Introduction
The first successful production of porcine somatic cell clones was reported in 2000 (Betthauser et al., Reference Betthauser, Forsberg, Augenstein, Childs, Eilertsen, Enos, Forsythe, Golueke, Jurgella, Koppang, Lesmeister, Mallon, Mell, Misica, Pace, Pfister-Genskow, Strelchenko, Voelker, Watt, Thompson and Bishop2000; Onishi et al., Reference Onishi, Iwamoto, Akita, Mikawa, Takeda, Awata, Hanada and Perry2000; Polejaeva et al., Reference Polejaeva, Chen, Vaught, Page, Mullins, Ball, Dai, Boonem, Walkerm, Ayares, Colman and Campbell2000), and since that time a large number of cloned pigs have been produced in many laboratories (Campbell et al., Reference Campbell, Fisher, Chen, Choi, Kelly, Lee and Xhu2007). The somatic cell nuclear-transfer (SCNT) technique is a novel tool for improving domestic animals, rescuing endangered species and producing transgenic animals for medical use. Because pigs and humans have similar physiologic characteristics, genetically modified pigs, such as knockout pig have been produced for xenotransplantation studies (Fujimura et al., Reference Fujimura, Takahagi, Shigehisa, Nagashima, Miyagawa, Shirakura and Murakami2008; Klymiuk et al., Reference Klymiuk, Aigner, Brem and Wolf2010).
Animals of several species have been cloned using SCNT techniques, but the cloning success rate is low (Campbell et al., Reference Campbell, Fisher, Chen, Choi, Kelly, Lee and Xhu2007). As SCNT technology includes different processes and the oocytes have species-specific characteristics, there are various problems that need to be solved. Among them, an appropriate in vitro culture system for recipient and SCNT oocytes is a crucial factor.
Preimplantation mammalian embryos incur damage due to the actions of reactive oxygen species (ROS) during in vitro culture (Goto et al., Reference Goto, Noda, Mori and Nakano1993), which results in damage to the organelles, particularly the mitochondria, and causes species-specific embryo developmental block and apoptosis (Yang et al., Reference Yang, Hwang, Kwon, Kim, Choi and Oh1998; Guerin et al., Reference Guerin, Mouatassim and Menezo2001; Kamjoo et al., Reference Kamjoo, Brison and Kimber2002). The protective effects of many different free-radical scavengers, such as hypotaurine (Fujitani et al., Reference Fujitani, Kasai, Ohtani, Nishimura, Yamada and Utsumi1997), thioredoxin (Bing et al., Reference Bing, Hirao, Takenouchi, Che, Nakamura, Yodoi and Nagai2003), beta-mercaptoethanol (Takahashi et al., Reference Takahashi, Nagai, Okamura, Takahashi and Okano2002), glutathione (Luvoni et al., Reference Luvoni, Keskintepe and Brackett1996) and cysteine (Ali et al., Reference Ali, Bilodeau and Sirard2003), against oxidative stress have been tested in in vitro-cultured embryos. Melatonin, 5-methoxy-N- acetyltryptamine, a hormone secreted from the pineal gland, regulates circadian rhythm and seasonal breeding (Reiter et al., Reference Reiter, Tan, Manchester, Paredes, Mayo and Sainz2009) and is a free radical scavenger that protects against cell damage in tissues such as porcine thyroid tissue (Karbownik & Lewinski, Reference Karbownik and Lewinski2003). Supplementation of the culture medium with melatonin increases the development of in vitro-fertilized (IVF) mouse (Ishizuka et al., Reference Ishizuka, Kuribayashi, Murai, Amemiya and Ito2000), porcine (Rodriguez-Osorio et al., Reference Rodriguez-Osorio, Kim, Wang, Kaya and Memili2007) and bovine (Papis et al., Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007) oocytes. Recently, Choi et al. (Reference Choi, Park, Lee, Kim, Jeong, Lee, Park, Kim, Hossein, Jeong, Kim, Hyun and Hwang2008) reported that supplementation with melatonin significantly increased the development of parthenogenetic porcine oocytes to blastocysts as well as the blastocyst cell number. They also demonstrated that the potential of porcine SCNT oocytes to develop into blastocysts in melatonin-supplemented medium is significantly higher than that of controls, but the observed difference was small (12.2% vs. 10.1%; Choi et al., Reference Choi, Park, Lee, Kim, Jeong, Lee, Park, Kim, Hossein, Jeong, Kim, Hyun and Hwang2008). Papis et al. (Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007) reported that the effects of melatonin on the development of IVF bovine oocytes are influenced by the oxygen concentration during in vitro culture; the development of IVF oocytes is significantly increased compared with that of controls under a high oxygen concentration, but decreased under low oxygen concentration. This finding suggests that the effectiveness of melatonin supplementation of the culture medium differs depending on the culture conditions.
