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Myonuclear apoptosis occurs during early posthatch starvation
Comparative Biochemistry and Physiology Part B 135 (2003) 677–681
Myonuclear apoptosis occurs during early posthatch starvation Simone Pophal, Juanita J. Evans, Paul E. Mozdziak* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695, USA Received 19 March 2003; received in revised form 16 May 2003; accepted 17 May 2003
Abstract Apoptosis is a naturally occurring process; it is important for the final shape and size of developing tissues, and it is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis. The objective of the study was to evaluate the occurrence of apoptosis in chickens immediately posthatch under fed and starved conditions. Male broiler chickens were or were not provided feed for the first 3 days posthatch. Chickens were killed immediately after hatch, at 1 day of age, at 2 days of age and at 3 days of age. The Pectoralis thoracicus was removed, fixed, dehydrated, cleared and embedded in paraffin. Muscle sections were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) for detection of apoptotic nuclei. Body weights were lower (P-0.05) in the starved compared to the fed group at 2 and 3 days posthatch. Myofiber cross-sectional area was only smaller (P-0.05) in the starved compared to the fed birds at 3 days posthatch. TUNEL-positive nuclei were present at all days for the fed and starved groups. The proportion of TUNEL-positive nuclei was higher (P-0.05) for the starved group at day 2 and day 3 posthatch compared to the fed group at 3 days posthatch. Apoptosis is a mechanism that contributes to the smaller myofiber size observed at 3 days posthatch. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Apoptosis; Starvation; Chickens; Skeletal muscle; Myonucleus; TUNEL
1. Introduction Chickens move from an embryonic metabolism based on nutrients derived from the yolk sac immediately posthatch to one based on nutrients derived from exogenous feed. Under commercial conditions, there is always a posthatch holding time, composed of (1) the time that chicks spend in the hatcher, (2) time required for procedures like sexing and vaccination, (3) period of transportation and (4) placement. Birds often do not *Corresponding author. Department of Poultry Science, North Carolina State University, Campus Box 7608yScott Hall, Raleigh, NC 27695, USA. E-mail address: [email protected] (P.E. Mozdziak).
receive feed or water until 48-h posthatch. Therefore, commercial birds may be in severe negative energy balance, which can lead to a considerable reduction in growth potential and meat yield (Pinchasov and Noy, 1993; Halevy et al., 2000). The reduction in body weight, between hatch and chick placement, can be partially explained by the decrease in the weight of the yolk sac, but twothirds of the body weight reduction is attributed to a loss of body tissue (Nir and Levanon, 1993). The compensatory growth for birds under a period of early posthatch starvation seems to be very poor or absent. A placement delay of 24-h posthatch adversely affects growth, and it could result in a significant reduction in body weight at 49 days of age (Vieira and Moran, 1999). Fur-
1096-4959/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959(03)00148-9
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S. Pophal et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 677–681
thermore, delayed chick placement affects the overall potential for protein deposition in the breast muscle (Vieira and Moran, 1999). The reduction in meat yield at market age that has been attributed to early posthatch starvation has been associated with a reduction in early posthatch satellite cell proliferation (Halevy et al., 2000), and a perturbation in satellite cell proliferation in juvenile muscle has also been associated with a reduction in mature muscle size (Mozdziak et al., 1997, 2000). Apoptosis is a regulated form of cell destruction that is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis, which is different from the cell swelling and membrane ruptures that is characteristic of necrosis. The difference between apoptosis and necrosis seems to be important to prevent damage to the surrounding cells (Smith et al., 2000; Primeau et al., 2002). Apoptosis may be a normal developmental process important for the final shape and size of developing tissues, and it may play an important role in embryonic or posthatch development of skeletal muscle. De Torres et al. (2002) have shown the existence of a period of naturally occurring cell death in rat skeletal muscle during postnatal development. Recently, Mozdziak et al. (2002a) has shown that the absence of feed during the first 3 days posthatch is associated with a significantly smaller myofiber cross-sectional area, and a higher level of apoptotic myonuclei compared to birds provided feed at hatching. Therefore, it appears that apoptosis might have a profound influence on ultimate muscle size because the myonuclei lost through starvation may not be regained after the resumption of feeding (Mozdziak et al., 2002b). The previous study (Mozdziak et al., 2002a) only examined apoptosis at 3 days posthatch and following refeeding. The objective of the current study was to examine apoptosis during the first 3 days of life to determine how quickly apoptosis becomes upregulated during early posthatch starvation. 