Scd1 Deficiency in Early Embryos Affects Blastocyst ICM Formation through RPs-Mdm2-p53 Pathway.

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.

Dynamic role of Scd1 gene during mouse oocyte growth and maturation.

Mammalian reproductive ability is regulated by many factors, among which the fatty acid metabolism network provides energy for oocyte growth and primordial follicle formation during early mouse oogenesis. But the mechanism behind that is still unknown. Stearoyl-CoA desaturase 1 (Scd1) gene expression is increased during the oogenesis process, supporting the oocyte's healthy growth. Taking advantage of gene-edited mice lacking stearoyl-Coenzyme A desaturase 1 gene (Scd1-/-), we analyzed relative gene expression in perinatal ovaries from wildtype, and Scd1-/- mice. Scd1 deficiency dysregulates expression of meiosis-related genes (e.g., Sycp1, Sycp2, Sycp3, Rad51, Ddx4) and a variety of genes (e.g., Nobox, Lhx8, Bmp15, Ybx2, Dppa3, Oct4, Sohlh1, Zp3) associated with oocyte growth and differentiation, leading to a lower oocyte maturation rate. The absence of Scd1 significantly impedes meiotic progression, causes DNA damage, and inhibits damage repair in Scd1-/- ovaries. Moreover, we find that Scd1 absense dramatically disrupts the abundance of fatty acid metabolism genes (e.g., Fasn, Srebp1, Acaca) and the lipid droplet content. Thus, our findings substantiate a major role for Scd1 as a multifunctional regulator of fatty acid networks necessary for oocyte maintenance and differentiation during early follicular genesis.

Goat FADS2 controlling fatty acid metabolism is directly regulated by SREBP1 in mammary epithelial cells.

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.

Malonyl/Acetyltransferase (MAT) Knockout Decreases Triacylglycerol and Medium-Chain Fatty Acid Contents in Goat Mammary Epithelial Cells.

Malonyl/acetyltransferase (MAT) is a crucial functional domain of fatty acid synthase (FASN), which plays a vital role in the de novo synthesis of fatty acids in vivo. Milk fatty acids are secreted by mammary epithelial cells. Mammary epithelial cells are the units of mammary gland development and function, and it is a common model for the study of mammary gland tissue development and lactation. This study aimed to investigate the effects of MAT deletion on the synthesis of triacylglycerol and medium-chain fatty acids. The MAT domain was knocked out by CRISPR/Cas9 in the goat mammary epithelial cells (GMECs), and in MAT knockout GMECs, the mRNA level of FASN was decreased by approximately 91.19% and the protein level decreased by 51.83%. The results showed that MAT deletion downregulated the contents of triacylglycerol and medium-chain fatty acids (p < 0.05) and increased the content of acetyl-Coenzyme A (acetyl-CoA) (p < 0.001). Explicit deletion of MAT resulted in significant drop of FASN, which resulted in downregulation of LPL, GPAM, DGAT2, PLIN2, XDH, ATGL, LXRα, and PPARγ genes in GMECs (p < 0.05). Meanwhile, mRNA expression levels of ACC, FASN, DGAT2, SREBP1, and LXRα decreased following treatment with acetyl-CoA (p < 0.05). Our data reveals that FASN plays critical roles in the synthesis of medium-chain fatty acids and triacylglycerol in GMECs.