In the present study, we examined the effect of melatonin addition to the culture medium on the in vitro maturation of porcine oocytes, and evaluated whether melatonin increased the in vitro development of parthenogenetic and SCNT porcine oocytes.
Materials and methods
All the chemicals used in the present study were purchased from Sigma-Aldrich Chemical Co. unless otherwise specified.
Preparation of recipient oocytes
Cumulus–oocyte complexes (COCs) were recovered from 3- to 6-mm ovarian follicles obtained from the slaughter house and washed with polyvinyl-alcohol (PVA)–phosphate-buffered saline (PBS)(–) or modified TL-HEPES-PVA according to previously reported procedures (Funahashi et al., Reference Funahashi, Cantley and Day1997; Kawakami et al., Reference Kawakami, Kato and Tsunoda2005), COCs were cultured with modified NCSU37 (mNCSU37) medium (Petters & Wells, Reference Petters and Wells1993) supplemented with 10% porcine follicular fluid, 1 mM dibutyryl-cAMP (dbcAMP) and hormones [1.3 μg/ml follicle stimulating hormone (FSH) and 0.6 μg/ml luteinizing hormone (LH)] for 20 h, and then cultured without dbcAMP and hormones for another 20 h under an atmosphere of 5% CO2 in air at 39 °C. After 40 h of in vitro maturation, cumulus cells were removed in PBS containing 0.1% hyaluronidase. The number of matured metaphase II oocytes was determined by evaluating the presence of a polar body. In some experiments, COCs were maintained with mNCSU37 medium supplemented with 10% porcine follicular fluid and 1 mM dbcAMP for 24 h before use (Kawakami et al., Reference Kawakami, Kato and Tsunoda2005), but the data were combined because there were no differences between fresh and preserved oocytes.
Parthenogenetic activation
Matured oocytes were activated with a direct current pulse (150 V/mm for 100 μs) in 0.28 M mannitol supplemented with 0.01% PVA, 0.1 mM MgCl2, and 0.05 mM CaCl2 (Kawakami et al., Reference Kawakami, Kato and Tsunoda2005) and then cultured in porcine zygote medium III (PZM-3) (Yoshioka et al., Reference Yoshioka, Suzuki, Tanaka, Anas and Iwamura2002) containing 3 mg/ml bovine serum albumin and 5 μg/ml cytochalasin B (CB) for 4 h (Hata et al., Reference Hata, Ohkoshi, Kato and Tsunoda1996). The activated oocytes were cultured in PZM-3 medium under an atmosphere of 5% CO2 in air at 39 °C for 6 days. Cleavage of the embryos was examined after 24 h and 48 h, and blastocyst formation was examined on day 6 and/or day 7.
Somatic cell nuclear transfer
Somatic cells from a fetus of unknown sex and age were prepared according to a previous report (Yin et al., Reference Yin, Tani, Yonemura, Kawakami, Miyamoto, Hasegawa, Kato and Tsunoda2002) and passaged fewer than 17 times before use. Before nuclear transfer, somatic cells were cultured in Dulbecco's modified Eagle medium (Nissui Co.) supplemented with 10% fetal bovine serum (FBS) for 7 days. For donor cell preparation, the cells were scraped from the bottom of the culture dish, mixed with HEPES-buffered mNCSU37 (hNCSU37) medium, centrifuged, and then mixed with 10% clinical grade polyvinylpyrrolidone solution (MediCult) after removing the supernatant.
