| Abstract | The five melanocortin receptors (MC (1-5)) are part of the G-coupled protein receptor (GPCR) family and have been reported to have a role in controlling appetite, modulating inflammation and stress response. The melanocortin receptor accessory proteins (MRAP1 and 2) influence MC receptor transport and signalling. The MCs are activated by proopiomelanocortin (POMC) derivatives: alpha-, beta-, and gamma-melanocyte stimulating hormones and adrenocorticotrophic hormone; while agouti signalling peptide (ASP) and agouti-related protein (AgRP) antagonise the peptide activity. The MCs were previously detected in the male mouse reproductive system, and their expression levels were found to be influenced by age.
This thesis aims to investigate the expression of the MC system in the female mouse reproductive system and to determine the influence of age and pregnancy on the expression levels of MCs, accessory proteins, POMC and AgRP.
The gene expression of the MC system in the reproductive axis using RT-qPCR revealed the differential MC expression in the female mouse hypothalamus, pituitary gland, ovary and uterus. Age influenced the expression of MC5 and POMC, whilst pregnancy altered the expression of MC2, MC4 and MC5. This research reports, for the first time, the characterisation of MRAP1 expression in the hypothalamus and the pituitary gland. The chromogenic RNA in situ hybridisation method (RNAscope®) enabled MC3, MC5 and MRAP2 localisation in the hypothalamus, in the preoptic (rostral and medial) areas and arcuate nucleus. Those areas are known to contain the gonadotropin-releasing hormone (GnRH) neuronal network in the female mouse hypothalamus. The RNAscope® studies also detected the expression of MC3 and MRAP2 in the gonadotrophs and the somatotrophs of the mouse anterior pituitary gland; these findings imply a modulatory role of MC3 in the anterior pituitary cellular network. The expression of MC3, MC5 and MRAP2 was influenced by age and pregnancy. The coexpression of MC3 and MRAP2 in the hypothalamus and the anterior pituitary gland, implicate the potential regulation of MC3 responsiveness by MRAP2.
An in vitro cell culture model of GPCR signalling “PRESTO-Tango” assay investigated the regulation of MC3 by the MRAPs and revealed a significant increase in the potency of MC3 to ACTH (1-39), and NDP-a-MSH MC3 in the presence of MRAP1. These studies also show a reduction in sensitivity of MC3 to ACTH (1-39) with 1:1 co-expression of MC3: MRAP2 whilst a 1:10 MC3: MRAP2 expression ratio increased the sensitivity of MC3 to both ACTH (1-39) and NDP-a-MSH. The data obtained in this thesis indicates that the members of the MC system are in close association within areas controlling female reproduction and that the MC system expression is affected by age and pregnancy. Finally, this research reports the regulation of MC3 by both MRAP1 and MRAP2 in vitro. |
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