Abstract
Maternal hyperthermia induces pre-implantation embryo death, which is accompanied by enhanced physiological oxidative stress. We evaluated whether the administration of dl-α-tocopherol acetate (TA) to hyperthermic mothers mitigated pre-implantation embryo death. Mice were exposed to heat stress (35 °C, 60% relative humidity) for 12 h or not heated (25 °C) on the day of mating. Twelve hours before the beginning of temperature treatment, TA was injected intraperitoneally at a dose of 1 g/kg body weight. After the treatment, zygotes were recovered and the developmental abilities and intracellular glutathione (GSH) levels were evaluated. Another set of mice, with or without TA treatment, was exposed to heat stress for 12, 24 and 36 h, and the urinary levels of the oxidative stress marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured. Heat stress significantly decreased the blastocyst development rate and the GSH content in zygotes, as compared with the non-heat-stressed embryos, while TA administration significantly mitigated the deleterious effects of heat stress with regard to both parameters. Moreover, although the urinary levels of 8-OHdG gradually increased according to the duration of heat exposure, with or without TA administration, the levels were lower in the TA-administered group than in the placebo-injected mice. These results suggest that heat stress enhances physiological oxidative stress, and that TA administration alleviates the hyperthermia-induced death of pre-implantation embryos by reducing physiological oxidative stress.
Introduction
The mammalian pre-implantation embryo is susceptible to stressors and its development is easily disturbed. Maternal hyperthermia, induced by either high ambient temperature or heightened metabolism, is known to be a major cause of serious damage to human embryos (reviewed by Edwards et al. 1997). This phenomenon has been reported in mammals, including large farm animals, such as cows (Ingraham et al. 1974), sheep (Woody & Ulberg 1964) and pigs (Wildt et al. 1975), and in experimental animals, such as mice (Ozawa et al. 2002). Although previous studies using in vitro models have shown that the early developing embryo is itself vulnerable to high temperature and readily degenerates when cultured at temperatures above 41 °C (Aréchiga & Hansen 1998, Rivera & Hansen 2001), recent studies using in vivo models have suggested that early embryonic death caused by maternal hyperthermia is the result not only of elevated temperature but also of interactions between the embryo and the maternal reproductive tract. For example, maternal hyperthermia on the day of artificial insemination causes a significant reduction in blastocyst development in vivo in cows (Ealy et al. 1993), whereas in vitro exposure to high temperatures, as mimicked by a fluctuating pattern of rectal temperature in a hyperthermic mother, of zygotes for 24 h does not compromise developmental ability to the blastocyst stage (Rivera & Hansen 2001). Similarly, we have reported that the exposure of murine zygotes in vitro to 39.5 °C, which is the average rectal temperature of heat-stressed mice, does not affect their development to the blastocyst stage, compared with the development of unstressed embryos, whereas maternal heat exposure on the day of mating for 12 h significantly compromises subsequent embryonic development (Ozawa et al. 2002). Furthermore, oxidative stress in the embryo is heightened in relation to maternal hyperthermia; for example, higher levels of reactive oxygen species (ROS) and decreased concentrations of glutathione (GSH), a major antioxidant in the embryo, have been observed in maternally heat-exposed embryos in mice (Ozawa et al. 2002).
Hyperthermia has been reported to increase oxidative stress in some tissues. For example, Salo et al. (1991) have reported that hyperthermia caused by exercise increases the generation of superoxide anion radicals and hydrogen peroxide in skeletal muscles and the heart. Similarly, heat stress-induced maternal hyperthermia enhances ROS production in the oviduct (Ozawa et al. 2004, Matsuzuka et al. 2005a, 2005b) and lipid peroxidation in the liver (Matsuzuka et al. 2005b). ROS are strongly reactive with cellular molecules and can cause serious dysfunction, such as enzyme inactivation (Halliwell & Gutteridge 1984), mitochondrial abnormalities (Kowaltowski & Vercesi 1999) and DNA fragmentation (Lopes et al. 1998, Takahashi et al. 1999).
Early developing embryos are susceptible to oxidative stress, and the exposure of cultured embryos to exogenous oxidative stress compromises their pre-implantation development (reviewed by Guérin et al. 2001). In contrast, the detrimental effects of in vitro oxidative stress on pre-implantation embryos can be reduced by the addition of antioxidant molecules to the culture medium. The addition of thioredoxin to the culture medium relieves the stage-dependent developmental arrest of mice embryos, known as ‘cell block’, which is triggered by oxidative stress (Natsuyama et al. 1993). Iwata et al. (1998) have also reported that superoxide dismutase administration improves the blastocyst development rate of porcine embryos under conditions of high glucose concentration in the culture medium, which are known to enhance oxidative stress (Catherwood et al. 2002, Karja et al. 2006). In addition to the in vitro studies, we have recently reported that in an in vivo model, administration of melatonin, which has antioxidant properties and is a free radical scavenger, to pregnant mice partially alleviates either hyperthermia-enhanced lipid peroxidation in the liver or reduction in the intracellular GSH levels in zygotes, resulting in an improved rate of embryonic development to the blastocyst stage, as compared with embryos exposed to heat stress plus placebo (Matsuzuka et al. 2005b).
