 1.5 C for 0e96 h. 2.5. Embryo culture Cold-stored two-cell embryos were washed and cultured to the blastocyst stage in a 100-mL drop of KSOM at 37 C in an atmosphere of 5% CO2/95% air. Developmental ability of cold-stored twocell embryos was evaluated according to developmental rates to four-cell embryos (24 h), morulae (48 h), and blastocysts (72 or 96 h) during embryo culture. 2.6. Quantification of intracellular GSH Intracellular GSH was measured using a thiol-selective fluorescent dye (ViVidFluor Cell Blue CMAC; 229e02131; Wako) [20]. Fresh or cold-stored two-cell embryos were collected, washed three times in phosphate-buffered saline (PBS) containing 1.0 mg/ mL polyvinyl alcohol (PBSePVA), stained with 50 mg/mL of ViVidFluor Cell Blue CMAC for 1 h in dark at room temperature, then washed again three times in PBSePVA, and observed under a fluorescence microscope (Biorevo BZ-9000, Keyence Co., Japan). Fluorescence intensity of intracellular GSH in the embryos was analyzed using image analyzing software (BZ-H2A version 1.42; Keyence Co., Japan). 2.7. Shipment of two-cell embryos at refrigerated temperatures Two-cell embryos were transported as described [46,47]. In brief, embryo storage boxes containing two-cell embryos were placed in a CARD Cold Transport Kit and transported from Asahikawa Medical College to the Center for Animal Resources and Development of Kumamoto University. During transport, the
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
Y. Horikoshi et al. / Cryobiology xxx (2016) 1e7
temperature in each embryo transport box was measured continuously using a temperature data logger (Thermochron iButtons; Maxim Integrated Products, Sunnyvale, CA). During transport, the temperature gradually dropped and was maintained at 4.0 C ± 2.8 C. The transported embryos were used for embryo transfer to confirm preservation of the developmental ability of the embryos.
3.2. Effect of cold-storage period on intracellular GSH of two-cell embryos
2.8. Embryo transfer
3.3. Effect of NAC on developmental ability of cold stored two-cell embryos in vitro
The cold-stored embryos were transferred into oviducts of ICR female mice (10 embryos/oviduct) on the day a vaginal plug was found (day 1 of pseudopregnancy). Embryos were transferred through the wall of the fallopian tube, as described previously [27]. After 19 days, the development of embryos into live young was examined (number of live young/number of transferred embryos 100%). 2.9. Statistical analysis Statistical analysis was performed using StatView-5.0 J for Windows software (SAS Institute Inc.). Group results were compared using analysis of variance. A probability (P) value of <0.05 was considered statistically significant. 3. Results 3.1. Effect of cold-storage period on the developmental ability of two-cell embryos in vitro As shown in Fig. 1, the developmental rate of cold-stored twocell embryos to blastocysts was decreased after 48 h. In addition, the rates of development of cold-stored embryos for 96 h to a fourcell embryo, morula, and blastocyst had decreased.
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Intracellular GSH levels of cold-stored two-cell embryos were significantly reduced after cold storage for 72 and 96 h (Fig. 2). The reduction in intracellular GSH was first demonstrated in coldstored two-cell embryos.
NAC improved the developmental rates of two-cell embryos stored at 4 C for 96 h (Fig. 3). The most effective concentration of NAC was 1.5 mM. Therefore, subsequent experiments were performed with 1.5 mM NAC. 3.4. Effect of NAC on intracellular GSH of cold-stored two-cell embryos Addition of NAC to cold storage medium alleviated the reduction in intracellular GSH of cold-stored two-cell embryos at 72 and 96 h (Fig. 4). 3.5. Developmental ability of transported two-cell embryos at refrigerated temperatures Offspring were obtained from transported two-cell embryos cold-stored for 96 h at refrigerated temperatures (Table 1). The rate of NAC-treated embryos was higher than that of control embryos cold-stored without NAC. 4. Discussion The results of this study demonstrated that cold storage of twocell embryos reduced developmental ability and intracellular GSH
Fig. 1. Effect of preservation period on the developmental ability of cold-stored two-cell embryos in vitro. After in vitro fertilization (IVF), two-cell embryos were transferred and stored in M2 medium for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the two-cell embryos were transferred and cultured in KSOM for 72 h. The embryos were observed at fourcell embryo, morula, and blastocyst stages at 24, 48, and 72 h, respectively. One hundred to 233 embryos were counted in each group. Developmental rates were calculated as the number of four-cell embryos, morulae, or blastocysts divided by the number of two-cell embryos 100 (%). Results are expressed as mean ± SD (n ¼ 3e6). *P < 0.05 compared with the control at 0 h.
