Progress 10/01/02 to 09/30/07
Outputs
OUTPUTS: This project focused on defining biological mechanisms which explain the mammogenic effects of the ovary and growth hormone in control of prepubertal heifer mammary development. Multiple cooperative experiments were completed, data analyzed, and summaries prepared. Primary outputs were: (1) publication of eight scientific journal articles, (2) ten presentations at professional meetings, (3) three major invited talks (national and international) and (4) thesis publication and graduation of two MS and dissertation publication and graduation of two Ph.D. students. The major audiences for this work are scientists and dairy industry professionals. Dissemination of our results has produced multiple contacts to pursue addition cooperative studies. Furthermore, three additional manuscripts are in review.
PARTICIPANTS: Collaborators: A. V. Capuco, Bovine Functional Genomics Laboratory, USDA-ARS, Beltsville MD 20705; S. E. Ellis, Department of Animal and Veterinary Sciences, Clemson University, Clemson SC 29634; H. Jiang, Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg VA 24061; M. E. Van Amburgh, Department of Animal Sciences, Cornell University, Ithaca, NY 14853; R. D. Howard, Virginia Maryland Regional College of Veterinary Medicine, Blacksburg, VA 24061; M. S. Weber Nielsen, Department of Animal Science, Michigan State University, East Lansing, MI 48824; M. J. VandeHaar, Department of Animal Science, Michigan State University, East Lansing, MI 48824; M. L. McGilliard, Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; R. E. Pearson, Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; Training and Professional Development: S. D. K.
Berry, Ph.D. Program Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; K. M. Daniels, Ph.D. Program Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; K. M. Daniels, MS Program Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; J.W. Forrest, MS Program Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Impacts
Results of this project partially define mechanisms explaining how the ovary and growth hormone (GH) control prepubertal heifer mammary development and interactions between these regulators and mammary tissue growth factors and extracelluar matrix proteins. Our hypothesis, that the period between birth and four months of age is critical and impacted by nutrition was confirmed. Growth of the mammary parenchymal tissue (the portion of the gland containing the mammary ducts) increased approximately 60-fold between 30 and 120 days of age. This finding clearly is contrary to the general view that mammary development in very young calves is relative inconsequential and unimportant. We were the first to apply proteomic techniques to determine the impact of nutrition on expression of mammary tissue proteins during mammary development in the bovine. Mammary tissue from heifers reared for (Moderate (M) 650 g/d or High (H) 950 g/d of daily gain) and slaughtered at one of two body
weights (BW, 200 or 350 kg) were used to determine effects on protein expression. Diet altered expression of 131 proteins, while heifer BW impacted the expression of 108 proteins. The 22 most highly impacted (statistically) spots were excised and submitted for mass spectrometry analyses. We concluded that a proteomic approach is an effective tool for the identification of proteins involved in bovine mammary development. In other experiments, we found that impaired mammary growth caused by ovariectomy of prepubertal heifers was associated with reduced synthesis of local mammary tissue insulin-like growth factor I (IGF-I) but increased local production of IGF-I binding protein 3. As anticipated the proportion of proliferating mammary epithelial cells was dramatically lower in ovariectomized animals but to our surprise the proportion of cells expressing estrogen receptor was greater in ovariectomized heifers. We also observed that reduced mammary cell proliferation in ovariectomized
heifers was accompanied by increased deposition of the extracellular matrix protein fibronectin (FN). In a follow-up study, we noted that growth of cultured bovine mammary cells was similar when stimulated by the addition of serum (5 percent) or IGF-I (5, 50 or 200 ng per ml) when cells plated on tissue culture plastic, laminin or collagen but growth rates were reduced for cells cultured on plates coated with FN. This finding further supports a role for FN in control of mammary development. In addition, changes in mammary development due to nutritional manipulation occurred in corresponding fashion with deposition of FN i.e. lower proliferation corresponding with greater FN deposition. Treatment with exogenous GH increased the proportion of proliferating epithelial cells but GH had no impact on the proportion of cells expressing estrogen receptor. In overview, these results illustrate the dynamic plasticity of prepubertal bovine mammary development. They also demonstrate opportunities
to modify development and function as well as offer insight into complex interactions between classic mammogenic hormones and local tissue regulators.
