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The key role of estrogens on osteoblastic cell function is well documented; however, the role of progesterone (P) and synthetic progestins remains controversial. While several reports indicate that P has no significant effects on bone cells, a number of clinical studies have shown that 19-norprogestins restore postmenopausal bone loss. The mechanisms by which 19-norprogestins induce estrogen-like effects on bone cells are not fully understood. To assess whether the actions of 19-norprogestins on osteoblasts are mediated by their non-phenolic metabolites, we studied the effects of norethisterone (NET), levonorgestrel (LNG), and two of their A-ring reduced derivatives upon cell proliferation and differentiation in neonatal rat osteoblasts. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin and calcium content, alkaline phosphatase activity, and mineral deposition. P failed to induce changes on osteoblasts, while NET and LNG exerted a number of actions. The most striking finding was that the 3beta,5alpha- and 3alpha,5alpha-tetrahydro derivatives of NET and LNG induced osteoblast proliferation and differentiation with higher potency than those exerted by their parent compounds, mimicking the effects of estradiol. Interestingly, osteoblast differentiation and mineral deposition induced by NET and LNG were abolished by finasteride, a 5alpha-reductases inhibitor, while the potent effect on osteoblast proliferation induced by progestin derivatives was abolished by a steroidal antiestrogen. Results demonstrate that A-ring reduced derivatives of NET and LNG exhibit intrinsic estrogen-like potency on rat osteoblasts, offering a plausible explanation for the mechanism of action of 19-norprogestins in bone restoration in postmenopausal women and providing new insights for hormone replacement therapy research.

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.

Norethisterone (NET) and its metabolite 5alpha-norethisterone (5alpha-NET) are competitors for the androgen receptor. The sensitivity of the rat vas deferens to the contractile action of methoxamine and serotonin is regulated by hormonal and anatomical factors. The aim of this study was to evaluate the ability of NET and 5alpha-NET to induce the androgen-regulated contractile response to methoxamine and serotonin in the epididymal and prostatic portions of rat vas deferens. Adult male rats either intact, castrated or steroid-treated castrated were used. The contractility was recorded isometrically, and non-cumulative concentration-response curves to either methoxamine or serotonin were obtained. NET and 5alpha-NET partially restored the sensitivity to methoxamine and serotonin in the epididymal portion of castrated rats. The maximal responses to both agonists were significantly higher than those observed in castrated rats, and significantly lower than the responses observed in either intact or androgen-treated castrated rats. The prostatic portion was less responsive to both agonists than the epididymal portion, in all groups but castrated rats, as castration induced sensitivity to both agonists. NET and 5alpha-NET displayed a partial though similar androgenic activity in the rat vas deferens. These results contrast with previous reports where a decrease of androgenic effect due to the 5alpha-reduction of NET has been found.

The putative anxiolytic action of the synthetic progestins, norethisterone (NET), levonorgestrel (LNG) and their respective 5 alpha-reduced metabolites, (5 alpha-NET), (3 alpha,5 alpha-NET), (3 beta,5 alpha-NET), (5 alpha-LNG), (3 alpha,5 alpha-LNG) and (3 beta,5 alpha-LNG), were studied in the burying behavior paradigm. From these compounds only 3 alpha,5 alpha-NET and 3 beta,5 alpha-LNG reduced burying behavior without modifying other parameters. Burying behavior latency was prolonged after administration of the highest dose (1.0 mg/rat) of NET and 5 alpha-NET. As a positive control 3 alpha,5 alpha-pregnanolone (allopregnanolone) was included at the same doses used for synthetic steroids. This steroid produced a clear dose-dependent reduction in burying behavior without affecting latency. No steroid with anxiolytic properties affected the general ambulatory behavior. All synthetic steroids with anxiolytic activity proved to be less potent than allopregnanolone. Results are discussed on the basis of the chemical structure requirements necessary to induce tranquilizing effects.