Progress 05/15/08 to 05/14/12
Outputs
OUTPUTS: Prolactin (PRL) is a critical pituitary-derived hormone for lactation and reproduction in all mammals, and milk production by sows limits piglet growth. The objective of this project was to resolve the structure and function of the prolactin receptor (PRLR) at the genetic and biochemical level in pigs. We first used 3'RACE to identify and clone a unique "short" form of the PRLR. This receptor acts as a dominant-negative that serves to suppress activity of the accompanying "long" form of the receptor. We next also identified several variants of this short PRLR that are genetically-distinct due to single nucleotide substitutions within the intracellular domain. Given the potential for these variants to confer different phenotypes, we examined the biochemical properties of all forms in vitro and found that all forms shared similar activities for PRL signaling and binding. Our analysis failed to detect any ability of this short PRLR to stimulate cell proliferation. We also determined the expression profile for this short PRLR during different developmental states. Similar to the long form, this short form PRLR increases its expression within target tissues such as the mammary glands and uterus during pregnancy, while it is also expressed in tissues such as the adrenals and ovaries. We also sought to establish how PRL confers its numerous biological effects by examining mechanisms behind how PRLR expression and function change during different physiological states and in different target tissues. We first used 5' RACE to identify the 5' ends of PRLR mRNA transcripts and the matching promoter regions across numerous tissues in the most thorough analysis to date for any mammal. We have identified several 5' transcriptional ends for PRLR mRNA. Most importantly, these 5' ends are expressed differently in various tissues as determined by comprehensive analysis of PRL target tissues during growth and gestation. Previously we had also identified that the long PRLR is differentially-regulated in tissues such as liver, kidney, mammary glands and uterus by hormones including estrogen, progesterone and PRL. We are now able to demonstrate that different promoters are active in these tissues and differentially respond to these hormones in a tissue-specific manner. Furthermore, we have been able to establish that certain regions of these promoters contain response elements for transcription factors responsible for promoter activation in response to hormones such as estrogen. PARTICIPANTS: We have collaborated with researchers at USDA-MARC, AgCanada, Iowa State, Southern Illinois University and Texas A&M to gain access to unique samples and methods. Training opportunities were provided for one postdoctoral fellow in animal genomics, and one PhD candidate from the UC Davis Animal Biology Graduate Group. Individuals working on project at UCDavis: Russell Hovey, PD, Associate Professor Josephine Trott, co-PD, Assistant Project Scientist Anke Schennink, postdoctoral fellow (postdoc training opportunity provided to Dr Schennink). Monica Van Klompenberg, a PhD candidate in the Animal Biology graduate group at UC Davis. TARGET AUDIENCES: We presented our work to the Swine Genome Sequencing consortium meeting in Hinxton, UK (2010) given the relevance of our gene sequence data to that effort. Given the reproduction and lactation relevance of this project, we reported our findings to the Joint Annual Meeting of the American Dairy Science and Animal Science Associations in New Orleans, July 2011 including a presentation to the Triennial International Lactation Workshop. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Prolactin is a hormone that is critical for numerous biological processes including lactation, reproduction, stress and behavior. By resolving the functionality of PRL at both the genetic and physiological level we have contributed to a body of data that can now be used to select animals for increased productivity while opening the door for new strategies including genetic testing and different management. Our identification of the full genetic sequence for the PRLR gene now presents opportunities for extended PRLR genotyping for performance traits in pigs. Previously, PRLR genotype variants based on the long form PRLR have been associated with traits such as litter size, piglet growth. Now that we have established additional sequence information, further relationships between genetic variants and production traits can be examined in promoters, coding regions, and untranslated regions. These data have also been provided to the pig genome sequencing consortium effort. Our data also indicate that specific hormones affect PRLR expression in different target tissues. This finding will impact the way that approaches such as reproduction management are viewed, in that opportunities to increase PRLR activation could be accomplished in target tissues by manipulating the endocrine axis.
Publications
- Trott, J.F., Horigan, K.C., Gloviczki, J.M., Costa, K.M., Freking, B.A., Farmer, C., Hayashi, K., Spencer, T., Morabito, J.E., and Hovey, R.C. (2009) Tissue-specific regulation of porcine (Sus scrofa) prolactin receptor expression by estrogen, progesterone and prolactin. Journal of Endocrinology 202(1): 153-166.