All procedures for nuclear transfer were performed using a piezo-actuated micromanipulator (PMA-CT150; Prime Tech Ltd) as previously reported (Wakayama et al., Reference Wakayama, Perry, Zuccotti, Johnson and Yanagimachi1998; Onishi et al., Reference Onishi, Iwamoto, Akita, Mikawa, Takeda, Awata, Hanada and Perry2000). The matured oocytes were incubated with 0.4 μg/ml demecolcine for 1 h according to a previous report (Yin et al., Reference Yin, Tani, Yonemura, Kawakami, Miyamoto, Hasegawa, Kato and Tsunoda2002) and then chromosomes from matured oocytes were removed in hNCSU37 supplemented with 5 μg/ml CB, 0.4 μg/ml demecolcine, 0.05 M sucrose and 0.4% bovine serum albumin.
The donor cells were injected into enucleated oocytes in hNCSU37 medium supplemented with 0.05 M sucrose and 10% FBS. After injection, SCNT oocytes were cultured in NCSU37 supplemented with 10% FBS for 2.5 h, and activated using two direct current pulses of 120 V/mm for 30 μs at 0.1-s intervals in 0.28 M mannitol supplemented with 0.01% PVA, 0.1 mM MgCl2 and 0.25 mM CaCl2. The oocytes were cultured in PZM-3 containing 5 μg/ml CB, 10 μg/ml cycloheximide and 50 nM trichostatin A (TSA) for 2 h, and then cultured in PZM-3 containing 50 nM TSA for 22 h (Zhang et al., Reference Zhang, Li, Villemoes, Pedersen, Purup and Vajta2007). The reconstructed oocytes were cultured in PZM-3 for 4 days and further cultured in PZM-3 with 10% FBS for 2 days under an atmosphere of 5% CO2 in air at 39 °C (Okada et al., Reference Okada, Krylov, Kren and Fulka2006).
Blastocyst cell number
Blastocyst cell numbers were determined using a double-staining method according to previously reported procedures (Papaioannou & Ebert, Reference Papaioannou and Ebert1988; Kawakami et al., Reference Kawakami, Kato and Tsunoda2005). Briefly, the zonae pellucidae of blastocysts were removed by treatment with 0.5% pronase at 37 °C and incubated with 25% rabbit anti-pig whole serum for 1 h, and then cultured in 10% guinea pig complement, 12 μg/ml Hoechst 33342 stain and 12 μg/ml propidium iodide for 40 min at 37 °C. The blastocyst cell numbers were counted under UV light using a fluorescence microscope.
ROS measurement
The ROS levels in parthenogenetic and SCNT embryos at the 4-cell stage were measured using the dichlorohydrofluorescein diacetate method (Hashimoto et al., Reference Hashimoto, Minami, Takakura, Yamada, Imai and Kashima2000). Briefly, embryos were transferred into PBS containing 10 μM 2′,7′-dichlorodihydrofluorescein diacetate, and then placed in the dark for 15 min at 37 °C. After washing with PBS, images of the embryos were recorded through a fluorescein isothiocyanate filter, and then measured by converting the number of pixels to grayscale images for analysis using Image J (NIH).
Assessment of apoptosis
The proportions of apoptotic cells in day 7 parthenogenetic and SCNT blastocysts were determined using an In Situ Cell Death Detection Kit (TUNEL Kit; Roche Molecular Biochemicals) as previously reported (Neuber et al., Reference Neuber, Luetjens, Chan and Schatten2002).
