Karen Plaut

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15–30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln125,232-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5′-flanking region of the ovine β-lactoglobulin gene directed the secretion of Gln125,232-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Effect of exogenous prolactin administration on lactational performance of dairy cows

Eight Holstein cows were utilized to examine the effect of prolactin on lactational performance prior to peak milk production (day 21-34 postpartum) and after peak milk production (day 60-73 postpartum). During each 14 day period, cows received daily intramuscular injections of pituitary-derived bovine prolactin (120 mg; 13.0 IU/mg protein) or excipient. Cows were housed in a controlled environment at 18.1C, 47.8% relative humidity and a 15 hr light: 9 hr dark cycle. In cows administered exogenous prolactin, circulating prolactin concentrations increased within one-half hr post injection, peaked within 2 to 6 hours and declined through the remainder of the day. Average prolactin concentration in the plasma was increased 2 to 5 fold over the 24 hr period in response to prolactin treatment. Yields of milk and milk components (fat, lactose and protein) were not affected by prolactin treatment in either period but the concentration of alpha-lactalbumin in milk was significantly increased (P less than .10) in both periods. Circulating concentrations of somatotropin, triiodothyronine, thyroxine, glucagon, nonesterified fatty acids and glucose were not altered. In prolactin-treated cows, the milking-stimulated prolactin release was decreased at both the PM milking, when circulating concentrations of prolactin were high, and the AM milking, when prolactin concentrations had returned to baseline. Concentration of prolactin in milk tended to increase but was not significantly altered by administration of exogenous prolactin. However, prolactin concentrations in plasma were correlated (r = .56) with milk concentrations. It is clear that postpartum administration of exogenous prolactin during the period of lactation prior to peak milk yield or after peak milk yield does not alter lactational performance in high producing dairy cows.

Temporal pattern of insulin-like growth factor-I response to exogenous bovine somatotropin in lactating cows☆

The effect of exogenous bovine somatotropin (bST) treatment on the temporal pattern of insulin-like growth factor-I (IGF-I) in serum of four multiparous Holstein cows was examined. Cows (190 +/- 24 days postpartum) were treated with daily subcutaneous injections of recombinant bST (40 mg) or excipient for 12-day periods in a crossover experimental design. During excipient treatment, concentrations of IGF-I in serum were relatively constant throughout the day and averaged 70 ng/ml. Following the first bST injection, serum IGF-I began increasing after a lag of 5 to 7 hr and progressively increased over the first 2 days of treatment. Serum IGF-I levels were approximately 2-fold greater than control values at the end of day 1 of bST treatment, with a 3-fold elevation observed at the end of day 2. Concentrations of IGF-I in serum plateaued by day 3 of bST treatment. Serum concentrations of IGF-I did not follow the oscillating pattern of bST in serum resulting from daily bST injections. Milk yield (3.5% fat-corrected) plateaued after 6 days of bST treatment and was increased 61% (+15.3 kg). Both IGF-I and milk yield remained essentially constant across days for the remainder of treatment. Following cessation of treatment, serum IGF-I and milk yield gradually declined, returning to control values after approximately 4 days. The temporal pattern of circulating concentrations of IGF-I is consistent with a role for IGF-I in mediating a portion of the effects of exogenous bST in lactating cows.

The Role of Glucocorticoids in Secretory Activation and Milk Secretion, a Historical Perspective

In this review we present our current understanding of the role of glucocorticoids in secretory activation and milk secretion by looking at the literature from a historical perspective. We begin with the early endocrine ablation experiments and continue from there to show that glucocorticoids are not just necessary for secretory activation and milk secretion—but mandatory. Specifically, we discuss the importance of glucocorticoids to: (1) induce the formation of ultrastructural components necessary to support milk synthesis and secretion, including rough endoplasmic reticulum and tight junction sealing; (2) regulate milk protein gene expression; and (3) prevent the second phase of involution, possibly by preventing the breakdown of the extracellular matrix.