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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Fig. 2. Effect of preservation period on intracellular GSH levels in cold-stored two-cell embryos. Two-cell embryos were stored in M2 medium for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the two-cell embryos were stained with ViVidFruor Cell Blue CMAC to visualize intracellular GSH. In each group, 29e31 embryos were observed by fluorescence microscopy (A: bright field, B: fluorescence image), and the amount of intracellular GSH was measured by the fluorescence intensity of the ViVidFluor Cell Blue CMAC. The amount of intracellular GSH in the two-cell embryos was calculated according to the fluorescence intensity of cold-stored embryos for 24, 48, 72, or 96 h divided by the fluorescence intensity of fresh embryos (0). Results are expressed as mean ± SD (n ¼ 4). *P < 0.05 compared with the control at 0 h. Scale bar ¼ 50 mm.
Fig. 3. Effect of adding NAC to cold-storage medium on the developmental ability of cold-stored two-cell embryos in vitro. Two-cell embryos were transferred into M2 medium with various concentrations of NAC and cold-stored for 96 h at 4 C. After cold storage, two-cell embryos were transferred and cultured in KSOM for 96 h. Developmental rates were calculated as the number of four-cell embryos, morulae, or blastocysts divided by the number of two-cell embryos 100 (%). Results are expressed as mean ± SD (n ¼ 7). *P < 0.05 compared with the control at 0 mM.
level in a time-dependent manner. Addition of NAC to the preservation solution recovered the reduction in developmental rate and the level of intracellular GSH in the cold-stored two-cell embryos. NAC-treated embryos had improved developmental ability for 96 h. In addition, offspring were obtained from transported embryos at refrigerated temperatures stored in preservation solution containing NAC. In mouse resource banks, more than 32,000 strains of genetically engineered mice have been archived in the form of
cryopreserved two-cell embryos or sperm [7]. The utilization of these mice will improve accessibility and quality control, avoiding duplicated production of animals to conduct efficient experiments [24]. Improved logistics of genetically engineered mice using reproductive technology is helpful to facilitate the use of mouse strains archived in mouse resource banks. In this study, we achieved prolonged cold storage of two-cell embryos by adding NAC to the preservation medium. Using the system of the cold storage of mouse embryos, researchers can easily obtain genetically
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
Y. Horikoshi et al. / Cryobiology xxx (2016) 1e7
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Fig. 4. Effect of adding NAC to cold-storage medium on the amount of intracellular GSH in cold-stored two-cell embryos. Two-cell embryos were transferred into M2 medium with or without 1.5 mM NAC and stored for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the level of intracellular GSH of two-cell embryos was measured according to the fluorescence intensity of ViVidFluor Cell Blue CMAC. In each group, 29e31 embryos were examined. Results are expressed as mean ± SD (n ¼ 4). *P < 0.05 compared with no treatment at each time point.
