孕酮受体介导哺乳动物雌性生殖活动的分子机理

孕酮作为一种甾体激素,在哺乳动物雌性生殖活动的调控中起着关键作用。孕酮的生理功能依赖于核孕酮受体介导的基因组效应和膜孕酮受体介导的非基因组效应,这两种效应共同介导了孕酮在各种雌性生殖活动中的不同作用,包括排卵、胚胎植入、妊娠维持、分娩启动和乳腺发育等。近年来,通过基因芯片技术筛选出大量的孕酮下游靶基因,但至今未能在这些基因的启动子区域上找到传统意义上的孕酮响应元件,故推测核孕酮受体调节下游靶基因转录活动的方式可能不同于传统的类固醇核受体。基于目前最新的研究成果,文章综述了在哺乳动物雌性生殖活动中,孕酮受体介导各种生理效应的分子机理。     

Progesterone receptor replenishment in T47D human breast cancer cells. Roles of protein synthesis and hormone metabolism

T47D are unusual human breast cancer cells that do not require estrogen to synthesize high levels of progesterone receptors. These cells can, therefore, be used to study the mechanisms by which progesterone, freed of estrogen interference, controls the synthesis of its receptors. In a recent paper we described progesterone receptor translocation and a subsequent very rapid nuclear processing step that results in an apparent loss of 60 to 80% of cellular progesterone receptors, 30 to 60 min after progesterone treatment. This paper deals with the replenishment of cellular receptors following processing. If progesterone is removed from cells after 60 min of treatment, cytoplasmic progesterone receptors replenish in 16 to 20 h. However, replenishment occurs even during chronic progesterone treatment; this is an artifact created by the extremely rapid (t1/2 approximately 2 h) metabolism of progesterone in media exposed to cells. If progesterone metabolism is blocked, then replenishment is not seen, probably because the hormone continuously retranslocates the newly replenished sites. There is an early protein synthesis-dependent step; cycloheximide in the first 4 h inhibits replenishment 24 h later, but if cycloheximide is slightly delayed (beyond 4 h), replenishment proceeds normally. In contrast to progesterone, the synthetic progestin R5020 completely suppresses progesterone receptor replenishment even 96 h after its removal from the medium. This compound can bind covalently to receptors and may be very difficult to remove from cells. Clearly, progestin treatment, and by analogy, circulating progesterone, will have profound effects on cytoplasmic and nuclear progesterone receptor levels when these are measured in biopsied human tumors as an adjunct to endocrine therapy.     

Estradiol and progesterone differentially regulate formalin-induced nociception in ovariectomized female rats

Clinical and preclinical studies have found sex-specific differences in the discrimination and perception of inflammatory stimuli. The emerging picture suggests that the biological basis of these differences resides in the regulatory activity of gonadal hormones in the central nervous system. This study describes the effects of ovarian hormones in inflammatory pain processes. Ovariectomized rats received estradiol and/or progesterone, and the number of paw flinches was measured after 1, 2.5 or 5% formalin administration. Both estradiol and progesterone altered the number of flinches only after 1% formalin administration. Estradiol significantly reduced the overall number of flinches during Phase II of the formalin nociceptive response while progesterone attenuated Phase I of the response. After co-administration of estradiol and progesterone, progesterone reversed estradiol's analgesic effect in Phase II, however, estradiol did not reverse progesterone's analgesic activity in Phase I. To determine if estradiol effects are receptor-mediated, tamoxifen (selective estrogen receptor mediator, 15 mg/kg) or alpha-estradiol (an inactive isomer of estradiol, 20 microg) were utilized. Tamoxifen decreased the number of formalin-induced flinches during Phase II while alpha-estradiol did not affect any formalin-induced responses. When co-administered with estradiol, tamoxifen failed to reverse estradiol's effect, suggesting both tamoxifen and estradiol activate similar intracellular mechanisms. Although Western blot analysis detected the presence of estradiol alpha and beta and progesterone B receptors in the spinal cord, hormone replacement treatments had no effects on the levels of these receptors. We postulate that the mechanisms by which estradiol and progesterone induce analgesia occur through the activation of their receptor at the spinal cord level.     

Reversible dissociation of steroid hormone x receptor complexes by mercurial reagents

Steroid hormone receptors contain a reactive sulfhydryl group (or groups) required for hormone binding. In the present study, the effects of several sulfhydryl-blocking reagents on hormone binding to aporeceptors and hormone x receptor complexes were compared, with the use of preparations of chick oviduct progesterone receptor and intestinal vitamin D receptor. N-Ethylmaleimide inhibited hormone binding to aporeceptors, whereas prior hormone binding protected against inactivation. In contrast, the mercurial reagent mersalyl both inhibited hormone binding to aporeceptors and dissociated hormone x receptor complexes. Complete dissociation of these complexes was achieved within 20 to 30 min at 0 degrees C. This process was a pseudo-first order reaction with a t 1/2 much less than the t 1/2 for hormone dissociation for either receptor at 0 degrees C. Hormone displacement was a general property of mercurial reagents; several organic mercurials as well as HgCl2 were effective. In contrast, sulfhydryl-alkylating agents (maleimides, iodoacetamide) and the disulfide 5,5'-dithiobis(2-nitrobenzoate) were ineffective in displacing bound hormone from either progesterone or vitamin D receptors. Finally, hormone displacement by mersalyl was reversible; addition of excess thiol reagent displaced the bound mersalyl and regenerated hormone binding activity in good yield. This result suggests that mercurial reagents should prove useful in further study of steroid hormone receptors, for example in elution of receptors from steroid-affinity adsorbents.     

