Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.     

Steroid regulation of progesterone synthesis in a stable porcine granulosa cell line: a role for progestins

The objective of this investigation was to determine the effect of steroid hormones on the synthesis of progesterone in a stable porcine granulosa cell line, JC-410. We also examined the effect of steroid hormones on expression of the genes encoding the steroidogenic enzymes, cytochrome P450-cholesterol side chain cleavage (P450scc) and 3β-hydroxy-5-ene steroid dehydrogenase (3β-HSD). We observed that 48 h exposure of the JC-410 cells to estradiol-17β (estradiol), androstenedione, 5-dihydrotestosterone, levonorgestrel, and 5-cholesten-3β, 25-diol (25-hydroxycholesterol) resulted in stimulation of progesterone synthesis. 25-Hydroxycholesterol augmented progesterone synthesis stimulated by estradiol, 5-dihydrotestosterone, levonorgestrel and 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP). This increase in progesterone synthesis was additive with estradiol, 5-dihydrotestosterone and levonorgestrel, and synergistic with 8-Br-cAMP. Cholera toxin, progesterone, levonorgestrel and androstenedione increased P450scc mRNA levels, whereas estradiol had no effect. Cholera toxin, progesterone and levonorgestrel increased 3β-HSD mRNA levels, but estradiol and androstenedione had no effect. The results were interpreted to mean that estrogens, androgens and progestins regulate progesterone synthesis in the JC-410 cells. The effect of androgens appears to be mediated by stimulation of P450scc gene expression while progestins stimulate both P450scc and 3β-HSD gene expression. Our results support the concept that progesterone is an autocrine regulator of its own synthesis in granulosa cells.     

Depletion of substrates for protein prenylation increases apoptosis in human periovulatory granulosa cells

Progesterone receptor (PR) stimulation promotes survival in human and rat periovulatory granulosa cells. PR antagonists, Org 31710 and RU 486, both increase apoptosis and decrease cholesterol synthesis in these cells. The decrease in cholesterol synthesis also causes decreased synthesis of other products branching from the cholesterol synthesis pathway, including substrates for protein prenylation. In this study we focus on the link between apoptosis and prenylation in human periovulatory granulosa cells. A decreased cholesterol synthesis and increased apoptosis was verified in experiments with human periovulatory granulosa cells treated with the PR antagonists Org 31710 or RU 486 by measuring caspase-3/7 activity and incorporation of 14C-acetate into cholesterol and progesterone. Correspondingly, specific inhibition of cholesterol synthesis in periovulatory human granulosa cells using HMG-CoA reductase inhibitors (lovastatin or simvastatin) increased apoptosis, measured as caspase-3/7 activity. The increase in apoptosis caused by simvastatin or Org 31710 was partially reversed by addition of the protein prenylation precursors farnesol or geranylgeraniol. In addition, the prenylation inhibitors FTI R115777 and GGTI 2147 increased apoptosis in these cells. In conclusion our data suggest that PR antagonists increase apoptosis and reduce cholesterol synthesis in periovulatory granulosa cells and that the resulting depletion of substrates for protein prenylation may contribute to the increased apoptosis sensitivity.     

miRNA介导PGR信号通路在雌性生殖功能调节中的作用机制

MicroRNA(miRNA)作为重要的后转录调节因子参与多种生理活动。孕酮(Progesterone,P4)是重要的甾类激素,通过结合特异性受体——孕酮受体(Progesterone receptors , PGR)发挥生理作用。PGR作为核受体超家族的一员参与调控生殖相关组织或非生殖相关组织的功能。P4/PGR和miRNA可单独在雌性生殖中发挥调控作用。然而,在雌性生殖过程中,miRNA和P4/PGR的相互作用对调控排卵等雌性生殖活动起到非常重要的作用,但作用机制还未阐明。本文综述了miRNA调节P4产生、PGR基因表达以及P4/PGR调节miRNA表达的可能作用方式,为更好地研究miRNA和P4/PGR在雌性生殖中的作用提供理论基础。     