Taken together, these previous studies have led us to hypothesise that maternal hyperthermia enhances physiological oxidative stress at the whole-body level, and that heightened oxidative stress may be a factor in the induction of pre-implantation fatalities. dl-α-tocopherol acetate (TA), which is generally known as vitamin E, is a common lipid-soluble antioxidant that has been administered to reduce oxidative stress in cancer patients (Valko et al. 2006) and pregnant women with pre-eclampsia (Holmes & McCance 2005). In the present study, we investigated the degree of physiological oxidative stress in heat-stressed mice by measuring the urinary levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), which is a predominant form of ROS-induced lesion in DNA and is widely used as a sensitive biomarker of oxidative stress (de Zwart et al. 1999, Wu et al. 2004). We also evaluated the effect of maternal administration of TA on hyperthermia-induced death of pre-implantation embryos by monitoring embryonic development and physiological and embryonic oxidative stress statuses after heat and TA treatments.
Results
Embryo developmental competence
The average rectal temperatures and parameters related to pre-implantation embryo development in each treatment group are shown in Table 1. The heat-stressed mice experienced a significant (P<0.01) increase in rectal temperature, as compared with the controls. Almost all zygotes underwent a first cleavage; no significant difference was detected between the treatment groups in this respect. In contrast, although the percentage of embryos that developed to the blastocyst stage decreased in both heat-stressed groups (P<0.01), as compared with the control, the percentage was significantly (P<0.05) improved, from 36.9±6.7 to 59.4±6.7%, by TA administration. In addition, the percentage of two-cell arrested embryos under heat stress conditions decreased significantly (P<0.01), from 27.0±5.2 to 11.7±3.7%, with TA administration. The mean cell numbers in the blastocysts recovered from heat-stressed mice treated with TA were not different from those in the control group, while the blastocyst cell numbers in the heat-with-placebo-treated group were significantly (P<0.01) lower than in the other two groups.
Effect of heat stress and dl-α-tocopherol acetate (TA) administration on embryo developmental competence.
| Percentages of embryo that | ||||||
|---|---|---|---|---|---|---|
| Treatment | Replication | Rectal temperature | Cleaved | Arrested at two-cell | Developed to blastocysts | Cell numbers in blastocyst |
| Control | 10 | 37.6±0.1b | 99.3±0.5a | 0.2±0.2a | 97.7±1.3a | 74.2±2.8a |
| Heat stress | 9 | 39.6±0.1a | 98.8±0.7a | 27.0±5.2a | 36.9±6.7c | 43.4±3.5b |
| Heat+TA | 10 | 39.5±0.1a | 97.9±1.0a | 11.7±3.7b | 59.4±6.7b | 66.9±1.0a |
Data are the mean±s.e.m. of animals per group. Different superscript letters indicate significantly different values (P<0.05, one-way ANOVA with Tukey's multiple comparisons test).
Intracellular GSH levels in zygotes
The GSH levels in the zygotes are presented in Fig. 1. The zygotes from TA-administered mice had significantly higher GSH levels (2.28±0.14 pmol/zygote, P<0.05) than the heat-stressed mice treated with placebo (1.89±0.08 pmol/zygote).
Mean intracellular GSH level in zygotes from each treatment group. Pregnant females were exposed to heat stress for 12 h on the day of mating and were administered TA (Heat+TA) or placebo (Heat) or were left unheated and given placebo (Control). The zygotes were recovered and GSH was measured using a DTNB-glutathione reductase recycling assay. The data are expressed as the means±s.e.m. of eight replicates. Different superscript letters indicate significantly different values (P<0.05).
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0379
TA concentrations in plasma and liver
The TA concentrations in the plasma and liver samples from each treatment group are presented in Fig. 2. The TA concentrations in heat-stressed mice (2.6±0.2 μg/ml in the plasma and 4.7±0.1 μg/g wet tissue in the liver) were significantly lower (P<0.01) than those in the control mice (4.6±0.2 μg/ml in the plasma and 26.9±1.3 μg/g wet tissue in the liver). In contrast, no significant difference was detected for plasma TA concentration between TA-administered, heat-stressed mice (5.0±0.2 μg/ml) and the controls. Moreover, the degree of TA content reduction in the liver was lower in the heat-stressed mice administered TA than in those administered the placebo, although the TA levels in both groups were significantly lower (P<0.01) than in the controls.