2. Materials and methods 2.1. Chicks The experimental procedures, involving animals, were approved by the North Carolina State Uni-
versity Institutional Animal Care and Use Committee. Male broiler chicks were obtained from North Carolina State University Farms. Immediately after hatching, the chicks were split into two groups. The first group (ns15) was provided with a standard starter diet (88% dry matter, 3% crude fat, 5% crude fiber, 6% ash and 18% crude protein) for the first 3 days posthatch. The second group (ns15) of chicks was not provided with any feed during the first 3 days posthatch. Another control group (ns5) was killed immediately after hatching. All birds were provided water ad libitum. Chicks were weighed and killed by an overdose of Euthasol䉸 (Delmarva Laboratories, Midlothian, VA; 0.25 mlykg body) at 24 h (ns5ygroup), 48 h (ns5ygroup) and 72 h (ns5ygroup) posthatch. Samples were harvested from the Pectoralis thoracicus, fixed in 4% paraformaldehyde in PBS overnight, dehydrated, cleared, infiltrated and embedded in paraffin. 2.2. Apoptosis detection Transverse sections (8 mm thick) were placed on poly-L-lysine treated slides, deparaffinzed, rehydrated and processed using an apoptosis detection kit (Promega, Madison, WI, catalog 噛 G3250). The apoptosis assay was performed as previously described (Mozdziak et al., 2002a). Briefly, apoptotic nuclei were identified through the histochemical assessment of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL), which uses the TdT enzyme to incorporate fluorescein isothiocyanate (FITC)labeled dUTP at the 39-OH end of fragmented DNA. Sections were counterstained with propidium-iodide (50 mgyl PBS). Positive controls were carried out with DNAse I and negative controls were processed in the absence of TdT. 2.3. Image analysis Tissue sections were observed with a Leica DMR microscope (Leica Microsystems, Bannockburn, IL) equipped with epi-fluorescence illumination. TUNEL-(q) nuclei were observed with a FITC filter set (Omega Optical, Brattleboro, VT), and all nuclei were visualized with a propidium iodide filter set (Omega Optical, Brattleboro, VT). Images of nuclei visualized with the FITC filters and nuclei visualized with the propidium iodide filters were acquired using a Spot-RT CCD Cam-
S. Pophal et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 677–681
679
Table 1 Apoptotic labeling, myofiber cross-sectional area (M) and average body weight (BW) size in chicks under feed (F) or starved (S) conditions during the first 3 days posthatch Day 0 C a
Apoptotic labeling M cross-sectional area Average BW (g)
Day 1
Day 2
F ab
26.99"12 15.29"1.3b
S ab
36.37"18 16.29"0.9b 49.6"1.2c
Day 3
F b
17.90"7 15.96"1.8b 45.5"1.9cd
S ab
38.23"17 16.51"1.0b 58.0"1.6b
F a
61.25"14 16.27"2.0b 43.0"2.8de
S b
13.95"9 27.58"5.0a 69.7"3.3a
61.53"13a 14.7"1.7b 38.3"1.5e
Values are means"S.E.M., ns5. Means in a row with different letters differ, P-0.05. Apoptotic labeling, number of TUNEL-labeled nuclei per 100 propidium iodine-labeled nuclei.
a
era (Diagnostic Instruments, Sterling Heights, MI). The number of FITC-labeled (apoptotic) nuclei, the number of propidium iodide-labeled nuclei and myofiber cross-sectional areas were determined for each muscle section using IMAGE-PRO PLUS software (Media Cybernetics, Silver Spring, MD). The criteria for concluding myonuclear analysis was counting at least 1000 propidium iodidelabeled nuclei from each tissue section for each muscle. An index of apoptotic labeling was calculated by expressing the number of FITC-TUNEL (q)-labeled nuclei relative to the number of propidium iodide-labeled nuclei. Myofiber cross-sectional area was evaluated for at least 100 myofibers from each muscle. 2.4. Statistical analysis
change for the fed and starved groups during the first 2 days posthatch. However, myofiber crosssectional area was larger (P-0.05) in the fed group at 3 days posthatch compared to all other groups (Table 1). 3.2. Apoptosis TUNEL-positive nuclei were found in the Pectoralis thoracicus of all groups. The index of apoptotic labeling was higher (P-0.05) in the starved group at 2 and 3 days posthatch compared to the fed group at 3 days posthatch. Similarly, there was an increase (P-0.05) in the index of apoptotic labeling, for the starved group, between 1 and 2 days posthatch (Table 1). 4. Discussion
