ABSTRACT

To investigate the participation of intracellular steroid hormone receptors in the sexual transformation process of the Harderian gland, a series of experiments were undertaken in adult golden hamsters. The in-vitro labelling of cytosolic steroid-binding sites with appropriate radioligands revealed the presence of androgen, oestrogen and glucocorticoid but not progestin receptors in the glands from animals of both sexes. The androgen receptor of the female gland was further characterized because it was found to be the predominant intracellular steroid receptor. Studies of binding kinetics using [³H]7α,17α-dimethyl-17β-hydroxy-4-oestren-3-one (DMNT) as ligand, demonstrated a high affinity androgen-binding site with an apparent dissociation constant (K _d) of 0·7 nmol/l and maximal saturation binding capacity of 84·0 ± 3·0 (s.d.) fmol/mg protein. Specificity of the androgen receptor was assessed by displacement analysis; DMNT, 5α-dihydrotestosterone, testosterone and 3α-androstanediol were efficient competitors for the androgen-binding site, while oestradiol-17β, progesterone and dexamethasone exhibited very little, if any, competitive potency. The sedimentation coefficient of the androgen receptor in sucrose density gradients was 8–9 S. These data indicate that the physicochemical characteristics of the androgen receptor from the female gland are similar to those previously described in the male gland. The striking observation of a complete lack of oestrogen-inducible and oestrogen-insensitive progestin receptors in glands cytosol, even after stimulation with cholera toxin, adds further support to the concept that the androgen receptor is the key molecule mediating the hormone-induced sexual transformation of the Harderian gland in this species.

Journal of Endocrinology (1989) 121, 149–156

ABSTRACT

Studies were conducted in castrated golden hamsters to assess whether sexual dimorphism and sensitivity to sex steroid hormones in the rodent Harderian gland are mediated by an interaction of androgens with specific intracellular receptors. Physical properties, binding kinetics and stereospecificity of the androgen receptor were analysed using [³H]mibolerone as the radioligand. The presence of [³H]mibolerone–androgen receptor complexes with a sedimentation coefficient of 7–8S was demonstrated in Harderian gland cytosol by a linear sucrose gradient ultracentrifugation technique using a vertical rotor. Kinetic analysis revealed an androgen-binding site with an apparent dissociation constant of 0·3±0·07 (s.d.) nmol/l and a saturation binding capacity of 113±15 fmol/mg protein. Displacement studies indicated that unlabelled mibolerone, methyltrienolone, 5α-dihydrotestosterone and testosterone were efficient competitors for the androgen-binding sites, while progesterone, 17β-oestradiol, dexamethasone, dehydroepiandrosterone, ethiocholanolone and 5α-16-androsten-3-one were not. Experiments in long-term castrated animals revealed that the Harderian gland androgen receptor concentration and sedimentation coefficient remained unmodified. The results of these studies were interpreted as demonstrating the presence of a specific high-affinity intracellular androgen receptor in the male hamster Harderian gland.

J. Endocr. (1987) 112, 3–8

Abstract

We have determined the presence and distribution of intracellular progesterone receptors (PRs) and glucocorticoid receptors (GRs) in the lung of adult female rabbits using immunohistochemistry. The effects of ovariectomy and administration of oestradiol benzoate (10 μg for 3 consecutive days) upon PR and GR immunoreactivity were also studied. The results demonstrated the presence of both steroid hormone receptors in the female rabbit lung. PR and GR immunoreactivity was predominantly nuclear and located in alveolar epithelial cells and various interstitial cells such as polymorphonuclear leucocytes. Tissue distribution of both receptors was similar in all cases. Oestradiol treatment induced a marked increase in the number of PR immunoreactive cells compared with intact and ovariectomized female animals. Neither ovariectomy nor oestradiol treatment modified the number of GR immunoreactive cells. The presence and localization of intracellular PRs and GRs in several lung cell types suggest that they may play an important role in mediating the effects of progesterone and glucocorticoids in various physiological processes in the rabbit lung. The data also indicated an oestrogen regulation of PRs in the rabbit lung.

Journal of Endocrinology (1994) 142, 311–316

Gestodene (17 alpha-ethynyl-13 beta-ethyl-17 beta-hydroxy-4, 15-gonadien-3-one) is the most potent synthetic progestin currently available and it is widely used as a fertility regulating agent in a number of contraceptive formulations because of its high effectiveness, safety and acceptability. The observation that contraceptive synthetic progestins exert hormone-like effects other than their progestational activities, prompted us to investigate whether gestodene (GSD) administration may induce oestrogenic effects, even though the GSD molecule does not interact with intracellular oestrogen receptors (ER). To assess whether GSD may exert oestrogenic effects through some of its neutral metabolites, a series of experimental studies were undertaken using GSD and three of its A-ring reduced metabolites. Receptor binding studies by displacement analysis confirmed that indeed GSD does not bind to the ER, whereas its 3 beta,5 alpha-tetrahydro reduced derivative (3 beta GSD) interacts with a relative high affinity with the ER. The 3 alpha,5 alpha GSD isomer (3 alpha GSD) also binds to the ER, though to a lesser extent. The ability of the A-ring reduced GSD derivatives to induce oestrogenic actions was evaluated by the use of two different molecular bioassays: (a) transactivation of a yeast system co-transfected with the human ER alpha (hER alpha) gene and oestrogen responsive elements fused to the beta-galactosidase reporter vector and (b) transactivation of the hER alpha-mediated transcription of the chloramphenicol acetyl transferase (CAT) reporter gene in a HeLa cells expression system. The oestrogenic potency of 3 beta GSD was also assessed by its capability to induce oestrogen-dependent progestin receptors (PR) in the anterior pituitary of castrated female rats. The results demonstrated that 3 beta GSD and 3 alpha GSD were able to activate, in a dose-dependent manner, the hER alpha-mediated transcription of both the beta-galactosidase and the CAT reporter genes in the yeast and HeLa cells expression systems respectively. In both assays the 3 beta derivative of GSD exhibited a significantly greater oestrogenic effect than its 3 alpha isomer, while unchanged GSD and 5 alpha GSD were completely ineffective. Neither 3 beta GSD nor 3 alpha GSD exhibited oestrogen synergistic actions. Interestingly, the pure steroidal anti-oestrogen ICI-182,780 diminished the transactivation induced by 3 beta GSD and 3 alpha GSD in the yeast expression system. Furthermore, administration of 3 beta GSD resulted in a significant increase of oestrogen-dependent PR in the anterior pituitaries of castrated rats in comparison with vehicle-treated animals. The characteristics of the 3 beta GSD-induced PR were identical to those induced by oestradio benzoate. The overall results demonstrate that 3 beta GSD and its 3 alpha isomeric alcohol specifically bind to the ER and possess a weak intrinsic oestrogenic activity, whereas unmodified GSD does not. The data contribute to a better understanding of the GSD mechanism of action and allow the hypothesis to be advanced that the slight oestrogenlike effects attributable to GSD are mediated by its non-phenolic, tetrahydro reduced metabolites.