TA concentrations in the (A) plasma and (B) liver of mice from each treatment group. Pregnant females were exposed to heat stress for 12 h on the day of mating and were administered TA (Heat+TA) or placebo (Heat), or left unheated and given the placebo (Control). The plasma and liver were sampled and the TA concentrations were measured by HPLC. The data are expressed as the means±s.e.m. of six replicates. Different superscript letters indicate significantly different values (P<0.01).
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0379
Urinary 8-OHdG levels
The patterns of urinary 8-OHdG levels for each treatment group during heat treatment are shown in Fig. 3. Significant differences were detected in the patterns of the urinary 8-OHdG concentrations either among the treatments, or time of sampling (P<0.05). The 8-OHdG level increased in relation to the duration of high-temperature treatment in the heat-stressed mice that were administered the placebo, and this level was significantly higher (1.14±0.09 μg/mg creatinine, P<0.05) at 48 h compared with that in the controls (0.70±0.12 μg/mg creatinine). In contrast, the 8-OHdG level in the TA-administered group after 12 h of heat treatment was far lower, less than one-third (0.18±0.08 μg/mg creatinine) of that in the controls (P<0.05). In addition, the 8-OHdG levels did not differ significantly from the controls, even though the TA-administered mice were exposed to heat stress for 48 h (0.70±0.12 μg/mg creatinine in controls versus 0.87±0.18 μg/mg creatinine in the TA-administered group), although the levels in the TA-administered group were also increased in line with the duration of heat exposure.
Urinary 8-OHdG levels in the Control, Heat and Heat+TA groups. Female mice were exposed to heat stress with a single administration of TA (Heat+TA) or placebo (Heat), or were left unheated and given the placebo (Control). During treatment, urine was collected three times at 12-h interval. The 8-OHdG levels were measured by ELISA and standardised by the urinary creatinine content. The data are expressed as the means±s.e.m. of six replicates. Different superscript letters indicate significantly different values (P<0.01).
Citation: REPRODUCTION 135, 4; 10.1530/REP-07-0379
Discussion
We investigated the degree of physiological oxidative stress during heat stress and the effects of maternal TA administration on physiological oxidative stress status and hyperthermia-induced pre-implantation embryonic death. To our knowledge, the results indicate for the first time that physiological oxidative stress in the whole body is increased according to the duration of maternal hyperthermia, as determined by urinary 8-OHdG levels. Moreover, TA administration to the mated female improved the developmental success of maternally heat-stressed embryos, and this was accompanied by a significant reduction in the physiological oxidative damage induced by hyperthermia.
Mortality and developmental malformations in pre-implantation embryos under conditions of maternal hyperthermia are well known in mammalian species (Mirkes 1997, Hansen et al. 2004). For example, heat exposure of cattle on the day of artificial insemination compromises pre-implantation embryo development in vivo (Ealy et al. 1993). Previous reports from our group have shown that maternal hyperthermia for 12 h soon after mating strongly disturbs normal embryonic development both in vitro (Ozawa et al. 2002, Matsuzuka et al. 2005a) and in vivo (Ozawa et al. 2003). The present results are consistent with those of previous studies, in that heat exposure of mated mice for 12 h severely compromised normal embryonic development, as determined by the blastocyst formation rate and the cell numbers in the blastocysts (Table 1). It is noteworthy that females with heightened metabolism were reported to have an increased risk of hyperthermia-induced pre-implantation embryo death. For example, exercise produces excess heat and increases at least a theoretical risk of embryo malformations in humans (Edwards 1986, Weissgerber & Wolfe 2006). Similarly, embryo mortality in dairy cattle has been reported to be pronounced, not only in the summer, but also into the fall in hot regions, such as Florida (Hansen et al. 2001), whereas the embryo mortality was less evident in heifers (Ingraham et al. 1974).
Although mammalian pre-implantation embryos are susceptible to exogenous stresses, such as heat stress, high temperature does not seem to be the only factor that compromises embryonic development during maternal hyperthermia; enhanced physiological oxidative stress is also implicated. In fact, recent reports have demonstrated that culturing zygotes at high temperatures, within the physiological range, in hyperthermic cows (40.5 °C) for 12 h did not alter subsequent development to the blastocyst stage (Ryan et al. 1992, Rivera & Hansen 2001). Our previous study has also indicated that in vitro exposure of zygotes to high temperature, mimicking the average rectal temperature of hyperthermic mice (39.5 °C), does not alter the blastocyst development rate, whereas maternal heat exposure for 12 h soon after mating drastically arrests subsequent embryonic development and is accompanied by increased H2O2 levels, decreased GSH levels (Ozawa et al. 2002) and damaged genomic DNA in the embryos, as assessed by elongation of the ladder tail in a comet assay, which is typical of ROS-mediated injury (Matsuzuka et al. 2005a).