Body weight data, myofiber cross-sectional area data and apoptotic labeling data were analyzed using the General Linear Models procedure of SAS (1985) to determine the effect of feed status and age on each variable. Means were separated using least significant differences (Ott, 1993). If variances were unequal, then the data were logarithmically transformed before analysis. 3. Results 3.1. Growth Body weights did not differ (P)0.05) between fed and starved groups at 1 day posthatch. However, body weights were significantly lower (P0.05) for the starved compared to the fed groups at 2 and 3 days posthatch. Body weight increased (P-0.05) for the fed group between 1 and 3 days posthatch. Body weights decreased (P-0.05) for the starved group between 1 and 3 days posthatch. Myofiber cross-sectional area did not (P)0.05)
The observed decrease in body weight for the starved group, from 1 day posthatch through 3 days posthatch, may indicate yolk sac mobilization and the utilization of other body tissues as a source of protein and energy for maintenance. Under the starved condition, the early posthatch chick may produce some energy from (1) the utilization of glycogen from the liver, (2) the utilization of the lipids from yolk sac and (3) the utilization of amino acids, derived from yolk sac, skeletal muscle and other cells, through gluconeogenesis. However, blood glucose levels cannot be maintained during prolonged starvation without further metabolic changes. Thus, major adjustments must be made to conserve glucose when other methods are used to produce ATP. The fatty acids, unlike glycerol, cannot be used to produce glucose, but cells process fatty acids through the Kreb’s cycle to produce ATP. Liver cells are able to oxidize amino acids to produce ATP. Fatty acids are converted to ketone bodies by the liver. The
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ketone bodies can be used by the heart, kidneys and other tissues to produce ATP (Riis, 1983). The yolk sac can serve as a temporary energy and protein source until food is available to furnish lipids and protein. However, the yolk is very low in carbohydrate, which can lead to ketosis with prolonged fasting. The yolk sac provides some protective effect to support life, but this protective effect may not be sufficient to prevent myonuclear loss. In the present study, the body weight of the chick decreased more quickly, under starvation conditions, than the Pectoralis thoracicus muscle, because myofiber cross-sectional area did not show any significant difference between the starved and fed groups until 3 days posthatch, while significant differences in body weights were observed at 2 and 3 days posthatch. Furthermore, myofiber crosssectional area did not change for the starved group from hatching through 3 days posthatch. Prolonged fasting may result in a decrease in concentration of all plasma amino acids because of a gradual decline in the rate of protein catabolism, which may spare, for a limited time, body protein. Skeletal muscle development in avian embryos depends on the proliferation, differentiation and fusion of embryonic myoblasts (Yablonka-Reuveni and Paterson, 2001). Satellite cells fuse with these myofibers and their nuclei direct the synthesis of new protein and function in the maturation of muscle. These developmental events are controlled by specific growth factors that are produced locally by satellite cells and other cells in the muscle (Dodson et al., 1996; McFarland, 1999). During early posthatch myofiber maturation, it is possible that some myonuclei, responsible for the synthesis of embryonic proteins, are no longer needed and are removed through an apoptotic pathway. Myofibers are centrally nucleated immediately posthatch, with myonuclei undertaking a peripheral position as myofibers mature. It is possible that the central nuclei are removed through apoptosis and then myofibers become peripherally nucleated through satellite cell fusion. Newlands et al. (1998) have shown that the total number of transcriptionally active nuclei for a particular gene is dynamic because it changes during fetal development to reflect the growth status of the myofiber. It is possible that gene transcription at particular stages of myofiber maturation occur in pulses along the myofiber (Newlands et al., 1998), which may be related to changes from synthesis of embryonic
