Molecular characterization of the melanocortin 3 receptor in the testes of mice

Monika M. Horanin, Kemi Akindele, Stephen J. Getting, Caroline Smith, Joanne Murray. University of Westminster, London, United Kingdom.

Melanocortins, such as ACTH, bind to a family of receptors: melanocortin receptors 1 to 5. Melanocortin 3 receptors (MC3-R) expressed in the brain are well characterized however the role of MC3-R in the periphery is unclear. We have recently described immunopositive staining for MC3-R in the testes of wild type mice and reported that the testicular histology of the MC3-R null mouse was abnormal (Wotherspoon et al 2008). The aims of this further work were to confirm that MC3-R is expressed in testes and determine if ACTH affects testicular steroidogenesis in vitro.RNA was extracted from the testes of sexually mature wild type (C57 B1.6) using Trizol (Invitrogen). MMLV-reverse transcriptase and random primers were used to generate cDNA, with no MMLV-RT in the non-template control. Primers for MC3-R were used as previously described (Getting et al 1999). To ensure that MC3-R was also expressed at the protein level, Western blotting of mouse testes lysate was done using a standard protocol with a previously characterized rabbit anti-MC3-R (Leoni et al 2008). In another series of experiments, testes collected from sexually mature wild type mice were hemi-sected and incubated in McCoy’s 5A media ± ACTH1-39 (10-16-10-8M) ± LH (2 ng/ml) for 5 hours at 35°C in air. At the end of the incubation the media were removed and frozen until assayed for testosterone by RIA. Each treatment was done in quadruplicate and each experiment was repeated 2 or 3 times. The results are presented as the mean of the means of each experiment ± SEM. *P<0.05 versus appropriate control.

PCR amplification of mouse testis cDNA yielded a single band of the expected PCR product size, 820 bp, on a 1.5% agarose gel. This band was absent in the non-template negative control. A band at the expected 40 kDa was detected by Western blotting with protein separated on 12% SDS-PAGE. In the absence of LH, the amount of testosterone released into the media over 5 hrs was 150±40 pg/mg of tissue (n = 3) irrespective of the concentration of ACTH1-39 used. Addition of LH significantly increased the amount of testosterone released into the media (620±80 pg/mg of tissue: *P<0.05) whilst the addition of ACTH1-39 modulated the amount of steroid produced there was a 55% inhibition of steroid produced in the presence of 10-10M ACTH1-39 (280±70 pg/mg of tissue: *P<0.05). In contrast to others (O’Shaughnessy et al 2003), we have demonstrated the presence of both mRNA and protein for MC3-R in adult mouse testes. Physiological concentrations of ACTH1-39 had very little effect on the basal production of testosterone, as has been previously shown by others (O’Shaughnessy et al 2003), but appears to inhibit LH stimulated production. ACTH signalling may have an important modulatory role in the male reproductive axis: further work is required.

Wotherspoon, G. et al. (2008) Proc. British Pharm. Soc. 6(4): abst067P.
Leoni, G. et al. (2008) FASEB J., 22, 4228-4238.
O’Shaughnessy, P.J. et al. (2003) Endo, 144, 3279-3284.
Getting, S.J. et al (1999) J. Immunol., 162, 7446-7453.

(MH and KA are joint first author).