Table 1 Developmental ability of transported two-cell embryos at refrigerated temperatures. Storage time (h)
NAC (mM)
No. of transferred two-cell embryos
No. of recipients
No. of offspring (%)
96
0 1.5
60 60
3 3
2 (3.3 ± 2.9) 28 (46.7 ± 10.4)a
Results are expressed as mean ± SD (n ¼ 3). a Value is significantly different (P < 0.05) from control at 0 mM.
engineered mice from international mouse resource banks. There have been some reports about cold storage of mouse embryos. For example, Whittingham and Wales [56] examined the developmental ability of two-cell embryos preserved in PBS with DL-lactate, sodium pyruvate, and bovine serum albumin at 0, 5, or 10 C for 3, 24, or 48 h. However, storage temperatures did not appear to maintain developmental ability. Moreover, Kasai et al. [17,18] reported that PBS containing 0.75 M sucrose improved survival and developmental ability of stored mouse morulae at 0 C. Herr and Wright [12,13] revealed that the developmental rates of various stage of one-cell embryos to blastocysts after cold storage at 4 C in modified Whitten' medium excluding sodium bicarbonate and supplemented with sodium lactate, sodium pyruvate, glucose, HEPES, and bovine serum albumin was most sensitive to cold storage in early stage embryos. Moreover, Nakamura and Tsunoda [31] reported that the addition of 0.5 M sucrose to PB1 medium improved the developmental ability of mouse two-cell embryos stored at 4 C. Miyoshi et al. [26] demonstrated that offspring were obtained from mouse two-cell embryos transported from Kochi to Sapporo over a 48-h period (birth rate of 14.1%). Moreover, our group reported success in obtaining offspring from vitrified/ warmed two-cell embryos or two-cell embryos produced from cryopreserved sperm following transport to distant Japanese laboratories for 48e72 h [46,47]. Afterward, this method was adopted to transport genetically engineered mice between facilities and research institutes [19]. In this study, we found that a reduction of intracellular GSH because of oxidative stress resulted in decreased developmental ability of cold-stored two-cell embryos, whereas
the reduction in developmental ability because of prolonged storage for 96 h was recovered by the addition of NAC to the preservation medium. NAC is a typical antioxidant with a thiol group that is used for GSH synthesis after being taken up by the cells [2,37,38]. NAC treatment of cells increased intracellular GSH concentrations and improved resistance to oxidative stress [16,57]. In general, intracellular GSH conveys a protective effect against oxidative stress and maintains a reductive environment within the cell [4,23,25]. We revealed that the addition of NAC recovered the developmental ability of cold-stored two-cell embryos by maintaining intracellular GSH levels during storage. In cold storage, treatment of NAC inhibited ischemia-reperfusion injury after transplantation by preventing oxidative stress and maintaining intracellular GSH levels in rat liver [22,36,53]. In addition, the antioxidant effects of NAC inhibited lipid peroxide generation during cold storage [40]. Hence, the protective effects of NAC in two-cell embryos may act against oxidative stress by maintaining intracellular GSH levels during storage. Intracellular GSH is a determining factor of the developmental ability of early-stage embryos. On average, the intracellular GSH concentration of mouse unfertilized oocytes is 7.0 mM, which is subsequently decreased by the process of fertilization and development, as the concentration is 3.5 mM in two-cell embryos and 0.7 mM in blastocysts [9]. A chemically induced reduction in intracellular GSH of two-cell embryos was found to decrease the developmental rates of in vitro culture [10,42]. Together, these results suggest that preservation of intracellular GSH may be
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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important to maintain the developmental ability of cold-stored two-cell embryos. In summary, we discovered that NAC improved the developmental ability of cold-stored two-cell embryos by maintaining intracellular GSH levels during storage. These results suggest that the application of preservation medium containing NAC for cold storage of two-cell embryos will enable efficient transportation of genetically engineered mice worldwide.
[19]
[20]
[21] [22]
Conflict of interest [23]
There are no conflicts of interest to declare. [24]
Statement of funding This work was funded by Kumamoto University.
[25] [26]
Acknowledgments
[27]
We thank Norihiko Shimizu and Chihiro Hino (Asahikawa Medical University, Japan) for providing and transporting mouse two-cell embryos.
