RESUMO
The effects of estradiol benzoate (EB) and progesterone (P4) upon progesterone receptor (PR) gene expression in the cerebral cortex and the hypothalamus of the rabbit were studied. Ovariectomized adult rabbits were subcutaneously treated with EB (25 micrograms/kg) for 2 days, and with EB (25 micrograms/kg) + a single dose of P4 (5 mg/kg) on day 3. Twenty-four hours after the last dose, the frontal cortex, the hypothalamus and the uterus were excised, total RNA was extracted and processed for reverse transcription-polymerase chain reaction. PR gene expression was induced by EB and down-regulated by P4 both in the frontal cortex and the hypothalamus in a manner similar to that observed in the uterus. The finding that PR gene transcription is regulated by steroid hormones in the cerebral cortex suggests that post-transcriptional processes are involved in the insensitivity of cortical PR protein to steroids regulation previously reported with binding techniques.
Assuntos
Córtex Cerebral/metabolismo , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/metabolismo , Progesterona/farmacologia , Receptores de Progesterona/genética , Animais , Regulação para Baixo , Feminino , Injeções Subcutâneas , Ovariectomia , Reação em Cadeia da Polimerase , Coelhos , Transcrição GênicaRESUMO
Progesterone (P4) and its metabolites are involved in several functions of the central nervous system (CNS). These steroids participate in neuronal excitability, reproduction and sexual behavior. P4 and its metabolites exert their effects on neurons and glial cells through several mechanisms that include the interaction of the steroids with: 1) intracellular specific receptors; 2) modulatory sites located in neurotransmitter receptors; and 3) ionic channels. By these mechanisms, modifications in gene expression, second messengers' production and ion conductance are induced. The activities of the P4 metabolites have been mainly related to membrane effects, whereas for P4, the transcriptional and translational effects are mediated by intracellular receptors. Thus, these steroids can modify the CNS functions at short (milliseconds), medium (minutes) or long term (hours or days) lapses. The knowledge of the molecular mechanisms involved in the actions of P4 and its metabolites in the CNS will contribute to the understanding of fundamental biological processes such as sexual behavior and reproduction, and it will open the possibility of alternative therapies in the treatment of some neurologic and psychiatric disorders such as epilepsy, anxiety, premenstrual syndrome, and cerebral tumors which possess hormonal regulation.
Assuntos
Sistema Nervoso Central/fisiologia , Progesterona/fisiologia , Animais , Estradiol/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Canais Iônicos/fisiologia , Progesterona/metabolismo , Receptores de Progesterona/fisiologia , Reprodução/fisiologia , Transdução de Sinais/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
Norethisterone (NET) is a synthetic progestin, used as a contraceptive agent, that is biotransformed at target tissues into 5 alpha-NET and 3 beta,5 alpha-NET, which possess different pharmacological properties. The effects of these metabolites on the expression of uteroglobin (UG) and progesterone receptor (PR) genes, both regulated by progesterone (P4), were evaluated in the uterus of prepubertal female rabbits that were simultaneously treated with P4 (1.0 mg) for 5 consecutive days. As determined by Western and Northern blot analyses, 5 alpha-NET inhibited the P4-induced UG gene expression in a dose-dependent manner. A similar inhibition was observed with the administration of RU-486. The estrogenic agent 3 beta,5 alpha-NET and estradiol at a dose of 1.0 mg also inhibited the UG gene expression induced by P4. Both 5 alpha-NET and 3 beta,5 alpha-NET blocked the PR down-regulation induced by P4 as assessed by Western and Northern blot methods. The inhibition of UG synthesis and PR down-regulation by 5 alpha-NET and 3 beta,5 alpha-NET indicates that these NET metabolites possess antiprogestational properties.
