All procedures were performed according to the guidelines established by the Sichuan Province Committee for the Protection of Laboratory Animals. Eleven tissues, namely, the ovary, spleen, rumen, liver, brain, heart, gastrocnemius muscle, biceps femoris muscle, longissimus dorsi muscle (LD), and mesenteric and subcutaneous adipose tissue, were dissected from three three-day-old female Nanjiang brown goats. All tissues were immediately frozen in liquid nitrogen and then stored at −80°C.

Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The integrity of total RNA was examined by agarose gel electrophoresis, and the purity and concentration were determined using a NanoDrop (Thermo Fisher, Waltham, MA, USA) instrument. First-strand complementary DNA (cDNA) was synthesized using 2 μg of total RNA and a PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s protocol.

Based on the conserved regions of the human and cattle AdipoQ, AdipoR1, and AdipoR2 gene sequences, primer pairs for gene cloning and quantitative polymerase chain reaction (qPCR) were designed by Primer Premier 5.0 (Table 1) and synthesized by Shanghai Sangon Biotechnology Company (Shanghai, China). The total reaction volume was 10 μL and included 1 μL cDNA, 5 μL 2× Taq PCR Master Mix, 0.8 μL (8 pM) primer pairs (ADF/R for AdipoQ, ADR1F/R for AdipoR1 and ADR2F/R for AdipoR2) and 3.2 μL ddH₂O. The PCR procedures were as follows: initial denaturation at 95°C for 3 min; 35 cycles of 95°C for 30 s, optimum annealing temperature (Table 1) for 30 s and 72°C for 1 to 2 min; and a final extension at 72°C for 5 min. All products were separated by agarose gel electrophoresis, purified using a gel extraction reagent kit (Tiangen, Beijing, China), cloned into the pMD19-T vector (TaKaRa, Japan) and sequenced by Shanghai Invitrogen Biotechnology Company (Shanghai, China).

To detect the mRNA tissue distributions of the AdipoQ, AdipoR1, and AdipoR2 genes, we performed semi-quantitative reverse transcription (RT)-PCR. The total reaction volume was 20 μL and included 2 μL cDNA, 10 μL 2× Taq PCR Master Mix, 1.2 μL (12 pM) primer pairs (ADQF/R for AdipoQ, ADR1QF/R for AdipoR1 and ADR2QF/R for AdipoR2) and 6.8 μL ddH₂O. The PCR procedure was as follows: initial denaturation at 95°C for 3 min; 26 to 32 cycles at 95°C for 30 s, optimum annealing temperature (Table 1) for 30 s and 72°C for 10 s; and a final extension at 72°C for 5 min. All products were examined by agarose gel electrophoresis to estimate mRNA abundance in the 11 tissues tested; β-actin was used as an internal control gene.

qPCR was carried out using a SYBR Green-based kit in 10-μL reaction volumes containing 5 μL SYBR Green real-time PCR Master Mix (TaKaRa, Japan), 0.8 μL cDNA and 0.4 μL each primer; a Bio-Rad CFX96 qPCR instrument (Bio-Rad, Hercules, CA, USA) was used. The qPCR procedure was as follows: initial denaturation at 95°C for 3 min; 40 cycles at 95°C for 30 s, alternative annealing for 20 s, and 72°C for 15 s; and a final extension for 5 min. Melting curve analysis was used to confirm specific PCR products. Gene expression levels were quantified relative to β-actin expression using the comparative Ct (2^−ΔΔCt) value method.

SMSCs were isolated using 1 mg/mL Pronase E (Sigma, St. Louis, MO, USA) from the longissimus dorsi muscles from 3-day-old female Nanjiang brown goats. After enzymatic digestion, cells were separated from tissue fragments by repeated centrifugation at 500×g for 5 min, followed by filtration through a 200-μm filter and then a 50-μm Nytex filter. SMSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin under conditions of 5% CO₂ and 37°C. When the cells had grown to 80% to 90% confluence, adipogenic differentiation of satellite cells was induced by transferring the cells into DMEM supplemented with an isobutylmethylxanthine, dexamethasone and insulin (MDI) cocktail (0.5 mM 1-methyl-3-isobutylxanthine, 1 μM dexamethasone, and 10 μg/mL insulin), followed by supplementation with 10 μg/mL insulin for 1 day. The medium was replaced every two days. Cells were visualized by staining with oil red O solution.

Using Primer Premier 5.0, a primer pair (Hind III-F, BamH I-R) was designed to amplify the coding region of AdipoQ for plasmid construction. PCR products and the pEGFP-N1 (Clontech, San Francisco, CA, USA) vector were digested with Hind III and BamH I restriction enzymes (TaKaRa, Japan), purified with a gel extraction reagent kit and ligated using T4 DNA ligase (TaKaRa, Japan). The resulting plasmid (pEGFP-N1-AD) was sequenced to confirm the correct orientation and reading frame.

Transient transfection was performed according to the manufacturer’s protocol: 4 μg plasmid and 10 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were mixed, cultured for 20 min and then transfected into cells. Twenty-four hours after transfection, the cells were rinsed with phosphate-buffered saline; a portion was fixed with 4% formaldehyde for 30 min at room temperature to detect the cellular distribution of the adiponectin fusion protein using a fluorescence microscope (Nikon, Tokyo, Japan); other cells were suspended in TRIzol reagent for RNA extraction and expression analysis. The results were normalized to the expression of β-actin, and the expression of the control group (pEGFP-N1) was set as 1 for expression analysis of each gene. The relative mRNA expression analysis was performed using SAS 9.0 software with the generalized linear model process. Duncan’s new multiple range test was applied to analyze statistical significance.

