2013 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.
For objective 1, this year has proven to be a highly productive one. The manuscript on the secretory activation of the lactose synthesis pathway was published. In addition, we published our investigation of unique microRNAs in human milk using next generation sequencing techniques. In the course of these studies we identified 6 unique microRNAs and found that some of these may be modulated by maternal diet. However, the role and regulation of the production and fate of these compounds remains largely, if not entirely unknown. We continued the analysis of the micro array data obtained from normal lactating women focusing on fatty acid metabolism within the human mammary gland and their temporal relationships to the composition and concentrations of fatty acids in human milk. The large body of data has been accepted in the Journal of Physiology-Endocrinology and Metabolism and will soon be available in the epublish release for the journal. Because of these and other previous publications, we were invited to write an editorial in Diabetes, the most prestigious journal in the area of diabetes and metabolism regarding the role of lactation on the risk of subsequent type 2 diabetes in the mother. We continue to analyze the data from this study of normal induction of lactation and will be analyzing the protein products and the changes in gene expression in the area of milk protein production and regulation. We have completed the isolation, production of cRNAs from the women who were obese, had premature infants and the teenage mothers that we successfully enrolled in the study. These data have only recently been available to us and it will take several months to analyze and compare these data from these groups and compare to the others and from our normal subjects. We anticipate that this will be completed within the next year. For objective 2, this year we have conducted RNA-seq on samples obtained during early lactation from mammary tissue of three high lactation mouse strains and three low lactation mouse strains in order to identify differentially expressed genes linked to high milk production. We also collected whole mammary biopsies from additional inbred mouse strains at two key times during postnatal mammary gland development making the total number of strains analyzed 43. Through image analysis we collected quantitative data for three mammary ductal development traits and then used this data to map genomic regions that are associated with variations in the traits. We conducted a comparison of mammary ductal development among lean and obese mice to determine if obesity is linked to decreased mammary ductal development. Lastly, we compared mammary cell apoptosis, JAK-STAT signaling, and mineralocorticoid receptor expression and localization among lean and obese mice during lactation. In objective 3, a cohort of animals in the appropriate genetic background were generated and are being analyzed for the effect of Dnmt1 deletion on:.
1)cell homeostasis by serial breeding, which initiates consecutive growth and regression cycles, 2k) stem progenitor and differentiated cell function by transplantation and cell culture.
Mapping of genes linked to mammary ductal development. The extent of genetic variation in mammary ductal development is unknown. Analysis of mammary ductal development among strains of mouse models has revealed greater variation in mammary ductal patterning than previously known. Using this data in conjunction with single nucleotide polymorphism (SNP) data, researchers at the Children's Nutrition Research Center in Houston, Texas, have identified several regions in the mouse genome that have many of the same SNPs associated with specific traits. The genes underlying these regions have been linked to breast cancer in humans or anomalies in mammary gland development. These results support the conclusion that variations in mammary ductal development can be attributable to specific regions within the genome, which could play a role in breast cancer and could possibly influence mammary gland function during lactation.
Lactation defects in obese mice are linked to increased inflammation. Maternal obesity is known to negatively affect lactation. By comparing mammary tissue among lean and obese mice during early lactation Scientists at the Children's Nutrition Research Center in Houston, Texas, have found that defects in lactation in response to maternal obesity are associated with elevated mammary tissue macrophages, increased mammary cell apoptosis, and increased phosphorylation of the transcriptional regulator STAT3. We have concluded that the mineralocorticoid receptor is abnormally activated in mammary tissue of obese females during early lactation. This data is significant because if provides the identity of pathways that could be targeted as a means to remedy the negative affects of maternal obesity on lactation.
Hormone and gene regulation of human lactation. Little is known about the gene regulation of milk production in humans. A number of women struggle to successfully breastfeed their infants; of specific note are those who are obese, teenage mothers, and mothers with premature infants. Using messenger RNA (molecular signal that causes the expression of genes) found in human milk, scientists at the Children's Nutrition Research Center in Houston, Texas, have been able to determine some of the factors that affect milk production and have traced the changes in gene expression over the first 42 days of lactation in normal women, and are now applying that knowledge to obese women. The primary trigger of the initiation of lactation in humans is unknown, but one of the factors is the withdrawal of progesterone (a hormone made in the placenta during pregnancy) with the removal of the placenta as a result of the birth process. As a result of these studies, therapeutic approaches may be available in the future to increase the success rates of breastfeeding.