proteins by centrally located embryonic myonuclei, that may be removed through apoptosis to protein synthesis from newly acquired peripheral myonuclei. TUNEL (q)-labeled cells, observed on day 0 and at 1 and 2 days posthatch for the fed group, may indicate an occurrence of natural myonuclear death during posthatch development of chicken skeletal muscle. Similar to the current experiment, myofibers display morphological features of apoptosis in rats during the first 9 postnatal days because TUNEL (q)-myonuclei were present, and apoptotic labeling peaked between 5 and 7 days of life (De Torres et al., 2002). Therefore, it appears that myonuclear apoptosis may play a role in early postnatal myofiber remodeling. The higher incidence of TUNEL (q)-labeled cells observed in the starved group at 3 days posthatch could precede detectable muscle atrophy or a decrease in myofiber cross-sectional area compared to chicks immediately posthatch. Muscle atrophy appears to be associated with the loss of myonuclei, and possibly through apoptotic-like mechanisms (Allen et al., 1997, 1999). Furthermore, the myonuclear domain (the amount of cytoplasm per myonucleus) is reduced following atrophy (Allen et al., 1999). Muscle atrophy is a physiological response to fasting. Low levels of insulin, low levels of IGF-1 and high levels of glucocorticoids activate muscle protein loss in fasting (Jagoe et al., 2002). Growth hormone and IGF-I treatment significantly attenuate the decrease in both myonuclear number and myofiber size during hindlimb unloading and decrease the number of apoptotic myonuclei (Edgerton et al., 2002). It appears that IGF-1 can mediate some aspects of the hypertrophy response via an increase in DNA that may maintain some critical DNA-to-protein ratio, possibly via incorporation of satellite cells. The low level of IGF-1, encountered during fasting, could be associated with a lower satellite cell incorporation to the myofiber, decreased DNA incorporation and reduced myonuclear domain, which could be associated with the myofiber crosssectional area changes observed between the starved and fed groups at 3 days posthatch. Furthermore, another consequence of prolonged starvation is reduction in total oxidative metabolism and basal metabolic rate. The metabolic change seems to regulate the destruction of some myonuclei contributing to the loss in myonuclear number during muscle atrophy (Hikida et al., 1997; Edger-
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ton et al., 2002). Another potential explanation for the apoptotic myonuclei in the muscle from the starved birds is the elimination of nuclei that are not needed because the myofibers are not growing. In summary, early posthatch starvation affects the body weight before detectable changes in myofiber size, even though it appears that early posthatch starvation reduces meat yield at market age (Halevy et al., 2000). Furthermore, the smaller myofiber size, observed at 3 days posthatch, may be related to the destruction of some myonuclei through an apoptotic process, suggesting that a reduction in myonuclei through apoptosis under an early posthatch starvation condition could be related to the reduction in meat yield at market age, and it is possible that apoptotic mechanisms become upregulated as early as 2 days posthatch. Acknowledgments Support was provided by funds under Project Number NC06590 (PEM) of North Carolina State University. A doctoral scholarship was provided by CAPES (SP). The authors thank the North Carolina State University Poultry Educational Unit II for supplying the chickens used in the study. The authors thank Darell McCoy of the Department of Poultry Science of North Carolina State University for assistance with the laboratory procedures. References Allen, D.L., Linderman, J.K., Roy, R.R., et al., 1997. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am. J. Physiol.—Cell Physiol. 273, C579–C587. Allen, D.L., Roy, R.R., Edgerton, V.R., 1999. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22, 1350–1360. De Torres, C., Munell, F., Roig, M., Reventos, J., Macaya, A., 2002. Naturally occurring cell death during postnatal development of rat skeletal muscle. Muscle Nerve 26, 777–783. Dodson, M.V., McFarland, D.C., Grant, A.L., Doumit, M.E., Velleman, S.G., 1996. Extrinsic regulation of domestic animal-derived satellite cells. Dom. Anim. Endocrinol. 13, 107–126.