Briefly, blastocysts were fixed with 4% paraformaldehyde (PFA) in PBS overnight at 4 °C and then immersed in 0.5% Triton X-100 in PBS at room temperature for 1 h. Permeabilized embryos were incubated in a terminal deoxynucleotidyl transferase dUTP nick end labeling reaction medium for 1 h at 37 °C in the dark. After fragmented DNA were labelled with fluorescein isothiocyanate, the embryos were washed three times in PBS supplemented with 0.01% PVA.
Stained blastocysts were mounted on slides and the nuclei were stained with 4′,6-diamidino-2-phenylindole solution. The number of positive apoptotic nuclei per blastocyst was counted under UV light using a fluorescence microscope.
Statistical analysis
The cleavage and the blastocyst formation proportions, and the proportion of apoptotic cells were compared using the chi-squared test, and cell numbers were compared using Student's t-test. A p-value of less than 0.05 was considered to indicate statistical significance.
Experimental design
Experiment 1
To examine the effect of adding to the maturation medium on the maturation rate and the ability of oocytes to develop into blastocysts after parthenogenetic activation, 10−4 M, 10−7 M, 10−10 M or 10−13 M melatonin was added to the maturation medium. Melatonin was dissolved in DMSO, and therefore 1% DMSO, which was equivalent to the concentration of DMSO in the 10−4 M melatonin solution (the stock solution of melatonin was 10−2 M in DMSO), was added to the culture medium. Medium without DMSO was also used as a control.
Experiment 2
In this experiment, the effect of melatonin addition to the culture medium on the developmental potential of parthenogenetic oocytes was examined. Parthenogenetic oocytes were cultured in melatonin-supplemented medium at the same concentrations used in Experiment 1.
Experiment 3
To examine the effect of melatonin addition to the culture medium on the developmental potential of SCNT oocytes, we used 10−7 M melatonin based on the results obtained in Experiments 1 and 2. For the control, 0.0001% DMSO, which was equivalent to the concentration of DMSO in the 10−7 M melatonin solution (the stock solution of melatonin was 10−1 M in DMSO), was added to the culture medium.
Experiment 4
The ROS levels were measured in parthenogenetic and SCNT embryos at the 4-cell stage developed from oocytes that were cultured for 2 days in PZM-3 supplemented with 10−7 M melatonin or 0.0001% DMSO. Parthenogenetic oocytes were produced by two different electric conditions: (1) a single direct current pulse of 150V/mm for 100 μs followed by treatment with CB for 4 h; or (2) two direct current pulses of 120 V/mm for 30 μs at 0.1-s intervals followed by treatment with CB, cycloheximide and TSA for 2 h.
Experiment 5
In this experiment, the apoptotic cell number and apoptotic cell rate were examined in blastocysts developed from parthenogenetic and SCNT oocytes that were cultured for 6 days in PZM-3 supplemented with 10−7 M melatonin or 0.0001% DMSO. The apoptotic cell number denotes the average number of apoptotic cells in each blastocyst, and apoptosis proportion denotes the number of apoptotic cells divided by the total cell number of all blastocysts examined.
Results
The effects of melatonin addition to the maturation medium on the maturation of porcine oocytes are shown in Table 1. The proportion of matured oocytes based on the presence of the first polar body in the 10−4 and 10−10 M melatonin-supplemented groups was significantly lower than that of the controls. The proportion of oocytes matured in the presence of melatonin that developed to the 2-cell, 4-cell and blastocyst stages after parthenogenetic activation was not different from that in the controls. The blastocyst cell number also did not differ among melatonin-treated and control groups (Table 2).
Table 1 Effect of melatonin addition to the maturation medium on the maturation of porcine oocytes
a –cValues with different superscripts in the same colum differ significantly (p < 0.05).
Table 2 Effect of melatonin addition to the maturation medium on the developmental potential of oocytes after parthenogenetic activation
a ,b Values with different superscripts in the same colum differ significantly (p < 0.05).
