Project description:RNA seq analysis was conducted to determine gene expression in the day 14 ovine conceptus. This was used in conjunction with the day 14 PPARG ChIP-seq analysis to identify genes bound by PPARG which were also expressed or not expressed in the day 14 conceptus. Understanding changes in gene expression during early pregnancy is critical to improving fertility and reproductive efficiency in ruminants. RNA seq analysis of 4 conceptuses from 4 individual Day 14 pregnant columbia/rambouillet crossbred ewes
Project description:PPARG ChIP seq analysis was conducted to determine genes bound by and potentially regulated by PPARG in the developing ovine conceptus. Determination of gene regulation by prostaglandins through PPARG helps to improve our understanding of early pregnancy events and provides a basis for strategies to improve fertility and reproductive efficiency in ruminants. PPARG ChIP seq analysis of 4 conceptuses from 4 individual Day 14 pregnant columbia/ramboulette crossbred ewes
Project description:RNA seq analysis of laser capture microdissected luminal and glandular epithelium from ewes on day of pregnancy 10, 12, 14, 16 and 20. As well as RNA seq of whole conceptuses, and trophectoderm tissue from day 12, 14, 16 and 20 of pregnancy. Determination of gene expression changes in the uterine epithelium and conceptus during early pregnancy helps to improve our understanding of early pregnancy events and provides a basis of new strategies to improve fertility and reproductive efficiency in ruminants.
Project description:Transcriptomics and functional bioinformatics were used to investigate the potential interactions of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on uterine indicators of metabolism and reproduction. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition during 28 days: maintenance energy intake (control; 5 cyclic, 6 pregnant ewes) providing 7.8 MJ of metabolisable energy per ewe and 0.5 maintenance intake (undernourished; 6 cyclic, 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. RNA from uterine tissue was harvested at slaughter on day 14 of estrus or pregnancy, and hybridized to the Agilent 15K Sheep Microarray chip. Functional bioinformatics analyses were performed using the Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis. Among metabolic pathways, citrate cycle, oxidative phosphorylation, pentose and glucuronate interconversions and biosynthesis of unsaturated fatty acids were upregulated in control pregnant compared with control cyclic ewes. However, these pathways were not altered in undernourished ewes. The presence of an embryo in undernourished ewes upregulated fatty acid and glycogenic amino acid metabolism. RIG-I and Toll like receptors and chemokine signaling pathways were upregulated by the presence of the embryo in both control and undernourished ewes, but in the latter group the impact was lower. Undernutrition alone upregulated carbohydrate metabolism, but different pathways were altered in cyclic versus pregnant ewes. Citrate cycle, glycolysis/gluconeogenesis, and pentose and glucuronate interconversions were upregulated in undernourished cyclic compared with control cyclic ewes. Glycolysis/gluconeogenesis, pyruvate and propanoate metabolism, beta-alanine and phenylalanine metabolism were upregulated in undernourished pregnant compared with control pregnant ewes, whereas biosynthesis of unsaturated fatty acids and replication and repair were downregulated. Undernutrition alone led to an overall weak activation of immune system pathways both in cyclic and pregnant ewes. However, undernourished cyclic compared with control cyclic ewes had a high activation of NOD-like and RIG-1 like receptor signaling pathways, whereas undernourished pregnant compared with control pregnant ewes only had a weak upregulation of T cell receptor signaling pathways. Overall, data revealed that metabolic and immune adaptations of the uterus to nutrient restriction are dependent on the presence of the conceptus.
