All animal experiments were performed according to the protocols approved by the South China Agriculture University Institutional Animal Care and Use Committee (approval number SCAU#0017). All animal procedures followed the regulations and guidelines established by this committee and minimized the suffering of animals.
For in vivo experiments, the Guangdong Wenshi Southern Poultry Breeding Co., Ltd. (Guangzhou, China), provided 21-d-old yellow-feather chickens. To isolate bone BMSCs, the Yuhe Agriculture and Animal Husbandry Co., Ltd. (Guangzhou, China), provided 3-d-old chickens.
The 21-d-old yellow-feather chickens included 15 SLD chickens and 15 normal chickens. To explore molecular mechanisms of GHR in vivo and determine the cause of fatty deposits in SLD chickens, mitochondrial function, mitochondrial biogenesis, and adipogenic differentiation in chicken BMSCs were examined in those chickens. The 3-d-old normal chickens were only used to isolate BMSCs, and the BMSCs were used to study GHR effects on mitochondria and adipogenic differentiation in vitro.
Paraffin sections and hematoxylin and eosin staining
The epiphyses of thighbone from the 21-d-old SLD and normal chickens were fixed with 10% neutral formalin for 5 d and then immersed in a hydrochloric acid/formic acid working solution to complete decalcification. After decalcification, samples were dehydrated in alcohol and transformed into a transparent state using xylene. After completing the transparency step, samples were soaked in wax and embedded in paraffin. A paraffin sectioning machine cut 7 to 10-μm-thick sections, which were stained with hematoxylin and eosin.
Frozen sections and oil red staining
Epiphyseal parts of femurs of the 21-d-old SLD and normal chickens were cut off and soaked in 4% paraformaldehyde for 48 h and then switched to decalcification solution for 30 d, with the solution changed every two days. After decalcification, tissues were placed in a 15% sucrose solution in a refrigerator at 4 °C to dehydrate and sink and then were transferred to a 30% sucrose solution at 4 °C to dehydrate and sink. Dehydrated tissue was placed cut side up on a sample tray and surrounded by drops of OCT embedding agent (Servicebio, Wuhan, China). The tray was placed on the quick-freeze table of a frozen sectioning machine, and the samples were sectioned after the OCT whitened and hardened. Section thickness was 8 to 10 μm. After sectioning, tissue sections were fixed on a slide and stained with oil red and hematoxylin.
Detection of triglyceride
Triglyceride was measured using a Triglyceride Assay Kit (Nanjing Jiancheng, Nanjing, China) according to the manufacturer’s protocol. Triglyceride was measured at 510 nm, and absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (Bio-Tek, Winooski, USA) according to the manufacturer’s protocol. Data were normalized to the control group and expressed as a percentage of the control.
Reverse-transcription quantitative PCR
RNA was extracted from tissues or cells using RNAiso reagent (Takara, Shiga, Japan) according to the manufacturer’s protocol. Concentration of RNA samples and optical density (OD) value of 260/280 were detected using a Nanodrop 2000c spectrophotometer (Thermo, Waltham, USA). Samples were stored at −80 °C for later use. For reverse-transcription quantitative PCR (RT-qPCR), cDNA was synthesized using MonScript™RTIII All-in-One Mix with dsDNase (Monad Co., Ltd., Guangzhou, China). ChamQ Universal SYBR qPCR Master Mix (Vazyme, Guangzhou, China) was used in RT-qPCRs run on a Bio-Rad CFX96 Real-Time Detection instrument (Bio-Rad, Hercules, USA) according to the manufacturer’s protocol. The reaction procedure included initial denaturation at 95 °C for 3 min, followed by denaturation at 95 °C for 10 s and annealing at 60 °C for 30 s, for a total of 40 cycles. At the end of the cycle, the dissolution curve was analyzed, and the detection temperature was 65 °C to 95 °C. Relative gene expression was measured using RT-qPCR twice for each reaction, and β-actin was used as the control. The primers used in RT-qPCR are listed in Table 1.
Extraction of chicken bone mesenchymal stem cells and cell culture
Bone mesenchymal stem cells were extracted using the appropriate separation kits (TBD science, Tianjin, China) following the manufacturer’s protocol.
Bone mesenchymal stem cells from 21-d-old SLD and normal chickens were extracted by cell separation kits and cultured in vitro to the appropriate density (the first generation). Then, assays were conducted on mitochondrial function and related gene expression and protein levels.
Bone mesenchymal stem cells from 3-d-old normal chickens were extracted by cell separation kits and were cultured in vitro and passaged to the third generation. Overexpression and knockdown of GHR were to explore the effects of GHR on mitochondrial and adipogenic differentiation in chicken BMSCs.
Bone mesenchymal stem cells were cultured in Gibco Dulbecco’s Modified Eagle Medium (DMEM): F-12 (Gibco, Waltham, USA) with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were cultured at 37 °C in a 5% CO2 humified atmosphere.
Induction of adipogenic differentiation
Bone mesenchymal stem cells were seeded into 6-well plates at 1.25 × 105 cells per cm2. Bone mesenchymal stem cells were induced with adipogenic medium containing DMEM/F12 (10% FBS), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, Darmstadt, Germany), 1 μM dexamethasone (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 200 μM indomethacin (Sigma-Aldrich). The medium was replaced every 2 d for 6 d.
