Dairy Goat Mammary Gland Research Articles

M6A Methylation Mediates the Function of the circRNA-08436/miR-195/ELOVL6 Axis in Regards to Lipid Metabolism in Dairy Goat Mammary Glands.

The nutritional value of goat milk is determined by the composition of its fatty acids, with particular importance placed on the role of unsaturated fatty acids in promoting human health. CircRNAs have been known to affect fatty acid metabolism through different pathways. In this study, high-throughput sequencing was employed to construct expression profiles of mammary tissue harvested during the dry period and peak lactation stages of dairy goats. Differentially expressed circRNAs and mRNAs were screened, revealing significantly higher expression levels of circRNA-08436 and ELOVL6 during the peak lactation period compared with the dry period. Thus, circRNA-08436 and ELOVL6 were chosen for subsequent studies. The findings demonstrated that circRNA-08436 not only promotes the synthesis of triglyceride (TAG) and cholesterol in goat mammary epithelial cells (GMECs), but also increases the concentrations of saturated fatty acids in the cells. Through the utilization of software prediction, the dual luciferase reporter system, and qRT-PCR, it was observed that circRNA-08436 binds to miR-195, with its overexpression reducing the expression levels of miR-195 and inhibiting TAG synthesis. In addition, circRNA-08436 upregulated the expression levels of the miR-195 target gene ELOVL6. The data also revealed that YTHDC1 facilitated the transport of circRNA-08436 from the nucleus to the cytoplasm, while YTHDC2 in the cytoplasm functioned as a "reader" to identify and degrade circRNA-08436. Taken together, these findings contribute to a better understanding of the molecular regulation of fatty acid metabolism in the mammary glands of dairy goats, thus offering a sound theoretical basis for the production of high-quality goat milk.

Knockdown of PROX1 promotes milk fatty acid synthesis by targeting PPARGC1A in dairy goat mammary gland

Goat milk is rich in various fatty acids that are beneficial to human health. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-seq analyses of goat mammary glands at different lactation stages revealed a novel lactation regulatory factor, Prospero homeobox 1 (PROX1). However, the mechanism whereby PROX1 regulates lipid metabolism in dairy goats remains unclear. We found that PROX1 exhibits the highest expression level during peak lactation period. PROX1 knockdown enhanced the expression of genes related to de novo fatty acid synthesis (e.g., SREBP1 and FASN) and triacylglycerol (TAG) synthesis (e.g., DGAT1 and GPAM) in goat mammary epithelial cells (GMECs). Consistently, intracellular TAG and lipid droplet contents were significantly increased in PROX1 knockdown cells and reduced in PROX1 overexpression cells, and we observed similar results in PROX1 knockout mice. Following PROX1 overexpression, RNA-seq showed a significant upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A) expression. Further, PPARGC1A knockdown attenuated the inhibitory effects of PROX1 on TAG contents and lipid-droplet formation in GMECs. Moreover, we found that PROX1 promoted PPARGC1A transcription via the PROX1 binding sites (PBSs) located in the PPARGC1A promoter. These results suggest a novel target for manipulating the goat milk-fat composition and improving the quality of goat milk.

Identification and Characterization of circRNAs in Non-Lactating Dairy Goat Mammary Glands Reveal Their Regulatory Role in Mammary Cell Involution and Remodeling.

This study conducted transcriptome sequencing of goat-mammary-gland tissue at the late lactation (LL), dry period (DP), and late gestation (LG) stages to reveal the expression characteristics and molecular functions of circRNAs during mammary involution. A total of 11,756 circRNAs were identified in this study, of which 2528 circRNAs were expressed in all three stages. The number of exonic circRNAs was the largest, and the least identified circRNAs were antisense circRNAs. circRNA source gene analysis found that 9282 circRNAs were derived from 3889 genes, and 127 circRNAs' source genes were unknown. Gene Ontology (GO) terms, such as histone modification, regulation of GTPase activity, and establishment or maintenance of cell polarity, were significantly enriched (FDR < 0.05), which indicates the functional diversity of circRNAs' source genes. A total of 218 differentially expressed circRNAs were identified during the non-lactation period. The number of specifically expressed circRNAs was the highest in the DP and the lowest in LL stages. These indicated temporal specificity of circRNA expression in mammary gland tissues at different developmental stages. In addition, this study also constructed circRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) regulatory networks related to mammary development, immunity, substance metabolism, and apoptosis. These findings help understand the regulatory role of circRNAs in mammary cell involution and remodeling.