It has been reported that hyperthermia and enhanced metabolic rate can cause increased oxidative stress in some tissues. For example, hyperthermia-enhanced ROS production in testicular germ cells (Ikeda et al. 1999) and excessive exercise produce H2O2 in rat vastus lateralis and cardiac muscle (Ji 1999). We have also observed that the TA concentration is decreased by maternal hyperthermia. Decreased liver TA levels have been reported to be induced by oxidative stress. For example, the administration of polychlorinated biphenyls (PCBs, oxidative stress inducers) decreases the TA concentration in the liver (Banudevi et al. 2006). In addition, in the present study, we showed that the urinary 8-OHdG levels were elevated, paralleling the duration of heat exposure, with or without TA administration (Fig. 2). The oxidised DNA damage product 8-OHdG is a predominant and stable form of ROS-induced DNA lesion. The level of urinary 8-OHdG should reflect whole-body formation of oxidised nucleobases (Pilger et al. 2002), and it has been used as a biomarker of the degree of oxidative stress (Cheng et al. 1992, Saito et al. 2000). Taken together, our results suggest that maternal hyperthermia increases oxidative stress, not only in certain tissue(s) but also at whole-body physiological levels.
In contrast, TA administration reduced the increased oxidative stress induced by hyperthermia, as determined by decreases in the 8-OHdG levels (Fig. 3) and increased liver TA concentrations (Fig. 2). In addition, the GSH levels in heat-stressed zygotes were significantly decreased by maternal heat stress, while they recovered to the unstressed levels after TA administration (Fig. 1). TA, which is a well-known lipid-soluble antioxidant, has been reported to inhibit NADPH oxidase-mediated superoxide anion generation and to protect cellular membranes against oxidative stress (Pascoe et al. 1987). In general, pre-implantation embryos are vulnerable to oxidative stress, and their development is easy to arrest or disturb by exposure to excessive oxidative stress (Guérin et al. 2001). GSH is synthesised and accumulated during the oocyte maturation period (Yoshida et al. 1993), and acts as an important antioxidant tripeptide, to maintain the balance of reduce-oxidative environment in the embryo (Guérin et al. 2001). The mouse embryo cannot synthesise GSH de novo until the blastocyst stage (Gardiner & Reed 1994, 1995). Therefore, the intracellular GSH level in the embryo reflects the environmental oxidative stress status and the developmental ability of the embryo. In fact, intracellular GSH levels in embryos are significantly decreased when the embryos are exposed to exogenous oxidative stress, and subsequent development is compromised (Nasr-Esfahani & Johnson 1991, Gardiner & Reed 1994). In contrast, the addition of the antioxidant thioredoxin to the culture medium has been shown to maintain intracellular GSH at higher levels and to increase the blastocyst development rate, as compared with embryos cultured without the antioxidant, in pigs (Ozawa et al. 2006). The addition of TA to a culture in which oxidative stress has been enhanced by the ROS inducer 12-phorbol-13-myristate acetate can restore the blastocyst development rate (Wang et al. 2002). Thus, it seems that TA administration protects the embryo against the excessive oxidative stress induced by hyperthermia, and alleviates the decline in developmental abilities of heat-stressed embryos, although further study is needed to clarify whether TA acts directly on the embryo or indirectly via the enhanced reducing status of the mother.
On the other hand, Ealy et al. (1994) have reported that i.m. administration of vitamin E to dairy cows at the time of AI during summer did not have a consistent beneficial effect on pregnancy rates in Florida. This conflict between the previous study and the present result may be attributed to the duration of the heat period; the cows were kept in hot environments before and after the TA administration (Ealy et al. 1994), whereas the female mice in the present study were exposed to heat stress for only 12 hours after mating. In addition, the embryos in the dairy cows were developed in vivo in the hyperthermic mother, while the embryos in the present study were cultured in vitro. Thus, the beneficial effect of TA administration on embryo development under hyperthermia may be less evident in the previous study using cows (Ealy et al. 1994).
Recently, we have reported that s.c. administration of melatonin, which acts as a free radical scavenger in vivo (Tan et al. 1993) and in vitro (Allegra et al. 2003), to mated female mice alleviates heat stress-induced pre-implantation embryo death, and this is accompanied by the maintenance of the reducing status in the oviduct (Matsuzuka et al. 2005b). On the other hand, melatonin is a water-soluble hormone, and it was necessary to inject it six times at 2-h interval to achieve a beneficial effect (Matsuzuka et al. 2005b). In addition, several studies have pointed out adverse effects of exogenous melatonin administration; melatonin injections in adult females disrupt the normal oestrus cycle by disturbing the secretion of LH-releasing hormone, resulting in low fertility in rats (Walker et al. 1982, Rivest, 1987). In contrast, since TA is a lipid-soluble vitamin, the effects of TA administration are longer lasting and even a single dose can be effective. For example, an injection of TA in dairy cattle increases the TA concentration in the milk for 6 days (Hidiroglou 1989). Furthermore, there are many results concerning TA administration in mammals, including human patients, with the aim of reducing physiological damage induced by oxidative stress, for example, in cancer (Valko et al. 2006), pre-eclampsia (Holmes & McCance 2005) and diabetic (Elliott et al. 1993) patients. Therefore, TA may be a beneficial and practical countermeasure against maternal hyperthermia-induced oxidation syndrome, including pre-implantation embryo mortality.
