Expression of mRNA for alpha1-adrenoceptor subtypes at the beginning of neurulation in rat embryos

We recently reported that alpha1-adrenoceptor agonists, administered at the beginning of neurulation (Stage 11a) in rat embryos grown in culture, interfere with normal development of the left/right body axis leading to situs inversus. Despite these pharmacological findings, expression of alpha1-adrenoceptor genes at such an early stage of development has not been demonstrated. In the present study, we examined the expression of mRNAs for cloned alpha1-adrenoceptor subtypes in rat embryos at Stage 11a. Timed-pregnant Sprague-Dawley rats were killed in the morning of gestational day 9 (vaginal plug day = day 0), and the implantation sites were removed. The implantation sites were separated into embryo, ectoplacental cone and decidua, only those at Stage 11a were selected, and these were immediately frozen on dry ice, and subsequently their total RNA was isolated. RNase protection assays were then performed for cloned alpha1a-, alpha 1b- and alpha1d-adrenocepter subtypes using 20-30 mug of total RNA for each hybridization. In all tissues, strong and weak signals were detected for alpha1b- and alpha1a-adrenoceptor subtype mRNAs, respectively. In contrast, a signal for alpha1d mRNA was not detected in any tissues. These results, together with previous pharmacological findings, suggest the existence of alpha1a- and alpha1b-adrenoceptor subtypes in rat embryos at Stage 11a.