The High Level of RANKL Improves IκB/p65/Cyclin D1 Expression and Decreases p-Stat5 Expression in Firm Udder of Dairy Goats.

Udder traits, influencing udder health and function, are positively correlated with lactation performance. Among them, breast texture influences heritability and impacts on the milk yield of cattle; however, there is a lack of systematic research on its underlying mechanism in dairy goats in particular. Here, we showed the structure of firm udders with developed connective tissue and smaller acini per lobule during lactation and confirmed that there were lower serum levels of estradiol (E2) and progesterone (PROG), and higher mammary expression of estrogen nuclear receptor (ER) α and progesterone receptor (PR), in dairy goats with firm udders. The results of transcriptome sequencing of the mammary gland revealed that the downstream pathway of PR, the receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) signal, participated in the formation of firm mammary glands. During the culture of goat mammary epithelial cells (GMECs), high RANKL level additions promote the Inhibitor kappaB (IκB)/p65/Cyclin D1 expression related to cell proliferation and decrease the phosphorylated signal transduction and transcription activator 5 (Stat5) expression related to milk-protein synthesis of GMECs, which is consistent with electron microscope results showing that there are fewer lactoprotein particles in the acinar cavity of a firm mammary. Furthermore, co-culturing with adipocyte-like cells for 7 d is beneficial for the acinar structure formation of GMECs, while there is a slightly negative effect of high RANKL level on it. In conclusion, the results of this study revealed the structure of firm udders structure and confirmed the serum hormone levels and their receptor expression in the mammary glands of dairy goats with firm udders. The underlying mechanism leading to firm udders and a decrease in milk yield were explored preliminarily, which provided an important foundation for the prevention and amelioration of firm udders and improving udder health and milk yield.

MiR-380-3p promotes β-casein expression by targeting αS1-casein in goat mammary epithelial cells.

αS1-Casein is more closely associated with milk allergic reaction than other milk protein components. microRNA (miRNA) is a class of small non-coding RNAs that modulate multiple biological progresses by the target gene. However, the post-transcriptional regulation of αS1-casein expression by miRNA in ruminants remains unclear. This study aims to explore the regulatory roles of miR-380-3p on αS1-casein synthesis in goat mammary epithelial cells (GMEC). αS1-Casein gene and miR-380-3p expression was measured in dairy goat mammary gland by quantitative real-time polymerase chain reaction (qRT-PCR). miR-380-3p overexpression and knockdown were performed by miR-380-3p mimic or inhibitor in GMEC. The effect of miR-380-3p on αS1-casein synthesis was detected by qRT-PCR, western blot, luciferase and chromatin immunoprecipitation assays in GMEC. Compared with middle-lactation period, αS1-casein gene expression is increased, while miR-380-3p expression is decreased during peak-lactation of dairy goats. miR-380-3p reduces αS1-casein abundance by targeting the 3'-untranslated region (3'UTR) of αS1-casein mRNA in GMEC. miR-380-3p enhances β-casein expression and signal transducer and activator of transcription 5a (STAT5a) activity. Moreover, miR-380-3p promotes β-casein abundance through target gene αS1-casein, and activates β-casein transcription by enhancing the binding of STAT5 to β-casein gene promoter region. miR-380-3p decreases αS1-casein expression and increases β-casein expression by targeting αS1-casein in GMEC, which supplies a novel strategy for reducing milk allergic potential and building up milk quality in ruminants.

Circ007071 Inhibits Unsaturated Fatty Acid Synthesis by Interacting with miR-103-5p to Enhance PPARγ Expression in the Dairy Goat Mammary Gland.

