PubMed1996
In this study we have examined the presence of mRNA encoding connexin 43 (Cx43) in bovine embryos derived in vivo and in vitro. Cumulus-oocyte complexes, immature and matured oocytes liberated from cumulus cells, zygotes, 2-4-cell and 8-16-cell embryos, morulae, blastocysts and hatched blastocysts were produced in 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 were exposed to bull spermatozoa for 19 h and then cultured in TCM 199 supplemented with 10% oestrous cow serum to the desired developmental stage. Morulae and blastocysts derived in vivo were collected from superovulated donor cows. Total RNA was extracted from pools of 60-200 bovine oocytes or embryos using a modified phenol-chloroform extraction method and analysed by reverse transcriptase polymerase chain reaction. Before reverse transcription, aliquots of DNase-digested embryonic RNA were tested by polymerase chain reaction using bovine-specific primers 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 reaction using Cx43 specific primers that define a 516 bp fragment of Cx43. The reverse transcriptase polymerase chain reaction product was verified by restriction enzyme analysis with Alu I and sequencing. Assays were repeated at least twice for each developmental stage and provided identical results between replicates. Cx43 transcripts were detected in bovine morulae and blastocysts grown in vivo. In contrast, whereas the early in vitro stages from cumulus-oocyte complexes to morulae expressed Cx43, blastocysts and hatched blastocysts did not have detectable concentrations of mRNA from this gene. Restriction enzyme cutting revealed three fragments of the predicted size (139, 177, 200 bp). The amplified product showed 100% identity with the published bovine genomic DNA sequence. Under our in vitro conditions the Cx43 gene either had never been activated, which would require that the maternal transcript was stable through early development, or embryonic gene expression that had been active was then terminated prematurely. The differences in transcription between bovine embryos derived in vivo or in vitro indicate that culture conditions affect gene expression. This affords a tool for the further optimization of in vitro production systems for bovine embryos and contributes towards physiological characterization by definition of transcription phenotype of bovine embryos produced in vitro.