Progesterone action through aggregation of a receptor on the sperm plasma membrane.

Rapid steroid effects, reported in several cell types, have pointed out the possibility of non-genomic mechanisms of action, presumably on cell surface receptors. Here we analyzed the effects of antibody-mediated aggregation of a novel type of progesterone receptor on the plasma membrane of human sperm cells. We report that aggregation of hormone-receptor complexes induces Ca2+ influx and a Ca(2+)-dependent exocytotic event in this system. These data suggest a possible mechanism for rapid steroid-induced events. Further research is warranted to examined if a similar mechanism is involved in rapid steroid effects in other cell types.     

Combined effects of levonorgestrel and quinestrol on reproductive hormone levels and receptor expression in females of the Mongolian gerbil (Meriones unguiculatus)

The effects of treatment with a combination of levonorgestrel and quinestrol (EP-1; ratio of 2:1) on reproductive hormone levels and the expression of their receptors in female Mongolian gerbils were examined. We show that serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) decreased, whereas serum estradiol (E2) and progesterone (P4) increased after EP-1 treatment. EP1 down-regulated mRNA expression of the follicle-stimulating hormone receptor (FSHR) and the estrogen receptor (ER) βin the ovary. EP-1 up-regulated the mRNA expression of the luteinizing hormone receptor (LHR) and the progesterone receptor (PR) in the ovary as well as ERα and PR in the uterus of Mongolian gerbils. The effects were time-dependent and dose-dependent. EP-1 had no obvious effects on ERα mRNA expression in the ovary. The current study demonstrates that the effect of EP-1 on the expression of ER subtypes is tissue-specific in Mongolian gerbils. EP-1 disrupted the reproductive endocrinology of the Mongolian gerbil. These findings suggest that the effects of EP-1 on reproductive hormone levels and their receptor expression in Mongolian gerbils may be the result of synergistic actions of levonorgestrel and quinestrol, with quinestrol playing the major role.     

Coactivation of an endogenous progesterone receptor by TIF2 in COS-7 cells

Transfection experiments, a powerful tool to study the function of steroid hormone receptors and their coregulators, are often performed in COS-7 cells, because of high transfection efficiencies and expression levels. Here we report on the presence in COS-7 cells of an endogenous steroid hormone receptor, which is highly responsive to progesterone and the synthetic steroids R1881 and ORG2058, but not to 5 alpha-DHT. A 10-fold excess of the progesterone antagonist RU486 abolishes the stimulation by progesterone, while cotransfection with the coactivator TIF2 increases its activity 6- to 7-fold. A comparison of the ligand specificity with transfected androgen or progesterone receptors indicates that the endogenous receptor is a progesterone receptor. Its presence is confirmed by steroid-binding experiments, RT-PCR and Northern blot analysis. Consequently, progesterone receptor function may be studied conveniently in COS-7 cells without cotransfection of receptor, but the endogenous receptor may interfere in studies of ligand specificity and coactivation of cotransfected receptors.     

Progesterone receptors on human spermatozoa

Progesterone, primarily recognized as a female steroid hormone, is reported to affect several sperm functions especially capacitation, motility and acrosome reaction. These effects of progesterone on the spermatozoa are mediated via the progesterone binding sites/progesterone receptor (PR) on the acrosomal membrane. These receptors in response to progesterone increase the intercellular Ca2+ levels and stimulate Ca2+ influx in the mature human spermatozoa via non-genomic mode of actions. Characterization of this receptor reveals that the sperm PR is masked protein and is exposed to the surface by some non-ionic detergents. Localized on to the acrosome region of the spermatozoa, these receptors are recognized by most antibodies directed towards the C-terminal region of the conventional PR. The estimated molecular weight of PR on spermatozoa varies from 27 kDa to 85 kDa. At the molecular level, sequences encoding for the entire DNA and hormone binding domains of the conventional PR are detected in the mRNA derived from spermatozoa. No insertions, deletions or mutations are detected in this region. These results are suggestive of the fact that at least the C terminal region of the conventional PR is conserved in the sperm. It is hypothesized that post-translational modifications or peptide splicing of the conventional PR in spermatozoa may possibly lead to the variant of the steroid hormone receptor. Detailed characterization of the sperm PR will be important in understanding the alternate non-genomic mode of action of steroid hormone receptors.     

Ovarian hormone receptors in human mammary stromal cells

Mesenchymal cells of the rodent breast express both estrogen and progesterone receptors. Searches for these molecules in the human breast have yielded conflicting results. Following immunohistochemical staining of samples of normal human breast tissue, the authors detected estrogen receptor alpha protein and progesterone receptor protein in extralobular (non-specialized) fibroblasts and estrogen receptor alpha protein in adipocytes. Tissues from young teenage girls and pregnant women contained the greatest number of receptor positive fibroblasts. These observations confirm prior reports of the presence of ovarian hormone receptors in mammary fibroblasts. The findings also illustrate similarities in the organization of the rodent and human breasts and thereby suggest that regulation of the gland by ovarian hormones involves similar mechanisms in both species.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.