Understanding more precisely the mechanisms controlling the metabolism of fatty acid in the mammary gland of dairy goats is essential for future improvements in milk quality. Particularly since recent data have underscored a key role for circular RNAs (circRNAs) in the mammary gland function, high-throughput sequencing technology was used to identify expression levels of circRNAs in the mammary tissue of dairy goats during early and peak lactation in the present study. Compared with early lactation, results demonstrated that the expression level of circ007071 during peak lactation was 12.02-fold up-regulated. Subsequent studies in goat mammary epithelial cells (GMECs) revealed that circ007071 stimulated the synthesis of triglycerides (TAG) and cholesterol, as well as increased the content of saturated fatty acids (C16:0 and C18:0). More importantly, using a double luciferase reporting system allowed us to detect the circ007071 sequence at a binding site of miR-103-5p, indicating that it targeted this miRNA. Overexpression of circ007071 significantly decreased the abundance of miR-103-5p and led to inhibition of TAG synthesis. In contrast, the abundance of peroxisome proliferator-activated receptor γ (PPARγ), a target gene of miR-103-5p, was reinforced with the overexpression of circ007071. Thus, we conclude that one key function of circ007071 in the regulation of milk fat synthesis is to attenuate the inhibitory effect of miR-103-5p on PPARγ via direct interactions with miRNA. As a result, the process of TAG and saturated fatty acid is able to proceed.

Circ003429 Regulates Unsaturated Fatty Acid Synthesis in the Dairy Goat Mammary Gland by Interacting with miR-199a-3p, Targeting the YAP1 Gene.

Fatty acid composition is a key factor affecting the flavor and quality of goat milk. CircRNAs are now recognized as important regulators of transcription, and they play an important role in the control of fatty acid synthesis. Thus, understanding the regulatory mechanisms controlling this process in ruminant mammary glands is of great significance. In the present study, mammary tissue from dairy goats during early lactation and the dry period (nonlactating) were collected and used for high-throughput sequencing. Compared to levels during the dry period, the expression level of circ003429 during early lactation was lower (12.68-fold downregulated). In isolated goat mammary epithelial cells, circ003429 inhibited the synthesis of triglycerides (TAG) and decreased the content of unsaturated fatty acids (C16:1, C18:1, and C18:2), indicating that this circRNA plays an important role in regulating lipid synthesis. A binding site for miR-199a-3p in the circ003429 sequence was detected, and a dual-luciferase reporter system revealed that circ003429 targets miR-199a-3p. Overexpression of circ003429 (pcDNA-circ003429) downregulated the abundance of miR-199a-3p. In contrast, overexpression of miR-199a-3p increased TAG content and decreased mRNA abundance of Yes-associated protein 1 (YAP1) (a target gene of miR-199a-3p), and TAG content was decreased and mRNA abundance was increased in response to overexpression of circ003429. These results indicate that circ003429 alleviates the inhibitory effect of miR-199a-3p on the mRNA abundance of YAP1 by binding miR-199a-3p, resulting in subsequent regulation of the synthesis of TAG and unsaturated fatty acids.

Transcriptome profiling of the nonlactating mammary glands of dairy goats reveals the molecular genetic mechanism of mammary cell remodeling