The effects of melatonin addition to the culture medium on the potential of oocytes to develop in vitro after parthenogenetic activation are shown in Table 3. The proportion of activated oocytes that developed into blastocysts in the 1% DMSO and 10−4 M melatonin-supplemented groups was significantly lower than that of control without DMSO (4% and 4% vs. 22%). This finding implies that the presence of 1% DMSO during 6 days of in vitro culture was detrimental to the development of parthenogenetic porcine oocytes. When 10−7 M melatonin was added to the medium, the proportion of oocytes that developed to the 2-cell and 4-cell stages was significantly higher than that of the control without DMSO (65% vs. 45% for the 2-cell stage, 61% vs. 35% for the 4-cell stage), and the proportion of oocytes that developed into blastocysts was slightly higher (31% vs. 22%). The blastocyst cell number, however, did not differ among the groups.
Table 3 Effect of melatonin addition to the culture medium on the developmental potential of parthenogenetic oocytes
a–cValues with different superscripts in the same colum differ significantly (p < 0.05).
The effects of melatonin on the developmental potential of SCNT oocytes are shown in Table 4. The proportion of SCNT oocytes that developed into the 2-cell and 4-cell stages did not differ between the 10−7 M melatonin- and 0.0001% DMSO-supplemented groups. The proportion of oocytes that developed into blastocysts on days 6 and 7 did not differ between groups. The blastocyst cell number also did not differ between the two groups.
Table 4 Effect of melatonin addition to the culture medium on the developmental potential of SCNT oocytes
As shown in Table 5, ROS levels in 4-cell parthenogenetic embryos were influenced by the activation procedures. When oocytes were activated by a single current pulse of 150 V/mm for 100 μs (the same method used for the results shown in Tables 2 and 3), ROS levels in the 10−7 M melatonin-supplemented group were significantly lower than those in the 0.0001% DMSO-supplemented group. When oocytes were activated by two direct current pulses of 120 V/mm for 30 μs at 0.1-s intervals following treatment with cycloheximide and TSA treatment (the same method used for nuclear transfer), however, the ROS levels did not differ between the melatonin- and DMSO-supplemented groups. The ROS levels in embryos developed from SCNT oocytes in melatonin-supplemented medium were also significantly lower than that in controls (Table 5).
Table 5 Effect of melatonin addition to the culture medium on ROS levels of parthenogenetic and SCNT embryos
a ,b Values with different superscripts in the same group, treatment and colum differ significantly (p < 0.05).
The effects of melatonin on apoptosis in parthenogenetic and SCNT blastocysts are shown in Table 6. The proportion of parthenogenetic and SCNT oocytes that developed to the 2-cell and 4-cell stages was not different between the 10−7 M melatonin- and 0.0001% DMSO-supplemented groups. There were also no significant difference between these groups in the proportion of oocytes that developed into blastocysts on days 6 and 7; the cell numbers of parthenogenetic and SCNT blastocysts; or the mean number of apoptotic cells or proportion of apoptotic cells in parthenogenetic blastocysts. When 10−7 M melatonin was added to the culture medium during in vitro culture of SCNT oocytes, the mean number of apoptotic cells and the proportion of apoptotic cells in SCNT blastocysts were both slightly lower than, but not significantly different from, those in the 0.0001% DMSO-supplemented group.
