The fertilized goose eggs used in the experiment were provided by Ningbo Langde Agriculture and Animal Husbandry Co., Ltd. (Ningbo, China). The eggs were put in the same incubator and incubated under the same conditions. The first day after hatching was set as E1. Five eggs were randomly removed at each of seven developmental stages, including embryonic stage of 7 days (E7), embryonic stage of 11 days (E11), embryonic stage of 15 days (E15), embryonic stage of 19 days (E19), embryonic stage of 23 days (E23), and embryonic stage of 27 days (E27) and 3 days post-hatching. Samples of LM were collected, snap frozen into liquid nitrogen and transfer to −80°C refrigerator for storage. All animal procedures used in this study were approved by the Ethics Committee for Animal Experiments of Zhejiang A&F University and were performed in accordance with the Guidelines for Animal Experimentation of Zhejiang A&F University (Hangzhou, China).

The 15 to 20 day fertilized goose eggs were disinfected with ethanol to isolate skeletal muscle satellite cells. The LM of the goose embryo was isolated and placed in a petri dish, washed with phosphate buffer solution (PBS) (HyClone, Logan, UT, USA) containing Penicillin-Streptomycin Liquid (Solarbio, Beijing, China) for 3 times and the skin, blood vessels, adipose tissue and connective tissue were removed. Then, the muscle tissues were cut into meat paste, and digested with 0.25% trypsin-ethylenediaminetetraacetic acid (Thermo Fisher, Shanghai, China) at 37°C for 20 min. Dulbecco’s modified eagle medium (DMEM)/F12 (HyClone, USA) containing 15% fetal bovine serum (FBS, Thermo Fisher, China) was added to terminate the digestion. The suspension was filtered through a 70 μm mesh sieve and centrifuged at 1,000 r/min for 8 min at room temperature. The supernatant was discarded, cells were resuspended with DMEM/F12 containing 15% FBS and cultured in the 5% CO₂ incubator at 37°C. One hour later, the fibroblasts had adhered to the bottom of cell culture flask, while the skeletal muscle satellite cells remained in the supernatant. The cell suspension was inhaled into a new cell petri dish and this process was repeated twice to enrich muscle satellite cells and eliminate fibroblasts. To induce cell differentiation, culture medium was switched to differentiation medium (DM) with 2% horse serum and 1% penicillin streptomycin (HyClone, USA).

Total RNA from tissues or cells was obtained using RNAiso reagent and treated with DNase I (Takara, Kyoto, Japan). The concentration and integrity of RNA were measured by spectrophotometer and denatured gel electrophoresis. The PrimeScript first Strand cDNA synthesis kit (Abcam, Shanghai, China) was used to synthesize the cDNAs. The cDNAs were stored at −20°C until use.

The cDNA was used as the template for quantitative real-time polymerase chain reaction (qRT-PCR) using the EvaGreen 2X qPCR Master Mix kit (abm, Suzhou, China). Each sample was tested in triplicate, and glyceraldehyde-3-phosphate dehydrogenase was used as a reference gene. Differential gene expression analysis was calculated using the 2^−ΔΔCT statistical analysis method. The primers were designed using PrimerPremier.5 software (Table 1) and synthesized by Hangzhou Youkang Biotechnology Co., Ltd. (Zhejiang, China).

Immunofluorescence was used to identify the isolated skeletal muscle satellite cells. Briefly, cells grown in 12-well plate were firstly washed with PBS for three times. and fixed with 4% paraformaldehyde for 15 min. After being washed three times with PBS, the cells were permeabilized with 0.25% Triton X-100 per well for 10 min and blocked at 4°C overnight. Afterwards, cells were incubated with 1:100 diluted primary anti-Desmin (Abcam, China) or anti-Pax7 (Abcam, China) for 1 hour at room temperature. After washing, 1:2,000 diluted fluorescent secondary antibody (Thermo Fisher, China) was used to incubate the cells for 1 hour at room temperature. The cells were then washed, added with 4′,6-diamidino-2- phenylindole (Invitrogen, Carlsbad, CA, USA) and incubated for 15 min at room temperature to stain the cell nuclei. In the end, samples were captured using a fluorescence microscope (OLYMPUS, Tokyo, Japan).

The RNA oligonucleotides, including the CTRP3 siRNAs and the negative control (NC) were all purchased from RiboBio Co., Ltd. (Guangzhou, China). Lipofectamine 3000 (Invitrogen, USA) was used for transfection following the manufacturer s instructions. All oligonucleotides sequences are listed in Table 2.

