EXPRESSION OF RNA FROM DEVELOPMENTALLY IMPORTANT GENES IN PREIMPLANTATION BOVINE EMBRYOS PRODUCED IN TCM SUPPLEMENTED WITH BSA

This study investigated the effects of a semi-defined culture system o n the temporal pattern of expression of RNA from genes involved in com paction and cavitation: gap junction protein connexin43 (Cx43), desmos omal glycoproteins desmoglein 1 (Dg 1), desmocollins I, II and III (Dc I, Dc II, Dc III), desmosomal protein plakophilin (Plako); metabolism glucosetransporter-1 (Glut-1); RNA processing poly(A)polymerase (Poly A); heat shock protein 70.1 (HSP); and trophoblastic function trophobl ast protein (TP) in bovine oocytes and embryos generated in vitro usin g TCM199 supplemented with BSA as the culture medium. Morulae and blas tocysts derived in vivo were collected from superovulated heifers and also used for this study. Poly(A)(+) RNA was extracted from pools of 2 0-50 oocytes or embryos, analysed by reverse transcription-polymerase chain reaction and the amplified fragments were verified by sequencing . Assays were repeated at least three times for each developmental sta ge and provided consistent results in all replicates. In bovine embryo s produced in vitro, mRNA encoding Cx43 was detectable up to the morul a stage, whereas blastocysts and hatched blastocysts did not express t his gene. No transcripts were found for Dg I and Dc I throughout the t ested preimplantation stages. Dc II and Dc III transcripts, were found from 2-4-cell embryos up to the hatched blastocyst stage. mRNA encodi ng Plako was detected in immature and mature oocytes and zygotes, whil e no transcripts were seen in 2-4-cell and 8-16-cell embryos. The gene was expressed again from the morulae to the hatched blastocyst stage. Oocytes and bovine embryos produced in vitro showed transcripts for G lut-1, PolyA and HSP throughout preimplantation development up to the hatched blastocyst stage. The gene encoding TP was transcribed only in blastocysts and hatched blastocysts. Morulae and blastocysts produced in vivo showed the same expression as their in vitro counterparts, wi th one exception: the in vivo embryos transcribed Cx43. The results of this study reveal for the first time the transcriptional pattern of a set of 'marker' genes involved in various processes in early bovine e mbryonic development. Transferable morulae and blastocysts produced in vitro expressed most genes similar to their in vivo counterparts. The se data contribute to the molecular characterization of this widely us ed in vitro culture system for bovine embryos and provide a major adva nce towards production of 'physiologically normal' embryos.