title: The effect of nitric oxide inhibition and temporal expression patterns of the mRNA and protein products of nitric oxide synthase genes during in vitro development of bovine pre-implantation embryos.
abstract: This study was conducted to determine the effect of Nitric oxide (NO) inhibition in bovine in vitro development and expression analysis of the three Nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), mRNA and protein in bovine oocytes and embryos. Selective inhibitor of NOS, N-omega-nitro-l-arginine methyl ester (l-NAME) was applied at different doses (0, 0.1, 1 and 10 mm) in maturation (experiment 1A), culture medium (experiment 1B) and in both maturation and culture media (experiment 1C). No significant differences were observed in cleavage and blastocyst rates when oocytes were matured in the presence of l-NAME as long as the inhibitor was omitted during fertilization and culture. However, significantly lower blastocyst rates were observed when l-NAME was present at higher level (10 mm) in culture medium alone and in both maturation and culture media. In experiment 2, mRNA isolated from triplicate pools of oocytes and embryos (n = 15-20) was subjected to quantitative real time reverse transcription polymerase chain reaction to investigate the expression of eNOS, iNOS and nNOS mRNA in normal IVP bovine oocytes and embryos. While eNOS and iNOS transcripts were detected at higher level in oocytes (immature and mature), two-cell and four-cell stage embryos, the nNOS was detected only in immature oocyte, two-cell and morula stages. In experiment 3, eNOS and iNOS protein expression analysis was performed in IVP oocytes and embryos and both proteins were detected in the cytoplasm and the nuclei (weak) of oocytes and embryos. These data provide the first evidence for the role of NO production and the presence of mRNA and protein products of NOS isoforms during bovine embryogenesis.
authors.affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Bonn, Germany. tesfaye@itz.uni-bonn.de
authors.forename: D
authors.initials: D
authors.lastname: Tesfaye
authors.forename: A
authors.initials: A
authors.lastname: Kadanga
authors.forename: F
authors.initials: F
authors.lastname: Rings
authors.forename: K
authors.initials: K
authors.lastname: Bauch
authors.forename: D
authors.initials: D
authors.lastname: Jennen
authors.forename: K
authors.initials: K
authors.lastname: Nganvongpanit
authors.forename: M
authors.initials: M
authors.lastname: Hölker
authors.forename: E
authors.initials: E
authors.lastname: Tholen
authors.forename: S
authors.initials: S
authors.lastname: Ponsuksili
authors.forename: K
authors.initials: K
authors.lastname: Wimmers
authors.forename: M
authors.initials: M
authors.lastname: Montag
authors.forename: M
authors.initials: M
authors.lastname: Gilles
authors.forename: G
authors.initials: G
authors.lastname: Kirfel
authors.forename: V
authors.initials: V
authors.lastname: Herzog
authors.forename: K
authors.initials: K
authors.lastname: Schellander
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000201:enzymology; D002417:Cattle / Q000196:embryology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D010064:Embryo Implantation / Q000502:physiology; D047108:Embryonic Development; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000201:enzymology; D011247:Pregnancy; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester