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<p> PEA3 is a member of the ets family of transcription factors. It is expressed throughout embryonic development and in mouse mammary adenocarcinomas induced by expression of the receptor tyrosine kinase Neu. Mice lacking PEA3 due to a targeted disruption of the gene, develop normally, however, male mice fail to mate for yet undetermined reasons. To further understand the role of PEA3 in mammary gland development and tumorigenesis, the effects of loss of function of PEA3 were examined in tumor formation and in mammary gland development. </p> <p> Analysis of tumor formation in PEA3 +I+ and PEA3 -/-animals failed to show a statistically significant difference in tumor onset. Loss of PEA3 did not affect the tumor morphology, nor did it inhibit metastasis of these tumors to the lung. These data indicate that PEA3 is not required for tumor formation or metastasis. </p> <p> PEA3 deficient animals displayed defects in branching morphogenesis in the mammary gland. Decreased ductal branching was observed in virgin and pregnant females. Mice with decreased levels of PEA3 expression also exhibited defects in branching morphogenesis, indicating a dosage effect. PEA3 is expressed in the myoepithelial cells during puberty and pregnancy. It is also express in the highly proliferative cap cell layer of the terminal end bud. In the embryonic mammary gland, PEA3 is expressed as early as 10.5 days in the mammary epithelium and continues late in embryogenesis. Expression in the male mammary gland is lost at approximately embryonic day 16. </p> / Thesis / Candidate in Philosophy

Dairy cows inefficiently convert dietary protein to milk protein causing economic and environmental costs. Amino acids (AA), insulin, and glucose significantly enhance muscle protein synthesis efficiencies. The objectives of this research project were 1) to investigate the regulatory effects of essential AA (EAA) and their interactions with insulin, glucose and acetate on mammary protein synthesis rates, 2) to investigate whether branched chain amino acids (BCAA): leucine , isoleucine , and valine , become limiting for milk protein synthesis when Met and Lys supply were not limiting, and 3) to develop a mathematical representation for the EAA and insulin effects on cellular signals for protein synthesis. MAC-T cells were treated with EAA, insulin, glucose, and acetate to observe their individual and interactive effects on phosphorylation of mTOR, rpS6, S6K1, 4EBP1, eEF2, eIF2α, Akt, and AMPK. These signaling effects on protein synthesis rates were examined with mammary tissue slices. A mathematical representation of the insulin and EAA effects was developed. The effects of supplementing BCAA on milk protein synthesis were investigated using nine Holstein cows, assigned to 7 d continuous jugular infusions of saline, Met and Lys, and Met and Lys plus BCAA. Multiple essential amino acids, Leu, Ile, Met, and Thr were able to substantially regulate protein synthesis rates in bovine mammary cells by increasing (P < 0.05) phosphorylation of mTOR, S6k1, 4EBP1, and decreasing (P < 0.10) eEF2 phosphorylation. Insulin considerably (P < 0.10) exerted similar signaling effects in MAC-T cells, independent of EAA. Supplementation of only acetate increased (P = 0.09) mammary cell energy status as indicated by reduced AMPK phosphorylation in MAC-T cells. Neither acetate nor glucose had substantial regulatory effects on mammary protein synthesis rates. Although Met and Lys supplementation increased (P < 0.01) milk protein yields and protein efficiencies, there were no apparent benefits of BCAA supplementation under the feeding circumstances of our study. The developed mathematical model adequately represented the regulatory effects of EAA and insulin. Such mathematical representations of regulatory effects of EAA and their interaction with other nutrients may improve our current AA requirement models to predict AA requirements of dairy cows with increased accuracy. / Ph. D.

Consumer demand for milk fat has declined due to the increased risk of cardiovascular disease associated with consuming a high saturated fat diet. Milk fat synthesis is energetically expensive for the dairy cow, especially during early lactation or periods of poor nutrition. Thus, manipulating milk fat production and composition may promote the synthesis of more market-valuable milk components and improve energy utilization in dairy cows during periods of increased energy demand. Therefore, the objective of the present studies was to identify molecular proteins that regulate fatty acid synthesis in bovine mammary epithelial cells. The regulation of lipogenic genes including acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) is controlled by transcription factors including sterol regulatory element binding protein-1 (SREBP1) and liver X receptor (LXR). In vivo, diet-induced milk fat depression or supplementing diets with polyunsaturated fatty acids inhibits milk fat synthesis by regulating SREBP1 expression. Results confirm that polyunsaturated fatty acids inhibit fatty acid synthesis in bovine mammary epithelial cells by regulating the expression of SREBP1. In hepatocytes, LXR can regulate the transcription of SREBP1 in addition to ACC and FAS. Results confirm that LXR activation enhanced synthesis of fatty acids in bovine mammary epithelial cells by promoting the transcription of FAS and SREBP1. Activation of LXR was unable to prevent the inhibitory effect of polyunsaturated fatty acids on fatty acid synthesis. In the lactating mammary gland, LXR may contribute to the synthesis of fatty acids by regulating the expression of SREBP1. In addition to modifying the expression of lipogenic genes, some enzymes can be phosphorylated by AMP-activated protein kinase (AMPK), an energy-sensing protein, inhibiting their activity. Presence of AMPK mRNA was identified in bovine mammary epithelial cells and activation of AMPK dramatically decreased fatty acid synthesis in bovine mammary epithelial cells. In the lactating mammary gland, AMPK may sense energy availability and regulate milk fat synthesis to control energy utilization. Identification of SREBP1, LXR, and AMPK as regulators of fatty acid synthesis in bovine mammary epithelial cells may lead to the development of technologies allowing dairy producers to modify milk fat production and composition to meet consumer demand and maximize profitability. / Ph. D.

