Progress 06/01/06 to 05/31/09
Outputs
OUTPUTS: Our overall objective was to identify key changes in mammary infrastructure during prepartum mammary remodeling and to determine the effect of transforming growth factor-beta (TGFB1) on remodeling of the gland during late gestation and the dry period. We report these findings in 2 manuscripts. The objective of the first study was to determine if mammary remodeling during the dry period of dairy cows is characterized by a transient increase in stromal fibronectin, myofibroblasts, stromelysin (MMP-3), transforming growth factor-beta1 (TGFB1) and its receptor, TGFBR2, as well as measure differential contribution of epithelial and stromal cell population to mammary modeling 3 weeks prepartum using comparative transcriptome analysis. The objective of the second study was to determine if TGFB1 increases mammary stromal remodeling during the dry period. Mammary biopsies were obtained from nine cows at: late lactation, 1 week after dry-off, 3 weeks before expected calving, and 1 week before expected calving. For objective 2, tissue was incubated for 2 h with 0 or 5 ng TGFB1/ml. Intralobular stromal area was greater at 1 week dry and 3 weeks before calving than at 1 week before calving (P=0.02). The number of fibroblasts was greater at one week dry than 1 week before calving (P=0.02). Stromal TGFB1 was 15% lower during the dry period than in late lactation (P=0.02). The number of myofibroblasts and the expression of fibronectin, MMP-3, or TGFBR2 did not differ. Epithelial and stromal tissue transcriptomes were significantly different and showed that remodeling of the gland was primarily due to transcriptional activity in stromal tissue including processes related to wound healing, organogenesis and cell proliferation. Incubation with TGFB1 had no effect on the percent intralobular stroma area expression of fibronectin or MMP-3. The number of myofibroblasts increased by 19% in the intralobular stroma (P=0.03) and the percent of myofibroblasts tended to increase by 4% (P=0.06) in tissue incubated with TGFB1. Based on these findings, TGFB1 may be important in the activation of stromal cells during involution and mammogenesis of the bovine mammary gland. PARTICIPANTS: Lindsey De Vries, a masters level graduate, defended her thesis based entirely on this study and was awarded a Master of Science degree in May 2009. Ms. De Vries is first author on the two manuscripts resulting from this study and she also presented 2 abstracts on data from this study during the 2008 and 2009 ADSA-ASAS annual meeting. At the 2009 ADSA-ASAS meeting, an undergraduate student, Kayla Weiss, also presented an abstract based on research she did for this study. She won second place in the undergraduate original research division. Additionally, Kayla presented a poster of this research at the 2009 University Undergraduate Research and Arts Forum at Michigan State University. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A dairy cow spends 15 percent of her life not lactating because she has a typical sixty day dry period. During this period, she is preparing for calving and her next lactation and she eats for maintenance and pregnancy but produces no milk. If the dry period was shortened without any impact on the subsequent lactation, animal wastes and land use per unit milk produced would be decreased leading to an increase in efficiency and profitability in dairy farming. However, research to date indicates that shortening the dry period results in decreased milk yield in subsequent lactations. Our long term goal is to understand the remodeling processes that occur in the mammary gland during the dry period so that successful strategies could be developed to shorten it. Firstly, our study showed that serial mammary biopsies during the dry period do not negatively impact milk production in the subsequent lactation. This has broad impacts on the dairy research community and indicates that there is no long term impact on animal health. Research animals can be returned to the production herd, with no negative impacts on subsequent milk production. Secondly, data from this study indicate that TGFB1 plays a role in remodeling during the first week following cessation of milking, and thus provides a tool for understanding dry period dynamics. Thirdly, Lindsey De Vries, a masters level graduate, defended her thesis based entirely on this study and was awarded a Master of Science degree in May 2009. Ms. De Vries is first author on the two manuscripts resulting from this study and she also presented 2 abstracts on data from this study during the 2008 and 2009 ADSA-ASAS annual meeting. Ms, De Vries is currently working in the dairy industry. At the 2009 ADSA-ASAS meeting, an undergraduate student, Kayla Weiss, also presented an abstract based on research she did for this study. Kayla won second place in the undergraduate original research division at the American Dairy Science Association national meeting in 2009.
Publications
- K Weiss, L De Vries, H Dover, T Casey, J Liesman, M VandeHaar, K Plaut. 2009. The Effect of TGFB-1 on Cell Proliferation in the Bovine Mammary Gland during Involution and Mammogenesis. Joint ADSA-ASAS Annual Meeting 2009. Abstract #33101
- L. De Vries, J. Liesman, K. Weiss, H. Dover, T. Casey, M. VandeHaar, K. Plaut. 2009. The Effects of TGF-B1 on Mammary Stroma during the Dry Period of Dairy Cows. Joint ADSA-ASAS Annual Meeting 2009. Abstract # 31669
- De Vries, L, M VandeHaar, T Casey, T Petzke, H Dover, J Liesman, K Plaut. Characterization of mammary stromal remodeling during the dry period. 2009 (in final revision).
