167 EXPRESSION PATTERN OF GAP JUNCTIONAL CONNEXINS DURING IN VITRO AND IN VIVO PREIMPLANTATION EMBRYO DEVELOPMENT IN BOVINE

Abstract

During preimplantation development, several connexin proteins are expressed and assembled into gap junctions in the plasma membrane at compaction but the functional significance of connexin diversity remains controversial. The present investigations were (i) to compare the expression pattern of a panel of gap junctional connexin (Cx) gene transcripts from in vitro-produced (IVP) under low (5%) and high (20%) oxygen (O₂) concentrations and in vivo-derived (IVD) bovine embryos during various preimplantation stages, and (ii) to evaluate the expression of the same set of gene transcripts in blastocysts derived from IVP (low O₂ concentration) and in vivo embryos following a conventional cryopreservation method using 1.5 m ethylene glycol (EG) (Hasler et al. 1997 Theriogenology 48, 563–579). Cumulus-oocyte complexes were matured in TCM199 supplemented with 10% FBS and hormones for 22 h at 39°C, 5% CO₂ in air, and then inseminated and cultured in SOF medium for 7 days. IVD embryos were collected from 18 superovulated and artificially inseminated cows. In Experiment I, five pooled embryos from each developmental stage (2-, 4-, 8-, 16-cell, morula, and blastocyst) and embryo source (IVP under low and high O₂ concentrations and IVD) and, in Experiment II, Day 7 IVP (low O₂ concentration) and IVD blastocysts following cryopreservation and storage for at least 1 week were used for analyzing the expression pattern of gap junctional connexin (Cx30, Cx31, Cx32, Cx36, Cx43, and Cx45) gene transcripts using real-time RT-PCR (four replicates). Normalization of mRNAs at each developmental stage of bovine preimplantation embryos was performed by employing a similar amount of RNA at the reverse transcription (RT) step. This was followed by analyzing by qRT-PCR the target genes using GAPDH as a reference gene. Significant differences in gene expression were analyzed byANOVA and Student's t-test. Relative abundances (RA) of Cx30, Cx31, and Cx32 of IVD embryos were significantly (P < 0.05) higher at all stages compared to that of IVP embryos, except at the blastocyst stage for Cx32. Differences in Cx36 and Cx45 were observed at all stages, with the levels being higher (P < 0.05) in IVD than in IVP embryos. However, the differences at the 4-cell stage between the two embryo sources were not significant. The RA of Cx43 transcript in IVD embryos at 4- and 16-cell stages was higher (P < 0.05) than in IVP embryos, but did not differ at the 2-cell stage. Furthermore, there were differences at the 8-cell and blastocyst stages among IVD, and low and high O₂ IVP embryos. Following cryopreservation, RA of all analyzed connexin transcripts of IVD blastocysts were significantly (P < 0.05) higher than that of the low O₂ concentration. However, the expression levels in both embryo sources were lower compared to blastocysts before cryopreservation, except for Cx36. To the best of our knowledge, this is the first report on the differences in the expression pattern of a panel of gap junctional connexin gene transcripts during the key developmental stages of IVP embryos with direct comparisons of IVD counterparts.