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We have previously demonstrated the existence of an adenosine 5',5"',P1,P4-tetraphosphate (Ap4A) receptor in mouse hart membrane fractions [Hilderman, R. H., Martin, M., Zimmerman, J. K., & Pivorun, E. P. (1991) J. Biol. Chem. 266, 6915-6918]. However, we did not determine the cellular localization or distribution of the receptor. In this report, the Ap4A receptor is shown to be on the cell surface of individual mouse heart cells by the following four methods: (1) intact cells show specific, saturable, and reversible binding of Ap4A; (2) monoclonal antibodies (Mabs) raised against the Ap4A receptor inhibit Ap4A binding to its receptor on intact heart cells; (3) bound Mabs are shown to be at the outer cell surface via reaction with a alkaline phosphatase conjugated goat anti-rat IgG; (4) when intact cells are labeled with the impermeable cell surface labeling reagent, (sulfosuccinimido) biotin, labeled receptor is immunoprecipitated with Mabs. Furthermore, subcellular fractionation of mouse hearts demonstrates that virtually all of the Ap4A receptor is associated with a membrane fraction with at least 77% of the active receptor on plasma membranes.

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Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.

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