The mammary gland redevelops to the prepregnancy state during involution, which shows the mammary cells have the characteristics of remodeling. The rapidity and degree of mammary gland involution vary across species (e.g., between model organism mice and dairy livestock). However, the molecular genetic mechanism of involution and remodeling of goat mammary gland has not yet been clarified. This work investigated the structural changes and transcriptome characteristics of the mammary gland tissue of nonlactating dairy goats during the late lactation (LL), the dry period (DP), and late gestation (LG). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed significant changes in the structure of the nonlactating goat mammary gland, and obvious cell apoptosis occurred at LL and DP. Sequencing identified 1,381 genes that are differentially expressed in mammary gland tissue at the 3 developmental stages. Genes related to cell growth, apoptosis, immunity, nutrient transport, synthesis, and metabolism exhibited adaptive transcriptional changes to meet the needs of a new set of mammary gland lactation functions. The significant enrichment of Gene Ontology terms such as humoral immune response, complement activation, and neutrophil-mediated immunity indicates that the innate immune system plays an important role in maintaining the health of degenerative mammary glands and eliminating apoptotic cells. The peroxisome proliferator-activated receptor signaling pathway plays an important regulatory role in lipid metabolism, especially the adaptive changes in expression of genes encoded lipid transport and enzymes, which promote the formation of milk fat during the lactation. The mammary gland development gene module revealed that pregnancy hormone receptors, cell growth factors and their receptors, and genes encoding insulin-like growth factor binding proteins regulate the physiological process of mammary gland involution through adaptive transcriptional changes. Interestingly, ERBB4 was identified as the hub gene of the network that regulates mammary gland growth and development. Overexpression of ERBB4 in mammary epithelial cells cultured in vitro can reduce cell cycle arrest in G1/S phase and apoptosis by regulating the PI3K/Akt signaling pathway and promote the proliferation of mammary epithelial cells. The gene ERBB4 also affects the expression of genes that initiate mammary gland involution and promote mammary gland remodeling. These findings contribute to an in-depth understanding of the molecular mechanisms involved in mammary gland involution and remodeling.

Comprehensive Transcriptome Profiling of Dairy Goat Mammary Gland Identifies Genes and Networks Crucial for Lactation and Fatty Acid Metabolism.

Milk fatty acids secreted by the mammary gland are one of the most important determinants of the nutritional value of goat milk. Unlike cow milk, limited data are available on the transcriptome-wide changes across stages of lactation in dairy goats. In this study, goat mammary gland tissue collected at peak lactation, cessation of milking, and involution were analyzed with digital gene expression (DGE) sequencing to generate longitudinal transcript profiles. A total of 51,299 unigenes were identified and further annotated to 12,763 genes, of which 9,131 were differentially expressed across various stages of lactation. Most abundant genes and differentially expressed genes (DEGs) were functionally classified through clusters of euKaryotic Orthologous Groups (KOG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A total of 16 possible expression patterns were uncovered, and 13 genes were deemed novel candidates for regulation of lactation in the goat: POLG, SPTA1, KLC, GIT2, COPS3, PDP, CD31, USP16/29/37, TLL1, NCAPH, ABI2, DNAJC4, and MAPK8IP3. In addition, PLA2, CPT1, PLD, GGA, SRPRB, and AP4S1 are proposed as novel and promising candidates regulating mammary fatty acid metabolism. “Butirosin and neomycin biosynthesis” and “Glyoxylate and dicarboxylate metabolism” were the most impacted pathways, and revealed novel metabolic alterations in lipid metabolism as lactation progressed. Overall, the present study provides new insights into the synthesis and metabolism of fatty acids and lipid species in the mammary gland along with more detailed information on molecular regulation of lactogenesis. The major findings will benefit efforts to further improve milk quality in dairy goats.

Sodium butyrate attenuated iE-DAP induced inflammatory response in the mammary glands of dairy goats fed high-concentrate diet.

Long-term high-concentrate (HC) diet feeding increased bacterial endotoxins, which translocated into the mammary glands of dairy goats and induced inflammatory response. γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP), bacterial peptidoglycan component, triggered inflammatory response through activating nucleotide oligomerization domain protein 1 (NOD1) signaling pathway. While dietary supplemented with sodium butyrate (SB) relieved inflammatory response and improved animal health and production. To investigate the effects and the mechanisms of action of SB on the inflammatory response in the mammary glands of dairy goats fed HC diet, 12 Saanen dairy goats were randomly assigned into HC group and SB regulated (BHC) group. The results showed that SB supplementation attenuated ruminal pH decrease caused by HC diet in dairy goats resulting in a decrease of proinflammatory cytokines and iE-DAP plasma concentration and the mRNA expression of NOD1 and other inflammation-related genes. The protein levels of NOD1, NF-κB p65 and NF-κB pp65 were decreased by the SB supplementation. The expression of histone deacetylase 3 (HDAC3) was also inhibited by the SB supplementation. Meanwhile, the chromatin compaction ratios and DNA methylation levels of NOD1 and receptor-interacting protein 2 (RIP2) of BHC group were upregulated. Collectively, the SB supplementation mitigated the inflammatory response in the mammary glands of dairy goats during HC-induced subacute ruminal acidosis (SARA) by inhibiting the activation of the NOD1/NF-κB signaling pathway through the decrease of the iE-DAP concentration in the rumen fluid and plasma and HDAC3 expression. DNA methylation and chromatin remodeling also contributed to the anti-inflammatory effect of SB. © 2020 Society of Chemical Industry.

