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

Smith, C.L., Nuri A. Berruien, N., Murray, J.F. and Le Tissier, P.R. 2016. Effect of age and pregnancy on the expression of melanocortin receptors 4 and 5 in the mouse female reproductive axis. BPS Pharmacology 2016. London, UK 12 - 14 Dec 2016 British Pharmacological Society.

TitleEffect of age and pregnancy on the expression of melanocortin receptors 4 and 5 in the mouse female reproductive axis
AuthorsSmith, C.L., Nuri A. Berruien, N., Murray, J.F. and Le Tissier, P.R.
TypeConference poster
Abstract

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, 6146–6151

KeywordsMelanocortin
Female
Reproduction
Melanocortin receptor
Year2016
ConferenceBPS Pharmacology 2016
PublisherBritish Pharmacological Society
Publication dates
Completed14 Dec 2016
Web address (URL)https://www.bps.ac.uk/BPSMemberPortal/media/BPSWebsite/Assets/Pharmacology-2016-programme-book.pdf

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Pathophysiological concentrations of ADMA alter human coronary artery endothelial cell gene expression: an insight into the pathophysiological significance of raised plasma ADMA levels
Smith, C.L., Anthony, S., Malaki, M., Hubank, M., Leiper, J.M. and Vallance, P. 2005. Pathophysiological concentrations of ADMA alter human coronary artery endothelial cell gene expression: an insight into the pathophysiological significance of raised plasma ADMA levels. in: Boger, R., Fleming, I. and Waltenberger, J. (ed.) Vascular Biology and Medicine: 3rd European Meeting, Hamburg, September 2005, abstracts Germany Karger.

Potential therapeutic benefit of novel DDAH inhibitors for the treatment of endotoxemia
Nandi, M., Rossiter, S., Torondel, B., Malaki, M., Smith, C.L., Stidwill, R., Leiper, J.M. and Vallance, P. 2005. Potential therapeutic benefit of novel DDAH inhibitors for the treatment of endotoxemia. in: Boger, R., Fleming, I. and Waltenberger, J. (ed.) Vascular Biology and Medicine: 3rd European Meeting, Hamburg, September 2005, abstracts Germany Karger.

Evidence of systemic and pulmonary endothelial dysfunction in the Dimethylarginine Dimethylaminohydrolase I (DDAH I+/-) heterozygous knockout mouse
Malaki, M., Nandi, M., Madhani, M., Gill, H., Smith, C.L., Leiper, J.M. and Vallance, P. 2005. Evidence of systemic and pulmonary endothelial dysfunction in the Dimethylarginine Dimethylaminohydrolase I (DDAH I+/-) heterozygous knockout mouse. in: Boger, R., Fleming, I. and Waltenberger, J. (ed.) Vascular Biology and Medicine: 3rd European Meeting, Hamburg, September 2005, abstracts Germany Karger.

Dimethylarginine dimethylaminohydrolase I (DDAH I) heterozygous knockout mice display a cardiovascular phenotype consisting of pulmonary and systemic endothelial dysfunction
Malaki, M., Nandi, M., Madhani, M., Gill, H., Smith, C.L., Leiper, J.M. and Vallance, P. 2005. Dimethylarginine dimethylaminohydrolase I (DDAH I) heterozygous knockout mice display a cardiovascular phenotype consisting of pulmonary and systemic endothelial dysfunction. British Pharmaceutical Society Summer Meeting. Cambridge, UK 06-08 Jul 2005

Leptin expression in the fetus and placenta during mouse pregnancy
Malik, N.M., Carter, N.D., Wilson, C.A., Scaramuzzi, R.J., Stock, M.J. and Murray, J.F. 2005. Leptin expression in the fetus and placenta during mouse pregnancy. Placenta. 26 (1), pp. 47-52. https://doi.org/10.1016/j.placenta.2004.03.009

Cardiovascular tests: use & limits of biochemical markers - therapeutic measurements of ADMA involved in cardiovascular disorders
Smith, C.L. and Vallance, P. 2005. Cardiovascular tests: use & limits of biochemical markers - therapeutic measurements of ADMA involved in cardiovascular disorders. Current Pharmaceutical Design. 11 (17), pp. 2177-2185. https://doi.org/10.2174/1381612054367364

Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase
Rossiter, S., Smith, C.L., Malaki, M., Nandi, M., Gill, H., Leiper, J.M., Vallance, P. and Selwood, D.L. 2005. Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase. Journal of Medicinal Chemistry. 48 (14), pp. 1670-1678. https://doi.org/10.1021/jm050187a

Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA
Smith, C.L., Anthony, S., Hubank, M., Leiper, J.M. and Vallance, P. 2005. Effects of ADMA upon gene expression: an insight into the pathophysiological significance of raised plasma ADMA. PLoS Medicine. 2 (10), pp. 1031-1043. https://doi.org/10.1371/journal.pmed.0020264

Effects of low dose ADMA on gene expression in human coronary artery endothelial cells
Smith, C.L., Leiper, J.M. and Vallance, P. 2004. Effects of low dose ADMA on gene expression in human coronary artery endothelial cells. Nitric Oxide: Biology and Chemistry. 11 (1), p. 59. https://doi.org/10.1016/j.niox.2004.07.003

Restoration of nitric oxide production by N-hydroxy-L-arginine in endothelial cells with NO deficiency triggered by elevated glucose
Crabtree, M.J., Smith, C.L. and Gross, S.S. 2004. Restoration of nitric oxide production by N-hydroxy-L-arginine in endothelial cells with NO deficiency triggered by elevated glucose. Nitric Oxide: Biology and Chemistry. 11 (1), p. 65. https://doi.org/10.1016/j.niox.2004.07.003

Overexpression of dimethylarginine dimethylaminohydrolase enhances tumour hypoxia: an insight into the relationship of hypoxia and angiogenesis In vivo
Kostourou, V., Troy, H., Murray, J.F., Cullis, E.R., Whitley, G.S.J., Griffiths, J.R. and Robinson, S. 2004. Overexpression of dimethylarginine dimethylaminohydrolase enhances tumour hypoxia: an insight into the relationship of hypoxia and angiogenesis In vivo. NeoPlasia. 6 (4), pp. 401-411. https://doi.org/10.1593/neo.04109

Effects of low dose ADMA on gene expression in human coronary artery endothelial cells
Smith, C.L., Leiper, J.M. and Vallance, P. 2004. Effects of low dose ADMA on gene expression in human coronary artery endothelial cells. 3rd International Conference on the Biology, Chemistry and Therapeutic Applications of Nitric Oxide: 4th Anuual Scientific Meeting of the Nitric Oxide Society of Japan. Nara, Japan 24-28 May 2004

Restoration of nitric oxide production by N-hydroxyarginine in endothelial cells with NO deficiency triggered by elevated glucose
Crabtree, M.J., Smith, C.L. and Gross, S.S. 2004. Restoration of nitric oxide production by N-hydroxyarginine in endothelial cells with NO deficiency triggered by elevated glucose. 3rd International Conference on the Biology, Chemistry and Therapeutic Applications of Nitric Oxide: 4th Anuual Scientific Meeting of the Nitric Oxide Society of Japan. Nara, Japan 24-28 May 2004

Neonatal 5HT activity antagonizes the masculinizing effect of testosterone on the luteinizing hormone release response to gonadal steroids and on brain structures in rats
Murray, J.F., Dakin, C.L., Siddiqui, A., Pellatt, L.J., Ahmed, S., Ormerod, L.J.A., Swan, A.V., Davies, D.C. and Wilson, C.A. 2004. Neonatal 5HT activity antagonizes the masculinizing effect of testosterone on the luteinizing hormone release response to gonadal steroids and on brain structures in rats. European Journal of Neuroscience. 19 (2), pp. 387-395. https://doi.org/10.1111/j.0953-816X.2003.03158.x

Central orexin A has site-specific effects on luteinizing hormone release in female rats
Small, C.J., Goubillon, M.L., Murray, J.F., Siddiqui, A., Grimshaw, S.E., Young, H., Sivanesan, V., Kalamatianos, T., Kennedy, A.R., Coen, C.W., Bloom, S.R. and Wilson, C.A. 2003. Central orexin A has site-specific effects on luteinizing hormone release in female rats. Endocrinology. 144 (7), pp. 3225-3236. https://doi.org/10.1210/en.2002-0041

