CCAAT/enhancer binding protein α (C/EBPα) is the key transcription factor involved in lipid metabolism, however, the role of C/EBPα in milk fat synthesis of dairy goats remains unknown. The objective of the present research was to clarify the function of C/EBPα in goat mammary epithelial cells (GMECs) and its impact on peroxisome proliferator-activated receptor gamma (PPARG) promoter activity. In this study, C/EBPα overexpression increased its mRNA and protein levels by 42-fold and 6-fold, respectively. In contrast, transfecting siRNA targeting C/EBPα decreased its mRNA level to 20% and protein abundance to 80% of the basal level. The contents of lipid droplets, triacylglycerol (TAG), and cholesterol were increased (P < 0.05) in C/EBPα-overexpressing GMECs, and knockdown of C/EBPα led to the opposite results. Overexpression of C/EBPα significantly increased the expression levels of genes involved in TAG synthesis (AGPAT6, DGAT2, P < 0.01), lipid droplet formation (PLIN2, P < 0.01), and fatty acid synthesis (FADS2, P < 0.05; ELOVL6, P < 0.01). Knockdown of C/EBPα decreased (P < 0.05) the expression levels of AGPAT6, DGAT1, DGAT2, PLIN2, FADS2, and ELOVL6. C/EBPα upregulated the expression level of PPARG (P < 0.05), and four C/EBPα binding regions were identified in the PPARG promoter at −1,112 to −1,102 bp, −734 to −724 bp, −248 to −238 bp, and −119 to −109 bp. Knockdown of C/EBPα reduced (P < 0.05) the PPARG promoter activity when the C/EBPα binding regions were mutated at −1,112 to −1,102 bp, −734 to −724 bp, and −248 to −238 bp locations of the promoter. However, the promoter activity did not change when the mutation was located at −119 bp. In conclusion, our results suggest that C/EBPα can promote TAG synthesis in GMECs through its effects on mRNA abundance of genes related to lipid metabolism and regulation of the PPARG promoter activity via C/EBPα binding regions. Goat milk is beneficial for human health because of its nutritional value, especially milk fat, which is plentiful in goat milk. The molecular mechanism of milk fat synthesis is of great importance for developing processing technology and using genetic approaches to improve goat milk quality. The purpose of this study was to identify the role of CCAAT/enhancer binding protein α (C/EBPα) in goat mammary gland epithelial cells (GMECs) to provide support for understanding the mechanism of milk fat synthesis. Overexpression of C/EBPα increased the contents of lipid droplets, triacylglycerol, and cholesterol in GMECs. The expression levels of genes related to lipid metabolism were influenced after C/EBPα overexpression or inhibition. The promoter transcriptional activity of peroxisome proliferator-activated receptor gamma, which is the key transcription factor in milk fat synthesis, was regulated by C/EBPα through its binding regions. Our results indicate that C/EBPα affects lipid metabolism in GMECs by regulating PPARG transcription. This study provides support for milk fat synthesis regulation and improvement of milk quality in goats.

Goat milk provides benefits to human health due to its richness in bioactive components, such as polyunsaturated fatty acids (PUFAs). The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in PUFAs biosynthesis. However, its role and transcriptional regulation mechanisms in fatty acid metabolism in dairy goat remain unclear. Here, our study revealed that the FADS2 gene was highly expressed during the peak lactation compared with the dry period, early lactation, and late lactation. The content of triacylglycerol (TAG) was enhanced with the increasing mRNA expression of TAG synthesis genes (diacylglycerol acyltransferase 1/2, DGAT1/2) in FADS2-overexpressed goat mammary epithelial cells (GMECs). Overexpression of FADS2 was positively correlated with the elevated concentrations of dihomo-gamma-linolenic acid (DGLA) and docosahexaenoic acid (DHA) in GMECs. BODIPY staining showed that FADS2 promoted lipid droplet accumulation in GMECs. To clarify the transcriptional regulatory mechanisms of FADS2, 2,226 bp length of FADS2 promoter was obtained. Deletion mutation assays revealed that the core region of FADS2 promoter was located between the −375 and −26 region, which contained SRE1 (−361/−351) and SRE2 (−191/−181) cis-acting elements of transcription factor sterol regulatory element-binding protein 1 (SREBP1). Overexpression of SREBP1 enhanced relative luciferase activity of the single mutant of SRE1 or SRE2, vice versa, and failed to alter the relative luciferase activity of the joint mutant of SRE1 and SRE2. Chromatin immunoprecipitation (ChIP) and site-directed mutation assays further demonstrated that SREBP1 regulated the transcription of the FADS2 gene by binding to SRE sites in vivo and in vitro. In addition, the mRNA levels of FADS2 were significantly decreased by targeting SRE1 and SRE2 sites in the genome via the CRISPR interference (CRISPRi) system. These findings establish a direct role for FADS2 regulating TAG and fatty acid synthesis by SREBP1 transcriptional regulation in dairy goat, providing new insights into fatty acid metabolism in mammary gland of ruminants. The fatty acid desaturase 2 (FADS2) is the first rate-limiting enzyme in polyunsaturated fatty acids (PUFAs) biosynthesis in mammals. This study aimed to investigate the function and transcriptional regulation mechanism of FADS2 in goat mammary epithelial cells (GMECs). The content of triacylglycerol (TAG) was enhanced with lipid droplet accumulation in FADS2-overexpressed GMECs. Overexpression of FADS2 was positively correlated with elevated concentrations of docosahexaenoic acid (DHA) in GMECs. Furthermore, site-directed mutation and chromatin immunoprecipitation (ChIP) assays simultaneously demonstrated that FADS2 was directly regulated by SREBP1 transcriptional factor binding to sterol regulatory element (SRE) in vitro and in vivo. In addition, genetic ablation of SRE1 and SRE2 in the genome resulted in a significant reduction in the mRNA levels of FADS2 via the CRISPR interference (CRISPRi) system. Altogether, this study discovered that the SREBP1 exerts control on FADS2 to regulate milk fatty acids, and provides a theoretical approach for improving milk quality via genetic approaches.