- Horigan, K.C., Trott, J.F., Barndollar, A.S., Scudder, J.M., Cupicha, L.E., Blauwiekel, R.M., and Hovey, R.C. (2009) Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine (Sus scrofa) mammary gland. Domestic Animal Endocrinology 37(2):124-138.
- Farmer, C., Palin, M.-F., and Hovey, R.C. (2010) Greater milk yield is related to increased DNA and RNA content but not to mRNA abundance of selected genes in sow mammary tissue. Canadian Journal of Animal Science 90: 379-388.
- VanKlompenberg, M.K., McMicking, H.F., and Hovey, R.C. (2011) A vacuum-assisted approach for biopsying the mammary glands of various species. Journal of Dairy Science 95(1):243-246.
- Trott, J.F., Schennink, A., Petrie, W.K., Manjarin, R., VanKlompenberg, M.K., and Hovey, R.C. (2011) Prolactin: The multi-faceted potentiator of mammary growth and function. Journal of Animal Science. 90(5):1674-1686.
- Trott, J.F., Identification of a short isoform of the porcine prolactin receptor and its variants. Joint Annual Meeting of the American Dairy/Animal Science Associations, New Orleans, July 10-14, 2011.
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Progress 05/15/10 to 05/14/11
Outputs
OUTPUTS: We have made strong progress regarding our goal of elucidating the role of prolactin (PRL) and its action via the PRL receptor (PRLR) during mammary growth and function in pigs. Previously we reported how PRL cooperates with the actions of estrogen on the mammary glands to induce proliferation of mammary epithelial cells. This proliferation corresponds to specific changes in the morphology of the mammary parenchyma. Combined, these data emphasize the role for PRL during mammary growth in the pig. We separately established that expression of the pig PRLR in the mammary glands increases during pregnancy, consistent with the essential role of PRL in this tissue prior to and during lactation. We also demonstrated a role for PRL in upregulating this expression of PRLR in the mammary glands, and showed that there is a distinct population of PRL-inducible PRLR in the stromal compartment of this organ, whereas PRLR in the epithelial cells is estrogen-induced. By contrast, PRLR in the endometrium was differentially regulated compared to PRLR in the mammary glands. Furthermore, we found that PRLR in the liver and kidney was insensitive to a female's endocrine status. During the reporting period we have completely resolved the 5' end of the pPRLR gene. This has demonstrated that pPRLR expression is controlled by 10 different alternative promoters. Such a finding is important because it highlights how different tissues can differentially control PRLR expression at the same within an animal exposed to the same endocrine environment. This finding underlies the importance of how tissue specificity is conferred during important events such as lactation and reproduction in the pig. Furthermore, we have begun to characterize the function of these different promoters by a) measuring their activation during different developmental states using qRT-PCR, and b) cloning the promoters and measuring their activation in response to hormone stimulation in a cell-based assay. These approaches will provide further insight into exactly how these different promoters function during various physiological states. Separately, we found that the 3' end of the pPRLR gene gives rise to a "short" form of the pPRLR protein that lacks an intracellular portion of the protein. We identified that this short form receptor can function as a "dominant negative" that suppresses the effects of PRL on the "long" form of the PRLR. This short form of the receptor is expressed in various PRL target tissues and is regulated moreso at the gene expression rather than the protein level, based on our analysis of various tissues. More recently we have identified that there are several variants of this short PRLR that are further investigating to determine whether these confer different responses to the effects of PRL in different breeds during states such as reproduction and lactation. Our work has made considerable contribution to understanding the genetic basis of PRL function in swine. We recently disseminated our findings to relevant audiences at the Joint Animal/Dairy Science Annual meetings in New Orleans (July, 2011) both in the Triennial Lactation workshop and as an oral abstract. PARTICIPANTS: We have collaborated with investigators from several institutions to realize progress to date. We have collaborated with researchers at USDA-MARC, AgCanada, Iowa State, Southern Illinois University and Texas A&M to gain access to unique samples and methods. Training opportunities are being provided for one postdoctoral fellow in animal genomics, and one PhD candidate from the UC Davis Animal Biology Graduate Group. Individuals working on project at UCDavis: Russell Hovey, PD, Associate Professor Josephine Trott, co-PD, Assistant Project Scientist Anke Schennink, postdoctoral fellow (postdoc training opportunity provided to Dr Schennink). Monica Van Klompenberg, a PhD candidate in the Animal Biology graduate group at UC Davis. TARGET AUDIENCES: Given the reproduction and lactation relevance of this project, we reported our findings to the Joint Annual Meeting of the American Dairy Science and Animal Science Assocations in New Orleans, July 2011. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The findings from this work will afford an understanding of how PRL, a hormone that is critical for lactation (and hence, milk production + piglet growth and survival) acts on its target tissues. The outcome from this work will potentially benefit all animal production that relies on milk production for either consumption (dairy) or offspring growth (meat, wool)
Publications
- Trott, J.F., Schennink, A., and Hovey, R.C. (2011) Cloning and expression of a unique short form of the porcine prolactin receptor. Journal of Molecular Endocrinology, 46(1):51-62.