Characterization of microRNA profiles in the mammary gland tissue of dairy goats at the late lactation, dry period and late gestation stages.

MicroRNAs (miRNAs) play an important role in regulating mammary gland development and lactation. We previously analyzed miRNA expression profiles in Laoshan dairy goat mammary glands at the early (20 d postpartum), peak (90 d postpartum) and late lactation (210 d postpartum) stages. To further enrich and clarify the miRNA expression profiles during the lactation physiological cycle, we sequenced miRNAs in the mammary gland tissues of Laoshan dairy goats at three newly selected stages: the late lactation (240 d postpartum), dry period (300 d postpartum) and late gestation (140 d after mating) stages. We obtained 4038 miRNAs and 385 important miRNA families, including mir-10, let-7 and mir-9. We also identified 754 differentially expressed miRNAs in the mammary gland tissue at the 3 different stages and 6 groups of miRNA clusters that had unique expression patterns. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that GO terms such as mammary gland development (GO:0030879) and mammary gland morphogenesis (GO:0060443) and important signaling pathways, including the insulin signaling pathway (chx04910), hippo signaling pathway (chx04390) and estrogen signaling pathway (chx04915), were enriched. We screened miRNAs and potential target genes that may be involved in the regulation of lactation, mammary gland growth and differentiation, cell apoptosis, and substance transport and synthesis and detected the expression patterns of important genes at the three stages. These miRNAs and critical target genes may be important factors for mammary gland development and lactation regulation and potentially valuable molecular markers, which may provide a theoretical reference for further investigation of mammary gland physiology.

Milk yield, milk composition, and milk metabolomics of dairy goats intramammary-challenged with lipopolysaccharide under heat stress conditions

Heat stress and mastitis are major economic issues in dairy production. The objective was to test whether goat’s mammary gland immune response to E. coli lipopolysaccharide (LPS) could be conditioned by heat stress (HS). Changes in milk composition and milk metabolomics were evaluated after the administration of LPS in mammary glands of dairy goats under thermal-neutral (TN; n = 4; 15 to 20 °C; 40 to 45% humidity) or HS (n = 4; 35 °C day, 28 °C night; 40% humidity) conditions. Milk metabolomics were evaluated using 1H nuclear magnetic resonance spectroscopy, and multivariate analyses were carried out. Heat stress reduced feed intake and milk yield by 28 and 21%, respectively. Mammary treatment with LPS resulted in febrile response that was detectable in TN goats, but was masked by elevated body temperature due to heat load in HS goats. Additionally, LPS increased milk protein and decreased milk lactose, with more marked changes in HS goats. The recruitment of somatic cells in milk after LPS treatment was delayed by HS. Milk metabolomics revealed that citrate increased by HS, whereas choline, phosphocholine, N-acetylcarbohydrates, lactate, and ß-hydroxybutyrate could be considered as putative markers of inflammation with different pattern according to the ambient temperature (i.e. TN vs. HS). In conclusion, changes in milk somatic cells and milk metabolomics indicated that heat stress affected the mammary immune response to simulated infection, which could make dairy animals more vulnerable to mastitis.

Transcriptome Analysis of Dairy Goat Mammary Gland Tissues from Different Lactation Stages.

