Gene expression in the preattachment bovine embryo: Regulation by retinoic acid and in vitro culture system

Methods and scope of study. In vitro bovine embryo production is a feasible way to generate embryos for commercial applications and basic research. The overall objective was to study the regulation of gene expression in in vitro produced preattachment bovine embryos by retinoids and the in vitro culture system. Retinoids, which include vitamin A and its active metabolite, retinoic acid are absolutely required for placental and embryo development. RT-PCR, immunohistochemistry, whole mount in situ hybridization and transient reporter assays were employed to investigate the retinoid signaling pathway. In vitro produced bovine embryos are inferior to their in vivo counterparts and therefore the efficiency of the in vitro embryo production system is poor. We hypothesized that aberrations in gene expression exist between in vivo and in vitro produced bovine embryos. Using SMART-PCR, SSH and real-time RT-PCR we compared global gene expression between in vivo and in vitro produced day 7 blastocysts. SSH was performed in both forward and reverse direction.; Findings and conclusions. mRNA and the immunoreactive protein for RBP, RARalpha, RARgamma, RXRalpha, RXRbeta, RALDH-2 and PPARgamma was detected in oocytes, all embryonic stages and cumulus-granulosa cells. The endogenous retinoid receptors expressed by the cumulus-granulosa cells can bind retinoic acid, recognize specific retinoic acid response elements present in the gene and transduce the signal downstream. We conclude that a functional retinoid signaling system is operational in cumulus-granulosa cells. In the second study, several differentially expressed genes were identified in in vivo and in vitro produced embryos. Galectin-1, fibronectin and filamin A were further characterized using real-time RT-PCR and the data agreed with the SSH analysis. Approximately 38% of the genes identified in in vitro produced embryos were related to transcription and this not only confirms our hypothesis but also tells us that altered gene expression patterns may be due to suboptimal culture conditions. We conclude that the in vitro culture system can alter gene expression during preattachment embryogenesis. These studies provide useful information about early embryo development and in the future may help improve in vitro culture systems so that superior quality embryos can be produced efficiently for various applications.