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.     

Enhancement of neutrophil function by ultrafiltered bovine whey

The objective of this work was to evaluate a proprietary ultrafiltered bovine whey product for its in vitro influence on the function of neutrophils from normal and dexamethasone-treated cattle, and for its in vivo influence on neutrophil function in periparturient dairy cows. The ultrafiltered bovine whey was produced by hyperimmunizing cows to various bacterial pathogens by intramammary injection, collecting and pooling the colostrum and milk for the first 3 wk after parturition, then separating and processing the whey through a filter with a nominal molecular mass cut off of 10,000 Da. In vitro treatment of neutrophils from normal calves with ultrafiltered bovine whey significantly increased neutrophil random migration, cytochrome C reduction, iodination activity, antibody-dependent cell-mediated cytotoxicity, and antibody-independent cell-mediated cytotoxicity. Cytochrome C reduction was the only neutrophil function parameter significantly enhanced by the in vitro treatment of neutrophils from dexamethasone-treated cattle with ultrafiltered bovine whey. In vivo treatment of periparturient cows with ultrafiltered bovine whey did not alter the total or differential leukocyte counts in the animals but did significantly increase the total erythrocyte counts. In vivo treatment with ultrafiltered bovine whey also significantly increased neutrophil iodination activity in the periparturient cows. Neutrophil iodination activity (a measure of the myeloperoxidase/hydrogen peroxide/halide antibacterial system) is a very potent bactericidal mechanism of neutrophils and has previously been shown to be suppressed in periparturient cows.     

Hyperactivation of mammalian spermatozoa: function and regulation

Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry. Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified. There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct. Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. The process may also involve increases in intracellular cAMP, which at least is required to support motility in general. Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways.     

Short communication: Glutamine modulates inflammatory responses to lipopolysaccharide in ex vivo bovine endometrium

Bacteria infect the endometrium lining the uterus of cattle after parturition, and clearance of these microbes depends on a robust innate immune response to bacterial molecules, such as the endotoxin lipopolysaccharide (LPS). Endometrial inflammation is characterized by secretion of the cytokines IL-1β and IL-6 and the chemokine IL-8. However, animals often fail to clear invading bacteria and develop uterine disease if they are in negative energy balance, with reduced abundance of glucose and glutamine, which are substrates for energy in tissues. Depletion of glucose blunts inflammatory responses in the endometrium, but the role of glutamine is not clear. The present study tested the hypothesis that depletion of glutamine compromises inflammatory responses to LPS in endometrial tissue. Ex vivo organ cultures of endometrium were challenged with LPS, and culture supernatants accumulated IL-1β, IL-6, and IL-8, as expected. However, reducing the availability of glutamine in culture medium containing glucose reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%. Surprisingly, in the absence of glucose, supplying increasing amounts of glutamine was not sufficient to augment inflammatory responses to LPS, whereas, in the absence of glutamine, supplying more glucose increased inflammation. Furthermore, inhibiting glycolysis reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%, even when glutamine and glucose were abundant. In conclusion, depletion of glutamine reduces inflammatory responses to LPS in the endometrium, and the activity of glutamine depends on glucose and glycolysis. These data provide mechanistic insights into how negative energy balance may be linked to postpartum uterine disease.     

Function and regulation of lymphocyte-mediated immune responses: relevance to bovine mastitis

Bovine mastitis is one of the most costly diseases to the dairy industry. Prospects for effective vaccines are limited by the variety of microorganisms capable of causing mastitis. An understanding of the physiologic and immunologic factors controlling the susceptibility of the cow to disease will lead to more rational approaches to prevention and control. In this paper, we describe the basic components of the immune system, drawing upon information derived from studies with rodents and humans. Some of these findings have been confirmed in the bovine and other domestic species, and it is likely that further study will reveal additional similarities between the immune systems of laboratory animals, humans, and domestic animals. Some important differences have already been identified, such as altered lymphocyte circulation patterns in ruminant versus non-ruminant species. These differences are discussed. We describe the structural and functional properties of major histocompatibility complex antigens and their role in regulation of immune responses. Finally, we discuss the consequences of antigen-induced activation of T-lymphocytes and the role of these cells in response to disease-causing microorganisms.     