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Edgerton, V.R., Roy, R.R., Allen, D.L., Monti, R.J., 2002. Adaptations in skeletal muscle disuse or decreased-use atrophy. Am. J. Phys. Med. Rehabil. 81, s127–s147. Halevy, O., Geyra, A., Bark, M., Uni, Z., Sklan, D., 2000. Early posthatch starvation decreases satellite cell proliferation and skeletal muscle growth in chicks. J. Nutr. 130, 858–864. Hikida, R.S., VanNostran, S., Murray, J.D., Staron, R.S., Gordon, S.E., Kraemer, W.J., 1997. Myonuclear loss in atrophied soleus muscle fibers. Anat. Rec. 247, 350–354. Jagoe, R.T., Lecker, S.H., Gomes, M., Goldenberg, A.L., 2002. Patterns of gene expression in atrophying skeletal muscle: response to food depravation. FASEB J. 16, 1697–1712. McFarland, D.C., 1999. Influence of growth factors on poultry myogenic satellite cells. Poultry Sci. 78, 747–758. Mozdziak, P.E., Schultz, E., Cassens, R.G., 1997. Myonuclear accretion is a major determinant of avian skeletal muscle growth. Am. J. Physiol. 272, C565–571. Mozdziak, P.E., Evans, J.J., McCoy, D.W., 2002a. Early posthatch starvation induces myonuclear apoptosis in chickens. J. Nutr. 132, 901–903. Mozdziak, P.E., Walsh, T.J., McCoy, D.W., 2002b. The effect of early posthatch nutrition on satellite cell mitotic activity. Poultry Sci. 81, 1703–1708. Mozdziak, P.E., Pulvermacher, P.M., Schultz, E., 2000. Unloading of juvenile muscle results in a reduced muscle size 9 wk after reloading. J. Appl. Physiol. 88, 158–164. Newlands, S., Levitt, L.K., Robinson, C.S., et al., 1998. Transcription occurs in pulses in muscle fibers. Genes Develop. 12, 2748–2758. Nir, I., Levanon, M., 1993. Effect of posthatch holding time on performance and on residual yolk and liver composition. Poultry Sci. 72, 1994–1997. Ott, L., 1993. An Introduction to Statistical Methods and Data Analysis. Duxbury Press, Belmont, CA. Pinchasov, Y., Noy, Y., 1993. Comparison of posthatch holding time and subsequent early performance of broiler chicks and turkey poults. Br. Poult. Sci. 34, 111–120. Primeau, A.J., Adhihetty, P.J., Hood, D.A., 2002. Apoptosis in heart and skeletal muscle. Can. J. Appl. Physiol. 27, 349–395. Riis, P.M., 1983. Dynamic Biochemistry of Animal Production. World Animal Science, A3. Elsevier Science Publishing Company Inc. Smith, H.K., Maxwell, L., Martyn, J.A., 2000. Nuclear DNA fragmentation and morphological alterations in adult rabbit skeletal muscle after short-term immobilization. Cell Tissue Res. 302, 235–241. Vieira, S.L., Moran Jr., E.T., 1999. Effect of egg of origin and chick post-hatch nutrition on broiler live performance and meat yields. World Poultry Sci. J. 56, 125–142. SAS Institute Inc., 1985. SAS User’s Guide: STATISTICS, Version 5 ed., SAS Institute, Cary, NC. Yablonka-Reuveni, Z., Paterson, B.M., 2001. MyoD and myogenin expression patterns in cultures of fetal and adult chicken myoblasts. J. Hist. Cyt. 49, 455–462.
Myonuclear apoptosis occurs during early posthatch starvation Simone Pophal, Juanita J. Evans, Paul E. Mozdziak* Department of Poultry Science, North Carolina State University, Raleigh, NC 27695, USA Received 19 March 2003; received in revised form 16 May 2003; accepted 17 May 2003
Abstract Apoptosis is a naturally occurring process; it is important for the final shape and size of developing tissues, and it is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis. The objective of the study was to evaluate the occurrence of apoptosis in chickens immediately posthatch under fed and starved conditions. Male broiler chickens were or were not provided feed for the first 3 days posthatch. Chickens were killed immediately after hatch, at 1 day of age, at 2 days of age and at 3 days of age. The Pectoralis thoracicus was removed, fixed, dehydrated, cleared and embedded in paraffin. Muscle sections were labeled using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) for detection of apoptotic nuclei. Body weights were lower (P-0.05) in the starved compared to the fed group at 2 and 3 days posthatch. Myofiber cross-sectional area was only smaller (P-0.05) in the starved compared to the fed birds at 3 days posthatch. TUNEL-positive nuclei were present at all days for the fed and starved groups. The proportion of TUNEL-positive nuclei was higher (P-0.05) for the starved group at day 2 and day 3 posthatch compared to the fed group at 3 days posthatch. Apoptosis is a mechanism that contributes to the smaller myofiber size observed at 3 days posthatch. 䊚 2003 Elsevier Science Inc. All rights reserved. Keywords: Apoptosis; Starvation; Chickens; Skeletal muscle; Myonucleus; TUNEL
1. Introduction Chickens move from an embryonic metabolism based on nutrients derived from the yolk sac immediately posthatch to one based on nutrients derived from exogenous feed. Under commercial conditions, there is always a posthatch holding time, composed of (1) the time that chicks spend in the hatcher, (2) time required for procedures like sexing and vaccination, (3) period of transportation and (4) placement. Birds often do not *Corresponding author. Department of Poultry Science, North Carolina State University, Campus Box 7608yScott Hall, Raleigh, NC 27695, USA. E-mail address: [email protected] (P.E. Mozdziak).
