Characterization and regulation of the gene expression of amino acid transport system A (SNAT2) in rat mammary gland

Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. beta-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells

Whey acidic protein (WAP) is the principal whey protein found in rodent milk, which contains a cysteine-rich motif identified in some protease inhibitors and proteins involved in tissue modeling. The expression of the Wap gene, which is principally restricted to the mammary gland, increases more than 1,000-fold around mid-pregnancy. To determine whether the expression of this major milk protein gene is a prerequisite for functional differentiation of mammary epithelial cells, we generated conventional knockout mice lacking two alleles of the Wap gene. Wap-deficient females gave birth to normal litter sizes and, initially, produced enough milk to sustain the offspring. The histological analysis of postpartum mammary glands from knockout dams does not reveal striking phenotypic abnormalities. This suggests that the expression of the Wap gene is not required for alveolar specification and functional differentiation. In addition, we found that Wap is dispensable as a protease inhibitor to maintain the stability of secretory proteins in the milk. Nevertheless, a significant number of litters thrived poorly on Wap-deficient dams, in particular during the second half of lactation. This observation suggests that Wap may be essential for the adequate nourishment of the growing young, which triple in size within the first 10 days of lactation. Important implications of these findings for the use of Wap as a marker for advanced differentiation of mammary epithelial cells and the biology of pluripotent progenitors are discussed in the final section.     

小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律,文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明,在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度(表达丰度>5%),脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%),与妊娠期乳腺基因的mRNA表达相比,在泌乳期这些基因的mRNA表达均有显著上调(P<0.05),并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高,而在泌乳初期(6 d)和泌乳末期(18 d)较低,呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加,在泌乳中期(12 d)表达有10倍上调,其变化规律与脂肪合成酶基因的表达模式相同,说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。     

小鼠不同泌乳期乳腺脂肪合成相关基因的mRNA表达

为了研究小鼠不同泌乳期乳脂肪合成相关基因的表达规律, 文章采用荧光定量PCR检测了小鼠乳腺中与脂肪合成和分泌相关20个基因的mRNA相对表达丰度和表达差异。结果表明, 在乳腺中脂蛋白脂酶(LPL)、乙酰辅酶A羧化酶(ACACA)、硬脂酰辅酶A去饱和酶(SCD)、黄嘌呤脱氢酶(XDH)、嗜乳脂蛋白(BTN)、脂肪酸分化蛋白(ADFP)基因都具有高mRNA表达丰度 (表达丰度>5%), 脂肪酸转运体(CD36)、脂肪酸合成酶(FASN)、1-酰基甘油磷酸酰基转移酶(AGPAT6)和甘油酰基转移酶(DGAT)基因具有中等mRNA表达丰度(5%>表达丰度>1%), 与妊娠期乳腺基因的mRNA表达相比, 在泌乳期这些基因的mRNA表达均有显著上调(P<0.05), 并且ACACA、SCD、FASN、AGPAT6和DGAT等脂肪合成酶基因的表达在泌乳中期(12 d)最高, 而在泌乳初期(6 d)和泌乳末期(18 d)较低, 呈现低-高-低的表达模式。转录因子固醇调节元件结合蛋白(SREBF)基因在泌乳开始时mRNA表达增加, 在泌乳中期(12 d)表达有10倍上调, 其变化规律与脂肪合成酶基因的表达模式相同, 说明SREBF基因在小鼠乳腺脂肪合成酶基因的表达调控中发挥重要调节作用。     

Metabolism of aminoacyl-p-nitroanilides by rat mammary tissue

We have examined the metabolism of aminoacyl-p-nitroanilides by rat mammary tissue isolated from rats during late pregnancy, peak lactation and late lactation. The rate of hydrolysis depended upon the chemical nature of the aminoacyl-p-nitroanilide compound and the physiological state of the donor animals. Thus, mammary tissue isolated from rats during late pregnancy and peak lactation hydrolysed aminoacyl-p-nitroanilides in the order L-met-p-nitroanilide=L-leu-p-nitroanilide>L-lys-p-nitroanilide>gamma- glu-p-nitroanilide. The order of activity was the same for mammary tissue taken from rats during late lactation except that L-lys-p-nitroanilide was hydrolysed at the same rate as the neutral aminoacyl-p-nitroanilides. Mammary tissue from peak lactating rats also hydrolysed alpha-L-glu-p-nitroanilide and alpha-L-asp-p-nitroanilide but to a lesser extent than the other compounds tested. The anionic aminoacyl-p-nitroanilides were able to trans-stimulate D-aspartate efflux from mammary tissue explants and the perfused mammary gland via the high-affinity anionic amino acid carrier. The clearance of gly-L-phe by the perfused mammary gland was markedly inhibited by L-phe. The results suggest that mammary tissue expresses a variety of dipeptidases at the basolateral aspect of the mammary epithelium which are capable of hydrolysing peptides extracellularly. These enzymes may be important for providing amino acids for milk protein synthesis and/or inactivating signal peptides.     