The mammary gland is a unique organ involved in lactation in dairy livestock, and its development and lactation ability are affected by both genetic and environmental factors. To explore the molecular regulatory mechanisms of these factors, this study used high-throughput sequencing technology and bioinformatics methods to systematically analyze the transcriptome of Laoshan dairy goat mammary gland tissues from different lactation stages. From three libraries, 36,336,892, 36,469,596, and 35,759,380, pure sequences were obtained, with 25,292, 23,665 and 27,220 expressed genes, respectively, resulting in a total of 14,892 nonredundant differentially expressed genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that these genes were annotated to biological regulation, cellular processes, metabolic processes, cells, organelles, binding, catalytic activity and transcriptional activity, and so on. The genes were mainly involved in cellular processes, environmental information processes, genetic information processes, human diseases, metabolism, and organ systems. Finally, 20 genes related to mammary gland development and lactation were screened to construct a gene regulatory network. These findings support the involvement of the constructed network in the regulation of mammary gland development and lactation, and they not only lay the foundation to further investigate and screen the main genes or molecular genetic markers controlling the development of the mammary gland and lactation ability of dairy goats but also increase the in-depth understanding of the lactation physiology of dairy goats.

The inhibitory effect of melatonin on mammary function of lactating dairy goats†.

The direct role of melatonin in mammary glands of dairy goats has remained obscure. This study aimed to evaluate the expression of melatonin membrane receptors (MT1 and MT2) in the pituitary and mammary glands of dairy goats during lactation, and to investigate the role of melatonin in mammary function. Both MT1 and MT2 were consistently expressed in the pituitary and mammary eight glands throughout the lactation period, and their levels were lower in 9 March (group I), June (group III), and September (group V) than in May (group II) and August (group IV). The expression patterns of pituitary and mammary MT1 and MT2 were consistent with those of blood melatonin during lactation. Furthermore, the mammary prolactin (PRL), and pituitary growth hormone (GH) and PRL mRNA expression showed an inverse trend in relation to blood melatonin levels. In mammary tissues, MT1 and MT2 immunoreactivity was predominantly located in the mammary epithelial cells (MECs). In addition, a dose- and time-dependent inhibition on cell viability was observed in cultured MECs. At the dose of 10 and 100 pg/ml, melatonin decreased mammary β-casein and PRL expression. Furthermore, the inhibitory effects of melatonin were blocked by luzindole, a nonselective MT1 and MT2 receptor antagonist. In addition, melatonin promoted MT1 and MT2 expression in cultured MECs. In conclusion, the presence of MT1 and MT2 in the pituitary and mammary glands and the inhibitory effects of melatonin on cell viability, β-casein, and PRL expression in MECs suggest the potential regulation by melatonin in goat mammary function.

Ghrelin is expressed in the pregnant mammary glands of dairy goats and promotes the cell proliferation of mammary epithelial cells

Little is known about ghrelin’s effects on cell proliferation in pregnant mammary epithelial cells (MECs) even though it is known to be a mitogen for a variety of other cell types. The objectives of this study were to evaluate the expression and localization of ghrelin and its functional receptor, GHSR-1a, in the mammary glands of dairy goats during pregnancy and to investigate the direct role of ghrelin in cell proliferation of primary cultured MECs. Compared to the early stage (days 30) of pregnancy, the abundance of transcripts and protein of ghrelin and GHSR-1a were significantly greater in mid- and late-phases (between days 90 and days 120) of pregnancy (p < .05). Immunohistochemistry analysis showed that ghrelin and GHSR-1a were predominantly localized in the alveolar and ductal mammary epithelial cells at various stages of pregnancy. In our in vitro experiments, ghrelin induced a dose- and time-dependent promotory effect on cell proliferation of MECs. At the dose of 103 pg/mL treatment 24 h, ghrelin augmented the expression of proliferation-related peptides (PCNA and cyclin B1). Furthermore, ghrelin promoted the expression of prolactin (PRL) and GHSR-1a in cultured MECs. Additionally, the stimulatory effects of ghrelin were blocked by d-Lys3-GHRP6, a selective antagonist of GHSR-1a. As the temporal changes in ghrelin and GHSR-1a expression in pregnant goat mammary glands coincided with the mammary growth and development during the pregnancy, activation of GHSR-1a signal transduction pathways by ghrelin may play a direct role in the regulation of mammary growth in dairy goats.