receive feed or water until 48-h posthatch. Therefore, commercial birds may be in severe negative energy balance, which can lead to a considerable reduction in growth potential and meat yield (Pinchasov and Noy, 1993; Halevy et al., 2000). The reduction in body weight, between hatch and chick placement, can be partially explained by the decrease in the weight of the yolk sac, but twothirds of the body weight reduction is attributed to a loss of body tissue (Nir and Levanon, 1993). The compensatory growth for birds under a period of early posthatch starvation seems to be very poor or absent. A placement delay of 24-h posthatch adversely affects growth, and it could result in a significant reduction in body weight at 49 days of age (Vieira and Moran, 1999). Fur-
1096-4959/03/$ - see front matter 䊚 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S1096-4959(03)00148-9
678
S. Pophal et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 677–681
thermore, delayed chick placement affects the overall potential for protein deposition in the breast muscle (Vieira and Moran, 1999). The reduction in meat yield at market age that has been attributed to early posthatch starvation has been associated with a reduction in early posthatch satellite cell proliferation (Halevy et al., 2000), and a perturbation in satellite cell proliferation in juvenile muscle has also been associated with a reduction in mature muscle size (Mozdziak et al., 1997, 2000). Apoptosis is a regulated form of cell destruction that is characterized by some morphological features such as plasma membrane blebbing, nuclear breakdown, chromosomal fragmentation and apoptotic bodies followed by phagocytosis, which is different from the cell swelling and membrane ruptures that is characteristic of necrosis. The difference between apoptosis and necrosis seems to be important to prevent damage to the surrounding cells (Smith et al., 2000; Primeau et al., 2002). Apoptosis may be a normal developmental process important for the final shape and size of developing tissues, and it may play an important role in embryonic or posthatch development of skeletal muscle. De Torres et al. (2002) have shown the existence of a period of naturally occurring cell death in rat skeletal muscle during postnatal development. Recently, Mozdziak et al. (2002a) has shown that the absence of feed during the first 3 days posthatch is associated with a significantly smaller myofiber cross-sectional area, and a higher level of apoptotic myonuclei compared to birds provided feed at hatching. Therefore, it appears that apoptosis might have a profound influence on ultimate muscle size because the myonuclei lost through starvation may not be regained after the resumption of feeding (Mozdziak et al., 2002b). The previous study (Mozdziak et al., 2002a) only examined apoptosis at 3 days posthatch and following refeeding. The objective of the current study was to examine apoptosis during the first 3 days of life to determine how quickly apoptosis becomes upregulated during early posthatch starvation. 2. Materials and methods 2.1. Chicks The experimental procedures, involving animals, were approved by the North Carolina State Uni-
versity Institutional Animal Care and Use Committee. Male broiler chicks were obtained from North Carolina State University Farms. Immediately after hatching, the chicks were split into two groups. The first group (ns15) was provided with a standard starter diet (88% dry matter, 3% crude fat, 5% crude fiber, 6% ash and 18% crude protein) for the first 3 days posthatch. The second group (ns15) of chicks was not provided with any feed during the first 3 days posthatch. Another control group (ns5) was killed immediately after hatching. All birds were provided water ad libitum. Chicks were weighed and killed by an overdose of Euthasol䉸 (Delmarva Laboratories, Midlothian, VA; 0.25 mlykg body) at 24 h (ns5ygroup), 48 h (ns5ygroup) and 72 h (ns5ygroup) posthatch. Samples were harvested from the Pectoralis thoracicus, fixed in 4% paraformaldehyde in PBS overnight, dehydrated, cleared, infiltrated and embedded in paraffin. 2.2. Apoptosis detection Transverse sections (8 mm thick) were placed on poly-L-lysine treated slides, deparaffinzed, rehydrated and processed using an apoptosis detection kit (Promega, Madison, WI, catalog 噛 G3250). The apoptosis assay was performed as previously described (Mozdziak et al., 2002a). Briefly, apoptotic nuclei were identified through the histochemical assessment of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL), which uses the TdT enzyme to incorporate fluorescein isothiocyanate (FITC)labeled dUTP at the 39-OH end of fragmented DNA. Sections were counterstained with propidium-iodide (50 mgyl PBS). Positive controls were carried out with DNAse I and negative controls were processed in the absence of TdT. 2.3. Image analysis Tissue sections were observed with a Leica DMR microscope (Leica Microsystems, Bannockburn, IL) equipped with epi-fluorescence illumination. TUNEL-(q) nuclei were observed with a FITC filter set (Omega Optical, Brattleboro, VT), and all nuclei were visualized with a propidium iodide filter set (Omega Optical, Brattleboro, VT). Images of nuclei visualized with the FITC filters and nuclei visualized with the propidium iodide filters were acquired using a Spot-RT CCD Cam-
S. Pophal et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 677–681
679
Table 1 Apoptotic labeling, myofiber cross-sectional area (M) and average body weight (BW) size in chicks under feed (F) or starved (S) conditions during the first 3 days posthatch Day 0 C a
Apoptotic labeling M cross-sectional area Average BW (g)
Day 1
Day 2
F ab
26.99"12 15.29"1.3b
S ab
36.37"18 16.29"0.9b 49.6"1.2c
Day 3
F b
17.90"7 15.96"1.8b 45.5"1.9cd
S ab
38.23"17 16.51"1.0b 58.0"1.6b
F a
61.25"14 16.27"2.0b 43.0"2.8de
S b
13.95"9 27.58"5.0a 69.7"3.3a
61.53"13a 14.7"1.7b 38.3"1.5e
Values are means"S.E.M., ns5. Means in a row with different letters differ, P-0.05. Apoptotic labeling, number of TUNEL-labeled nuclei per 100 propidium iodine-labeled nuclei.