Assuntos
Expressão Gênica/efeitos dos fármacos , Noretindrona/metabolismo , Receptores de Progesterona/genética , Uteroglobina/genética , Animais , Northern Blotting , Western Blotting , Regulação para Baixo/efeitos dos fármacos , Feminino , Noretindrona/farmacologia , Progesterona/antagonistas & inibidores , Progesterona/farmacologia , RNA Mensageiro/metabolismo , Coelhos , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
Norethisterone (NET) has been used as a contragestational postcoital agent. It is biotransformed to 5 alpha dihydro-NET (5 alpha-NET) and 3 beta,5 alpha tetrahydro-NET (3 beta,5 alpha-NET) in target tissues. The participation of these metabolites in NET effects is unknown. We have examined the antiimplantation and antiprogestational effects of NET and its metabolites, in adult mated female rabbits, by assessing the number of implantation sites and the expression products of the uteroglobin (UTG) gene in the uterus, and by comparing them with those of RU-486 and estradiol. Steroids were daily administered s.c. at several doses for 7 consecutive days, starting 24 hr after coitus. To assure that fertilization occurred in all animals, the presence of early pregnancy factor was determined. The results demonstrated that high doses (5 mg/kg) of NET reduced both implantation and the expression of the UTG gene. On the other hand, lower doses (1.5 mg/kg) of 5 alpha-NET produced an antiimplantation effect and suppressed UTG synthesis and its mRNA. These effects were similar to those of RU-486. At lower doses (1 mg/kg), both estradiol and the estrogenic metabolite 3 beta,5 alpha-NET were also effective in inhibiting implantation and UTG gene expression. The overall results suggest that NET metabolites exert antiimplantation and antiprogestational effects through their interaction with progesterone and estrogen receptors, and provide an explanation for the molecular mechanisms involved in the postcoital contraceptive action of NET.
Assuntos
Implantação do Embrião/efeitos dos fármacos , Endométrio/metabolismo , Noretindrona/análogos & derivados , Noretindrona/farmacologia , Uteroglobina/biossíntese , Útero/fisiologia , Animais , Biotransformação , Relação Dose-Resposta a Droga , Endométrio/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Fertilização , Masculino , Mifepristona/farmacologia , Noretindrona/metabolismo , Gravidez , Coelhos , Fatores de Tempo , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0-5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1-4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4-5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period.
Assuntos
Desenvolvimento Embrionário/genética , Endométrio/metabolismo , Receptores de Progesterona/genética , Uteroglobina/genética , Animais , Sondas de DNA , Feminino , Expressão Gênica , Gravidez , RNA Mensageiro/genética , CoelhosRESUMO
Enzyme-mediated A-ring reduction of norethisterone (NET) results in the transformation of a molecule with potent intrinsic progestational activity into neutral derivatives with estrogen-like effects. To ascertain whether these structural modifications of NET are able to modify the uteroglobin (U) gene (G) expression, a series of experiments assessing the UG products after the administration of NET and its reduced A-ring metabolites were conducted in prepubertal female rabbits. Synthesis of endometrial uteroglobin and its specific mRNA were studied in animals following the administration of NET, 5 alpha-dihydro NET,3 beta,5 alpha-tetrahydro NET and progesterone. Animals treated with either estradiol or vehicle alone served as controls. The uteroglobin content in uterine flushings and cytosols was determined by immunodiffusion and polyacrilamide gel electrophoresis techniques and by a specific double-antibody radioimmunoassay, while the U mRNA synthesis was assessed by its molecular hybridization to [alpha 32P]d-ATP uteroglobin cDNA. NET induced a significant increase of the uterine content of uteroglobin similar to that observed with progesterone with a simultaneous increase on U mRNA synthesis. On the contrary, 5 alpha-NET and 3 beta,5 alpha-NET induced very little, if any uteroglobin synthesis with a concomitantly low U mRNA production as compared with NET; thus exhibiting a similar effect to that observed in estradiol-treated animals. The overall results were interpreted as demonstrating that the enzyme mediated structural changes of NET which occur at the target organs induce variable expression of the uteroglobin gene. The data indicate that the rabbit uteroglobin gene products are suitable molecular markers to evaluate the hormonal potency of contraceptive synthetic progestins and their derivatives.