The highly regulated maturation of the mammary gland is poorly understood. Our studies were designed to further characterize the role of ovarian hormones, growth hormone (GH)/IGF axis proteins and extracellular matrix (ECM) in the growth and development of prepubertal mammary glands. Prepubertal heifers were injected with either exogenous GH or subjected to ovariectomy (OVX). Mammary parenchyma (PAR) and mammary fat pad (MFP) were harvested for DNA, protein, lipid, and western blot analysis. Remaining tissues were preserved for histological staining or snap frozen for quantitative real-time PCR. We examined 13 genes that work in conjunction with the extracellular matrix to regulate mammary proliferation and morphogenesis. Administration of GH, while impacting composition of MFP, had no effect on expression of the selected genes; there was a decrease in expression of fibronectin in PAR. Ovariectomy had no effect on gene expression in MFP but decreased expression of epimorphin, a potent regulator of morphogenesis, in PAR. In both experiments, the presence of a 55 kDa band corresponding to androgen converting enzyme aromatase was detected but its expression was unaffected. In another study, we used in vitro cell culture to evaluate the role of ECM in mammary gland maturation and employed quantitative real-time PCR to evaluate gene expression profiles of select genes involved in proliferation and differentiation. Expression of Rac1 was decreased in response to bovine insulin (BI) but increased on collagen I (Col). Expression of aldehyde dehydrogenase was decreased in BI and serum on plastic and on Col in the presence of BI. Expression of IGF binding proteins (BP) 3, -4, and -6 were decreased in the presence of serum on laminin (LM). Also, IGF-BP2 expression was decreased on Col while IGF-BP6 was increased on LM with BI. Clusterin, a ubiquitous non-adhesive ECM protein was not affected by ECM substrate but did increase over time. In conclusion, we propose that the mammary gland is not able to respond to GH at this age and that while OVX did effect the expression of some genes, the presence of aromatase maintained local estrogen concentrations. Furthermore, ECM alone is insufficient to regulate mammary gland development and growth. / Ph. D.

Current data indicates that feeding dairy calves more nutrients in early life allows them to produce more milk in the future. Mechanisms responsible are poorly understood. Thirty-six Holstein heifer calves were fed either a restricted (R; 20.2% crude protein [CP], 19.8% fat, dry matter (DM) basis, fed at 0.44 kg/hd/d, DM basis) or an enhanced (EH; 28.9% CP, 26.2% fat, DM basis, fed at 1.08 kg/hd/day, DM basis) milk replacer (MR) and given either a placebo or estradiol (E2) implant to assess differential responses to E2. Our underlying hypothesis was that calves fed more nutrients are better able to respond to mammogenic stimuli and will have a more developed mammary gland as a result of imposed treatments. Enhanced-fed calves grew at a faster rate, were heavier at weaning, and had more functional mammary tissue (i. e., parenchyma; PAR) mass in the mammary gland at weaning (7.3-fold). Additionally, biochemical composition of the PAR was not impacted by the dietary treatments imposed. Furthermore, EH-fed calves had an increase in the number of actively dividing cells throughout the mammary PAR as well as increased intensity of estrogen receptor expression in the population of cells expressing the estrogen receptor. Enhanced-fed calves had an up-regulation of genes and pathways in the PAR related to metabolism, cellular signaling, and cellular growth. When given E2, EH-fed calves experienced the greatest overall mammary gland development and had the greatest PAR mass without compromised composition. When comparing EH- and R-fed calves given E2, differential expression of genes and pathways related to cell growth, cell signaling, and metabolism was observed. In summary, data indicates that enhanced feeding of calves in early life allows increased responsiveness to mammogenic stimuli and a corresponding increase in mammary development. We suggest that this may at least partly explain the improved future milk production in calves fed in this manner. / Ph. D.