Project description:Ovine interferon-tau (IFNT) is released from the conceptus by Day 12 of pregnancy and disrupts pulsatile release of endometrial prostaglandin F2 alpha (PGF), thereby protecting the corpus luteum (CL). IFNT may also have endocrine action through inducing interferon stimulated genes (ISGs) in the CL. The hypothesis that gene expression differs in CL collected from pregnant (P) and non-pregnant (NP) ewes by Day 14 due to the lytic action of PGF during the estrous cycle or the presence of a conceptus was tested. RNA was isolated on Days 12 and 14 in NP or P ewes (n = 3 ewes/group) and analyzed using the Affymetrix bovine microarray (24,000 targets). Differential gene expression (>1.5 fold, P < 0.05) was confirmed using semi-quantitative real time PCR (RTPCR). Serum progesterone concentrations decreased (P < 0.05) from 1.7 ng/ml on Day 12 to 1.3 ng/ml by Day 14 in NP ewes suggesting initiation of luteolysis; and remained > 1.7 ng/ml in Day 12 and 14 P ewes indicating that the conceptus protected the CL from luteolysis. Early luteolysis from Day 12 to 14 NP was associated with differential expression of 683 genes, including SERPINE1 and THBS1. Presence of a conceptus from Day 12 to 14 also induced expression of 743 genes, i.e., ISGs (ISG15, MX1), PTX3, and IL-6 and stabilized expression of VEGF and LHR genes. In conclusion, pregnancy circumvents luteolytic pathways, and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, angiogenic and epithelial to mesynchymal transition pathways in the CL. There are 12 samples that were analyzed for the microarray, no duplicates and we increased the sample size to 50 for the RTPCR. This Series contains the Affymetrix array data only (not RT-PCR data).
Project description:RNA seq analysis was conducted to determine gene expression in the day 14 ovine conceptus. This was used in conjunction with the day 14 PPARG ChIP-seq analysis to identify genes bound by PPARG which were also expressed or not expressed in the day 14 conceptus. Understanding changes in gene expression during early pregnancy is critical to improving fertility and reproductive efficiency in ruminants.
Project description:Previoulsly expression profiling of the whole mammary gland across different stages of pregnancy and lactation has been performed on different strains of mice. Since mammary gland has both epithelial and stromal compartments, to specifically identify the genes involved in the transition from pregnancy to lactation a process termed as secretory activation, expression profiling of isolated mammary epithelial cells (MECs) from four CD1 mice each at Pregnancy day 14 (P14) and Lactation day 2 (L2) was performed in the current study. Statistical analysis of the mRNA changes between P14 and L2 identified 5,499 unique genes as being differentially expressed (5% FDR), of which, 2,902 genes and 2,604 genes were higher in P14 or L2 stages, respectively.
Project description:Ovine interferon-tau (IFNT) is released from the conceptus by Day 12 of pregnancy and disrupts pulsatile release of endometrial prostaglandin F2 alpha (PGF), thereby protecting the corpus luteum (CL). IFNT may also have endocrine action through inducing interferon stimulated genes (ISGs) in the CL. The hypothesis that gene expression differs in CL collected from pregnant (P) and non-pregnant (NP) ewes by Day 14 due to the lytic action of PGF during the estrous cycle or the presence of a conceptus was tested. RNA was isolated on Days 12 and 14 in NP or P ewes (n = 3 ewes/group) and analyzed using the Affymetrix bovine microarray (24,000 targets). Differential gene expression (>1.5 fold, P < 0.05) was confirmed using semi-quantitative real time PCR (RTPCR). Serum progesterone concentrations decreased (P < 0.05) from 1.7 ng/ml on Day 12 to 1.3 ng/ml by Day 14 in NP ewes suggesting initiation of luteolysis; and remained > 1.7 ng/ml in Day 12 and 14 P ewes indicating that the conceptus protected the CL from luteolysis. Early luteolysis from Day 12 to 14 NP was associated with differential expression of 683 genes, including SERPINE1 and THBS1. Presence of a conceptus from Day 12 to 14 also induced expression of 743 genes, i.e., ISGs (ISG15, MX1), PTX3, and IL-6 and stabilized expression of VEGF and LHR genes. In conclusion, pregnancy circumvents luteolytic pathways, and activates or stabilizes genes associated with interferon, chemokine, cell adhesion, cytoskeletal, angiogenic and epithelial to mesynchymal transition pathways in the CL.