The mammary gland undergoes intensive remodeling during the lactation cycle, and the involution process of mammary gland contains extensive epithelial cells involved in the process of autophagy. Our studies of mice mammary glands suggest that miR-30a-3p expression was low during involution compared with its high expression in the mammary glands of lactating mice. Then, we revealed that miR-30a-3p negatively regulated autophagy by autophagy related 12 (Atg12) in mouse mammary gland epithelial cells (MMECs). Restoring ATG12, knocking down autophagy related 5 (Atg5), starvation, and Rapamycin were used to further confirm this conclusion. Overexpression of miR-30a-3p inhibited autophagy and altered mammary structure in the involution of the mammary glands of mice, which was indicative of alteration in mammary remodeling. Taken together, these results elucidated the molecular mechanisms of miR-30a-3p as a key induction mediator of autophagy by targeting Atg12 within the transition period between lactation and involution in mammary glands.

The emergence of the jacquard loom in the early 19th century was one of the most important mechanical inventions among the technological innovations of the French silk weaving industry. Since then, France continuously optimized and improve jacquard loom, resulting in many technological changes. Based on the technical principle of different types of jacquard looms in France in the 19th century, and by referring to literature and conducting field visits to France and other places, our study systematically analyzed and summarized the technological changes of jacquard in French silk weaving in the 19th century. These changes mainly included the modification of crochet shape, direction and thickness of placement, the change of the shape and material of pattern card, the size and arrangement of its holes, and the addition of harness cord and shaft monture, so as to promote the continuous, rapid and steady progress of the whole silk weaving industry.

The prolactin/STAT5 and AKT1/mTOR pathways play a key role in milk protein transcription and translation, respectively. However, the correlation between them in bovine mammary epithelial cells remains unclear. Here, mRNA and protein expression levels of AKT1, STAT5, and mTOR and the phosphorylation of these proteins were determined. Cell proliferation and viability were examined using the CASY-TT assay. Purified bovine mammary epithelial cells were cultured in differentiation media for different periods. The basic differentiation medium contained a lactogenic hormone cocktail of insulin (5 μg/mL), hydrocortisone (1 μg/mL), and prolactin (5 μg/mL). The cells cultured in this medium grew slowly and expressed higher levels of p-STAT5, p-AKT1, and p-mTOR (activated form) than those of control cells. Although the phosphorylation ratio was not increased, transcription and translation of these proteins were upregulated by the addition of insulin-like growth factor-1 or growth hormone, which further increased β-casein mRNA expression. Furthermore, the three proteins were upregulated or downregulated synchronously, even after RNA interference silencing of either Stat5 or Akt1. These findings indicate that a few hormones and other factors play lactogenic and galactogenic roles by promoting two key lactogenic signaling associated with milk protein expression. We provide evidence of prolactin/STAT5 and AKT1/mTOR synchronization, establishing a direct correlation between transcription regulation and translation regulation of milk protein in bovine mammary epithelial cells.

Cover: The cover image is based on the Original Research Article Let-7g-5p regulates mouse mammary cells differentiation and function by targeting PRKCA by Lei Tian et al., DOI: 10.1002/jcp.27676.

本实验以健康的泌乳期中国荷斯坦奶牛的乳腺上皮细胞为实验模型,通过使用DNA去甲基化药物5-氮杂-脱氧胞苷(5-Aza)对奶牛的乳腺上皮细胞进行处理,采用CASY细胞分析仪测定细胞活力,最后采用western blotting检测mTOR蛋白和β-酪蛋白的表达量.实验结果显示,0.1 μmol/L 5-Aza处理后的奶牛乳腺上皮细胞中的mTOR蛋白及β-酪蛋白的表达量显著增加,说明一定浓度的5-Aza可以提高乳腺泌乳信号通路中关键蛋白mTOR的表达水平,最后提高β-酪蛋白的表达水平,表明DNA甲基化也参与了奶牛泌乳调控.本实验试图从DNA甲基化方面揭示影响奶牛乳品质差异的原因,进一步完善奶牛泌乳的分子机制.

