Expression of retinol-binding protein messenger RNA and retinoic acid receptors in preattachment bovine embryos.

Abstract

In cattle, retinoic acid (RA) has been indirectly associated with developmental potential of the embryo. RA is transported by retinol-binding protein (RBP) and actions of RA are mediated by several subtypes of nuclear retinoic acid receptors (RAR). Bovine embryos, produced in vitro from oocytes harvested from ovaries collected at a local abattoir, were frozen in liquid nitrogen at the oocyte, 2-, 4-, 8-, 16 to 20-cell, morula, blastocyst, and hatched blastocyst stages. Employing reverse transcription polymerase chain reaction (RT-PCR) we investigated mRNA expression for RBP, RARalpha, RARbeta, RARgamma, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Total RNA was extracted from 25 pooled embryos at each stage and RT-PCR analysis was repeated thrice. GAPDH transcript was detected in all stages. Transcripts for RBP, RARalpha, and RARgamma were also detected in all stages from the oocyte through to the hatched blastocyst. Expression of RARbeta was not detected at any stage. Whole-mount immunohistochemistry was performed with intact and hatched blastocysts using polyclonal antibodies against RARalpha and RARgamma2 to investigate if these embryonic mRNAs were translated to the mature protein. Strong immunostaining was observed for both RARalpha and RARgamma2 in the inner cell mass and trophectoderm of intact and hatched blastocysts. Expression of mRNA for RBP, RARalpha, RARgamma, and of the RARalpha and RARgamma2 receptor proteins in the bovine embryo suggests that RA is likely to directly regulate gene expression during preimplantation development in that species.