Symposium review: Oxylipids and the regulation of bovine mammary inflammatory responses

Inflammation is a critical aspect of the innate immune system that can determine the outcome of several economically important diseases of dairy cattle such as mastitis. The purpose of the inflammatory response is to eliminate the source of tissue injury and then return tissues to normal function. Aggressive or uncontrolled inflammatory responses, however, can damage host tissues and contribute significantly to the pathophysiology associated with mastitis. A precarious balance between pro-inflammatory and pro-resolving mechanisms is needed to ensure optimal pathogen clearance and a prompt return to immune homeostasis. Therefore, inflammatory responses must be tightly regulated to avoid bystander damage to the milk-synthesizing tissues of the mammary gland. Oxylipids are potent lipid mediators that can regulate all aspects of the inflammatory response. The biosynthetic profiles of oxylipids are dependent on both the availability of diverse polyunsaturated fatty acids substrates and their subsequent metabolism through various oxidizing pathways. Changes in lipid metabolism in dairy cows around parturition can profoundly change the composition and concentration of oxylipids in the mammary gland that may be responsible for dysfunctional inflammatory responses during this time. This review will provide a brief overview of the bovine inflammatory response and the role that oxylipids play in contributing to the onset and resolution of inflammation especially as it pertains to mastitis. Factors associated with periparturient cows that can contribute to dysfunctional regulation of inflammation as a function of altered oxylipid biosynthesis and metabolism also will be described. Understanding the role that oxylipids may play in the development of mastitis is key to developing novel prevention and control programs for the dairy industry.     

Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non–membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein–protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis.     

15-Hydroxyprostaglandin dehydrogenase in the bovine endometrium during the oestrous cycle and early pregnancy

Prostaglandins (PG) are primary regulators of reproductive function. In ruminants, the relative production of PGE2 and PGF2alpha determines the return to a new oestrous cycle or to the establishment of pregnancy in response to a viable embryo. PG action depends on biosynthesis, transport and interaction with their receptors, which are all expressed differentially during the oestrous cycle. PGs are, however, local mediators and thus the onsite degradation by enzymes such as 15-hydroxyprostaglandin dehydrogenase (HPGD), also known as 15-PGDH, is another factor to consider in the regulation of physiological action. Little information is available on PG catabolism in the endometrium during the oestrous cycle or early pregnancy. The purpose of this study was to clone the bovine 15-PGDH, produce the recombinant protein and generate a specific antibody to study its activity and its expression in the endometrium during the oestrous cycle. We have found that the bovine 15-PGDH is highly homologous to the rat and human isoforms. 15-PGDH is localized principally in the glandular epithelium and to a lesser extent in stromal and luminal epithelial cells. The enzyme expression is regulated during the oestrous cycle and it reaches its maximal level on days 16-18. Transient expression is observed in luminal epithelial and trophoblast cells on day 21 of pregnancy. The mRNA is expressed at a constant high level throughout the cycle. The activity of the recombinant 15-PGDH was also tested and was found comparable for PGF2alpha and PGE2. These data suggest that 15-PGDH contributes to the tight regulation of PG action in the endometrium especially at the critical period of recognition of pregnancy.     

Expression of inter-alpha-trypsin inhibitor heavy chains in endometrium of cyclic and pregnant gilts

Attachment of the placenta to the uterus in pigs involves extracellular interaction between the expanding trophoblastic membrane and the thick glycocalyx present on the uterine epithelial microvilli. Formation of complexes between members of inter-alpha-trypsin inhibitor family may function in the maintenance of the extracellular matrix. This study investigated the change in the inter-alpha-trypsin inhibitor heavy chains (ITIH1, ITIH2, ITIH3 and ITIH4) during the oestrous cycle and early pregnancy in pigs. Gene expression of ITIH1, ITIH2, ITIH3 and ITIH4 was detected in the endometrium of cyclic and pregnant gilts; however, gene expression of ITIH was not altered throughout the oestrous cycle or early pregnancy. Western blot analysis with an ITIH antiserum identified the possible linkage forms of ITIH with the serine protease inhibitor, bikunin. Pregnancy altered the release of the various inter-alpha-inhibitor forms from the endometrium during the period of trophoblastic attachment. The results from this study indicate that the inter-alpha-trypsin inhibitor family plays an important role in maintenance of the uterine surface glycocalyx during placental attachment in pigs.     