miRNAs play an important role in the processes of cell differentiation, biological development, and physiology. Here we investigated the molecular mechanisms regulating milk secretion and quality in dairy cows via transcriptome analyses of mammary gland tissues from dairy cows during the high-protein/high-fat, low-protein/low-fat or dry periods. To characterize the important roles of miRNAs and mRNAs in milk quality and to elucidate their regulatory networks in relation to milk secretion and quality, an integrated analysis was performed. A total of 25 core miRNAs were found to be differentially expressed (DE) during lactation compared to non-lactation, and these miRNAs were involved in epithelial cell terminal differentiation and mammary gland development. In addition, comprehensive analysis of mRNA and miRNA expression between high-protein/high-fat group and low-protein/low-fat groups indicated that, 38 miRNAs and 944 mRNAs were differentially expressed between them. Furthermore, 38 DE miRNAs putatively negatively regulated 253 DE mRNAs. The putative genes (253 DE mRNAs) were enriched in lipid biosynthetic process and amino acid transmembrane transporter activity. Moreover, putative DE genes were significantly enriched in fatty acid (FA) metabolism, biosynthesis of amino acids, synthesis and degradation of ketone bodies and biosynthesis of unsaturated FAs. Our results suggest that DE miRNAs might play roles as regulators of milk quality and milk secretion during mammary gland differentiation.

MicroRNAs (miRNAs) are closely related with the posttranscriptional regulation of gene expression. Our past study showed that let-7g-5p decreased during pregnancy and lactation in mouse mammary gland, what suggested the prospective regulating role of let-7g-5p in mammary epithelial cells. In this study, to unravel the role of let-7g-5p, we found PRKCA (PKC-alpha, PKC-α) might serve as potential targets of let-7g-5p by bioinformatics. Then let-7g-5p was knocked down by an antisense oligonucleotide in mouse primary mammary epithelial cells. As predicted, PRKCA gene expression and mammary epithelial cells growth were increased by anti-let-7g-5p regulation in vitro. By using reporter constructs, it showed that the let-7g-5p could direct binding with 3'-the untranslated regions (3'-UTR) of PRKCA mRNA. Furthermore, ectopic overexpression of let-7g-5p in vivo reduced PRKCA and β-casein protein expression as well as inhibited mammary gland growth. These results suggested that let-7g-5p could inhibit the mammary epithelial cells differentiation and β-casein protein synthesis and expression through suppression of PRKCA on the cycle of mammary cell differentiation and development and that let-7g-5p may be a novel important regulated target in mammary cells function.

MicroRNAs have important roles in many biological processes. However, the role of miR-139 in healthy mammary gland remains unclear. The objective of this study was to investigate the effects of miR-139 on lactation in dairy cows.

Adequate lipid synthesis by the mammary gland during lactation is essential for the survival of mammalian offspring. Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) is a lipid droplet-associated protein and functions to promote lipid accumulation and inhibit lipolysis in mice and human adipocytes. However, the function of CIDEC in regulation of milk lipid synthesis in dairy cow mammary gland remains largely unknown. In this study, 6 multiparous Holstein cows (parity = 3) in early lactation were allocated to high-fat milk (milk yield 33.9 ± 2.1 kg/d, milk fat >3.5%, n = 3) and low-fat milk (milk yield 33.7 ± 0.5 kg/d, milk fat <3.5%, n = 3) groups according to their milk fat content. Lactating cows were slaughtered at 90 d in milk and mammary tissues were collected to detect CIDEC localization. Immunofluorescence staining of sections of lactating mammary glands with high- and low-fat milk showed that CIDEC was expressed in the cytoplasm of epithelial cells and localized to lipid droplets. Lipid droplets and CIDEC protein were also detected in isolated lactating mammary epithelial cells of dairy cows. Immunostaining of CIDEC in isolated mammary epithelial cells also confirmed its presence in the nucleus. The knockdown of CIDEC in cultured bovine mammary epithelial cells decreased milk lipid content and reduced expression of genes associated with mammary de novo fatty acid synthesis, short- and long-chain intracellular fatty acid activation, triacylglycerol synthesis, and transcription regulation. These genes included those for acetyl-CoA carboxylase (ACC, -60%), fatty acid synthase (FASN, -65%), acyl-CoA synthetase short-chain family member 2 (ACSS2, -50%), acyl-CoA synthetase long-chain family member 1 (ACSL1, -30%), diacylglycerol acyltransferase 1 (DGAT1, -60%), sterol regulatory element-binding protein 1 (SREBP1, -45%), and SREBP cleavage activating protein (SCAP, -66%). Conversely, in cells overexpressing CIDEC, triacylglycerol content was increased, and transcription of those genes involved in milk lipid synthesis was coordinately upregulated. These results suggest that CIDEC plays an important role in regulating milk lipid synthesis in dairy cow mammary gland via a mechanism involving gene expression, which provides further insight into the mechanisms regulating mammary lipogenesis in ruminants.