Publications
- Daniels, K. M., S. R. Hill, K. F. Knowlton, R. E. James, M.L. McGilliard, and R.M. Akers. 2007. Effects of milk replacer composition on selected blood metabolites and hormones in pre-weaned Holstein heifers. J. Dairy Sci. 90: (Suppl 1):84.
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Progress 10/01/05 to 09/30/06
Outputs
In a first study, Holstein heifer calves were fed one of four milk replacer diets varying in protein and fat content. Calves were fed either 450 g of a milk replacer powder containing 20 percent protein and 20 percent fat (20:20), 930 g of a 28 percent protein:20 percent fat replacer (28:20), 940 g of a 28 percent protein: 28 percent fat (28:28L) or 1,500 g of a 28 percent protein: 28 percent fat milk replacer powder (28:28H). A calf starter containing 20 percent whole cottonseed hulls was offered free choice. At age 30d calves were injected with 5-Bromo-2'-deoxyuridine (BrdU; 5 mg/kg) for 4 consecutive days to label all cells in the S-phase of the cell cycle. This protocol allowed for the tracking of label-retaining cell frequency, thereby aiding the identification of mammary stem-like cells. Calves were sacrificed at 8 weeks. On going measurements will evaluate body composition, nutrient excretion, and concentrations of circulating metabolites and hormones. Data
confirm lowest average daily gain (kg/d) for 20:20; 0.38 kg/d and highest for 28:28H; 0.76 kg/d. Mammary tissue samples were collected in three regions (distal, medial, and proximal) oriented along both a central and lateral axis positioned from the teat to the body wall through the parenchymal tissue of a randomly selected mammary gland. Specific to mammary development, our working hypothesis is that faster rates of gain alter the development, persistence, or activity of populations of putative mammary epithelial stem cells and thereby impact mammary development. We further propose that changes in cell populations are modified by local mammary tissue expression of selected regulatory proteins. Preliminary results demonstrate the presence of small numbers of BrdU label retaining epithelial and stromal cells at the time of slaughter. This supports use of the approach to identify populations of stem-like cells in the developing udder. We will determine the percentage of cells
(epithelial and stromal) that express Ki67 and/or PCNA as an acute measure of rates of cell proliferation at the time of slaughter. In addition we have validated real time polymerase chain reaction methods to measure expression of mRNA for insulin-like growth factor one (IGF-I), IGF-I receptor, IGF-I binding proteins 1-6, growth hormone receptor, and estrogen receptor. We have confirmed presence of aromatase in mammary tissue stroma via immunocytochemistry and are currently validating a method to directly measure enzyme activity. Conformation of enzyme activity would support our secondary hypothesis that local mammary tissue estrogen synthesis impacts mammary development in prepubertal heifers. We will also utilize immunocytochemical methods to relative expression and localization of selected extracellular matrix molecules (fibronectin, collagen IV, and laminin) in mammary tissue samples.
Impacts
About 3 million heifers are needed each year to replace culled dairy cows and to provide the foundation for increased production per animal. Changes in expression of local tissue proteins in mammary glands of prepubertal heifers are modified by alterations in diet composition, rate of gain, ovarian activity and levels of growth hormone. These proteins include insulin-like growth factor one (IGF-I), IGF-I receptor, IGF-I binding proteins, estrogen receptor, extracellular matrix proteins, aromatase and others. Moreover, impacts of these local proteins on the growth of mammary ducts are likely manifested by changes in selected populations of putative bovine mammary stem cells.