Alpha-lactalbumin in bovine serum: relationships with udder development and function

Concentrations of alpha-lactalbumin in blood from cattle in various physiological states were measured as an index of udder development and function. Included were primiparous heifers during gestation and the peripartum period, nonlactating, nonpregnant cows hormonally induced into lactation, and cows milked two or three times daily in early, middle, or late lactation. Concentrations of alpha-lactalbumin in serum increased in two phases during gestation. Initial values (7.3 ng/ml, up to 120 d prepartum) rose, then leveled at 29.9 ng/ml on d 120 to 30 prepartum. Concentrations subsequently increased, averaging 133.2 ng/ml over the 30 d prior to parturition. During the peripartum period, alpha-lactalbumin rose from 221.2 ng/ml on d 4 prepartum, peaked at calving (918.8 ng/ml), then declined, stabilizing at approximately 500 ng/ml (1.5 to 3 d postpartum). Concentrations of alpha-lactalbumin in cows induced to lactate were low on d 1 to 9 of hormone treatment (15.7 ng/ml), rose to a maximum on d 17 (803.4 ng/ml), then fell to a plateau (185.8 ng/ml) on d 21 to 25. alpha-Lactalbumin concentrations were higher in early (101.9 ng/ml) than in middle or late lactation (81.4 and 79.2 ng/ml, respectively). Concentrations were also greater in twice versus thrice milked cows (101.9 vs. 73.0 ng/ml). Changes in alpha-lactalbumin concentrations in serum are associated with developmental and functional status of the udder. The measurement provides a noninvasive method to assess mammary gland activity.     

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1β, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection.     

Postmortem regulation of glycolysis by 6-phosphofructokinase in bovine M. Sternocephalicus pars mandibularis

This experiment addressed the hypothesis that 6-phosphofructokinase (6-PFK) regulates glycolysis in postmortem in M. sternocephalicus pars mandibularis. In two separate experiments, muscle samples were excised from randomly-selected steers that would typically be found on a commercial slaughter floor. In the first experiment, two samples were obtained from each of 6 steers immediately post-exsanguination; one sample was immersed immediately in liquid nitrogen and the other was stored at 4 °C for 4 d, to compare 6-PFK enzyme activity and glycolytic intermediate concentrations between fresh and d 4 postmortem samples. The greatest activity of 6-PFK was measured in fresh muscle extracts at pH 7.4, whereas little activity was detectable at pH 7.0. 6-PFK activity measured at pH 7.4 in d 4 samples also was barely detectable. Hill coefficient values for 6-PFK in fresh samples measured at pH 7.4 or 7.0, and d 4 samples measured at pH 7.4 were 2.9, 0.8, and 0.7, respectively, indicating loss of cooperativity with both lowered pH during assay and with time postmortem. Glycogen concentrations decreased 45% from d 0 to d 4, to 39.6 μmol glycogen/g muscle. Muscle concentrations of free glucose increased (P < 0.001) from 0.84 μmol/g at d 0 to 6.54 μmol/g at d 4. Fructose-6-phosphate and glucose-6-phosphate increased (P < 0.001) from d 0 to d 4 (2.8-fold and 4.7-fold, respectively). Lactate began accumulating immediately (3.33 μmol/g) and was elevated to 45.9 μmol/g by d 4. In the second experiment, conversion of [U-¹⁴C]glucose to lactate, glycogen, and CO₂ was measured in vitro at pH 7.4 and 7.0 in fresh M. sternocephalicus pars mandibularis strips from four steers. Total [U-¹⁴C]glucose was less in muscle strips incubated at pH 7.0 than in those incubated at pH 7.4 (55.5 vs. 123 nmol glucose utilized per 100 mg muscle per h; P = 0.04), due primarily to a reduction in glucose conversion to lactate. The conversion of glucose to glycogen or CO₂ in vitro was unaffected by media pH. These results suggest that the postmortem decline in pH in M. sternocephalicus pars mandibularis ultimately inactivates 6-PFK; this occurs prior to the depletion of glycogen reserves.     

Expression of bovine trypsin in Lactococcus lactis

Bovine trypsin is widely used in the production of bioactive peptides from milk proteins. The objective of this study was to express bovine trypsin in Lactococcus lactis to produce peptides by fermentation. The bovine trypsin with bias codon of L. lactis was synthesized, cloned into a secretory expressive vector pSEC-E7, and then introduced to L. lactis strain NZ9000 by electroporation. The expression of trypsin induced by nisin was identified by Western blot. Western blot analysis revealed a band in the protoplast fraction corresponding to SP_Usp45 (signal peptide of usp45)-trypsin. Biological activity of expressed trypsin was confirmed by the single substrate overlay assay. These results suggest that the expression of biologically active bovine trypsin has been detected in the protoplast fraction of L. lactis strain NZ9000 (pSEC:trypsin).