The effects of acute cold exposure on morphology and gene expression in the heart of neonatal chicks.

Cold exposure induces an increase in blood flow and blood pressure, and long-term exposure to cold causes cardiac hypertrophy. Neonatal chicks (Gallus gallus domesticus) are highly sensitive to cold exposure, because their capacity for thermogenesis is immature until 1 week after hatching. Hence, we hypothesized that the heart of chicks at around 1 week of age acutely responds to cold environment. To investigate the effect of acute (24 h) and long-term (2 weeks) cold on the heart of chicks, 7-day-old chicks were exposed to cold temperature (4 °C) or kept warm (30 °C). Chicks exposed to the cold showed cardiac hypertrophy with marked left ventricular (LV) chamber dilation and wall thickening. On the other hand, long-term cold exposure (2 weeks from 7-day-old) induced an increase in total ventricular mass, but not in LV morphological parameters. Then, we investigated the details of acute cardiac hypertrophy in chicks. Electron microscopy revealed that cardiomyocytes in the hypertrophied LV had enlarged mitochondria with less dense cristae. Although the mRNA expression of lipoprotein lipase in the LV of the cold-exposed chicks significantly increased, the mRNA expression of genes involved in fatty acid ß-oxidation did not change in response to cold exposure. In addition, the mRNA expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha, which enhances mitochondrial biogenesis and function under physiological cardiac hypertrophy, increased in LV of cold-exposed chicks. The study found that acute cold exposure to neonatal chicks induces LV hypertrophy. However, these results suggest that acute cold exposure to chicks might induce both adaptive and maladaptive responses of the LV.

Role of prolactin-like protein (PRL-L) in cold-induced increase of muscle mass in chicks.

This study examined the hypothesis that a novel prolactin-like protein gene (PRL-L) is involved in cold-induced growth of skeletal muscle in chicks. Six-day-old chicks (Gallus gallus domesticus) were exposed to cold at 4°C or kept warm at 30°C for 24h. Cold exposure induced significant increases in PRL-L expression that coincided with increases in the weight of the sartorius muscle, which comprises both fast- and slow-twitch fibers. Meanwhile, no induction of PRL-L mRNA was observed in the heart, liver, kidney, brain, or fat. Myoblast cells that expressed PRL-L mRNA grew faster than untransduced cells in media containing 2% serum. These results suggested that PRL-L might be involved in in controlling cold-induced muscle growth of chicks.

In vivo gene transfer into skeletal muscle of neonatal chicks by electroporation.

Chicks (Gallus gallus domesticus) show considerable growth of skeletal muscle during the neonatal period. The in vivo gene transfer method is useful for studying gene function and can be employed to elucidate the molecular mechanisms of skeletal muscle growth in chicks. We evaluated the following conditions for gene transfer to the skeletal muscle of neonatal chicks by electroporation: (i) voltage; (ii) age of the chick; (iii) plasmid DNA injected amount; and (iv) duration of gene expression. The results obtained from this study indicate that the most efficient gene transfer condition was as follows: 75 µg of plasmid DNA encoding ß-galactosidase was injected into the gastrocnemius muscle of chicks at 4 days of age electroporated at 50 V/cm. In addition, peak transferred gene expression was observed from 3 days to 5 days after electroporation. Our results provide optimal electroporation conditions for elucidating the gene function related to skeletal muscle growth and development in neonatal chicks.

Increased expression of NOR-1 mRNA in the skeletal muscles of cold-exposed neonatal chicks.

Nuclear receptor subfamily 4, group A (NR4A) subgroup orphan receptors are rapidly induced by various physiological stimuli and have been suggested to regulate oxidative metabolism and muscle mass in mammalian skeletal muscle. The results showed that the NR4A subgroup orphan receptor, NOR-1 (NR4A3), was acutely increased in skeletal muscles of neonatal chicks in response to short-term cold exposure. The increased NOR-1 gene expression was concomitant with cold-induced changes in gene expression of both myostatin and proliferator-activated receptor-gamma coactivator-1 (PGC-1α), and the increase in skeletal muscle mass. These observations suggest that NOR-1 might play a role in controlling skeletal muscle growth in cold-exposed neonatal chicks.