- Trott, J.F., Identification of a short isoform of the porcine prolactin receptor and its variants. Joint Annual Meeting of the American Dairy/Animal Science Associations, New Orleans, July 10-14, 2011.
- Hovey, R.C., Trott, J.F., Schennink, A., Petrie, W.K., Van Klompenberg, M.K. (2011) Prolactin - the multi-faceted potentiator of mammary growth and function. Joint Annual Meeting of the American Dairy/Animal Science Associations, New Orleans, July 10-14, 2011.
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Progress 05/15/09 to 05/14/10
Outputs
OUTPUTS: We have made strong progress regarding our goal of elucidating the role of prolactin (PRL) and its action via the PRL receptor (PRLR) during mammary gland growth and function in pigs. Previously we reported how PRL cooperates with the actions of estrogen on the mammary glands to induce maximum proliferation of mammary epithelial cells. This proliferation corresponds to specific changes in the morphology of the mammary parenchyma. Combined, these data emphasize the important role for PRL during mammary gland growth in the pig. We separately established that expression of the pig PRLR in the mammary glands increases during pregnancy, consistent with the essential role of PRL in this tissue prior to and during lactation. We also demonstrated a role for PRL in upregulating this expression of PRLR in the mammary glands, and showed that there is a distinct population of PRL-inducible PRLR in the stromal compartment of this organ, whereas PRLR in the epithelial cells is estrogen-induced. By contrast, PRLR in the endometrium was differentially regulated compared to PRLR in the mammary glands. Furthermore, we found that PRLR in the liver and kidney was insensitive to a female's endocrine status. Together, these findings have strongly positioned us to further investigate the molecular mechanisms by which PRLR expression is differentially controlled across tissues. During the reporting period we have resolved the 5' end of the pPRLR gene to determine how differential control is realized to regulate pPRLR gene expression. From these analyses we have identified that multiple gene promoters are utilized to direct expression of the pPRLR gene. Furthermore, we are currently resolving the 3' end of the pPRLR gene to identify different variants of the mature protein that are generated by alternative mRNA transcript splicing. This work has enabled us to identify receptor protein variants that differ in their capacity to modulate PRL signaling and function. Our work has made considerable contribution to understanding the genetic basis of PRL function in swine. We recently disseminated our preliminary findings regarding the genetics and functional genomics of PRL action in the pig mammary glands at the Swine Genome meeting in Hinxton, UK. PARTICIPANTS: e have collaborated with investigators from several institutions to realize progress to date. We have collaborated with researchers at USDA-MARC, AgCanada, Iowa State, Southern Illinois University and Texas A&M to gain access to unique samples and methods. Training opportunities are being provided for one postdoctoral fellow in animal genomics, and one PhD candidate from the UC Davis Animal Biology Graduate Group. Individuals working on project at UCDavis: Russell Hovey, PD, Associate Professor Josephine Trott, co-PD, Assistant Project Scientist Anke Schennink, postdoctoral fellow (postdoc training opportunity provided to Dr Schennink who recently received her PhD from Univ of Wageningen in the Netherlands in the area of animal genetics). Monica Van Klompenberg, a PhD candidate in the Animal Biology graduate group at UC Davis. TARGET AUDIENCES: Given the gene discovery part of this project, we reported our findings to the Swine Genome Sequencing Consortium and the PigNet Genome Conference
at the Sanger Institute. This collection of researchers is focused on understanding the genetics of swine biology and was an excellent audience for our genetic findings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The findings from this work will afford an understanding of how PRL, a hormone that is critical for lactation (and hence, milk production + piglet growth and survival) acts on its target tissues. The outcome from this work will potentially benefit all animal production that relies on milk production for either consumption (dairy) or offspring growth (meat, wool)
Publications
- Farmer, C., Palin, M-F., and Hovey, R.C. (2010). Greater milk yield is related to increased DNA and RNA content but not to mRNA abundance of selected genes in sow mammary tissue. Canadian Journal of Animal Science, In Press.