旨在构建牛生长激素受体(Growth hormone receptor,GHR)基因3'非翻译区(Untranslated region,UTR)双荧光素酶载体,并确定miR-139与牛GHR基因的靶向关系.本研究采用生物信息学方法预测miR-139与牛GHR基因3'UTR的结合位点;人工合成牛GHR基因3'UTR及突变型片段并克隆至双荧光素酶载体psi-CHECK-2上,构建野生型(psiCHECK2-GHR-W 3'UTR)及突变型(psiCHECK2-GHR-M 3'UTR)双荧光素酶报告质粒.将培养的Hela细胞分为4组,分别共转染miR-139mimic或阴性对照和psiCHECK2-GHR-W 3'UTR重组质粒或psiCHECK2-GHR-M 3'UTR重组质粒,然后用双荧光素酶检测试剂盒检测4组细胞中荧光素酶活性.之后培养奶牛乳腺上皮细胞,分别设对照组、阴性对照组、miR-139 mimic及miR-139 inhibitor组,对阴性对照组、miR-139 mimic组及miR-139 inhibitor组分别转染阴性对照、miR-139 mimic或miR-139 inhibitor,利用qPCR(Real-time quantitative PCR)进一步检测各组miR-139及GHR基因的mRNA表达.结果,通过双酶切试验证实成功构建了野生型及突变型重组双荧光素酶报告质粒psiCHECK2-GHR-W 3'UTR和psiCHECK2-GHR-M 3'UTR;当野生型重组质粒psiCHECK2-GHR-W 3'UTR和miR-139 mimic共转染Hela细胞后,双荧光素酶报告质粒表达的荧光素酶活性显著低于其他处理组(P＜0.05).qPCR进一步证实miR-139能显著下调奶牛乳腺上皮细胞中GHR基因mRNA的表达(P＜0.05).本研究成功构建了牛野生型及突变型GHR基因3'UTR双荧光素酶报告质粒;双荧光素酶试验及qPCR证实miR-139与牛GHR基因3'UTR存在靶向关系.

Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells.

A dairy cow mammary epithelial cell line was established through culture method of tissue block and detection of cell biological characters. Effects of methionine and lysine on viabilities and β-casein of DCMECs were evaluated by CASY and HPLC, and determined the optimal dose of methionine and lysine was 0.6mmol/L and 1.2mmol/L, the best time was 24h. A nuclear phosphoproteomics of DCMECs was successfully established, five proteins for which expression was significantly increased in methionine-treated DCMECs were selected, The 5 up-regulated expressed phosphoproteins included Staphylococcal nuclease domain-containing protein 1(SND1), Septin-6, Glycyl-tRNA synthetase (GARS), Twinfilin-1 and eukaryotic elongation factor1-beta (eEF1B); Six proteins for which expression was significantly increased in lysine-treated DCMECs were selected, The 6 up-regulated expressed phosphoproteins included SKIV2L2、sec-related protein D、T-complex protein 1 subunit delta、protein disulfide-isomerase A3、coronin actin binding protein 1C and mitogen-activated protein kinase 1(MAPK1); the expression levels of these were verified by quantitative real-time PCR (qRT-PCR) and Western blotting analysis, which were consisted with the results of 2-DE. In this research, eukaryotic expression vector pGCMV-IRES-EGFP-MAPK1 and pGCMV-IRES-EGFP-eEF1B were constructed by stably transfected into DCMECs after geneticin (G418) selection. The gene functions of MAPK1 and eEF1B were identified by the RNA interference and gene overexpression, methionine and lysine as energy substrates can promote expression of Stat5a gene and increase lactation of DCMECs by MAPK1 and eEF1B. MAPK1 and eEF1B also regulates the expression of key mediators of the PRLR/JAK2/STAT5 and mTOR signaling pathway. We used Co-immunoprecipitation and GST-pull down assay here to identify the interacting proteins of MAPK1, including ARPC4、β-enolase、Annexin A2、Small G protein signaling modulator 1and Polymerase I and transcript release factor, the interacting proteins of eEF1B are 14-3-3theta、Heat shock protein、eEF1A1、Small nuclear ribonucleoprotein polypeptide A and 40S ribosomal protein S4. The results of this research have enriched the study content of nutritional genomics of dairy cow, and have important basic theory and research methods.

