Project description:We aimed to study the effects of CSF2 treatment on the methylome of the Day 15 female and male embryo. Methylomic profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females
Project description:We aimed to study the effects of CSF2 treatment on the transcriptome of the Day 15 female and male embryo. Within the raw files, channel 1= Cy3 and channel 2= Cy5 Transcriptional profiling of bovine extra-embryonic membrane at Day 15 post-insemination produced by IVF. Oocytes were matured and fertilized in vitro using a single Holstein bull. After 5 days in vitro culture, the embryos were either treated with 10 ng/ml bovine recombinant CSF2 or the Control (DPBS/BSA) until Day 7. At Day 7, the embryos were transferred into recipients. At day 15 following insemination, embryos were flushed and collected for total RNA and gDNA extraction. The gender of the embryos were determined by PCR prior to microarray analysis. CSF2 males were compared to Control males while CSF2 females were compared to Control females. Control females were also compared to control males.
Project description:Exposure of bovine conceptuses to colony stimulating factor 2 (CSF2) from Day 5 to 7 of development can increase the percent of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from Day 5 to 7 after insemination, transferred into cows, and flushed from the uterus at Day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2 treated conceptus (35% for control vs. 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-{tau} (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.06) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at Day 15 of pregnancy. Experimental conditions: CSF2 treated vs. CSF2 nontreated bovine in vitro produced preimplantation embryos were tranfered to a receptor cow and recovered at Day 15 of embryo development. The embryonic disc (ED) and the trophectoderm (Tr) were used for the expression analysis separately. Biological replicates: CSF2 treated vs. nontreated bovine preimplantation embryos were used in a dye switch two-color microarray experimental design.
Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps.
Project description:Exposure of bovine conceptuses to colony stimulating factor 2 (CSF2) from Day 5 to 7 of development can increase the percent of transferred conceptuses that develop to term. The purpose of this experiment was to understand the mechanism by which CSF2 increases embryonic and fetal survival. Conceptuses were produced in vitro in the presence or absence of 10 ng/ml CSF2 from Day 5 to 7 after insemination, transferred into cows, and flushed from the uterus at Day 15 of pregnancy. There was a tendency (P=0.07) for the proportion of cows with a recovered conceptus to be greater for those receiving a CSF2 treated conceptus (35% for control vs. 66% for CSF2). Antiviral activity in uterine flushings, a measure of the amount of interferon-{tau} (IFNT2) secreted by the conceptus, tended to be greater for cows receiving CSF2-treated conceptuses than for cows receiving control conceptuses. This difference approached significance when only cows with detectable antiviral activity were considered (P=0.07). In addition, CSF2 increased mRNA for IFNT2 (P=0.06) and keratin 18 (P<0.05) in extraembryonic membranes. Among a subset of filamentous conceptuses that were analyzed by microarray hybridization, there was no effect of CSF2 on gene expression in the embryonic disc or extraembryonic membranes. Results suggest that the increase in calving rate caused by CSF2 treatment involves, in part, more extensive development of extraembryonic membranes and capacity of the conceptus to secrete IFNT2 at Day 15 of pregnancy.