The role of relaxin family receptor 1 and 2 (RXFP1 and RXFP2) in gender- specific physiology and pathology

In most female mammals the peptide hormone relaxin plays an important role in reproductive physiologies, such as decidualisation, lactation, implantation, birth, uterine vascularization, and uterine quiescence, as well as maintaining systemic osmolarity during pregnancy. Both relaxin and the structurally related hormone INSL3 (insulin-like peptide 3) are specific ligands for the large GPCR receptors RXFP1 and RXFP2, respectively. At a low physiological nanomolar range, both peptide hormones can bind and activate only their cognate receptors, though in humans, relaxin may interact with RXFP2 at the high supra-physiological concentration. In ruminants, such as sheep or cows, the gene for relaxin has been deleted during recent evolution, though the receptors RXFP1 and RXFP2 appear to be still present. The aim of this research was to determine whether these receptors were normally expressed in the ruminant uterus, whether they were still functional in a physiological context, and therefore whether the development of relaxin analogues as a potential therapeutic agent for cows and sheep would be a plausible option. Firstly, the expression of gene transcripts for the ruminant RXFP1 and RXFP2 receptors was assessed in different uterine cells. It was found that RXFP1 was expressed in a sheep glandular epithelial cell-line (OGE), as well as in primary cultures of bovine uterine stromal and myometrial cells. Additionally, RXFP1 appears to be weakly expressed in bovine endometrial epithelial cells. RXFP2 gene transcripts were expressed at low levels in bovine myometrial cells and ovine stromal cells. As receptor signalling end-point, cAMP production by uterine cells was assessed. cAMP production was increased by 100ng/ml relaxin in the presence or absence of IBMX or forskolin in bovine stromal cells. However, cAMP production was inhibited by relaxin in the OGE cell-line and in primary bovine myometrial cells. Also, cAMP production was inhibited by INSL3 in bovine myometrial cells and the ovine stromal cell-line (OST). We also tested the small molecule agonist ML290 and its analogues, which had been shown to activate human RXFP1 in an allosteric manner. None of the ML290-related compounds were found to have any effect on cAMP production at the bovine RXFP1 receptor. As alternative end-points to test for relaxin functionality at its specific receptor RXFP1, specific gene expression was assessed in the sheep glandular epithelial (OGE) cell-line. Genes were chosen which are involved in the maternal recognition of pregnancy, using relaxin, oxytocin (OT) and interferon-t (IFN-tau) as effectors. It was found that the expression of the oxytocin receptor (OTR) at 8 hours and the progesterone receptor (PR) at 24 hours were significantly inhibited by 100nM relaxin. There were also significant effects in synergy with IFN-tau and/or OT on the expression of PDGS2, AKR1A1, and ER. Similar indicator genes were also assessed for expression in response to relaxin in primary cultures of myometrial smooth muscle cells. Effects were seen on both OTR (stimulatory) and ER(inhibitory) gene expression. No other genes tested appeared to be influenced by relaxin, though could be affected by either IFN-tau or OT. In order to determine further relaxin-dependent end-points that might be important in the context of uterine physiology, archived RNA samples from mouse and human were utilized which derived from previous experiments in the Anand-Ivell laboratory. These samples were re-analyzed using either modern RNA-Seq technology or microarray analysis in order to obtain a global profile of transcriptome changes consequent upon relaxin stimulation. Although the species and experimental paradigms were quite different, there were also marked similarities in the signaling pathways that were activated by relaxin. In particular, pathways associated with cytokine production, inflammation, as well as the cell cycle were significantly altered. Future research in the bovine should additionally focus on gene products typical for these pathways. Altogether, in spite of the absence of the natural hormone relaxin from sheep and cows, my research has indicated that numerous cells and signaling pathways within the ruminant uterus can still be influenced by exogenous hormone. This opens up the possibility in the future of developing relaxin analogues as potential therapeutic agents in the treatment of uterine dysfunction in sheep and cows. Unfortunately, because of the Covid-19 epidemic, my research had to be truncated and further experimentation restricte