Identification, sequence analysis and mRNA tissue distribution of the bovine sterol transporters ABCG5 and ABCG8

The family of ATP-binding cassette (ABC) transporters consists of several transmembrane proteins that use ATP hydrolysis as an energy source for the transport of a variety of substances through cellular membranes. Two members of this family, ABCG5 and ABCG8, are implicated in the intestinal absorption and biliar excretion of sterols. Cholesterol content in milk is highly variable among species, breeds, and individuals of the same species, but a potential application of these genes in lipid homeostasis in the mammary gland has never been addressed. In the present work, expression of ABCG5 and ABCG8 in the bovine was demonstrated for the first time and characterized by quantitative PCR. The entire coding region and promoter area were sequenced and screened for motifs involved in lipid homeostasis. Both ABCG5 and ABCG8 presented a high level of length and sequence identity with other mammalian species. In the intergenic promoter region, 2 GATA boxes, a liver receptor homolog-1 response element, and a nuclear factor-kappaB response element, important factors in other lipid regulatory processes, were identified. As expected, high expression levels of both ABCG5 and ABCG8 were present in liver and digestive tract samples, and interestingly, in the mammary gland, opening new avenues for further investigation about their potential role in lipid trafficking and excretion during lactation.     

共轭亚油酸对乳腺脂类代谢和乳腺发育的作用及其机理

共轭亚油酸(Conjugated Linoleic Acid,CLA)是亚油酸具有共轭双键的一组同分异构体,由瘤胃细菌在生物氢化过程中合成,具有抗癌、抗炎症、抗动脉粥样硬化等多种生物学功能。CLA在代谢方面也有调节作用,能影响全身代谢,影响肝脏、乳腺以及富含脂肪的相关组织代谢。在此介绍了CLA的来源、结构、对动物乳腺脂类代谢和乳腺发育的作用及其机理。     

Identification of ABCA1 and ABCG1 in milk fat globules and mammary cells--implications for milk cholesterol secretion

The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play an important role in cellular cholesterol homeostasis, but their function in mammary gland (MG) tissue remains elusive. A bovine MG model that allows repeated MG sampling in identical animals at different functional stages was used to test whether 1) ABCA1 and ABCG1 protein expression and subcellular localization in mammary epithelial cells (MEC) change during the pregnancy-lactation cycle, and 2) these 2 proteins were present in milk fat globules (MFG). Expression and localization in MEC were investigated in bovine MG tissues at the end of lactation, during the dry period (DP), and early lactation using immunohistochemical and immunofluorescence approaches. The presence of ABCA1 and ABCG1 in MFG isolated from fresh milk was determined by immunofluorescence. The ABCA1 protein expression in MEC, expressed as arbitrary units, was higher during the end of lactation (12.2 ± 0.24) and the DP (12.5 ± 0.22) as compared with during early lactation (10.2 ± 0.65). In contrast, no significant change in ABCG1 expression existed between the stages. Throughout the cycle, ABCA1 and ABCG1 were detected in the apical (41.9 ± 24.8 and 49.0 ± 4.96% of cows, respectively), basal (56.2 ± 28.1 and 54.6 ± 7.78% of cows, respectively), or entire cytoplasm (56.8 ± 13.4 and 61.6 ± 14.4% of cows, respectively) of MEC, or showed combined localization. Unlike ABCG1, ABCA1 was absent at the apical aspect of MEC during early lactation. Immunolabeling experiments revealed the presence of ABCA1 and ABCG1 in MFG membranes. Findings suggest a differential, functional stage-dependent role of ABCA1 and ABCG1 in cholesterol homeostasis of the MG epithelium. The presence of ABCA1 and ABCG1 in MFG membranes suggests that these proteins are involved in cholesterol exchange between MEC and alveolar milk.     