Reduced representation bisulfite sequencing (RRBS) of dairy goat mammary glands reveals DNA methylation profiles of integrated genome-wide and critical milk-related genes.

DNA methylation (DNAm), a major element of epigenetics, plays critical roles in individual development. Reduced representation bisulfite sequencing (RRBS) is an effective and economical method for analyzing the DNA methylation of a single base. The aims of this study were to determine the DNAm profiles of the methylation contexts (CGs and non-CGs) of lactation and dry periods of goat mammary glands using the RRBS, and to identify potential milk-related genes. The proportion of CG was the highest among all the sequence contexts. The highest CG levels (72.44% to 75.24%) occurred in the 3′ UTR region, followed by the gene body region (61.14% to 65.45%). The non-CG levels were low compared to the CG levels. Bioinformatic analysis demonstrated that the CGs were mainly enriched at high methylation levels (>90%), while non-CGs were enriched at low methylation levels. Methylation levels of 95 and 54 genes in the lactation period were up- or downregulated, respectively, relative to the dry period, such as PPARα, RXRα and NPY genes. The bisulfite sequencing PCR results showed that the methylation level of goat PPARα gene during the lactation period was significant lower than in the dry period, while the methylation level of the RXRα gene was lower in the dry period than in the lactation period. Meanwhile, the methylation levels of human PPARα and NPY genes were significantly higher in MCF-7 than in MCF-10A cells. These findings provide essential information for DNA methylation profiles of goat mammary gland and detect some potential milk-related genes in dairy goats.

Scd1 Contributes to Lipid Droplets Formation in GMEC via Transcriptional Regulation of Tip47 and Adrp

Sterol‐CoA desaturase 1 (Scd1) plays a pivotal role in the synthesis and metabolism of fatty acids (FA). However, whether Scd1 contributes to lipid accumulation in dairy goat mammary epithelial cells (GMEC), and how it affects FA synthesis and metabolism remains unexplored. The aim of this study was to detect the effect of Scd1 on lipid droplets (LD) formation and uncover its regulatory mechanism in GMEC. In this study, a novel fluorescence reporter system was developed to confirm the expression of SCD1 protein in GMEC plasma. In addition, a mammalian expression vector (pCAG‐SCD1) and siRNA targeting Scd1 were constructed and confirmed to be efficient by qRT‐PCR. Notably, the amount and the size of LD varied with the alteration of Scd1. Furthermore, Scd1 induced the mRNA expression of genes involved in de novo FA synthesis, triacylglycerol (TAG) biosynthesis, lipid droplets formation, and milk fat lipolysis. In particular, changes in Scd1 led to a consistent change in LD formation‐associated genes (tail‐interacting protein of 47 kDa (Tip47) and adipose differentiation‐related protein (Adrp)). Overall, the results suggest that Scd1 affects lipid accumulation by altering the FA metabolic profile in GMEC.Practical Applications: As is known, FA metabolism closely associates with human health. The results of our research firstly exposed the metabolic profile associated with SCD1 in GMEC. It will be helpful for our understanding about the regulatory role of Scd1 in FA metabolism in mammary gland of dairy goats. It also provides a reference for the studies on Scd1‐related FA metabolic pathways, which is of great significance for human health. Furthermore, this study lays the foundation for engineering the FA content via SCD1 in transgenic goat milk products.Sterol‐CoA desaturase 1 (Scd1) differentially activates signaling pathways associated with de novo fatty acid (FA) synthesis (Acaca and Fasn), TAG biosynthesis (Gpam, Agpat6, and Dgat1), lipid droplets (LD) formation (Tip47 and Adrp), and milk fat lipolysis (Atgl and Hsl) in GMEC; in particular, it contributes to the LD formation through transcriptional regulation of Tip47 and Adrp.

