title: Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct.
abstract: In this study, a combination of RNAi and endoscopic transfer to the oviduct of synchronized heifers has been used to investigate the effect of suppression of Cx43 and E-cadherin on the development, mRNA and protein expression of bovine blastocysts cultured in vitro or in vivo. In vitro matured and fertilized bovine zygotes were randomly assigned to one of four groups namely: Connexin43 dsRNA-injected (n = 790), E-cadherin dsRNA-injected (n = 775), water-injected (n = 774), and noninjected controls (n = 652). Following 2 days in vitro culture, 4- and 8-cell stage embryos from each treatment group were used for culture in vitro or in vivo. About half of the 4-8-cell stage embryos from each treatment group were transferred to the oviduct of synchronized heifers, while the remainder were further cultured in vitro. Embryos from in vivo culture were flushed from recipients on the fourth day post transfer (= Day 7 post insemination). Blastocyst stage embryos from both culture systems were used for mRNA and protein expression analysis. Irrespective of treatment or culture conditions, microinjection resulted in a decline in the proportion of embryos reaching the blastocyst stage. Significantly, lower blastocyst development was observed in E-cadherin and water-injected embryos following in vivo culture compared to the noninjected controls, while intermediate results were obtained following injection with Cx43 dsRNA. Both mRNA and protein products of the target genes were suppressed but the efficiency of suppression of the target genes varied depending on the initial level of transcript abundance, which is known to be greatly affected by the culture environment.
authors.affiliation: Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Bonn, Germany. tesfaye@itz.uni-bonn.de
authors.forename: Dawit
authors.initials: D
authors.lastname: Tesfaye
authors.forename: Patrick
authors.initials: P
authors.lastname: Lonergan
authors.forename: Michael
authors.initials: M
authors.lastname: Hoelker
authors.forename: Franca
authors.initials: F
authors.lastname: Rings
authors.forename: Korakot
authors.initials: K
authors.lastname: Nganvongpanit
authors.forename: Vitezslav
authors.initials: V
authors.lastname: Havlicek
authors.forename: Urban
authors.initials: U
authors.lastname: Besenfelder
authors.forename: Danyel
authors.initials: D
authors.lastname: Jennen
authors.forename: Ernst
authors.initials: E
authors.lastname: Tholen
authors.forename: Karl
authors.initials: K
authors.lastname: Schellander
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D005260:Female; D005307:Fertilization in Vitro; D010057:Oviducts; D034622:RNA Interference; D012330:RNA, Double-Stranded; D011897:Random Allocation
chemical_list: D015820:Cadherins; D018031:Connexin 43; D012330:RNA, Double-Stranded; D004247:DNA