Expression and localization of extracellular matrix-degrading proteinases and their inhibitors in the bovine mammary gland during development, function, and involution

In degrading the extracellular matrix, matrix metalloproteinases (MMP) and the plasminogen activator (PA) system may play a critical role in extensive remodeling that occurs in the bovine mammary gland during development, lactation, and involution. Therefore, the aim of our study was to investigate the mRNA expression of MMP-1, MMP-2, MMP-14, MMP-19, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, urokinase-type PA, tissue-type PA, urokinase-type PA receptor, and PA inhibitor-1 by quantitative PCR and to localize with immunohistochemistry MMP-1, MMP-2, MMP-14, and TIMP-2 proteins in the bovine mammary gland during pubertal mammogenesis, lactogenesis, galactopoiesis, and involution. Expression of mRNA for each of the studied factors was relatively lower during galactopoiesis and early involution but was markedly increased during mammogenesis and late involution, 2 stages in which tissue remodeling is especially pronounced. The localization of proteins for MMP-1, MMP-14, and TIMP-2 showed a similar trend with strong staining intensity in cytoplasm of mammary duct and alveolar epithelial cells during pubertal mammogenesis and late involution. Interestingly, MMP-2 protein was localized only in the cytoplasm of endothelial cells during late involution. Our study demonstrated clearly that expression of extracellular matrix-degrading proteinases coincides with a concomitant expression of their inhibitors. High expression levels of MMP, TIMP, and PA family members seem to be a typical feature of the nonlactating mammary gland.     

Short communication: Staphylococcus aureus infection modulates expression of drug transporters and inflammatory biomarkers in mouse mammary gland

Mastitis is the most common disease in dairy herds worldwide and is often caused by Staphylococcus aureus. Little is known about the effect of mastitis on transporters in the mammary gland and the effect on transporter-mediated secretion of drugs into milk. We studied gene expressions of ATP-binding cassette and solute carrier transporters in S. aureus-infected mammary glands of mice. On d 7 of lactation, NMRI mice were inoculated with 1,000 cfu of S. aureus in 2 mammary glands and with a saline vehicle in 2 control glands. Gene expression of the transporters, Bcrp, Mdr1, Mrp1, Oatp1a5, Octn1, and Oct1, and of Csn2, the gene encoding β-casein, were determined in mammary glands at 72 h after treatment. As biomarkers of the inflammatory response gene, expressions of the cytokines Il6, Tnfα, and the chemokine Cxcl2 were measured. Despite a high individual variation between the 6 animals, some characteristic patterns were evident. The 3 inflammatory biomarkers were upregulated in all animals; Csn2 was downregulated compared with controls in all animals, although not statistically significantly. Both Mrp1 and Oatp1a5 were statistically significantly upregulated and Bcrp was downregulated. Gene expression of Bcrp followed the expression of Csn2 in each of the animals, indicating a possible co-regulation. The findings demonstrate that S. aureus infection has an effect on expression of drug transporters in the mammary gland, which may affect secretion of drugs into milk and efficacy of drug therapy.     

Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period

The incidence and severity of mastitis can be high during the period of transition from pregnancy to lactation when dairy cattle are susceptible to oxidative stress. Oxidative stress may contribute to the pathogenesis of mastitis by modifying the expression of proinflammatory genes. The overall goal of this study was to determine the relationship between critical antioxidant defense mechanisms and proinflammatory markers in normal bovine mammary tissue during the periparturient period. Mammary tissue samples were obtained from 12 cows at 35, 20, and 7 d before expected calving and during early lactation (EL, 15 to 28 d in milk). Enzyme activities for cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase were relatively low during the dry period, but increased during EL, whereas activity of thioredoxin reductase 1 did not change significantly as a function of time. In contrast, gene expression for these antioxidant selenoproteins and for heme oxygenase-1 gradually decreased as parturition approached and then increased during EL. The expression of intercellular vascular adhesion molecule-1 and vascular cell adhesion molecule-1 followed a similar trend where mRNA abundance gradually declined as parturition approached with a slight rebound in EL. Gene expression of the pro-oxidant, 15-lipoxygenase 1, which is known to increase during times of oxidative stress, also increased dramatically in mammary tissue from EL cows. Expression of the proinflammatory cytokines, IL-1β, IL-6, and IL-8 did not change significantly during the periparturient period. Strong positive correlations were found between several antioxidant enzymes (cytosolic glutathione peroxidase, thioredoxin reductase 1, and heme oxygenase-1) and vascular adhesion molecules (intercellular vascular adhesion molecule-1, vascular cell adhesion molecule-1) suggesting a protective response of these antioxidants to an enhanced proinflammatory state. Ability to control oxidative stress through manipulation of key antioxidant enzymes in the future may modify the proinflammatory state of periparturient cows and reduce incidence and severity of some diseases such as mastitis.     