Plasmid construction, small interfering RNA, and transfection
Third generation BMSCs were plated onto 6-well plates, and transfection began when the density reached approximately 80%. After 6 h of transfection, the DMEM/F12 medium was changed to adipogenic induction medium to induce adipogenic differentiation of BMSCs.
GeneCreate (Wuhan, China) synthesized the plasmid pcDNA3.1-GHR. Plasmid transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Waltham, USA) following the manufacturer’s protocol, and nucleic acids were diluted in OPTI-MEM (Gibco). All cells were analyzed 72 h after transfection.
Guangzhou RiboBio (Guangzhou, China) synthesized small interfering RNAs (siRNA) used for GHR knockdown. In preliminary experiments, four siRNAs were designed to interfere with GHR, and the si-GHR with the highest interference efficiency was used. The siRNA sequence is provided in Table 2. The si-GHR sequence was transfected in BMSCs to a final concentration of 100 nM using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s protocol. Cells were analyzed at 72 h after transfection.
Detection of reactive oxygen species
Production of ROS in mitochondria was measured using an ROS assay kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Dichlorofluorescein (DCF) fluorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (Bio-Tek). Data were normalized to the control group and are expressed as a percentage of the control.
Detection of ATP content
ATP levels were measured using an ATP assay kit (Beyotime) according to the manufacturer’s protocol. A Fluorescence/Multi-Detection Microplate Reader (BioTek) was used to determine ATP levels. Data were normalized to the control group and are expressed as a percentage of the control.
Detection of mitochondrial membrane potential
Mitochondrial membrane potential (ΔΨm) was measured using a JC-1 kit (Beyotime) according to the manufacturer’s protocol. Mitochondria were fixed with JC-1, and after cells were incubated with JC-1 for 20 min at 37 °C, fluorescence was determined using a Fluorescence/Multi-Detection Microplate Reader (Bio-Tek). Rotenone, 10 μmol/L, was used as a standard inhibitor of ΔΨm. Data (the ratio of aggregated and monomeric JC-1) were normalized to the control group and are expressed as a percentage of the control.
Detection of enzymatic activity of mitochondrial oxidative phosphorylation complexes
Commercial assay kits (Solarbio, Beijing, China) were used to measure enzyme activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes in BMSCs according to the manufacturer’s protocol. Complex I enzyme activity was determined by the change in absorbance of NADH at 340 nm. Complex II enzyme activity was determined by the change in absorbance of 2,6-dichlorophenol indophenol at 600 nm. Enzyme activity of complex III and complex IV was determined by the change in absorbance of reduced cytochrome c at 550 nm. Absorbance was determined using a Fluorescence/Multi-Detection Microplate Reader (Bio-Tek). Data were normalized to the control group and are expressed as a percentage of the control.
Mito-tracker green staining and Hoechst 33342 staining
Mito-tracker green staining and Hoechst 33342 staining were used to label mitochondria and nuclei in BMSCs, respectively. At 72 h after transfection, cells were washed twice with phosphate buffered saline (PBS) and incubated with Mito-tracker green (Beyotime) for 30 min. Cells were then suspended in PBS, and 10 µL of Hoechst 33342 dye was added (Beyotime). After washing twice with PBS, a fluorescence microscope (Nikon TE2000-U, Tokyo, Japan) was used to capture five randomly selected fields that were analyzed with NIS-Elements software.
Oil red O staining and quantification
Bone mesenchymal stem cells were seeded into 6-well culture plates. After transfection and differentiation for 5 d, differentiated BMSCs were washed with PBS and then fixed with 4% formaldehyde for 30 min. Differentiated BMSCs were dyed with oil red O working solution (BBI, Shanghai, China) for 60 min at room temperature and then washed three times with PBS, according to the manufacturer’s specification. After washing, a fluorescence inverted light microscope (Leica DMi8, Wetzlar, Germany) was used to capture images. At the end, stain in cells was extracted by isopropanol and absorbance was measured at 510 nm with a Fluorescence/Multi-Detection Microplate Reader (Bio-Tek).
Western blot analysis
Radio-immune precipitation assay buffer (Beyotime) with phenylmethane sulfonyl fluoride protease inhibitor (Beyotime) was used to lyse tissue and cellular proteins. The homogenate was centrifuged at 13,000 ×g for 10 min at 4 °C. The supernatant was collected, and protein concentration was determined immediately using a bicinchoninic acid assay protein quantification kit (Beyotime). Proteins were separated in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, and then probed with antibodies following standard procedures.
The following antibodies and their dilutions were used in western blot: mouse anti-PGC1 alpha antibody (1C1B2, 1:5,000; Proteintech, Rosemont, USA), rabbit anti-NRF1 antibody (AF7620, 1:1,000; Beyotime), rabbit anti-TOMM20 antibody (AF1717, 1:1,000; Beyotime), rabbit anti-PPAR gamma polyclonal antibody (bs-0530R, 1:1,000; Bioss, Beijing, China), rabbit anti-CEBP alpha polyclonal antibody (bs-24540R, 1:1,000; Bioss), rabbit anti-beta-actin antibody (bs-0061R, 1:5,000; Bioss), goat anti-rabbit IgG-HRP (BS13278, 1:10,000; Bioworld, Minnesota, USA), and goat anti-mouse IgG-HRP (BS12478, 1:10,000; Bioworld).
All experiments were performed at least three times. Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using Student’s t-test, with statistical significance indicated as *P < 0.05, **P < 0.01, and ***P < 0.001.