Milk protein gene expression in the rat mammary gland

For the studies of the expression of milk proteins during the functional development of the rat mammary gland and in mammary tumor MTW9, milk proteins were purified, their mRNAs isolated, and the cDNA sequences complementary to these mRNAs cloned in E. coli cells. Results of such studies show: (1) rat α‐LA is unique in that it is larger than any known α‐LAs and is glycosylated; (2) rat milk contains unique whey phosphoproteins not found in other milks; (3) more than one plasmid DNA with differences in the restriction maps have been identified for several of these milk proteins, suggesting eithera presence of a family of genes or allelic differences for these proteins; (4) the expression of individual milk proteins is dependent on the functional stage of the gland; (5) there is an inverse relationship between the expression of milk proteins and the methylation of their gene sequences; (6) mammotrophic hormones required for synthesis and stability of milk proteins and their mRNAs, when withdrawn arrest the synthesis of α‐LA in the mammary tumor MTW9 at 6 hr or earlier of withdrawal but without any measurable effect on other proteins of the tumor.     

乳腺特异性人α-乳白蛋白真核表达载体的构建

从正常人血液中提取人基因组DNA.以其为模板,扩增人α-乳白蛋白(α-LA)2.36kb的总DNA序列,经测序鉴定后,将其连接到PSV载体SV40启动子后.构建真核表达载体LA-DNA-psv.扩增牛XS1-酪蛋白5'调控序列约1.2kb的片段.测序鉴定.切去LA-DNA-psv栽体原来的SV40启动子,连接牛αS1-酪蛋白5'调控序列约1.2kb的片段为启动子,构建真核表达载体αS1-LA-DNA-psv.为在动物乳腺中特异高效表达人α-乳白蛋白做准备研究.     

Glucose transporter and hypoxia-associated gene expression in the mammary gland of transition dairy cattle

Glucose is an important energy substrate, especially needed by dairy cows postpartum to support the onset of lactation. The prioritization and regulation of glucose uptake is accomplished, in part, by changes in expression of cellular glucose transport molecules (GLUT) within the mammary gland. The objectives of this study were to (1) evaluate the expression and cell-type specific localization of GLUT and hypoxia-associated genes that may regulate GLUT expression over the transition period and through lactation in bovine mammary tissue and (2) determine functionality of GLUT on primary bovine mammary endothelial cells (BMEC). Mammary tissue biopsies were taken from cows at 15 d before calving and again at 1, 15, 30, 60, 120, and 240 d post-parturition for quantitative real-time PCR analysis of GLUT and hypoxia-associated genes. Additional mammary tissue samples were used to localize GLUT within the cells of the lobulo-alveolar system via fluorescence microscopy. Cultures of primary bovine mammary endothelial cells were used to confirm the functionality of GLUT with a fluorescent glucose analog uptake assay. Significant increases in GLUT1 gene expression were observed during early lactation, whereas both GLUT3 and GLUT4 gene expression increased during late lactation. The gene expression for 2 receptors of vascular endothelial growth factor increased significantly during early lactation and remained increased throughout lactation when compared with gene expression during the transition period. All GLUT were detected on cultured BMEC and were capable of internalizing glucose through GLUT-mediated mechanisms. These data suggest mammary vascular tissues express GLUT during lactation and BMEC express functional glucose transporters. A better understanding of glucose uptake at the endothelial level may prove to be critical to improve glucose absorption from the blood for utilization by mammary epithelial cells.     

Expression and regulation of glucose transporters in the bovine mammary gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.     

Developmental and nutritional regulation of the prepubertal heifer mammary gland: I. Parenchyma and fat pad mass and composition

Prior to puberty, elevated nutrient intake has been shown to negatively affect prepubertal mammary development in the heifer. The objective of this study was to evaluate the effects of increased nutrient intake on mammary development in Holstein heifers at multiple body weights from birth through puberty. Specifically, this study evaluated the effects of nutrient intake and body weight at harvest on 1) total weight and DNA content of the parenchyma (PAR) and mammary fat pad (MFP) and 2) PAR and MFP composition. Starting at 45 kg of body weight, heifers (n = 78) were assigned to either a restricted (R) or elevated (E) level of nutrient intake supporting 650 (R) or 950 (E) g/d of body weight gain. Heifers were harvested at 50-kg increments from 100 to 350 kg of body weight. Mammary fat pad weight and DNA content were greater in E- than in R-heifers. Additionally, E-heifers had a greater fraction of lipids and a smaller fraction of protein in their MFP than did R-heifers. Parenchyma weight and DNA were lower in E- than in R-heifers; however, when analyzed with age as a covariate term, treatment was no longer a significant term in the model. Level of nutrient intake had no effect on the lipid, protein, or hydroxyproline composition of the PAR. Collectively, these data demonstrate that PAR is refractory to the level of nutrient intake whereas MFP is not. Furthermore, the covariate analysis demonstrated that age at harvest, not the level of nutrient intake, was the single greatest determinant of total PAR DNA content.     