a
era (Diagnostic Instruments, Sterling Heights, MI). The number of FITC-labeled (apoptotic) nuclei, the number of propidium iodide-labeled nuclei and myofiber cross-sectional areas were determined for each muscle section using IMAGE-PRO PLUS software (Media Cybernetics, Silver Spring, MD). The criteria for concluding myonuclear analysis was counting at least 1000 propidium iodidelabeled nuclei from each tissue section for each muscle. An index of apoptotic labeling was calculated by expressing the number of FITC-TUNEL (q)-labeled nuclei relative to the number of propidium iodide-labeled nuclei. Myofiber cross-sectional area was evaluated for at least 100 myofibers from each muscle. 2.4. Statistical analysis
change for the fed and starved groups during the first 2 days posthatch. However, myofiber crosssectional area was larger (P-0.05) in the fed group at 3 days posthatch compared to all other groups (Table 1). 3.2. Apoptosis TUNEL-positive nuclei were found in the Pectoralis thoracicus of all groups. The index of apoptotic labeling was higher (P-0.05) in the starved group at 2 and 3 days posthatch compared to the fed group at 3 days posthatch. Similarly, there was an increase (P-0.05) in the index of apoptotic labeling, for the starved group, between 1 and 2 days posthatch (Table 1). 4. Discussion
Body weight data, myofiber cross-sectional area data and apoptotic labeling data were analyzed using the General Linear Models procedure of SAS (1985) to determine the effect of feed status and age on each variable. Means were separated using least significant differences (Ott, 1993). If variances were unequal, then the data were logarithmically transformed before analysis. 3. Results 3.1. Growth Body weights did not differ (P)0.05) between fed and starved groups at 1 day posthatch. However, body weights were significantly lower (P0.05) for the starved compared to the fed groups at 2 and 3 days posthatch. Body weight increased (P-0.05) for the fed group between 1 and 3 days posthatch. Body weights decreased (P-0.05) for the starved group between 1 and 3 days posthatch. Myofiber cross-sectional area did not (P)0.05)
The observed decrease in body weight for the starved group, from 1 day posthatch through 3 days posthatch, may indicate yolk sac mobilization and the utilization of other body tissues as a source of protein and energy for maintenance. Under the starved condition, the early posthatch chick may produce some energy from (1) the utilization of glycogen from the liver, (2) the utilization of the lipids from yolk sac and (3) the utilization of amino acids, derived from yolk sac, skeletal muscle and other cells, through gluconeogenesis. However, blood glucose levels cannot be maintained during prolonged starvation without further metabolic changes. Thus, major adjustments must be made to conserve glucose when other methods are used to produce ATP. The fatty acids, unlike glycerol, cannot be used to produce glucose, but cells process fatty acids through the Kreb’s cycle to produce ATP. Liver cells are able to oxidize amino acids to produce ATP. Fatty acids are converted to ketone bodies by the liver. The
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ketone bodies can be used by the heart, kidneys and other tissues to produce ATP (Riis, 1983). The yolk sac can serve as a temporary energy and protein source until food is available to furnish lipids and protein. However, the yolk is very low in carbohydrate, which can lead to ketosis with prolonged fasting. The yolk sac provides some protective effect to support life, but this protective effect may not be sufficient to prevent myonuclear loss. In the present study, the body weight of the chick decreased more quickly, under starvation conditions, than the Pectoralis thoracicus muscle, because myofiber cross-sectional area did not show any significant difference between the starved and fed groups until 3 days posthatch, while significant differences in body weights were observed at 2 and 3 days posthatch. Furthermore, myofiber crosssectional area did not change for the starved group from hatching through 3 days posthatch. Prolonged fasting may result in a decrease in concentration of all plasma amino acids because of a gradual decline in the rate of protein catabolism, which may spare, for a limited time, body protein. Skeletal muscle development in avian embryos depends on the proliferation, differentiation and fusion of embryonic myoblasts (Yablonka-Reuveni and Paterson, 2001). Satellite cells fuse with these myofibers and their nuclei direct the synthesis of new protein and function in the maturation of muscle. These developmental events are controlled by specific growth factors that are produced locally by satellite cells and other cells in the muscle (Dodson et al., 1996; McFarland, 1999). During early posthatch myofiber maturation, it is possible that some myonuclei, responsible for the synthesis of embryonic proteins, are no longer needed and are removed through an apoptotic pathway. Myofibers are centrally nucleated immediately posthatch, with myonuclei undertaking a peripheral position as myofibers mature. It is possible that the central nuclei are removed through apoptosis and then myofibers become peripherally nucleated through satellite cell fusion. Newlands et al. (1998) have shown that the total number of transcriptionally active nuclei for a particular gene is dynamic because it changes during fetal development to reflect the growth status of the myofiber. It is possible that gene transcription at particular stages of myofiber maturation occur in pulses along the myofiber (Newlands et al., 1998), which may be related to changes from synthesis of embryonic