Higher Stromal Expression of Transforming Growth Factor-beta Type II Receptors is Associated with Poorer Prognosis Breast Tumors

Transforming growth factor-beta (TGFB) is a potent inhibitor of normal epithelial cell proliferation, and may be one of the regulatory factors that are perturbed during tumor development. While many tumor cell lines no longer respond to the inhibitory effects of TGFB due to a reduction or absence of the type II receptor (TGFBR2), the role of TGFBR2 in tumors from patients with breast cancer is less clear. The objective of this study was to screen human breast tumors to determine if there was a TGFBR2 mutation and/or altered expression of TGFBR2 protein. Using 10 unique primers, SSCP-PCR was used to detect heterozygosity in the complete coding sequence from 72 tumors and normal DNA from 20 individuals. One region of the promoter was also examined. Expression of TGFBR2 in the same breast tumors was examined by immunohistochemistry. Sequence variations were identified among normal and tumor tissue samples by SSCP-PCR within coding regions of exon 4 (1/72 samples) and within non-coding regions of intron 2 (1/72), intron 3 (72/72), and intron 6 (1/72). A new polymorphism was identified in intron 3. Observed allele frequencies were consistent with Hardy–Weinberg equilibrium in both the tumors and normal DNA. TGFBR2 was expressed in the epithelium and stroma of tumor tissue. The percentage of cells expressing TGFBR2 in stroma was higher in patients that had a positive lymph node status and/or negative estrogen and progesterone receptor expression. There was no relationship between TGFBR2 expression in the epithelium and these variables.

Molecular signatures reveal circadian clocks may orchestrate the homeorhetic response to lactation.

Genes associated with lactation evolved more slowly than other genes in the mammalian genome. Higher conservation of milk and mammary genes suggest that species variation in milk composition is due in part to the environment and that we must look deeper into the genome for regulation of lactation. At the onset of lactation, metabolic changes are coordinated among multiple tissues through the endocrine system to accommodate the increased demand for nutrients and energy while allowing the animal to remain in homeostasis. This process is known as homeorhesis. Homeorhetic adaptation to lactation has been extensively described; however how these adaptations are orchestrated among multiple tissues remains elusive. To develop a clearer picture of how gene expression is coordinated across multiple tissues during the pregnancy to lactation transition, total RNA was isolated from mammary, liver and adipose tissues collected from rat dams (n = 5) on day 20 of pregnancy and day 1 of lactation, and gene expression was measured using Affymetrix GeneChips. Two types of gene expression analysis were performed. Genes that were differentially expressed between days within a tissue were identified with linear regression, and univariate regression was used to identify genes commonly up-regulated and down-regulated across all tissues. Gene set enrichment analysis showed genes commonly up regulated among the three tissues enriched gene ontologies primary metabolic processes, macromolecular complex assembly and negative regulation of apoptosis ontologies. Genes enriched in transcription regulator activity showed the common up regulation of 2 core molecular clock genes, ARNTL and CLOCK. Commonly down regulated genes enriched Rhythmic process and included: NR1D1, DBP, BHLHB2, OPN4, and HTR7, which regulate intracellular circadian rhythms. Changes in mammary, liver and adipose transcriptomes at the onset of lactation illustrate the complexity of homeorhetic adaptations and suggest that these changes are coordinated through molecular clocks.

Evaluation of interference by insulin-like growth factor I (IGF-I) binding proteins in a radioimmunoassay for IGF-I in serum from dairy cows

Insulin-like growth factor I (IGF-I) circulates in serum bound to a number of different binding proteins (BPs). With antibodies currently available, BPs must be dissociated and inactivated or removed from serum prior to measurement of IGF-I by radioimmunoassay (RIA). Serum samples which spanned a 13-fold range in IGF-I concentration were obtained from lactating dairy cows and used to develop conditions for assay of IGF-I with minimal interference from BPs. Removal of BPs from serum by acid-ethanol extraction resulted in interference in the RIA. Therefore, serum was incubated with 0.1 M glycyl-glycine HCl to inactivate BPs as suggested by Underwood et al. Time, temperature and pH were optimum when serum was incubated for 48 hr at 37 C, pH 3.7. Binding protein inactivation was evaluated by ability of glycyl-glycine incubated serum to reassociate with 125I-IGF-I. In addition, BPs isolated by gel filtration of glycyl-glycine incubated serum were tested for interference in the RIA. The concentration of IGF-I in serum where inactivated BPs were removed by acid gel filtration was compared to corresponding glycyl-glycine incubated serum. There was a 1:1 relationship which intersected at zero indicating that total IGF-I could be measured. Therefore, incubation of serum with glycyl-glycine is a reliable method for measuring total IGF-I in serum from dairy cows.

