Expression of microRNA and microRNA processing machinery genes during early quail (Coturnix japonica) embryo development.

MicroRNA (miRNA) are small regulatory RNA molecules that are implicated in regulating and controlling a wide range of physiological processes including cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis. The aim of this study was to determine the expression pattern of 32 miRNA and 18 miRNA processing machinery genes during somite formation in quail embryos. The embryos were collected at stages HH (Hamburger and Hamilton) 4, 6, and 9 of embryo development (19, 24, and 30 h of incubation, respectively). Total RNA including miRNA was isolated from 4 groups of embryos (each group consisting of 6 to 8 embryos) were collected at each of the 3 stages (19, 24, and 30 h). The expression pattern of candidate miRNA and miRNA processing machinery genes was performed using quantitative real-time PCR. The results demonstrated that 7 miRNA (let-7a, mir-122, mir-125b, mir-10b, P < 0.01; let-7b, mir-26a, and mir-126, P < 0.05) were differentially expressed during early quail embryo development. In addition, the expression profile of 18 miRNA processing machinery genes was not significantly increased at 30 h of incubation compared with both 19 and 24 h. Our results suggest that machinery genes for miRNA biogenetic pathways are functional, and hence, miRNA may be involved in the regulation of early quail development. These 7 differentially expressed miRNA are suggested to play critical roles in quail embryo somite formation.

Follistatin alters myostatin gene expression in C2C12 muscle cells.

The objective of the present study was to determine the effects of follistatin addition on myostatin and follistatin gene expression patterns in C2C12 muscle cells. C2C12 cells were administered with 100 ng/ml recombinant human (rh) follistatin in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 4 mM glutamine and antibiotics daily for three days. Rh follistatin was not added in the control wells. Follistatin and myostatin gene cDNAs were synthesised by reverse transcriptase polymerase chain reaction (RT-PCR). The time course of follistatin gene expression pattern was similar in both the control and the follistatin-treated group. Myostatin mRNA level significantly increased in the follistatin-treated group after 24 h of culture (Fig. 3, P < 0.01). Amounts then sharply decreased (Fig. 3, P < 0.01) at 48 h of culture, whereas there was no significant difference between the control and the follistatin-treated group at 72 h of culture. Our results demonstrated that myostatin and follistatin mRNA were expressed in C2C12 cells and rh follistatin changed the myostatin expression pattern.

Myostatin expression and possible functions in animal muscle growth.

Myostatin (also known as growth/differentiation factor-8) is a recently identified member of the transforming growth factor-beta family of secreted regulatory factors. Mice having targeted disruption of the myostatin gene displayed a marked increase in muscle mass, up to three times normal size. Additionally, a myostatin mutation has been linked to double muscled cattle breeds characterized by a visible, generalized increase in muscle mass. Therefore, it is suggested that myostatin in muscle may be one of the long sought inhibitors that specifically control the growth of individual tissues or organs. In the present paper, we review involvement of myostatin in muscle growth of different species.

Temporal expression of growth factor genes during myogenesis of satellite cells derived from the biceps femoris and pectoralis major muscles of the chicken.

The expression of mRNAs for transforming growth factors (TGF-beta2, myostatin, activin-B, and follistatin), insulin-like growth factors (IGF-I and -II), and fibroblast growth factor (basic, bFGF) was investigated in satellite cells derived from chicken pectoralis major (PM) and biceps femoris (BF) muscles in the stages from initiation of proliferation to fusion. These growth factor gene cDNAs were synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). No myostatin, activin-B, follistatin or bFGF expression was detected in either cell culture at 0 h. TGF-beta2 mRNA level increased at 48 h (P < 0.01) and remained constant through 144 h in both PM and BF satellite cell cultures. The ontogeny of myostatin gene expression with the exception of a sharp increase in BF culture at 72 h (P < 0.01), was nearly identical in both cell cultures. Activin-B mRNA level in PM satellite cells was higher than that in BF satellite cells at 72 h and 120 h (P < 0.01). Follistatin mRNA in PM satellite cells was higher than that in BF satellite cells at 24, 96, and 120 h culture (P < 0.01). No IGF-I gene expression was detected in cell cultures at any time point. IGF-II gene expression in BF satellite cells declined at 96 h (P < 0.01) and remained reduced until 144 h. bFGF mRNA in both satellite cell cultures increased at 24 h (P < 0.05) and remained at this level in BF satellite cells through 144 h.

Effect of in ovo administration of insulin-like growth factor-I on composition and mechanical properties of chicken bone.

The influence of in ovo administration of insulin-like growth factor-I (IGF-I) on long bone growth (tibiae and femora) of 42-d-old broiler chickens was investigated. Eggs were divided into three groups: uninjected control, vehicle-injected control, and recombinant human (rh) IGF-I. Eggs were injected once with 100 microL vehicle (10 mM acetic acid and 0.1% BSA) per embryo or vehicle containing 100 ng rh IGF-I/100 microL per embryo (n = 555 eggs total) on Days 1, 2, 3, or 4 of embryonic development. Males had greater bone length and moment of inertia than did females for the tibia and the femur (P < or = 0.01 for all). Although fracture load was significantly affected by gender (P < or = 0.02 and P < or = 0.006 for the femur and tibia, respectively), there was no treatment effect on these variables. However, when the fracture load was normalized with body weight of the animal, treatment and gender effects were found for femora (P < or = 0.04). Hydroxyproline concentrations of bones from male broilers were increased by the treatment (P < or = 0.02), whereas it had no effect on female broilers. There was no treatment effect on ash content, stiffness, yield load, yield deflection, and ultimate deflection and elastic, plastic, and total work for the femur or the tibia. We suggest that the effect of in ovo administration of IGF-I on bone mechanical properties was site-specific, and treated femora tended to have a lower fracture load relative to increased body weight.