Effect of alpha-tocopherol deprivation on the involution of mammary gland in sheep

The objective of this study was to investigate the effect of alpha-tocopherol deprivation on mammary gland involution and apoptosis in sheep. Two groups of four single lamb ewes were used. The control group received 100 mg/d of RRR-alpha-tocopherol supplementation and the experimental group received no vitamin E supplementation. After 3 mo of suckling, ewes were dried off, and blood samples from the jugular vein and tissue biopsies from the mammary gland were collected at d 1, 3, 5, and 8 after dry-off. The experimental group had lower plasma concentrations of alpha-tocopherol (1.8 vs. 4.2 micromol/L), lower glutathione peroxidase activity in erythrocytes, and higher concentration of malondialdehyde in plasma than the control group. Immunohistochemical analysis of tissue samples resulted in marked differences of bcl-2 and bax protein expressions during involution and between groups. The bax expression was decreased by alpha-tocopherol deprivation at 1, 3, and 5 d, but not at 8 d, while the bcl-2 score was higher only at 8 d (1.5 vs. 0.0 for experimental and control groups, respectively). As a result, the bcl-2 to bax ratios were increased for the experimental group at 1 and 8 d. During involution, apoptotic counts increased (from 0.12 to 4.06%), but no effects were detected in relation to bcl-2 to bax ratio and alpha-tocopherol. These results indicate that alpha-tocopherol can control bcl-2 expression, but not apoptosis in cells of the mammary gland during involution.     

Aminopeptidase N gene expression and abundance in caprine mammary gland is influenced by circulating plasma peptide

This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation.     

Disrupted secretory activation of the mammary gland after antenatal glucocorticoid treatment in sheep

Antenatal glucocorticoids are administered to women at risk of preterm delivery to prevent neonatal respiratory morbidity. The effects of exogenous glucocorticoids on the development of lactation are unknown. This study investigated the effects of a single dose of antenatal glucocorticoids on secretory activation in sheep before and after parturition. Pregnant ewes (N=36) were randomised to receive either medroxyprogesterone acetate (MPA) at 118 days of pregnancy and betamethasone at 125 days (BETA group), MPA at 118 days and saline at 125 days (MPA group) or saline at 118 and 125 days (SALINE group). The concentration of lactose, progesterone, cortisol and prolactin in maternal plasma was measured during pregnancy. After term parturition, the concentration of lactose in milk and maternal plasma was measured daily for 5 days. Lambs were weighed at birth and at 5 days of age; milk volume was measured on day 5. The concentration of lactose in maternal plasma increased significantly after betamethasone administration, corresponding to a fall in plasma progesterone. No changes in lactose were observed in MPA or SALINE ewes. Transient decreases in cortisol and increases in prolactin were observed in the BETA group, but not in either the MPA or SALINE group. After parturition, BETA ewes experienced reduced milk yield and lamb weight gain, and delayed increases in milk lactose levels compared with MPA and saline controls. This study demonstrated that, in sheep, antenatal glucocorticoid administration disrupted secretory activation, causing precocious mammary secretion before parturition and compromising postpartum milk production and lamb growth.     

Synthesis of lactoferrin and transport of transferrin in the lactating mammary gland of sheep.

Two iron-binding proteins, lactoferrin and transferrin, are present in ruminant milk. Lactoferrin commonly has been assumed to be a product of mammary synthesis, but the origin of milk transferrin has not been elucidated. The objective of this experiment was to study the synthesis and distribution of these two proteins in the mammary gland of sheep. Explants from lactating mammary gland of sheep have been cultured in the presence of [3H]leucine to determine rates of synthesis of lactoferrin and transferrin. After incubation, [3H]lactoferrin was found, but labeled transferrin was not. The capacity of the mammary gland to synthesize lactoferrin decreased markedly in the first 24 h of lactation. Immunohistochemical techniques were utilized to identify the locations of lactoferrin and transferrin in the mammary gland. Transferrin was found in the colostrum contained in the alveolar lumen, in the cytoplasm of the secretory cells, and in the connective tissue between the mammary acini. High concentration of transferrin was found in the basal membrane of the secretory alveolar cells, mainly in those near capillary vessels. Lactoferrin was found in the colostrum and in the cytoplasm of secretory cells with a more homogeneous distribution than transferrin. The connective tissue stained negative for lactoferrin. These results suggest that, although lactoferrin is synthesized by mammary gland of the sheep, transferrin comes from blood serum, probably by a receptor-mediated mechanism of transcytosis.     

