Effect of age and pregnancy on the expression of melanocortin receptors 4 and 5 in the mouse female reproductive axis

Introduction: There are five melanocortin receptors (MC1-5): these are G-protein-coupled receptors expressed in the central nervous system and in peripheral tissues. MC4 and MC5 have roles in controlling appetite, immuno-modulation, exocrine function, erectile dysfunction and grooming behaviour1. The melanocortin receptor accessory proteins (MRAP1 and 2) influence MC receptor transport. Proopiomelanocortin (POMC) is the precursor for the alpha-melanocyte stimulating hormones and adrenocorticotrophic hormone, which bind to MC4 and MC5 receptors1. Aims: 1. To characterise MC4, MC5 and MRAP1 distribution in the reproductive tissues of female mice (hypothalamus, pituitary gland, uterus and ovary). 2. To investigate if MC4, MC5 and MRAP1 expression changes with age or pregnancy. Method: Virgin C57BL/6 female mice aged 2, 6, 9, 10 and 14 weeks and pregnant (aged 9 weeks plus 13 days post coitus) were sacrificed by schedule 1. Tissue RNA was extracted by TRIzol/RNA cleanup (Qiagen) and cDNA synthesised using SuperScript II reverse transcriptase (Thermoscientific). Appropriate reference genes were determined using GeNorm selection kit and software (Primerdesign; Biogazelle respectively). The following genes were the most stable and used in SYBRgreen RTqPCR according to precisionPLUS master mix protocol (Primerdesign for normalisation2 of MC4, MC5 and MRAP1 expression: Qiagen primers). Tissue Reference gene 1 Reference gene 2 Hypothalamus YWHAZ CANX Pituitary gland EIF4A2 CANX Ovary GAPDH ATP5B Uterus CYC1 RPL13A Results: MC4 and MC5 were expressed in the hypothalamus, pituitary, ovary and uterus. MC4 and MC5 expression was higher in 2 week old mice in the hypothalamus (n=3, p=0.0056 and <0.0001) and uterus (n=3, p=0.0119 and <0.0001, respectively) compared to all other age groups using one-way ANOVA and Tukey’s post-hoc test. Pregnancy did not affect either MC4 or MC5 expression. MRAP1 was expressed in the hypothalamus, ovary and pituitary and was unaffected by age. MRAP1 expression was greater in the ovary and decreased in the hypothalamus of pregnant compared to non-pregnant mice (n=3, p=0.0004). Conclusion: The role of the two MC in the development of the hypothalamus and uterus in young mice requires further investigation. The changes in MRAP1 expression with pregnancy could result in changes in the signalling ability of both MC4 and MC5 since MRAP1 has been shown to alter cAMP production resulting from activation of these two receptors in vitro3. References: 1. Gantz I. and Fong TM. (2003). Am J Physiol Endocrinol Metab. 284, E468-E474 2. Vandesompele J. (2002). Genome Biology 3,–research0034.11 3. Chan et al., (2009). PNAS. 106, 6146–615