Symposium review: The influences of heat stress on bovine mammary gland function

Heat stress reduces cow milk yield and results in a significant economic loss for the dairy industry. During lactation, heat stress lowers milk production by 25 to 40% with half of the decrease in milk synthesis resulting from the reduced feed intake. In vitro studies indicate that primary bovine mammary epithelial cells display greater rates of programmed cell death when exposed to high ambient temperatures, which may lead to a decrease in the total number of mammary epithelial cells in the mammary gland, partially explaining the lower milk production of lactating cows under heat stress. The function of mammary cells is also altered by heat stress. In response to heat stress, mammary cells display higher gene expression of heat shock proteins, indicating a need for cytoprotection from protein aggregation and degradation. Further, heat stress results in increased gene expression without altering protein expression of mammary epithelial cell junction proteins, and does not substantially influence the integrity of mammary epithelium. These data suggest that the mammary gland strives to maintain cell-to-cell junction integrity by synthesizing more proteins to compensate for protein losses induced by heat stress. During the dry period, heat stress negatively affects mammary gland development by reducing mammary cell proliferation before parturition, resulting in a dramatic decrease in milk production in the subsequent lactation. In addition to mammary growth, the mammary gland of the heat-stressed dry cow has reduced protein expression of autophagic proteins in the early dry period, suggesting heat stress influences mammary involution. Emerging evidence also indicates that heifers born to cows that experience late-gestation heat stress have lower milk yield during their first lactation, implying that the maternal environment may alter mammary gland development of the offspring. It is not clear if this is due to a direct epigenetic modification of prenatal mammary gland development by maternal heat stress. More research is needed to elucidate the effect of heat stress on mammary gland development and function.     

奶牛乳腺中瘦素及其受体的表达与定位

为探讨瘦素在奶牛乳腺发育、泌乳及退化各时期的表达变化规律及在乳腺组织中的具体位置,采用免疫印迹(western blot-ting)技术及激光共聚焦技术检测奶牛乳腺组织中瘦素及瘦素受体(OB-Rb)的表达变化及其定位。结果表明,瘦素在青春期表达量较高,在泌乳期表达量最低。瘦素受体在青春期和妊娠期表达量较高,泌乳期表达量相对较低,退化期逐渐恢复到妊娠期水平。     

奶牛不同发育时期乳腺细胞凋亡规律的研究

为系统地研究奶牛乳腺不同发育时期细胞凋亡的规律,应用TUNEL法对奶牛不同发育时期正常乳腺组织(冰冻切片)的乳腺细胞凋亡进行了系统检测。结果表明,青春期乳腺发育缓慢,结构变化较小,乳腺细胞凋亡量相对较少;妊娠期乳腺导管持续发育,凋亡量上升,其中妊娠初期2月,乳腺腺泡大量出现,脂肪细胞凋亡随之增加,出现了一个高峰;泌乳期乳腺的结构和功能最为完善,乳腺结构变化很小,细胞凋亡量维持在很低水平;退化期腺泡瓦解,大量细胞发生凋亡,其中退化初期乳腺细胞凋亡持续增加,退化3 d达到最大值,之后凋亡量逐渐降低,退化30 d后乳腺已经基本恢复到青春期状态,凋亡细胞量也随之减少。     

Leptin affects prolactin action on milk protein and fat synthesis in the bovine mammary gland

Leptin, a protein hormone produced and secreted predominantly by white adipose tissue, has a critical role in the regulation and coordination of energy metabolism. Identification of leptin in the milk of several mammals, including humans, led us to investigate its presence and regulatory effect in the cow mammary gland. The expression of leptin receptor in tissue culture of lactating mammary gland was augmented approximately 25 times by prolactin, but had no effect on virgin calf mammary tissue. Expression of leptin in tissue culture from mammary glands of lactating cows was enhanced 2.2-fold by prolactin. No effect of prolactin on leptin and leptin receptor expression was found in mammary gland tissue culture from calves. Leptin-enhanced fatty acid synthesis in the presence of prolactin, but had no effect without presence of prolactin. A similar pattern was found in the expression of alpha-casein and beta-lactoglobulin in mammary gland explants from a lactating cow. Our findings indicate that leptin plays an important role in mammary gland lactogenesis, and that the expression of leptin requires the presence of prolactin.     