奶山羊青春期乳腺发育重要基因RelA的全长克隆

从本实验室已建立的关中奶山羊7个时期乳腺Long-SAGE标签库中筛选出在青春期高表达的RelA基因,用RACE方法克隆并获得关中奶山羊RelA基因的全长,将全长序列与NCBI GenBank中人(H.sapiens)和牛(Bos taurus)数据库进行同源性比对,证实用所选EST标签引物克隆出的基因全长是奶山羊RelA基因.     

Developmental and nutritional regulation of the prepubertal bovine mammary gland: II. Epithelial cell proliferation, parenchymal accretion rate, and allometric growth

It is well documented that elevated nutrient intake prior to puberty reduces prepubertal mammary development in the bovine. The companion paper demonstrated that age at harvest is a primary determinant of parenchymal (PAR) mass and that any effects of elevated energy intake on mechanisms regulating mammary development are dwarfed by this effect of time. Therefore, it is hypothesized that while causing a decrease in prepubertal PAR mass, elevated nutrient intake will have no effect on growth characteristics of the mammary gland. The objectives of this experiment were to evaluate the effects of increased nutrient intake from early in life on 1) mammary epithelial cell proliferation, 2) mammary PAR DNA accretion rates, and 3) the dynamics of prepubertal allometric PAR growth. Holstein heifers (n = 78) were fed from 45 kg of body weight either elevated (E) or restricted (R) levels of nutrients to support 950 (E) or 650 (R) g/d of body weight gain. Six heifers per treatment were harvested at 50-kg increments from 100 to 350 kg of body weight. Heifers on the E plane of nutrition had higher plasma leptin and less PAR DNA than their body weight-matched R-intake cohorts. Despite this reduction in PAR DNA, treatment did not negatively influence mammary epithelial cell proliferation or the PAR DNA accretion rate. Dynamics of allometric and isometric mammary growth were also unaffected by the level of nutrient intake, as was exit from allometric growth. This work represents the first demonstrating that the level of nutrient intake and the concomitant increase in plasma leptin have no measurable influence on 1) the rate of PAR DNA accretion, 2) mammary epithelial cell proliferation, or 3) total PAR mass and, by default, the local or systemic controls that coordinate these processes.     

Spatial and temporal expression of insulin-like growth factor-I, insulin-like growth factor-II and the insulin-like growth factor-I receptor in the sheep fetal mammary gland

The mammary gland is an example of a tissue of epidermal origin that depends for the development of its characteristic morphology on underlying mesenchymal cells. The interaction between mesenchyme and epithelium appears to be mediated by polypeptide growth factors. In situ hybridization has been used to study, in the mammary gland of female sheep fetuses, the distribution of mRNA for the mammary mitogens, insulin-like growth factor (IGF)-I and IGF-II, and the IGF-I receptor, from 10 to 20 weeks of intrauterine life (term is approximately 22 weeks). At 10 weeks, secondary ducts had formed from the primary duct. By week 20, the gland had increased in volume and complexity, showing primitive lobules embedded in intralobular connective tissue disposed around main ducts. IGF-I and IGF-II mRNA were expressed in cells of the intralobular connective tissue underlying the epithelium, while the IGF-I receptor was expressed in epithelium. Quantitation by absorbance measurements showed that mRNA expression increased with pregnancy stage for IGF-I and IGF-II, but not significantly for the IGF-I receptor, and that IGF-II was more highly expressed than IGF-I. A role for the IGF system in mediating mesenchymal epithelial interactions in mammary development is indicated.     

Basigin expression and hormonal regulation in the rat uterus during the peri-implantation period

Basigin is essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of the basigin gene in the rat uterus during the peri-implantation period. Basigin mRNA was localized strongly in the luminal epithelium on day 1 of pregnancy and gradually decreased to a basal concentration from day 3 to day 5 of pregnancy. Basigin mRNA and protein were expressed strongly in the implanting blastocyst and primary decidua on day 6 of pregnancy. A similar expression pattern was also induced in the uterus after delayed implantation was terminated by oestrogen treatment and the embryo implanted, whereas expression was not detected during delayed implantation. Basigin expression was not detected on day 6 of pseudopregnancy. Basigin mRNA was expressed strongly in the decidua on days 7 and 8 of pregnancy. Furthermore, both basigin mRNA and protein were induced in the decidua during artificial decidualization. In addition, oestrogen stimulated strong expression of basigin mRNA in the uterine epithelium of ovariectomized rats. These findings indicate that basigin may play a role during implantation and decidualization in rats.     

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.