Measurement of cardiac troponin I in striated muscle using three experimental methods
Fredericks, S., Bainbridge, K., Murray, J.F., Collinson, P.O., Carter, N.D. and Holt, D.W. 2003. Measurement of cardiac troponin I in striated muscle using three experimental methods. Annals of Clinical Biochemistry. 40 (3), pp. 244-248. https://doi.org/10.1258/000456303321610547

Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype
Smith, C.L., Birdsey, G.M., Anthony, S., Arrigoni, F.I., Leiper, J.M. and Vallance, P. 2003. Dimethylarginine dimethylaminohydrolase activity modulates ADMA levels, VEGF expression, and cell phenotype. Biochemical and Biophysical Research Communications. 308 (4), pp. 984-989. https://doi.org/10.1016/S0006-291X(03)01507-9

Cardiac troponin T and creatine kinase MB content in skeletal muscle of the uremic rat
Fredericks, S., Murray, J.F., Carter, N.D., Chesser, A.M.S., Papachristou, S., Yaqoob, M.M., Collinson, P.O., Gaze, D. and Holt, D.W. 2002. Cardiac troponin T and creatine kinase MB content in skeletal muscle of the uremic rat. Clinical Chemistry. 48 (6), pp. 859-868.

The redox status of bound pterin cofactor determines whether eNOS produces NO or superoxide anion: [3H] - BH4 binding studies provide insights into vascular pathophysiology
Jones, C.L., Vasquez-Vivar, J., Kalyanaraman, B., Griscavage-Ennis, J.M., Gross, S.S. and Smith, C.L. 2002. The redox status of bound pterin cofactor determines whether eNOS produces NO or superoxide anion: [3H] - BH4 binding studies provide insights into vascular pathophysiology. in: Milstien, S., Kapatos, G., Levine, R.A. and Shane, B. (ed.) Chemistry and biology of pteridines and folates: proceedings of the 12th International Symposium on Pteridines and Folates, National Institutes of Health, Bethesda, M.D. Boston, USA Kluwer Academic Publishers. pp. 271-276

Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding
Weissman, B.A., Jones, C.L., Liu, Q., Gross, S.S. and Smith, C.L. 2002. Activation and inactivation of neuronal nitric oxide synthase: characterization of Ca2+-dependent [125I]Calmodulin binding. European Journal of Pharmacology. 435 (1), pp. 9-18. https://doi.org/10.1016/S0014-2999(01)01560-6

Leptin requirement for conception, implantation, and gestation in the mouse
Malik, N.M., Carter, N.D., Murray, J.F., Scaramuzzi, R.J., Wilson, C.A. and Stock, M.J. 2001. Leptin requirement for conception, implantation, and gestation in the mouse. Endocrinology. 142 (12), pp. 5198-5202.

Cardiac troponin T and creatine kinase MB are not increased in exterior oblique muscle of patients with renal failure
Fredericks, S., Murray, J.F., Bewick, M., Chang, R., Collinson, P.O., Carter, N.D. and Holt, D.W. 2001. Cardiac troponin T and creatine kinase MB are not increased in exterior oblique muscle of patients with renal failure. Clinical Chemistry. 47 (6), pp. 1023-1030.

Chapter 10: Tetrahydrobiopterin: An Essential Cofactor of Nitric Oxide Synthase with an Elusive Role
Smith, C.L. 2000. Chapter 10: Tetrahydrobiopterin: An Essential Cofactor of Nitric Oxide Synthase with an Elusive Role. in: Ignarro, L.J. (ed.) Nitric Oxide: Biology and Pathobiology Elsevier. pp. 167-185

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS
Thorup, C., Jones, C.L., Gross, S.S., Moore, L.C. and Goligorsky, M.S. 1999. Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS. American journal of physiology. 277 (6), pp. F882-F889.

An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase
Salerno, J.C., Harris, D.E., Irizarry, K., Patel, B., Morales, A.J., Smith, S.M.E., Martasek, P., Roman, L.J., Masters, B.S.S., Jones, C.L., Weissman, B.A., Lane, P., Liu, Q., Gross, S.S. and Smith, C.L. 1997. An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase. Journal of Biological Chemistry. 272 (47), pp. 29769-29777.

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