In conclusion, maternal heat exposure enhances physiological oxidative stress, and TA administration to hyperthermic mice can alleviate hyperthermia-induced pre-implantation embryo death by reducing the elevated physiological oxidative stress. These findings may help in the development of a new and practical strategy to overcome embryo mortality in pregnant females exposed to hyperthermia.
Materials and Methods
Animals
All experimental protocols and procedures were reviewed and approved by the Animal Care and Use Committee of the University of Tsukuba, Japan.
Female ICR (8–12 weeks old) and male BDF1 (>8 weeks old) mice were purchased from Charles River Japan (Yokohama, Japan). All animals were raised at 25 °C and 50% relative humidity (RH) with a 12 h light/12 h darkness photoperiod (lights on at 0600 h).
Chemicals
All reagents were purchased from Sigma Chemical Co., unless otherwise specified. Equine chorionic gonadotrophin (eCG; Serotrophin) and human chorionic gonadotrophin (hCG; Gonatrophin) were purchased from Teikokuzouki Pharmaceutical Co. (Tokyo, Japan).
Superovulation, TA administration and heat exposure
Females were superovulated by i.p. injection of eCG (5 IU), followed by hCG (5 IU), 48 h apart. Then, each female was mated with a male during the dark phase of the light/darkness cycle. At the time of start of mating, females were injected intraperitoneally with TA dissolved in peanut oil (0.01% (w/v) at 1 g/kg body weight) or with the oil alone (placebo control). Mating was evaluated by the presence of a vaginal plug at 0600 h the next morning (day 1 of pregnancy). The mated mice in each group were then heat stressed (35 °C, 60% RH) between 0600 and 1800 h on day 1 (heat stress) or were kept at 25 °C and 50% RH (Control). The rectal temperature of each mouse was measured in an environmental chamber at 1800 h on day 1 with a thermistor instrument (D611; Takara Thermistor Co., Tokyo, Japan) by inserting a 1 cm long probe into the rectum for 20 s.
Embryo collection and evaluation of developmental ability in vitro
Mated females were killed by diethyl ether anaesthesia, followed by decapitation at the end of the temperature treatment (1800 h). Embryos were immediately recovered from the killed mother by flushing the oviducts with potassium simplex optimised medium (KSOM) and cultured as a group from each female in 50 μl KSOM under mineral oil at 37 °C in 5% CO2, in a humidified incubator for 84 h, and the blastocyst developmental rate was observed. In addition, blastocyst-stage embryos were fixed with acetic alcohol (1:3) in whole-mount preparations for more than 48 h. Then, the fixed blastocysts were stained with 1% (w/v) aceto-orcein and the total cell number in each blastocyst was scored. The mean blastocyst development rate or cell number of the blastocysts in a group from each female was designated as one replicate, and nine (Control or Heat+TA group) or ten (heat group) replicate trials were carried out.
Assay for glutathione in zygotes
Another set of zygotes from each treatment group was used for GSH measurements. Immediately after recovery from the oviduct, zygotes were washed five times with GSH assay buffer (0.2 M phosphate buffer (pH 7.4), 10 mM Na2-EDTA). Then, the zygotes were sampled in groups of 15–20 in 5 μl GSH assay buffer plus 5 μl of 1.25 M phosphoric acid, and stored at −80 °C until assayed. Intracellular GSH concentrations in embryos were measured according to Anderson (1985), with some modifications. Briefly, 350 μl assay buffer that contained 0.33 mg/ml β-NAD (reduced form, NADPH), 50 μl of 6 mM 5,5′-dithio-bis-2-nitrobenzoic acid and 90 μl distilled water were added to the sample tube and mixed. After warming at room temperature for 15 min, 5 μl of 125 U/ml glutathione reductase were added to the reaction mix to start the reaction. Absorbance at 412 nm was recorded six times at 30-s interval using a spectrophotometer (u.v.-160A; Shimadzu Co., Kyoto, Japan). The GSH standards and a blank were also assayed under the same conditions. Eight replicate trials were conducted.
