Project description:Exosomes/microvesicles (hereafter referred to as extracellular vesicles) were isolated from the ULF of day 14 cyclic and pregnant ewes using ExoQuick-TC. Extracellular vesicle RNA was pooled (n=4 per status) and analyzed for small RNAs by sequencing on the Ion Torrent PGM platform and analysis with CLC Genomics Workbench small RNA workflow based on the miRBase (Release 19) Bos taurus database. Small RNA analysis of day 14 uterine luminal fluid extracellular vesicles isolated from pregnant and cyclic ewes.
Project description:The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy. Transcriptional profiles from day 14 conceptus extracellular vesicles isolated from 24 hour conceptus-conditioned culture media (n=3) were generated by sequencing on the Illumina HiSeq 2500 platform.
Project description:We have completed the high quality reference genome for domestic sheep (Oar v3.1). Early-stage Illumina GA sequence platform sequenced less reads in high GC content regions than in other regions. To read through higher GC content regions, we generated 2 Gb MeDIP-seq data for filling gaps in sheep reference genome assembly.
Project description:Progesterone (P4) acts via the endometrium to modify the uterine environment and promotes conceptus growth for elongation and pregnancy establishment. Ewes were ovariectomized and treated with P4 for 14 days or P4 for 14 days and RU486, a progesterone receptor antagonist, from days 8 to 14. Small RNA sequencing of endometrium and EVs from the uterine lumen detected expression of 768 miRNAs and P4 regulation of 9 endometrial and 7 extracellular vesicle miRNAs. Overall design: Endometrium from P4-treated (n=5) or P4+RU486-treated ewes (n=5); Extracellular vesicles from the uterine lumen of P4-treated (n=4) or P4+RU486-treated ewes.
Project description:In the present study, we studied the effect of dietary selenium (Se) supplementation on the transcriptomic profile of sheep. The main objective was to evaluate the effect of Se-supplementation on the overall transcriptome of sheep, the altered pathways, and the biological processes related to it . A custom oligo microarray platform (AMADID: 070119) was designed, then used to profile gene expression from 20 samples from 10 sheep at two time points (T0; before Se-supplementation, and T40; at the end of a 40-d Se-supplementation period). Isolated and purified total RNAs were individually hybridized to the custom (4x44k) DNA microarray. The comparison of control and treated animal transcriptomes revealed a large set of differentially expressed genes. After functional analysis and qPCR validation, the result showed several pathways and biological processes that have been altered following Se-supplementation to the diet. Overall design: Ten lactating crossbred ewes (3 to 4 y of age and 55 to 65 kg BW) were assigned to a before-and-after experimental design, where each animal served as its own control. The sheep have been acclimatized (4-wk) on a basal diet containing a maintenance level of Se (0.13 mg Se/kg DM). At the end of the 4-wk acclimatization period the sheep started to receive the supra Se-supplemented diet (0.45 mg Se/kg DM) for a 40-d period. Blood samples were collected at 2 time points [T0; one day before the supra Se-supplementation, and T40; at the end of the 40-d supra Se-supplementation phase]. Blood samples were collected from each individual animal directly into Paxgene blood collection tubes, processed and stored at -20˚C till further analyses. All samples yielded high quality RNA that was tested prior the microarray setup.