Progress 10/01/05 to 09/30/09
Outputs
OUTPUTS: This project is to determine the functions and the uderlying molecular mechanism of how estrogen receptors mediate mammary epithelial cell proliferation. Estrogen is one of the most important regulators for mammary gland development, and the function of estrogen is mediated by two types of estrogen receptors - ERa and ERb. Lack of any of the estrogen receptors will impair the mammary gland development to some extent, suggesting that both ERa and ERb are required for the full development of mammary gland. In the past year we evaluated the functions of bovine ERa and ERb. Both bovine ERa and ERb are localized in the nucleus and can bind to the ERE sequences to stimulate the expression of reporter genes. In Mac-T cells overexpressing bovine ERa, we did not observe significant effect on cell proliferation. In Mac-T cells overexpressing bovine ERb, we found that cell proliferation was significantly inhibited. FACS analysis on cell cycle showed that more cells were in the G2/M phase, suggesting that bovine ERb may induce G2 arrest in Mac-T cells. Moreover, the inhibitory function of bovine ERb is independent of estrogen binding. PARTICIPANTS: Sivashankar Ramakrishnan, M.S. student, UVM Huining Tan, Postdoc, UVM Yili Zhong, M.S. Student, UVM TARGET AUDIENCES: Researchers in mammary gland biology and lactation. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The general goal was to understand how mammary epithelial cell proliferation is regulated by estrogen receptors. Bovine ERa and ERb full-length cDNAs were subcloned into different protein expression vectors and lentiviral vectors. Bovine ERb is highly expressed during late lactation and involution stages. The inhibitory role of ERb suggests that ERb may accounts for the dramatic apoptosis during these stages.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: This project is to determine the molecular mechanism of how estrogen receptors mediate mammary epithelial cell proliferation. Estrogen is one of the most important regulators for mammary gland development, and the function of estrogen is mediated by the estrogen receptors (ER) - ERa and ERb. Lack of any of the estrogen receptors will impair the mammary gland development to some extent, suggesting that both ER-alpha and ER-beta are required for the full development of mammary gland. In the last report we were not successful in cloning the full length cDNAs for bovine ERa and ERb. In the past year we have had the full length cDNAs for bovine ERa and ERb subcloned into different exprssion vectors. The functions of bovine ERa and ERb on cell proliferation are under evaluation. Preliminary studies indicated that ERb might inhibit cell proliferation, which makes it difficult to analyze using conventional transfection. To overcome this problem, we are in the process to have bovine ERb expression under an inducible system to control ERb expression. PARTICIPANTS: Yili Zhong, Graduate Student, 70% effort. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: The bovine ERalpha and ERbeta cDNAs were subcloned into the Tet-ON inducible system, and will also be subcloned into the lentiviral vectors for lentivirus production. These two systems will be used to evaluate the regulation of cell proliferation by bovine ERalpha and ERbeta in MacT cells.
Impacts
The general goal was to understand how mammary epithelial cell proliferation is regulated by estrogen receptors. Bovine ERa and ERb full-length cDNAs were subcloned into different protein expression vectors. These plasmid constructs will be used to access the common and disctint functions of ERa and ERb.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: This project is to determine the molecular mechanism of how estrogen receptors mediate mammary epithelial cell proliferation. Estrogen is one of the most important regulators for mammary gland development, and the function of estrogen is mediated by the estrogen receptors (ER) - ERalpha and ERbeta. Lack of any of the estrogen receptors will impair the mammary gland development to some extent, suggesting that both ER-alpha and ER-beta are required for the full development of mammary gland. We stained the bovine mammary gland tissues obtained from the slaughter-house and assessed the expression of bovine estrogen receptors in the bovine mammary gland. We demonstrated ERalpha expression in about 30% of the mammary epithelial cells from the non-lactating and non-pregnant cows. We did not detect the expression of ERbeta in the mammary epithelial cells from these cows. We modified our experiment plan for the next year since we were unable to get mammary gland biopsy samples from
the UVM Miller Research Farm for some reason beyond our control. Rather than looking at the expression of estrogen receptors in the different phases of the estrous cycle, we plan to focus on the ERalpha and see whether ERalpha cells can proliferate by co-staining ERalpha with a couple of cell proliferation markers including Ki-67. We used RT-PCR to get the cDNA of bovine estrogen receptors. We obtained several PCR products for ERalpha, from 250bp to about 2kb. We did not obtain any PCR product for ERbeta. The PCR fragments were sequenced and we found that only the 250bp fragment contains sequences matching the bovine ERalpha sequence in the GenBank, but the 2kb and 900bp fragments did not match any genes in the databases. We are going to continue the cloning of the genes. In the meanwhile, we are going to over-express the estrogen receptors from other species into the bovine mammary epithelial cells and evaluate the proliferation of the bovine mammary epithelial cells.