- De Vries, L., J. Liesman, K. Weiss, H. Dover, T. Casey, M. VandeHaar, K. Plaut. The effects of TGFβ-1 on Mammary Stromal Remodeling During the Dry Period. 2009 (in progress). Casey, T., Dover, H., Liesman, J., Steibel, J.P., DeVries, L., Weiss, K., Kiupel, M., VandeHaar, M., and Plaut, K. Transcriptome Analysis of Epithelial and Stromal Contributions to Mammogenesis in Prepartum Dry Cows. 2009 (in progress).
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Progress 06/01/07 to 05/31/08
Outputs
OUTPUTS: Our overall objective is to identify key changes in mammary infrastructure and gene signaling pathways during prepartum mammary remodeling and to determine the effect of transforming growth factor-beta (TGF-beta) on remodeling of the gland during late gestation. Specifically during this reporting period we: a) measured the effect of serial mammary gland biopsies during the dry period on milk production; b) identified changes during the dry period in intralobular stromal area, fibronectin expression, and percent activated fibroblasts (expressing smooth muscle alpha actin [SMA]); and c) established a protocol for isolating total RNA from laser capture microdissected stromal and epithelial tissue. Briefly, serial mammary biopsies were completed on the 9 pregnant, multigravid Holsteins used in this study. Approximately 1 g of mammary tissue was biopsied at 275-290 days in milk (biopsy 1), at 7 days dry (biopsy 2), and at 3 weeks (biopsy 3) and 1 week before expected calving date (biopsy 4). Immediately after biopsies 1 and 4, a teat cannula was inserted into the biopsied quarter to drain any accumulated blood. Cows were hand stripped at each milking until blood clots were no longer observed in milk. Analysis of milk yields during the lactation prior to and following biopsies indicated that milk production in biopsied cows was not different from the herd. Further the use of teat cannula after biopsy appears to improve cow comfort. Secondly, we have begun to measure the changes that occur in the stroma during the dry period by cutting 7 micron sections from formalin-fixed paraffin embedded biopsied tissue. Tissue was stained with H&E to examine changes in mammary morphology and measure changes in intralobular stromal area and used for immunohistochemistry to measure fibronectin expression and to detect activated fibroblasts expressing SMA. We found that: a) the intralobular stromal area decreased relative to the lumen and epithelial area from 48% to 32% between biopsy 3 and 4 (P<0.01), b) percent activated fibroblasts was similar for the first 3 biopsies but increased from 20% to 33% between biopsies 1 and 4 (P=0.04), and c) fibronectin expression did not change across time points. These two studies were reported at the 2008 combined ADSA-ASAS meeting. Thirdly, biopsied tissue exposed to explant culture were used to isolate epithelial and stromal tissue for microarray analysis. At completion of a 2 hour culture in media supplemented with insulin, hydrocortisone and 0 or 2.5 ng/ml TGF-beta1, tissue was placed in OCT, submerged in liquid nitrogen and stored at -80C. Tissue was cryosectioned into 7 micron sections, placed on glass slides, and stained using RNase free techniques. Epithelial and stromal tissue were microdissected using laser capture and total RNA was isolated. Bioanalysis of RNA showed that adequate amounts of good quality RNA is being isolated using these methods. RNA will be used for microarray analysis of gene expression changes in these two tissues during the dry period and to measure the effect of TGF-beta on gene expression in these tissues at the varying time points. PARTICIPANTS: PI: Karen Plaut; PD: Michael VandeHaar; Lindsey De Vries; Heather Dover; Osman Patel; Theresa Casey; Jim Liesman; Tracy Petzke; Monica Vanklompenberg TARGET AUDIENCES: This project provided experiential learning opportunities for several undergraduate students, who assisted with biopsies and stripping the cows following biopsies. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A dairy cow spends 15 percent of her life not lactating during the typical sixty day dry period. During this period, she is preparing for calving and her next lactation and she eats for maintenance and pregnancy but produces no milk. If the dry period was shortened without any impact on the subsequent lactation, animal wastes and land use per unit milk produced would be decreased leading to an increase in efficiency and profitability in dairy farming. However, research to date indicates that shortening the dry period results in decreased milk yield in subsequent lactations. Our long term goal is to understand the remodeling processes that occur in the mammary gland during the dry period so that successful strategies could be developed to shorten it. Our preliminary data show that mammary composition in terms of cell type, tissue content, and extracellular protein content change from late lactation throughout the dry period. We expect our explant culture to show that TGF-beta accelerates changes during the dry period, having minimal effects during late lactation and just prior to calving and maximal effects during early and the mid-dry period. Therefore the potential impact of this work is to shorten the dry period without negatively affecting milk production and thus increasing profits for the dairy farmer. Further our study shows that serial mammary biopsies during the dry period do not negatively impact milk production in the subsequent lactation has broad impacts on the dairy research community. Research animals can be returned to the production herd, at little to no cost to the research project, as following a week or so of respite, their productions appears to return to normal levels. Serial biopsies also should decrease variability in studies due to inter-animal differences. In terms of outreach and teaching, several undergraduate students have partaken in this study as part of their undergraduate research program, and the thesis project of 1 graduate student (Lindsey De Vries) is focused on identifying key changes in mammary infrastructure using histochmeistry and immunohistochemistry. This graduate student presented her findings at the joint ADSA-ASAS 2008 meeting.