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Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
N-acetyl cysteine prolonged the developmental ability of mouse twocell embryos against oxidative stress at refrigerated temperatures Yuka Horikoshi, Toru Takeo, Naomi Nakagata* Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2016 Received in revised form 25 April 2016 Accepted 4 May 2016 Available online xxx
Cold storage of two-cell embryos at refrigerated temperatures is a useful means to ship genetically engineered mice. We previously reported that M2 medium maintained the developmental ability of twocell embryos for 48 h at 4 C, and offspring were obtained from embryos transported by a courier service under refrigerated temperatures. The limitation of 48 h practically restricts the shipping destination of the embryos. To enhance the applicability of the cold-storage technique, prolonging the time to maintain developmental ability of the embryos is required. Oxidative stress may be a cause of the declining developmental ability of cold-stored embryos. However, the effect of oxidative stress on developmental ability of embryos has not been investigated. We examined intracellular glutathione (GSH) levels of coldstored two-cell embryos to evaluate the effect of oxidative and investigated the efficacy of adding Nacetyl cysteine (NAC) to the preservation medium on the developmental ability of cold-stored embryos and transported two-cell embryos at refrigerated temperatures. Intracellular GSH levels of two-cell embryos decreased by cold storage for longer than 72 h, whereas NAC recovered this reduction and improved the developmental ability of embryos cold-stored for 96 h. In the transport experiment, the developmental rate of transported two-cell embryos to offspring was increased by adding NAC to the preservation medium. We found that NAC prolonged the storage period of two-cell embryos and maintained the developmental ability by alleviating the reduction of intracellular GSH. These findings will improve the technique of cold-storage of two-cell embryos to facilitate efficient transport of genetically engineered mice worldwide. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cold storage Two-cell embryo Mouse N-acetyl cysteine Intracellular GSH
1. Introduction Mouse resource banks that produce, preserve, and supply genetically engineered mice have been established worldwide [5,30]. At present, more than 32,000 strains of genetically engineered mice that have been archived in mouse resource banks are available from banks via the International Mouse Strain Resource website [7,8]. Advances in reproductive technology made it possible to efficiently manage genetically engineered mice in mouse resource banks [11,50]. In vitro fertilization and embryo transfer have enabled the efficient production of genetically engineered animals [44,45,48,49]. Cryopreservation of embryos, oocytes, and sperm is a common method to preserve mouse lines and also reduces labor,
* Corresponding author. E-mail address: [email protected] (N. Nakagata).
space, and cost [28,29,32]. In addition, transportation of fresh embryos and sperm at refrigerated temperatures is useful for the shipment of genetically engineered mice alternative to the shipment of live animals [6,43,46,47,51]. In the shipment of genetically engineered mice, many factors must be considered, such as various laws and regulations, animal loss, microbiological control, working efficiency, and cost [33,55]. Transport of live animals should basically be avoided because of stress to animals, risk of death or escape of animals, and microbial infection during transport [41]. Experience with different freezing and thawing protocols is necessary to transport cryopreserved embryos and sperm, and special containers are required to keep the specimens at a constant temperature of 80 or 196 C [39]. On the other hand, fresh embryos and sperm can be transported without the above risks and drawbacks [46,51]. Hence, shipment of fresh embryos or sperm is the simplest and most suitable method for transport of genetically engineered mice. Early stages of mouse embryos maintain developmental ability
