Canine preprorelaxin: Nucleic acid sequence and localization within the canine placenta

Employing uteroplacental tissue at Day 35 of gestation, we determined the n ucleic acid sequence of canine preprorelaxin using reverse transcription- a nd rapid amplification of cDNA ends-polymerase chain reaction. Canine prepr orelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino a cids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding r egion in the N'-terminal part of the canine relaxin B domain GRDYVR contain ed two substitutions from the classical motif (E-->D and L-->Y). Canine pre prorelaxin shared highest homology with porcine and equine preprorelaxin. N orthern analysis revealed a l-kilobase transcript present in total RNA of c anine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in si tu hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin , cytokeratin, vimentin, and von Willebrand factor was performed on uteropl acental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin o r vimentin but was immunopositive for cytokeratin and identified as cytotro phoblast cells. The cell layer surrounding the chorionic villi displayed sp ecific hybridization signals for relaxin mRNA and immunoreactivity for rela xin and cytokeratin but not for vimentin, and was identified as syncytiotro phoblast. Those areas of the chorioallantoic tissue with most intense relax in immunoreactivity were highly vascularized as demonstrated by immunoreact ive von Willebrand factor expressed on vascular endothelium. The uterine gl ands and non-placental uterine areas of the canine zonary girdle placenta w ere devoid of relaxin mRNA and relaxin. We conclude that the syncytiotropho blast is the source of relaxin in the canine placenta.