Screening of miRNA profiles and construction of regulation networks in early and late lactation of dairy goat mammary glands

In recent years, studies related to the expression profiles of miRNAs in the dairy goat mammary gland were performed, but regulatory mechanisms in the physiological environment and the dynamic homeostasis of mammary gland development and lactation are not clear. In the present study, sequencing data analysis of early and late lactation uncovered a total of 1,487 unique miRNAs, including 45 novel miRNA candidates and 1,442 known and conserved miRNAs, of which 758 miRNAs were co-expressed and 378 differentially expressed with P < 0.05. Moreover, 76 non-redundant target genes were annotated in 347 GO consortiums, with 3,143 candidate target genes grouped into 33 pathways. Additionally, 18 predicted target genes of 214 miRNAs were directly annotated in mammary gland development and used to construct regulatory networks based on GO annotation and the KEGG pathway. The expression levels of seven known miRNAs and three novel miRNAs were examined using quantitative real-time PCR. The results showed that miRNAs might play important roles in early and late lactation during dairy goat mammary gland development, which will be helpful to obtain a better understanding of the genetic control of mammary gland lactation and development.

MiR-2478 inhibits TGF\u03b21 expression by targeting the transcriptional activation region downstream of the TGF\u03b21 promoter in dairy goats

In a previous study, miR-2478 was demonstrated to be up-regulated in dairy goat mammary glands during peak lactation compared with the dry period. However, the detailed mechanisms by which miR-2478 regulates physiological lactation and mammary gland development in dairy goats remain unclear. In this study, we used bioinformatics analysis and homologous cloning to predict the target genes of miR-2478 and selected INSR, FBXO11, TGFβ1 and ING4 as candidate target genes of miR-2478. Subsequently, by targeting the 5′UTR of the TGFβ1 gene, we verified that miR-2478 significantly inhibited TGFβ1 transcription and the Pearson’s correlation coefficient between miR-2478 expression and TGFβ1 expression was −0.98. Furthermore, we identified the potential promoter and transcription factor binding regions of TGFβ1 and analyzed the potential mechanisms of interaction between miR-2478 and TGFβ1. Dual-luciferase reporter assays revealed that two regions, spanning from −904 to −690 bp and from −79 to +197 bp, were transcription factor binding regions of TGFβ1. Interesting, the miR-2478 binding sequence was determined to span from +123 to +142 bp in the TGFβ1 gene promoter. Thus, our results have demonstrated that miR-2478 binds to the core region of the TGFβ1 promoter and that it affects goat mammary gland development by inhibiting TGFβ1 transcription.

Computational identification and characterization of novel microRNA in the mammary gland of dairy goat (Capra hircus).

Many studies have indicated that microRNAs (miRNAs) influence the development of the mammary gland by posttranscriptionally affecting their target genes. The objective of this research was to identify novel miRNAs in the mammary gland of dairy goats with a bioinformatics approach that was based on expressed sequence tag (EST) and genome survey sequence (GSS) analyses. We applied all known major mammals, miRNAs to search against the goat EST and GSS databases for the first time to identify new miRNAs. We, then, validated these newly predicted miRNAs with stem-loop reverse transcription followed by a SYBR Green polymerase chain reaction assay. Finally, 29 mature miRNAs were identified and verified, and of these, 14 were grouped into 13 families based on seed sequence identity and 85 potential target genes of newly verified miRNAs were subsequently predicted, most of which seemed to encode the proteins participating in regulation of metabolism, signal transduction, growth and development. The predicting accuracy of the new miRNAs was 70.37%, which confirmed that the methods used in this study were efficient and reliable. Detailed analyses of the sequence characteristics of the novel miRNAs of the goat mammary gland were performed. In conclusion, these results provide a reference for further identification of miRNAs in animals without a complete genome and thus improve the understanding of miRNAs in the caprine mammary gland.