Measurements of plasma and liver TA contents by HPLC
Quantification of TA in plasma and liver was performed by HPLC methods using a u.v. detector (Li et al. 1991) with some modifications. Plasma and livers were collected from the killed mice. Collected plasma (0.5 ml) was mixed with 0.5 ml internal standard solution (2,2,5,7,8-pentamethyl-6-chromanol in isopropyl alcohol) and 1 ml absolute ethyl alcohol, and was then used for the following TA extraction procedure. Approximately, 0.1 g liver was homogenised on ice in 0.5 ml internal standard solution. Then, 1 ml ethyl alcohol that contained 3% (w/v) pyrogallol and 50 μl of 1% (w/v) NaCl were added into the homogenate and heated at 70 °C for 2 min. Subsequently, 100 μl of 60% (w/v) KOH were added to the sample and heated at 70 °C for 30 min. Liver samples were then snap-cooled on ice and 2 ml of 1% (w/v) NaCl were added, and this mixture was used in the following TA extraction procedure.
Extraction of TA from plasma or liver samples was carried out by adding 1.5 ml ethyl acetate:n-hexane (1:9) to each sample, vortexing for 5 min and centrifuging at 910 g for 5 min. After centrifugation, 1.3 ml supernatant that contained the TA were collected in another tube, 1 ml isopropanol was added and then the solvent was removed by vacuum extraction at 35 °C. The sample was reconstituted in 100 μl of 100% methanol and injected into the HPLC. The HPLC system was equipped with dual pumps (DP-8020; Tosoh Corp., Tokyo, Japan), a reverse-phase column (4.6×250 mm, TSK gel ODS-80Ts; Tosoh), and a u.v. detector (u.v.-8020; Tosoh). TA was detected at a wavelength of 290 nm and the peak heights were evaluated using a computer integrator (n=7 for each treatment).
Urinary 8-OHdG and creatinine measurements
The other sets of mice were used for measurement of urinary 8-OHdG levels. Each female mouse was housed in a metabolic cage (CM-10S; CLEA Japan, Tokyo, Japan) without superovulation treatment or mating, and exposed to heat stress for 36 h or not subjected to heat stress. During temperature treatment, mice were fed Lab Animal Diet (0.75 g/animal per day; MF, Oriental Yeast Co., Tokyo, Japan) and urine was collected three times at 12-h interval. Water was available ad libitum. TA was administered once to the females at the same dose and for the same time as in the earlier experiment in the present study. Collected urine was centrifuged (1000 g for 15 min at room temperature) and the supernatants were stored at −80 °C until analysis. The 8-OHdG levels in the collected urine were measured with a commercial 8-OHdG ELISA kit (Japan Institute for the Control of Aging, Shizuoka, Japan), in accordance with manufacturer's protocol, using a microplate reader (Model 680; Bio-Rad Laboratories) at 450 nm. Urinary creatinine was also measured by Jaffe's reaction (Bartels et al. 1972). Briefly, 100 μl deproteinised urine sample were added to 700 μl alkaline picric solution (saturated picric acid solution, distilled water and 2.5 M NaOH; v:v:v=5:5:2), mixed well, and incubated for 15 min at room temperature. The absorbance at 500 nm of each sample was measured using a spectrophotometer. Creatinine standards and a blank were also assayed under the same conditions. The urinary 8-OHdG levels were standardised by the creatinine content of each sample. Six replicate trials were conducted.
Statistical analysis
Data are expressed as means±s.e.m. All percentage data were initially arcsin-transformed. The rectal temperature, embryo developmental status, intra-embryo GSH and TA content were analysed using one-way ANOVA, followed by Tukey's multiple comparisons test. Changes in urinary 8-OHdG concentrations were analysed using the ANOVA for repeated measures, followed by Tukey's multiple comparisons test.
Acknowledgements
The authors thank Dr H Honda for general advice concerning the use of the fluorescence microplate reader, and Ms K Neath for proofreading. Part of this study was supported by grant-in-aid (no. 16658102) from The Japan Society for the Promotion of Science to Y K. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
References
Allegra M, Reiter RJ, Tan DX, Gentile C, Tesoriere L & Livrea MA 2003 The chemistry of melatonin's interaction with reactive species. Journal of Pineal Research 34 1–10.
Anderson ME 1985 Determination of glutathione and glutathione disulfide in biological samples Meister AIn Glutamate, Glutamine, Glutathione, and Related Compounds New York:Academic Press:548–555.
Aréchiga CF & Hansen PJ 1998 Response of preimplantation murine embryos to heat shock as midified by developmental stage and glutathione status. In Vitro Cellular & Developmental Biology. Animal 34 655–659.
Banudevi S, Krishnamoorthy G, Venkataraman P, Vignesh C, Aruldhas MM & Arunakaran J 2006 Role of alpha-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats. Food and Chemical Toxicology 44 2040–2046.
Bartels H, Böhmera N & Heterli C 1972 Serum creatinine determination without protein precipitation. Clinica Chimica Acta 37 193–197.
