The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

The chick embryo is a classical model to study embryonic development. However, most researchers have not studied the effect of embryonic manipulation on chick hatchability. The objective of this study was to determine the effect of egg orientation and type of sealing film on the hatchability of cultured embryos. Windows were made in the small end of recipient surrogate chicken eggshells, and donor embryos were placed into the recipient eggshell for the first 3 d of incubation. Survival over the first 3 d was maximized (P < 0.05) when windowed eggs sealed with Saran Wrap were positioned with the window-end down compared with window-end up. Three-day-old cultured embryos were transferred into recipient turkey eggshells, sealed with cling film, and cultured until hatch. Water weight loss of the surrogate eggshell cultures regardless of cling film type was not significantly different from control intact eggs. The embryos cultured in turkey eggshells and sealed with Handi Wrap exhibited higher hatchability (75% +/- 10.2%) than cultures sealed with Saran Wrap (45.2% +/- 13.8%). Hatchability of control intact eggs (86.4% +/- 5.3%) was not significantly (P > 0.05) different from the hatchability of eggs sealed with Handi Wrap, which suggested that Handi Wrap was an excellent sealant for chick embryos cultured after 3 d of incubation.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same (P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly (P < 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly (P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same (P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.

The role of satellite cells and DNA unit size in determining skeletal muscle growth was studied after mitotic activity was inhibited in the left pectoralis thoracicus of 2-wk-old tom turkeys by means of a 25-Gy dose of irradiation. Toms were killed and muscle weights were obtained 1 (n = 5), 4 (n = 6), 7 (n = 6), and 15 (n = 4) wk after irradiation. Satellite cell mitotic activity and DNA unit size were determined using enzymatically isolated myofiber segments and image analysis. Irradiated and nonirradiated muscle weights increased (P < 0.01) between all ages examined, but irradiated muscle weights were significantly (P < 0.01) lower than nonirradiated muscle weights at 4, 7, and 15 wk after irradiation. Satellite cell mitotic activity was lower (P < 0.01) in irradiated than in nonirradiated muscles at 1 and 4 wk after irradiation and resulted in a significant reduction (P < 0.05) in the number of myofiber nuclei per millimeter at 4 and 7 wk after irradiation. Satellite cell mitotic activity was higher (P < 0.05) in irradiated than in nonirradiated muscles at 7 wk after irradiation, but at 15 wk after irradiation it had fallen to low levels in both muscles. There was no significant (P > 0.10) difference in DNA unit size between muscles at any time, but there was an age-related increase (P < 0.01) for both muscles. Irradiation reduced muscle growth through a transient reduction in myonuclear production at a critical time (3-6 wk of age) in posthatch skeletal muscle development. The age-related increase in DNA unit size was not accelerated to compensate for the reduction in myonuclear accretion. Thus it appears that muscle growth potential is governed mostly by myonuclear accretion and to a lesser extent by DNA unit size.

The relationship between satellite cell mitotic activity and skeletal myofiber growth was examined in Pectoralis thoracicus and Biceps femoris muscles of Large White tom turkeys (Nicholas strain) at 3, 6, 9, 18, and 26 wk of age. Mitotically active satellite cells were labeled with 5-bromo-2'-deoxyuridine (BrdU). Labeled satellite cells were identified on enzymatically isolated myofiber segments using mouse anti-BrdU followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibodies. Myofiber nuclei (satellite cell nuclei + myonuclei) were counterstained with propidium iodide (PI). Myofiber segment diameter, myofiber segment length, and number of FITC- and PI-labeled nuclei were determined for each segment. At each age interval there was an increase in myofiber diameter, suggesting that the myofibers were growing during the entire experimental period. There was an age-related (P < .001) decrease in satellite cell mitotic activity and an age-related increase (P < .001) in the cytoplasmic volume to nucleus ratio (CNR) from 3 to 26 wk of age. An early phase of myofiber growth, between 3 and 6 wk of age, was characterized by a high level of satellite cell mitotic activity and increased CNR. Between 6 and 9 wk of age, satellite cell mitotic activity decreased, but the CNR showed no change (P > .05). During a late phase of myofiber growth, beyond 9 wk of age, satellite cell mitotic activity continued to decrease and myofiber growth occurred by an increased CNR. This study demonstrated that both Pectoralis thoracicus and Biceps femoris undergo a significant late phase of growth without appreciable production of myonuclei by satellite cell proliferation.

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.

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