Meat quality traits, particularly WHC and tenderness, are pivotal for consumer satisfaction and economic value in the sheep industry. However, their genetic regulatory mechanisms remain unclear. We used RNA-Seq and WGCNA to identify genes regulating WHC and tenderness. Sixty longissimus thoracis samples were classified into high/low WHC (HWHC vs. LWHC) and high/low tenderness (HTN vs. LTN) groups. Comparative transcriptomics identified 270 differentially expressed genes (DEGs) linked to WHC, enriched in pathways like the regulation of the ATP metabolic process and the inhibition of canonical Wnt signaling. Key DEGs (e.g., SORBS1, FOXO1, PDE4B, CDH1) correlated significantly with WHC-associated traits. For tenderness, 165 DEGs were identified, including LEP, FABP4, PLIN1, and GLP1R, enriched in PPAR signaling, fat cell differentiation, and cAMP signaling pathways. WGCNA revealed modules associated with WHC and tenderness, with hub genes (ATP2C1, GSKIP, PATL1, PPARA, CYLD) involved in ATP metabolism, lipid biosynthesis, and myofibril assembly. Tissue-specific gene integration prioritized muscle-enriched candidates (METTL21C and ACTC1) with strong trait correlations. Our findings unveil interconnected gene networks governing WHC and tenderness, highlighting some candidate genes as potential biomarkers for precision breeding. This study provides novel insights into the molecular determinants of meat quality, offering actionable targets to enhance mutton production sustainability and consumer appeal.

In nonruminants, microRNA (miRNA)-24 plays an important role in lipid metabolism in adipose tissue and the liver. Although the abundance of miR-24 in ruminant mammary glands is the highest during peak lactation, its potential role in regulating the synthesis and secretion of fat into milk is unclear. This study aimed to identify the function of miR-24 in these processes using CRISPR/Cas9 technology in primary goat mammary epithelial cells (GMEC). A single clone containing a 66-nucleotide deletion between two sgRNAs mediating double-strand break (DSB) sites was obtained. The abundance of miR-24-3p and miR-24-5p encoded by the deleted sequence was decreased, whereas the target genes INSIG1 and FASN increased. In addition, miR-24 knockout reduced the gene abundance of genes associated with fatty acid and TAG synthesis and transcription regulator. Similarly, the content of cholesterol and monounsaturated fatty acid (MUFA) C18:1 decreased, whereas that of polyunsaturated fatty acids (PUFA) C18:2, C20:3, C20:4 and C20:5 increased. Subsequently, knocking down of INSIG1 but not FASN reversed the effect of miR-24 knockout, indicating that miR-24 modulated cholesterol and fatty acid synthesis mainly by targeting INSIG1. Overall, the present in vitro data demonstrated a critical role for miR-24 in regulating lipid and fatty acid synthesis and highlighted the possibility of manipulating milk components in dairy goats.

Embryos contain a large number of lipid droplets, and lipid metabolism is gradually activated during embryonic development to provide energy. However, the regulatory mechanisms remain to be investigated. Stearoyl-CoA desaturase 1 (Scd1) is a fatty acid desaturase gene that is mainly involved in intracellular monounsaturated fatty acid production, which takes part in many physiological processes. Analysis of transcripts at key stages of embryo development revealed that Scd1 was important and expressed at an increased level during the cleavage and blastocyst stages. Knockout Scd1 gene by CRISPR/Cas9 from zygotes revealed a decrease in lipid droplets (LDs) and damage in the inner cell mass (ICM) formation of blastocyst. Comparative analysis of normal and knockout embryo transcripts showed a suppression of ribosome protein (RPs) genes, leading to the arrest of ribosome biogenesis at the 2-cell stage. Notably, the P53-related pathway was further activated at the blastocyst stage, which eventually caused embryonic development arrest and apoptosis. In summary, Scd1 helps in providing energy for embryonic development by regulating intra-embryonic lipid droplet formation. Moreover, deficiency activates the RPs-Mdm2-P53 pathway due to ribosomal stress and ultimately leads to embryonic development arrest. The present results suggested that Scd1 gene is essential to maintain healthy development of embryos by regulating energy support.