PcDNA-3.1 CTRP3 was the expression vector. The primers were designed to amplify the entire coding sequence region of CTRP3 and were digested with BamHI and EcoRI restriction sites. The primers’ sequences were as follows: 5′-CGCG GATCCATGGCAGAGAAGGATTTCATC-3′and 5′-CCG GAATTCTTACTTGGTTTCAAAGAGA-3′. The PCR product was then cloned into the pcDNA-3.1 vector.

Total proteins of the cells were prepared and treated as previously described [19]. Cells were homogenized in radio immunoprecipitation assay buffer with 1% phenylmethanesulfonyl fluoride and incubated on ice for 30 min to extract total proteins. Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred onto the polyvinylidene fluoride membranes (Millipore, Bllerica, MA, USA). After blocking with 5% bovine serum albumin, the membranes were incubated with primary antibodies for MyHC (sc-32732; Santa Cruz Biotechnology, Santa Cruz, CA, USA), CTRP3 (ab36870; Abcam, China), β-actin (ab40854; Abcam, USA). Thereafter, the membranes were incubated with secondary antibody for horseradish peroxidase-labeled anti-rabbit/mouse immunoglobulin G (A0208/A0216; Beyotime, Shanghai, China). Finally, the blots were detected through the chemiluminesence detection system (Amersham, Piscataway, NJ, USA) using chemiluminescence (ECL) reagent (Thermo Scientific, Waltham, MA, USA). Image J software was used for gray value analysis.

All results are shown as the mean±standard error of the mean. Each treatment was repeated for three times. Unpaired Student’s t-test was performed to test statistical significance using SPSS 20.0 software. Two-tailed t tests were used in the analysis. p<0.05 was considered as significant.

The isolated goose skeletal muscle satellite cells were cultured in growth medium and began to adhere to plates 12 h later. After 24 h, the cells began to grow dispersedly, and some of them had completely adhered. After 36 h, all the cells were completely adhered and gradually extended into elongated shape. The cell confluence approximately reached 90% after 48 h (Figure 1).

Desmin and Pax7 are special markers of skeletal muscle satellite cells [20], and are often used to distinguish skeletal muscle satellite cell from other cells. After immunofluorescence staining, expression of Pax7 and Desmin were observed in the isolated cells. Pax7 was distributed in the nucleus and Desmin was in the cytoplasm (Figure 2). This result further confirmed the isolated cells were goose skeletal muscle satellite cells.

To investigate the expression profile of CTRP3 during LM development, qPCR assay was performed. The mRNA expression level at E19 was regarded as a control and assigned a value of 1. The results show that the expression of CTRP3 reached its highest level in E11 (Figure 3A). The expression level rose from E7 to E11, then decreased sharply from E15 to P3 (Figure 3A), which indicated that CTRP3 might have a potential role in goose skeletal myogenesis.

Next, we set up a cell model to detect the function of CTRP3 during myoblast differentiation. Both CTRP3 and two marker genes of myogenic differentiation, MyHC and MyoG, were up-regulated during this period (Figure 3B–3C). Thus, we could confirm that the in vitro cell model was successfully established.

To assess the role of CTRP3 gene in goose skeletal muscle satellite cell differentiation, we detected muscle-specific gene expression after transfection with siRNA-CTRP3. CTRP3 was significantly down-regulated in muscle cells at 3 day of DM (Figure 4A–4B). The mRNA level of MyHC and MyoG were decreased remarkably (Figure 4C–4D), and the protein level of MyHC was decreased as well (Figure 4B). Thus, these results indicated that CTRP3 knockdown might attenuate goose myogenesis.

To further investigate the role of CTRP3 in goose myoblast differentiation, the cells were transfected with the expression vector pcDNA3.1-CTRP3. As a result, the expression of CTRP3 was much higher than the control at 3 days post-transfection (Figure 5A–5B). The mRNA level of MyHC and MyoG were increased significantly (Figure 5C-5D). Meanwhile, the protein level of MyHC was also increased (Figure 5B). These results suggested that overexpression of CTRP3 might induce goose skeletal muscle satellite cell differentiation.

Notch signaling pathway plays an important role in muscle cell differentiation [21]. Previous studies have found that CTRP3 gene could suppress the expression of Notch signaling pathway during renal cell fibrosis [22]. As shown in Figure 6A, the mRNA level of Notch signaling pathway-related genes, notch receptor 1 (Notch1), notch receptor 2 (Notch2), and hairy/enhancer-of-split related with YRPW motif 1 (Hey1), were significantly induced after transfecting with siRNA-CTRP3 in goose skeletal muscle satellite cell. Nevertheless, over-expression of CTRP3 in DM reduced their expression (Figure 6B). Therefore, we illustrated that CTRP3 might also regulate the Notch signaling pathway in goose skeletal muscle cells.