proteins by centrally located embryonic myonuclei, that may be removed through apoptosis to protein synthesis from newly acquired peripheral myonuclei. TUNEL (q)-labeled cells, observed on day 0 and at 1 and 2 days posthatch for the fed group, may indicate an occurrence of natural myonuclear death during posthatch development of chicken skeletal muscle. Similar to the current experiment, myofibers display morphological features of apoptosis in rats during the first 9 postnatal days because TUNEL (q)-myonuclei were present, and apoptotic labeling peaked between 5 and 7 days of life (De Torres et al., 2002). Therefore, it appears that myonuclear apoptosis may play a role in early postnatal myofiber remodeling. The higher incidence of TUNEL (q)-labeled cells observed in the starved group at 3 days posthatch could precede detectable muscle atrophy or a decrease in myofiber cross-sectional area compared to chicks immediately posthatch. Muscle atrophy appears to be associated with the loss of myonuclei, and possibly through apoptotic-like mechanisms (Allen et al., 1997, 1999). Furthermore, the myonuclear domain (the amount of cytoplasm per myonucleus) is reduced following atrophy (Allen et al., 1999). Muscle atrophy is a physiological response to fasting. Low levels of insulin, low levels of IGF-1 and high levels of glucocorticoids activate muscle protein loss in fasting (Jagoe et al., 2002). Growth hormone and IGF-I treatment significantly attenuate the decrease in both myonuclear number and myofiber size during hindlimb unloading and decrease the number of apoptotic myonuclei (Edgerton et al., 2002). It appears that IGF-1 can mediate some aspects of the hypertrophy response via an increase in DNA that may maintain some critical DNA-to-protein ratio, possibly via incorporation of satellite cells. The low level of IGF-1, encountered during fasting, could be associated with a lower satellite cell incorporation to the myofiber, decreased DNA incorporation and reduced myonuclear domain, which could be associated with the myofiber crosssectional area changes observed between the starved and fed groups at 3 days posthatch. Furthermore, another consequence of prolonged starvation is reduction in total oxidative metabolism and basal metabolic rate. The metabolic change seems to regulate the destruction of some myonuclei contributing to the loss in myonuclear number during muscle atrophy (Hikida et al., 1997; Edger-
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ton et al., 2002). Another potential explanation for the apoptotic myonuclei in the muscle from the starved birds is the elimination of nuclei that are not needed because the myofibers are not growing. In summary, early posthatch starvation affects the body weight before detectable changes in myofiber size, even though it appears that early posthatch starvation reduces meat yield at market age (Halevy et al., 2000). Furthermore, the smaller myofiber size, observed at 3 days posthatch, may be related to the destruction of some myonuclei through an apoptotic process, suggesting that a reduction in myonuclei through apoptosis under an early posthatch starvation condition could be related to the reduction in meat yield at market age, and it is possible that apoptotic mechanisms become upregulated as early as 2 days posthatch. Acknowledgments Support was provided by funds under Project Number NC06590 (PEM) of North Carolina State University. A doctoral scholarship was provided by CAPES (SP). The authors thank the North Carolina State University Poultry Educational Unit II for supplying the chickens used in the study. The authors thank Darell McCoy of the Department of Poultry Science of North Carolina State University for assistance with the laboratory procedures. References Allen, D.L., Linderman, J.K., Roy, R.R., et al., 1997. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am. J. Physiol.—Cell Physiol. 273, C579–C587. Allen, D.L., Roy, R.R., Edgerton, V.R., 1999. Myonuclear domains in muscle adaptation and disease. Muscle Nerve 22, 1350–1360. De Torres, C., Munell, F., Roig, M., Reventos, J., Macaya, A., 2002. Naturally occurring cell death during postnatal development of rat skeletal muscle. Muscle Nerve 26, 777–783. Dodson, M.V., McFarland, D.C., Grant, A.L., Doumit, M.E., Velleman, S.G., 1996. Extrinsic regulation of domestic animal-derived satellite cells. Dom. Anim. Endocrinol. 13, 107–126.
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