Publications
- Akers, R.M. 2006. Major advances with hormone and growth factor regulation of mammary growth and lactation in dairy cows. Journal Dairy Sci. 89: 1222-1234.
- Daniels, K. M., K. E. Webb, Jr., M. L. McGilliard, M. J. Meyer, M. E. Van Amburgh and R. M. Akers. 2006. Effects of body weight and nutrition on mammary protein expression profiles in Holstein heifers. Journal Dairy Science 89: 4276-4288.
- Akers, R.M, A.V. Capuco, and J.E. Keys. 2006. Mammary histology and alveolar cell differentiation during late gestation and early lactation in mammary tissue of beef and dairy heifers. Livestock Science 155: 44-49.
- Porter, R.M., R.M. Akers, R.D. Howard, and K. Forsten-Williams. 2006. Transcriptional and proteolytic regulation of the insulin-like growth factor-I system of equine articular chrondrocytes by recombinant equine interleukin-1 beta. Journal Cellular Physiology 209: 542-550.
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Progress 10/01/04 to 09/30/05
Outputs
Mammary tissue from heifers reared for (Moderate (M) 650 g/d or High (H) 950 g/d of daily gain) and slaughtered at one of two body weights (BW, 200 or 350 kg) were used to determine effects on protein expression. Cytosolic mammary gland extracts were used to generate two-dimensional protein maps. Proteome maps were constructed using PDQuest software (Biorad). Densities of 820 protein spots were analyzed using the Mixed Procedure of SAS. Protein spots were characterized by changes in expression in response to increased BW and/or dietary treatment. Diet altered expression of 131 protein spots, while heifer BW impacted the expression of 108 spots. The 22 most highly impacted (statistically) spots were excised and submitted for mass spectrometry analyses. Returned protein names and accession numbers were used in National Center for Biotechnology Information database searches to obtain information on the identified proteins. For example, one of the proteins that differed
by dietary treatment, transferrin, a binding protein (BP) of insulin-like growth factor BP-3, was identified via these methods. Possible roles of this and other proteins in mammary development were described. We conclude that a proteomic approach is an effective tool for the identification of proteins involved in bovine mammary development. Mammary tissue morphology in samples from 54 heifers reared on M or H treatments slaughtered at 150, 200, 250, 300, or 350 kg BW was evaluated as well. Mean percentages of mammary parenchyma occupied by interlobular stroma, epithelium, lumen, and intralobular stroma averaged 28, 20, 7, and 45 percent, respectively. Percentages did not differ by BW or treatment, nor was there an interaction between treatment and BW. However, number of epithelial (31 vs. 47) and luminal (24 vs. 38) structures per image increased between 150 and 350 kg BW. There were dramatic changes in ductal development between 150 and 350 kg BW but differences in rate of gain,
accomplished by feeding varying amounts of the same diet, did not impact tissue characteristics. But this does not address the possibility that there might be specific effects of individual diet formulations or interactions of diet with rate of gain.
Impacts
About one third of the 9.3 million lactating dairy cows are replaced as heifers each year. Our data indicated that local tissue factors within the developing udder regulate growth of mammary ducts and therefore impact future productive potential. Altering diet composition and rate of gain provide possible management tools to positively impact milk production potential of these replacement heifers.
Publications
- Brown, E. G., M. J. VanderHaar, K. M. Daniels, J. S. Liesman, L. T. Chapin, J. W. Forrest, R. M. Akers, R. E. Pearson, and M. S. Weber Nielsen. 2005. Effect of increasing energy and protein intake on mammary development in heifer calves. J. Dairy Sci. 88: 595-603.
- Akers, R. M., S. E. Ellis, S. D. Berry. 2005. Ovarian and IGF-I control of mammary development in prepubertal heifers. Domestic Animal Endocrinology. 29: 259-267.
- Daniels, K. M., 2004 Effects of bodyweight and plane of nutrition on mitogenic capacity of mammary extracts in cell culture, mammary growth and development, and protein expression profiles of mammary tissue in Holstein heifers, Virginia Polytechnic Institute and State University, MS thesis.