Publications
- Dover, H, M VandeHaar, J Liesman, O Patel, L De Vries, K Plaut. 2008. Serial Mammary Biopsies in Cows Do Not Alter Overall Milk Production. Joint ADSA-ASAS Annual Meeting. 2008. Abstract #: TH115.
- De Vries, L, M VandeHaar, T Casey, T Petzke, H Dover, J Liesman, K Plaut. Stromal Changes in the Bovine Mammary Gland During Involution and Mammogenesis. Joint ADSA-ASAS Annual Meeting. 2008. Abstract #687.
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Progress 06/01/06 to 05/31/07
Outputs
The overall objective of this project is to identify key changes in mammary infrastructure and gene signaling pathways during prepartum mammary remodeling and to determine the effect of transforming growth factor-beta (TGF-beta) on remodeling of the gland during late gestation. Specifically we will 1) Characterize mammary tissue remodeling from lactation through the dry period. 2) Measure if TGF-beta1 stimulates remodeling in mammary explant cultures during lactation, involution and prepartum mammogenesis. 3) Determine if TGF-beta1 stimulates expression of regulatory genes in mammary explants and develop a model that elucidates TGF-beta's action. To accomplish these objectives we have begun to collect mammary tissue by serial biopsy of gravid, multiparous Holstein cows in late lactation and the dry period. Biopsy tissues was taken at approximately 275 days in milk , 1 week dry, 3 weeks prepartum, and 1 week prepartum. Tissue from the biopsies were cut into explants and
cultured for 2 h in the presence of insuiln, hydrocortisone and 0 or 2.5 ng/ml TGF-beta1. Bromodeoxyuridine (BrdU) is added to measure rate of cell proliferation. Other tissue sections were placed in 1) buffered formalin for histochemical analysis of ultrastructural and morphological changes, or 2) OCT and plunged in liquid nitrogen to preserve for laser capture microdissection of stromal and epithelial tissues followed by isolation of total RNA for microarray analysis of changes in gene expression during the dry period. Following culture, tissue was placed in buffered formalin or OCT and will be used as described above. We have completed biopsies and explant culture on 2 groups of cows with 2 and 4 cows in each group and plan to start the third group late summer 2007. Thus far we have developed the methodology to biopsy mammary quarters and obtain approximately 1gm of tissue at each time point. Our preliminary data on explant culture conditions from the first group of cows allowed us
to optimize culture conditions for measuring the effect of TGF-beta on remodeling. Protocols to evaluate mammary morphology and ultrastructure have been developed using sections stained with hematoxylin and esoin, BrdU labeling of proliferating cells, immunohistochemistry for expression of smooth muscle actin, fibronectin, laminin and vimentin. Preliminary data from the first groups of cows demonstrate: 1) The 4 biopsy time points selected show distinct developmental changes that occur in the mammary gland during the dry period. 2) The gland is being actively remodeled during the dry period as is evident by changes in percent myofibroblasts, and proliferation rates of epithelial and stromal cells, as well as amounts of extracellular matrix proteins at each time point.
Impacts
A dairy cow spends 15 percent of her life not lactating during the typical sixty day dry period. During this period, she is preparing for calving and her next lactation and she eats for maintenance and pregnancy but produces no milk. If the dry period was shortened without any impact on the subsequent lactation, animal wastes and land use per unit milk produced would be decreased leading to an increase in efficiency and profitability in dairy farming (Rastani et al., 2005). However, research to date indicates that shortening the dry period results in decreased milk yield in subsequent lactations. Our long term goal is to understand the remodeling processes that occurs in the mammary gland during the dry period so that successful strategies could be developed to shorten it. Our preliminary data show that mammary composition in terms of cell type, cell growth and death, and extracellular protein content change from late lactation throughout the dry period. We expect our
explant culture to show that TGF-beta accelerates changes during the dry period, having minimal effects during late lactation and just prior to calving and maximal effects during early and the mid-dry period. Therefore the potential impact of this work is to shorten the dry period without negatively affecting milk production and thus increasing profits for the dairy farmer.
Publications
- No publications reported this period
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