http://dx.doi.org/10.1016/j.cryobiol.2016.05.002 0011-2240/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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at refrigeration temperatures [3,12,13,17,18,26,46,47]. Previously, we reported that M2 medium maintained the developmental ability of two-cell embryos for up to 48 h at 4 C and demonstrated that two-cell embryos transported at refrigerated temperatures developed into live young via embryo transfer in receiving laboratories [46,47]. The technique for the cold storage of embryos is useful for short-term storage or shipment of two-cell embryos both domestically and internationally. However, the time limit of 48 h restricts the ability to transport embryos. Oxidative stress has been suggested as a possible cause of inhibition of the developmental ability of two-cell embryos during cold storage. In general, cold storage of cells and organs incur declining intracellular glutathione (GSH) levels because of oxidative stress [54]. Reduction of intracellular GSH plays a role in the decreased developmental ability of early stage mouse embryos [9,15]. However, the relationship between levels of intracellular GSH and the developmental ability of cold-stored two-cell embryos remains unclear. Moreover, the effect of addition of antioxidants, such as N-acetyl cysteine (NAC), to embryo preservation medium on the developmental ability of two-cell embryos has not been examined. In this study, we measured intracellular glutathione (GSH) levels of cold-stored two-cell embryos to monitor oxidative stress during cold storage. In addition, we investigated the effect of NAC on the developmental ability of cold-stored two-cell embryos and transported two-cell embryos at refrigerated temperatures. 2. Materials and methods 2.1. Animals C57BL/6 J mice, purchased from CLEA Japan, were used to obtain sperm (from 12 to 16-week-old male mice) or oocytes (from 8 to 12-week-old female mice). ICR mice (8e12 weeks old) were used as recipients of two-cell embryos. All animals were housed under a 12-h darkelight cycle (light from 07:00 to 19:00) at 22 C ± 1 C with free access to food and water. The Animal Care and Use Committee of Kumamoto University School of Medicine approved the protocols for animal experiments. 2.2. Media Modified Krebs-Ringer bicarbonate solution (TYH) containing 1.0 mg/mL of polyvinyl alcohol and 0.75 mM of methyl-b-cyclodextrin (Sigma-Aldrich) (cTYH) was used as sperm pre-incubation medium [45,52]. Modified human tubal fluid (mHTF) with a high concentration of calcium (5.14 mM) was used as fertilization medium [21,35]. M2 embryo storage medium (Sigma-Aldrich) was used for cold storage of two-cell embryos [34]. To examine the efficacy of NAC, various concentrations (0, 0.5, 1.0, 1.5, 2.0 or 3.0 mM) of NAC were added to M2 cold storage medium before use. Potassium simplex optimized medium (KSOM, embryo culture medium) was used to culture two-cell embryos to the blastocyst stage [1,14]. 2.3. In vitro fertilization Female mice were superovulated by intraperitoneal injection of 7.5 IU of equine chorionic gonadotropin (ASKA Pharmaceutical Co. Ltd.) and then injected with 7.5 IU of human chorionic gonadotropin (hCG; ASKA Pharmaceutical Co. Ltd.) 48 h later. At 14e16 h after hCG injection, the mice were euthanized by cervical dislocation, then their oviducts were quickly removed and transferred to a fertilization dish containing paraffin oil. Under microscopic observation, a needle and forceps were used to remove 8e10
cumuluseoocyte complexes (COCs) from the ampulla of the fallopian tubes from four to five mice. The COCs were then introduced into a 200-mL drop of mHTF and covered with paraffin oil. After male mice were euthanized by cervical dislocation, twotailed caudal epididymides were collected. Each caudal epididymis was transferred into paraffin oil, and the epididymis ducts were cut using a pair of micro-dissecting scissors. Next, using a dissecting needle, the mass of sperm released from the caudal epididymis was introduced into a 100-mL drop of cTYH and incubated at 37 C for 60 min under an atmosphere of 5% CO2/95% air. After incubation, an aliquot of the sperm suspension was added to a 200-mL drop of mHTF containing COCs and co-incubated at 37 C for 60 min in an atmosphere of 5% CO2/95% air. At 3 h after insemination, the oocytes were washed four times in 80-mL drops of mHTF, covered with paraffin oil, and cultured at 37 C in an atmosphere containing 5% CO2/95% air. At 24 h postinsemination, fertilization rates were calculated as the total number of two-cell embryos divided by the total number of inseminated oocytes multiplied by 100. The fertilization rate was more than 90% in all experiments. Two-cell embryos were used for cold storage, in vitro culture, quantification of intracellular GSH, transport experiments, or embryo transfer. 