Expression of adiponectin in SMSCs was detected by immunocytochemical staining. Briefly, cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized in 0.25% Triton X-100 with shaking at room temperature for 30 min, followed by blocking with 1% bovine serum albumin for 1 h. The cells were then incubated overnight at 4°C with an anti-adiponectin primary antibody (1:100) (Boster Company, Wuhan, China). The secondary antibody, horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:200), was incubated with the cells for 30 min. Cells incubated without the primary antibody were used as the negative control.

ORF Finder (http://www.ncbi.nlm.nihgov/gorf/gorf.html) and DNASTAR were used to predict the open reading frames of the three genes. The domains of the three proteins were predicted by ProtParam (http://cn.expasy.org/tools/protparam.html) and SMART (http://smart.embl-heidelberg.de/), and the functional domain diagram was generated with DOG 2.0 software [21]. Amino acid sequences were aligned using ClustalX (http://www.ebi.ac.uk/clustalw/).

The full-length coding sequences of the goat AdipoQ, AdipoR1, and AdipoR2 genes were obtained (accession numbers: JX573539, KC286912 and JX573540). The coding regions of the AdipoQ, AdipoR1, and AdipoR2 are 720 bp, 1,128 bp, and 1,176 bp, respectively. The AdipoQ gene encodes a 239-aa protein with a molecular weight of 25.95 kDa. The AdipoR1 gene encodes a 375-aa protein with a molecular weight of 42.41 kDa, and the AdipoR2 gene encodes a 391-aa protein with a molecular weight of 43.81 kDa. These three genes share high similarity with other mammalian orthologous genes (Supplementary Table S1). Goat AdipoR1 shows the highest homology with other mammals, with the amino acid sequence sharing more than 96% identity with other mammalian sequences. Goat AdipoQ and AdipoR2 exhibit relatively high homology with other mammalian sequences. The amino acid sequence of AdipoQ is 81.17%, 87.45%, 81.59%, and 99.16% identical to those of Homo sapiens, Bos taurus, Mus musculus, and Ovis aries, respectively. The amino acid sequence of AdipoR2 shows similar identity to those of humans (86.27%), Bos taurus (92.49%), Mus musculus (84.20%), and Ovis aries (94.04%). Prediction of the deduced protein structure showed that adiponectin harbors a 137-aa C1Q domain; although the receptors both contain a 220-aa seven-transmembrane domain, these receptors are not identical (Figure 1).

The mRNA tissue distribution of these three genes was detected by semi-quantitative RT-PCR, and the agarose gel electrophoresis results are shown in Figure 2. AdipoQ expression was predominantly observed in mesenteric and subcutaneous adipose tissues but was weakly expressed in the gastrocnemius muscle, LD muscle and biceps femoris muscle; expression was not detected in other tissues. AdipoR1 mRNA was found to be expressed in almost all tissues, with high expression in skeletal muscles and the rumen, followed by adipose tissue; its expression in other tissues was relatively weak. Similarly, AdipoR2 was expressed in all tissues but was most abundant in adipose and skeletal muscle tissues; it was weakly expressed in other tissues. The expression patterns of these three genes were further quantitated by qPCR (Figure 3). AdipoQ mRNA was predominantly expressed in adipose tissue, whereas its expression in skeletal muscles was relatively weak. AdipoR1 and AdipoR2 mRNA were expressed in all tissues examined, with the highest expression in adipose and skeletal muscle tissues.

Fluorescence analysis showed a green fluorescent signal throughout cells of the control group transfected with the pEGFP-N1 plasmid; In contrast, the green fluorescence of the adiponectin fusion protein was only observed in the cytoplasm of cells transfected with the pEGFP-AD plasmid (Figure 4), suggesting that adiponectin is localized to the cytoplasm. Furthermore, immunostaining in SMSCs using an antibody against the human adiponectin protein revealed strong positive staining within the goat SMSC cytoplasm (Figure 5A) compared with the controls (Figure 5B).

To determine the role of adiponectin in the adipogenic differentiation of goat MSCs, cells were transfected with the pEGFP-N1-AD plasmid to overexpress the goat AdipoQ gene. To evaluate effective overexpression of adiponectin, whole-cell lysates were harvested for analysis of expression levels. In SMSCs, adiponectin was overexpressed approximately 80-fold compared with that in control cells transfected with the pEGFP-N1 vector (Figure 6). These results demonstrate that cells transfected with the pEGFP-N1-AD plasmid can overexpress adiponectin compared with the expression levels of control goat SMSCs expressing green fluorescent protein (GFP).

Next, satellite cells were induced with an MDI adipogenic cocktail and stained with oil red O seven days later. As shown in Figure 7, more oil red O-positive cells were observed in the pEGFP-N1-AD-transfected group than in the control group, indicating that adiponectin promoted MSC differentiate into adipocytes. In addition, several markers of adipogenic differentiation were detected by qPCR. The results showed that adiponectin overexpression significantly (p<0.01) up-regulated the mRNA expression levels of AdipoR2, ACC, FAS, and SREBP-1 (Figure 8), which were 1.85, 4.55, 4.88, and 4.25 times higher, respectively, than those of the control group. Conversely, no significant changes in expression were found for C/EBPα, PPARγ, and AdipoR1 (data not shown).