In this study, we performed high throughput RNA sequencing at the primary bovine preadipocyte (Day-0), mid-differentiation (Day-4), and differentiated adipocyte (Day-9) stages in order to characterize the transcriptional events regulating differentiation and function. The preadipocytes were isolated from subcutaneous fetal bovine adipose tissues and were differentiated into mature adipocytes. The adipogenic characteristics of the adipocytes were detected during various stages of adipogenesis (Day-0, Day-4, and Day-9). We used RNA sequencing (RNA-seq) to investigate a comprehensive transcriptome information of adipocytic differentiation. Compared to the pre-differentiation stage (Day-0), 2510 genes were identified as differentially expressed genes (DEGs) at the mid-differentiation stage (Day-4). We found 2446 DEGs in the mature adipocytic stage relative to the mid-differentiation stage. Some adipogenesis-related transcription factors, CCAAT-enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) were differentially expressed at Day-0, Day-4, and Day-9. We further investigated the adipogenic function of 5-hydroxytryptamine receptor 2A (HTR2A) in adipogenesis. Overexpression of HTR2A stimulated the differentiation of preadipocytes, and knockdown of HTR2A had opposite effects. Furthermore, functional enrichment analysis of DEGs revealed that the PI3K-Akt signaling pathway was the significantly enriched pathway, and HTR2A regulated adipogenesis by activating or inhibiting phosphorylation of phospho-AKT (Ser473). In summary, the present study provides the first comparative transcription of various periods of adipocytes in cattle, which presents a solid foundation for further study into the molecular mechanism of fat deposition and the improvement of beef quality in cattle.

Important candidate genes that regulate lipid metabolism have the potential to increase the content of intramuscular fat (IMF) and improve meat quality. Secreted protein acidic and rich in cysteine like 1(SPARCL1) is a secreted glycoprotein with important physiological functions and is involved in the proliferation and differentiation of various cells. However, the role of the SPARCL1 gene in sheep preadipocytes and its regulatory mechanism is still unclear. In this study, we explored the effect of SPARCL1 on the proliferation and differentiation of sheep preadipocytes. The results showed that the expression level of the SPARCL1 gene is higher in fat tissue than in other tissues, and the gene was significantly increased on the 6^th day of preadipocyte differentiation. In the preadipocyte proliferation stage, interference of SPARCL1 gene reduced cell viability and increased cell apoptosis. In preadipocyte differentiation stage, SPARCL1 overexpression significantly inhibited lipid droplets accumulation and triglyceride content by increasing Wnt10b, Fzd8, IL6, and β-catenin and inhibiting PPARγ, C/EBPα, LPL, and IGF1 genes expression, whereas SPARCL1 deficiency significantly promoted cell differentiation by inhibiting β-catenin and increasing GSK3β, PPARγ, C/EBPα, and LPL. The results of this study suggest that SPARCL1 plays a negative role during preadipocyte differentiation and may become a novel target for regulating preadipocyte differentiation and improving IMF. Abbreviations: IMF: Intramuscular fat SPARCL1: Secreted protein acidic and rich in cysteine like 1 PPARγ: Peroxisome proliferator-activated receptor γ C/EBPα: CCAAT/enhancer-binding protein-α LPL: Lipoprotein lipase IGF1: Insulin-like growth factor 1 Wnt10b: Wnt family member 10B Fzd8: Frizzled class receptor 8 IL6: Interleukin 6 β-catenin: Catenin beta interacting protein 1 GSK3β: Glycogen synthase kinase 3 beta LRP5/6: Low-density lipoprotein receptor-related protein 5/6

Puerarin has the potential of regulating the differentiation of preadipocytes, but its mechanism of action has not yet been elucidated. Adipocytes found in adipose tissue, the main endocrine organ, are the main sites of lipid deposition, and are widely used as a cell model in the study of in vitro fat deposition. This study aimed to investigate the effects of puerarin on adipogenesis in vitro. Puerarin was added to the culture medium during the process of adipogenesis. The proliferation and differentiation of bovine preadipocytes was measured through cell viability and staining with Oil Red O. The content of triacylglycerol (TG) was measured using a triglyceride assay kit. The mRNA and protein expression levels of adipogenic genes, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα), were measured using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. The addition of puerarin significantly increased adipogenesis of bovine preadipocytes and enhanced the mRNA and protein level expression of PPARγ (p<0.01). The expression of P-Akt increased after adipogenic hormonal induction, whereas puerarin significantly increased PPARγ expression by promoting the Akt signaling component, P-Akt. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473, which may activate the downstream signaling of the Akt pathway. Puerarin was able to promote the differentiation of preadipocytes and improve fat deposition in cattle. The mechanism of adipogenesis was found to be related to the phosphorylation level of Ser473.