2.4. Cold storage of two-cell embryos Cold storage of two-cell embryos was performed as described previously [46,47]. After in vitro fertilization (IVF), two-cell embryos were transferred, washed three times using an 80-mL drop of M2 medium each time, and then transferred into 0.6-mL tubes (Fisher brand Microcentrifuge Graduated, Flat Cap Tubes; Fisher Scientific, Pittsburgh, PA) containing 600 mL of M2 medium with various concentrations (0, 0.5, 1.0, 1.5, 2.0, or 3.0 mM) of NAC. The tubes were packed in a CARD Cold Transport Kit (Kyudo Co. Ltd) and stored in a refrigerator at 4.0 C ± 1.5 C for 0e96 h. 2.5. Embryo culture Cold-stored two-cell embryos were washed and cultured to the blastocyst stage in a 100-mL drop of KSOM at 37 C in an atmosphere of 5% CO2/95% air. Developmental ability of cold-stored twocell embryos was evaluated according to developmental rates to four-cell embryos (24 h), morulae (48 h), and blastocysts (72 or 96 h) during embryo culture. 2.6. Quantification of intracellular GSH Intracellular GSH was measured using a thiol-selective fluorescent dye (ViVidFluor Cell Blue CMAC; 229e02131; Wako) [20]. Fresh or cold-stored two-cell embryos were collected, washed three times in phosphate-buffered saline (PBS) containing 1.0 mg/ mL polyvinyl alcohol (PBSePVA), stained with 50 mg/mL of ViVidFluor Cell Blue CMAC for 1 h in dark at room temperature, then washed again three times in PBSePVA, and observed under a fluorescence microscope (Biorevo BZ-9000, Keyence Co., Japan). Fluorescence intensity of intracellular GSH in the embryos was analyzed using image analyzing software (BZ-H2A version 1.42; Keyence Co., Japan). 2.7. Shipment of two-cell embryos at refrigerated temperatures Two-cell embryos were transported as described [46,47]. In brief, embryo storage boxes containing two-cell embryos were placed in a CARD Cold Transport Kit and transported from Asahikawa Medical College to the Center for Animal Resources and Development of Kumamoto University. During transport, the
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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temperature in each embryo transport box was measured continuously using a temperature data logger (Thermochron iButtons; Maxim Integrated Products, Sunnyvale, CA). During transport, the temperature gradually dropped and was maintained at 4.0 C ± 2.8 C. The transported embryos were used for embryo transfer to confirm preservation of the developmental ability of the embryos.
3.2. Effect of cold-storage period on intracellular GSH of two-cell embryos
2.8. Embryo transfer
3.3. Effect of NAC on developmental ability of cold stored two-cell embryos in vitro
The cold-stored embryos were transferred into oviducts of ICR female mice (10 embryos/oviduct) on the day a vaginal plug was found (day 1 of pseudopregnancy). Embryos were transferred through the wall of the fallopian tube, as described previously [27]. After 19 days, the development of embryos into live young was examined (number of live young/number of transferred embryos 100%). 2.9. Statistical analysis Statistical analysis was performed using StatView-5.0 J for Windows software (SAS Institute Inc.). Group results were compared using analysis of variance. A probability (P) value of <0.05 was considered statistically significant. 3. Results 3.1. Effect of cold-storage period on the developmental ability of two-cell embryos in vitro As shown in Fig. 1, the developmental rate of cold-stored twocell embryos to blastocysts was decreased after 48 h. In addition, the rates of development of cold-stored embryos for 96 h to a fourcell embryo, morula, and blastocyst had decreased.
3
Intracellular GSH levels of cold-stored two-cell embryos were significantly reduced after cold storage for 72 and 96 h (Fig. 2). The reduction in intracellular GSH was first demonstrated in coldstored two-cell embryos.