Effects of photoperiod modulation and melatonin feeding around drying-off on bovine mammary gland involution

The risk for a dairy cow to acquire new intramammary infections is high during the transition from lactation to the dry period, because of udder engorgement and altered immune functions. Once the gland is fully involuted, it becomes much more resistant to intramammary infections. Therefore, strategies to depress milk yield before drying-off and accelerate the involution process after drying-off could be beneficial for udder health. The objective of this study was to assess the effect of photoperiod manipulation and melatonin feeding from 14 d before to 14 d after drying-off on the speed of the involution process. Thirty Holstein cows in late lactation were randomly allocated to one of the following treatments: (1) a long-day photoperiod (16 h of light: 8 h of darkness), (2) a short-day photoperiod (8 h of light: 16 h of darkness), and (3) a long-day photoperiod supplemented by melatonin feeding (4 mg/kg of body weight). Milk and blood samples were collected on d ?26, ?19, ?12, ?5, ?1, 1, 3, 5, 7, 10, and 14 relative to the last milking to determine concentrations of mammary gland involution markers and serum prolactin. Additional blood samples were taken around milking on d ?15, before the start of the treatments, and on d ?1, before drying-off, to evaluate the treatment effects on milking-induced prolactin release. The short-day photoperiod slightly decreased milk production and basal prolactin secretion during the dry period. The milking-induced prolactin surge was smaller on d ?1 than on d ?15 regardless of the treatments. Lactoferrin concentration, somatic cell count, and BSA concentration as well as matrix metalloproteinase-2 and -9 activities increased in mammary secretions during the first 2 wk of the dry period, whereas milk citrate concentration and the citrate:lactoferrin molar ratio decreased. The rates of change of these parameters were not significantly affected by the treatments. The long-day photoperiod supplemented by melatonin feeding did not affect milk production, prolactin secretion, or mammary gland involution. Under the conditions in this study, photoperiod modulation and melatonin feeding did not appear to affect the rate of mammary gland 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.     

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24 h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24 h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.     

Early mammary gland metabolic and immune responses during natural-like and forceful drying-off in high-yielding dairy cows

The present work compared metabolic and immune responses in genetically high-producing cows that produced a low amount of milk before expected involution and in cows with the same genetic potential that produced copious amounts of milk before their scheduled drying-off. Ten multiparous lactating Israeli Holstein cows producing approximately 10,500 L in the current lactation, without bacterial infection and scheduled for drying-off approximately 60 d before their expected parturition, were studied. Five of the cows that exhibited a sharp, spontaneous reduction in milk yield at the end of their lactation and produced less than ~14 L/d were defined as cows approaching natural involution (ANI), and 5 cows that produced between 25 and 35 L/d were defined as cows approaching forced involution (AFI). Three days before scheduled drying-off, milking was stopped and milk samples were collected from each quarter. After milking cessation, only modest swelling was observed in the udders of the ANI cows. In the ANI cows, lactose and fat concentrations decreased and the fat:lactose concentration ratio indicated that on d 1 and 2 fat concentrations decreased faster than lactose concentration, whereas on d 3, the rate of reduction was about the same for lactose and fat. In contrast, in AFI cows, fat concentrations increased on d 1 and the fat:lactose ratio indicated that changes in fat secretion were minor compared with those of lactose secretion. Rennet clotting time of milk after drying-off in the ANI cows increased, whereas curd firmness decreased rapidly, such that mammary secretions did not coagulate on d 3. In the AFI cows, such significant changes were observed only on d 3. The inflammatory response increased in both groups, but at each stage the increase was greater in ANI cows than in AFI cows. On d 1, the increase in leukocyte numbers in the ANI cows was made up of mononuclear cells (i.e., T lymphocytes and macrophages). In contrast, in the AFI cows, we observed a marked increase in leukocyte numbers, mainly in the form of polymorphonuclear cells. Our data indicate that the abrupt mammary involution induced in AFI cows provoked signs of distress, which were associated with neutrophilia in milk. In contrast, in the ANI cows, cessation of milking occurred without evidence of engorgement of the udder. Physiological differences in ANI and AFI cows are distinct and are reflected in the differences in the leukocyte populations in milk.