Effects of 17-hydroxyprogesterone caproate (17-OHPC) administration to pregnant squirrel monkeys (Saimiri boliviensis boliviensis)

Poor reproductive performance of the squirrel monkey (Saimiri boliviensis boliviensis) in captivity and a relative progesterone (P) deficiency in pregnancy have been reported. Since premature births may contribute to pregnancy wastage, we attempted to measure the effectiveness of 17-hydroxyprogesterone caproate (17-OHPC) treatment of pregnant squirrel monkeys to prevent early deliveries. Based on clearance studies of nonpregnant animals, 25 mg of 17-OHPC was administered at 6-day intervals to a test group of 31 pregnant monkeys while the control group of 29 received saline. Venous blood was obtained at 6- to 12-day intervals for measurement of 17-hydroxyprogesterone (17-OHP), P, 17-B estradiol (E), and androstenedione (A), and dihydroepiandrosterone (DHEA) levels by radioimmunoassays. The treated group had a significant increase in serum 17-OHP (P < 0.001), P (P < 0.01), and DHEA (P < 0.05) levels compared to controls. The numbers of live births, stillbirths, or neonatal deaths did not differ significantly between groups. Although 17-OHPC administration appeared to increase P and 17-OHP levels, this did not alter the duration of pregnancy nor delay the onset of labor. A significant fall in 17-OHP, P, and E levels was observed 6–12 days before delivery.     

Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor

The functional characteristics of membrane progesterone receptors (mPRs) have been investigated using recombinant mPR proteins over-expressed in MDA-MB-231 breast cancer cells. Although these cells do not express the full-length progesterone receptor (PR), it is not known whether they express N-terminally truncated PR isoforms which could possibly account for some progesterone receptor functions attributed to mPRs. In the present study, the presence of N-terminally truncated PR isoforms was investigated in untransfected and mPR-transfected MDA-MB-231 cells, and in MDA-MB-468 breast cancer cells. PCR products were detected in PR-positive T47D Yb breast cancer cells using two sets of C-terminus PR primers, but not in untransfected and mPR-transfected MDA-MB-231 cells, nor in MDA-MB-468 cells. Western blot analysis using a C-terminal PR antibody, 2C11F1, showed the same distribution pattern for PR in these cell lines. Another C-terminal PR antibody, C-19, detected immunoreactive bands in all the cell lines, but also recognized α-actinin, indicating that the antibody is not specific for PR. High affinity progesterone receptor binding was identified on plasma membranes of MDA-MB-468 cells which was significantly decreased after treatment with siRNAs for mPRα and mPRβ. Plasma membranes of MDA-MB-468 cells showed very low binding affinity for the PR agonist, R5020, ≤1% that of progesterone, which is characteristic of mPRs. Progesterone treatment caused G protein activation and decreased production of cAMP in MDA-MB-468 cells, which is also characteristic of mPRs. The results indicate that the progestin receptor functions in these cell lines are mediated through mPRs and do not involve any N-terminally truncated PR isoforms.     

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。     

Structural Features of Pregna-D′-pentaranes Determining Their Interaction with Three Rat Proteins

Competition of a number of progesterone 16,17-cycloalkane derivatives with ³H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16,17-cyclopropanoprogesterone, and 16,17-cyclopent-3-enoprogesterone and the selective ligands for serum pentaranophilin are 6-methyl-16,17-cyclohexanopregna-1,4-diene-3,20-dione and 3-hydroxy-16,17-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D we studied decreased the affinity of ligands for all the three proteins. The substitution of the ⁵-3-hydroxy grouping for the ⁴-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3,4-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets.     