- Daniels, K. M., K. E. Webb, Jr., M. L. McGilliard, M. J. Meyer, M. E. Van Amburgh and R. M. Akers. 2005. A proteomic approach to evaluate the effects of body weight and plane of nutrition on protein expression profiles of mammary gland extracts from Holstein heifers. J. Dairy Sci. 88:(Suppl 1):365).
- Daniels, K. M., M. L. McGilliard, P. L. Boyle, M. J. Meyer, M. E. VanAnburgh, A. V. Capuco, and R. M. Akers. 2005. Effects of bodyweight and plane of nutrition on histological development of mammary tissue in Holstein heifers. J. Dairy Sci. 88(Suppl 1):288.
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Progress 10/01/03 to 09/30/04
Outputs
Our broad objectives are to determine the biological mechanisms which explain mammogenic effects of the ovary and growth hormone and interactions with locally mammary tissue produced growth factors, including insulin-like growth factor (IGF-I), insulin-like growth factor binding proteins (IGFBP) and extracellular matrix (ECM) proteins. We hypothesize that the period between birth and four months of age, during which time ovarian and GH stimulation of mammary ductal development occurs, is critical and is impacted by nutrition. A first experiment was designed to determine if increased energy and protein intake from 2 to 14 wk of age would affect mammary development in heifer calves. At 2 wk of age, Holstein calves were assigned to 1 of 4 treatments (HH, HL, LH, and LL) with 2 levels of energy intake (High or Low) and 2 periods of growth (2 to 8 and 8 to 14 wk of age). At 14 wk, parenchyma at the stromal interface, mid-gland, and above the gland cistern were collected,
fixed, and embedded in paraffin. Digital images of stained sections were used to determine tissue composition (percentage epithelium, lumen, and stroma). Immunochemistry revealed estrogen receptor (ER) and Ki67 (nuclear proliferation antigen) positive cells, type IV collagen, fibronectin (FN), and laminin. Images representing 4 increasing grades were used to quantify ECM protein deposition. As a percent lumenal and stromal were 3.5 units lower and 4.0 units higher, respectively, in HH and HL heifers. Percent Ki67 labeled cells in terminal ductular units and subtending ducts was 2.1 and 1.4 units lower for the same feeding level combination. FN deposition was also increased in HH and HL heifers. Changes in FN deposition were most closely related to tissue growth differences. In a second experiment we showed that growth of bovine mammary cells in culture was similar when stimulated by the addition of serum (5 percent) or IGF-I (5, 50 or 200 ng per ml) for cells cultured on tissue
culture plastic, laminin, collagen, or poly-lysine coated plates but growth rates were substantially reduced for cells cultured on plates coated with FN. These results support our earlier report (Berry et al., 2003) indicating poor mammary growth in ovariectomized heifers was associated with increased tissue abundance of FN.
Impacts
About one third of the 9.3 million lactating dairy cows are replaced as heifers each year. Our data indicated that local tissue factors within the developing udder regulate growth of mammary ducts and therefore impact future productive potential. Specifically, local tissue synthesis of extracellular matrix proteins likely modulates the capacity of growth factors and hormones to stimulate growth of mammary epithelial cells.
Publications
- Forrest, J. W., 2003. Effect of varying energy intakes on mammary growth and development in prepubertal heifers, Virginia Polytechnic Institute and State University, MS thesis.
- Elhadj, S., R. M. Akers, K. Forsten-Williams. 2003. Chronic pulsatile shear stress alters insulin-like growth factor-I (IGF-I) binding protein release in vitro. Annals Biomedical Engineering 31: 163-170.
- Berry, S. D. K., R. D. Howard, and R. M. Akers. 2003. Mammary localization and abundance of laminin, fibronectin, and collagen IV proteins in prepubertal heifers. J. Dairy Sci. 86: 2864-2874.