LACTATION BIOLOGY SYMPOSIUM: Circadian clocks as mediators of the homeorhetic response to lactation

The transition from pregnancy to lactation is the most stressful period in the life of a cow. During this transition, homeorhetic adaptations are coordinated across almost every organ and are marked by changes in hormones and metabolism to accommodate the increased energetic demands of lactation. Recent data from our laboratory showed that changes in circadian clocks occur in multiple tissues during the transition period in rats and indicate that the circadian system coordinates changes in the physiology of the dam needed to support lactation. Circadian rhythms coordinate the timing of physiological processes and synchronize these processes with the environment of the animal. Circadian rhythms are generated by molecular circadian clocks located in the hypothalamus (the master clock) and peripherally in every organ of the body. The master clock receives environmental and physiological cues and, in turn, synchronizes internal physiology by coordinating endocrine rhythms and metabolism through peripheral clocks. The effect of the circadian clock on lactation may be inferred by the photoperiod effect on milk production, which is accompanied by coordinated changes in the endocrine system and metabolic capacity of the dam to respond to changes in day length. We have shown that bovine mammary epithelial cells possess a functional clock that can be synchronized by external stimuli, and the expression of the aryl hydrocarbon receptor nuclear translocator-like gene, a positive limb of the core clock, is responsive to prolactin in bovine mammary explants. Others showed that 7% of genes expressed in breasts of lactating women had circadian patterns of expression, and we report that the diurnal variation of composition of bovine milk is associated with changes in expression of mammary core clock genes. Together these studies indicate that the circadian system coordinates the metabolic and hormonal changes needed to initiate and sustain lactation, and we believe that the capacity of the dam to produce milk and cope with metabolic stresses in early lactation is related to her ability to set circadian rhythms during the transition period.

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three‐week‐old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo‐alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo‐alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo‐alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones. J. Cell. Physiol. 180:298–304, 1999.

Characterization of mammary stromal remodeling during the dry period.

During the dry period between successive lactations, the mammary gland of dairy cows undergoes extensive remodeling that is marked by phases of involution and mammogenesis. Changes in the mammary epithelium during the dry period have been well characterized; however, few studies have examined the changes that occur in stromal tissue. The objective of this study was to characterize changes that occur in mammary stroma during the dry period. Mammary biopsies were taken from 9 multigravid Holstein cows in late lactation, at 1 wk after dry-off, 3 wk before expected calving date, and 1 wk before expected calving date. Tissue was fixed in formalin, embedded in paraffin, and cut into 5-mum sections. Sections were stained with hematoxylin and eosin or with immunohistochemistry for expression of smooth muscle alpha actin (SMA), fibronectin, stromelysin-1 (MMP-3), transforming growth factor-beta1 (TGF-beta1), and TGF-beta receptor 2 (TGF-betaR2). Images of tissues were captured with light microscopy, and imaging software was used to measure intralobular stromal area, number of activated fibroblasts, as identified by expression of SMA, and percentage of intralobular stromal area expressing fibronectin, MMP3, TGF-beta1, and TGF-betaR2. Analyses of variance were conducted and statistical differences were based on the least squares means of biopsy stage. Number of activated fibroblasts was greater at 1 wk dry than at 1 wk before calving (2,720 vs. 1,800 cells/mm(2)), percentage intralobular stromal area was greater at 1 wk dry (32%) and 3 wk before calving (37%) than at 1 wk before calving (25%), and TGF-beta1 expression decreased 15% from late lactation to the dry period. The percentages of stromal area expressing fibronectin, MMP-3, and TGF-betaR2 and the percentage of myofibroblasts were not different across biopsy stages. These results support the concept that stromal expression of transforming growth factor-beta1 and fibroblast proliferation may be important for remodeling during the dry period.