本实验将中国荷斯坦牛泌乳期高乳品质奶牛(H)和泌乳期低乳品质奶牛(L)乳腺组织作为实验对象,利用高通量测序技术进行了miRNA测序,与miRNA数据库比对,获得已知miRNA,整合miREvo和mirDeep2这两个miRNA预测软件,进行新miRNA分析,通过差异表达分析筛选组间差异miRNAs,获得56个差异表达miRNA (P＜0.05,FDRq＜0.05)并对差异表达miRNA进行靶基因预测;利用DAVID对靶基因进行GO(Gene Ontology)和信号通路富集分析.经过对靶基因筛选,发现了4个已报道与乳蛋白、乳脂紧密相关的功能基因:CSN3、SCD、LALBA和DGAT2.靶基因聚集的生物学功能多数参与了蛋白质和脂肪代谢,乳腺发育和分化,以及免疫功能.靶基因主要富集在MAPK信号通路、甘油磷酸脂质代谢、缺氧诱导因子1和磷脂酰肌醇3激酶-蛋白激酶B信号转导通路.结果显示,靶基因主要富集在糖类代谢、脂肪代谢、蛋白质代谢、细胞凋亡以及免疫相关通路.

Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows.

The 14-3-3γ protein participates in many biological processes; however, its regulatory mechanism in milk protein synthesis is not well studied. We hypothesized that 14-3-3γ might affect eIF5 (an initiation factor) to regulate β-casein synthesis in dairy cows. In this study, a possible interaction between 14-3-3γ and eIF5 was investigated using bovine mammary epithelial cells (BMECs). The expression levels of 14-3-3γ and eIF5 in the mammary gland tissues from cows producing higher quality milk were higher than those from cows producing low-quality milk. Moreover, the expression of 14-3-3γ, eIF5, and β-casein were increased at both mRNA and protein levels in BMECs cultured in vitro with methionine (Met) supplementation. Coimmunoprecipitation, colocalization, and FRET analysis further showed the evidences that 14-3-3γ physically bound to eIF5 in BMECs. Gene function studies revealed that 14-3-3γ positively regulated eIF5 through alteration of eIF2α/p-eIF2α ratio. Collectively, our data suggest that 14-3-3γ regulates β-casein translation in BMECs through interaction with eIF5.

Estrogen and prolactin can regulate mammary gland development and epithelial cell growth as well as lactation. Meanwhile, the Wnt signaling pathway and subsequent upregulation of b-catenin driven by downstream target of cyclinD1 also play a role in development of mammary gland. This study aimed to assess the possible involvement of estrogen and prolactin in regulation of cell growth in mammary gland. Bovine mammary gland epithelial cells (MECs) were treated with estrogen and prolactin (5 µg mL-1) respectively for 48 h and then measured for cell viability. mRNA and protein expression level of genes (E-cadherin, CyclinD1 and b-catenin) related with Wnt pathway were measured by qRT-PCR and Western blot respectively while sub-cellular localizations of the proteins in MECs were further monitored by immunofluorescence. Expression of E-cadherin and CyclinD1 were most highly expressed at 36 h (P