- Burk, R., W. B. Staniar, D. S. Kronfeld, R. M. Akers, and P. A. Harris. 2003. Insulin-like growth factor binding proteins fluctuate with age in growing Thoroughbred foals. Proc. Equine Nutr. Phys. Soc, East Lansing, MI 18: 84.
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Progress 10/01/02 to 09/30/03
Outputs
We determined the effect of ovariectomy (OVX) and epithelial-stromal interactions on mammary development and local expression of insulin-like growth factor I (IGF-I) and insulin-like growth factor I binding proteins (IGFBP) mRNA in prepubertal heifers. Epithelium-free fat-pads were prepared in two glands in each of fourteen 1 to 3 month old Holstein heifers. Eight of the calves were also ovariectomized. Serum concentrations of GH, IGF-I and PRL were not affected by OVX. At age six months, calves were sacrificed to provide mammary samples of parenchyma (PAR), epithelium-free fat pad (CFP) and intact fat pad (MFP). Mammary mass (g) was reduced in OVX calves (130 vs. 304). Uterus weight (g) was also reduced by OVX (14.5 vs. 30.4). In support of our hypothesis that local IGF-I mediates prepubertal mammary development, mRNA expression of IGF-I was lower in OVX than control calves (62.1 vs. 91.6 density untis). Specific binding of IGF-I to mammary parenchymal microsomes was
also reduced by OVX vs. 868 dpm), suggesting decreased sensitivity to IGF-I. Expression of IGFBP-3 and IGFBP-5 mRNA were not influenced by OVX. Expression of IGF-I, IGFBP-3 and IGFBP-5 mRNA did not differ between CFP and MFP suggesting that expression of these factors was not influenced by interactions between stroma and developing PAR. However, interactions between the ovary and the local IGF-I axis act to optimize the availability and effectiveness of IGF-I within the gland to stimulate mammary growth. The objectives of a second study were 1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells to mammary tissue extracts from high and low-fed heifers, and 2) to determine whether mammary tissue extracts from (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to
a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged nine months, stimulated significantly more proliferation of MAC-T cells, as measured by thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (80,600 vs. 41,900 dpml). These observations suggested that MAC-T cells would be a suitable alternative to primary mammary epithelial cells for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of
complex interactions between local growth factors and systemic hormones.
Impacts
IGF-I and its family of related proteins (IGFBP) and IGF receptor are critical in regulation of mammary development and function. About one third of the 9.3 million lactating dairy cows are replaced as heifers each year. Our data indicate that local tissue factors within the developing udder regulate growth of mammary ducts and therefore impact future productive potential.
Publications
- Berry, S. D., R. D. Howard, P. M. Jobst, and R. M. Akers. 2003. Interactions between the ovary and the local IGF-I axis modulate mammary development in prepubertal heifers. J. Endocrinology 177: 295-304.
- Berry, S. D. K., M. S. Weber Nielsen, P. L. Boyle, and R. M. Akers. 2003. Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic capacity of extracts of heifer mammary tissue: Effects of nutrition and ovariectomy. Domestic Animal Endocrinol. 25: 245-253.
- Forrest, J. W., R. M. Akers, R. E. Pearson, E. G. Brown, M. J. VanderHaar, and M. S. Weber Nielsen. 2003. Effect of varying energy intakes on estrogen receptor, cell proliferation, and tissue composition in mammary tissue of pre-pubertal heifers. J. Dairy Sci. 86: (Suppl 1, page 468).
- Forrest, J. W., R. M. Akers, R. E. Pearson, E. G. Brown, M. J. VanderHaar, and M. S. Weber Nielsen. 2003. Effect of varying energy intakes on the deposition of type IV collagen (Col IV) and fibronectin (FN) in the mammary tissue of pre-pubertal heifers. J. Dairy Sci. 86: (Suppl 1, page 300).
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