Table 6 Effect of melatonin addition to the culture medium on apoptosis of parthenogenetic and SCNT embryos
Discussion
Recent studies have demonstrated that melatonin, which is mainly secreted from the mammalian pineal grand, enhances in vitro oocyte maturation and embryo development in mice (Ishizuka et al., Reference Ishizuka, Kuribayashi, Murai, Amemiya and Ito2000), sheep (Abecia et al., Reference Abecia, Forcada and Zuniga2002), heifers (Papis et al., Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007), sows (Rodriguez-Osorio et al., Reference Rodriguez-Osorio, Kim, Wang, Kaya and Memili2007) and goats (Berlinguer et al., Reference Berlinguer, Leoni, Succu, Spezzigu, Madeddu, Satta, Bebbere, Contreras-Solis, Gonzalez-Bulnes and Naitana2009). The present study demonstrated that addition of melatonin to the maturation medium of porcine oocytes for 40 h, however, did not increase the oocyte maturation rates (53–60% vs. 65% and 69%) or the potential of oocytes to develop into blastocysts after parthenogenetic activation compared with the levels in the controls (17–22% vs. 17% and 22%). The total blastocyst cell number was not significantly different among groups. Kang et al.(Reference Kang, Koo, Kwon, Park, Jang, Kang and Lee2009) reported that supplementation of the culture medium with 10 ng/ml (4.4 × 10−8 M) melatonin significantly increased the proportion of porcine oocytes that matured, and supplementation with 50 ng/ml (2.2 × 10−7 M) melatonin also significantly increased the proportion of oocytes that developed into blastocysts after parthenogenetic activation (21.4% vs. 13.3%). The main reason for these discrepancies might be the differences in the culture media used. NCSU37 medium supplemented with 10% porcine follicular fluid was used in the present study because the addition of follicular fluid to the maturation medium promotes oocyte quality (Naito et al., Reference Naito, Fukuda and Toyoda1988), but Kang et al. (Reference Kang, Koo, Kwon, Park, Jang, Kang and Lee2009) used TCM 199 without follicular fluid for oocyte maturation. Because porcine follicular fluid contains approximately 10−11 M melatonin (Shi et al., Reference Shi, Tian, Zhou, Wang, Gao, Zhu, Zeng, Tian and Liu2009), the beneficial effect of supplementation with melatonin, if these is one, might not have been observed in the present study. Shi et al. (Reference Shi, Tian, Zhou, Wang, Gao, Zhu, Zeng, Tian and Liu2009) reported that TCM199 with follicular fluid supplemented with 10−9 M melatonin, however, enhanced cleavage and blastocyst rates after parthenogenetic activation. The difference in the atmosphere during in vitro culture of activated oocytes –5% CO2 and 5% O2 in the report of Shi et al. (Reference Shi, Tian, Zhou, Wang, Gao, Zhu, Zeng, Tian and Liu2009) and 5% CO2 in air in the present study – is a possible reason for the difference in the proportion of oocytes cleaving and developing into blastocysts.
The present study demonstrated that supplementation of the culture medium with 10−7M melatonin significantly increased the proportion of parthenogenetically activated oocytes that developed to the 2-cell and 4-cell stages compared with controls (65% vs. 45% for 2-cell and 61% vs. 35% for 4-cell). Preimplantation mammalian embryos incur some level of oxidative stress during in vitro culture, which leads to a reduced developmental potential of embryos. Supplementation with melatonin, a scavenger of nitric oxide radicals (Noda et al., Reference Noda, Mori, Liburdy and Packer1999), reduces the oxidative stress and improves the developmental potential of embryos in mice (Ishizuka et al., Reference Ishizuka, Kuribayashi, Murai, Amemiya and Ito2000), sheep (Abecia et al., Reference Abecia, Forcada and Zuniga2002), heifers (Papis et al., Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007) and sows (Rodriguez-Osorio et al., Reference Rodriguez-Osorio, Kim, Wang, Kaya and Memili2007). The improved development of porcine parthenogenetic oocytes to the 2-cell and 4-cell stages in the present study might also have been due to the low ROS levels, as shown in Table 5. The difference in the parthenogenetic activation procedures, however, affected the ROS levels in the embryos.
