Endocrine Abstracts

MC3-R expressed in the brain are well characterized however the role of MC3-R in the periphery is unclear. Recently, we 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. The aims of this further work were to confirm that MC3-R is expressed in testes and determine if ACTH_1–39 affects testicular steroidogenesis in vitro.

RNA and protein were extracted from the testes of adult wild-type mice (C57 B1.6) for use in RT-PCR and western blotting, respectively. Testes were also hemi-sected and incubated ± ACTH_1–39 (10⁻¹⁶–10⁻⁸ M) ± LH (2 ng/ml) for 5 h at 35 °C in air. The media were removed and frozen until assayed for testosterone by RIA. Treatments were 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 ±S.E.M.

A single band of the expected PCR product size, 820 bp, was obtained: this band was absent in the negative control. A band, as expected at 40 kDa, was detected by western blotting. Testes incubated in the absence of LH, released 150±40 pg testosterone/mg of tissue (n=3) into the media over 5 h and this was not modified by the addition of ACTH_1–39 regardless of the concentration 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 ACTH_1–39 resulted in the inhibition of steroid production (55% inhibition in the presence of 10⁻¹² M ACTH_1–39: P<0.05).

Both mRNA and protein for MC3-R have been detected in adult mouse testes. ACTH_1–39 has no effect on the basal production of testosterone but appears to inhibit LH-stimulated production.