NAC improved the developmental rates of two-cell embryos stored at 4 C for 96 h (Fig. 3). The most effective concentration of NAC was 1.5 mM. Therefore, subsequent experiments were performed with 1.5 mM NAC. 3.4. Effect of NAC on intracellular GSH of cold-stored two-cell embryos Addition of NAC to cold storage medium alleviated the reduction in intracellular GSH of cold-stored two-cell embryos at 72 and 96 h (Fig. 4). 3.5. Developmental ability of transported two-cell embryos at refrigerated temperatures Offspring were obtained from transported two-cell embryos cold-stored for 96 h at refrigerated temperatures (Table 1). The rate of NAC-treated embryos was higher than that of control embryos cold-stored without NAC. 4. Discussion The results of this study demonstrated that cold storage of twocell embryos reduced developmental ability and intracellular GSH
Fig. 1. Effect of preservation period on the developmental ability of cold-stored two-cell embryos in vitro. After in vitro fertilization (IVF), two-cell embryos were transferred and stored in M2 medium for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the two-cell embryos were transferred and cultured in KSOM for 72 h. The embryos were observed at fourcell embryo, morula, and blastocyst stages at 24, 48, and 72 h, respectively. One hundred to 233 embryos were counted in each group. Developmental rates were calculated as the number of four-cell embryos, morulae, or blastocysts divided by the number of two-cell embryos 100 (%). Results are expressed as mean ± SD (n ¼ 3e6). *P < 0.05 compared with the control at 0 h.
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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Fig. 2. Effect of preservation period on intracellular GSH levels in cold-stored two-cell embryos. Two-cell embryos were stored in M2 medium for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the two-cell embryos were stained with ViVidFruor Cell Blue CMAC to visualize intracellular GSH. In each group, 29e31 embryos were observed by fluorescence microscopy (A: bright field, B: fluorescence image), and the amount of intracellular GSH was measured by the fluorescence intensity of the ViVidFluor Cell Blue CMAC. The amount of intracellular GSH in the two-cell embryos was calculated according to the fluorescence intensity of cold-stored embryos for 24, 48, 72, or 96 h divided by the fluorescence intensity of fresh embryos (0). Results are expressed as mean ± SD (n ¼ 4). *P < 0.05 compared with the control at 0 h. Scale bar ¼ 50 mm.
Fig. 3. Effect of adding NAC to cold-storage medium on the developmental ability of cold-stored two-cell embryos in vitro. Two-cell embryos were transferred into M2 medium with various concentrations of NAC and cold-stored for 96 h at 4 C. After cold storage, two-cell embryos were transferred and cultured in KSOM for 96 h. Developmental rates were calculated as the number of four-cell embryos, morulae, or blastocysts divided by the number of two-cell embryos 100 (%). Results are expressed as mean ± SD (n ¼ 7). *P < 0.05 compared with the control at 0 mM.
level in a time-dependent manner. Addition of NAC to the preservation solution recovered the reduction in developmental rate and the level of intracellular GSH in the cold-stored two-cell embryos. NAC-treated embryos had improved developmental ability for 96 h. In addition, offspring were obtained from transported embryos at refrigerated temperatures stored in preservation solution containing NAC. In mouse resource banks, more than 32,000 strains of genetically engineered mice have been archived in the form of
cryopreserved two-cell embryos or sperm [7]. The utilization of these mice will improve accessibility and quality control, avoiding duplicated production of animals to conduct efficient experiments [24]. Improved logistics of genetically engineered mice using reproductive technology is helpful to facilitate the use of mouse strains archived in mouse resource banks. In this study, we achieved prolonged cold storage of two-cell embryos by adding NAC to the preservation medium. Using the system of the cold storage of mouse embryos, researchers can easily obtain genetically
Please cite this article in press as: Y. Horikoshi, et al., N-acetyl cysteine prolonged the developmental ability of mouse two-cell embryos against oxidative stress at refrigerated temperatures, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.05.002
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Fig. 4. Effect of adding NAC to cold-storage medium on the amount of intracellular GSH in cold-stored two-cell embryos. Two-cell embryos were transferred into M2 medium with or without 1.5 mM NAC and stored for 0, 24, 48, 72, or 96 h at 4 C. After cold storage, the level of intracellular GSH of two-cell embryos was measured according to the fluorescence intensity of ViVidFluor Cell Blue CMAC. In each group, 29e31 embryos were examined. Results are expressed as mean ± SD (n ¼ 4). *P < 0.05 compared with no treatment at each time point.
