2010 Annual Report
1a.Objectives (from AD-416)
Objective 1: Determine gene expression in human lactating mammary epithelium.
Subobjective 1A: Determine the pattern of mammary epithelial gene expression using milk fat globule mRNA from delivery through the first 4 weeks of lactation. Compare these results with those in mothers of premature infants and teenage mothers over a similar period of time.
Subobjective 1B: Characterize the mRNA response to exogenous lactogenic hormones.
Objective 2: Characterize inbred mouse strains for lactation performance, gene expression and weight gain among offspring in lean and obese animals, making use of a cross-fostering design where appropriate.
Subobjective 2A. Identify genes in which strain-dependent differences in mammary gland gene expression, and SNP haplotype, are correlated with strain-dependent differences in milk production, lactation persistence, mammary gland development, or milk composition.
Subobjective 2B. Determine the extent to which genes identified from the whole genome scan and microarray work described in 2A are responsible for the lactation defect in mice with maternal obesity.
Objective 3: Study the effect of nutrients on mammary gland development and function in mouse models. Define the critical window for effects on mammary gland development and function.
Subobjective 3A1. Determine effect of exposure to low protein diet by analyzing mammary gland development, milk production, and milk composition, as well as gene expression and gene promoter methylation in mammary gland tissue of dams exposed to diets with low protein content during gestation.
Subobjective 3A2. Use a mouse model for tissue-specific alteration of Dnmt1 levels to confirm role of DNA methylation in effects of low protein diet on mammary gland development.
Subobjective 3B. Define critical window for effect of low protein diet on mammary gland function by limiting nutritional intervention to specific developmental windows.
Subobjective 3C. Determine impact of low protein diet on genetic variants for mammary gland development and lactation capacity as identified in objective 2.
1b.Approach (from AD-416)
Children's Nutrition Research Center researchers will determine gene expression in human lactating mammary epithelium by isolating mRNA from human colostrum or milk over the first 4 weeks post partum and the expression arrays measured to determine the relative gene express over this period of time. Data from groups of mothers will be assessed to prove or disprove our hypotheses. A variety of potential lactogenic hormones will be administered short term (over 3 days) to normal women with established lactation between 6 and 12 weeks post partum. The hormones initially to be tested are prolactin, cortisol, and IGF-1. Breast milk will be collected every 3 hr and RNA isolated for measurement of expression of mRNA expression using microchip technology. The data will be compared to that already obtained from similar studies in women prior to and following the administration of recombinant human growth hormone. Additionally, a panel of lactation traits will be measured in 32 inbred strains of mice. The data from these measurements will be used as phenotype data in combination with whole genome SNP data to conduct a statistical association analysis across the entire mouse genome. The Viable yellow agouti (Avy) mouse will be used as a model of maternal obesity. Gene expression will be determined by microarray analysis of mammary tissue samples collected from obese and lean Avy females during early lactation. Genes that are differentially expressed between lean and obese females will then be compared to the list of genes identified to test for overlap. The lactation traits, as well as gene expression and epigenetic profiles will be measured in transgenic animals containing the conditional allele for Dnmt1 (dnmt1-lox2) and a mammary gland specific Cre recombinase to determine the effects of deletion in the mammary gland of Dnmt1. The data will be compared to those of low protein diets.
We obtained IRB approval, recruited 7 normal women, and have completed milk collection in 6. For this study, we are collecting samples at 12, 24, 36, 48, 60, 72, and 96 hr and at 7, 14, 21, 28, 35, 42 weeks. We have completed studies in 2 obese women. Depending on results of the gene expression patterns we may modify the timing and number of samples collected. The micro array chip we originally used to measure the transcription of specific genes in the mothers' breast is no longer manufactured, but a newer chip should give us additional gene targets to assess in addition to all of the ones previously measured. We have established that there is a large amount of RNA in human colostrum, but the isolation of high quality RNA has been challenging. With a number of modifications in our collection technique and processing protocol, we have substantially improved the quality of the RNA. We are now measuring pattern of genes that are expressed in these women. The milk-derived RNA has been isolated, quantitated, and the cDNA and cRNA produced. We have established contacts and collaboration with local hospitals and have carried out a number of nursing in-service presentations. Hopefully this will provide us with sufficient subjects over the next 18 months to complete the sample collection.
We measured traits associated with milk production in 32 inbred strains of mice and collected milk, blood, urine, and mammary tissue for future use. We compared maternal behavior, food intake, and body composition among these 32 strains and measured maternal ability to mobilize body fat during early lactation.
We have begun experimental animal breeding to be used for our experiments, but the extensive breeding needed has limited our ability to expand this population.
For this subobjective it was necessary to breed together 3 separate mouse lines into 1 mouse line to obtain certain transgenes. Currently we have the first limited group of experimental animals. We have started collecting phenotypic data and samples for further analysis. It has become clear that the current approach is limited by unforeseen deficiencies in the used model. The inactivating gene we used was supposed to be expressed specifically in mammary gland tissue and at specific developmental stages. In addition, expression of this gene was supposed to be under control of an activator that is regulated by the administration of an inducing substance. We found that gene expression of the inactivating gene is not developmental-stage-specific, it is not exclusively expressed in the mammary gland but is also expressed in some other tissues, and it is expressed regardless of administration of the inducing substance. This model might provide preliminary data, but we will have to adjust our strategy. We plan to use a transplant model, which is an established procedure used frequently in mammary gland biology. Based on recently published studies by other investigators this will better address the anticipated effects of deletion of the gene of interest on mammary gland development.
The ADODR monitors activities for the project by routine site visits, and review of major purchases and use of SCA funds.
The effect of nutrients on mammary gland development and function. The effect of undernutrition on mammary gland development is not known, and this is important as it impacts the biological processes of the mother and offspring. As part of their research, scientists at the Children's Nutrition Research Center in Houston, Texas, have established that a mouse model system intended to result in mammary-gland-specific inactivation of genes of interests, is not functioning as intended. These findings are important for researchers, as this data implies that this particular mouse model is not suitable for these particular studies. As a result, alternative research methods will need to be identified and used.
Functional genomics of lactation. Researchers at the Children's Nutrition Research Center in Houston, Texas, have established a unique method to isolate and measure mRNA from human milk. mRNA is the material that takes the information from the DNA to different parts of cells to cause specific proteins to be made. We have completed the collection of mRNA from the time of delivery through the first 6 weeks of life of the infant in 6 normal and 2 obese women. With these data we will have established for the first time the post-partum pattern of gene expression in normal lactating women and compared this to women who traditionally struggle with breastfeeding (teenage mothers, mothers with premature infants, and obese mothers).