Progesterone and allopregnanolone improves stroke outcome in male mice via distinct mechanisms but neither promotes neurogenesis

Based on the outcome of a number of experimental studies, progesterone (PROG) holds promise as a new therapy for stroke. To understand more about the mechanisms involved, we administered PROG (or the major metabolite, allopregnanolone, ALLO), intra‐peritoneally, for a period of 24 h after transient middle cerebral artery occlusion to male mice variably expressing intracellular progesterone receptors (iPR) A/B. Effects on infarct volume and neurogenesis were then assessed up to 1 month later. Predictably, infarct volume in wild‐type mice receiving either drug was significantly smaller. However, mice heterozygous for iPRs A/B showed protection by ALLO but not by PROG. There was robust amplification of cell division in the wall of the lateral ventricle on the injured side of the brain, these cells migrated into the striatum and lateral cortex, and a significant number survived for at least 3 weeks. However, very few doublecortin‐positive cells emerged from the subventricular zone and subsequent expression of NeuN in these newborn neurons was extremely rare. Neither PROG nor ALLO amplified the rate of neurogenesis, suggesting that the long‐term benefits of acute drug administration results from tissue preservation.

     

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.     

哺乳类动物妊娠晚期孕激素撤退的三种机制

内分泌激素是维持妊娠和启动分娩的重要因素。孕激素是静息子宫、维持妊娠的主要激素,而糖皮质激素、前列腺素和雌激素等激素则与分娩启动密切相关。孕激素水平的下降是很多哺乳类动物分娩启动的前提条件,然而有些哺乳类动物包括灵长类在整个妊娠过程包括分娩中均维持着高水平的孕激素,此现象令人费解。越来越多的证据表明,人类分娩启动时孕激素同样出现了撤退,但是发生在孕激素的受体水平,主要表现为孕激素受体亚型表达比值和孕激素受体转录辅助因子表达的改变。本文比较了人类和其它哺乳类动物分娩启动时孕激素撤退的三种模式,即黄体溶解、胎盘P450c17羟化酶上调和孕激素受体功能改变,旨在进一步阐明人类分娩启动机制,从而为防治早产提供新的思路。     

Progesterone as a neurosteroid: synthesis and actions in rat glial cellsProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

The central nervous system (CNS) and the peripheral nervous system (PNS) are targets for steroid hormones where they regulate important neuronal functions. Some steroid hormones are synthesized within the nervous system, either de novo from cholesterol, or by the metabolism of precursors originating from the circulation, and they were termed ‘neurosteroids'. The sex steroid progesterone can also be considered as a neurosteroid since its synthesis was demonstrated in rat glial cell cultures of the CNS (oligodendrocytes and astrocytes) and of the PNS (Schwann cells). Both types of glial cells express steroid hormone receptors, ER, GR and PR. As in target tissue, e.g. the uterus, PR is estrogen-inducible in brain glial cell cultures. In the PNS, similar PR-induction could not be seen in pure Schwann cells derived from sciatic nerves. However, a significant PR-induction by estradiol was demonstrated in Schwann cells cocultured with dorsal root ganglia (DRG), and we will present evidence that neuronal signal(s) are required for this estrogen-mediated PR-induction. Progesterone has multiple effects on glial cells, it influences growth, differentiation and increases the expression of myelin-specific proteins in oligodendrocytes, and potentiates the formation of new myelin sheaths by Schwann cells in vivo. Progesterone and progesterone analogues also promotes myelination of DRG-Neurites in tissue culture, strongly suggesting a role for this neurosteroid in myelinating processes in the CNS and in the PNS.     

Estrogen-Inducible Progesterone Receptors in the Rat Lumbar Spinal Cord: Regulation by Ovarian Steroids and Fluctuation across the Estrous Cycle