In the aim of detectting the role of Pten gene in the mammary gland of dairy cow, dairy cows mammary epithelial cells (DCMECs) in mid-lactation period were used as models to investigate the relationship of Pten expression and mammary glands development and lactation, which provides basic data for the study of ruminant mammary gland development and lactation mechanisms, and the theoretical support for milk production and milk quality of the artificial regulation at the same time. In this research, Holstein dairy cows were used as experimental animals, applying to qRT-PCR, Western blotting, and immunofluorescence triple staining technology, Pten mRNA&nbsp;and protein expression at different development stages and various milk qualities of dairy cows mammary gland tissue were detected. Furthermore, DCMECs as research objects in vitro were used to study the function of Pten gene. Recombinant plasmid pGCMV-Pten-IRES-EGFP was constructed and transient transfected into cells to prosue the Pten gene overexpression experiment. Meanwhile, RNAi method was used to transfect Pten siRNA in the Pten gene inhibition experiment. We determined concentrations of β-casein, triglyceride, and lactose following Pten gene overexpression and inhibition by specific kits. To determine whether Pten gene affected DCMEC viability and proliferation, cells were analyzed by CASY-TT and flow cytometry. Genes involved in lactation-related signaling pathways were detected by qRT-PCR and Western blotting. After prolactin and glucose were added to the cell cultures, concentrations of β-casein, triglyceride, and lactose were detected, and Pten gene expression was also assessed. Thus investigating the role of Pten gene in the process of glucose transform into lactose induced by prolactin. In summary, we showed that Pten gene is specifically involved in lactation of dairy cow mammary epithelial cells, and down-regulates DCMEC viability, proliferation ability, and the cell cycle along with β-casein, triglyceride, and lactose secretion. Pten gene targets and regulates the PI3K/AKT pathway, which in turn regulates other lactation-related signaling genes. Moreover, the expression of Pten gene can be down-regulated by prolactin, but the introduction of glucose to culture medium revealed no significant difference in Pten gene expression level in DCMECs.

Peroxisome proliferator-activated receptor gamma (PPARγ) participates in lipogenesis in rats, goats, and humans. However, the exact mechanism of PPARγ regulation on milk fat synthesis in dairy cow mammary epithelial cells (DCMECs) remains largely unexplored. The aim of this study was to investigate the role of PPARγ regarding milk fat synthesis in DCMECs and to ascertain whether milk fat precursor acetic acid and palmitic acid could interact with PPARγ signaling to regulate milk fat synthesis. For this study, we examined the effects of PPARγ overexpression and gene silencing on cell growth, triacylglycerol synthesis, and the messenger RNA (mRNA) and protein expression levels of genes involved in milk fat synthesis in DCMECs. In addition, we investigated the influences of acetic acid and palmitic acid on the mRNA and protein levels of milk lipogenic genes and triacylglycerol synthesis in DCMECs transfected with PPARγ small interfering RNA (siRNA) and PPARγ expression vector. The results showed that when PPARγ was silenced, cell viability, proliferation, and triacylglycerol secretion were obviously reduced. Gene silencing of PPARγ significantly downregulated the expression levels of milk fat synthesis-related genes in DCMECs. PPARγ overexpression improved cell viability, proliferation, and triacylglycerol secretion. The expression levels of milk lipogenic genes were significantly increased when PPARγ was overexpressed. Acetic acid and palmitic acid could markedly improve triacylglycerol synthesis and upregulate the expression levels of PPARγ and other lipogenic genes in DCMECs. These results suggest that PPARγ is a positive regulator of milk fat synthesis in DCMECs and that acetic acid and palmitic acid could partly regulate milk fat synthesis in DCMECs via PPARγ signaling.

GSK3β为AKT1下游信号分子,受PI3K/AKT调控.GSK3β磷酸化后被抑制,通过调节下游分子elF2B等促进乳蛋白合成.mTOR在调节乳蛋白合成中发挥重要作用,近来发现GSK3β和mTORC1均可通过调节SREBP1促进乳脂合成.但尚不清楚GSK3β和mTORC1在调节乳蛋白和乳脂合成方面的相互作用关系.该研究通过对GSK3β进行超表达和抑制,观察其对体外培养的奶牛乳腺上皮细胞细胞增殖、乳蛋白和乳脂肪合成的影响,及对mTOR途径的影响.结果发现GSK3β是奶牛乳腺上皮细胞细胞增殖、乳蛋白和乳脂肪合成的负调控分子,通过抑制mTORC1发挥作用.蛋氨酸可以抑制GSK3β和激活mTORC1,促进细胞增殖、乳蛋白和乳脂肪合成.该研究结果进一步揭示了GSK3β在奶牛乳腺上皮细胞细胞增殖、乳蛋白和乳脂肪合成中的负调控分子机理.