Postnatal growth of broilers in response to in ovo administration of chicken growth hormone.

The effect of in ovo administration of chicken growth hormone (cGH) on growth rate and efficiency of gain, organ, and long bone growth of 42-d-old broiler chickens was investigated. Eggs were injected once with 100 microL vehicle (0.03 M NaHCO3, 0.15 M NaCl, pH 8.3) per embryo or vehicle containing 100 ng cGH/100 microL per embryo (n = 630 eggs total) on one of the following Days: 1, 4, or 7 through 18 of embryogenesis. There was no significant difference in hatchability between control and cGH treatment groups on any given injection day. Cumulative feed conversion of all treatment groups was improved relative to their respective control groups (P < 0.05). In ovo administration of cGH on Day 15 or 16 of incubation increased body weights (P < 0.01) of female broilers. On the other hand, body weights of male broilers were significantly increased by treatment on Day 1 (P < 0.04). Breast weights of female broilers from treatment groups Day 15 or 16 were increased (P < 0.01, P < 0.05, respectively). Liver weights of female broilers from treatment groups Day 1 and 15 were increased (P < 0.05, P < 0.01, respectively). In contrast, in ovo administration of cGH on Day 11 of incubation increased liver weights of male broilers (P < 0.03). There was no significant difference between control and treatment groups, in terms of heart or leg weights, or in Warner-Bratzler shear force of Pectoralis profundus muscle. Hydroxyproline concentration and cross-sectional area of female broiler tibias from treatment groups Day 11 or Day 16 were increased (P < 0.05), and ultimate breaking strength (stress) of tibias from the same groups was reduced (P < 0.05). In ovo administration of cGH altered growth and tissue development of broiler chickens in a time by sex dependent fashion.

The ontogeny of myostatin, follistatin and activin-B mRNA expression during chicken embryonic development.

The developmental pattern of myostatin, follistatin and activin-B genes in chick embryonic development was investigated. Total RNA was isolated from whole embryos on each of embryonic days (E) 0 to 6, from cranial halves of the embryo at E7 to 8, and from pectoralis muscle tissues at E9 to 20. Myostatin, follistatin and activin-B cDNAs were synthesized by reverse-transcription polymerase chain reaction (RT-PCR). Myostatin expression was first detected in embryos as early as the blastoderm stage (unincubated embryo, stage 1, E0). Myostatin mRNA concentration declined approximately 5 fold by E2 and remained lower through E6. Levels then increased 3 fold on E7 and plateaued through E16. Follistatin mRNA was first detected in the blastoderm stage of chick embryos. Overall follistatin mRNA increased 6 fold from E1 to E20 of development. Follistatin levels declined on E1 (approximately 2 fold) and remained low through E9. Follistatin mRNA reached the highest level prior to hatching. Activin-B mRNA from the whole embryo preparations (E0-E6) varied as the embryo matured. Overall activin-B gene expression from E11 to E20 appeared to decline (approximately 3.5 fold). This pattern is opposite of follistatin during the same period which is consistent with the opposing functions of these two proteins. We suggest that follistatin, activin-B and myostatin play an important role in embryogenesis and skeletal muscle development of the chick embryo. This study represents the first comprehensive report of myostatin mRNA patterns in chicken embryos.

In ovo administration of recombinant human insulin-like growth factor-I alters postnatal growth and development of the broiler chicken.

Two experiments assessed the efficacy of in ovo administration of insulin-like growth factor-I (IGF-I) to enhance skeletal muscle development and improve feed efficiency of broilers. Hatching eggs were divided into three groups: uninjected control, vehicle-injected control, and recombinant human (rh) IGF-I (100 ng per embryo). Eggs in Experiment 1 were injected on Day 1, 4, or one of Day 7 through 18 of incubation. Growth rates for Days 1 and 4 resulted in the greatest response to treatment (P < 0.01, P < 0.06 respectively). Based on these results, Experiment 2 focused on Days 1 to 4 of incubation. Results from Experiment 2 showed that there was no significant difference in hatchability among control and rh IGF-I treatment groups. Injection on Day 3 resulted in the greatest response for increased live (P < 0.035) and leg (P < 0.02) weights in both sexes. Feed efficiencies of all rh IGF-I groups were significantly (P < 0.01) improved for the first 3 wk. In ovo administration of rh IGF-I on Day 3 increased feed efficiency (6.65%; P < 0.009) in pens of mixed-sex broilers. In addition, live weights (12.3%; P < 0.002), leg weights (11.7%; P < 0.01), breast weights (9.9%; P < 0.04), and heart weights (11.4%; P < 0.02) were increased in males. These results demonstrate that in ovo administration of rh IGF-I alters feed efficiency, growth, and tissue development. This finding lends itself to significant improvements in broiler production efficiency and profitability.