Table 1 Developmental ability of transported two-cell embryos at refrigerated temperatures. Storage time (h)
NAC (mM)
No. of transferred two-cell embryos
No. of recipients
No. of offspring (%)
96
0 1.5
60 60
3 3
2 (3.3 ± 2.9) 28 (46.7 ± 10.4)a
Results are expressed as mean ± SD (n ¼ 3). a Value is significantly different (P < 0.05) from control at 0 mM.
engineered mice from international mouse resource banks. There have been some reports about cold storage of mouse embryos. For example, Whittingham and Wales [56] examined the developmental ability of two-cell embryos preserved in PBS with DL-lactate, sodium pyruvate, and bovine serum albumin at 0, 5, or 10 C for 3, 24, or 48 h. However, storage temperatures did not appear to maintain developmental ability. Moreover, Kasai et al. [17,18] reported that PBS containing 0.75 M sucrose improved survival and developmental ability of stored mouse morulae at 0 C. Herr and Wright [12,13] revealed that the developmental rates of various stage of one-cell embryos to blastocysts after cold storage at 4 C in modified Whitten' medium excluding sodium bicarbonate and supplemented with sodium lactate, sodium pyruvate, glucose, HEPES, and bovine serum albumin was most sensitive to cold storage in early stage embryos. Moreover, Nakamura and Tsunoda [31] reported that the addition of 0.5 M sucrose to PB1 medium improved the developmental ability of mouse two-cell embryos stored at 4 C. Miyoshi et al. [26] demonstrated that offspring were obtained from mouse two-cell embryos transported from Kochi to Sapporo over a 48-h period (birth rate of 14.1%). Moreover, our group reported success in obtaining offspring from vitrified/ warmed two-cell embryos or two-cell embryos produced from cryopreserved sperm following transport to distant Japanese laboratories for 48e72 h [46,47]. Afterward, this method was adopted to transport genetically engineered mice between facilities and research institutes [19]. In this study, we found that a reduction of intracellular GSH because of oxidative stress resulted in decreased developmental ability of cold-stored two-cell embryos, whereas
the reduction in developmental ability because of prolonged storage for 96 h was recovered by the addition of NAC to the preservation medium. NAC is a typical antioxidant with a thiol group that is used for GSH synthesis after being taken up by the cells [2,37,38]. NAC treatment of cells increased intracellular GSH concentrations and improved resistance to oxidative stress [16,57]. In general, intracellular GSH conveys a protective effect against oxidative stress and maintains a reductive environment within the cell [4,23,25]. We revealed that the addition of NAC recovered the developmental ability of cold-stored two-cell embryos by maintaining intracellular GSH levels during storage. In cold storage, treatment of NAC inhibited ischemia-reperfusion injury after transplantation by preventing oxidative stress and maintaining intracellular GSH levels in rat liver [22,36,53]. In addition, the antioxidant effects of NAC inhibited lipid peroxide generation during cold storage [40]. Hence, the protective effects of NAC in two-cell embryos may act against oxidative stress by maintaining intracellular GSH levels during storage. Intracellular GSH is a determining factor of the developmental ability of early-stage embryos. On average, the intracellular GSH concentration of mouse unfertilized oocytes is 7.0 mM, which is subsequently decreased by the process of fertilization and development, as the concentration is 3.5 mM in two-cell embryos and 0.7 mM in blastocysts [9]. A chemically induced reduction in intracellular GSH of two-cell embryos was found to decrease the developmental rates of in vitro culture [10,42]. Together, these results suggest that preservation of intracellular GSH may be
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important to maintain the developmental ability of cold-stored two-cell embryos. In summary, we discovered that NAC improved the developmental ability of cold-stored two-cell embryos by maintaining intracellular GSH levels during storage. These results suggest that the application of preservation medium containing NAC for cold storage of two-cell embryos will enable efficient transportation of genetically engineered mice worldwide.
[19]
[20]
[21] [22]
Conflict of interest [23]
There are no conflicts of interest to declare. [24]
Statement of funding This work was funded by Kumamoto University.
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Acknowledgments
[27]
We thank Norihiko Shimizu and Chihiro Hino (Asahikawa Medical University, Japan) for providing and transporting mouse two-cell embryos.
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