Identification of tamaraw (Bubalus mindorensis) from natural habitat-derived fecal samples by PCR-RFLP analysis of cytochrome b gene.

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit-Baco National Park and species-known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species-unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus- and tamaraw-specific primers for PCR of cytochrome b gene were newly designed. The Bubalus-specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw-specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR-RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR-RFLP analysis also showed that the species-unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.

Increased mass of slow-type skeletal muscles and depressed myostatin gene expression in cold-tolerant chicks.

Temperature is maintained in birds by skeletal muscle shivering as well as by non-shivering thermogenesis in a cold environment because they lack brown adipose tissue, which is a mammalian thermogenic organ. Chicks acquire cold tolerance after their skeletal muscles mature. Here, we found that muscle fibers transformed to the slow-twitch type with increasing gene expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), and that the mass increased with decreasing myostatin gene expression, in the leg muscles of 7-day-old and younger chicks within 24 h of cold exposure. Muscle fibers did not transform and the mass did not increase within 24 h of cold exposure in muscles from chicks older than 8 days of age. Myostatin mRNA expression remained depressed in cold-tolerant muscles for 24 h, whereas cold-enhanced growth of the muscle continued for 48 h. Myostatin expression was depressed and muscle mass was increased only in chick leg muscles that comprised both fast- and slow-twitch fibers. These results suggest that the acute regulation of PGC-1alpha and myostatin gene expression in leg muscles is required for chicks to acquire cold tolerance up to 7 days of age.

Possible roles of myostatin and PGC-1alpha in the increase of skeletal muscle and transformation of fiber type in cold-exposed chicks: expression of myostatin and PGC-1alpha in chicks exposed to cold.

This study examined the hypothesis that myostatin and PGC-1alpha are involved in the increase in skeletal muscle mass and transformation of fiber type in cold-exposed chicks. One-week-old chicks were exposed to acute (24h) or long-term (8d) cold at 4 degrees C or kept warm at 30 degrees C. Acute cold exposure induced a significant increase in the skeletal muscle weight and the ratio of slow- to fast-fiber specific troponin I expression (sTnI/fTnI), accompanied by a significant decrease in lactate dehydrogenase activity. Expression of myostatin mRNA in the muscle was significantly lower in cold-exposed chicks than in the controls, whereas PGC-1alpha mRNA expression was significantly enhanced. These changes in the gene expression rapidly returned to the levels of the control chicks after the end of cold exposure, whereas the changes in fiber type and enzymatic activity were not resumed within 24h after removal of cold exposure. On the other hand, long-term exposure to cold resulted in a remarkable increase in skeletal muscle weight, accompanied by a significant increase in the ratio of sTnI/fTnI and the enzymatic activities of cytochrome oxidase and lactate dehydrogenase. However, the expression level of myostatin mRNA in cold-exposed chicks was not different from that in their age-matched control chicks and that of PGC-1alpha mRNA was significantly lower than in the controls. These results indicate that myostatin and PGC-1alpha expression in the skeletal muscle rapidly change in response to acute cold, suggesting the possibility that these two genes could be involved in the increase in muscle mass and transformation of fiber type, respectively, at the initial stage of adaptation in cold-exposed chicks.

DL-alpha-tocopherol acetate mitigates maternal hyperthermia-induced pre-implantation embryonic death accompanied by a reduction of physiological oxidative stress in mice.

Maternal hyperthermia induces pre-implantation embryo death, which is accompanied by enhanced physiological oxidative stress. We evaluated whether the administration of DL-alpha-tocopherol acetate (TA) to hyperthermic mothers mitigated pre-implantation embryo death. Mice were exposed to heat stress (35 degrees C, 60% relative humidity) for 12 h or not heated (25 degrees 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.

Alleviation of maternal hyperthermia-induced early embryonic death by administration of melatonin to mice.

