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Expression of developmentally important genes in preimplantation bovine embryos generated in vitro
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Theriogenology
EXPRESSION OF DEVELOPMENTALLY IMPORTANT GENES IN PREIMPLANTATION BOVINE EMBRYOS GENERUED IN VITRO C. Wrenzycki, D. Herrmann, E. Lemme, J. Eckert, J.W. Camwath and H. Niemann Institut fi.ir Tierzucht und Tierverhalten (FAL), Mariensee, 3 1535 Neustadt, Germany Preimplantation embryo development is characterized by the three distinct morphological steps compaction, cavitation, and blastocoel expansion which require the well-orchestrated expression of genes derived corn the maternal (mat.) and/or embryonic (embr.) genome. In in vitro produced bovine embryos it is thought that embryonic gene expression starts at the 2- to 4-cell stage. The improvement of in vitro production of bovine embryos (IVP) and the development of the reverse transcriptase polymerase chain reaction (RT-PCR) made it feasible to analyse the transcription of developmentally important genes in cattle. The purpose of our present study was to investigate the temporal pattern of transcription of the following genes involved in early embryonic development employing RT-PCR: desmocollin type I, II, III (Dc I, II, BI), desmoglein 1 (Dg I), plakophilii (Plako), connexin43 (Cx43), heat shock protein (Hsp) 70.1, poly(A)polymerase (PolyA), glucose transporter 1 (Glut-l), and trophoblast protein (TP). Embryos were produced in vitro f+om slaughterhouse ovaries as described recently (Theriogenology 43, 1211-1225, 1995) employing TCM 199 supplemented with 0.1% bovine serum albumin (BSA, fraction V) but lacking serum. Immature (iooc.) and matured oocytes (m.ooc.), zygotes (zyg.), 2- to 4-cell (2-Q 8- to 16-cell (8-16) stages, morulae (mo.), blastocysts (bla.) and hatched blastocysts (h.bla.) were produced for this study. Poly(A)RNA was prepared using a Dynabead oligo-dT mRNA purification kit (Dynal, Oslo, Norway). As positive control total RNA was also extracted from bovine tongue and as negative controls RNA or reverse transcriptase were omitted during RT-reaction. After RT-PCR the products were separated via electrophoresis and visualized by ethidium bromide staining. The identity of the fragments was confirmed by sequencing. Assays were repeated at least twice with diierent embryo batches and provided identical results in all samples. The results are shown in the table. Table: Expression of developmentally important genes in bovine embryos
Dg I/DC I Hsp/PolyA/Glut-1 Plako cx43 DcII/DcBI TP
iocc. m.ooc. + + + + + + _ _ +: transcripts
8-16 mo. zyg. 2-4 bla. h.bla. + + + + + + + +--+ + + + + + _ + + + + + + + detectable; -: no transcripts detectable
expression pattern no mat./embr. mat./embr. mat./embr. embr. embr.
In the case of the Cx43 gene the transcripts could be also only of maternal origin due to the persistence of maternal RNA The same phenomenon was observed in embryos cultured in medium containing 10% ECS (e&us cow serum; Wrenzycki et al., 1996, JRF, in press). For the first time the transcription pattern of these developmentally important genes in preimplantation bovine embryos has been demonstrated. Our tindings will aid to compare embryos corn diierent culture systems as well as of in vivo origin to obtain a detailed insight into the transcriptional activity of preimplantation bovine embryos.
Theriogenology
EXPRESSION OF DEVELOPMENTALLY IMPORTANT GENES IN PREIMPLANTATION BOVINE EMBRYOS GENERUED IN VITRO C. Wrenzycki, D. Herrmann, E. Lemme, J. Eckert, J.W. Camwath and H. Niemann Institut fi.ir Tierzucht und Tierverhalten (FAL), Mariensee, 3 1535 Neustadt, Germany Preimplantation embryo development is characterized by the three distinct morphological steps compaction, cavitation, and blastocoel expansion which require the well-orchestrated expression of genes derived corn the maternal (mat.) and/or embryonic (embr.) genome. In in vitro produced bovine embryos it is thought that embryonic gene expression starts at the 2- to 4-cell stage. The improvement of in vitro production of bovine embryos (IVP) and the development of the reverse transcriptase polymerase chain reaction (RT-PCR) made it feasible to analyse the transcription of developmentally important genes in cattle. The purpose of our present study was to investigate the temporal pattern of transcription of the following genes involved in early embryonic development employing RT-PCR: desmocollin type I, II, III (Dc I, II, BI), desmoglein 1 (Dg I), plakophilii (Plako), connexin43 (Cx43), heat shock protein (Hsp) 70.1, poly(A)polymerase (PolyA), glucose transporter 1 (Glut-l), and trophoblast protein (TP). Embryos were produced in vitro f+om slaughterhouse ovaries as described recently (Theriogenology 43, 1211-1225, 1995) employing TCM 199 supplemented with 0.1% bovine serum albumin (BSA, fraction V) but lacking serum. Immature (iooc.) and matured oocytes (m.ooc.), zygotes (zyg.), 2- to 4-cell (2-Q 8- to 16-cell (8-16) stages, morulae (mo.), blastocysts (bla.) and hatched blastocysts (h.bla.) were produced for this study. Poly(A)RNA was prepared using a Dynabead oligo-dT mRNA purification kit (Dynal, Oslo, Norway). As positive control total RNA was also extracted from bovine tongue and as negative controls RNA or reverse transcriptase were omitted during RT-reaction. After RT-PCR the products were separated via electrophoresis and visualized by ethidium bromide staining. The identity of the fragments was confirmed by sequencing. Assays were repeated at least twice with diierent embryo batches and provided identical results in all samples. The results are shown in the table. Table: Expression of developmentally important genes in bovine embryos
Dg I/DC I Hsp/PolyA/Glut-1 Plako cx43 DcII/DcBI TP
iocc. m.ooc. + + + + + + _ _ +: transcripts
8-16 mo. zyg. 2-4 bla. h.bla. + + + + + + + +--+ + + + + + _ + + + + + + + detectable; -: no transcripts detectable
expression pattern no mat./embr. mat./embr. mat./embr. embr. embr.
In the case of the Cx43 gene the transcripts could be also only of maternal origin due to the persistence of maternal RNA The same phenomenon was observed in embryos cultured in medium containing 10% ECS (e&us cow serum; Wrenzycki et al., 1996, JRF, in press). For the first time the transcription pattern of these developmentally important genes in preimplantation bovine embryos has been demonstrated. Our tindings will aid to compare embryos corn diierent culture systems as well as of in vivo origin to obtain a detailed insight into the transcriptional activity of preimplantation bovine embryos.