Nucleic Acid Sequence of Feline Preprorelaxin and Its Localization within the Feline Placenta1

Abstract

The cat placenta is known to secrete large amounts of relaxin. We employed uteroplacental tissue at approximately Day 35 of gestation to determine the nucleic acid sequence of feline preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Feline preprorelaxin cDNA was found to consist of 540 base pairs encoding a protein of 180 amino acids (aa). We identified a signal peptide of 25 aa, a B domain of 33 aa, a C domain of 98 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the B domain contained one substitution from the classical GRELVR motif (L-->F). Feline preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a specific 1-kilobase transcript present in total RNA of feline uteroplacental tissue but not of liver tissue. Nonradioactive in situ hybridization was used to localize relaxin mRNA, and immunohistochemistry was used to localize the relaxin hormone and cytokeratin, in tissues of the feto-maternal interface recovered from two queens at Day 35 of gestation. Specific hybridization signals for relaxin mRNA were exclusively detected in cells located in the lamellar placental labyrinth but were absent from other placental and nonplacental uterine parts. The cells expressing relaxin mRNA also displayed immunoreactivity for cytokeratin and were, therefore, identified as trophoblast cells. Immunoreactive relaxin colocalized in those placental areas expressing relaxin mRNA. Trophoblast cells located at the villous chorioallantoic tips invading the endometrium and extravillous trophoblast cells in the junctional placental zone were devoid of relaxin.

The molecular detection of relaxin and its receptor RXFP1 in reproductive tissue of Felis catus and Lynx pardinus during pregnancy

“…We found relaxin signals not only in the same cellstrophoblast cells of the placental labyrinth -as described previously by Klonisch et al (1999a) but also in trophoblast cells of villous tips, extravillous trophoblast cells of placental junctional zone, giant cells, blood vessel cells, and cells that could not be further specified.…”

“…The origin of these placental cells was verified by immunoreactions against vimentin, which expresses specificity toward fetal mesenchyme cells, maternal endothelial cells, giant cells, and other maternal cells (as described here and by Walter & Schonkypl (2006)) and against E-cadherin, which detects epithelial cell. Klonisch et al (1999a) confirmed their immunostaining by in situ hybridization experiments, indicating that the source of relaxin was within the placenta. In some other species, the localization of relaxin was also analyzed in placental tissue.…”

“…This was confirmed by a study on the domestic cat, where relaxin immunoreacitvity was detected by RIA mainly in placental tissues and in lower levels in CL, fetus, and uterus (Addiego et al 1987). In addition, the cellular localization of placental relaxin was also shown by in situ hybridization and immunohistochemistry (Klonisch et al 1999a).…”

“…We detected relaxin signals in more cat placenta cell types than Klonisch et al (1999a). So we suggest that our more specific antibody (with an assumed higher affinity to feline relaxin) was also able to detect relaxin at target cells.…”

“…Although antibodies against canine and/or porcine relaxin are able to recognize domestic cat relaxin (Addiego et al 1987, Klonisch et al 1999a (Harris et al 2008), the Witness relaxin test, which is based on a canine antibody, worked well for domestic cats and Pallas cat but failed in others, e.g. cheetahs, lions, and sand cat (Harris et al 2008).…”