- Schennink, A., Trott, J.F., Bond, J.P., Famula, T.R., and Hovey, R.C. (2009). Functional genomics of mammary gland growth in a unique pig model of breast development. Swine Genome Sequencing Consortium and PigNet Genome Conference,
The Wellcome Trust Sanger Institute, Hinxton, UK
November 2 to 4, 2009.
- Trott, J.F., Schennink, A., and Hovey, R.C. (2009). Genomic organization and transcriptional regulation of the porcine prolactin receptor gene. Swine Genome Sequencing Consortium and PigNet Genome Conference
The Wellcome Trust Sanger Institute, Hinxton, UK
November 2 to 4, 2009
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Progress 05/15/08 to 05/14/09
Outputs
OUTPUTS: This project was initiated May 2008 after relocation of our laboratory to UC Davis. A new postdoctoral fellow has been recruited to work on this project and commenced 1/2009. We have made strong progress regarding our goal of elucidating the role of prolactin (PRL) and its action via the PRL receptor (PRLR) during mammary gland growth and function in pigs. In a recent publication (Horigan et al., 2009) we have reported how PRL cooperates with the actions of estrogen on the mammary glands to induce maximum proliferation of mammary epithelial cells. This proliferation corresponds to specific changes in the morphology of the mammary parenchyma. Combined, these data emphasize the important role for PRL during mammary gland growth in the pig. In a separate publication (Trott et al., 2009), we have established that expression of the pig PRLR in the mammary glands increases during pregnancy, consistent with the essential role of PRL in this tissue prior to and during lactation. We also demonstrated a role for PRL in upregulating this expression of PRLR in the mammary glands, and showed that there is a distinct population of PRL-inducible PRLR in the stromal compartment of this organ, whereas PRLR in the epithelial cells is estrogen-induced. By contrast, PRLR in the endometrium was differentially regulated compared to PRLR in the mammary glands. Furthermore, we found that PRLR in the liver and kidney was insensitive to a female's endocrine status. Together, these findings have strongly positioned us to further investigate the molecular mechanisms by which PRLR expression is differentially controlled across tissues. We have also started to classify the 5' end of the pPRLR gene to determine how this differential control is realized. Furthermore, we are currently cataloging the 3' end of the pPRLR gene, with the hypothesis that tissue-specific responses to PRL are potentially mediated by specific PRLR isoforms. PARTICIPANTS: We have collaborated with investigators from several institutions to realize progress to date. We have collaborated with researchers at USDA-MARC, AgCanada and Texas A&M to gain access to unique samples and methods. Individuals working on project at UCDavis: Russell Hovey, PD Josephine Trott, co-PD, assistant project scientist Anke Schennink, postdoc (postdoc training opportunity provided to Dr Schennink who recently received her PhD from Univ of Wageningen in the Netherlands). Monica Van Klompenberg, a PhD candidate in the Animal Biology graduate group at UC Davis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The findings from this work will afford an understanding of how PRL, a hormone that is critical for lactation (and hence, milk production + piglet growth and survival) acts on its target tissues. The outcome from this work will potentially benefit all animal production that relies on milk production for either consumption (dairy) or offspring growth (meat, wool)
Publications
- Trott, J.F., Horigan, K.C., Gloviczki, J.M., Costa, K.M., Freking, B.A., Farmer, C., Hayashi, K., Spencer, T., Morabito, J.E., and Hovey, R.C. (2009) Tissue-specific regulation of porcine prolactin receptor expression by estrogen, progesterone and prolactin. Journal of Endocrinology, In Press.
- Horigan, K.C., Trott, J.F., Barndollar, A.S., Scudder, J.M., Blauwiekel, R.M., and Hovey, R.C. (2009) Hormone interactions confer specific proliferative and histomorphogenic responses in the porcine mammary gland. Domestic Animal Endocrinology, In Press.
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