The potential of parthenogenetic oocytes to develop into blastocysts in 10−7 M melatonin-supplemented medium was slightly higher than, but not significantly different from, that of the controls (31% vs. 22%). The potential of porcine oocytes to develop into blastocysts after parthenogenetic activation has been reported to be significantly higher than that of controls when oocytes are cultured in the presence of melatonin under a low oxygen atmosphere (Choi et al., Reference Choi, Park, Lee, Kim, Jeong, Lee, Park, Kim, Hossein, Jeong, Kim, Hyun and Hwang2008: Kang et al., Reference Kang, Koo, Kwon, Park, Jang, Kang and Lee2009; Shi et al., Reference Shi, Tian, Zhou, Wang, Gao, Zhu, Zeng, Tian and Liu2009). In contrast, however, Rodriguez-Osorio (Reference Rodriguez-Osorio, Kim, Wang, Kaya and Memili2007) reported that the potential for IVF porcine oocytes to cleave was significantly higher than that of controls, but the potential to develop into blastocysts was not different from that of controls when oocytes were cultured in medium supplemented with 10−9 M melatonin under 5% CO2 in air. One possible reason for this disagreement regarding the effectiveness of melatonin to improve the development into blastocysts is the difference in the gas atmosphere during in vitro culture of the embryos. Papis et al. (Reference Papis, Poleszczuk, Wenta-Muchalska and Modlinski2007) demonstrated that the potential of IVF bovine oocytes to develop into blastocysts in the presence of melatonin was significantly higher than that of controls if the oocytes were cultured under a high oxygen atmosphere, but lower than that of controls when the oocytes were cultured under low oxygen atmosphere.
The present study demonstrated that supplementation of the maturation medium with 1% DMSO, which was used to dissolve the melatonin, did not inhibit the oocyte maturation or the developmental potential of oocytes after parthenogenetic activation, but supplementation of the culture medium with 1% DMSO significantly inhibited the development of activated oocytes. Because inhibitory effects of ethanol and DMSO on nuclear and cytoplasmic maturation have been reported (Avery & Greve, Reference Avery and Greve2000), the effect of the solvent on the developmental potential of oocytes should be taken into account.
The ROS levels in 4-cell embryos developed from SCNT oocytes in the presence of 10−7 M melatonin were significantly lower than that in controls. The mean number of apoptotic cells of blastocysts developed from SCNT oocytes in the presence of melatonin was low but not significantly different from that of controls. The beneficial effects of melatonin supplementation on the ROS levels, however, were not reflected by the potential of SCNT oocytes to develop to the 2-cell, 4-cell and blastocyst stages, the developmental rate, or the total blastocyst cell numbers. The results of the present study did not confirm the report of Choi et al. (Reference Choi, Park, Lee, Kim, Jeong, Lee, Park, Kim, Hossein, Jeong, Kim, Hyun and Hwang2008), which is the only report that has demonstrated that the supplementation of the culture medium with 10−10 M melatonin slightly but significantly improved the potential of SCNT porcine oocytes to develop into blastocysts (12.2% vs. 10.1%). In the present study, SCNT oocytes were treated with a histone deacetylase inhibitor, TSA, for 24 h to enhance the developmental potential (Kishigami, Reference Kishigami, Wakayama, Thuan, Ohta, Mizutani, Hikichi, Bui, Balbach, Ogura, Boiani and Wakayama2006; Rybouchkin et al., Reference Rybouchkin, Kato and Tsunoda2006; Zhao et al., Reference Zhao, Hao, Ross, Spate, Walters, Samuel, Rieke, Murphy and Prather2010). Because TSA induces apoptosis in bovine 2-cell embryos (Carambula et al., Reference Carambula, Oliveira and Hansen2009), it is possible that TSA treatment offset the anti-apoptotic effects of melatonin on the embryos (Choi et al., Reference Choi, Park, Lee, Kim, Jeong, Lee, Park, Kim, Hossein, Jeong, Kim, Hyun and Hwang2008).
Although the present study revealed that parthenogenetic and SCNT melatonin-treated embryos had significantly lower ROS levels than controls, the potential of melatonin-treated embryos to develop into blastocysts was not significantly improved, in contrast to previous reports. The effectiveness of supplementation with melatonin on the developmental potential of oocytes might depend on the culture conditions.
Acknowledgement
We thank Dr T. Tani for his assistance. This work was supported by a grant from the Ministry of Education, Science, and Culture (no. 21028022).