Context: Muscle fibre characteristics are important internal factors that can directly affect pork quality. Especially muscle-fibre types can interconvert with the influence of certain factors. Aims: The purpose of this experiment was to study the differences in meat quality among Songliao black pigs, Jilin Hua pigs and Dongliao black pigs, and the molecular mechanism of the differences. Methods: First, the conventional meat-quality traits of each breed were determined. Second, the muscle-fibre characteristics of different muscles were analysed by the haematoxylin–eosin staining and immunohistochemistry. Next, the activity of several key oxidative/glycolytic enzymes was detected with kits. Finally, quantitative polymerase chain reaction was used to analyse the expression abundance of myosin heavy-chain (MyHC) genes and key genes related to muscle fibre-type transformation. Key results: The results of meat-quality measurement showed that Songliao black pigs were superior to Jilin Hua pigs and Dongliao black pigs in water-holding capacity, tenderness and intra-muscular fat; and Jilin Hua pigs were superior to Dongliao black pigs in water-holding capacity. The diameter and area of muscle fibres were the smallest, the density was the largest, the proportion of oxidative muscle fibres, oxidative enzyme activity and expression abundance of oxidative genes were the highest in Songliao black pigs, followed by Jilin Hua pigs and Dongliao black pigs. The proportions of oxidative muscle fibres ranged from 10.37% to 33.6% in Songliao black pigs, from 6.96% to 26.42% in Jilin Hua pigs, and from 5.86% to 17.42% in Dongliao black pigs. The psoas major exhibited the smallest muscle-fibre diameter, the highest density, followed by triceps brachii, biceps femoris and longissimus thoracis. The oxidative muscle fibre proportions of the psoas major and triceps brachii were significantly greater than those of the biceps femoris and longissimus thoracis. The expression abundances of AMP-activated protein kinase (AMPK), silencing information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) (Sirt1/AMPK/PGC-1α) pathway and mitochondrial function-related genes were the highest in Songliao black pigs, followed by Jilin Hua pigs and Dongliao black pigs. Conclusions: The results showed that Songliao black pigs had the best meat quality, followed by Jilin Hua pigs and Dongliao black pigs. The meat quality of psoas major and triceps brachii was significantly greater than that of biceps femoris and longissimus thoracis. This experiment suggests that transformation of oxidative muscle fibres can be promoted through the Sirt1/AMPK/PGC-1α pathway. Implications: By analysing the meat-quality traits and the molecular mechanism of meat-quality differences, it can provide data reference and direction for further meat-quality improvement of three breed pigs.

The differentiation of embryonic stem cells (ESCs) into germ cells in vitro could have very promising applications for infertility treatment and could provide an excellent model for uncovering the molecular mechanisms of germline generation. This study aimed to investigate the differentially expressed miRNAs (DEMs) during the differentiation of chicken ESCs (cESCs) into male germ cells and to establish a profile of the DEMs. Cells before and after induction were subjected to miRNA sequencing (miRNA-seq). A total of 113 DEMs were obtained, including 61 upregulated and 52 downregulated DEMs. GO and KEGG enrichment analyses showed that the target genes were enriched mainly in the MAPK signaling pathway, HTLV infection signaling pathway, cell adhesion molecule (CAM)-related pathways, viral myocarditis, Wnt signaling pathway, ABC transporters, TGF-β signaling pathways, Notch signaling pathways and insulin signaling pathway. The target genes of the miRNAs were related to cell binding, cell parts and biological regulatory processes. Six DEMs, let-7k-5p, miR-132c-5p, miR-193a-5p, miR-202-5p, miR-383-5p and miR-6553-3p, were assessed by qRT-PCR, and the results were consistent with the results of miRNA-seq. Based on qRT-PCR and western blot verification, miR-383-5p and its putative target gene STRN3 were selected to construct an STRN3 3'-UTR dual-luciferase gene reporter vector and its mutant vector. The double luciferase reporter activity of the cotransfected STRN3-WT + miR-383-5p mimics group was significantly lower (by approximately 46%) than that of the other five groups (p < 0.01). There was no significant difference in luciferase activity among the other 5 groups. This study establishes a DEM profile during the process of cESC differentiation into male germ cells; illustrates the mechanisms by which miRNAs regulate target genes; provides a theoretical basis for further research on the mechanisms of the formation and regulation of male germ cells; and provides an important strategy for gene editing, animal genetic resource protection and transgenic animal production.