Catherwood MA, Powell LA, Anderson P, McMaster D, Sharpe PC & Trimble ER 2002 Glucose-induced oxidative stress in mesangial cells. Kidney International 61 599–608.
Cheng KC, Cahill DS, Kasai H, Nishimura S & Loeb LA 1992 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G→T and A→C substitutions. Journal of Biological Chemistry 267 166–172.
Ealy AD, Drost M & Hansen PJ 1993 Developmental changes in embryonic resistance to adverse effects of maternal heat stressing cows. Journal of Dairy Science 76 2899–2905.
Ealy AD, Aréchiga CF, Bray DR, Risco CA & Hansen PJ 1994 Effectiveness of short-term cooling and vitamin E for alleviation of infertility induced by heat stress in dairy cows. Journal of Dairy Science 77 3601–3607.
Edwards MJ 1986 Hyperthermia as a teratogen: a review of experimental studies and their clinical significance. Teratogenesis, Carcinogenesis, and Mutagenesis 6 563–582.
Edwards MJ, Walsh DA & Li Z 1997 Hyperthemia, teratogenesis, and the heat shock response in mammalian embryos in culture. International Journal of Developmental Biology 41 345–358.
Elliott RB, Pilcher CC, Stewart A, Fergusson D & McGregor MA 1993 The use of nicotinamide in the prevention of type 1 diabetes. Annals of the New York Academy of Sciences 30 333–341.
Gardiner CS & Reed DJ 1994 Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo. Biology of Reproduction 51 1307–1314.
Gardiner CS & Reed DJ 1995 Status of glutathione in the preimplantation mouse embryo. Archives of Biochemistry and Biophysics 318 30–36.
Guérin P, El Mouatassim S & Ménézo Y 2001 Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Human Reproduction Update 7 175–189.
Halliwell B & Gutteridge MC 1984 Oxidative toxicity, oxygen radicals, transtion metals and disease. Biochemical Journal 219 1–14.
Hansen PJ, Drost M, Rivera RM, Paula-Lopes FF, Al-Katanani YM, Krininger CE III & Chase CC Jr 2001 Adverse impact of heat stress on embryo production: causes and strategies for mitigation. Theriogenology 55 91–103.
Hansen PJ, Soto P & Natske RP 2004 Mastitis and fertility in cattle – possible involvement of inflammation or immune activation in embryonic mortality. American Journal of Reproductive Immunology 51 294–301.
Hidiroglou M 1989 Mammary transfer of vitamin E in dairy cows. Journal of Dairy Science 72 1067–1071.
Holmes VA & McCance DR 2005 Could antioxidant supplementation prevent pre-eclampsia? Proceedings of the Nutrition Society 64 491–501.
Ikeda M, Kodama H, Fukuda J, Shimizu Y, Murata M, Kumagai J & Tanaka T 1999 Role of radical oxygen species in rat testicular germ cell apoptosis induced by heat stress. Biology of Reproduction 61 393–399.
Ingraham RH, Gillette DD & Wagner WD 1974 Relationship of temperature and humidity to conception rate of Holstein cows in subtropical climate. Journal of Dairy Science 57 476–481.
Iwata H, Akamatsu S, Minami N & Yamada M 1998 Effect of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose. Theriogenology 50 365–375.
Ji LL 1999 Antioxidants and oxidative stress in exercise. Proceedings of the Society for Experimental Biology and Medicine 222 283–292.
Karja NW, Kikuchi K, Fahrudin M, Ozawa M, Somfai T, Ohnuma K, Noguchi J, Kaneko H & Nagai T 2006 Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions. Reproductive Biology and Endocrinology 4 54.
Kowaltowski AJ & Vercesi AE 1999 Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biology and Medicine 26 463–471.
Li DH, Wang YM, Nath RG, Mistry S & Randerath K 1991 Modulation by dietary vitamin E of I-compounds (putative indigenous DNA modifications) in rat liver and kidney. Journal of Nutrition 121 65–71.
Lopes S, Jurisicova A, Sun JG & Casper RF 1998 Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa. Human Reproduction 13 896–900.
Matsuzuka T, Ozawa M, Nakamura A, Ushitani A, Hirabayashi M & Kanai Y 2005a Effects of heat stress on the redox status in the oviduct and early embryonic development in mice. Journal of Reproduction and Development 51 281–287.
Matsuzuka T, Sakamoto N, Ozawa M, Ushitani A, Hirabayashi M & Kanai Y 2005b Alleviation of maternal hyperthermia-induced early embryonic death by administration of melatonin to mice. Journal of Pineal Research 39 217–223.
Mirkes PE 1997 Molecular/cellular biology of the heat stress response and its role in agent-induced teratogenesis. Mutation Research 396 163–173.
Nasr-Esfahani MH & Johnson MH 1991 The origin of reactive oxygen species in mouse embryos cultured in vitro. Development 113 551–560.
