EXPRESSION OF THE GAP JUNCTION GENE CONNEXIN43 (CX43) IN PREIMPLANTATION BOVINE EMBRYOS DERIVED IN-VITRO OR IN-VIVO

In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocy te complexes, immature and matured oocytes liberated from cumulus cell s, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and h atched blastocysts were produced iir vitro from ovaries obtained from an abattoir using TCM 199 supplemented with hormones and 10% oestrous cow serum for maturation. Cumulus-oocyte complexes matured for 24 h we re exposed to bull spermatozoa for 19 h and then cultured in TCM 199 s upplemented with 10% oestrous cow serum to the desired developmental s tage. Morulae and blastocysts derived in vivo were collected from supe rovulated donor cows. Total RNA was extracted from pools of oo-zoo bov ine oocytes or embryos using a modified phenol-chloroform extraction m ethod and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RN A were tested by polymerase chain reaction using bovine-specific prime rs to control for residual genomic DNA contamination. DNA-free, total RNA was reverse transcribed after preincubation with the Cx43 specific 3' primer. The resultant cDNA was amplified by polymerase chain react ion using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verif ied by restriction enzyme analysis with Alu I and sequencing. Assays w ere repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected i n bovine morulae and blastocysts grown in vivo. In contrast, whereas t he early in vitro stages from cumulus-oocyte complexes to morulae expr essed Cx43, blastocysts and hatched blastocysts did not have detectabl e concentrations of mRNA from this gene. Restriction enzyme cutting re vealed three fragments of the predicted size (139, 177, 200 bp). The a mplified product showed 100% identity with the published bovine genomi c DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcrip t was stable through early development, or embryonic gene expression t hat had been active was then terminated prematurely. The differences i n transcription between bovine embryos derived in vivo or in vitro ind icate that culture conditions affect gene expression. This affords a t ool for the further optimization of in vitro production systems for bo vine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in v itro.