Ovarian hormones influence the physiology of the spinal cord through incompletely understood cellular mechanisms. To date, there has been little compelling evidence for progesterone receptors in spinal cord neurons. Using two antibodies specific for progesterone receptors in an immunohistochemical investigation, we now report the presence of estrogen-inducible progesterone receptors in the spinal cord. Estrogen-inducible progesterone receptors were observed in the neurons of lamina X and the interomedialateral cell column, which are also known to express estrogen receptors. Estrogen-inducible progesterone receptors similar to those observed in females were also apparent in lamina X and interomediolateral cell column neurons in the spinal cords of males treated with estradiol. Furthermore, the density of progesterone receptors in lamina X was observed to fluctuate across the estrous cycle in female rats, with the highest progesterone receptor expression levels occurring late in proestrus, following the estradiol surge and coincident with high circulating progesterone levels. The lowest progesterone receptor expression levels were observed late in estrus following the progesterone surge. Together, these results demonstrate that estrogen-sensitive progestin targets exist in the spinal cord, and their possible role in the nervous control of reproduction and ovarian steroid modulation of nociception is discussed.     

Four-year study of controlled timed breeding of rhesus monkeys (Macaca mulatta)

As part of the timed breeding colony at Tulane National Primate Research Center, exogenous progesterone administration (5 mg/day for 10 days) has been used to select conception dates by inducing artificial luteal phases in female rhesus monkeys. A retrospective analysis of data obtained during four breeding seasons (1998-2001) revealed that conceptions occurred an average of 18 days after the last administration of progesterone. The age of the female to be bred, previous pregnancy history, and timing of breeding during the breeding season were determined to be critical factors in the success of the procedure. The benefit of this method of timed breeding is that it does not require tracking of menstrual cycles, which can be labor-intensive and requires that animals be monitored several months in advance of breeding to determine each female's individual cycle length. It also provided an efficient use of breeding-age males.     

雌、孕激素联合应用对豚鼠心室乳头肌细胞动作电位及收缩力的影响

目的:观察不同浓度17β雌二醇(E2)与孕酮(P)联合运用时豚鼠心室乳头肌细胞动作电位及心肌收缩力的影响.方法:采用常规玻璃微电极技术记录豚鼠心室乳头肌细胞动作电位,生理二道记录仪与示波器连接记录收缩力.结果:①E2与P单独作用可使动作电位时程(APD90、APD)延长,APD20缩短,心肌收缩力降低,APA、Vmax降低.②不同浓度E2与P联合应用,P可加强E2的效应.结论:E2与P可能抑制Ca2 通道、K 通道、Na 通道,不同浓度E2与P联合应用,P可加强E2的效应,这种作用可能呈现浓度依赖性,提示在临床应用中加用孕激素应以低浓度为益.     

Identification of membrane progestin receptors in human breast cancer cell lines and biopsies and their potential involvement in breast cancer

Novel membrane progestin receptors (mPRs) coupled to G proteins recently identified in several species, including humans, are potential intermediaries in rapid, nongenomic progestin actions observed in a wide variety of tissues. Here we demonstrate mPR mRNA and protein expression and specific membrane-associated progestin binding in MCF-7 and SK-BR-3 human breast cancer cells. Interestingly, human mPRalpha mRNA expression was higher in breast tumor biopsies than in normal tissue from the same breast. Recent studies indicate intracellular signaling pathways initiated by the mPRs are broadly similar to those induced during breast cancer growth and development. Taken together these results suggest a potential involvement of mPRs during the development or progression of breast cancer.     

The binding of progesterone in different parts of the rabbit uterus during implantation

Progesterone receptors, both nuclear and cytosolic, were determined in the embryonic and inter-embryonic segments of the rabbit uterus at 6, 7 and 8 daypost-coitum. At day6 postcoitum a higher concentration of nuclear receptor in the embryonic segment was observed compared with that in the inter-embryonic segment. A reverse situation was observed in the case of cytoplasmic receptors. On the 7th daypost-coitum, no significant alteration in the concentration of either kind of the receptors was observed. However, on day 8, a higher concentration of both nuclear and cytosolic receptors at the embryonic site was observed compared to that in inter-embryonic segment. Since receptors are influenced only in the immediate vicinity of the blastocyst, it can be suggested that the blastocyst plays a role in the induction of its own implantation. Further, at day 8 increase in receptor concentration at the embryonic site may be related to the presence of decidual tissue at this site. CDRI Communication no. 2741.