PARTICIPANTS: Sivashankar Ramakrishnan, M.S. student, UVM Huining Tan, Postdoc, UVM
PROJECT MODIFICATIONS: We modified our experiment plan for the next year since we were unable to get mammary gland biopsy samples from the UVM Miller Research Farm for some reason beyond our control. Rather than looking at the expression of estrogen receptors in the different phases of the estrous cycle, we plan to focus on the ERalpha and see whether ERalpha cells can proliferate by co-staining ERalpha with a couple of cell proliferation markers including Ki-67. We tried to use RT-PCR to get the cDNA of bovine estrogen receptors but did get the full-length bovine ERalpha and ERbeta cDNA. We are going to continue the cloning of the genes. In the meanwhile, we are going to over-express the estrogen receptors from other species into the bovine mammary epithelial cells and evaluate the proliferation of the bovine mammary epithelial cells.
Impacts
The general goal was to understand how mammary epithelial cell proliferation is regulated by estrogen receptors. We developed immunohistochemical and immunofluorescent assays for bovine ERalpha and determined the expression of ERalpha in the mammary gland of non-lactating non-pregnant cows. The expression of ERalpha were confirmed by Western-blotting and RT-PCR. We subcloned a fragment of the bovine ERalpha. Using different detection methods, we demonstrated that ERalpha and ERbeta have different expression patterns in the mammary gland of non-lactating and non-lactation cows. These results suggest that ERalpha and ERbeta might be differentially involved in bovine mammary gland development. These results are important to the researchers in the dairy cow field in particular and will expand the knowledge on the functions of estrogen receptors across different species in general.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
This project is to determine the molecular mechanism of how ER-alpha and ER-beta stimulate mammary gland development. Estrogen is one of the most important regulators for mammary gland development, and the function of estrogen is mediated by its receptors ER-alpha and ER-beta. Lack of any of the estrogen receptors will impair the mammary gland development to some extent, suggesting that both ER-alpha and ER-beta are required for the full development of mammary gland. In the past year, we initiated Specific Aims (1) and (2). The Specific Aims in the proposal are (1) To determine the expression patterns of ER-alpha and ER-beta mRNA and protein at different stages of bovine mammary gland development and the effect of estrogen on their expression patterns; (2) To determine the function and mechanism of ER-alpha and ER-beta, respectively, in mammary epithelial cell proliferation. Specific Aim (1). We got a few mammary gland tissue samples, none of them showed expression
for the ER-alpha and ER-beta, as evaluated by immuno-blotting. As ER-alpha and ER-beta could be expressed in the uterine and ovarian tissues, as a positive control we could see expression of ER-alpha in one of the uterine tissue samples. We did not see ER-beta expression in any of the samples we collected. The lack of ER-alpha and ER-beta expression in the few samples evaluated so far raised the concern that the staging of the samples could be very important for the interpretation of their expression patterns. For this reason, we modified this specific aim to evaluate the expression of ER-alpha and ER-beta in all the four phases of the estrous cycle. In this modification, we plan to get mammary biopsy from 4-6 post-pubertal heifers (10-12 month old) for each phase of the estrous cycle. To minimize the variation, we plan to synchronize the animals for estrus using Lutalyse (Dinoprost) so that we can collect the samples from all the 4-6 animals on the same day for each phase of the
estrous cycle. The modified animal protocol was approved by UVM IACUC and is in the reviewing process by the Dairy Committee of UVM ASCI department and Miller Research Farm. Next year we will evaluate the expression of bovine ER-alpha and ER-beta in the different phases of the estrous cycle. Specific Aim (2). While in the process of getting approval for the modified animal protocols, we started to clone the bovine ER-alpha and ER-beta cDNA. We got some fragments for ER-alpha, but have not find a good source of sample for high expression of ER-beta. Next year we will continue to get the full-length of the bovine ER-alpha and ER-beta cDNA and evaluate their functions.
Impacts
Full development of the mammary gland is very important for lactation and mile yield. Understanding how ER-alpha and ER-beta regulate mammary gland development will provide insights into how to enhance mammary gland function and milk production.
Publications
- No publications reported this period
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