Maternal hyperthermia induces early embryonic death via increased oxidative stress to the embryo. In this study, we examined whether melatonin administered to heat-stressed pregnant mice would reduce hyperthermia-induced embryonic death. Mice were heat stressed (12 hr at 35 degrees C, 60% relative humidity) on the day of mating and melatonin (3 mg/kg body weight) was injected subcutaneously every 2 hr during heat exposure. Thereafter, zygotes were collected, and in vitro developmental ability and intracellular glutathione (GSH) content were assessed. In addition, reactive oxygen species (ROS) levels and free radical scavenging activity (FRSA) in the oviduct as well as lipid peroxidation in the liver were measured. Melatonin administration was associated with a tendency for higher intracellular GSH content in zygotes (1.67 pmol/zygote) and a significantly higher percentage of embryos that developed to the morula or blastocyst stage (47.91%; P < 0.01) compared with the parameters in heat-stressed mice that were administered a placebo (1.48 pmol GSH/zygote and 14.78% development). Lipid peroxidation levels in the liver and ROS levels in the oviduct were the same in melatonin-treated stressed mice and the controls, while these parameters were significantly higher in heat-stressed mice that were not treated with melatonin. Furthermore, FRSA in the oviduct was significantly (P < 0.05) higher in the melatonin-treated mice than in the controls. These results suggest that administration of melatonin to heat-stressed mice alleviates hyperthermia-induced early embryonic death and that this is accomplished in part by maintaining a neutral redox status within the mother.

Effects of heat stress on the redox status in the oviduct and early embryonic development in mice.

This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .

Transformation of Skeletal Muscle from Fast- to Slow-Twitch during Acquisition of Cold Tolerance in the Chick.

Although birds lack brown adipose tissue, a thermogenic organ found in mammals, they possess other thermogenic mechanisms. In the current studies, we examined the molecular mechanisms of avian thermogenesis by studying how chicks acquire cold tolerance. We found that the acquisition of cold tolerance corresponded with an increase in the redness of the skeletal muscle, suggesting an increase in slow-twitch muscle fiber. This was confirmed by histological analysis. In addition, in chicks acquiring cold tolerance, there was an enhanced expression of the chicken homologue of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, a protein involved in adaptive thermogenesis in mammalian brown adipose tissue and in slow-twitch fiber formation in mammalian skeletal muscle. Subtraction and differential display techniques further showed that, when chicks acquired cold tolerance, the expression of genes associated with slow-twitch fibers increased, whereas those associated with fast-twitch fibers decreased. There was also an enhanced expression of mitochondrial oxidative genes. Together, these results suggest that transformation of skeletal muscle fiber from fast-twitch to slow-twitch is involved in the acquisition of thermogenesis in chicks.

Redox status of the oviduct and CDC2 activity in 2-cell stage embryos in heat-stressed mice.

Mammalian preimplantation embryos are vulnerable to heat stress. However, the mechanisms by which maternal heat stress compromises embryonic development are unclear. We hypothesized that the loss of developmental competence in maternally heat-stressed embryos results from enhanced oxidative stress in the oviducts. In experiment 1, oviducts and zygotes were collected from mice that were heat-stressed at 35 degrees C and 60% relative humidity for 12 h on the day of pregnancy as well as from control mice. The zygotes were cultured for 84 h to assess their development, and the H(2)O(2) level, glutathione concentration, and free radical scavenging activity (FRSA) were measured in the oviduct. In experiment 2, zygotes were cultured for 22 h to reach the late G(2) phase in the 2-cell stage, and Cdc2 activity was assessed using immunoblotting. A high percentage (87.6%) of control embryos developed to morulae or blastocysts, whereas the majority (67.4%) of the heat-stressed group arrested at the 2-cell stage. Although heat stress did not alter the FRSA or glutathione concentration in the oviducts, the H(2)O(2) level (P < 0.01) and its ratio to the FRSA (P < 0.05) significantly increased in the heat-stressed group. The Cdc2 activation at the 2-cell stage, as shown by the ratio of the dephosphorylated form to the phosphorylated form, was evident in control embryos but absent in heat-stressed embryos, and the level was similar to that in embryos blocked at the 2-cell stage (positive control). These results indicate that maternal heat stress enhances oxidative stress in the oviducts and that loss of developmental competence in maternally heat-stressed embryos correlates with a defect in Cdc2 activity at the 2-cell stage.