The Dietary Protein/Carbohydrate Ratio Differentially Modifies Lipogenesis and Protein Synthesis in the Mammary Gland,Liver and Adipose Tissue during Gestation and Lactation

During gestation and lactation, a series of metabolic changes that are affected by the diet occurs in various organs of the mother. However, little is known about how the dietary protein (DP)/carbohydrate (DCH) ratio regulates the expression of metabolic genes in the mother. Therefore, the purpose of this work was to study the effect of consuming different percentages of DP/DCH, specifically 10/73, 20/63 and 30/53%, on the expression of genes involved in lipogenesis and protein synthesis in the mammary gland, liver and adipose tissue during gestation and lactation in dams. While the amount of weight gained during gestation was similar for all groups, only dams fed with 30/53% DP/DCH maintained their weight during lactation. In the mammary gland, the expression of the genes involved in lipogenesis, specifically SREBP1 and FAS, was dramatically increased, and the expression of the genes involved in protein synthesis, such as mTOR1, and the phosphorylation of its target protein, S6K, were also increased throughout pregnancy and lactation, regardless of the concentration of DP/DCH. In the liver and adipose tissue, the expression of the genes and proteins involved in lipid metabolism was dependent on the proportion of DP/DCH. The consumption of a low-protein/high-carbohydrate diet increased the expression of lipogenic genes in the liver and adipose tissue and the amount of lipid deposition in the liver. Conversely, the consumption of a high-protein/low-carbohydrate diet increased the expression of genes involved in amino acid oxidation in the liver during gestation. The metabolic adaptations reflected by the changes in the expression of metabolic genes indicate that the mammary gland has a priority for milk synthesis, whereas the adaptations in the liver and adipose tissue are responsible for providing nutrients to the mammary gland to sustain milk synthesis.     

Transcriptional activity of the promoter region of rat frizzled-related protein gene

We cloned two new paralogous genes that encode proteins homologous to seminal vesicle autoantigen (SVA) in rodents. The open reading frame of one mouse gene encodes a polypeptide consisting of 151 amino acid residues which has 43% identity to SVA. RT-PCR analysis showed selective expression in the colon, and thus the protein was tentatively named "SVA-like protein in the colon (SLP-C)". The other mouse gene has an open reading frame encoding 144 amino acid residues with 46 and 65% identity to SVA and SLP-C, respectively. Expression of this gene was detected in the mammary, submaxillary, parotid, and lacrimal glands, and this protein was named "SLP in the mammary gland (SLP-M)". Orthologs of both genes were also found in rats. The three homologous genes coding for SVA, SLP-C, and SLP-M may have been generated by gene duplication with divergence of tissue expression in the course of evolution. They comprise a unique structurally-related gene family. Moreover, these genes share significant sequence homology with that of another secretory glycoprotein, prolactin-inducible protein.     

牦牛DBI基因的分子特征及其在乳腺组织中的表达与定位

地西泮结合抑制因子(Diazepam binding inhibitor,DBI)与酰基辅酶A具有高亲和力,在动物组织中广泛存在,与脂肪酸代谢、类固醇激素合成密切相关。为研究DBI基因的分子特征及该基因在乳腺发育中的作用,对牦牛DBI基因编码区进行克隆,进行生物信息学分析;采用实时荧光定量PCR (Quantitative real-time PCR,qPCR)、蛋白免疫印迹技术(Western blotting,WB)和免疫组织化学(Immunohistochemistry,IHC)方法对牦牛泌乳前期、泌乳期和干乳期的乳腺组织中DBI的相对表达量和表达部位进行研究。DBI序列分析显示：牦牛DBI基因编码区序列长264 bp,编码87个氨基酸残基,与牛的同源性高达99.62％;qPCR数据表明：牦牛泌乳前期乳腺组织中DBI基因的相对表达量显著高于泌乳期和干乳期(P< 0.05);WB结果显示：牦牛泌乳前期乳腺组织中DBI蛋白的表达量最高,干乳期次之,泌乳期最低(P< 0.05);IHC结果表明：不同发育时期的牦牛乳腺组织中DBI的表达部位并无明显差异,主要表达于乳腺腺泡上皮细胞、导管上皮细胞及小叶间质细胞。DBI在不同发育时期牦牛乳腺组织中的相对表达量具有明显差异(P< 0.05),揭示DBI可能参与牦牛乳腺发育的过程,这为进一步探究DBI基因在生物体中的作用提供相应的理论参考。     

Molecular characterization and expression analysis of osteopontin cDNA from lactating mammary gland in yak (<Emphasis Type="Italic">Bos grunniens</Emphasis>)

Osteopontin (OPN) is a secreted phosphorylated glycoprotein. It has an important role in mammary gland development and lactation, as well as, is thought to be a potential candidate gene for lactation traits. In the present work, we isolated and characterized a full-length open reading frame (ORF) of yak OPN cDNA from lactating mammary tissue, and examined its expression pattern in mammary gland during different stages of lactation, as well as, the recombinant OPN protein of yak was expressed successfully in E. coli. The sequencing results indicated that the isolated cDNA was 1132-bp in length containing a complete ORF of 837-bp. It encoded a precursor protein of yak OPN consisting of 278 amino acid with a signal peptide of 16 amino acids. Yak OPN has a predicted molecular mass of 29285.975 Da and an isoelectric point of 4.245. It had an identity of 65.50–99.16% in cDNA, identity of 52.06–98.56% and similarity of 65.40–98.56% in deduced amino acids with the corresponding sequences of cattle, buffalo, sheep, goat, pig, human, and rabbit. The phylogenetic analysis indicated that yak OPN had the closest evolutionary relationship with that of cattle, and next buffalo. In mammary gland, yak OPN was generally transcribed in a declining pattern from colostrum period to dry period with an apparent increase of OPN expression being present in the late period of lactation compared with peak period of lactation. Western blot analysis indicated that His-tagged yak OPN protein expressed in E. coli could be recognized not only by an anti-His-tag antibody but also by an anti-human OPN antibody. These results from the present work provided a foundation for further insight into the role of OPN gene in yak lactation.