Natsuyama S, Noda Y, Yamashita M, Nagahama Y & Mori T 1993 Superoxide dismutase and thioredoxin restore defective p34cdc2 kinase activation in mouse two-cell block. Biochimica et Biophysica Acta 1176 90–94.
Ozawa M, Hirabayashi M & Kanai Y 2002 Development competence and oxidative state of mouse zygotes heat-stress maternally or in vitro. Reproduction 124 683–689.
Ozawa M, Yamasaki Y, Hirabayashi M & Kanai Y 2003 Viability of maternal heat-stressed mouse zygotes in vivo and in vitro. Animal Science Journal 74 181–185.
Ozawa M, Matsuzuka T, Hirabayashi M & Kanai Y 2004 Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice. Biology of Reproduction 71 291–296.
Ozawa M, Nagai T, Fahrudin M, Karja NW, Kaneko H, Noguchi J, Ohnuma K & Kikuchi K 2006 Addition of glutathione or thioredoxin to culture medium reduces intracellular redox status of porcine IVM/IVF embryos, resulting in improved development to the blastocyst stage. Molecular Reproduction and Development 73 998–1007.
Pascoe GA, Fariss MW, Olafsdottir K & Reed DJ 1987 A role of vitamin E in protection against cell injury. Maintenance of intracellular glutathione precursors and biosynthesis. European Journal of Biochemistry 166 241–247.
Pilger A, Ivancsits S, Germadnik D & Rüdiger HW 2002 Urinary excretion of 8-hydroxy-2′-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection. Journal of Chromatography. B. Analytical Technologies in the Biomedical and Life Sciences 778 393 – 401.
Rivera RM & Hansen PJ 2001 Developmental of cultured bovine embryos after exposure to high temperatures in the physiological range. Reproduction 121 107–115.
Rivest RW 1987 The female rat as a model describing patterns of pulsatile LH secretion during puberty and their control by melatonin. Gynecological Endocrinology 1 279–293.
Ryan DP, Blakewood EG, Lynn JW, Munyakazi L & Godka RA 1992 Effect of heat-stress on bovine embryo development in vitro. Journal of Animal Science 70 3490–3497.
Saito S, Yamauchi H, Hasui Y, Kurashige J, Ochi H & Yoshida K 2000 Quantitative determination of urinary 8-hydroxydeoxyguanosine (8-OH-dg) by using ELISA. Research Communications in Molelcular Pathology and Pharmacology 107 39–44.
Salo DC, Donovan CM & Davies KJA 1991 HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise. Free Radical Biology & Medicine 11 239–246.
Takahashi M, Saka N, Takahashi H, Kanai Y, Schultz RM & Okano A 1999 Assessment of DNA damage in individual hamster embryos by comet assay. Molecular Reproduction and Development 54 1–7.
Tan DX, Pöeggeler B, Reiter RJ, Chen LD, Chen S, Manchester LC & Barlow-Walden LR 1993 The pineal hormone melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in vivo. Cancer Letters 70 65–71.
Valko M, Rhodes CJ, Moncol J, Izakovic M & Mazur M 2006 Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chemico-Biological Interactions 160 1–40.
Walker RF, McCamant S & Timiras PS 1982 Melatonin and the influence of the pineal gland on timing of the LH surge in rats. Neuroendocrinology 35 337–342.
Wang X, Falcone T, Attaran M, Goldberg JM, Agarwal A & Sharma RK 2002 Vitamin E and Vitamin C supplementation reduce oxidative stress-induced embryo toxicity and improve the blastocyst development rate. Fertility and Sterility 78 1272–1277.
Weissgerber TL & Wolfe LA 2006 Physiological adaptation in early human pregnancy: adaptation to balance maternal–fetal demands. Applied Physiology, Nutrition, and Metabolism 31 1–11.
Wildt DE, Regle GD & Dukelow WR 1975 Physiological temperature response and embryonic mortality in stressed swine. American Journal of Physiology 229 1471–1475.
Woody CO & Ulberg LC 1964 Viability of one-cell sheep ova as affected by high environmental temperature. Journal of Reproduction and Fertility 7 275–280.
Wu LL, Chiou CC, Chang PY & Wu JT 2004 Urinary 8-OHdG: a marker of oxidative stress to DNA and a risk factor for cancer, atherosclerosis and diabetics. Clinica Chimica Acta 339 1–9.
Yoshida M, Ishigaki K, Nagai T, Chikyu M & Pursel VG 1993 Glutathione concentration during maturation and after fertilization in pig oocytes: relevance to the ability of oocytes to form male pronucleus. Biology of Reproduction 49 89–94.
De Zwart LL, Merman JH, Commandeur JN & Vermeulen NP 1999 Biomarkers of free radical damage applications in experimental animals and in humans. Free Radical Biology and Medicine 26 202–226.
